Abstract: Background and Objective: Cyperus rotundus (C. rotundus) has been used in medicine years ago. The aim of the study was to investigate the possible anti-angiogenic and antioxidant activity and to find the different phytochemicals present in Cyperus rotundus ethanol extract that may has anti-angiogenic activity. Methodology: Qualitative analysis of various secondary metabolites by specific chemical tests was carried out on the ethanol extract. The ex vivo rat aorta ring assay was used to screen the extract for possible anti-angiogenic activity, this assay was also used to determine the dose-response effect of the active extract. Six concentrations of crude extract were tested (100, 50, 25, 12.5, 6.25 and 3.125 μg mL–1) on rat aortic rings to determine the dose response curve. Free radical scavenging activity of extract was determined by 1, 1-Diphenyl-2-picrylhydrazyl (DPPH) assay. Results: Cyperus rotundus ethanol extract showed significant dose-dependent blood vessels inhibition in comparison to the negative control (p<0.05). Ethanol extract showed significant free radical scavenging activity. Phytochemical investigation of alcoholic extract indicated the presence of various chemical constituents like alkaloids, glycosides, steroids, tannins, carbohydrates and flavonoids. Conclusion: The anti- angiogenic activity showed by the ethanol extract may be due to the presence of antioxidant compounds.
INTRODUCTION
Cyperus rotundus, the Arabic common name is Saed, Sajal, Seil and in English it is called nut grass, purple nutsedge, Nagarmotha and in China known as Xiang Fu. Cyperus rotundus is widely distributed in tropical, subtropical and temperate climates such as in Iraq, Egypt, Tunisia, China and India1. The plant fruits were used as carminative, diuretic, tonic, stomachic, anti-bilious and refrigerant2. While the tubular part utilized for the treatment of dysmenorrhoea and irregular menstrual cycle. Different pharmacological and biological activities including anti-Candida3, anti-inflammatory4, antidiabetic5, antidiarrhoeal, antioxidant6, cytoprotective7, antimutagenic8, antimicrobial, antibacterial9, cytotoxic and apoptotic, anti-pyretic and analgesic activities have been reported for Cyperus rotundus10. Angiogenesis is a process that involves the formation of new blood vessels from pre-existing ones, this process is typically initiated within hypoxic tissues where additional new blood vessels are required to maintain oxygenation and nutritional supply11. These processes are activated only under certain conditions when there is a physiological demand for increase in the blood supply as in wound healing or implantation of the fertilized egg in the endometrium12. Strict regulation of this system is very important for the human being, because both excessive and insufficient development of blood vessels lead to serious diseases13. Normal angiogenesis is limited to perhaps a few days or weeks. However, in a disease setting, angiogenesis can persist for months or years14. The aim of this study was assessing of anti-angiogenic effect, phytochemical investigation and the free radical scavenging activity of Cyperus rotundus.
MATERIALS AND METHODS
The study was carried out from March-June, 2017, in Al-Nahrain University, College of Pharmacy and in University of Baghdad.
Plant material: The C. rotundus collected from local market in Baghdad and authenticated by the National Herbarium at Abu-Graib, Baghdad.
Chemicals: Ethanol 98% (BDH), ascorbic acid (Sigma-Aldrich, USA), dimethyl sufoxide (DMSO) Romil, UK, aprotinin (Sigma-Aldrich, USA), fibrinogen (Sigma-Aldrich, USA), thrombin 100 IU vial (Sigma-Aldrich, USA), foetal bovine serum (Sigma-Aldrich, USA), L-glutamine (Sigma-Aldrich, USA), gentamicin 80 mg/2 mL vial (Al-Hikma, Jordan), aminocaproic acid (Sigma-Aldrich, USA), amphotericin B vial (Bristol Myers Squibb, England), Earls salt M199 solution (Sigma-Aldrich, USA).
Extraction of Cyperus rotundus: The dried and powdered whole plant parts (which includes roots, rhizomes, flowers and leaves) of Cyperus rotundus (200 g) extracted with 85% ethanol using Soxhlet apparatus until exhaustion for 16 h. Alcoholic extract was evaporated under reduced pressure at a temperature not exceeding 40°C using rotary evaporator to produce crude extract.
Preliminary qualitative phytochemical analysis: Chemical tests were carried out using ethanol extracts of Cyperus rotundus and standard procedures15 were carried out to identify the active constituents. Alkaloids identified by Mayer’s test, 10 mL of ethanol extract was mixed with 5 mL of 1% HCL on a steam bath then added the reagent, the result is white precipitate indicating +ve test for alkaloids. Flavonoids identified by lead acetate: 5 mL of ethanol extract mix with 1 mL of 10% lead acetate to yield yellowish- white precipitate indicating the presence of flavonoids. Steroids Liebermann-Burchard identified via taking 3 mL of extract stirred with chloroform, acetic anhydride and few drops of sulphuric acid to yield dark pink or red colour indicate the presence of steroids. Anthraquinones identified by Borntrager’s test through taking 3 mL of extract was shaken with 3 mL of benzene. Then, filtration was performed and 5 mL of 10% ammonia solution was added to the filtrate. The mixture was shaken well, the development of a pink, red or violet colour in the ammonia (lower) phase indicates the presence of anthraquinones. Tannins been discovered by FeCl3 solution (5% w/v) through taking 3 mL of distilled leaves extract (in water) mixed with 3 mL of FeCl3 solution (5% w/v) to produce dark green or blue black precipitate indicate the presence of tannins. Terpenoids identification done by taking ethanol extract (2 mL) and dissolved in chloroform (2 mL) and evaporated to dryness. Concentrated sulphuric acid (2 mL) was then added and heated for about 2 min. A greyish colour was considered an indication for the presence of terpenoids.
Rat aorta ring anti-angiogenic assay: The rat aortic ring assay experiment was conducted after the experimental procedures were revised and approved by Ethics Committee of Al-Nahrain University, College of Medicine. The assay was performed according to the standard protocol16. Albino male rats were humanely sacrificed via cervical dislocation under anaesthesia with diethyl ether. Thoracic aorta was excised, rinsed with serum free media, cleaned from the fibro-adipose tissue and was cross sectioned into thin rings of 1 mm thickness. A 300 μL of M199 growth medium (prepared by addition of fibrinogen 3 mg mL–1 and aprotinin 5 μg mL–1 to M199) was used as lower layer and loaded in 48-well plate and one aortic ring was seeded in each well. To each well, 10 μL of thrombin was added and then was incubated and allowed to solidify at 37°C in 5% CO2 for 30-60 min. The top layer medium was prepared by adding 20% of heat inactivated fetal bovine serum (HIFBS), 1% L-glutamine, 0.1% aminocaproic acid, 1% amphotericin B and 0.6% gentamicin to M199 medium. A stock solution of Cyperus rotundus extract was prepared by dissolving the sample in dimethyl sulfoxide (DMSO) and diluted in M199 growth medium to make the final DMSO concentration 1%. Serial dilutions of the active extract were prepared in the following concentrations: 100, 50, 25, 12.5, 6.25 and 3.125 μg mL–1. Wells without test samples were received medium with 1% DMSO used as the negative control. The tissue rings were incubated at 37°C, 5% CO2 in a humidified incubator. The results examined on day 5 under inverted microscope and the extent of blood vessel growth was quantified under 10X magnification with aid of camera and software package. The magnitude of blood vessel growth inhibition was determined according to the technique developed by Nicosia et al.17. The experiment was repeated 3 times using six replicate per sample and the percentage of blood vessels inhibition was determined according to the following formula:
| A0 | = | Distance of blood vessels growth for the test substance in mm |
| A | = | Distance of blood vessels growth in the control in mm. The concentration that inhibits 50% of the growing blood vessels "IC50" was calculated using the logarithmic equation for the extract18 |
Antioxidant activity (DPPH radical scavenging assay): Free radical scavenging activity of the active extract was measured by using the DPPH method. Two hundred microliters of 0.1 mM DPPH dissolved in methanol was added to 100 μL of the active extract in the following concentrations (200, 150, 100, 50 and 25 μg) and incubated for 30 min. This procedure was executed using 96 well plates and each concentration was tested in triplicate, then the absorbance was measured at 517 nm using an ELISA reader. Ascorbic acid (Vitamin C) was used as a positive control and methanol alone as blank. The negative control was made of 100 μL of methanol and 200 μL DPPH. The percentage of antioxidant activity (AA) was calculated according to the formula19:
| AS | = | Absorbance of sample |
| AB | = | Absorbance of blank |
| AC | = | Absorbance of control |
Statistical analysis: Statistical analysis was performed using Statistical Package for the Social Sciences SPSS 20, IBM, Armonk, NY, United States of America Software (version 20.0) data presented were Mean±SD from three different experiments. Statistical significance between different groups was determined using paired t-test. A value of p<0.05 was considered statistically significant.
RESULTS
Dose response curve of extract on rat aortic ring model: Six serial concentrations of extracts (100, 50, 25, 12.5, 6.25 and 3.125 μg mL–1) was prepared and added to the embedded rat aortic rings to determine the dose response curve. Extract showed significant dose dependent inhibition of blood vessels growth when compared to the negative control (DMSO 1%) (p<0.05) at 5 days of the experiment as found in Table 1. Also the dose response effect of each extract on blood vessels growth is shown in Fig. 1. The IC50 was determined from the logarithmic equation that is shown in Fig. 2 and it was found to be 15.39 μg mL–1.
| Table 1: | Qualitative analysis of Cyperus rotundus extract |
| Fig. 1(a-g): | Dose response effect of Cyperus rotundus ethanolic extract in rat aortic rings assay, represent the blood vessels growth inhibition which treated with (a) 1% DMSO, (b) 12.5, (c) 100, (d) 6.25, (e) 50, (f) 3.125 and (g) 25 μg mL–1, respectively |
Antioxidant activity: The free radical scavenging activity of extracts was measured using the DPPH assay. Five serial concentrations were used to determine the scavenging activity as shown in Table 2. Ascorbic acid had significantly higher scavenger activity compared to Cyperus extract at concentrations 25, 50, 100 and 150 μg mL–1. However, at concentration 200 μg mL–1 there was no significant difference as shown in Fig. 3. The IC50 equal to 129 mg mL–1 according to Fig. 4.
Phytochemical analysis: Phytochemical analysis indicates the presence of alkaloids, flavonoids, anthraquinones, tannins and steroids as shown in Table 3. The presence of these groups provides a scientific rational for the supposed etheno-medical uses of Cyperus rotundus in the treatment of indicated diseases. The presence of tannins, flavonoids, alkaloids and terpenoids seems to be significant.
| Table 2: | Serial concentrations and their respective percentage of blood vessels inhibition |
| Table 3: | Percentage of DPPH free radical scavenging activity for ethanol extract of Cyperus rotundus |
Scavenger activity presented as mean±SD, Sig.: Significant difference, NS: Not significant |
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| Fig. 2: | Dose response curve of ethanol extract of Cyperus rotundus in rat aortic rings model |
| Fig. 3: | Relationship between scavenger activity and concentration of both agents |
| Fig. 4: | Semilog of the relationship between scavenger activity of Cyperus extract and log concentration |
DISCUSSION
This study showed that Cyperus rotundus ethanol extract has robust anti-angiogenic activity, because angiogenesis process linked to many important and serious diseases and linked to important physiological process as well many scientists have focused on this process and targeting its steps as mean to stop or control it20. Further investigations were mandatory to understand the possible mechanism of action which may elucidate the pharmacologic action and the possible interaction may have as well. Free radical scavenging activity was tested for the extract in comparison to vitamin C which approved as antioxidant drug, the data showed that this extract has significantly scavenge the free radicals and this finding agreed with previous study conducted by Soumaya et al.21, moreover it was important to identify the possible bioactive constituents and the results showed that there are many agents with potent antioxidant activity. Several studies demonstrated that antioxidants were able to inhibit angiogenesis process both, in vitro and in vivo22. These compounds include natural substances (catechins, resveratrol, polyphenols, flavonoids, isoflavones, lycopene, pigment epithelium-derived factor, glutathione), nutritional components (vitamins C, D, E, β-carotene and selenium) and semi-synthetic and synthetic compounds (Nacetylcysteine, L-NAME, L-NIO, sodium pyruvate, pyrrolidine dithiocarbamate and organoselenium compounds)23. In this concern, agent with antioxidant activity strongly inhibit vascular endothelial growth factor VEGF expression and H2O2-induced release of VEGF in vascular smooth muscle cells in concentrations that are likely to be achieved in blood after moderate consumption of antioxidant activity24. Moreover, antioxidant agents showed ability to scavenge ROS such as hydroxyl radical and superoxide anion to inhibit the expression of xanthine oxidase to diminish adhesion and invasion of tumoural cells induced by ROS from hypoxanthine/xanthine oxidase system and to increase activity of catalase and glutathione peroxidase24. This study considers the first step in identifying the pharmacological activity, further study required to identify the responsible agents by using high performance liquid chromatography. Moreover, in vivo study very important to start the formulation prior conducting clinical trial.
CONCLUSION
Phytochemical investigation of Iraqi Cyperus rotundus was done and the results revealed the presence of alkaloids, flavonoids, steroids, tannins and terpenoids in the whole plant extract and the absence of anthraquinones in this plant parts, also this study demonstrates the positive anti-angiogenic effect of Cyperus rotundus, ethanol extract showed significant reduction of free radicals by the DPPH assay and in a concentration-dependent manner. Free radical scavenging activity was very important in order to better understand the possible mechanism of action behind their ability to suppress blood vessels growth.
SIGNIFICANCE STATEMENT
This study discovers the significance anti-angiogenic activity of Cyperus rotundus ethanol extract-that can be beneficial for targeting many pathological process such as rheumatoid arthritis, tumor and psoriasis. This study may help researchers in future to formulate it and test it clinically to be prescribe later on. This study will help the researchers to uncover the critical areas of fighting tumor and other angiogenesis related disease and possibly other combinations may be arrived at. Regarding to the novelty of this study no one has conducted such study on this extract.
ACKNOWLEDGMENT
The authors are thankful to University of Baghdad and Al- Nahrain University for providing necessary facilities to carry out the study.