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In vitro antioxidant potentials of Cyperus rotundus L. rhizome extracts and their phytochemical analysis
Arunagiri Kamala1, Sushil Kumar Middha2, Chitra Gopinath3, HS Sindhura3, Chandrakant S Karigar4
1 Department of Studies and Research in Biochemistry, Tumkur University, Tumkur, Karnataka, India
2 Department of Biotechnology, Maharani Lakshmi Ammanni College for Women, Bengaluru, Karnataka, India
3 Department of Biochemistry, Maharani Lakshmi Ammanni College for Women, Bengaluru, Karnataka, India
4 Department of Studies and Research in Biochemistry, Tumkur University, Tumkur; Department of Biochemistry, Bangalore University, Bengaluru, Karnataka, India
| Date of Submission | 14-Jun-2017 |
| Date of Acceptance | 28-Aug-2017 |
| Date of Web Publication | 10-Apr-2018 |
Correspondence Address:
Chandrakant S Karigar
Department of Biochemistry, Bangalore University, Bengaluru - 560 056, Karnataka
India
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/pm.pm_228_17
 Abstract | | |
Background: Cyperus rotundus L. (family Cyperaceae), native to India, is a multivalent medicinal plant widely used in conventional medicine. The research reports on bioactive components from C. rotundus L. are scanty. Objective: The objective of the study was to optimize the best solvent system and bioprospect the possible phytochemicals in C. rotundus L. rhizome (CRR). Materials and Methods: The phytochemicals were extracted from the rhizomes of C. rotundus L. by successive Soxhlet technique with solvents of increasing polarity. The resultant extracts were analyzed for their total flavonoid content (TFC), total phenolic content (TPC), total proanthocyanidin content (TPAC), in vitro antioxidant potential, and inhibition of lipid peroxidation. The 70% acetone extract of CRR was analyzed using gas chromatography–mass spectrometry (GC-MS) for probable phytochemicals. Results and Discussion: The TPC, TFC, and TPAC estimates ranged from 0.036 ± 0.002 to 118.924 ± 5.946 μg/mg extract, 7.196 ± 0.359 to 200.654 ± 10.032 μg/mg extract, and 13.115 ± 0.656 to 45.901 ± 2.295 μg/mg extract, respectively. The quantities of TPC, TFC, and TPAC were found to be the highest in 70% acetone extract. The 70% acetone and 70% methanol extracts revealed best radical scavenging effect. GC-MS analysis of CRR extract revealed the presence of a novel compound 1 (2)-acetyl-3 (5)-styryl-5 (3)-methylthiopyrazole. Conclusion: The study indicated that 70% acetone and 70% methanol extracts of CRRs can be a potential source of antioxidants.
Abbreviations used: ACRE: Acetone C. rotundus L. rhizome extract; AlCl3: Aluminum chloride; AQRE: Aqueous C. rotundus L. rhizome extract; CE: Catechin Equivalent; CHRE: Chloroform C. rotundus L. rhizome extract; CRR: C. rotundus L. rhizome; DPPH: 2,2 diphenyl-1-picrylhydrazyl; ETRE: Ethanolic C. rotundus L.rhizome extract; EARE: Ethyl acetate C. rotundus L.rhizome extract; FRP: Ferric reducing power; GAE: Gallic acid equivalent; GC-MS: Gas chromatography-mass spectrometry; HERE: Hexane C. rotundus L.rhizome extract; MERE: Methanolic C. rotundus L.rhizome extract; PERE: Petroleum ether C. rotundus L.rhizome extract; QE: Quercetin equivalent; RNS: Reactive nitrogen species; ROS: Reactive oxygen species; TFC: Total flavonoid content; TPC: Total phenolic content; TPAC: Total proanthocyanidin content.
Keywords: Alkaloids, antioxidants, Cyperus rotundus L. Rhizome, gas chromatography-mass spectrometry analysis, successive solvent extraction
How to cite this article:
Kamala A, Middha SK, Gopinath C, Sindhura H S, Karigar CS. In vitro antioxidant potentials of Cyperus rotundus L. rhizome extracts and their phytochemical analysis. Phcog Mag 2018;14:261-7 |
How to cite this URL:
Kamala A, Middha SK, Gopinath C, Sindhura H S, Karigar CS. In vitro antioxidant potentials of Cyperus rotundus L. rhizome extracts and their phytochemical analysis. Phcog Mag [serial online] 2018 [cited 2021 Oct 22];14:261-7. Available from: http://www.phcog.com/text.asp?2018/14/54/261/229686 |
SUMMARY
- The studies suggest 70% methanol and acetone as the suitable solvents for the extraction of phytochemicals
- Novel compound 1(2).Acetyl.3(5).styryl.5(3).methylthiopyrazole was detected in 70% acetone extract.
| Introduction | |  |
Natural products engross enormous chemical diversity. These phytochemicals find tremendous applications in agriculture, cosmetics, food, and medicine.[1],[2],[3] The minimal side effects related to their use make them more popular than synthetic drugs.[4] The Indian subcontinent, “The land of Ayurveda,” is known for its biological abundance of medicinal plants.[5],[6]
Cyperus rotundus L. (family: Cyperaceae) is popularly known as “Nut grass” in English, “musta moola churna” in ancient Ayurveda Charaka Samhita,[7] and “Xiangfu or Xiangfuzi” in Chinese Traditional Medicine.[8] It is a multivalent herb reported for its pharmacological properties such as an analgesic, antibacterial, antidiabetic, antidiarrheal, anti-inflammatory, antioxidant, antipruritic, antisaturative, appetizer, diaphoretic, digestant, lactodepurant, thirst relieving, and tranquilizing effect.[9],[10],[11],[12],[13] The phytochemical studies of C. rotundus L. led to the isolation of sesquiterpenes,[11],[14] flavonoids,[15],[16],[17] phenylpropanoids,[18],[19] phenolic acids,[18],[20] alkaloids,[21] and saponins.[22] Investigations on C. rotundus L. rhizome (CRR) extract reported the presence of methyl 3,4-dihydroxybenzoate, ipolamiide, 6-β-hydroxyipolamiide, and rutin and also stated the uses of ethyl acetate fraction coupled with methanolic fraction in treatment of CCl4-induced hepatic injury in rats.[13] Lydia J and Sudarsanam (2014) identified 15-Hydroxy-4-oxo-10-pentadecynoic acid lactone from C. rotundus L.[12] Recently, rotunduside, a phenolic compound from methanol extract of CRR, was isolated and its potential antidepressant activity with murine models was demonstrated.[23]
However, there is limited information in the selection of the best solvent system to extract active molecules from CRR. Therefore, the current study aims to optimize the best solvent system and bioprospect the possible phytochemicals in CRR.
| Materials and Methods | | |
Chemicals and reagents
Aluminum chloride, ascorbic acid, chloroform, ethanol, ethyl acetate, ferric chloride, ferrous sulfate, gallic acid, glacial acetic acid, hexane, hydrogen peroxide, α-naphthol, petroleum ether, potassium dihydrogen phosphate, potassium hydrogen phosphate, potassium chloride, potassium ferricyanide, sodium bicarbonate, trichloroacetic acid (TCA), and vanillin were procured from SD Fine Chemicals Limited, Mumbai, India. Acetone, hydrochloric acid, methanol, sodium hydroxide, and sulfuric acid were acquired from Spectrum Chemical Private Limited, Cochin. Acetic anhydride, Dragendorff reagent, Folin–Ciocalteu reagent, lead acetate, Mayer's reagent Sodium nitrate, and Wagner's reagent were purchased from MERCK Specialties Private Limited, Mumbai. 2,2-diphenyl-1-picrylhydrazyl (DPPH) was procured from SISCO Research Laboratories, Maharashtra, India. Tris base was from NR Chemicals Private Limited, Mumbai, India. Thiobarbituric acid (TBA) was bought from Loba Chemie Private Limited, Mumbai, India. All chemicals and reagents were of analytical grade.
Collection and identification of plant material
The CRRs were obtained from the regional market, Bengaluru and authenticated by National Ayurveda Dietetics Research Institute, Bengaluru. A specimen copy was deposited in the herbarium of the Regional Research Centre (RRCBI-AP. 77). The cleaned and shade dried rhizomes were finely powdered using a mechanical blender (Kenstar, India) and were subjected to successive soxhlation using various solvents.
Preparation of C. rotundus L. rhizome extract
The powdered CRR was directed to successive Soxhlet extraction using eight different solvents, i.e., hexane, petroleum ether, ethyl acetate, chloroform, 70% acetone, 70% ethanol, 70% methanol, and water in their increasing polarity (1:12 w/v ratio) for 24 h at their respective boiling temperatures [Schema 1]. The lyophilized (LyoQuest, Telstar, Spain) samples were stored at 4°C for further use.
Quantification of C. rotundus L. rhizome extract yield
The obtained percentage yield of CRR extracts using various solvents was calculated using the following formula: % Yield = (W1/W2) × 100; where W1 = the weight of the extract after solvent evaporation and W2 = the weight of the dry plant material.
Preliminary screening of phytochemicals in C. rotundus L. rhizome
Qualitative phytochemical analysis for the detection of alkaloids, carbohydrates, glycosides, flavonoids, resins, saponins, steroids, tannins, and phenols was carried for all the solvent extracts using standard methods.[24] Each solvent extract was redissolved using Millipore water to obtain various concentrations and filtered. The filtrates were employed for further uses. All the tests were performed thrice.
Quantitative phytochemical assay
Determination of total phenolic content
The total phenolic content (TPC) was quantified by Folin–Ciocalteau method [25] using gallic acid as standard for hexane C. rotundus L. rhizome extract (HERE), petroleum ether C. rotundus L.rhizome extract (PERE), ethyl acetate C. rotundus L. rhizome extract (EARE), chloroform C. rotundus L. rhizome extract (CHRE), 70% acetone C. rotundus L.rhizome extract (ACRE), 70% ethanolic C. rotundus L. rhizome extract (ETRE), 70% methanolic C. rotundus L.rhizome extract (MERE), and aqueous C. rotundus L.rhizome extract (AQRE). The absorbance of standard and test samples was read at 765 nm. TPC of the resulting successive extract was determined using standard curve and expressed as μg gallic acid equivalent (GAE)/mg extract using T = [C × V]/M formula. Where T is the TPCs in μg/mg of the extracts as GAE, C is the concentration of gallic acid in μg/mL, V is the volume of the extracts in mL, and M is the weight in mg of the extract.
Determination of total flavonoid content
The estimation of total flavonoid content (TFC) was carried out for HERE, PERE, EARE, CHRE, ACRE, ETRE, MERE, and AQRE using AlCl3 method [26] with standard quercetin. The absorbance was read at 510 nm. The total amount of flavonoid was expressed as μg of quercetin equivalents (QEs)/mg of extract.
Determination of total proanthocyanidin content
The total proanthocyanidin content (TPAC) of HERE, PERE, EARE, CHRE, ACRE, ETRE, MERE, and AQRE was quantified using vanillin–hydrochloride method as detailed by Usha et al.[27] The absorbance was measured at 500 nm using vanillin–hydrochloride as blank. The standard curve was constructed by preparing catechin solutions at concentrations of 5–25 μg/mL in methanol. TPAC contents were expressed as μg catechin equivalents/mg of extract.
EARE, ACRE, ETRE, and MERE extracts were continued for further studies since they showed the better results.
In vitro assays
2,2-diphenyl-1-picrylhydrazyl radical scavenging assay
The free radical scavenging activity of EARE, ACRE, ETRE, and MERE was evaluated according to the modified method described by Goyal et al.[24] using DPPH. The free radical scavenging potentials are indicated by the degree of discoloration of DPPH caused due to the hydrogen-donating ability of the extract which was measured at 517 nm using methanol as blank. Quercetin was used as a reference standard. For control, only DPPH prepared in 95% methanol was taken without any extract.
The percentage of free radical scavenging capacity of the extracts was calculated using the following equation:
DPPH radical scavenging effect (%) = (A 0 − A 1)/A 0 × 100
where, A 0: Absorbance of the control,
A 1: Absorbance of the sample
The effectual inhibitory concentration of the sample required to scavenge DPPH radical by 50% (IC50 value) was obtained by linear regression analysis of dose–response curve plotted between %inhibition (y-axis) and concentrations (x-axis).[28]
Determination of ferric reducing power
The reducing power of CRR extracts (EARE, ACRE, ETRE, and MERE) was estimated by a reported method of Goyal et al.[24] Absorbance was read at 700 nm using 0.2 M phosphate buffer (pH 6.6) as blank. Ascorbic acid was used as reference standard. The increase in absorbance of the reaction mixture indicated the increased reducing power of the samples.
Lipid peroxidation inhibition assay
The lipid peroxidation potential of extract was determined by TCA and TBA following the method of Okhawa et al.[29] 200 mg of goat liver was homogenized with 10 mL of Tris-HCl buffer (40 mM, pH-7) and the homogenate was centrifuged for 10 min at 3000 rpm. Supernatant was collected. A volume of 0.5 and 1.0 mL aliquots of extract (1 mg/mL) was pipetted out into various tubes, and the volume was made up to 1 mL with Millipore water. 0.5 mL of supernatant was added to each tube, followed by addition of 200 μL KCl (0.15 M), 1 mL FeSO4(15 mM), and 1 mL ascorbic acid (6 mM). The control was prepared without using the supernatant. The test tubes were incubated at 37°C for 1 h. 1 mL of TCA (10%) was added and the tubes were centrifuged at 3000 rpm for 20 min at 4°C. The supernatant in each tube was collected and treated with 1 mL TBA (0.8%). The test tubes were then heated at 90°C in water bath for 20 min and cooled. The absorbance of the TBA-malondialdehyde (MDA) complex was read at 532 nm after adding 2 mL of butanol. The percentage of lipid peroxidation potential by EARE, ACRE, ETRE, and MERE extracts was calculated as follows:
% lipid peroxidation potential (MDA) = [A 0–A 1/A 0] ×100
where, A 0: Absorbance of the control
A 1: Absorbance of the extract
Gas chromatography–mass spectrometry analysis of C. rotundus L. rhizome extracts
Gas chromatography–mass spectrometry (GC-MS) for ACRE extract was recorded with Thermo GC-Trace Ultra 5.0, Thermo MS DSQ II (Thermo Fisher Scientific, USA). TR 5-MS capillary standard nonpolar column with 30 m dimension, Id: 0.25 mm, 0.25 mm film was used. Helium gas was used as a carrier gas with flow rate of 1 mL/min.
Statistical analysis
All the experiments were carried out in triplicates and the results were expressed as mean ± standard error of the mean. The data were statistically analyzed using Microsoft Office Excel 2007.
| Results | | |
Yield of C. rotundus L. rhizome extracts
The yield of CRR extracts obtained after extraction with each solvent are given in [Table 1]. The highest yield of the extract was found to be in ETRE (22.72%) followed by MERE (3.22%). The lowest yield was obtained in PERE (0.133%). | Table 1: Yield of Cyperus rotundus L. rhizome extracts after successive soxhlation
Click here to view |
Preliminary phytochemical screening of CRR extracts
HERE, PERE, and EARE extracts revealed the presence of alkaloids, carbohydrates, glycosides, and steroids. Flavonoids were not traced in CHRE extract. Saponins were present in ETRE, MERE, and AQRE whereas resins were present only in PERE and EARE extracts. CHRE showed the presence of tannins only. ACRE, ETRE, and MERE showed the presence of carbohydrates, glycosides, flavonoids, saponins, steroids, tannins, and phenols. Except HERE, PERE, and CHRE phenols were found in all the extracts [Table 2]. | Table 2: Preliminary phytochemical screening of Cyperus rotundus. L rhizome
Click here to view |
Quantitative phytochemical assay of C. rotundus L. rhizome extracts
Different extracts of CRR were quantitatively analyzed for TPC, TFC, and TPAC [Table 3]. According to the results, TPC of the CRR extract ranged from 0.0358 ± 0.002 to 118.924 ± 5.946 μg GAE/mg, TFC ranged from 7.196 ± 0.359 to 200.654 ± 10.032 μg QE/mg, and TPAC ranged from 13.115 ± 0.656 to 45.901 ± 2.295 μg CE/mg. ACRE showed the highest value of TPC, TFC, and TPAC. The lowest TFC and TPAC were observed in hexane extract whereas the lowest TPC was detected in PERE extract. | Table 3: Quantitative phytochemical analysis of rhizome of Cyperus rotundas L. extracts
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In vitro assays
2,2 diphenyl-1-picrylhydrazyl radical scavenging assay
[Figure 1]a indicated an increasing trend in DPPH radical scavenging activity with an increase in concentration of extract. It was found that ACRE showed highest scavenging activity compared to other extracts. Lower the IC50 value better is the extract. IC50 of the ACRE was found to be the lowest (0.901) and more effective than other extracts [Figure 1]b. The descending order of IC50 values was found to be EARE > MERE > ETRE > ACRE. | Figure 1: (a) Free radical scavenging activity of Cyperus rotundus L. rhizome extracts and (b) IC50 for 2,2 diphenyl-1-picrylhydrazyl radical scavenging assay
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Reducing power of C. rotundus L. rhizome extracts
Ferric reducing power (FRP) assay determines the reducing power of the extracts. It was found that ACRE extract showed a greater reducing property compared to other solvent extracts [Figure 2].
Lipid peroxidation inhibition assay
Ascending order of lipid peroxidation inhibition was found as followed: ACRE > EARE > ETRE > MERE [Figure 3]. ACRE (23.65%) followed by EARE (11.18%) extracts showed the highest inhibition as compared to other extracts. | Figure 3: Inhibition of lipid peroxidation by Cyperus rotundus L rhizome extracts
Click here to view |
Gas chromatography–mass spectrometry analysis of C. rotundus L. rhizome extracts
The detected compounds were 3, 3, 5, 5,3',3',5',5'-Octamethyl-di-(delta- pyrazolinylidene), dimethyl-2- [o-(Ethoxycarbonyl) benzoyl]-1, 2-di hydro-1-iso quinolylphosphonate, 2-amino-cyclopentanemethanamine, cyclopentane, 1-nitro-2- (2-propenyl)-cis, i-Propyl-3- (phenylamino)-2- (phenylseleno)-3-(phenyl) propanoate, 1-cyclopropyl-2, 3-diazabicyclo [2.2.1] hept-2-ene. The 1 (2)-Acetyl-3 (5)-styryl-5 (3)- methylthiopyrazole was detected for the first time from the ACRE [Figure 4]. | Figure 4: Gas chromatography–mass spectrometry analysis of phytochemicals from acetone rhizome extract of Cyperus rotundus L
Click here to view |
| Discussion | | |
Different solvents are used to extract the secondary metabolites from plant materials. The most widely used solvents for extraction are water, ethanol, methanol, acetone, and solvent mixtures of different ratios with water, with or without acid.[24],[30],[31],[32] The extraction of the secondary metabolites or phenolic constituents is based on the solvent used and its polarity. In this study, CRR extract was prepared using eight solvents based on their increasing order of polarity. Since EARE, ACRE, ETRE, and MERE extracts showed the best quantitative results, they were used for in vitro antioxidant and free radical scavenging activity studies.
The phenolics being the major group of secondary metabolites, also act as primary antioxidants and free radical inhibitors. Flavonoid, a polyphenolic compound, is one of the most numerous phenolics and widely spread in the plant kingdom.[33] Various reports have shown the diverse functions of flavonoid such as UV protection, disease resistance, pigmentation, and nitrogen fixation stimulation in the nodules.[4],[34],[35] Proanthocyanidins, a subclass of the most complex flavonoids, are the nonpolar, condensed tannins, and polymer of flavan-3-ols. ACRE extract contained the highest TPC, TFC, and TPAC, whereas PERE contained the least TPC and HERE contained the lowest TFC and TPAC. The principle antioxidants such as 3-hydroxy-4-methoxy-benzoic acid, galloylquinic acid, ferulic acid, quercetin, luteolin, afzelechin, and catechin are the phenolic compounds present in C. rotundus L. as reported by Kilani-Jaziri et al.[36] Boeing et al.[37] have reported 70% acetone as an efficient solvent for extracting phenolic compounds which is similar to the results obtained in the present study. Singh et al.[10] had reported that total oligomeric flavonoid extract of C. rotundus L. possessed a broad spectrum of pharmacological properties such as antioxidant, antimutagenic, antigenotoxic, antimicrobial, anticancer, and neuroprotective properties.
Further, the effect of CRR on free radical scavenging was investigated using DPPH assay. Among different solvent extracts, ACRE showed better and higher radical scavenging activity than other extracts. The increased scavenging activity of the ACRE extract may be accredited to its potent hydrogen donating ability.[28] The IC50 values of the EARE, ACRE, ETRE, and MERE were 2.7, 0.8, 1.9, and 2, respectively. The scavenging activity is inversely proportionate to the IC50 value.[6] The lowest IC50 value of ACRE indicated its high free radical scavenging activity, indeed an indication of high antioxidant activity. In situ, Viuda-Martos et al.[38] have shown a linkage between polyaromatic hydrocarbon cations and carcinogenesis. Thus, ACRE can also exhibit in vivo free radical scavenging activity similar to in vitro DPPH free radical scavenging activity.
FRP assay is a reliable and simple method for determining the reducing power of antioxidants. In the presence of an antioxidant, potassium ferricyanide and ferric chloride are converted into potassium ferrocyanide and ferrous chloride, respectively. ACRE showed the highest reducing power than other solvent extracts. The reducing power is dependent on the concentration of the extracts. Phenolic content (118.9 μg GAE/mg extract) may be responsible for the ferric reducing ability of ACRE extract in accordance with Siddhuraju et al.[39] Goyal et al.[31] reported that the FRP could be mainly due to the antioxidative compounds.
Total flavonoids contents (200.6 μg QE/mg extract) were found to be more than phenols in this study. Since flavonoids are polyphenols, it is usually observed that the phenolic content is greater than the flavonoid content in the given plant extract. However, sometimes, flavonoid content may be greater than that of the phenols. This may be due to the formation of complex ring structures between various phenolic compounds which may not be measured in the assay for phenols and thus goes unaccounted. These results are seen to be in compliance with the results as reported by Murugan et al.[40]
Lipid peroxides formed as a product of oxidative stress in the body are unstable reactive products. MDA is frequently used as an indicator of lipid peroxidation. Under conditions of oxidative stress, the reactive oxygen species produced, cause lipid peroxidation in cell membranes, particularly through oxidation of polyunsaturated fatty acids.[4] ACRE exhibited a stronger inhibitory effect on lipid peroxidation, while MERE exhibited a lower inhibitory effect.
GC-MS analysis of ACRE has revealed the presence of alkaloids such as 3, 3, 5, 5,3',3',5',5'-Octamethyl-di-(delta-pyrazolinylidene), dimethyl-2-[o-(Ethoxycarbonyl) benzoyl]-1,2-di hydro-1-iso quinolylphosphonate,2-amino-cyclopentanemethanamine, cyclopentane,1-nitro-2-(2-propenyl)-cis, i-Propyl-3-(phenylamino) -2-(phenylseleno)-3-(phenyl)propanoate,1-cyclopropyl-2, 3-diazabicyclo [2.2.1]hept-2-ene. The 1(2)-Acetyl-3(5)-styryl- 5(3)-methylthiopyrazole was detected for the first time from the CRR. There are no reports found associated with all these compounds previously. Ambiguity found in earlier reports prompted this study on the antioxidant activity of CRR. Hence, it is very tricky to compare our findings with that of prior studies. The consumption of CRR may prevent human aliments associated with free radicals.
| Conclusion | | |
This work is an effort to understand the medicinal potential of C. rotundus L.cultivated in Karnataka region, India, for the first time. Successive soxhlation of C. rotundus L was carried out to identify the suitable solvent system for the extraction of numerous secondary metabolites and acetone was found to be a better solvent. The crude extract of the rhizome of CRR was observed to harbor substantial antioxidant activity, and the ACRE extract showed remarkable activity. The novel compound 1 (2)-Acetyl-3 (5)-styryl-5 (3)-methylthiopyrazole, an alkaloid is reported for the first time. This work could provide a better understanding of phytochemicals present in C. rotundus and its antioxidative properties.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3]
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