Cyperus iria linn. Roots ethanol extract: its phytochemicals, cytotoxicity, and anti-inflammatory activity

ABSTRACT Inflammatory diseases are normally treated with non-steroidal anti-inflammatory drugs which may cause adverse effects if used for prolonged periods. Thus, there is a need to search for alternative anti-inflammatory medicine. This study investigated the phytochemical characteristics, cytotoxic, and anti-inflammatory activities of C. iria Linn. roots ethanol extract. The extract was subjected to preliminary phytochemical screening using standard procedures. The anti-inflammatory activities were determined using the egg albumin denaturation assay and the human red blood cell membrane stabilization method while the cytotoxic activity was investigated using the brine shrimp lethality assay. Preliminary phytochemical screening revealed the presence of flavonoids, phenolic acids, terpenoids, and saponins in the extract. Anti-inflammatory assays revealed that the extract has comparable results with the positive control (250 µg/ml Celecoxib) while being non-toxic. These results suggest that the synergetic actions of identified phytochemicals may contribute to the anti-inflammatory effect on egg albumin and human red blood cells.


Introduction
Inflammation is a natural and complex process associated with vascular permeability, membrane alteration, and an increase in protein denaturation. Immune cells play important roles in inflammatory responses and are activated by the release of chemical mediators from damaged tissues [1]. However, if this process is not properly directed, inflammation can result in more adverse tissue damage leading to allergy, rheumatoid arthritis, or pancreatic and prostate cancers [2,3]. Inflammation is commonly medicated with either steroidal anti-inflammatory drugs (SAIDs) or non-steroidal antiinflammatory drugs (NSAIDs). However, most of these drugs are expensive and may cause gastrointestinal (GI) and renal adverse effects in long-term use [4]. GI adverse effects may include minor symptoms such as heartburn, dyspepsia and nausea, and serious gastrointestinal hemorrhaging. Renal adverse effects include the inability to detoxify the blood and some serious kidney diseases [5]. Thus, there is an increasing interest in discovering new non-toxic, anti-inflammatory agents that originate from natural products [6].
The role of cytotoxicity test in the development of drugs is very important, it is a critical factor in drug development from natural products [7]. Cytotoxicity studies are widely used for the toxicity of substances and drug screening. It is an important initial study in determining the tolerable level of a substance such as natural products in cells. Substances may have different cytotoxic mechanisms including cell lysis, malfunctioning of binding receptors, and protein synthesis inhibition [8,9]. Cytotoxic assays are widely used for preliminary in vitro toxicity screening before further experiments on animal models. Testing the cytotoxicity level of a natural product is an initial step in developing it into an alternative medically important substance [10]. Thus, there is a need to look for alternative medicine which is non-toxic, has fewer adverse side effects, and is yet effective.
Natural products are an important source of novel drugs for the treatment of many diseases [11][12][13][14][15]. Plants that are known to have medicinal value have potential applications in biomedical [16][17][18]. Nowadays, most pharmaceutical drugs originated from traditional alternative medicine [19]. The Food and Drug Administration (FDA) approved more than one-third (39.1%) of drugs that are of natural origin [20]. Up to this date, natural products are still important sources of drug discovery [21][22][23][24]. The diversity of secondary metabolites illustrates the importance of natural products for the development of new drugs [25]. The ability of plants to develop a complex defense system resulted in a complex range of chemicals. This plant's ability makes it one of the important sources of novel active compounds that have pharmaceutical importance [26]. Compared to NSAIDs and SAIDs, natural products are believed to be reasonable, environment-friendly, costefficient, and have fewer adverse side effects [27].
Traditionally, Cyperus plants had been used as a remedy to treat different human ailments involving digestive and inflammatory diseases [28]. Several Cyperus species such as Cyperus rotundus [29,30], Cyperus esculentus L. [31], and Cyperus articulatus [32] had been proven to have medicinal value due to the presence of different bioactive constituents. This only proved that several Cyperus spp. had promising pharmacological effects and biological activities that may be beneficial for various disease treatments [33]. Cyperus iria Linn. (C. iria Linn.), or payong-payong, is a common annual sedge weed belonging to the same family. It is widely distributed on irrigated rice fields in the different parts of the Philippines and is considered an invasive species that reduce crop yield and quality [34,35]. But in traditional Chinese medicine, C. iria Linn. has been used as a treatment for fracture and rheumatism [36]. A review of the phytochemical composition of Cyperus spp. revealed that several of its members are rich in biological compounds that exhibited pharmacological properties [33] and this may also be present in C. iria Linn. Currently, there is no published research on the anti-inflammatory and cytotoxic activities of C. iria Linn.
The phytochemical properties of C. iria Linn. have not been reported except for the presence of terpenoids [36]. However, several species from the Cyperus genus have well-documented phytochemistry [33]. Cyperus species such as C. rotundus, and C. esculentus L. have been found to harbor the following phytochemicals: alkaloids, flavonoids, phenols, and tannins [37,38]. Thus, the chances of these phytochemicals being present in C. iria Linn. is likely to be high. The presence of phytochemicals, cytotoxic effects, and anti-inflammatory potential of the C. iria Linn. roots ethanol extract (CREE) are determined in this study. This research hopes to reduce the presence of C. iria Linn, an invasive weed in rice agriculture, by utilizing it as a possible source of alternative anti-inflammatory medicine. This study may also provide information to other researchers who have a scientific interest in C. iria Linn. particularly its anti-inflammatory and cytotoxic properties.

Chemicals
Phosphate buffered saline (Sigma-Aldrich) and isotonic saline (0.9%) were obtained from local distributors. All the other chemicals used in the study were analytical grade and were readily available in the Chemistry Analytical Research Laboratory of Ateneo de Davao University.

Preparation of C. iria Linn. Roots Ethanol Extract (CREE)
The Cyperus iria Linn. (payong-payong) whole plant was collected from Maguindanao province, Philippines, and was authenticated at the Ateneo de Davao University (AdDU) Biological Collections, Philippines. The voucher specimen (AdDU2019.02) was kept in the AdDU Herbarium for future reference. The roots of C. iria Linn. plants were immediately washed with running tap water to remove dirt and other debris. The roots were cut into small pieces (1 cm) and were oven-dried at 40°C using a precision compact heating and drying oven (BIO-BASE BOV-T105F, Metro Manila, Philippines). Small pieces of rough dry roots were weighed using an analytical balance (Essae PG-34000 Precision Weighing Balance) every 24 h until a constant mass was reached. The ovendried sample (250 g) was soaked in a 1.5 L ethanol solution (95%) for 48 h. The extract was filtered through Whatman filter paper No. 1 and was concentrated using a rotary evaporator (IKA RV-3, Selangor, Malaysia) set at 60°C. The crude ethanolic extracts of C. iria Linn. roots weighed 41 g.

Phytochemical screening
The presence of secondary metabolites such as flavonoids, tannins, phenolic acids, alkaloids, terpenoids, and saponins of the CREE was evaluated using the standard methods by Harborne [39] and Guevarra [40].

Egg albumin denaturation assay
CREE's in vitro anti-inflammatory potential was estimated using the egg albumin denaturation assay with some modifications as described by Alam et al. [41]. This assay was conducted to determine if the CREE can inhibit protein denaturation which can cause inflammation [2]. A mixture of 200 μL of egg albumin (from fresh hen's egg), 2800 μL of phosphate-buffered saline (pH 6.4), and 2000μL of the samples or controls in different test tubes were prepared. These mixtures were incubated at 37 0 C for 15 min, then heated in a water bath at 70 0 C for 5 min. The mixtures were allowed to cool to room temperature for 5 min. Five hundred microliters (500 μL) of every mixture were transferred in their respective microwell on a 24-well plate. The absorbance of the samples was read at 660 nm using Epoch Microplate Spectrophotometer (Biotek, Winooski, USA). The experiment was conducted in triplicates and in three independent trials. The percent inhibition of protein denaturation was determined using the formula:

Human Red Blood Cell Membrane (HRBCM) stabilization method
HRBCM stabilization method was conducted to determine if CREE can protect cells from lysis which is one of the causes of inflammation [42,43]. Fresh whole human blood (5cc) was extracted by a licensed Medical Technologist from healthy volunteers who have not consumed any steroidal or non-steroidal anti-inflammatory drugs before the collection. The extracted blood was immediately transferred to heparin tubes and used for the bioassay. The collected blood sample was mixed in a conical tube with an equal volume of isotonic saline (0.9%) and was set aside for one minute to allow the blood cells to settle. The blood was centrifuged (Fisher Scientific, Waltham, USA) at 3000 rpm for 10 min and the supernatant was discarded. The packed cells were mixed with an equal volume of 0.9% isotonic saline (pH 7.2) for washing. This was done three times. The volume of the blood was determined and transformed as 10% v/v suspension with isotonic saline. The suspension was used immediately for the heat-induced hemolysis.
Heat-induced hemolysis was conducted by mixing 1000 μL of 0.15M phosphate buffer (pH 7.4), 2000μL isotonic saline (0.9% NaCl), 500 μL HRBC suspension (10% v/v), and 500 μL of the samples or controls in conical tubes. The mixtures were gently shaken and heated at 40 0 C using an incubator for 60 min. The tubes were allowed to cool for 10 min and centrifuged at 1500 rpm for another 10 min. The resulting supernatant in every reaction mixture was transferred using a single channel adjustable pipette to its respective microwell plates. The absorbance was read at 560 nm using an Epoch Microplate Spectrophotometer (Biotek, Winooski, USA) to determine the percent inhibition of hemolysis of the human red blood cells. The experiment was conducted in triplicates and in three independent trials. Percent inhibition of hemolysis was determined using the formula:

Brine shrimp lethality assay
Brine shrimp lethality assay was conducted to determine the cytotoxicity of the different concentrations of CREE [44]. A complete randomized design by drawing lots was applied in the assignment of samples and control. Three thousand microliters (3000 μL) of the different samples or control (artificial seawater) were placed in the respective well using a Pasteur pipette. Ten brine shrimps were pipetted in every microwell. The number of alive, impaired, and dead brine shrimps were counted after 30 min, 6, and 24 h of exposure to the samples and control. The experiment was conducted in five replicates and three independent trials.

Statistical analysis
All values for the anti-inflammatory assays were analyzed with ANOVA (SPSS vs. 26) followed by Tukey's HSD post hoc test. The values of p < 0.05 were considered statistically significant. This test was used to determine if the results of the anti-inflammatory assays have significant differences compared to the results of positive control.

Phytochemical screening
Phytochemical screening was conducted to evaluate the presence of secondary metabolites in CREE ( Table 1). The presence of flavonoids, phenolic acids, terpenoids, and saponins was confirmed using the Wilstatter "Cyanidin" test, ferric chloride test, Salkowski test, and froth test, respectively. On the other hand, tannins and alkaloids were absent when tested using the gelatin test and Dragendorff's and Mayer's test, respectively. Figure 1 shows the percent inhibition of protein denaturation of the different concentrations of CREE relative to the positive control (250 μg/mL Celecoxib). The observed IC50 is 434 μg/mL, indicating that this is the amount of concentration that inhibits the process in protein denaturation assay by half [45]. Results (Table 2) showed that 1000 μg/mL CREE exhibited comparable protein denaturation inhibition activity with the positive control (250 μg/mL Celecoxib) (p < 0.999). Similar to other Cyperus species such as C. rotundus [29,30,[89][90][91] and C. esculentus [31], this implies that CREE may have a potent anti-inflammatory property [33]. The potential of CREE to protect proteins from denaturation may be attributed to its flavonoid contents [46]. Flavonoids possess a significant binding affinity for proteins [47,48]. The non-covalent attraction between protein and flavonoids may give rise to multiple hydrogen bonds that influence the strength of the formed protein-flavonoids complexes [49,50]. In the study of Zhang et al. [51], it is indicated that proteins that interact with flavonoids have structures that are more conformationally open and flexible. Seczyk et al. [52] emphasized that phenolic molecules can also link with Notes: Values are mean ± SD (n = 9). Mean values with an asterisk ( * ) in the same column are significantly different from the positive control (250 μg/mL Celecoxib) using one-way ANOVA.

Egg albumin denaturation assay
different fragments of the polypeptide chain of protein molecules making it stable even if exposed to a stressor such as heat.
The presence of flavonoids in the CREE could also have potentially increased the thermal stability of the protein making it more stable. This observation is supported by several studies conducted [53][54][55][56]. Particularly, studies conducted by Ali et al. [53] and Hu et al. [55], demonstrated that the thermal stability of proteins increased as a result of interaction with flavonoids. In the study conducted by Ali et al., [53], the aggregation bands were not observed among complexes of the protein bovine serum albumin (BSA) and flavonoids. The inhibition of aggregation in BSA suggests that the hydroxyl groups of flavonoids can bind simultaneously at more than one site on this molecule preventing its unfolding [57]. Higher thermal stability was also observed in the complexes of protein glycinin and flavonoids [53,58]. The denaturation temperature of glycinin increased in the presence of the flavonoids indicating conformational structural changes in the protein [59][60][61][62], possibly resulting in the formation of proteinflavonoids complexes through hydrogen bonding and hydrophobic interaction.
The presence of saponins may also contribute to the anti-denaturation activity of CREE [63,64]. A study conducted by Grabowski et al. [64], showed that saponins were potent inhibitors of BSA molecules that were denatured via increasing temperature. Another phytochemical that may also contribute to the inhibited denaturation of albumin is the terpenoids [65,66]. A study conducted by Truong et al. [66] showed that terpenoids have the potential to inhibit the denaturation of protein albumin exposed to heat.

Human red blood cell membrane (HRBCM) stabilization method
The percent inhibition of hemolysis of the different concentrations of CREE relative to the positive control (250 μg/mL Celecoxib) is shown in Figure 2. In this assay, the observed IC50 is 355 μg/mL, indicating that this is the amount of concentration that inhibits the process in the HRBCM stabilization method by half [45]. Results from the HRBCM stabilization method ( Table 2) showed that 1000 μg/mL CREE exhibited comparable percent inhibition of hemolysis with the positive control (250 μg/mL Celecoxib) (p < 0.900). This implies that CREE may have a potent anti-inflammatory property [33].
The potential of CREE to inhibit hemolysis may be attributed to its flavonoid contents [67,68]. Some flavonoid contents such as naringenin, rutin, and genistein [69] had been known to interact with lipid bilayers and can alter the membrane's physicochemical properties [70]. Flavonoids usually partition the unilamellar vesicles and decrease the membrane fluidity of cells [70]. The decrease in the membrane fluidity in cells leads to an increase in membrane rigidity and strength, thus making the membrane more stable [71]. Considering the structural similarity of human RBC and the lysosomal membranes, the results of this study suggest that CREE may stabilize lysosomal membranes thereby preventing the release of lysosomal components that may contribute to inflammatory responses [72].
A possible mechanism in which plant extracts may inhibit membrane lysis is by reducing lipid peroxidation [73][74][75]. The deterioration of lipids in plasma and the   organellar membrane is due to the presence of free radicals [76][77][78][79][80]. These free radicals such as oxygen-derived free radicals particularly the hydroxide (OH) may cause oxidative damage by linking to the double bonds in the unsaturated fatty acids [81]. This interaction can produce unstable and reactive peroxide leading to an extensive membrane, organellar and cellular damage [80]. Lipid peroxidation due to oxidative destruction of polyunsaturated fatty acids is harmful because it may alter the integrity of membranes [82]. The presence of phenolic acids in CREE may reduce lipid peroxidation [83][84][85]. Thus, phenolic acids can stabilize cell membranes by reducing lipid peroxidation [86][87][88]. The presence of phenolic acids in CREE implies a potent reduction of lipid peroxidation as a possible mechanism for stabilizing membranes thereby preventing inflammatory responses.
The findings of this study suggest that CREE may have a potential source of anti-inflammatory compounds similar to other Cyperus species such as C. rotundus [29,30,[89][90][91] and C. esculentus [31]. A study conducted by Fenanir et al. [91], revealed that several essential oil molecules from the roots of C. rotundus may have potent inhibitory activity against 5-LO and LTA4H, which plays an important role in inflammatory responses. Since C. iria and C. rotundus are closely related, the chances of having similar compounds present in their system are possibly high [92]. And to establish the potential of CREE for its anti-inflammatory activity, the Quantitative Structure-Activity Relationship (QSAR) and molecular docking of potential new active compounds towards acetylcholinesterase enzyme must also be considered in the future studies [93][94][95].

Brine shrimp lethality assay
The percent mortality of brine shrimps after 24 h of exposure to the various CREE concentrations was shown in Figure 3. Results showed that the CREE concentrations are non-toxic since the rate of mortality of brine shrimps in all concentrations did not reach 50%. Data of BSLA shows an LC 50 of 2250 μg/mL CREE concentration. The absence of alkaloids in CREE may have contributed to the non-toxicity of the extract. Alkaloids can penetrate the cell membrane of larval brine shrimps, thus causing damage and disrupting the membrane's permeability leading to brine shrimps' death [96].
This low cytotoxicity of CREE is consistent with the data obtained from studies using other species of the genus Cyperus. In the study of Khojaste et al. [97], results of the cytotoxicity test suggested that Cyperus rotundus tuber alcoholic extract showed no toxic effects on human gingival fibroblast (HGF) cells after 24 h of exposure to the samples while in the study of Hu et al. [55], C. rotundus rhizome showed an effective decrease in the development of nicked DNA and elevated supercoiled form of DNA. This indicates a potential to prevent DNA damage in cells.

Conclusions
The CREE at a concentration of 1000 ug/mL showed potential anti-inflammatory activity using egg albumin denaturation assay and human red blood cell membrane stabilization method. The synergistic effect of the phytochemicals such as the flavonoids, phenolic acids, terpenoids and saponins in CREE may have contributed to its anti-inflammatory activity. Moreover, all the concentrations of CREE are non-toxic to brine shrimps. The non-toxicity of CREE may be attributed to the absence of alkaloids. Thus, this implies that CREE at 1000 ug/mL is non-toxic to cells and may have contributed to the stability of the protein albumin and stabilizes biological membranes, thus having a potent anti-inflammatory property. This discovery of the potential medicinal use of C. iria may elevate its value from being an agricultural pest to being pharmaceutically significant. For further studies, the actual mechanism of membrane stabilization and enhancement of protein stability by CREE be determined to verify further its anti-inflammatory property. The conduct of in vivo assays, such as the paw edema test and ear carrageenan test can be used to further test the anti-inflammatory property of CREE.

Disclosure statement
No potential conflict of interest was reported by the author(s).