The increased use of traditional anti-inflammatory drugs, which have many side effects, scientists and researchers have turned towards natural products to avoid the side effects of traditional chemical drugs. Plants are used in ancient times due to their association with humans as food and medicine. Different parts of plant have been common among people and pharmaceutical industry used in food and medical industries for production of drugs used in treatment of most diseases a variety of active constituents with wide range of pharmacological actions have been approval in recent years. The analgesic and anti- inflammatory effect of some of these plants has also been found. Among these plants, watercress (Eruca sativa) was used as food in different area of the world. The present study aimed to investigate the analgesic and antiinflammatory properties of watercress (Eruca sativa) leaves extract (WE) using rats. Evaluated of analgesic activities were done through hot plate, tail immersion, and writhing assay tests at different WE doses (100,200 and 300 mg/kg) using male albino rats. Antiinflammatory assay was performed by carrageenan induced paw edema of WE at different doses (100,200 and 300 mg/kg). Morphine and aspirin were employed as a standard for analgesic tests while Indomethacin was used for anti-inflammatory studies, The present study demonstrated that watercress (Eruca sativa) leaves extract (WE) have analgesic and anti-inflammatory properties through inhibition the release mediators. In the current study, the higher dose of WE (300mg/kg) exhibited greater analgesic and antiinflammatory activities compared to the control and standard drugs. WE also showed good effects on pain and inflammation and therefore it can be said that WE can be used as a good alternative to relieve the effects of pain and inflammation without side effects of NSAIDs.

Pain and inflammation remain among the most common global health problems that humans face socially and economically throughout the world, regardless of the availability of medicines or their high prices [1,2]. Inflammation and pain are common manifestations for many diseases of various endogenous and or exogenous mediators and considered as nature defense mechanism aimed to remove the injury and the tissue healing [3]. Pain brought on by tissue injury or probable occur is considered a source of anxiety and discomfort for humans due to the constant unpleasant sensory impact on a location in the human body and may affect the movement and activity of the entire human body in many cases [4], Pains consider mechanism forces these injured to quickly seek treatment for these injuries and recover from them [5]. Many people is influenced by pain and inflammation, which are difficult health problems over the world particularly developing countries [6]. Many Opioids and non-opioids materials were found as anti-inflammatory drugs to treat both acute and chronic pains [7,8] reported the acute and chronic pains leads to the development and increase of the disease condition in people infected with these infections. Inflammation is considered a natural response to movement and vital processes taking place within the body’s cells, and it is a signal of the biological line of defense to confront the dangers of movement, infection, damage to cells and tissues, such as redness, swelling, high body temperature, and pain[8-10]. Moreover, inflammation produced from endogenous metabolic processes are consider the most important sources of free radicals react with lipids, proteins, carbohydrates and damage all types of biomolecules [11-13]. Therefore, it is necessary to rapid combat pain and inflammation and also in need to search using new sources produce analgesics and anti-inflammatory drugs [14,15]. Recent experimental studies of pain and inflammation has been shown that many types of medications used as antidepressants, such as amitriptyline and nortriptyline, Imipramine, fluoxetine, clomipramine, maprotiline, desipramine and doxepin widely used to treat various types of pain and inflammation [8,10,16,17]. Long-term use of conventional anti-inflammatories causes stunted growth, high blood sugar, high blood pressure, and osteoporosis. [18,19]. Other studies reported that the long-term use of anti-inflammatory drugs causes toxicity to the heart, digestive system, kidneys, and liver, in addition to addiction and imbalanced hormonal disorders [20,21]. Antiinflammatory drugs, especially opioids and non-steroidal anti-inflammatory drugs (NSAIDs) are used as analgesics to reduce and eliminate pain completely [15,22]. NSAIDs are among the most clinically widely used in medicine due to their efficacy for a wide range of pain and inflammatory conditions and used for treatment of inflammation-related diseases like arthritis, asthma, and cardiovascular disease [10,23]. However, the long-term administration of NSAID may induce gastro-intestinal ulcers, bleeding, and renal disorders. Different pharmacological approaches were used for treated pain and inflammation as opioids analgesics corticosteroids and some combination therapy to enhance efficacy [16,17], indicated these drugs having side effects,\ including bronchospasm, gastric ulcer, cardiac abnormalities and renal damage. Therefore, researchers are now focusing on plants and their products/compounds [7,15]. such as polyphenols, polysaccharides, alkaloids, flavonoids, steroids, and terpenes as they have different pharmacological activities including analgesic, antipyretic and anti-inflammatory effects [24,25,26].These medications are used with extreme caution because they are narcotic substances and their effectiveness in treating pain and inflammation is questionable [16]. Therefore, new anti-inflammatory and analgesic drugs lacking those effects are being searched all over the world as alternatives to NSAIDs and opiates [23,27,28].Some studies showed the use of biosynthetic drug as treatment of analgesic and inflammation produces disturbances in human body organs and other side effects [18,29]and hence different substances of plant origin are gaining importance for the treatment of analgesic and inflammation. Plants are used in ancient times due to their association with humans as food and medicine for many diseases for many years [30,31]. Different parts of plant have been common among people and pharmaceutical industry were found extensively utilized in food and medical industries to synthesize and produce drugs of biological activity in the treatment of most diseases [32,33]. Other investigators [34,35], showed that the most population in the developing countries depends on traditional medicines for treatment of diseases due to inexpensive of herbal medicines availability and the lack of medical facilities [35,36]. Various plant products as alkaloids, saponins, triterpenes, glycosides, polysaccharides, polyphenols and flavonoids have shown anticancer properties [26,37]. Other phytoconstituents such as tannins and anthraquinone were heals wounds and inflammations [34,38]. Plants are an important main source for the production of new chemicals [39,40,41] that have therapeutic effects for various diseases and necessary research strategy to search for new analgesic and anti-inflammatory drugs [42,43,44]. Plants and their components were also used to prevent epidemics and various diseases, as it was found most of them have antioxidant, anti-inflammatory and anti-parasitic properties [36,45,46]. According to the increases required for treatment for analgesic and anti- inflammatory this leads to scientists have intensified their efforts to search for other sources to produce plant derived materials and compounds that can be used to treat pain and inflammation [10,47,48]. Recently, many natural compounds such as terpenoids, phenolic, flavonoids, lignans and others were discovered from plant sources [34,49,50], they manifested that these compounds have anti-inflammatory, antitumor and anticarcinogenic activities. Natural products have been regarded as important sources of potential chemotherapeutic agents and drugs have shown antiinflammatory activity [46,51,52]. Among natural products, plant extract, have antimicrobial, antifungal, anti-diarrhoea, anti- viral, antitumor, antifertility and anti-inflammatory activities [49-56]. Different studies reported different biological activities of substances or compounds were isolated from plants origin used as antidiabetic [13,57], antihypertensive [58], cancer chemopreventive effects [59] anticancer activities, [60], and antiepileptic properties [46,61] due to their phytochemical constituents. Other investigators reported some plants have been use, in treatment of disorders and other biological activities including antifungal and antibacterial effects due to its antioxidant properties [62]. Polysaccharides were isolated from Portulaca oleracea prevents vascular inflammation, and dysfunction in mice [13,63]. Plant extract are non-toxic and biodegradable that consequently suitable for different pharmaceutical and biomedical uses [64]. Recent study has shown that some plant extract intake cause improve in some biochemical parameters [60]. Different plants extract have been shown the potential health impacts in preventing some diseases and have antiinflammatory activities [64,65]. Current researchers concentrate to examine new kinds of natural products shown maintenance health and analgesic and anti-inflammatory [66,67]. Phytochemicals such as polyphenols, flavonoids and fatty acids have considerable interest in the field of food chemistry, pharmacy and medicine due to their biological effects including analgesic and inflammation properties [46,60]. Plant-based substances may act as a physiological modulator for the treatment of pain and inflammation. Plant contains different compounds which exert different kinds of pharmacological activity [59,64,68] more than those of synthetic compounds. Many plant extracts and their constituents were found to be used for treatment of many kinds of human diseases [60,69]. Different studies were done for extraction of pure bioactive compounds from plant [70]. Most studies have been shown the beneficial effects of diets rich in plant in reducing the inflammation [71] correlate increased phenolic compounds levels in foods with reduced disease mortality. Flavonoids consider a major group of polyphenolic compounds that considered are essential constituents of plant cells possess [62,72,73]. Therefore, watercress (Eruca sativa) extract can be used as strong sources of novel analgesic and ant-inflammatory agents [74,75,76]. The present research was done to investigate the effects of watercress (Eruca sativa) extract (WE) against pain, analgesic and anti-inflammatory activities using male albino rats.

Materials

·  A fresh watercress (Eruca sativa) as whole plants were obtained from local vegetable market in Egypt. The plants were cleaned carefully and damaged plants were removed, washed with tap-water followed by distilled water and cut into small pieces and stored at 4 º C till used.

· Chemicals used in the present study, Morphine, aspirin, Indomethacin, carrageenan, and acetic acid solution were obtained from Sigma Chemicals Co., St Louis, USA.

Thirty five male albino rats (Rattus norvgicus), weighing 160±1.02 g/kg were purchased from Biological Products of National Research Center, Cairo, Egypt. Rats were kept under controlled temperature with 12±1h light/dark cycle at the Institute of Biological Products of National Research Center. Rats were fed with standard commercial pellet diet and given water ad libitum. Further, the animals were under investigation up to a period of 2 weeks for mortality and behaviors. After two week of acclimatization, the rats were then divided into seven groups, 5 rats each, on the basis of their body weight, housed in wire screen cages. Rat group administered saline (10 mg / kg) only was used as control group. Three rat groups orally administered the standard drugs for analgesic and anti-inflammatory assays (morphine,10 mg/kg for the hot plate, aspirin 150 mg/kg for the acetic acid-induced writhing, and indomethacin, 25 mg/kg for the carrageenan-induced paw edema).The remaining three rat groups were administered WE at different doses of 100, 200 and 300 mg per kg body weight respectively, Following a period of one hour after oral administration of saline, standard drugs or extracts, rats were intraperitoneally injected. Study was carried out in agreement with NRC and approval was done by ethical review committee of NRC, Dokki, and Cairo, Egypt.

Preparation of Extract

A known weight of fresh watercress (Eruca sativa), was ground in a food grinder (mincer) and mixed well with hot water (1:1 V/V) twice using a homogenizer for 5 min. The homogenate was filtered through cheesecloth and Whatman No.1 filter paper. The obtained watercress (Eruca sativa) extract (WE) was used for chemical analysis and oral administration to the rats [40].

Analytical methods

Protein concentration was measured using bovine serum albumin as a standard [77]. Lipids were extracted with chloroform-methanol mixture (2:1 V/V), according to the method previous described [78]. Total carbohydrate value was also estimated [79]. Ashes were quantified gravimetrically after incineration in a muffle oven at 550 °C. Phenolic and flavonoid were estimation [80].Total phenolic content (TPC) was determined [81,82]. Total flavonoid contents (TFC) was estimated spectrophotometrically [83]. Flavonoids was identified using apigenine, quercetin and catechin as standard [84,85]. Fatty acids composition of WE were also estimated [86,87] using gas liquid chromatography (GLC).

Cytotoxicity

Cytotoxicity test of the present extract (WE) was measurement according to the method described previous [88,89], Watercress extract (Eruca sativa) extract (WE) were administered orally to overnight fasted rat at the doses of 100, 200 and 300 mg/kg body weight (b. w.). After administration WE, the rats were observed continuously for 72 hours. Rats were under investigation for a period of two weeks for following their general behavior, toxicity, physiologically reaction and mortality [90,91]. The doses were selected based on cytotoxicity study.

Anti-inflammatory and analgesic assay

For the analgesic and anti-inflammatory activity tests, rats were divided into seven groups, each consisting of 5 animals [40,88]. One group was orally administered distilled water and used as control group, three group were designated as the standard (morphine 20 mg/kg for the hot plate method, aspirin 150 mg/kg for the acetic acid-induced writhing, and indomethacin 10 mg/kg for the carrageenan-induced paw edema). The remaining three treated groups were given orally three different doses of WE (100, 200, and 300 mg/kg b.w. respectively).

Hot plate method

Hot plate test was done to evaluate the analgesic activities of extract used in the present study. Pain was induced by placing the rat on a hot plate at 55 ±1 °C and the response time was recorded as an index of analgesic activity. Rats were reacted to the thermal pain by licking of their hind paw and jumping where the reaction time was measured in a regular time interval and the reaction strength of each rat was determined in a regular intervals of at 0 min, 30, 60, 90 and 120 min and the time increase was taken as an index of analgesic activity. Analgesia was compared the groups ertr administered WE doses with control and standard drug groups [26,90].

Tail immersion test

The method described previous [92] was used in this test. Rats were divided into seven groups of 5 rats each. The lower end portion of rats tail 5 cm were immersed in hot water maintained at 55 ± 0.5? C [93,94]. Within a few minutes, the rats reacted by withdrawing the tail from hot water. The time for tail withdrawal from the hot water was taken as the reaction time and recorded using stopwatch. The time reaction was measured in seconds for the rat withdrawal their tail from the water and was taken as the reaction time. The reaction time was measured [23]. Oral administration of WE doses (100, 200 and 300 mg/kg, b. w.), saline and standard drug, 30min before the immersion of the tail. The time reaction is taken at 0, 30, 60, 90 and 120 minutes.

Acetic Acid-Induced Writhing Assay

Analgesic activity of extract was determined by acetic acid-induced writhing test [95,96], Rat of either five were given WE doses, One hour later, rats in their respective groups received intraperitoneal injections of 1 % acetic acid (10 mL/kg b.w.). The analgesic activities of the WE doses were measured five minutes after the acetic acid injection by counting the numbers of writhing, which is characterized by contraction of the abdominal muscle together with stretching of the hind limbs for 30 minutes. The numbers of abdominal writhing were counted for 20 min observation beginning at 5 min after the injection. The rats were treated with WE (100, 200 and 300 mg/kg b. w.), 30 min before administration of acetic acid. The percent reduction in the number of writhes relative to the control group was used as an index of analgesia [95,97,98].The significant reduction in number of writhes of treated groups was compared to that of the control and standard groups. The percentage inhibition of abdominal constrictions was calculated [90].

Carrageenan induction test

Anti-inflammatory activity was investigated using paw edema method [98,99] using carrageenan and plethysmometer for anti-inflammatory assay. Rats were in fasting condition before start the experiment, For induction of edema, an hour after oral administration of control, standard drug and WE tests and the paw edema volumes were recorded after 30 min, 60 min, 90 min. and 120 min s of carrageenan injected using plethysmometer [99,100]. Acute inflammation was induced half an hour after treatment by injection of carrageenan (0.1 mL freshly prepared) in left hind paw of rats [99,101]. The rat paw volume was measured at 1, 2, 3, 4 and 5hrs after the carrageenan injection [102].The difference between the rats paw volumes indicated to the degree of inflammation [103]. Edema was expressed as percent increase in rat paw volume due to administration carrageenan compared to rat paw given the control and standard drugs. The average increase in paw volume of each group was calculated and compared with the control and the standard groups [8].

Statistical Analysis

Data were analyzed using ANOVA statistical and the results were presented as mean value

± Standard error (SE).

Chemical composition of watercress (E. sativa) extract (WE)

Natural products have been widely used in treat of pain and inflammation. Watercress (E. sativa) is among natural products widely used between ancient people societies for treating some diseases [104,105]. Little scientific reports were found in the literature about activities of watercress (E. sativa) in analgesic and anti-inflammatory using experimental animal. Phytochemicals an evidence for the pharmacological potential of the plant secondary metabolites having different activities in fighting cytotoxicity and diseases [106]. Watercress (Eruca sativa) extract sample (WE) was obtained and used for chemical composition. Results in (table 1) revealed the chemical composition of watercress (E. sativa) extract (WE). Chemical analysis of WE revealed the presence of different contents of protein, lipid, carbohydrate, phenolic and flavonoid as shown in (Table 1). WE contain different percentages of protein, lipid and carbohydrates (24.40, 22.60 and 48.20% respectively) Similar results were obtained by other investigators [31,76], they found higher protein content in watercress (Eruca sativa) seeds than the other plant seeds. Other investigators found the protein content of plant extracts was 21.7% [32,107]. Results also showed the lipid contents in WE (22.60%) which are in the range with those reported by other investigators [37,108] using different plant extracts. Higher level of carbohydrates was observed in WE (48.20%) as shown in Table (1). Similar results were obtained by several investigators [32,107,109]. Data in (Table 1) showed the WE contain suitable content of phenol (28.20 mg/g) and flavonoid (22.80 mg/g).These results are higher than those reported by other studies [76,109,110], they reported the total phenolic content was ranged from 12.7 to 25.6mg/100g in plant extract. Other investigators [111,112,113] reported the phenolic content was varied from 15.9mg/g to 22.7mg/g in different plant extracts These results are in agreement with the finding of other investigators [107.114,115] found the phenolic contents ranged from 0.98 to 3.35mg gallic acid equivalents per gram. Moreover, several studies [13,116,117] determined the content of phenolic compounds at a level of 0.44mg/g as gallic acid equivalents. Results in Table (1) showed higher flavonoid content in WE (22.80 mg/g). The present results are in a good agreement with those reported by other investigators [110,113]. Other investigators [46,60,76] found the flavonoid contents in three samples of Eurca sativa was ranged from 23 to 25%. Other studies [111,118] found lower flavonoid contents in radish, and Lettuce extracts. Lower flavonoid contents in safflower and coriander extracts was also obtained [32,119]. The present results showed significant and correlation with flavonoids and phenolic, suggest their involvement in various important characteristics such as analgesic and anti- inflammatory activities [46,117,120] reported phenolics and flavonoids have potential activities in biological systems, However, the highest flavonoid and phenolic contents were found in WE, have potentially analgesic, anti-inflammatory and antioxidant [23,62,121]. Therefore, WE used in the present study are recognized as dietary elements may be effects on human health and could be used as pharmaceutical and drug for treatment of different diseases. Results also showed the presence of lipids (22.60%) in WE sample as shown in (Table 1). The present results are in agreement with those reported by other investigators [65,122,123] using extract for production of safflower oils. Lower level (8.50%) was obtained from coriander [32]. The lipid content explains potential used of US as natural sources for higher lipid production recognized as dietary elements play an important role in human health. WE lipid in present study was used for analyses and determination of fatty acids (FAs) composition using Gas Liquid Chromatographic (GC). Generally, lipids have saturated fatty acids (SFAs) and unsaturated fatty acids (USFAs) as major contents. Different values of USFAs and SFAs were found in different plant lipids [31,124]. The percentages of SFAs, monounsaturated fatty acids (MUSFA) and polyunsaturated fatty acids (PUSFAs) were 14.40%, 26.80% and 58.80% respectively in the obtained WE sample as shown in (Table 1). Results also showed the PUSFAs were predominant than that of SFAs. These results are in agreement with those obtained by other investigators [86,125] found the USFAs were predominant than that of SFAs in different types of plant extracts. The analyses of WE sample using GC revealed the presence of of 7 main fatty acids linolinic acid C18:3 ?-3), linoleic acid (C18:2 ?-6) and oleic acid (C18:1 ?-9), palmitic acid (C16:0), stearic acid (C18::0), arachidic acid (C20:0) and behenic acid (C22::0) as shown in Figure (1).These variations induced differences in SFAs, MUSFA and PUSFAs as shown in Table (1). Five main fatty acids were detected in purslane extract [126]. Results showed higher percentage of PUSFAs, linolenic acid C18:3 (22.60 %) in WE, These results were quite close to the finding of other investigators [115,127], they detected the content of C18:3 was ranged from 23.65% to 37.5%while other study found the C18:3content was

10.56 [118]. Other workers found the C18:3 contents in rapeseed and coriander extracts was ranged from 5.2%to15.0% [32,107,128]. The WE analyses showed higher C18:2 content (36.20%), these results are higher than those reported by other investigators using coriander and watercress extracts [32,108,129] found C18:2 content was ranged from 12.3% to 16.60%. The level of C18:2 content was ranged from 5.9% to 14.5% in rapeseed extract [52,123]. The higher level of C18:1(26.80%) was found in WE as shown in Figure(1). These results are quite close in agreement to the previous finding [128,130]. Other studies [13,31] found the content of C18:1 was 22.2% in chickpeas extract while. 20.9% of C18:1 was obtained from radish extract [127,128]. Lower levels (9-15%) were obtained of C18:1 in dill and safflower extracts [107,119]. Lower level (9.90%) of C18:1 was found in watercress extracts [132] but lowest level (7.5%) obtained from coriander extract [32,62] while. Results showed percentages of SFAs (14.40%) in WE as shown in Figure (1). SFAs contents in rapeseed extract was 9.20% [128]. Many investigators [52,108,118] found the percentages of SFAs was ranged from 9.0 to 9.6 % in in different plant extract samples Similar results were obtained by other investigators [31,133] they found SFA content was ranged from 6.9% to 12.40% when they were used different plant extracts. These results are lower than those reported by other investigators [32,107,132] found 22.4% SFAs content in safflower extract. Results showed C16:0 and C18::0, C20:0 and C22::0 were identified in WE (Figure 1). Results also showed the percentages of C16:0 (6.80%)) and C18:0 (3.20%) were predominant in SFAs followed by C20:0 and C22:0(2.40 and 1.60% respectively). These results are in agreement with those obtained by other investigators [37,131] used different plant extracts. These results are in accordance with those finding by previous studies [31,128,130] obtained similar results of C16:0 and C18:0 percentages. C16:0 content was 5.7% while the C18:0 content was 5.0% in safflower extract [115]. Other investigators [31,32,132] reported the C16:0 content was 3.8% and 4.08% in watercress and coriander respectively. The C20:0 content was higher in US (2.4%). Many investigators obtained similar results of SFAs with coriander, lettuce and dill seed extracts [32,107,108]. These results are higher than those obtained by other investigators [107,118,132] found different ranges in the content of C16:0 (20–22%), C18:0 (1.5%) and C20:0(1.4%) using radish and watercress extracts [76,127]. Different variations (0.2-1.4% and 1.4-2.6%) were observed in the conent of C20:0 on using chickpeas [109,118,133] and purslane samples [118,131]. C22:0 level was 1.60 % as shown in (Figure1). Zambiazi et al. [134] found C22:0 in the samples of sunflower extract was 0.8%. However, it was excellent for the extraction of bioactive compounds involved in the prevention and treatment of different diseases. In this work, the effect of WE containing bioactive compounds possessing analgesic and anti-inflammatory activities was investigated by well-known pharmacological methods [13,19,135]. It seems that in WE, more phenolic, flavonoids and fatty acids with analgesic and anti-inflammatory activities [101,136,137] may account for more pronounced pharmacological properties with analgesic and anti-inflammatory activities [22,23,98]. In addition, polyphenols with analgesic and anti-inflammatory activities in WE is the reason for its pharmacological effects, which can reconfirm more effective role of fatty acids compared to polyphenols for decreasing pain and inflammation [19,135]. 

Table 1: Chemical composition of watercress (Eruca sativa) extract (WE). (Mean values of three samples).

Hot plate test

Hot plate method is the most significant method suitable to evaluate and determine the central analgesic activity including spinal reflexes of WE in rats. The paws of rats are very sensitive to heat at temperatures which are not damaging the skin. The responses are jumping, withdrawal and licking of the paws. The time until these responses occur is prolonged after administration of centrally acting analgesics [138,139] indicated the Jumping and paw licking are considered supraspinally integrated behavioral responses, and the time of the latency to the onset of this reaction following injection is a measure of the analgesic activity [121]. In this study, we used morphine (10 mg/kg body weight) as a standard and three doses of WE (100,200 and 300 mg/kg body weight) compared to control. The effect of WE doses in hot plate method was shown in (Table 2). Results showed significant analgesic effect by delaying the reaction time intervals (0 -120 minutes). The reaction time (paw licking / jumping response) in rat groups with different doses of WE (100, 200 and 300 mg/kg) were found to be increase started from 30 min. and remains significant increases till 120 min. as compared to control rat group (Table 2). The duration of analgesic effect was more in WE dose, 300 mg/kg compared to other two doses (100 and 200 mg/kg). Hot-plate test was also used to distinguish between peripheral and central effects [46,135,139]. The maximum analgesic activities of all doses of the WE (100, 200 and 300 mg/kg) were observed at 60 and 120 minutes of observation as compared with the s tandard drug as shown in table (2). These results are in accordance with previous study [121,140], reported the hot plate test is an easy and suitable method for determination of central analgesic activity. These results of analgesic activity of WE are attributed due to the presence of high levels of phenolic, flavonoids and FAs in WE (Table 1 and Figure 1 respectively). Thus, we containing phenolic, flavonoids and FAs have analgesic effect. WE exhibited significant increase in reaction time compared to control in rats showing central analgesic effect. Other findings [19,140,141] showed reduced of prostaglandins due to the presence of flavonoids might be responsible for analgesic activity as prostaglandins are involved in the process of pain perception [31,142] previously reported the presence of phenolic and flavonoids in some plant seeds are considered to have a role in biological system due to their capability of quenching free-radicals [60,120]. Other investigators [143,144] reported the presence of these flavonoids halt key enzyme prostaglandin synthetase involved in the process of pain perception. The reduced availability of prostaglandins is due to the presence of flavonoids might be responsible for analgesic activity [137,141]. The inhibition was significant at the dose of 200 and 300mg/kg body weight as compared to that of the standard drug, (Morphine 10 mg/kg b.w.) at 30 minutes after treatment. These results indicate that, WE at the dose of 200 and 300 mg/kg showed the significant increase in time as compared to control, produce significant analgesic activity. The WE showed a rapid onset of analgesic action at all WE doses than standard. The highest analgesic activity was observed at 60 and 120 minutes. WE showed highest significant analgesic activity at the dose of 300 mg/kg as compared to other two doses [19,40,145]. Based on the increased rat response times in the hot-plate test, it can be concluded that the WE has a central analgesic activity [139]. Tail immersion test

Tail immersion test was considered to determine the analgesics activities of WE in rats. Results showed significant elongation of reaction time of the WE doses after 60 and 120 minutes (Table 3). Maximum elongation in the reaction time percentage was observed at WE dose of 300mg/kg body weight after 60 and 120 minutes (76.86 % and 70.74% respectively) as compared to control rat group. Results also showed the reaction time percentage was observed at standard drug after 60 and 120 minutes (78.48 % and 72.22 % respectively) as compared to control rat group. WE at a dose of 200 mg/kg body weight showed elongation in the reaction time percentage (63.90 % and 67.56 % respectively) after 60 and 120 minutes. Lower elongation reaction time percentage (38.33 and 42.86 %) was observed at WE dose of 100 mg/kg body weight after 60 and 120 minutes respectively. The present WE with various compounds may act through a central and peripheral analgesic mechanism while NSAIDs block only peripheral pain [146]. Nonsteroidal anti-inflammatory drugs inhibit only peripheral pain while extract inhibit both peripheral and central mechanism of pain, suggesting that the plant extract may act as a narcotic analgesic [138,147]. Flavonoids may increase the amount of endogenous serotonin which may be involved in the mechanism of central analgesic activity [146,147]. Moreover, WE showed highest analgesic activity in all the experimental rats which may be due to the presence of high level of flavonoid which are responsible for free radical scavenging activity, as these free radicals are involved during pain stimulation, and antioxidants showed reduction in such pain [148]. The results of the present study have shown that the WE exhibited very high analgesic activities. These activities may be due to the presence of polyphenol and flavonoids compounds WE. Flavonoids and saponins are well known for their ability to inhibit pain perception as well as anti-inflammatory properties due to their inhibitory effects on enzymes involved in the production of the chemical mediator of inflammation [149]. Thus, the presence of phenolic and flavonoids in WE might be responsible for the analgesic activity in rats [19.22,23].

 Acetic acid induced writhing Assay

Acetic acid induced writhing test is commonly used for peripherally acting agents [150] that simulates visceral pain, and the writhing accompanied by abdominal muscular contraction [19,23].The acetic acid induced writhing response is a sensitive procedure to evaluate peripherally acting analgesics [15,146,147]. In the present study, significant analgesic activity was observed by using WE at different doses. In this test pain occurs by the liberation of endogenous substances like arachidonic acid and prostaglandin causes activation of peritoneal receptors leads to abdominal constriction [44,152]. Intra-peritoneal injection of acetic acid causes intense pain and acute inflammation in that area. The endogenous substances stimulate nerve endings and causes the indirect stimulation of peritoneal nociceptors [114,128].Writhing is manifested by the abdominal muscles constriction, extension of the forelimbs and elongation of the body. WE significantly decrease the abdominal constriction, indicated that the WE had dose-dependent effect but has lower effectiveness than standard drug [142,116].Therefore, it can be suggested that WE contain some pharmacologically active compounds which block the endogenous substances and arachidonic acid metabolites which cause excitation of the pain nerve endings [153]. The effect of WE at different doses (100, 200 and 300 mg/kg) on writhing rats by induction of acetic acid solution (1%) as shown in Table 4. Results showed the administration of WE doses (100, 200, and 300 mg/kg) has significantly lowered the number of acetic acid-induced writhing (abdominal constrictions of rats), compared to the standard and control rat group. The effect of these doses reached a maximum inhibition of 63.34% at the dose of 300 mg/kg. However, the WE extract dose (300 mg/kg) response was lower as compared to the standard drug (65.22 %) rat group. These results indicate protective effect of WE due to the presence of different levels of FAs (Figure 1).Similar results were obtained by other investigators [19,23,142]. Thus the higher dose of the WE extract had greater protection of writhing in rats group [19,22] than other rat groups given other doses of the same extract (Table 4). Thus, WE possessed antinociceptive effect against acetic acid-induced abdominal writhing response in rats.

Anti-Inflammatory Activity

Carrageenan-induced hind paw edema test (inflammation test) was performed to estimate the anti-inflammatory effect of WE doses (100, 200 and 300 mg/kg body weight) in rats [135]. Carrageenan-induced paw edema is being an in-vivo investigational model for acute inflammation which have been extensively used to determine the anti-inflammatory effect of new investigational agents [19,136]. The development of edema in paw of rat after the injection of carrageenan is due to release of some inflammatory mediators elevated level of prostaglan-dins, proteases and other mediators cause pain [135,154]. Result of anti- inflammatory effects of WE doses (100,200 and 300 mg/kg) were illustrated (Figure 2). In the present study carrageenan administration in rats causes inflammation that induce edema [106], Endomethacine used as anti-inflammatory standard drug were compared with control [138].

Results showed the paw volume of carrageenan induced rat was increased till 5 hour due to the inflammation development and then paw volume was decreased (Figure 2). The paw volumes and inhibition by the WE doses and standard drugs are shown in Figure (2). Carrageenan injection was administered one hour after treatment of extracts at three doses (100,200 and 300 mg/kg) and indomethacin. Measurement of paw size was taken before carrageenan injection and then 1,2,3,4 and 5 hours after carrageenan injection. Results showed the inhibition inflammation induced by carrageenan is much pronounced one hour after injection of carrageenan with maximum values observed at 5 hours after administration (Figure 2). The WE tested doses (100, 200 and 300 mg/kg) showed a significant reduction in paw edema with various degrees [134,136,148] that starts from 1 to 5 hr. after induction as compared to the control in carrageenan-induced paw edema rat groups. These results suggest that it interferes with the effects which inhibit the release of mediators involved inflammation [19,135,155] Since prostaglandins involved in pain perception are inhibited by flavonoids, it could be suggested that reduced availability of prostaglandins by flavonoids, phenol and FAs were present in WE might be responsible for its analgesic and anti-inflammatory effect [19,46,135]. However, the inhibition was the highest at 3h at 200mg/kg WE dose which was slightly lower than indomethacin effect. Indomethacin significantly reduced the paw volume at 2, 3, 4 and 5 hours as compared to the control and with the lower dose of WE (100 mg/kg). The medium dose (200 mg/kg) and higher (300mg/kg) doses of WE had comparable anti-inflammatory activity to control at all observations. The maximum edema inhibition of WE doses (100, 200, and 300 mg/kg) was accomplished 5 hours %. (Figure 2) after induction, with the values of 23.8%, 31.9%, and 32.5. [154]. There are considerable value of percent inhibition of inflammation was found by determining the percentage of change in paw volume after treatment. 

Figure 1: SFAs and USFAs contents in we.

Figure 2: Effect of WE on Carrageenan - induced rat paw edema.

The largest value of percentage inhibition of inflammation from control was found at 5hour after treatment (Figure 2). The inhibition was significant at the dose of 300 mg/kg (52.625%) to that of the standard drug (Figure 2), These results are in the range (58.985%) reported previous [19,135].The significant anti-inflammatory activity exhibited by WE at a dose used (300 mg/kg body weight) against edema induced by carrageenan in rats compared to the control group is an indication that, WE have pharmacological activity. Anti-inflammatory activity of standard and WE were observed in the present study may be due to the inhibition of release of inflammatory mediators. The present study demonstrated that oral dose of WE repressed the edema throughout all stages of inflammation (1 -5 hrs.) due to inactivation of inflammation mediator substances [144], reported L. sativa as an analgesic and anti- inflammatory agents. Phytochemical constituents of WE were illustrated in Tables (1) and (Figure1) revealed the presence of phenols, flavonoids and fatty acids (FAs) in different concentrations [31,46,138] stated the anti-inflammatory properties of these phytochemicals Therefore, it seems that analgesic and anti-inflammatory potential of WE. Similar with the effects of non-steroidal anti-inflammatory medications, such as indomethacin, the observed edema inhibition was higher implying that the anti-inflammatory activity is mediated by inhibition of mediators. WE displayed significant antiinflammatory activity against carrageenan-induced paw edema [19,22].The presence of phenolic, flavonoids, and FAs levels present in WE (Table 1 and Figure 1) indicated to analgesic and anti-inflammatory effects of WE [19,138,156]. Moreover, the present results revealed a significant increase reaction time to heat stimuli, strongly suggests that the mechanism of the WE may be mediators inhibition [19,135], reported the phenolic and flavonoids are known to inhibit prostaglandin synthetase and possess antiinflammatory analgesic activity [135,156]. Thus, the results of the present study indicates that WE were found to be effective as anti- inflammatory and analgesc agents and can be effective as therapeutic agent to treat inflammatory and analgesc conditions [156].

The present study concluded that the aqueous extract of watercress (Eureka sativa) at certain doses (100, 200 and 300 mg/kg) possesses analgesic and anti-inflammatory activities in rats due to their effect in inhibition the release of inflammatory mediators. Moreover, this is a preliminary study and further study is needed to find out the potential mechanism of action and isolate the active compounds responsible for analgesic and anti- inflammatory activities in rats. Thus, plant extracts particularly watercress (Eureka sativa) extract, consider better way than conventional, biosynthetic and chemical treatment of pain and inflammation.

1. World Health Organization WHO. Guidelines on the Pharmacological Treatment of Persisting Pain in Children with Medical Illnesses. Thieme New York: World Health Organization. 2012.
2. Calati R, Bakhiyi CL, Artero S, Ilgen M, Courtet P. The impact of physical pain on suicidal thoughts and behaviors: Meta-analyses. J psychiatric research. 2015; 71: 16-32.
3. Maldini M, Sosa SI, Montoro P, Giangaspero A, Balick MJ, Pizza C, et al. Screening of the topical anti-inflammatory activity of the bark of Acacia cornigera Willdenow, Byrsonima crassifolia Kunth, Sweetia panamensis Yakovlev and the leaves of Sphagneticola trilobata Hitchcock. Journal of ethnopharmacology. 2009; 122: 430-3.
4. Raja SN, Carr DB, Cohen M, Finnerup NB, Flor H, Gibson S, et al. The revised International Association for the Study of Pain definition of pain: concepts, challenges, and compromises. Pain. 2020; 161: 1976-82.
5. Javed F, Jabeen Q, Aslam N, Awan AM. Pharmacological evaluation of analgesic, anti-inflammatory and antipyretic activities of ethanolic extract of Indigofera argentea Burm. f. J ethnopharmacology. 2020; 259: 112966.
6. Beal BR, Wallace MS. An overview of pharmacologic management of chronic pain. Medical Clinics. 2016; 100: 65-79.
7. Sontakke SD, Hire R, Rayasum S. Experimental evaluation of analgesic and anti-inflammatory potential of Oyster mushroom Pleurotus florida. Indian J Pharmacology. 2013; 45: 541-2.
8. Fullerton JN, Gilroy DW. Resolution of inflammation: a new therapeutic frontier. Nature reviews Drug discovery. 2016; 15: 551-67.
9. Kumar S, Bajwa BS, Kuldeep S, Kalia AN. Anti-inflammatory activity of herbal plants: a review. Int J Adv Pharm Biol Chem. 2013; 2: 272-81.
10. Arome D, Sunday AI, Onalike EI, Amarachi A. Pain and inflammation: Management by conventional and herbal therapy. Indian J Pain. 2014; 28: 5-12.
11. Conforti F, Sosa S, Marrelli M, Menichini F, Statti GA, Uzunov D, et al. The protective ability of Mediterranean dietary plants against the oxidative damage: The role of radical oxygen species in inflammation and the polyphenol, flavonoid and sterol contents. Food Chemistry. 2009; 112: 587-94.
12. Mahendran G,Thamotharan G, Sengottuvelu S, NarmathaBai V. Antidiabetic activity of Swertia corymbosa (Griseb.) Wight ex C.B. Clarke aerial parts extract in streptozotocin induced diabetic rats J. Ethnopharmacol. 2014; 151: 1175–1183.
13. Lee JI, Oh JH, Kong CS, Seo Y. Evaluation of anti-adipogenic active homoisoflavonoids from Portulaca oleracea. Zeitschrift für Naturforschung C. 2019; 74: 265-73.
14. Allahmoradi E, Taghiloo S, Omrani-Nava V, Shobeir SS, Tehrani M, Ebrahimzadeh MA, et al. Anti-inflammatory effects of the Portulaca oleracea hydroalcholic extract on human peripheral blood mononuclear cells. Medical j Islamic Republic of Iran. 2018; 32: 80.
15. Miao L, Tao H, Peng Y, Wang S, Zhong Z, El-Seedi H, et al. The anti-inflammatory potential of Portulaca oleracea L.(purslane) extract by partial suppression on NF-?B and MAPK activation. Food chemistry. 2019; 290: 239-45.
16. Varrassi G, Alon E, Bagnasco M, Lanata L, Mayoral-Rojals V, Paladini A, Pergolizzi JV, Perrot S, Scarpignato C, Tölle T. Towards an effective and safe treatment of inflammatory pain: a Delphi-guided expert consensus. Advances in Therapy. 2019; 36: 2618-37.
17. Ohadoma SC, Akah PA, Okolo CE. Okoro EP, Michael HU. Limitations of non-steroidal anti-inflammatory drugs and the utility of natural products for antinociceptive and antiexudative effects. European J Pharma Medical Research.2020; 2: 86-98.
18. Bayramoglu G, Senturk H, Bayramoglu A, Uyanoglu M, Colak S, Ozmen A, et al. Carvacrol partially reverses symptoms of diabetes in STZ-induced diabetic rats. Cytotechnology. 2014; 66: 251-7.
19. Ashagrie G, Girmaw F, Tarekegn A, Baye T, Dagne A. Evaluation of Analgesics and Anti-Inflammatory Activity of the Root Extract of Impatiens rothii (Balsaminaceae) in Rodents. J  Experimental Pharmacology. 2023: 207-14.
20. Bindu S, Mazumder S, Bandyopadhyay U. Non-steroidal anti-inflammatory drugs (NSAIDs) and organ damage: A current perspective. Biochemical pharmacology. 2020; 180: 114147.
21. Pires Jr EO, Caleja C, Garcia CC, Ferreira IC, Barros L. Current status of genus Impatiens: Bioactive compounds and natural pigments with health benefits. Trends in Food Science & Technology. 2021; 117: 106-24.
22. Fan SH, Ali NA, Basri DF. Evaluation of analgesic activity of the methanol extract from the galls of Quercus infectoria (Olivier) in rats. Evidence?Based Complementary and Alternative Medicine. 2014; 1-5: 976764.
23. Kaushik D, Kumar A, Kaushik P, Rana AC. Analgesic and Anti?Inflammatory Activity of Pinus roxburghii Sarg. Advances in Pharmacological and Pharmaceutical Sciences. 2012; 1-6: 245431.
24. Shah AS, Alagawadi KR. Anti-inflammatory, analgesic and antipyretic properties of Thespesia populnea Soland ex. Correa seed extracts and its fractions in animal models. J  Ethnopharmacology. 2011; 137: 1504-9.
25. Meng Y, Ying Z, Xiang Z, Hao D, Zhang W, Zheng Y, Gao Y, Ying X. The anti-inflammation and pharmacokinetics of a novel alkaloid from Portulaca oleracea L. J Pharmacy and Pharmacology. 2016; 68: 397-405.
26. Meng D, Wang L, Du J, Chen J, Chen C, Xu W, Li C. The analgesic activities of Stauntonia brachyanthera and YM11 through regulating inflammatory mediators and directly controlling the sodium channel prompt. Scientific Reports. 2017; 7: 7574.
27. Kumara N. Identification of strategies to improve research on medicinal plants used in Sri Lanka, in Proceedings of the WHO Symposium, University of Ruhuna, Galle. Sri Lanka. 2001; 12-14.
28. Dharmasiri MG, Jayakody JR, Galhena G, Liyanage SS, Ratnasooriya WD. Anti-inflammatory and analgesic activities of mature fresh leaves of Vitex negundo. Journal of ethnopharmacology. 2003 ; 87: 199-206.
29. Meng XL, Xue L, Zhang ZW, Gui XJ, Chen YH, Tang JF, et al. Effect of Portulaca oleracea polysaccharide on immunological function in mice with cyclophosphamide-induced immunosuppression, Chin. J. New Drugs. 2017; 26: 1296-1300.
30. Karunamoorthi K, Jegajeevanram K, Vijayalakshmi J, Mengistie E. Traditional medicinal plants: a source of phytotherapeutic modality in resource-constrained health care settings. J Evid Based Complementary Altern Med. 2013; 18: 67 -74.
31. Moharib SA, Tadrus PH. Anticancer and cytotoxic activities of the produced seed oils against various cancer cell lines. Palgo J.Med. Medical Sci. 2020; 1: 1-18.
32. Sriti J, Bettaieb I, Bachrouch O, Talou T. Chemical composition and antioxidant activity of the coriander cake obtained by extrusion. Arab. J. of Chemist. 2014; 30: 1-9.
33. Fabian CJ, Kimler BF, Hursting SD. Omega-3 fatty acids for breast cancer prevention and survivorship. Breast cancer res. 2015; 17: 1-1.
34. Maduka HC, Okpogba AN, Ugwu CE, Dike CC, Ogueche PN, Onwuzurike DT, et al. Phytochemical, antioxidant and microbial inhibitory effects of Spondias mombin leaf and stem bark extracts. J Pharm Biol Sci. 2014; 9: 14-7.
35. Khan MS, Ahmad I. Herbal medicine: current trends and future prospects. InNew look to phytomedicine. Academic Press. 2019; 3-13.
36. Yohannis SW, Asfaw Z, Kelbessa E. Ethnobotanical study of medicinal plants used by local people in menz gera midir district, north shewa zone, amhara regional state, Ethiopia. Journal of Medicinal Plants Research. 2018; 12: 296-314.
37. Guo G, Yue L, Fan S, Jing S, Yan LJ. Antioxidant and antiproliferative activities of purslane seed oil. Journal of hypertension: open access. 2016; 5: 218.
38. Ayoka AO, Akomolafe RO, Akinsomisoye OS, Ukponmwan OE. Medicinal and economic value of Spondias mombin. African J Biomedical Res. 2008; 11.
39. Vongtau HO, Amos S, Binda L, Kapu SD, Gamaniel KS, Kunle OF, Wambebe C. Pharmacological effects of the aqueous extract of Neorautanenia mitis in rodents. J Ethnopharmacology. 2000; 72: 207-14.
40. Moharib SA. Antidiabetic and antioxidant effects of parsley (Petroselinum sativum) extract in streptozocin-induced diabetic rats. 2016; 38: 22-34.
41. He Y, Long H, Zou C, Yang W, Jiang L, Xiao Z, Li Q, Long S. Anti-nociceptive effect of Portulaca oleracea L. ethanol extracts attenuated zymosan-induced mouse joint inflammation via inhibition of Nrf2 expression. Innate Immunity. 2021; 27: 230-9.
42. Winter CA, Porter CC. Effect of alterations in side chain upon anti-inflammatory and liver glycogen activities of hydrocortisone esters. Journal of the American Pharmaceutical Association (Scientific ed.). 1957; 46: 515-9.
43. Gupta M, Mazumder UK, Gomathi P, Selvan VT. Antiinflammatory evaluation of leaves of Plumeria acuminata. BMC Complementary and alternative medicine. 2006; 6: 1-6.
44. Lu TC, Ko YZ, Huang HW, Hung YC, Lin YC, Peng WH. Analgesic and anti-inflammatory activities of aqueous extract from Glycine tomentella root in mice. J ethnopharmacology. 2007; 113: 142-8.
45. Sayyah M, Hadidi N, Kamalinejad M. Analgesic and anti-inflammatory activity of Lactuca sativa seed extract in rats. Journal of Ethnopharmacology. 2004; 92: 325-9.
46. Alam MA, Nadirah TA, Mohsin GM, Saleh M, Moneruzzaman KM, Aslani F, et al. Antioxidant compounds, antioxidant activities, and mineral contents among underutilized vegetables. International J Vegetable Sci. 2021; 27: 157-66.
47. Chekole G. Ethnobotanical study of medicinal plants used against human ailments in Gubalafto District, Northern Ethiopia. J ethnobiology ethnomedicine. 2017; 13: 1-29.
48. Nigussie D, Legesse BA, Davey G, Fekadu A, Makonnen E. Ethiopian medicinal plants used for their anti-inflammatory, wound healing or anti-infective activities: protocol for systematic literature review and meta-analysis. BMJ open science. 2020; 4: 100064.
49. Maha I A, Wafa S A, Eman A I, Manal EA E. Anti-oxidant, anti-inflammatory and anti-cancer activities of avocado (Persea americana) fruit and seed extract. J. of King Saud Univ. Sci. 2018; 30, 1-5.
50. Ebtesam S A. Anticancer potential of seed extract and pure compound from Phoenix dactylifera on human cancer cell lines. Phcog Mag. 2019; 15: 494-499.
51. Neto AG, Costa JM, Belati CC, Vinholis AH, Possebom LS, Da Silva Filho AA, et al. Analgesic and anti-inflammatory activity of a crude root extract of Pfaffia glomerata (Spreng) Pedersen. Journal of Ethnopharmacology. 2005; 96: 87-91.
52. Kulaitien? J, ?erniauskien? J, Jarien? E, Danil?Enko H, Levickien? D. Antioxidant activity and other quality parameters of cold pressing pumpkin seed oil. Notulae Botanicae Horti Agrobotanici Cluj-Napoca. 2018; 46: 161-6.
53. Li Y, Guo C, Yang J, Wei J, Xu J, Cheng S. Evaluation of antioxidant properties of pomegranate peel extract in comparison with pomegranate pulp extract. Food chemistry. 2006; 96: 254-60.
54. Bostancio glu RB, K urkc M¸ Baseruo glu KHC, Koparal AT. “Assessment of anti-angiogenic and anti-tumoral potentials of Origanum onites L. essential oil,” Food and Chem. Toxicol. 50: 2010.
55. Shirazi MT, Gholami H, Kavoosi G, Rowshan V, Tafsiry A. Chemical composition, antioxidant, antimicrobial and cytotoxic activities of T agetes minuta and O cimum basilicum essential oils. Food science & nutrition. 2014; 2: 146-55.
56. Moharib SA, Adly RS. Antimicrobial Activities of Polysaccharides Isolated from Some Plant Leaves. Journal of Virology Research & Reports. 2025; 6: 1-8.
57. Balamurugan K, Nishanthini A, Mohan VR. Antidiabetic and antihyperlipidaemic activity of ethanol extract of Melastoma malabathricum Linn. Leaf in alloxan induced diabetic rats. Asian Pacific j tropical biomedicine. 2014; 4: 442-8.
58. Menezes IA, Moreira ÍJ, Carvalho AA, Antoniolli AR, Santos MR. Cardiovascular effects of the aqueous extract from Caesalpinia ferrea: involvement of ATP-sensitive potassium channels. Vascular pharmacology. 2007; 47: 41-7.
59. Moharib SA, Adly RS. Hypoglycemic and hepatoprotective activities of Coriander (Coriandrum sativum) extract in streptozotocin-induced diabetic rats. J Advances Biology Biotechnology. 2024; 27: 15-38.
60. Sorial AM. Anticancer and Antioxidant Effects of Brassica napus and Raphanus sativus Seed Oils against Chemically Induced Liver Cancer in Rats. EC Veterinary Science. 2022; 7: 16-36.
61. Duke JA. Handbook of Medicinal Herbs, CRC Press, New York, NY, USA. 2001.
62. Moharib SA. Hypolipidemic activities and nutritive values of Brassica napus and Eruca sativa seed supplementation in rats fed a high cholesterol diet. EC Veterinary Science. 2021; 6: 29-40.
63. Lee AS, Lee YJ, Lee SM, Yoon JJ, Kim JS, Kang DG, Lee HS. An aqueous extract of Portulaca oleracea ameliorates diabetic nephropathy through suppression of renal fibrosis and inflammation in diabetic db/db mice. The American journal of Chinese medicine. 2012; 40: 495-510.
64. Aboulthana WM, Youssef AM, El-Feky AM, Ibrahim NE, Seif MM, Hassan AK. Evaluation of antioxidant efficiency of Croton tiglium L. seeds extracts after incorporating silver nanoparticles. Egypt J Chem. 2019; 62: 181-200.
65. Itani WS, El-Banna SH, Hassan SB, Larsson RL, Bazarbachi A, Gali- Maha I, et al. Anti-oxidant, anti-inflammatory and anti-cancer activities of avocado. Journal of King Saud University – Science. 2018.
66. Nilnakara S, Chiewchan N, Devahastin S. Production of antioxidant dietary fibre powder from cabbage outer leaves. Food and Bioproducts Process. 2009; 87: 301-307.
67. Zhang J, Wu Y, Wang C, Xu W, Zhang Z, Zhang S, Guan X, Wang X. The antioxidant, anti-inflammatory and analgesic activity effect of ethyl acetate extract from the flowers of Syringa pubescens Turcz. Journal of Ethnopharmacology. 2024; 322: 117561.
68. Veeresham C. Natural products derived from plants as a source of drugs. J advanced pharmaceutical technology research. 2012; 3: 200-1.
69. Alam F, Shafique Z, Amjad ST, Bin Asad MH. Enzymes inhibitors from natural sources with antidiabetic activity: A review. Phytotherapy Research. 2019; 33: 41-54.
70. Lefebvre T, Destandau E, Lesellier E. Selective extraction of bioactive compounds from plants using recent extraction techniques: A Review. J Chromatography A. 2020; 461770.
71. Hervert-Hernández D, García OP, Rosado JL, Goñi I. The contribution of fruits and vegetables to dietary intake of polyphenols and antioxidant capacity in a Mexican rural diet: Importance of fruit and vegetable variety. Food Research International. 2011; 44: 1182-9.
72. Abdelmoaty MA, Ibrahim MA, Ahmed NS, Abdelaziz MA. Confirmatory studies on the antioxidant and antidiabetic effect of quercetin in rats. Indian Journal of Clinical Biochemistry. 2010; 25: 188-92.
73. Kedir WM, Geletu AK, Weldegirum GS, Sima MF. Antioxidant activity of selected plants extract for palm oil stability via accelerated and deep frying study. Heliyon. 2023; 9.
74. Zhao Jun ZJ, Yan Ming YM, Huang Yi HY, He WenYi HW, Zhao Yu ZY. Flavonoids from the leaves of Sabina vulgaris Antoine.
75. Yu JQ, Lei JC, Zhang XQ, Yu HD, Tian DZ, Liao ZX, Zou GL. Anticancer, antioxidant and antimicrobial activities of the essential oil of Lycopus lucidus Turcz. Var. hirtus Regel. Food Chemistry. 2011; 126: 1593-8.
76. Zeb A. Phenolic profile and antioxidant potential of wild watercress (Nasturtium officinale L.). SpringerPlus. 2015; 4: 714.
77. Lowry OH, Rosebrough NJ, Farr AL, Randall, RJ. Protein measurement with the Folin phenol reagent. J. Biol. Chem.1951; 193, 256-275.
78. Folch J, Lees M, Stanley GS. A simple method for the isolation and purification of total lipides from animal tissues. J biological chemistry. 1957; 226: 497-509.
79. DuBois M, Gilles KA, Hamilton JK, Rebers PA, Smith F. Colorimetric method for determination of sugars and related substances. Analytical chemistry. 1956; 28: 350-6.
80. Ribarova F, Atanassova M, Marinova D, Ribarova F, Atanassova M. Total phenolics and flavonoids in Bulgarian fruits and vegetables. JU Chem. Metal. 2005; 40: 255-60.
81. Singleton VL, Orthofer R, Lamuela-Raventós RM. [14] Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent. InMethods in enzymology 1999; 299: 152-178.
82. Nabavi SM, Ebrahimzadeh MA, Nabavi SF, Hamidinia A, Bekhradnia AR. Determination of antioxidant activity, phenol and flavonoids content of Parrotia persica Mey. Pharmacologyonline. 2008; 2: 560-7.
83. Zhishen J, Mengcheng T, Jianming W. The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. Food chemistry. 1999; 64: 555-9.
84. Adam JH, Ramian O, Wilcock CC. Phytochmeical screening of flavonoids in three hybrids of Napenthes (Napenthaceae) and their putative parental species from Sarawak and Sabah. Online J. boil. sci. 2002; 2: 623-625.
85. Peng J, Fan G, Wu Y. Preparative separation and isolation of three flavonoids and three phloroglucinol derivatives from Hypericum japonicum thumb. Using high?speed countercurrent chromatography by stepwise increasing the flow rate of the mobile phase. Journal of liquid chromatography & related technologies. 2006; 29: 1619-32.
86. Scapin G, Abaide ER, Nunes LF, Mazutti MA, Vendruscolo RG, Wagner R, da Rosa CS. Effect of pressure and temperature on the quality of chia oil extracted using pressurized fluids. The Journal of Supercritical Fluids. 2017; 127: 90-6.
87. Lepage G, Roy CC. Direct transesterification of all classes of lipids in a one-step reaction. Journal of lipid research. 1986; 27: 114-20.
88. Zhao J, Maitituersun A, Li C, Li Q, Xu F, Liu T. Evaluation on Analgesic and Anti?Inflammatory Activities of Total Flavonoids from Juniperus sabina. Evidence?Based Complementary and Alternative Medicine. 2018; 1: 7965306.
89. Couto M, Cates C. Laboratory guidelines for animal care. Vertebrate Embryogenesis: Embryological, Cellular, and Genetic Methods. 2019: 407-30.
90. Tasleem F, Azhar I, Ali SN, Perveen S, Mahmood ZA. Analgesic and anti-inflammatory activities of Piper nigrum L. Asian Pacific j tropical medicine. 2014; 7: 461-8.
91. Huo X, Zhang L, Gao L, Guo Y, Zhang L, Li L, Si J, Cao L. Antiinflammatory and analgesic activities of ethanol extract and isolated compounds from Millettia pulchra. Biological and Pharmaceutical Bulletin. 2015; 38: 1328-36.
92. Aydin S, Demir T, Ozturk Y, Ba?er KH. Analgesic activity of Nepeta italica L. Phytotherapy Research: An International Journal Devoted to Pharmacological and Toxicological Evaluation of Natural Product Derivatives. 1999; 13: 20-3.
93. Janssen PA, Niemegeers CJ, Dony JG. The inhibitory effect of fentanyl and other morphine-like analgesics on the warm water induced tail withdrawl reflex in rats. Arzneimittel-forschung. 1963; 13: 502-7.
94. Taïwe GS, Bum EN, Talla E, Dimo T, Weiss N, Sidiki N, Dawe A, Okomolo Moto FC, Dzeufiet PD, Waard MD. Antipyretic and antinociceptive effects of Nauclea latifolia root decoction and possible mechanisms of action. Pharmaceutical biology. 2011; 49: 15-25.
95. Koster R, Anderson M, De Beer EJ. Acetic acid for analgesic screening. Fed Proc. 1959; 18: 412-415.
96. Robert, A. Antisecretory, antiulcer, cytoprotective and dia rrheogenic properties of prostaglandins, Advances in Prostaglandin Thromboxane Research. 1976; 2: 507-520.
97. Tahiri O, Atmani-Kilani D, Sanchez-Fidalgo S, Aparicio-Soto M, Alarcón-de-la-Lastra C, Barrajón-Catalán E, et al. The flavonol-enriched Cistus albidus chloroform extract possesses in vivo anti-inflammatory and anti-nociceptive activity. J  Ethnopharmacology. 2017; 209: 210-8.
98. Demsie DG, Yimer EM, Berhe AH, Altaye BM, Berhe DF. Anti-nociceptive and anti-inflammatory activities of crude root extract and solvent fractions of Cucumis ficifolius in mice model. Journal of Pain Research. 2019: 1399-409.
99. Winter CA, Risley EA, Nuss GW. Carrageenin-induced edema in hind paw of the rat as an assay for antiinflammatory drugs. Proceedings of the society for experimental biology and medicine. 1962; 111: 544-7.
100. Kamanyi A, Mbiantcha M, Nguelefack TB, Ateufack G, Watcho P, Ndontsa BL, et al. Anti-nociceptive and anti-inflammatory activities of extracts from the stem bark of Croton macrostachyus (Euphorbiaceae) in mice and rats. Journal of Complementary and Integrative Medicine. 2009; 6.
101. Tatiya AU, Saluja AK, Kalaskar MG, Surana SJ, Patil PH. Evaluation of analgesic and anti-inflammatory activity of Bridelia retusa (Spreng) bark. Journal of traditional and complementary medicine. 2017; 7: 441-51.
102. Mujumdar AM, Misar AV. Anti-inflammatory activity of Jatropha curcas roots in mice and rats. J ethnopharmacology. 2004; 90: 11-5.
103. Adeyemi OO, Okpo SO, Ogunti OO. Analgesic and anti-inflammatory effects of the aqueous extract of leaves of Persea americana Mill (Lauraceae). Fitoterapia. 2002; 73: 375-80.
104. Ameya G, Gure A, Dessalegn E. Antimicrobial activity of Echinops kebericho against human pathogenic bacteria and fungi. African Journal of Traditional, Complementary and Alternative Medicines. 2016; 13: 199-203.
105. Tamrat Y, Nedi T, Assefa S, Teklehaymanot T, Shibeshi W. Anti-inflammatory and analgesic activities of solvent fractions of the leaves of Moringa stenopetala Bak.(Moringaceae) in mice models. BMC complementary and alternative medicine. 2017; 17: 1-0.
106. Sajid M, Khan MR, Shah NA, Shah SA, Ismail H, Younis T, Zahra Z. Phytochemical, antioxidant and hepatoprotective effects of Alnus nitida bark in carbon tetrachloride challenged Sprague Dawley rats. BMC complementary and alternative medicine. 2016; 16: 1-7.
107. Stanojevi? LP, Radulovi? NS, Djoki? TM, Stankovi? BM, Ili? DP, Caki? MD, Nikoli? VD. The yield, composition and hydrodistillation kinetics of the essential oil of dill seeds (Anethi fructus) obtained by different hydrodistillation techniques. Industrial Crops and Products. 2015; 65: 429-36.
108. Al Nomaani RS, Hossain MA, Weli AM, Al-Riyami Q, Al-Sabahi JN. Chemical composition of essential oils and in vitro antioxidant activity of fresh and dry leaves crude extracts of medicinal plant of Lactuca sativa L. native to Sultanate of Oman. Asian Pacific j tropical biomedicine. 2013; 3: 353-7.
109. Rezig L, Chouaibi M, Meddeb W, Msaada K, Hamdi S. Chemical composition and bioactive compounds of Cucurbitaceae seeds: Potential sources for new trends of plant oils. Process Safety and Environmental Protection. 2019; 127: 73-81.
110. Ayaz FA, Hay?rl?oglu-Ayaz S, Alpay-Karaoglu S, Grúz J, Valentová K, Ulrichová J, Strnad M. Phenolic acid contents of kale (Brassica oleraceae L. var. acephala DC.) extracts and their antioxidant and antibacterial activities. Food Chemistry. 2008; 107: 19-25.
111. Harsha SN, Anilakumar KR, Mithila MV. Antioxidant properties of Lactuca sativa leaf extract involved in the protection of biomolecules. Biomed. Prev. Nutr. 2014; 3: 367 - 373.
112. Bachir RG, Bellil A. Preliminary Phytochemical Screening of Five Commercial Essential Oils. World J. of Appl. Chemist. 2017; 2: 145-151.
113. Can-Cauich CA, Sauri-Duch E, Moo-Huchin VM, Betancur-Ancona D, Cuevas-Glory LF. Effect of extraction method and specie on the content of bioactive compounds and antioxidant activity of pumpkin oil from Yucatan, Mexico. Food chemistry. 2019; 285: 186-93.
114. Yu SY, Lee YJ, Kim JD, Kang SN, Lee SK, Jang JY, Lee HK, Lim JH, Lee OH. Phenolic composition, antioxidant activity and anti-adipogenic effect of hot water extract from safflower (Carthamus tinctorius L.) seed. Nutrients. 2013; 5: 4894-907.
115. Ahmadzadeh S, Kadivar M, Saeidi G. Investigation of oil properties and seed composition in some safflower lines and cultivars. J Food Biochemistry. 2014; 38: 527-32.
116. Yu LL, Zhou KK, Parry J. Antioxidant properties of cold-pressed black caraway, carrot, cranberry, and hemp seed oils. Food chemistry. 2005; 91: 723-9.
117. Afshar FH, Delazar A, Nazemiyeh H, Esnaashari S, Moghadam SB. Comparison of the total phenol, flavonoid contents and antioxidant activity of methanolic extracts of Artemisia spicigera and A. splendens growing in Iran. Pharmaceutical sciences. 2019; 18: 165-70.
118. Delfan-Hosseini S, Nayebzadeh K, Mirmoghtadaie L, Kavosi M, Hosseini SM. Effect of extraction process on composition, oxidative stability and rheological properties of purslane seed oil. Food Chemistry. 2017; 222: 61-6.
119. Longoria-Sanchez A, Morales MV, Oomah BD, Ochoa-Espinoza XM, Góngora-Gómez AM, Espinosa-Alonso LG. Characteristics and antioxidant properties of cold pressed high oleic and linoleic oils from Mexican safflower varieties. Emirates Journal of Food and Agriculture. 2019; 31: 679-87.
120. Kim HJ, Chen F, Wang X, Choi JH. Effect of methyl jasmonate on phenolics, isothiocyanate, and metabolic enzymes in radish sprout (Raphanus sativus L.). Journal of agricultural and food chemistry. 2006; 54: 7263-9.
121. Younus I, Ismail H, Rizvi CB, Dilshad E, Saba K, Mirza B, Tahir M. Antioxidant, analgesic and anti-inflammatory activities of in vitro and field-grown Iceberg lettuce extracts. J. Pharm. Pharmacogn. Res. 2019; 7: 343-55.
122. Rezig L, Chouaibi M, Msaada K, Hamdi S. Chemical composition and profile characterisation of pumpkin (Cucurbita maxima) seed oil. Industrial Crops and Products. 2012; 37: 82-7.
123. Aydeniz B, Güne?er O, Y?lmaz E. Physico?chemical, sensory and aromatic properties of cold press produced safflower oil. Journal of the American Oil Chemists' Society. 2014; 91: 99-110.
124. Daniewski M, Jacórzy?ski B, Filipek A, Balas J, Pawlicka M, Mielniczuk E. Fatty acids content in selected edible oils. Roczniki Panstwowego Zakladu Higieny. 2003; 54: 263-7.
125. Sargi SC, Silva BC, Santos HM, Montanher PF, Boeing JS, Santos Júnior OO, Souza NE, Visentainer JV. Antioxidant capacity and chemical composition in seeds rich in omega-3: chia, flax, and perilla. Food Science and Technology. 2013; 33: 541-8.
126. Stroescu M, Stoica-Guzun A, Ghergu S, Chira N, Jipa I. Optimization of fatty acids extraction from Portulaca oleracea seed using response surface methodology. Industrial Crops and Products. 2013; 43: 405-11.
127. Gutiérrez RM, Perez RL. Raphanus sativus (Radish): their chemistry and biology. The scientific world j. 2004; 4: 811-37.
128. Adamska E, Cegielska-Taras T, Kaczmarek Z, Sza?a L. Multivariate approach to evaluating the fatty acid composition of seed oil in a doubled haploid population of winter oilseed rape (Brassica napus L.). J Applied Genetics. 2004; 45: 419-25.
129. Tian J, Ban X, Zeng H, Huang B, He J, Wang Y. In vitro and in vivo activity of essential oil from dill (Anethum graveolens L.) against fungal spoilage of cherry tomatoes. Food Control. 2011; 22: 1992-9.
130. Kujawski R, Baraniak JU, Kania M, Bartkowiak-Wieczorek J, Bogacz A, Ozarowski M, Cichocki MI, Spoz E, Mikolajczak PL. Diet based on oil of seeds of Brassica napus. Implications for the prevention and treatment of prostate diseases. Herba Polonica. 2014; 60.
131. Zia-Ul-Haq M, Iqbal S, Ahmad S, Imran M, Niaz A, Bhanger MI. Nutritional and compositional study of desi chickpea (Cicer arietinum L.) cultivars grown in Punjab, Pakistan. Food Chemistry. 2007; 105: 1357-63.
132. Edziri HÁ, Smach MA, Ammar S, Mahjoub MA, Mighri Z, Aouni M, Mastouri M. Antioxidant, antibacterial, and antiviral effects of Lactuca sativa extracts. Industrial Crops and Products. 2011; 34: 1182-5.
133. Jukanti AK, Gaur PM, Gowda CL, Chibbar RN. Nutritional quality and health benefits of chickpea (Cicer arietinum L.): a review. British J Nutrition. 2012; 108: S11-26.
134. Zambiazi RC, Przybylski R, Zambiazi MW, Mendonca CB. Fatty acid composition of vegetable oils and fats. B. ceppa, curitiba. 2007; 25: 111-20.
135. Ahmadi A, Khalili M, Salimi M, Mirsistani N, Niksirat A, Nazirzadeh S. The effects of solvent polarity on analgesic and anti-inflammatory activities of Securigera securidaca (L.), Achillea eriophora DC, and Portulaca oleracea extracts. Pharmaceutical Chemistry J. 2019; 53: 248-63.
136. Arfan M, Amin H, Khan N, Khan I, Saeed M, Khan MA. Analgesic and anti-inflammatory activities of 11-O-galloylbergenin. J Ethnopharmacology. 2010; 131: 502-4.
137. Masroor D, Baig SG, Ahmed S, Ahmad SM. Analgesic, anti-inflammatory and diuretic activities of Cicer arietinum L. Pakistan J Pharmaceutical Sci. 2018; 31.
138. Ismail H, Mirza B. Evaluation of analgesic, anti-inflammatory, anti-depressant and anti-coagulant properties of Lactuca sativa (CV. Grand Rapids) plant tissues and cell suspension in rats. BMC complementary and alternative medicine. 2015; 15: 1-7.
139. Jan S, Khan MR. Antipyretic, analgesic and anti-inflammatory effects of Kickxia ramosissima. J ethnopharmacology. 2016; 182: 90-100.
140. Yimer T, Birru EM, Adugna M, Geta M, Emiru YK. Evaluation of analgesic and anti-inflammatory activities of 80% methanol root extract of Echinops kebericho M.(Asteraceae). J Inflammation Research. 2020: 647-58.
141. Kayani WK, Dilshad E, Ahmed T, Ismail H, Mirza B. Evaluation of Ajuga bracteosa for antioxidant, anti-inflammatory, analgesic, antidepressant and anticoagulant activities. BMC Complementary and Alternative Medicine. 2016; 16: 1-3.
142. Al-Enazi MM, Ahamd R, Rahiman S. Antinociceptive and Anti-inflammatory Activities of Eruca Sativa L. Leaves Extract. Adv. Biores.2014; 5: 145-50.
143. Hugar et al., 2010 Hugar MH, Hosamani KM, Ahmed ML. Phytochemical and pharmacological studies of ethanol extract of Dalbergia sissoo seeds: an approach for the in-vivo analgesic and antipyretic activities. Int J Pharma Bio Sci. 2010; 1: 272–280.
144. Ismail H, and Mirza B (2015). Evaluation of analgesic, anti-inflammatory, anti- depressant and anti-coagulant properties of Lactuca sativa (CV. Grand Rapids) plant tissues and cell suspension in rats. BMC Complement Alt Med. 15: 199.
145. Younus I, Siddiq A. Raphanus sativus L. Var. caudatus as an analgesic and antipyretic agent in animal models. Pakistan J. Zool. 2022; 54: 1643-8.
146. Voilley N. Acid-sensing ion channels (ASICs): new targets for the analgesic effects of non-steroid anti-inflammatory drugs (NSAIDs). Current Drug Targets-Inflammation & Allergy. 2004; 3: 71-9.
147. Annegowda HV, Mordi MN, Ramanathan S, Mansor SM. Analgesic and antioxidant properties of ethanolic extract of Terminalia catappa L. leaves. Int j pharmacology. 2010; 6: 910-5.
148. Kim HK, Park SK, Zhou JL, Taglialatela G, Chung K, Coggeshall RE, Chung JM. Reactive oxygen species (ROS) play an important role in a rat model of neuropathic pain. Pain. 2004; 111: 116-24.
149. Sawadogo, W. R., Boly, R., Lompo, M. et al. Anti-inflammatory, analgesic and antipyretic activities of Dicliptera verticillata,” International J Pharmacology. 2006; 2: 435-438.
150. Gené RM, Segura L, Adzet T, Marin E, Iglesias J. Heterotheca inuloides: anti-inflammatory and analgesic effect. Journal of Ethnopharmacology. 1998; 60: 157-62.
151. Hossain MM, Ali MS, Saha A, Alimuzzaman M. Antinociceptive activity of whole plant extracts of Paederia foetida. Dhaka University Journal of Pharmaceutical Sciences. 2006; 5: 67-9.
152. Bennett RN, Mellon FA, Botting NP, Eagles J, Rosa EA, Williamson G. Identification of the major glucosinolate (4-mercaptobutyl glucosinolate) in leaves of Eruca sativa L.(salad rocket). Phytochemistry. 2002; 61: 25-30.
153. Khan H, Saeed M, Gilani AU, Khan MA, Khan I, Ashraf N. Antinociceptive activity of aerial parts of Polygonatum verticillatum: attenuation of both peripheral and central pain mediators. Phytotherapy Research. 2011; 25: 1024-30.
154. Araruna K, Carlos B. Anti-inflammatory activities of triterpene lactones from Lactuca sativa. Phytopharmacology. 2010; 1: 1-6.
155. Itou RD, Sanogo R, Ossibi AW, Ntandou FG, Ondelé R, Pénemé BM, et al. Anti-inflammatory and analgesic effects of aqueous extract of stem bark of Ceiba pentandra Gaertn. Pharmacology & Pharmacy. 2014; 5: 1113-1118.
156. Rao AS, Latha P, Dhakate OM, Gunda SA, Ramachandra M. Anti-inflammatory and analgesic activity of Cicer arietinum L. Plant Extracts in Rats. Interna J Pharmaceutical Res. 2014; 6: 74-78.