In Vivo Antimalarial Activity of Cyperus rotundus and Its Combination with Dihydroartemisinin against Plasmodium berghei

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Introduction
Malaria remains one of the most serious diseases and is a signifcant public health concern.Malaria is a disease that is attributed to the transmission of a Plasmodium parasite through female Anopheles mosquitoes.In humans, there are fve specifc Plasmodium species that have the ability to infect individuals, namely, P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi [1].In 2021, an estimated 247 million cases of malaria and 619,000 deaths were reported in 84 malaria-endemic nations with children under the age of 5 and pregnant women constituting high-risk groups [2].Even though an efective malaria vaccine is the most efective long-term control option, current research on vaccine development remains at the preclinical phase.Tus, the malaria control strategy mainly focuses on the use of antimalarial medications, although Plasmodium strains resistant to antimalarial drugs have rapidly emerged [3].Te World Health Organization recommends artemisinin-based combination therapy (ACT) as the initial treatment in malaria-endemic countries.Nonetheless, artemisininresistant parasites have been reported to be spreading in the Greater Mekong subregion of Southeast Asia over the past decade [3][4][5].Tis has driven the creation of antimalarial drugs with novel mechanisms of action, and medicinal plants have become regarded as potential agents that could be used in ACT.However, the claimed efcacy of medicinal plants must be scientifcally evaluated, and toxicity assays must be conducted.
Te nutgrass, Cyperus rotundus L., a sedge of the Cyperaceae family, is a colonial and perennial herb cultivated in India and commonly used as in Asian countries to cure stomach and bowel ailments, as well as infammatory conditions.Several studies have documented that C. rotundus extract has pharmacological properties, including antioxidant, antiinfammatory, antipyretic, antidiarrheal, antiarthritic, antihyperglycemic, antihyperlipidemic, antiulcer, antimicrobial, antihelmintic, antiallergic, neuroprotective, gastroprotective, hepatoprotective, wound healing, and analgesic efects [6,7].Te phytochemical components of C. rotundus include alkaloids, favonoids, phenols, terpenoids, glycosides, tannins, saponin, steroids, starch, and a large number of novel sesquiterpenoids [8].In addition, C. rotundus extract has demonstrated potent antimalarial activity against P. falciparum in culture [9,10].However, the antimalarial activity of C. rotundus alone and in combination therapy with artemisinin derivatives has not yet been investigated in animal models.Terefore, the aim of this study was to evaluate the efectiveness of the aqueous crude extract of C. rotundus, both alone and in combination with dihydroartemisinin, in treating mice that were infected with P. berghei.

Plant Material and Extraction.
Fresh C. rotundus rhizomes were gathered in the Chiang Mai province of Tailand in October 2022.Botanical identifcation was performed, and a voucher specimen (NRU65/09-010) was stored at the Research Institute for Health Sciences, Chiang Mai University, Chiang Mai, Tailand.Plant materials were washed with water and air-dried at room temperature.Te dried material was then reduced to coarse powder and subsequently prepared as a fne powder using an electric blender (Mettler-Toledo, AB204-S, SN 1120510763).For aqueous crude extraction, 30 g of dried powdered plant material was extracted in 150 mL of boiled distilled water (DW) at room temperature for 20-24 h using an orbital shaker (SUSPA lifeline, AFB1224SHE, SN MU-E70-91031) and fltered through a Whatman no.1 flter paper.Te fltrate was then centrifuged (Hettich Zentrifugen, ROTANTA 460RF, SN 0000813-06) at 4,000 g for 10 min, and supernatant was collected.Te supernatant was lyophilized (Martin Chist, Alpha 1-4 LSCpkus, SN 20464) to obtain an aqueous crude extract of C. rotundus (CRE), which was stored at 4 °C [11].CRE was dissolved in DW at chosen doses for oral administration by gavage to mice.

Preparation of Dihydroartemisinin.
DHA was purchased from Sigma-Aldrich (Sigma-Aldrich, Inc., St. Louis, MO, USA).Fresh doses of DHA were prepared in DW and stored at 4 °C.

Experimental Mice.
Four-week-old male BALB/c mice (22-25 g) were purchased from Nomura Siam International, Tailand.Mice were kept for a minimum of one week in a standard laboratory environment and were given standard pellets (CP diet 082, Perfect Companion Company, Tailand) and access to clean water ad libitum.Te National Institutes of Health Guidelines for Care and Use of Laboratory Animals were followed for all mouse-related experiments (2011).Te Walailak University Animal Care and Use Committee approved all experimental protocols (WU-ACUC-65077).

Acute Toxicity
Testing.Te acute toxicity of the CRE was determined in accordance with the Organization for Economic Cooperation and Development (OECD) Guideline 423 [12].BALB/c mice were randomly divided into two groups (n � 5).Te frst group received a single dose of 2,000 mg/kg of CRE orally by gavage, whereas the second group received DW.During the frst 30 min and daily thereafter, mice were observed for any clinical signs of toxicity (paw licking, salivation, stretching of the entire body, weakness, respiratory distress, and coma) or mortality.

Subacute Toxicity Testing. Te procedures described in
Guide 407 of the OECD guidelines were performed using the limit test with 1,000 mg/kg of CRE [13].BALB/c mice (n � 5) were administered a daily dose of 1,000 mg/kg of CRE orally by gavage for 30 days.Te control group received 10 mL/kg of DW.At the end of the 30-day observation period, the mice were anesthetized, and blood was then collected by cardiac puncture in heparinized vacuum tubes for biochemical marker measurement.

Biochemical Marker Measurement.
After centrifuging blood samples at 3,000 g for 10 min, plasma was collected for biochemical marker analysis.Using an automated clinical chemistry analyzer (Cobas c111), the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), blood urea nitrogen (BUN), and creatinine were measured.2.7.Rodent Malaria Parasite.P. berghei ANKA strain (PbANKA), obtained from the Malaria Research and Reference Reagent Resource Center, was used in this study.Parasites were maintained in mice by sequential blood passage through intraperitoneal (IP) injection with a 5-day interval.Infected mouse blood with parasitemia of 15-30% was collected via cardiac puncture and diluted with normal saline to 5 × 10 7 parasitized erythrocytes per mL.Each mouse was then inoculated with 0.2 mL of this diluted blood via IP injection.[15].On D0, 25 BALB/c mice were inoculated via IP injection with 1 × 10 7 PbANKA-parasitized erythrocytes.On D4, the experimental groups were treated with CRE (100, 200, and 400 mg/kg) orally by gavage.On the other hand, the untreated and positive controls received 10 mL/kg of DW and 5 mg/kg of DHA, respectively.Treatment was performed once a day for 4 consecutive days (D4-D7).On D8, parasitemia, PCV, BW, rectal temperature, and MST were then measured.Percent inhibition was also calculated.

Prophylactic Test.
Te method described by Peters (1965) was used to assess the prophylactic activity [16].
Twenty-two BALB/c mice were administered with 100, 200, and 400 mg/kg of CRE orally by gavage once a day for four consecutive day (D0-D3).Mice were then inoculated with 1 × 10 7 PbANKA-parasitized erythrocytes via IP injection on D4 and monitored for 72 h (D4-D7).Untreated and positive controls received 10 mL/kg of DW and 5 mg/kg of DHA, respectively.On D8, parasitemia, PCV, BW, rectal temperature, and MST were measured, and percent inhibition was calculated.

Evaluation of Efective Dose.
Te standard 4-day suppressive test was used to determine the efective dose (ED 50 ) of the drugs (CRE and DHA) [14].BALB/c mice (n � 5) were injected intraperitoneally with 1 × 10 7 parasitized erythrocytes of PbANKA.At 2 h after infection, mice were orally administered with CRE (50, 100, 200, 400, and 600 mg/kg) and DHA (0.1, 1, 5, 10, and 20 mg/kg) daily for four days (D0-D3).Infected mice without treatment received 10 mL/ kg of DW.On day 4, the parasitemia was measured, and percent inhibition was then calculated.Moreover, the optimal ED 50 value was determined by using a nonlinear regression for estimating the dose-response variable slope.

Combination Antimalarial Treatment.
Te ED 50 values of CRE and DHA were used to evaluate the combination treatment.Te CRE and DHA were combined at a fxed ratio (1 : 1) of ED 50 , ED 50/2 , ED 50/4 , and ED 50/8 .Te combination was tested using the standard 4-day suppressive test [14].On D4, parasitemia was measured, and percent inhibition was calculated.Points above the joint line indicated synergism while those around the line or below indicated additive or antagonistic interactions, respectively.A combination index (CI) value was also performed.
Advances in Pharmacological and Pharmaceutical Sciences 2.17.Statistics.Results are displayed as the mean ± standard error of the mean (SEM).Using nonlinear regression for the dose-response variable slope, the optimal ED 50 value was determined.A one-way ANOVA with Tukey's post hoc test was used to compare the means of control and treatment groups.Signifcance was considered with 95% confdence intervals and p < 0.05.Data were analyzed with GraphPad Prism 9.5.0 (GraphPad Software, Inc., San Diego, CA, USA).CompuSyn software (ComboSyn, Inc., USA) was used to calculate the CI value to indicate synergism (CI < 1), additive efect (CI � 1), and antagonism (CI > 1).

Toxicity Test of CRE in Mice.
Te acute toxicity test revealed that oral administration of CRE at a single dose of 2,000 mg/kg in mice did not cause mortality or produce any signs of toxicity in treated mice, and all mice exhibited normal behavior throughout the 14-day observation period.
In the subacute toxicity test, administration of 1,000 mg/kg of CRE daily for 30 days did not induce any toxic symptoms or cause mortality in mice, which behaved normally throughout the experiment.No signifcant changes in the plasma levels of AST, ALT, ALP, BUN, or creatinine were observed in biochemical tests of liver or renal injuries (Table 1).Consequently, the LD 50 value for CRE was >2,000 mg/kg, and CRE was subsequently considered safe for oral administration.
In addition, BW and rectal temperature were considerably decreased in PbANKA-infected mice (Figure 1(b)), and all infected mice died within two weeks (Figure 1(c)).Hence, PbANKA infection induced malaria-associated hemolysis, BW loss, and hypothermia.

Discussion
Recent resistance to artemisinin and its derivatives has emerged in Southeast Asia.Consequently, new antimalarial drug candidates, such as those derived from medicinal plants, are urgently required.Tis study therefore investigated the antimalarial activity of CRE and its combination with DHA against PbANKA infection in mice.Te acute toxicity test in mice evaluates the adverse efects over a short period following administration of a single, high dose of a substance.No signs of toxicity, behavioral change, or mortality were observed in mice administered with 2,000 mg/kg of CRE.Tus, CRE with an LD 50 >2,000 mg/kg can be considered nontoxic.Toxicity evaluation after repeated dosing provides evidence of dose response with potential health risks after 30 days of subacute toxicity testing.Here, dosing of mice at 1,000 mg/kg of CRE for 30 days did not produce any abnormal signs, toxicity, or death or any changes in biochemical markers.After CRE treatment, the plasma levels of AST, ALT, ALP, BUN, and creatinine levels remained within physiological limits, indicating that CRE did not adversely afect the liver or kidneys.Tus, CRE can be regarded as relatively harmless in terms of acute and subacute toxicity.If the LD 50 of the test substance is >3-fold, the minimum efective dose (400 mg/ kg), the drug may be an appropriate candidate for further study [17].
During ongoing PbANKA infection in mice, malariaassociated hemolysis, BW loss, and a decrease in rectal temperature were observed.PCV reduction is a defning characteristic of both human and rodent malaria.Mice with a high parasitemia experience a rapid destruction of parasitized and uninfected erythrocytes, suppression of erythropoietin, and dyserythropoiesis and may develop severe anemia [18,19].In addition, an increase in the amount of free radicals and infammatory cytokines, followed by oxidative stress and lipid peroxidation, may contribute to hemolysis during malaria infection [20].BW loss is a common Advances in Pharmacological and Pharmaceutical Sciences symptom of P. berghei infection in mice and is associated with an increase in parasitemia.BW loss may be caused by a catabolic action on stored lipids, a hypoglycemic efect of the parasite, or an anorexigenic efect on mouse appetite that suppresses food consumption [21,22].Mice infected with P. berghei experienced a decline in rectal temperature because of the presence of the parasite.Malarial infections can decrease the metabolic rate and cause a decrease in rectal temperature.Terefore, these results agreed with those of other studies [23][24][25].
Te antimalarial activity of CRE was investigated using the 4-day suppressive, curative, and prophylactic tests.PbANKA-infected mice were evaluated for schizontocidal activity against early, established, and residual infections.In the 4-day suppressive test, CRE signifcantly reduced the level of parasitemia, and the highest parasite inhibition (55.3%) was found at 400 mg/kg.Tus, CRE may exert an antimalarial activity that potentially mitigates the early infection by PbANKA.General extracts that inhibit parasitemia by ≥30% are considered active [26].In vivo antimalarial activity can be classifed as moderate, good, or very good if the percentage of inhibition is ≥50% at doses of 500, 250, and 100 mg/kg, respectively [27].Consequently, this study showed that CRE possesses active and moderate 4-day suppressive antimalarial activity.Te antimalarial activity of crude extracts of medicinal plants is due to the presence of active compounds.Several studies have implicated secondary metabolites, such as favonoids, phenols, terpenoids, alkaloids, saponins, tannins, glycosides, and anthraquinone, in this antimalarial activity [28][29][30].Phytochemical analysis has revealed that CRE contains alkaloids, favonoids, phenols, terpenoids, glycosides, tannins, saponin, steroids, starch, and many novel sesquiterpenoids [8].α-Cyperone and β-selinene autoxidation products derived from C. rotundus are highly promising antimalarial compounds [31].Furthermore, sesquiterpenoids, such as patchoulenone, caryophyllene α-oxide, 10,12-peroxycalamenene, and 4,7-dimethyl-1-tetralone, have been reported to exhibit antimalarial activity against P. falciparum in culture [10].Terefore, the antimalarial activity observed in the CRE may be attributable to such secondary active compounds, which may act singly or in combination.However, it is necessary to identify the compounds responsible for the parasite inhibition.Te antimalarial activity of CRE might be mediated by various mechanisms, including the response of the antioxidant and immunomodulatory systems, suppression of protein synthesis suppression, inhibition of enzyme activity, interference with 6 Advances in Pharmacological and Pharmaceutical Sciences erythrocyte invasion by parasites, inhibition of parasite growth and multiplication, blocked entry of nutrients into parasite, inhibition of heme polymerization, or other unidentifed mechanisms [30].Parasite inhibition was not observed at the lower doses (100 and 200 mg/kg) of CRE.Tis might be because the active compounds in the extract were only present at a low level, and their activity may not be detectable at these doses.In both curative and prophylactic studies, no signifcant diferences in inhibition of parasitemia by CRE at any of the three doses were observed compared with that of the untreated control.Tese results suggest that CRE has a greater chemosuppressive efect on early infection than on either established or residual infections.Tis lack of efect may be associated with the metabolic processing of CRE following administration and reduction of its concentration in the body, and it could also be related to the rapid multiplication of the parasite in an established infection, where the parasite is growing exponentially.In addition, the absence of efect of low concentrations may be due to the rapid hepatic metabolism or metabolic inactivation and clearance of the CRE active compounds before parasite inoculation in the prophylactic test.
PbANKA infection is typically manifested by a decrease in PCV, loss of BW, and decline in rectal temperature, and treatment with CRE with antimalarial activity could therefore protect against these abnormalities.In all test models, the highest CRE dose (400 mg/kg) signifcantly (p < 0.01) inhibited the reduction of PCV, BW, and rectal temperature compared with those in untreated controls.CRE treatment could ameliorate anemia by preventing the destruction of erythrocytes caused by PbANKA.Te antioxidant favonoids and tannins in CRE may signifcantly help to protect erythrocytes from oxidative stress and infammation during infection [32].In addition, the polyphenolic compounds in the CRE may increase the survival rate of both uninfected and infected erythrocytes [8].Surprisingly, a signifcant (p < 0.01) decrease in PCV was observed in all tested models in mice treated with DHA.Artemisinin drugs and derivatives are known to kill malaria parasites by inducing oxidative stress following the activation of the peroxide bridge, which generates reactive metabolites that cause hemolysis [33].In addition, artemisinin-induced and postartemisinin-delayed hemolysis have been reported [34,35].Tis fnding is consistent with other reports on artemisinin-induced hemolysis in PbANKA-infected mice [36][37][38].Te mechanism by which CRE prevents BW loss may involve the reduction of parasitemia in PbANKA-infected mice to enable normal metabolism and growth to continue unimpeded.In addition, this is probably due to the activation of an appetite stimulant and the addition of vitamin B [30].CRE treatment stabilized temperatures in PbANKA-infected mice.Tis may be due to  Advances in Pharmacological and Pharmaceutical Sciences the CRE suppressing parasites and regulating pathological and immune processes in infected mice, thereby compensating for the decrease in metabolic rate that causes a decrease in rectal temperature.Furthermore, the presence of active metabolites, including polyphenols, favonoids, terpenoids, steroids, tannins, glycosides, and saponins, which tend to stabilize temperature, may prevent the PbANKA-induced decrease in rectal temperature [3].However, the activity of 100 and 200 mg/kg of CRE was insufcient to prevent these abnormalities in PbANKA-infected mice.MST is an additional criterion used to evaluate the antimalarial activity of plant extracts, and extracts that produce a longer MST than that of the untreated control are considered active.In this study, PbANKA-infected mice treated with 400 mg/kg of CRE lived signifcantly longer than untreated controls in all three antimalarial test models.Tis may be due to the antimalarial properties of the CRE and the prevention of PCV depletion, BW loss, and a decrease in rectal temperature.However, the MST of mice treated with CRE was shorter than that of mice treated with DHA.Te current fndings were consistent with those of previous studies [23,37,38].
Combination strategies are preferred over single strategies in treating malaria, and we therefore tested the combination of CRE and DHA on PbANKA-infected mice.Te combination of CRE and DHA at doses of ED 50/1 and ED 50/2 showed signifcant antimalarial activity compared with that of the untreated control, although only ED 50/2 exerted a signifcant efect compared with that of the CRE or DHA treatment alone.As indicated by the CI value <1.0, a synergistic interaction was observed.However, the lower doses (ED 50/4 and ED 50/8 ) of the combination had no signifcant efect on parasitemia.Te antimalarial mechanism for this combination treatment is not readily apparent from our current research, as both compounds could exert an antimalarial synergistic efect via their own individual actions or via a novel pathway induced by the combination.Terefore, we consider that the combination of CRE and DHA may provide an alternative antimalarial therapeutic approach.

Conclusions
Our study clearly indicates that CRE has potent antimalarial activity with the highest parasitemia inhibition observed at 400 mg/kg in the 4-day suppressive test.Combining CRE with DHA at doses of ED 50/2 showed more successful chemotherapeutic treatment in the PbANKA-infected mouse model.In addition, CRE in combination with DHA could inhibit PCV reduction, BW loss, and hypothermia induced by malaria infection and prolong MST.Tese fndings also support the traditional claim of C. rotundus for malarial treatment.However, further investigations should be conducted to isolate and identify the active compounds and elucidate the mechanism of action for antimalarial activity from this extract.

ACT:
Artemisinin-based combination therapy ALP: Alkaline phosphatase ALT: Alanine aminotransferase ANOVA: Analysis of variant AST: Aspartate aminotransferase

Figure 1 :
Figure 1: PbANKA infection in Balb/c mice.Mice were infected with PbANKA and parameters including (a) parasitemia and PCV, (b) BW and rectal temperature, and (c) survival time were then investigated as previously described in Methods section.Results were expressed as the mean ± SEM (n � 5).

Figure 3 :
Figure 3: Curative antimalarial activity of CRE against PbANKA infection in Balb/c mice.Mice were inoculated with PbANKA for 4 days and administered orally with 100, 200, and 400 mg/kg of CRE for 4-consecutive days as previously described in Methods section.(a) Parasitemia, (b) PCV (c) BW, and (d) rectal temperature were measured.Moreover, (e) MST was also investigated for 30 days.Results were expressed as the mean ± SEM (n � 5).* * p < 0.01, and * * * p < 0.001 compared to UN or H. UN: untreated control; H: healthy control; 100, 200, and 400 CRE: aqueous crude extract of C. rotundus at the doses of 100, 200, and 400 mg/kg, respectively; DHA: 5 mg/kg of dihydroartemisinin.

Figure 4 :Figure 5 :
Figure 4: Prophylactic antimalarial activity of CRE against PbANKA infection in Balb/c mice.Mice were administered orally with 100, 200, and 400 mg/kg of CRE for 4-consecutive days and then infected with PbANKA for 4 days as previously described in Methods section.(a) Parasitemia (b) PCV (c) BW, and (d) rectal temperature were measured.Moreover, (e) MST was also investigated for 30 days.Results were expressed as the mean ± SEM (n � 5).* * p < 0.01, and * * * p < 0.001 compared to UN or H. UN: untreated control; H: healthy control; 100, 200, and 400 CRE: aqueous crude extract of C. rotundus at the doses of 100, 200, and 400 mg/kg, respectively; DHA: 5 mg/kg of dihydroartemisinin.

Table 1 :
Te parasitemia level did not signifcantly decrease at any of the three CRE doses utilized to treat mice (Figure3(a)).In comparison to the healthy control group, mice that were administered with 400 mg/kg of CRE did not exhibit statistically signifcant decreases in PCV (48.8 ± 0.7%), BW (23.6 ± 0.8 g), or rectal temperature (34.2 ± 0.6 °C).Efect of oral administration of CRE on biochemical markers in mice.