Previous studies conducted by our team revealed the presence of catechin, afzelechin and galloyl quinic acid in TOF-enriched extract from the aerial parts of C. rotundus, and luteolin, ferulic acid, quercetin, 3-hydroxy, 4-methoxybenzoic acid and 6,7 dimethoxycoumarin in the ethyl acetate extract . Among these secondary compounds, belonging to either tannin, polyphenol or flavonoid families, several are known to exhibit antinociceptive and anti-inflammatory effects [34, 35], and we believe that the analgesic and anti-inflammatory activities we observed with extracts from the aerial parts of Cyperus rotundus should be ascribed to such compounds. Besides, i.p. administration of different doses (125 mg/kg to 1 g/kg b.w.) of Cyperus rotundus extracts to mice gives no toxic effect. This finding suggests that the plant extracts are, at the tested doses, safe in mice.
The peripheral analgesic effect may be mediated through the inhibition of cyclo-oxygenases and/or lipoxygenases (and other inflammatory mediators) . This hypothesis is in accordance with those of Adeyemi et al.  and Zhang et al. , who have postulated that acetic acid-induced writhing is a highly sensitive and useful test for analgesic drug development, especially peripherally acting analgesics. Acetic acid induces pain by liberating endogenous substances (bradykinin, serotonin, histamine, substance P) , which in turn excite the pain nerve endings. In our observation, aqueous, methanolic, ethyl acetate and TOF-enriched extracts significantly (P < 0.001) reduced the abdominal constriction response induced by the acetic acid in a dose-dependent manner. TOF-enriched extract exhibited the most analgesic potency, whereas methanolic extract showed the weakest one. This result could be explained by the presence of active compounds diluted and/or masked by various components in the methanolic extract and which are more accessible and/or more concentrated in the TOF- enriched extract. Besides, this test is useful for the evaluation of mild analgesic non steroidal, anti-inflammatory compounds [40, 41]. This suggests a peripherally induced mechanism of the analgesic action for Cyperus rotundus extracts . Therefore, one possible mechanism of the analgesic activity by C. rotundus extracts could be due to the blockage of the effect, or the release of endogenous substances (arachidonic acid metabolites) that excite pain nerve endings by the pharmacologically active principles of C. rotundus extracts. From a mechanistic point of view, the lack of specificity in acetic acid-induced writhing test suggests the involvement of different nociceptive mechanisms in the reduction of muscular constriction, such as sympathetic system, through the release of biogenic amines, cyclooxygenases and their metabolites inhibition, and through opioids receptors mechanisms .
In evaluating the anti-inflammatory effect, it is important to estimate the activities of the extracts in the acute phase as well as in the chronic phase of inflammation. Xylene-induced ear oedema in mice was selected to evaluate acute anti-inflammatory activity and is a good in vivo test useful for evaluating lipoxygenase inhibitors and partially associated with substance P . The results in Table 3 showed that the different extracts of C. rotundus caused significant inhibition of oedema as compared to the control group. These results may suggest that the plant extracts exert significant anti-inflammatory activity, especially in the acute inflammatory response. The suppression of this response is a likely indication of the antiphlogistic effect . The topical anti-inflammatory effect suggests that constituents of the extract may relieve rheumatism and offer the additional advantage of suppressing inflammatory response initiated by tissue injury when the leaves are used in folkloric treatment of arthritis and wounds respectively . In fact, the activity may be related to the inhibition of inflammatory mediators such as histamine, serotonin, bradykinin, and prostaglandins.
On the other hand, the anti-inflammatory and analgesic activities of many plant extracts have been attributed to their high sterol/triterpene  or flavonoid contents . Other studies have demonstrated that various flavonoids such as rutin, quercetin, luteolin, hesperidin and biflavonoids produced significant antinociceptive and/or anti-inflammatory activities . On the basis of these results, it can be concluded that the different extracts of C. rotundus possess antinociceptive and anti-inflammatory activities and this may be due to any one or a combination of their phytochemical constituents .
The difference in significance between ethyl acetate and TOF-enriched extracts concerning the analgesic and anti-inflammatory activities may be due to their different constituents. Several compounds obtained from C. rotundus extracts were identified as catechin, luteolin, quercetin and ferulic acid . Previous reports indicated that various flavonoids have an anti-inflammatory activity  suggesting that flavonoids should be responsible for the anti-inflammatory and analgesic effects obtained with the different C. rotundus tested extracts. In addition, we previously showed that ethyl acetate extract contains sterols , which are documented as anti-inflammatory constituents [50, 51].
Several medicinal plants are considered immunomodulatory as they display a variety of anti-inflammatory, antimicrobial and antitumoral effects . The modulation of immune cell activities by molecules from medicinal plant origin is an area of active interest for inflammation, autoimmunity and cancer therapy. Investigating the effects of substances that promote or inhibit lymphocyte inflammatory response represents a potent means to study immunomodulation and drug discovery. In the present study, lymphocytes isolated from mice were used as models because of their critical role in controlling innate and immune inflammatory responses . Lymphocytes are the key effector cells of mammalian adaptive immune system and our studies show that the different subpopulations of lymphocytes are differentially activated by Cyperus rotundus extracts at varying levels. The extracts significantly increased the proliferation of splenocytes at concentrations of 1–1000 μg/ml, and these effects were markedly enhanced in the presence of LPS. The concomitant presence of lectin and extracts (1 mg/ml) enhance splenocyte proliferation, except with the methanolic extract. Lectin acts directly on lymphocyte T cells, which are involved in cell mediated immunity, while LPS acts on lymphocyte B cells that are responsible for humoral immune response . Therefore, C. rotundus extracts could enhance both cellular and humoral immunity depending on the concentrations. A dose of 1 mg/ml stimulates cellular and humoral immunity. In fact, lymphocyte B cell proliferation as well as lymphocyte T cell response are stimulated by both high and low extract concentrations. We believe that phenolic compounds, especially flavonoids, might contribute to the immunomodulatory effects of C. rotundus extracts and their anti-inflammatory activity.
Injury of the mucous membranes cells may provoke the production of active oxygen species. The latter can directly injure the surrounding cells and extracellular matrices, such as hyaluronic acid, and produce lipid peroxides and metabolites of arachidonic acid. In general, active oxygen species are thought to promote inflammation through these processes . On the other hand, antioxidants, such as superoxide dismutase and catechins, are known to suppress inflammation. Antioxidants may offer resistance against the oxidative stress by scavenging the free radicals, inhibiting the lipid peroxidation, and/or by other mechanisms, and thus preventing some diseases . The different extracts of C. rotundus exhibited important antioxidant activities. The results obtained in the present study may be attributed to several reasons viz: the inhibition of ferryl-perferyyl complex formation; scavenging of OH or superoxide radicals or by changing the ratio of Fe3+ / Fe2+; reducing the conversion rate of ferrous to ferric iron or by chelating of the iron itself. The antioxidant activity of the extracts may probably be due to the rapid and extensive degradation of the oxidant principles in an ex vivo state. In our study, there seems to be a good correlation between the phenolic content and antioxidant activity of the extracts, since the TOF-enriched extract with higher phenolic content showed the highest antioxidant activity. However, it is known that nonphenolic antioxidants could also contribute to the antioxidant activity of an extract. Thus, we strongly believe that the anti-inflammatory activity revealed by C. rotundus plant extracts, should be ascribed, at least in part, to their antioxidant activity as far as radical-induced actions play a central role in the inflammatory condition. Thus, the antioxidant and free radical scavenging flavonoids could prevent generation of inflammatory mediators .
Based on these results, we chose to perform the genotoxicity assay on mice by numbering the chromosome aberrations in sample treated animal cells, and comparing them to control animal cells. The chromosomal aberrations (CA) are changes in chromosome structure resulting from a break or exchange of chromosomal material. Most of the CA observed in cells are lethal, but there are many aberrations that are viable and can cause genetic effects, either somatic or inherited . Our study showed no induction of chromosome or chromatid aberrations in the cells obtained from animals treated with each tested extract. To explain this result, we propose the following hypotheses:  mutagenic compounds were deactivated in the animal body,  mutagenic compounds did not reach bone marrow cells, C. rotundus extracts does not cause genetic damage at all, or does not cause genetic damage detected by using the chromosome aberrations assay.