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Prev Nutr Food Sci 2022; 27(2): 198-211

Published online June 30, 2022 https://doi.org/10.3746/pnf.2022.27.2.198

Copyright © The Korean Society of Food Science and Nutrition.

Chemical Composition, In Silico and In Vitro Antimutagenic Activities of Ethanolic and Aqueous Extracts of Tigernut (Cyperus esculentus)

Olumide David Olukanni1 , Temitope Abiola1 , Adedayo Titilayo Olukanni1 , and Abosede Victoria Ojo2

1Department of Biochemistry, Faculty of Basic Medical Sciences, Redeemer’s University, Ede, Osun State 232101, Nigeria
2Department of Chemical Sciences, Biochemistry Unit, College of Natural and Applied Sciences, Oduduwa University, Ile-Ife, Osun State 220101, Nigeria

Correspondence to:Temitope Abiola, Email: abiolat@run.edu.ng

Received: January 12, 2022; Revised: May 3, 2022; Accepted: May 20, 2022

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Tigernut, also known as Cyperus esculentus, is said to be high in nutritional and medicinal value. The purpose of this study was to determine the C. esculentus’s antimutagenic activity. The ethanolic and aqueous extracts of the nut were analyzed for chemical constituents, antioxidants, ultraviolet-visible, and gas chromatography-mass spectrometry using standard procedures. The extracts contained a total of 17 major compounds that were docked against human RecQ-like protein 5 (RECQL5) helicase protein. The antimutagenic property of the ethanolic extract in vitro was assessed using the Allium cepa chromosome assay. Onion bulbs were pre-treated with 200 mg/kg of ethanolic extract of C. esculentus for 24 h and then, grown in NaN3 (250 μg/L) for 24 h; onion bulbs were also first exposed to NaN3 (250 μg/L) for 24 h before treatment with 100 mg/kg and 200 mg/kg of the ethanolic extract respectively. Standard methods were used to determine the mitotic index and chromosomal aberrations. Results revealed that C. esculentus ethanolic extract contained flavonoids (22.47 mg/g), tannins (0.08 mg/g), alkaloids (19.71 mg/g), glycosides, phenol, and tannin and showed high scavenging activity against 2,2-diphenyl-1-picrylhydrazyland H2O2. Docking with RECQL5 showed good binding energies (ΔG>−7) of five compounds in C. esculentus ethanolic extract. The A. cepa assay results revealed a significant (P<0.05) reduction in chromosomal aberrations and a higher mitotic index in groups treated with the C. esculentus ethanolic extract. The antimutagenic activity of C. esculentus ethanolic extract was attributed to its high levels of phytosterols and phenolic compounds.

Keywords: Allium cepa chromosome assay, antimutagenic, Cyperus esculentus, sodium azide

INTRODUCTION

Cancer is a group of disorders caused by mutations in the genes that control cell growth and differentiation, resulting in tissue growth dysregulation (Croce, 2008). Environmental factors that cause mutations, such as carcinogens and mutagens have been linked to the etiology of cancer because they play a role in the induction and progression of numerous diseases that affect humans including cancer (Tomasetti et al., 2017). Mutagens can be of physical origins such as ultraviolet rays, chemical origins such as reactive oxygen species, or biological origins that can induce mutations, thereby producing different breaks in DNA and the formation of base dimers (Hockberger, 2002). Sodium azide (NaN3) is a chemical mutagen because it is an azide salt that causes plant mutations (Owais and Kleinhofs, 1988). Numerous biological assays can be used to assess the mutagenicity of various chemical compounds. Plants are commonly used in some of these assays because of their low cost, accessibility, and compatibility with other animal-based assays (Fiskesjö, 1985). Levan (1938) first proposed the use of onions (Allium cepa) as an effective test system for toxicity assessment. Plants are commonly used to detect the causative agents of DNA damage or agents that can protect against such effects because they are less expensive and easier to use. As a result, some species, such as onion bulbs (A. cepa), are used in mutagenesis studies to predict risks in higher eukaryotes such as mammals. This is because of benefits such as sensitivity to complex mixtures, distinct chromosomes (2n=16), affordability, and availability (Levan, 1938). After being exposed to a known mutagen and antimutagen, onion bulbs are examined microscopically to determine the mitotic index (MI) and the number of chromosomal aberrations (CA) caused by the mutagenic chemical. The MI is used to assess the cytotoxicity of a mutagen (Akinboro et al., 2011). The antimutagenicity of a compound is confirmed by a significant increase and decrease in the MI and CA after exposure to a mutagen, respectively.

Antimutagens are substances that can prevent the conversion of mutation-inducing substances into mutagenic compounds, render mutagenic compounds inactive, or inhibit the association of DNA with mutagen (Bhattacharya, 2011). Plants with antimutagenic properties could be very useful for preventing human cancer without harming normal cells (De Flora, 1998). Phytochemicals with antioxidant properties are abundant in fruits, vegetables, and plants (Luximon-Ramma et al., 2003). These plant compounds are effective in neutralizing the mutagenicity of various mutagens by removing reactive oxygen and nitrogen species, thereby ameliorating some of the resulting consequences to genetic material (Mazzio and Soliman, 2009; Akinboro et al., 2011). The antimutagenic effect of phytochemicals may be greater than flavonoids’ quenching effects. Their ability to bind and activate a family of proteins known as genome guardians may be an unexplored mechanism. The RecQ-like protein 5 (RECQL5), which is found in both the cytoplasm and nucleus, is prominent among these proteins (Luong and Bernstein, 2021).

RECQL5 is a helicase that aids in the preservation of gene integrity in organisms through the processes of DNA metabolism and repair (Bachrati and Hickson, 2003). RECQL5 Knockdown has been linked to increased apoptosis in DPP-resistant A549 cells (Xia et al., 2021). Certain phytochemicals in tigernut were considered to be able to bind to and activate RECQL5.

Tigernut (Cyperus esculentus), as depicted in Fig. 1, is a plant that resembles a perennial grass in the Cyperaceae family and is surrounded by a fiber-like covering (Takhatajah, 1992). Tigernuts come in a variety of colors, including black, yellow, and brown (Belewu and Abodunrin, 2006). The yellow and brown varieties are widely available in Nigeria, with the yellow variety preferred over others because of its larger size, higher protein content, and lower fat and anti-nutrients content (Okafor et al., 2003). The nuts contain significant amounts of starch, dietary fiber, digestible carbohydrate (mono-, di-, and polysaccharide), and a moderate amount of minerals (Temple et al., 1990; Salem et al., 2005; Sanful, 2009). Tigernut is known to have antioxidant and antidiabetic properties (Owusu and Owusu, 2016). The oil derived from the nut has been shown to increase high-density lipoprotein cholesterol levels while decreasing low-density lipoprotein cholesterol (Belewu and Aboudurin, 2006), lowering the risk of arteriosclerosis. There is still little information on tigernut’s antimutagenic potential, so this study was conducted to assess tigernut’s antimutagenic potential in silico and in vitro.

Figure 1. Image of a tigernut (Cyperus esculentus).

MATERIALS AND METHODS

Collection and identification of the plant

The yellow variety of C. esculentus dried tubers (tigernuts) was purchased in the Oja Oba area of Kwara State, Nigeria. The nuts were authenticated at the Botany Department of Obafemi Awolowo University in Ile-Ife, Nigeria, and were given the identification number ILE-IFE-17678.

Plant extracts preparation

The crude extraction protocol was followed in accordance with established procedures (Feyisayo and Oluokun, 2013). A manual engine blender was used to pulverize dried tubers into powdery form. The fine powder of CE was dissolved in 1,000 mL each of 80% ethanol and water. The samples were macerated for 72 h and shaken every 6 h. The residues in the sample were removed after 72 h by filtering through filter paper into conical flasks. The filtrates were dried into a colloid form using a rotary evaporator at 45°C under low pressure inside a vacuum and stored at 4°C.

Phytochemical analysis of the ethanolic extract

Determination of tannin content: In a plastic container, 500 mg of the sample was mixed with 50 mL of distilled water for 1 h using a shaker. The sample was then filtered into a 50 mL volumetric flask and adjusted to the mark. In a test tube, 5 mL of the filtrate was mixed with 2 mL of 0.1 M iron(III) chloride in 0.1 N hydrochloric acid and 0.008 M potassium ferrocyanide. Within 10 min, the absorbance was measured at 120 nm (Van Buren and Robinson, 1969).

Determination of alkaloid: In a separating funnel, 1 mL of the extract (1 mg/mL) was mixed with 1 mL of 2 N HCl and filtered before adding 5 mL of bromocresol green solution and phosphate buffer. Drop by drop, chloroform was added to the mixture, which was then shaken before being collected in a 10 mL volumetric flask and adjusted with chloroform to the mark. Atropine solutions were prepared in known concentrations (20, 40, 60, 80, and 100 μg/mL). At 470 nm, the absorbance of the test and standard solutions were measured using a ultraviolet-visible (UV-VIS) spectrophotometer against the reagent blank. The total alkaloid content was calculated in mg of atropine equivalents/g of extract (Shamsa et al., 2008).

UV-VIS spectrum analysis

The aqueous and ethanolic extracts of tigernut were analyzed using a UV-VIS spectrophotometer (Spectroquant Pharo 300, Merck KGaA, Darmstadt, Germany) with a wavelength range of 190 to 900 nm. This was done to detect the common peaks in the extracts.

Antioxidant assay

2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity: This was done exactly as described by Alhakmani et al. (2013). One mL of tigernut extract was added to a test tube containing 2 mL of 1 mM methanolic DPPH. The resulting mixture was thoroughly shaken and allowed to stand for 30 min at 37°C in an incubator. There was also a blank that did not contain either the standard or the extract. The difference in absorbance was then measured at 515 nm against gallic acid as a standard and expressed as a percentage inhibition using the formula below:

Inhibition (%)=[(A515 Control-A515 Sample)/A515 Control]×100

Hydrogen peroxide (H2O2) scavenging activity: At 230 nm, the absorbance of a 40 mM H2O2 solution prepared in 50 mM phosphate buffer (pH 7.4) was measured. Following that, 1 mL of sample extract or standard was added to 2 mL of hydrogen peroxide solution. After allowing it to stand for 10 min, the absorbance was compared to that of a blank solution containing only phosphate buffer. H2O2 scavenging activity was then calculated as a percentage (Nabavi et al., 2008).

Total antioxidant capacity: This was determined as described by Prieto et al. (1999). The total antioxidant capacity was then calculated using the formula below:

[(A695 Control-A695 Sample)/A695 Control]×100

Proximate composition determination

The aqueous and ethanolic extracts of C. esculentus were subjected to proximate analysis in accordance with the Association of Official Analytical Chemists methods (Helrich, 1990). The nitrogen content of crude protein was calculated and expressed as (nitrogen×6.25). The carbohydrate content in percentage was calculated by subtracting the sum of all other nutrients deducted from 100.

Mineral composition analysis

The mineral content of tigernut ethanolic and aqueous extracts, including zinc, copper, magnesium, iron, nickel, and calcium, was determined using an atomic absorption spectrophotometer (PerkinElmer Inc., Waltham, MA, USA).

Gas chromatographymass spectrometry (GC-MS) analysis

This was done with the PerkinElmer Clarus 680 system of tigernut aqueous and ethanolic extracts (PerkinElmer Inc.). At a constant movement rate of 1 mL/min, the carrier gas was unalloyed helium gas (99.99%). During the injection of 1 L of the extract, the temperature of the injector was kept at 250°C. The chemical compounds in the tigernut extract were identified by comparing their retention time (min), peak area, peak height, and mass spectral patterns to those found in the library (Linstrom et al., 2008).

In silico evaluation of antimutagenic activity by molecular docking

A total of 17 major phytochemical compounds present in both the ethanolic and aqueous extracts of tigernut were docked with RECQL5 Helicase Apo form, and the binding energies were computed using AutoDock Vina (Trott and Olson, 2010).

Protein preparation: The crystal structure of human RECQL5 Helicase Apo form in Protein Data Bank (PDB) format (Fig. 2), which was assigned PDB ID 5LB8, was obtained from the PDB (Newman et al., 2017). PyMOL was used to analyze the active site and remove water and impurities; however, the zinc metal was retained in the structure because it has been shown to be efficient for enzymatic activity (Ren et al., 2008). Docking grid maps were created using MGLTools 1.5.4 (Center for Computational Structural Biology, La Jolla, CA, USA). The enzyme’s grid box was assigned based on the amino acids at the enzyme’s active site, as previously reported by Newman et al. (2017). This was then used to generate a three-dimensional grid box map with the coordinates (center_x=−14.636; center_y=6.322; center_z= −25.85; size_x=30; size_y=80; size_z=38; exhaustiveness=8). The protein was prepared for docking analysis using the deter MGLTools 1.5.4; polar hydrogens were added to the protein, along with the assignment of bond orders.

Figure 2. Human RECQL5 protein has a three-dimensional structure (Protein Data Bank ID: 5LB8).

Ligand preparation: The three-dimensional configurations of 17 major phytochemicals present in tigernut aqueous and ethanolic extracts were obtained in structural data files form from PubChem (2020, http://pubchem.ncbi.nlm.nih.gov), before being converted into PDB files using PyMOL. MGLTools 1.5.4 was used to generate the ligands’ pdbqt files, with the number of bonds that can be rotated set to maximum.

Molecular docking: Both the protein and the ligands that had already been modified as described earlier were used in molecular docking studies with AutoDock Vina 1.1.2 (Trott and Olson, 2010). PyMOL (The PyMOL Molecular Graphics System, version 4.40, Schrödinger LLC., New York, NY, USA) and discovery studio were used to perform docking analyses. The binding energy and interaction of the molecules were determined using the BIOVIA discovery studio visualizer (version 19, BIOVIA, San Diego, CA, USA). The ligands were docked within the RECQL5 active site. For the in vitro evaluation of antimutagenicity, the extract containing the compound with the highest binding energies was chosen.

Antimutagenicity assay

Experimental design: The A. cepa assay was used to evaluate antimutagenicity, and it was done according to the Fiskesjö method (1985). Five onion bulbs ranging in size from 40 g to 60 g were purchased from a local market and sun-dried for 5 days till the layers on the outside could easily be peeled off and the root ring remained intact. Chemicals and herbicides that may have been on the bulbs were thoroughly rinsed away with tap water. The experimental grouping was done as follows:

•Control group: Onion bulbs (40 g) were placed in tap water (positive control) for 48 h.

•Mutagenicity control: Onion bulbs were grown in an aqueous solution of 250 μg/L of NaN3 for 48 h.

•Pre-treatment group: Onion bulbs of roots with 2 to 3 cm were suspended in 200 mg/kg of tigernut ethanolic extract for 24 h before being transferred into an aqueous solution containing 250 μg/L of NaN3.

•Post-treatment group: Onion bulbs of root with 2 to 3 cm were first suspended in an aqueous solution of 250 mg/L NaN3 for 24 h and were then transferred into tigernut ethanolic extract solution of 100 mg/kg and 200 mg/kg, respectively, and maintained for another 24 h.

Microscopic analysis

After 48 h, the onion roots were collected and suspended in a mixture of ethanol and acetic acid (3:1, v/v) and kept for 24 h at 4°C. According to Akinboro and Bakare’s method (2007), 1 N HCl was used to hydrolyze the fixed root. Following that, the roots were rinsed with distilled water before slides were prepared. Two to three cm rootlets were homogenized and smeared on a glass slide before being stained with two drops of acetoorcein. The surplus stain was blotted with filter paper, and the stained smear was covered with a coverslip so that the stained cell did not overlap. On each slide, an oil immersion objective lens (×100) of a Nikon Eclipse E400 microscope (Nikon, Tokyo, Japan) was used to view and observe three different stages of the MI and chromosomal aberration. A total of 1,000 normal and deviant dividing cells were scored and recorded. Statistical analysis was carried out on the results of MI, the number of CA, and the percentage reduction (Akinboro et al., 2011).

The damage reduction (DR) percentage was calculated as follows:

DR (%)=(A-B)(A-C)×100

where A is number of CA stimulated by NaN3, B is number of CA gotten from a mixture of NaN3 and the extract, and C is number of CA gotten from the positive control.

The MI was calculated as follows:

MI=Total dividing cellsTotal number of cells counted×100

The frequency of CA was calculated as follows:

Frequency of CA (%)=Average number of aberrant cellsTotal number of cells counted×100

Statistical analysis

The obtained results were presented as mean±standard deviation of triplicate determinations. At a significance level of P<0.05, one-way ANOVA was used to compare mean differences statistically.

RESULTS

Phytochemical analysis

Table 1 shows that tigernut ethanolic extract contains flavonoids, alkaloids, phenol, tannin, and glycosides but no resin. Table 2 shows the quantity of some phytochemical present in the extract, with flavonoids being the most abundant.

Table 1 . Tigernut ethanolic extract was subjected to a qualitative phytochemical screening.

PhytochemicalFlavonoidAlkaloidTanninResinGlycosidePhenol
Tigernut extract+++++

+, present; −, not detected..



Table 2 . Cyperus esculentus ethanolic extract phytochemical analysis.

Phytochemical Tigernut extract (mg/g)
Flavonoids22.47±0.63
Alkaloid19.71±0.75
Tannin0.08±0.06

Values are presented as mean±SD of three determinations..



UV-VIS analysis

UV absorption for both the ethanolic and aqueous extracts of tigernut was measured at wavelengths ranging from 198 nm to 900 nm to cover an extensive range of values as shown in Fig. 3. Table 3 also shows the absorption bands for both the ethanolic and aqueous extracts. The UV spectrum of the ethanolic extract showed peaks at 213, 230, and 292 nm corresponding to absorbance values of 0.597, 0.562, and 3.706, respectively, while that of the aqueous extract profile showed peaks at 212, 220, and 298 nm, which correspond to absorbance values of 0.881, 0.639, and 3.634, respectively.

Table 3 . Ultraviolet-visible peak values of tigernut ethanolic and aqueous extracts.

NoEthanolic extractAqueous extract


Wavelength (nm)AbsorbanceWavelength (nm)Absorbance
12120.8812130.597
22200.6392300.562
32983.6342923.706


Figure 3. Ultraviolet-visible spectra of ethanolic and aqueous tigernut extracts.

Antioxidant analysis

As shown in Table 4, the percentage of DPPH inhibition of the aqueous extract at a concentration of 0.002 g/mL was greater than that obtained for the ethanolic extract at a concentration of 0.01 g/mL, with the aqueous extract achieving 86.17%. Similarly, the total antioxidant activity of the aqueous extract was approximately 3.6 times greater than that of the ethanolic extract. However, the ethanolic extract outperformed the aqueous extract in terms of hydrogen peroxide radical scavenging activity.

Table 4 . Antioxidant activities of tigernut ethanolic and aqueous extracts.

SampleDPPH (% inhibition)H2O2 (% inhibition)Total antioxidant activity
Ethanolic extract76.1361.922.56
Aqueous extract86.1748.382.17


Proximate composition

Table 5 shows that the aqueous extract had significantly (P<0.05) higher crude protein, moisture, ash, and carbohydrate content than the ethanolic extract. However, the ethanolic extract contained a significantly (P<0.05) higher content of crude lipid than the aqueous extract. Although the aqueous extract had a higher fiber content, the difference in crude fiber content between the two extracts was not significant.

Table 5 . Proximate composition of ethanolic and aqueous tigernut extracts.

Parameter (%)Ethanolic tigernut extractAqueous tigernut extract
Crude protein3.48±0.02b6.13±0.01a
Crude fiber0.007±0.01a0.01±0.00a
Crude lipid60.01±0.01a15.99±0.04b
Moisture6.0±0.00b34.03±0.03a
Ash0.013±0.006b2.01±0.006a
Carbohydrate30.49±0.01b41.83±0.03a

Values are presented as mean±SD of triplicate determinations..

Different letters (a,b) within the same row are significantly different at P<0.05..



Mineral composition

Table 6 shows that the aqueous tigernut extract has significantly (P<0.05) higher zinc and copper contents than the ethanolic extract. The magnesium content of the ethanolic extract was found to be higher than that of the aqueous extract, although this difference was not statistically significant (P>0.05).

Table 6 . The mineral content of ethanolic and aqueous tigernut extracts.

Mineral (ppm)Ethanolic tigernut extractAqueous tigernut extract
Zinc6.233±0.112b10.864±1.092a
Copper5.049±0.139b7.793±0.034a
Magnesium17.396±0.0154ns15.163±28.000
Iron6.523±0.171b7.5163±0.0341a
Manganese8.2474±0.001a7.0448±0.0061b

Values are presented as mean±SD of triplicate determinations..

Different letters (a,b) within the same row are significantly different at P<0.05. nsNot significant..



GC-MS analysis

Table 7 displays the phytochemical constituents identified by GC-MS in the ethanolic extract. The GC-MS revealed 103 constituents in total, but only those with a percentage abundance greater than 0.50% were presented in the table. These accounted for 90.29% of the compounds present. The major compounds present include trans-13-octadecenoic acid, γ-sitosterol, hexadecanoic acid ethyl ester, stigmasterol, campesterol, octadecanoic acid, vitamin E, 2,4-di-tert-butylphenol, 3H-[1,2,3]triazole, 3-cyclohexyl-5-(1H-pyrazol-3-yl)-4-cyclohexylamino-, and benzo[H]quinoline, 2,4-dimethyl-. The chromatogram of the ethanolic tigernut extract (Fig. 4) revealed 7 prominent peaks as 2,4-di-tert-butylphenol with a retention time of 11.5245 min and peak area of 2.64%, hexadecanoic acid, ethyl ester (15.7924 min, 12.68%), trans-13-octadecenoic acid (17.1323 min 23.69%), vitamin E (23.3177 min, 3.54%), stigmasterol (24.3456 and 24.5824 min in total, 7.58% and 0.90% in total), and γ-sitosterol (24.8654 min, 14.13%). Other smaller peaks could also be seen in the GC-chromatogram spectrum.

Table 7 . Phytochemical components of ethanolic tigernut extract.

Serial No.Retention time (min)Library/IDArea (%)Qual
117.1323trans-13-Octadecenoic acid23.6990
224.8654γ-Sitosterol14.1399
315.7924Hexadecanoic acid, ethyl ester12.6898
424.3456Stigmasterol7.5899
524.0973Campesterol5.8799
617.2709Octadecanoic acid4.4699
723.3177Vitamin E3.5499
811.52452,4-Di-tert-butylphenol2.6497
924.98093H-[1,2,3]Triazole, 3-cyclohexyl-5-(1H-pyrazol-3-yl)-4-cyclohexylamino-2.1035
1026.0494Benzo[H]quinoline, 2,4-dimethyl-2.0825
1116.5721Behenic alcohol1.2894
1225.443Lanosterol1.1545
1327.8975Pyrido[2,3-D]pyrimidine, 4-phenyl-1.0925
1422.7401β-Tocopherol1.0299
1527.4239Benzenepropanoic acid, 3,5-bis(1,1-dimethylethyl)-4-hydroxy-, octadecyl ester0.9997
1624.5824Stigmasterol0.9056
1712.21175-Octadecene, (E)-0.8998
1815.92531H-1,3-Benzimidazole, 5,6-dimethyl-1-[(2,3,5,6-tetramethylphenyl)methyl]-0.8055
1921.15771-Methoxy-3-(2-hydroxyethyl)nonane0.6325
2025.29289,10-Methanoanthracen-11-ol, 9,10-dihydro-9,10,11-trimethyl-0.6153
2121.4753Squalene0.6087
2219.2114Diltiazem0.5259
2317.41539-Octadecenoic acid0.5297
2428.71181H-Indole, 5-methyl-2-phenyl-0.5125
Total90.29


Figure 4. Major components in the gas chromatography-mass spectrometry spectra of tigernut ethanolic extract. a, 2,4-di-tert-butylphenol; b, hexadecanoic acid, ethyl ester; c, trans-13-octadecenoic acid; d, octadecanoic acid; e, vitamin E; f, campesterol; g, stigmasterol; h, γ-sitosterol.

The phytochemical constituents in the aqueous extract are shown in Table 8. According to GC-MS, 92 constituents were present. However, only those with a percentage abundance greater than 0.50% were shown in the table, accounting for 92.23% of the total. Major compounds in the aqueous extract include propionic acid, 2-mercapto-, allyl ester; propanenitrile, 3-(methylthio)-; 2-chloro-ethyl methyl sulfide; 1,4-bis(trimethylsilyl)benzene; octadec-9-enoic acid, 1,2-bis(trimethylsilyl)benzene, furan, 2,5-dimethyl-; p-dioxane-2,3-diol; and 1,2,3,4-butanete-trol, [S-(R*,R*)]-, and cyclotrisiloxane, hexamethyl- (Table 8). The chromatogram of the aqueous tigernut extract (Fig. 5) portrays about 7 prominent peaks along with many small peaks indicating the presence of major compounds such as propanenitrile, 3-(methylthio)- with a retention time of 3.5373 min and peak area of 8.49%, p-dioxane-2,3-diol (5.0216 min, 3.10%), 1,2,3,4-butanetetrol, [S-(R*,R*)]- (5.2237 min, 3.01%), cyclotrisiloxane, hexamethyl- (5.5125 min, 2.57%), cyclotetrasiloxane, octamethyl- (7.6493 min, 1.40%), n-hexadecanoic acid (6.194 and 15.6366 min in total, 2.33%), octadec-9-enoic acid (4.3401 and 17.0804 min in total, 4.46%), and 1,2- bis(trimethylsilyl)benzene (4.5711 and 28.0188 min in total, 4.35%).

Table 8 . Phytochemical components of aqueous tigernut extract.

PeakRetention time (min)Library/IDArea (%)Qual
13.3294Propionic acid, 2-mercapto-, allyl ester13.31342
23.5373Propanenitrile, 3-(methylthio)-8.48657
33.76262-Chloroethyl methyl sulfide6.729325
43.86071,4-Bis(trimethylsilyl)benzene5.471730
54.2419Butanoic acid, 3-methyl-5.128953
64.3401Octadec-9-enoic acid4.463460
74.57111,2-Bis(trimethylsilyl)benzene4.345338
84.7732Furan, 2,5-dimethyl-3.483822
95.0216p-Dioxane-2,3-diol3.103272
105.22371,2,3,4-Butanetetrol, [S-(R*,R*)]-3.006838
115.5125Cyclotrisiloxane, hexamethyl-2.568370
125.7839Triethylenediamine2.453432
135.90521,2-Bis(trimethylsilyl)benzene2.336427
146.194n-Hexadecanoic acid2.325815
156.56942(5H)-Furanone, 5-methyl-2.309835
166.7831,2,3,4-Butanetetrol, [S-(R*,R*)]-2.276614
176.9101Thiodiglycol2.109738
187.0025Benzamide, 4-ethyl-N,N-dimethyl-1.935825
197.2566Benzenemethanol, 3-fluoro-1.648245
207.401Pyrido[2,3-D]pyrimidine, 4-phenyl-1.423440
217.6493Cyclotetrasiloxane, octamethyl-1.403232
227.95541,1,1,3,5,5,5-Heptamethyltrisiloxane1.30340
238.53291,2,3,4-Butanetetrol, [S-(R*,R*)]-1.178850
249.0585Formaldehyde, methyl(2-propenyl)hydrazine1.177838
259.3993Boronic acid, ethyl-, bis(2-mercaptoethyl ester)1.11443
269.50321,2,3,4-Butanetetrol, [S-(R*,R*)]-1.025938
279.78621,2-Bis(trimethylsilyl)benzene0.923445
289.94792-Furanmethanol0.922638
2910.3984Pyvalic acid, 3-chlorophenyl ester0.863935
3010.67561H-Imidazole-4-carboxylic acid, methyl ester0.829935
3110.8721,4-Bis(trimethylsilyl)benzene0.804138
3211.0221Carbamic acid, N-(4-tolyl)-, 2-(3-methylpyrazol-1-yl)ethyl ester0.62238
3311.1145Benzenamine, 4-(2-phenylethenyl)-N-(3,5-dimethyl-1-pyrazolylmethyl)-0.613746
3411.24162(1H)-Pyrimidinone0.596552
Total92.2981


Figure 5. Major components in the gas chromatography-mass spectrometry spectra of tigernut aqueous extract. a, propanenitrile, 3-(methylthio); b, p-dioxane-2,3-diol, ethyl ester; c, 1,2,3,4-butanetetrol; d, cyclotrisiloxane, hexamethyl; e, cyclotetrasiloxane, octamethyl; f, n-hexadecanoic acid; g, bis(trimethylsilyl)benzene; h, boronic acid, ethyl-, bis(2-mercaptoethyl ester); i, n-hexadecanoic acid; j, octadec-9-enoic acid.

In silico/molecular docking studies

The binding energies obtained from the molecular docking of the major compounds in both extracts are shown in Table 9. As shown from the table, only 5 out of the 17 compounds showed a good binding affinity with the RECQL5 protein (∆G≥7) and the compounds are γ-sitosterol, benzo[H]quinoline, 2,4-dimethyl-, stigmasterol, campesterol, and vitamin E (Fig. 6), which are all present in the ethanolic extract, with the highest binding energy obtained from the interaction of γ-sitosterol (−11.0 kcal/mol). Fig. 7A∼7C and 7D, 7E depict the specific interactions of the five ligands that demonstrated high binding affinity with some of the amino acid residues of the RECQL5 protein.

Table 9 . Table displaying the binding energy and major protein residues interacting with the ligand within 4 Å.

LigandBinding energy (kcal/mol)Key residues interacting within 4 Å
1,2-Bis(trimethylsilyl)benzene−4.7PHE 169, THR 395
1,4-Bis(trimethylsilyl)benzene−4.8GLU 378, LYS 381
2-Chloroethyl methyl sulfide−2.5LYS 381, THR 196, GLU 378, GLU 131, ARG 175, ARG 269, GLU 384, TRP 165, PRO 78, ASP 172
Benzo[h]quinoline, 2,4-dimethyl-−7.3ARG 267, ILE 317, LYS 338, ALA 337
Butanoic acid-3-methyl−3.8LYS 385, ILE 317, LYS 338, GLY 342, SER 339
Campesterol−8.6PHE 169, PRO 171, LEU 382, THR 395
Furan-2,5 dimethyl-−4.3LYS 389,PRO 130, ALA 193, SER 357, THR 194,
γ-Sitosterol−11.0LEU 382, GLN 383, ALA 380, ILE 396, GLU 378, ILE 375
Hexadecanoic ethyl ester−5.8ILE 317, HIS 167, ARG 269, LYS 381
Octadec-9-enoic acid−5.0LYS 385, LYS 338, ILE 317, THR 395, SER 391
Octadecanoic acid−6.1LEU 382, PHE 169, TYR 419, ILE 317, LYS 381, GLU 384, GLU 201, GLN 164
Propane nitrile, 3-(methylthio)−2.9GLY 342, ILE 317, LEU 79
Propionic acid, 2-mercapto-, allyl ester−3.4ILE 336, ILE 317, ARG 267, ALA 337
Squalene−5.4LYS 385, PHE 169, ARG 386, PRO 171, LEU 382
Stigmasterol−9.5PRO 171, LEU 382, PHE 169, GLY 166
trans-13-Octadecenoic acid−5.4LEU 382, PHE 169, PRO 171, GLN 164, SER 391
Vitamin E−7.6PRO 171, ARG 386, LYS 385, PHE 169, THR 395, ALA 394, GLN 164, SER 391


Figure 6. Compound structures in the ethanolic extract that have a high affinity for the RECQL5 protein.

Figure 7. Interactions between RECQL5 protein and campesterol (A), benzo[H]quinoline, 2,4-dimethyl- (B), vitamin E (C), γ-sitosterol (D), and stigmasterol (E).

Antimutagenicity assay

Fig. 8 depicts the antimutagenicity assay results. When compared to the positive control, the treated groups had a significant (P<0.05) reduction in the total number of CA. Furthermore, all treated groups had a higher percentage of mitotic indices than the untreated group. In the post-treatment group, DR percentages were higher than in the pre-treated group.

Figure 8. Heatmap indicating chromosomal aberrations and mitotic indices observed in Allium cepa treated with sodium azide (NaN3) and ethanolic extract of Cyperus esculentus (EECE). Values with different superscripts (a-c) across row indicate statistically different result at P<0.05. NC, negative control (distilled water); PC, positive control (250 μg/L NaN3); PR, pre-treatment with 200 mg/kg EECE after initial exposure to 250 μg/L NaN3; PO1, post-treatment with 100 mg/kg EECE after initial exposure to 250 μg/L NaN3; PO2, post-treatment with 200 mg/kg EECE after initial exposure to 250 μg/L NaN3; SE, standard error; MN, micro nucleus; AB, anaphasic bridge; MA, multipolar anaphase; NB, nucleus bud; BM, chromosomal break in metaphase; BA, chromosomal break in anaphase; BT, chromosomal break in telophase; CBM, chromosomal bridge in metaphase; CBA, chromosomal bridge in anaphase; CBT, chromosomal bridge in telophase; CA, chromosomal aberrations; TFA, total frequency of aberrations; MI, mitotic index; DR, damage reduction.

DISCUSSION

The UV-VIS analysis was performed to identify some of the phytoconstituents found in tigernut ethanolic and aqueous extracts. It also identifies σ-bonds, π-bonds, and lone pairs of electrons, chromophores, and aromatic rings containing chemical compounds. In the UV-VIS spectra, distinct peaks form between 200 and 400 nm, indicating the presence of unsaturated groups and numerous atoms such as sulfur, nitrogen, and oxygen (Njoku et al., 2013). The spectra for tigernut ethanolic and aqueous extracts show one major peak each at positions 292 nm and 298 nm, respectively. This demonstrates the presence of organic UV light-absorbing molecules in the extract, which was further confirmed specifically using GC-MS.

The preliminary phytochemical study on the tigernut ethanolic extract revealed that it contained chemicals with medicinal uses such as alkaloids, flavonoids, tannin, and phenol but no resin. This report is also consistent with the findings of Imam et al. (2013) and Chukwuma et al. (2010). However, varying amounts of the phyto-chemicals were reported, which could be attributed to differences in soil constituents, washing away of the topmost soil layer, and climatic factors. The most abundant phytochemical in the extract was discovered to be flavonoids. Flavonoids are the largest class of phytochemicals (Du et al., 2016). They have prominent antiviral, anti-inflammatory, cytotoxic, antimicrobial, and antioxidant effects. Like flavonoids, certain tannins and alkaloids with medicinal values have been reported (Okaka et al., 1991; Chukwuma et al., 2010).

DPPH free radicals scavenging assay determines the extract’s ability to mop up free radicals, which is demonstrated by the inhibition and repair of oxidative stress in organisms. In this study, aqueous extract demonstrated a higher DPPH scavenging percentage inhibition compared to the ethanolic extract. However, the result obtained for the DPPH scavenging activity of the ethanolic extract (76.13%) in this study is still higher compared to the result obtained by Elom and Ming (2019). This could be because of a variety of factors such as climatic and geographical conditions, nature, and the type of cultivar of the tigernut used. A similar result was obtained for total antioxidant capacity, indicating a higher content of phenolics and flavonoids in the aqueous extract as corroborated by Willis et al. (2019). The amount of phenolics in the extract that can decompose H2O2 to water by contributing electrons determines hydrogen peroxide scavenging activity (Bhatti et al., 2015). The ethanolic extract of tigernut demonstrated greater H2O2 scavenging activity, implying that the ethanolic extract has a higher amount of phenolics.

The aqueous tigernut extract had higher crude protein, ash, moisture, and carbohydrate content than the ethanolic extract in the proximate analysis. However, when compared to the results obtained by Imo et al. (2019), our study yielded lower values. This could be attributed to the loss of some of the nutrients during the process of extraction. However, the aqueous extract contains a significant amount of nutrients, particularly carbohydrates. Carbohydrates are energy-giving foods that are required for organism nutrition (Edeoga et al., 2005). Crude fats are a dense nutrient source that, when broken down, provides a lot of calories. They also serve as a form of energy storage (Potter and Hotchkiss, 1995). The ethanolic extract’s high lipid content could be attributed to its higher content of phytosterols such as stigmasterol, campesterol, sitosterol, etc., as revealed in the GC-MS results.

In proximate analysis, the ash residue content is regarded as an index of mineral composition (Onwuka, 2005). The higher levels of zinc, copper, and iron in the aqueous extract corroborated the higher ash content of the extract. Copper is a vital element that is required in minute amounts and functions as a cofactor for many redox enzymes and iron metabolism, antioxidant protection, production of neuropeptides, and immune processes (Uriu-Adams and Keen, 2005). Magnesium aids in the development of strong bones and the proper functioning of certain enzymes, the nervous and the cardiovascular system. Zinc (Zn) also plays important roles in metabolism as a cofactor for some enzymes, modulator of protein configuration, and expression of genes. Magnesium is relatively present in both the aqueous and the ethanolic extracts. It is vital to the normal functioning of muscle, heart, gland secretion, and nerve transmission (Castiglione et al., 2018). Because iron is a major component of hemoglobin, it plays an important role in oxygen transport (Andreini et al., 2018). Manganese is an essential cofactor for many enzymes involved in growth and maturation, absorption, reproduction, scavenging of free radicals, generation of energy, and functioning of the immune and nervous system (Chen et al., 2018).

As shown in Tables 7 and 8, GC-MS analysis was performed to further recognize the specific compounds present in both the ethanolic and aqueous extracts. The appearance of minor peaks demonstrated that some of the compounds are only present in trace amounts and that others are by-products of the disintegration of the main compounds. The peaks with short retention times are mostly phytochemicals with small retention times that can interact with water (Satapathy et al., 2009). Table 10 displays the bioactivities of 14 prominent constituents of tigernut ethanolic extract identified using GC-MS (Kumar et al., 2010; Mondul et al., 2015; Varsha et al., 2015; Zhao et al., 2015; Kurano et al., 2018; Senyilmaz-Tiebe et al., 2018; Nna et al., 2019; Asbaghi et al., 2020; Purkait et al., 2020; Antoci et al., 2021; Maurya et al., 2022).

Table 10 . Bioactivity of 11 major components of a tigernut ethanolic extract was determined using gas chromatography-mass spectrometry.

Serial No.Compound nameReported bioactivityReference
1trans-13-Octadecenoic acidAntifungal activityPurkait et al. (2020)
2γ-SitosterolHypolipidemic, antiviralMauryaet al. (2022)
3StigmasterolAntioxidant, antimicrobial, anticancerNna et al. (2019)
4CampesterolAntiviral, reduced apoptosisKurano et al. (2018)
5Octadecanoic acid (stearic acid)Mitofusin activity, antihypertensive activitySenyilmaz-Tiebe et al. (2018)
6Vitamin EAntioxidant, anti-inflammatoryAsbaghi et al. (2020)
72,4-Di-tert-butylphenolAntioxidant, antifungal activityVarsha et al. (2015)
8Benzo[h]quinoline, 2,4-dimethyl-General antimicrobialAntoci et al. (2021)
9LanosterolAnti-cataractogenic activityZhao et al. (2015)
10Hexadecanoic acid, ethyl esterAntioxidants, hypocholesterolemic nematicide, pesticides, anti-androgenic flavor, hemolytic, 5-α reductase inhibitorKumar et al. (2010)
11β-TocopherolAnticancer activityMondul et al. (2015)


Helicases are enzymes that use ATP as a source of energy to unwind complementary strands of DNA. They play important roles in DNA metabolisms such as replication, recombination, transcription, and repair; all of which are necessary steps in preventing mutagenesis, which could ultimately lead to cancer (Bachrati and Hickson, 2003). The beta isoform of the RECQL5 enzyme contains a conserved Zn2+ binding sub-domain (residues 365∼437) essential for efficient helicase activity (Ren et al., 2008). The binding conformation of two associating molecules with known chemical configurations is revealed by molecular docking. It thus provides a theoretical prediction of the most stable receptor-ligand complex’s preferred conformation (Madeswaran et al., 2012). The results of this study revealed that, of the 13 compounds docked with the human RECQL5 protein, only 5 of the compounds which were identified in the ethanolic extract of tigernut showed a very strong binding affinity for the protein-based on their docking scores. This is supported by other studies that found that using an alcoholic solvent such as ethanol is preferable to other solvents such as water when extracting medicinal phytochemicals (Gberikon et al., 2015). The five compounds γ-sitosterol, campesterol, benzo[H]quinoline 2,4 dimethyl, and vitamin E are all associated with specific amino acid residues at the active site within 4 Å of the enzyme protein, which might be responsible for their strong binding affinity. The compound with the highest binding energy was γ-sitosterol (−11.0 kcal/mol). These five compounds are well-known anticancer, antioxidant, and antimicrobial agents, implying their functions.

Molecular docking enabled compounds with high binding energy values to bind to human RECQL5 protein. This suggests that some compounds present in the ethanolic extract can activate this enzyme thereby preventing the onset of mutagenesis that can lead to carcinogenesis. Thus, the antimutagenicity assay was only performed using the ethanolic extract because the compounds revealed by the GC-MS analysis of the ethanolic extract, that is, the phytosterols were the ones that showed very good binding affinities for the helicase enzyme. The results of the in vitro antimutagenic functional tests performed in this study confirmed the ethanolic extract’s antimutagenic activity. Our findings showed that NaN3 caused CA at the selected dosage and was cytotoxic by lowering the percentage MI. The MI can be used for evaluating cytotoxicity, whereas CA are a yardstick for determining the mutagenicity of chemicals in organisms’ cells (Akinboro et al., 2011). Where NaN3 is exposed to cells, its mutagenicity is demonstrated by the action of its by-product-L-azidoadenine, which interacts with the genetic material, thereby inducing changes in the DNA particularly base substitutions (Gulluce et al., 2010).

The pre-treatment protocol in which the bulbs were first treated with the ethanolic extract of CE and then exposed to NaN3 resulted in a lower percentage reduction in DNA damage, whereas a much higher percentage reduction in DNA damage was observed in the post-treatment protocol in which A. cepa cells were first exposed to NaN3, and then to the extract. As a result, the findings suggest that tigernut extract has antimutagenic properties. Other plants like Origanum majorana L. and Ruta chalepensis have been shown to have antimutagenic activity in the A. cepa chromosomal assay (Khatab and Elhaddad, 2015).

The precise mechanism by which tigernut ethanolic extract exhibits antimutagenicity is unknown. However, it can be inferred that by the time the extract’s compounds percolated into the cell, particularly the phytosterols–that is, the γ-sitosterol, campesterol, stigmasterol, and vitamin E–they may have moderated the repair system of the cell by binding with a high affinity to the RECQ helicase enzyme. This is because plants have several RECQ homologs in the family of helicases, and recent studies have revealed a link between RECQ helicases of plant and their orthologs in man (Wiedemann et al., 2018). The ligands are thought to have activated the helicase enzyme in the onion plant, which acted to repair the NaN3-induced DNA damage in the post-treatment protocol, explaining the higher percentage reduction in the CA. Phytosterols are innate plant molecules that constitute the cell membrane. They have been shown to be anticancer by inhibiting cell division, initiating tumor cell death, and modulating some of the hormones involved in tumor growth (Awad and Fink, 2000). In addition to immune-modulatory effects, they have also been revealed to act against inflammation and oxidative stress (Ogbe et al., 2015). This most likely resulted in a decrease in the frequency of mutation, implying that the extract demonstrated chemoprotection via bioantimuta-genesis. Compounds in the extract may have the capacity of enhancing the efficacy of the repair system, as our findings revealed that the total number of CA in the treated groups was significantly reduced (P<0.05) compared to the untreated group.

Haraguchi et al. (1995) discovered that compounds in tigernut with many phenol groups in tigernut can effectively protect biological systems against diseases orchestrated by free radicals. Mutagenesis is known to be induced by the production of excess free radicals, which can have a negative effect on DNA, resulting in gene changes and chromosomal abnormalities (Degtyareva et al., 2008). In conclusion, both in silico and in vitro studies revealed that the ethanolic tigernut extract has antimutagenic activity, which could be attributed to the presence of vitamin E and other phytosterols, which have antioxidant properties and can activate repair enzymes. Because antimutagenic agents are potential anti-carcinogenic agents, compounds from tigernut extract can be isolated and used as nutraceuticals for cancer prevention. More research is needed, however, to confirm the antimutagenic activity in an in vivo system.

FUNDING

None.

AUTHOR DISCLOSURE STATEMENT

The authors declare no conflict of interest.

AUTHOR CONTRIBUTIONS

Conceptualization: ODO, TA. Methodology: all authors. Validation: ODO. Formal analysis: all authors. Investigation: TA, AVO. Writing–original draft preparation: TA, AVO. Writing–review and editing: ODO, TA. Supervision: ODO, TA, ATO. Project administration: ODO, TA, ATO. All authors have read and agreed to the published version of the manuscript.

Fig 1.

Figure 1.Image of a tigernut (Cyperus esculentus).
Preventive Nutrition and Food Science 2022; 27: 198-211https://doi.org/10.3746/pnf.2022.27.2.198

Fig 2.

Figure 2.Human RECQL5 protein has a three-dimensional structure (Protein Data Bank ID: 5LB8).
Preventive Nutrition and Food Science 2022; 27: 198-211https://doi.org/10.3746/pnf.2022.27.2.198

Fig 3.

Figure 3.Ultraviolet-visible spectra of ethanolic and aqueous tigernut extracts.
Preventive Nutrition and Food Science 2022; 27: 198-211https://doi.org/10.3746/pnf.2022.27.2.198

Fig 4.

Figure 4.Major components in the gas chromatography-mass spectrometry spectra of tigernut ethanolic extract. a, 2,4-di-tert-butylphenol; b, hexadecanoic acid, ethyl ester; c, trans-13-octadecenoic acid; d, octadecanoic acid; e, vitamin E; f, campesterol; g, stigmasterol; h, γ-sitosterol.
Preventive Nutrition and Food Science 2022; 27: 198-211https://doi.org/10.3746/pnf.2022.27.2.198

Fig 5.

Figure 5.Major components in the gas chromatography-mass spectrometry spectra of tigernut aqueous extract. a, propanenitrile, 3-(methylthio); b, p-dioxane-2,3-diol, ethyl ester; c, 1,2,3,4-butanetetrol; d, cyclotrisiloxane, hexamethyl; e, cyclotetrasiloxane, octamethyl; f, n-hexadecanoic acid; g, bis(trimethylsilyl)benzene; h, boronic acid, ethyl-, bis(2-mercaptoethyl ester); i, n-hexadecanoic acid; j, octadec-9-enoic acid.
Preventive Nutrition and Food Science 2022; 27: 198-211https://doi.org/10.3746/pnf.2022.27.2.198

Fig 6.

Figure 6.Compound structures in the ethanolic extract that have a high affinity for the RECQL5 protein.
Preventive Nutrition and Food Science 2022; 27: 198-211https://doi.org/10.3746/pnf.2022.27.2.198

Fig 7.

Figure 7.Interactions between RECQL5 protein and campesterol (A), benzo[H]quinoline, 2,4-dimethyl- (B), vitamin E (C), γ-sitosterol (D), and stigmasterol (E).
Preventive Nutrition and Food Science 2022; 27: 198-211https://doi.org/10.3746/pnf.2022.27.2.198

Fig 8.

Figure 8.Heatmap indicating chromosomal aberrations and mitotic indices observed in Allium cepa treated with sodium azide (NaN3) and ethanolic extract of Cyperus esculentus (EECE). Values with different superscripts (a-c) across row indicate statistically different result at P<0.05. NC, negative control (distilled water); PC, positive control (250 μg/L NaN3); PR, pre-treatment with 200 mg/kg EECE after initial exposure to 250 μg/L NaN3; PO1, post-treatment with 100 mg/kg EECE after initial exposure to 250 μg/L NaN3; PO2, post-treatment with 200 mg/kg EECE after initial exposure to 250 μg/L NaN3; SE, standard error; MN, micro nucleus; AB, anaphasic bridge; MA, multipolar anaphase; NB, nucleus bud; BM, chromosomal break in metaphase; BA, chromosomal break in anaphase; BT, chromosomal break in telophase; CBM, chromosomal bridge in metaphase; CBA, chromosomal bridge in anaphase; CBT, chromosomal bridge in telophase; CA, chromosomal aberrations; TFA, total frequency of aberrations; MI, mitotic index; DR, damage reduction.
Preventive Nutrition and Food Science 2022; 27: 198-211https://doi.org/10.3746/pnf.2022.27.2.198

Table 1 . Tigernut ethanolic extract was subjected to a qualitative phytochemical screening

PhytochemicalFlavonoidAlkaloidTanninResinGlycosidePhenol
Tigernut extract+++++

+, present; −, not detected.


Table 2 . Cyperus esculentus ethanolic extract phytochemical analysis

Phytochemical Tigernut extract (mg/g)
Flavonoids22.47±0.63
Alkaloid19.71±0.75
Tannin0.08±0.06

Values are presented as mean±SD of three determinations.


Table 3 . Ultraviolet-visible peak values of tigernut ethanolic and aqueous extracts

NoEthanolic extractAqueous extract


Wavelength (nm)AbsorbanceWavelength (nm)Absorbance
12120.8812130.597
22200.6392300.562
32983.6342923.706

Table 4 . Antioxidant activities of tigernut ethanolic and aqueous extracts

SampleDPPH (% inhibition)H2O2 (% inhibition)Total antioxidant activity
Ethanolic extract76.1361.922.56
Aqueous extract86.1748.382.17

Table 5 . Proximate composition of ethanolic and aqueous tigernut extracts

Parameter (%)Ethanolic tigernut extractAqueous tigernut extract
Crude protein3.48±0.02b6.13±0.01a
Crude fiber0.007±0.01a0.01±0.00a
Crude lipid60.01±0.01a15.99±0.04b
Moisture6.0±0.00b34.03±0.03a
Ash0.013±0.006b2.01±0.006a
Carbohydrate30.49±0.01b41.83±0.03a

Values are presented as mean±SD of triplicate determinations.

Different letters (a,b) within the same row are significantly different at P<0.05.


Table 6 . The mineral content of ethanolic and aqueous tigernut extracts

Mineral (ppm)Ethanolic tigernut extractAqueous tigernut extract
Zinc6.233±0.112b10.864±1.092a
Copper5.049±0.139b7.793±0.034a
Magnesium17.396±0.0154ns15.163±28.000
Iron6.523±0.171b7.5163±0.0341a
Manganese8.2474±0.001a7.0448±0.0061b

Values are presented as mean±SD of triplicate determinations.

Different letters (a,b) within the same row are significantly different at P<0.05. nsNot significant.


Table 7 . Phytochemical components of ethanolic tigernut extract

Serial No.Retention time (min)Library/IDArea (%)Qual
117.1323trans-13-Octadecenoic acid23.6990
224.8654γ-Sitosterol14.1399
315.7924Hexadecanoic acid, ethyl ester12.6898
424.3456Stigmasterol7.5899
524.0973Campesterol5.8799
617.2709Octadecanoic acid4.4699
723.3177Vitamin E3.5499
811.52452,4-Di-tert-butylphenol2.6497
924.98093H-[1,2,3]Triazole, 3-cyclohexyl-5-(1H-pyrazol-3-yl)-4-cyclohexylamino-2.1035
1026.0494Benzo[H]quinoline, 2,4-dimethyl-2.0825
1116.5721Behenic alcohol1.2894
1225.443Lanosterol1.1545
1327.8975Pyrido[2,3-D]pyrimidine, 4-phenyl-1.0925
1422.7401β-Tocopherol1.0299
1527.4239Benzenepropanoic acid, 3,5-bis(1,1-dimethylethyl)-4-hydroxy-, octadecyl ester0.9997
1624.5824Stigmasterol0.9056
1712.21175-Octadecene, (E)-0.8998
1815.92531H-1,3-Benzimidazole, 5,6-dimethyl-1-[(2,3,5,6-tetramethylphenyl)methyl]-0.8055
1921.15771-Methoxy-3-(2-hydroxyethyl)nonane0.6325
2025.29289,10-Methanoanthracen-11-ol, 9,10-dihydro-9,10,11-trimethyl-0.6153
2121.4753Squalene0.6087
2219.2114Diltiazem0.5259
2317.41539-Octadecenoic acid0.5297
2428.71181H-Indole, 5-methyl-2-phenyl-0.5125
Total90.29

Table 8 . Phytochemical components of aqueous tigernut extract

PeakRetention time (min)Library/IDArea (%)Qual
13.3294Propionic acid, 2-mercapto-, allyl ester13.31342
23.5373Propanenitrile, 3-(methylthio)-8.48657
33.76262-Chloroethyl methyl sulfide6.729325
43.86071,4-Bis(trimethylsilyl)benzene5.471730
54.2419Butanoic acid, 3-methyl-5.128953
64.3401Octadec-9-enoic acid4.463460
74.57111,2-Bis(trimethylsilyl)benzene4.345338
84.7732Furan, 2,5-dimethyl-3.483822
95.0216p-Dioxane-2,3-diol3.103272
105.22371,2,3,4-Butanetetrol, [S-(R*,R*)]-3.006838
115.5125Cyclotrisiloxane, hexamethyl-2.568370
125.7839Triethylenediamine2.453432
135.90521,2-Bis(trimethylsilyl)benzene2.336427
146.194n-Hexadecanoic acid2.325815
156.56942(5H)-Furanone, 5-methyl-2.309835
166.7831,2,3,4-Butanetetrol, [S-(R*,R*)]-2.276614
176.9101Thiodiglycol2.109738
187.0025Benzamide, 4-ethyl-N,N-dimethyl-1.935825
197.2566Benzenemethanol, 3-fluoro-1.648245
207.401Pyrido[2,3-D]pyrimidine, 4-phenyl-1.423440
217.6493Cyclotetrasiloxane, octamethyl-1.403232
227.95541,1,1,3,5,5,5-Heptamethyltrisiloxane1.30340
238.53291,2,3,4-Butanetetrol, [S-(R*,R*)]-1.178850
249.0585Formaldehyde, methyl(2-propenyl)hydrazine1.177838
259.3993Boronic acid, ethyl-, bis(2-mercaptoethyl ester)1.11443
269.50321,2,3,4-Butanetetrol, [S-(R*,R*)]-1.025938
279.78621,2-Bis(trimethylsilyl)benzene0.923445
289.94792-Furanmethanol0.922638
2910.3984Pyvalic acid, 3-chlorophenyl ester0.863935
3010.67561H-Imidazole-4-carboxylic acid, methyl ester0.829935
3110.8721,4-Bis(trimethylsilyl)benzene0.804138
3211.0221Carbamic acid, N-(4-tolyl)-, 2-(3-methylpyrazol-1-yl)ethyl ester0.62238
3311.1145Benzenamine, 4-(2-phenylethenyl)-N-(3,5-dimethyl-1-pyrazolylmethyl)-0.613746
3411.24162(1H)-Pyrimidinone0.596552
Total92.2981

Table 9 . Table displaying the binding energy and major protein residues interacting with the ligand within 4 Å

LigandBinding energy (kcal/mol)Key residues interacting within 4 Å
1,2-Bis(trimethylsilyl)benzene−4.7PHE 169, THR 395
1,4-Bis(trimethylsilyl)benzene−4.8GLU 378, LYS 381
2-Chloroethyl methyl sulfide−2.5LYS 381, THR 196, GLU 378, GLU 131, ARG 175, ARG 269, GLU 384, TRP 165, PRO 78, ASP 172
Benzo[h]quinoline, 2,4-dimethyl-−7.3ARG 267, ILE 317, LYS 338, ALA 337
Butanoic acid-3-methyl−3.8LYS 385, ILE 317, LYS 338, GLY 342, SER 339
Campesterol−8.6PHE 169, PRO 171, LEU 382, THR 395
Furan-2,5 dimethyl-−4.3LYS 389,PRO 130, ALA 193, SER 357, THR 194,
γ-Sitosterol−11.0LEU 382, GLN 383, ALA 380, ILE 396, GLU 378, ILE 375
Hexadecanoic ethyl ester−5.8ILE 317, HIS 167, ARG 269, LYS 381
Octadec-9-enoic acid−5.0LYS 385, LYS 338, ILE 317, THR 395, SER 391
Octadecanoic acid−6.1LEU 382, PHE 169, TYR 419, ILE 317, LYS 381, GLU 384, GLU 201, GLN 164
Propane nitrile, 3-(methylthio)−2.9GLY 342, ILE 317, LEU 79
Propionic acid, 2-mercapto-, allyl ester−3.4ILE 336, ILE 317, ARG 267, ALA 337
Squalene−5.4LYS 385, PHE 169, ARG 386, PRO 171, LEU 382
Stigmasterol−9.5PRO 171, LEU 382, PHE 169, GLY 166
trans-13-Octadecenoic acid−5.4LEU 382, PHE 169, PRO 171, GLN 164, SER 391
Vitamin E−7.6PRO 171, ARG 386, LYS 385, PHE 169, THR 395, ALA 394, GLN 164, SER 391

Table 10 . Bioactivity of 11 major components of a tigernut ethanolic extract was determined using gas chromatography-mass spectrometry

Serial No.Compound nameReported bioactivityReference
1trans-13-Octadecenoic acidAntifungal activityPurkait et al. (2020)
2γ-SitosterolHypolipidemic, antiviralMauryaet al. (2022)
3StigmasterolAntioxidant, antimicrobial, anticancerNna et al. (2019)
4CampesterolAntiviral, reduced apoptosisKurano et al. (2018)
5Octadecanoic acid (stearic acid)Mitofusin activity, antihypertensive activitySenyilmaz-Tiebe et al. (2018)
6Vitamin EAntioxidant, anti-inflammatoryAsbaghi et al. (2020)
72,4-Di-tert-butylphenolAntioxidant, antifungal activityVarsha et al. (2015)
8Benzo[h]quinoline, 2,4-dimethyl-General antimicrobialAntoci et al. (2021)
9LanosterolAnti-cataractogenic activityZhao et al. (2015)
10Hexadecanoic acid, ethyl esterAntioxidants, hypocholesterolemic nematicide, pesticides, anti-androgenic flavor, hemolytic, 5-α reductase inhibitorKumar et al. (2010)
11β-TocopherolAnticancer activityMondul et al. (2015)

References

  1. Akinboro A, Bakare AA. Cytotoxic and genotoxic effects of aqueous extracts of five medicinal plants on Allium cepa Linn. J Ethnopharmacol. 2007. 112:470-475.
    Pubmed CrossRef
  2. Akinboro A, Mohamed KB, Asmawi MZ, Sofiman OA. Mutagenic and antimutagenic potentials of fruit juices of five medicinal plants in Allium cepa L.: Possible influence of DPPH free radical scavengers. Afr J Biotechnol. 2011. 10:10520-10529.
    CrossRef
  3. Alhakmani F, Kumar S, Khan SA. Estimation of total phenolic content, in-vitro antioxidant and anti-inflammatory activity of flowers of Moringa oleifera. Asian Pac J Trop Biomed. 2013. 3:623-627.
    Pubmed KoreaMed CrossRef
  4. Andreini C, Putignano V, Rosato A, Banci L. The human iron-proteome. Metallomics. 2018. 10:1223-1231.
    Pubmed CrossRef
  5. Antoci V, Oniciuc L, Amariucai-Mantu D, Moldoveanu C, Mangalagiu V, Amarandei AM, et al. Benzoquinoline derivatives: A straightforward and efficient route to antibacterial and antifungal agents. Pharmaceuticals. 2021. 14:335. https://doi.org/10.3390/ph14040335.
    Pubmed KoreaMed CrossRef
  6. Asbaghi O, Sadeghian M, Nazarian B, Sarreshtedari M, Mozaffari-Khosravi H, Maleki V, et al. The effect of vitamin E supplementation on selected inflammatory biomarkers in adults: A systematic review and meta-analysis of randomized clinical trials. Sci Rep. 2020. 10:17234. https://doi.org/10.1038/s41598-020-73741-6.
    Pubmed KoreaMed CrossRef
  7. Awad AB, Fink CS. Phytosterols as anticancer dietary components: Evidence and mechanism of action. J Nutr. 2000. 130:2127-2130.
    Pubmed CrossRef
  8. Bachrati CZ, Hickson ID. RecQ helicases: Suppressors of tumorigenesis and premature aging. Biochem J. 2003. 374:577-606.
    Pubmed KoreaMed CrossRef
  9. Belewu MA, Abodunrin OA. Preparation of Kunnu from unexploited rich food source: Tiger nut (Cyperus esculentus). WJDFS. 2006. 1:19-21.
    CrossRef
  10. Bhattacharya S. Natural antimutagens: A review. Res J Med Plants. 2011. 5:116-126.
    CrossRef
  11. Bhatti MZ, Ali A, Ahmad A, Saeed A, Malik SA. Antioxidant and phytochemical analysis of Ranunculus arvensis L. extracts. BMC Res Notes. 2015. 8:279. https://doi.org/10.1186/s13104-015-1228-3.
    Pubmed KoreaMed CrossRef
  12. Castiglione D, Platania A, Conti A, Falla M, D'Urso M, Marranzano M. Dietary micronutrient and mineral intake in the Mediterranean healthy eating, ageing, and lifestyle (MEAL) study. Antioxidants. 2018. 7:79. https://doi.org/10.3390/antiox7070079.
    Pubmed KoreaMed CrossRef
  13. Chen P, Bornhorst J, Aschner M. Manganese metabolism in humans. Front Biosci. 2018. 23:1655-1679.
    Pubmed CrossRef
  14. Chukwuma ER, Obioma N, Christopher OI. The phytochemical composition and some biochemical effects of Nigerian tigernut (Cyperus esculentus L.) tuber. Pak J Nutr. 2010. 9:709-715.
    CrossRef
  15. Croce CM. Oncogenes and cancer. N Engl J Med. 2008. 358:502-511.
    Pubmed CrossRef
  16. De Flora S. Mechanisms of inhibitors of mutagenesis and carcinogenesis. Mutat Res. 1998. 402:151-158.
    Pubmed CrossRef
  17. Degtyareva NP, Chen L, Mieczkowski P, Petes TD, Doetsch PW. Chronic oxidative DNA damage due to DNA repair defects causes chromosomal instability in Saccharomyces cerevisiae. Mol Cell Biol. 2008. 28:5432-5445.
    Pubmed KoreaMed CrossRef
  18. Du G, Sun L, Zhao R, Du L, Song J, Zhang L, et al. Polyphenols: Potential source of drugs for the treatment of ischaemic heart disease. Pharmacol Ther. 2016. 162:23-34.
    Pubmed CrossRef
  19. Edeoga HO, Okwu DE, Mbaebie BO. Phytochemical constituents of some Nigerian medicinal plants. Afr J Biotechnol. 2005. 4:685-688.
    CrossRef
  20. Elom SA, Ming TO. Antioxidant screening and cytotoxicity effect of tigernut (Cyperus esculentus) extracts on some selected cancer-origin cell lines. Euromediterranean Biomed J. 2019. 14:1-6.
  21. Feyisayo AK, Oluokun OO. Evaluation of antioxidant potentials of Monodora myristica (Gaertn) dunel seeds. Afr J Food Sci. 2013. 7:317-324.
    CrossRef
  22. Fiskesjö G. The Allium test as a standard in environmental monitoring. Hereditas. 1985. 102:99-112.
    Pubmed CrossRef
  23. Gberikon GM, Adeoti II, Aondoackaa AD. Effect of ethanol and aqueous solutions as extraction solvents on phytochemical screening and antibacterial activity of fruit and stem bark extracts of Tetrapleura tetrapteraon Streptococcus salivarus and Streptococcus mutans. Int J Curr Microbiol App Sci. 2015. 4:404-410.
  24. Gulluce M, Agar G, Baris O, Karadayi M, Orhan F, Sahin F. Mutagenic and antimutagenic effects of hexane extract of some Astragalus species grown in the eastern Anatolia region of Turkey. Phytother Res. 2010. 24:1014-1018.
    Pubmed CrossRef
  25. Haraguchi H, Saito T, Okamura N, Yagi A. Inhibition of lipid peroxidation and superoxide generation by diterpenoids from Rosmarinus officinalis. Planta Med. 1995. 61:333-336.
    Pubmed CrossRef
  26. Helrich K. Official methods of analysis. 15th ed. Association of Official Analytical Chemists, Arlington, TX, USA. 1990. p 212.
  27. Hockberger PE. A history of ultraviolet photobiology for humans, animals and microorganisms. Photochem Photobiol. 2002. 76:561-579.
    Pubmed CrossRef
  28. Imam TS, Aliyu FG, Umar HF. Preliminary phytochemical screening, elemental and proximate composition of two varieties of Cyperus esculentus (tiger nut). Nig J Basic Appl Sci. 2013. 21:247-251.
    CrossRef
  29. Imo C, Uhegbu FO, Arowora KA, Ezeonu CS, Opara IJ, Nwaogwugwu CJ, et al. Chemical composition of Cyperus esculentus nut and Phoenix dactylifera fruit. Afr J Biotechnol. 2019. 18:408-415.
    CrossRef
  30. Khatab HA, Elhaddad NS. Evaluation of mutagenic effects of monosodium glutamate using Allium cepa and antimutagenic action of Origanum majorana L. and Ruta chalepensis medical plants. Biotechnol J Int. 2015. 8:1-11.
    CrossRef
  31. Kumar PP, Kumaravel S, Lalitha C. Screening of antioxidant activity, total phenolics and GC-MS study of Vitex negundo. Afr J Biochem Res. 2010. 4:191-195.
  32. Kurano M, Hasegawa K, Kunimi M, Hara M, Yatomi Y, Teramoto T, et al. Sitosterol prevents obesity-related chronic inflammation. Biochim Biophys Acta Mol Cell Biol Lipids. 2018. 1863:191-198.
    Pubmed CrossRef
  33. Levan A. The effect of colchicine on root mitoses in Allium. Hereditas. 1938. 24:471-486.
    CrossRef
  34. Linstrom PJ, Mallard WG; National Institute of Standards and Technology. The NIST Chemistry WebBook. 2008 [cited 2021 Aug 15]. Available from: https://webbook.nist.gov.
  35. Luximon-Ramma A, Bahorun T, Crozier A. Antioxidant actions and phenolic and vitamin C contents of common Mauritian exotic fruits. J Sci Food Agric. 2003. 83:496-502.
    CrossRef
  36. Madeswaran A, Umamaheswari M, Asokkumar K, Sivashanmugam T, Subhadradevi V, Jagannath P. Computational drug discovery of potential phosphodiesterase inhibitors using in silico studies. Asian Pac J Trop Dis. 2012. 2:S822-S826.
    CrossRef
  37. Maurya VK, Kumar S, Bhatt MLB, Saxena SK. Antiviral activity of traditional medicinal plants from Ayurveda against SARS-CoV-2 infection. J Biomol Struct Dyn. 2022. 40:1719-1735.
    Pubmed KoreaMed CrossRef
  38. Mazzio EA, Soliman KF. In vitro screening for the tumoricidal properties of international medicinal herbs. Phytother Res. 2009. 23:385-398.
    Pubmed KoreaMed CrossRef
  39. Mondul AM, Moore SC, Weinstein SJ, Karoly ED, Sampson JN, Albanes D. Metabolomic analysis of prostate cancer risk in a prospective cohort: The alpha-tocopherol, beta-carotene cancer prevention (ATBC) study. Int J Cancer. 2015. 137:2124-2132.
    Pubmed KoreaMed CrossRef
  40. Nabavi SM, Ebrahimzadeh MA, Nabavi SF, Jafari M. Free radical scavenging activity and antioxidant capacity of Eryngium caucasicum Trautv and Froripia subpinnata. Pharmacologyonline. 2008. 3:19-25.
  41. National Center for Biotechnology Information. PubChem. 2020 [cited 2021 Sep 22]. Available from: https://pubchem.ncbi.nlm.nih.gov.
  42. Newman JA, Aitkenhead H, Savitsky P, Gileadi O. Insights into the RecQ helicase mechanism revealed by the structure of the helicase domain of human RECQL5. Nucleic Acids Res. 2017. 45:4231-4243.
    Pubmed KoreaMed CrossRef
  43. Njoku DI, Chidiebere MA, Oguzie KL, Ogukwe CE, Oguzie EE. Corrosion inhibition of mild steel in hydrochloric acid solution by the leaf extract of Nicotiana tabacum. Adv Mater Corros. 2013. 2:54-61.
  44. Nna PJ, Tor-Anyiin T, Muluh E. Stigmasterol and β-sitosterol from the root of Mangifera indica and their biological activities against some pathogens. J Complement Altern Med Res. 2019. 7:1-10.
    CrossRef
  45. Ogbe RJ, Ochalefu DO, Mafulul SG, Olaniru OB. A review on dietary phytosterols: Their occurrence, metabolism and health benefits. Asian J Plant Sci Res. 2015. 5:10-21.
  46. Okafor JNC, Mordi JI, Ozumba AU, Solomon HM, Olatunji O. Preliminary studies on the characterization of contaminants in tigernut (yellow variety). Proceedings of 27th Annual Nigerian Institute of Food Science and Technology (NIFST) Conference. 2003 Oct 13. Kano, Nigeria. p 210-211.
  47. Okaka JC, Enoch NJ, Okaka NC. Human Nutrition: An Integrated Approach. ESUT Publications, Agbani, Nigeria. 1991. p 57-58.
  48. Onwuka GI. Food analysis and instrumentation: Theory and practice. Napthali Prints, Lagos, Nigeria. 2005. p 219-230.
  49. Owais WM, Kleinhofs A. Metabolic activation of the mutagen azide in biological systems. Mutat Res. 1988. 197:313-323.
    Pubmed CrossRef
  50. Owusu NR, Owusu BP. Tiger nuts: A healthier pseudo-nut of all nuts in the tropics. IJIRMF. 2016. 2:307-312.
  51. Potter NN, Hotchkiss JH. Food science. 5th ed. Springer Science+Business Media, New York, NY, USA. 1995. p 52.
    CrossRef
  52. Prieto P, Pineda M, Aguilar M. Spectrophotometric quantitation of antioxidant capacity through the formation of a phosphomolybdenum complex: Specific application to the determination of vitamin E. Anal Biochem. 1999. 269:337-341.
    Pubmed CrossRef
  53. Purkait A, Worede RE, Baral D, Dipak KH, Hazra DK, Panja BN, et al. Development of nanoemulsion formulation of mustard oil, its chemical characterization and evaluation against post harvest anthracnose pathogens. Indian Phytopathol. 2020. 73:449-460.
    CrossRef
  54. Ren H, Dou SX, Zhang XD, Wang PY, Kanagaraj R, Liu JL, et al. The zinc-binding motif of human RECQ5b suppresses the intrinsic strand-annealing activity of its DExH helicase domain and is essential for the helicase activity of the enzyme. Biochem J. 2008. 412:425-433.
    Pubmed CrossRef
  55. Salem ML, Zommara M, Imaizumi K. Dietary supplementation with Cyperus esculentus L (tiger nut) tubers attenuated atherosclerotic lesion in apolipoprotein E knockout mouse associated with inhibition of inflammatory cell responses. Am J Immunol. 2005. 1:60-67.
    CrossRef
  56. Sanful RE. The use of tiger-nut (Cyperus esculentus), cow milk and their composite as substrates for yoghurt production. Pak J Nutr. 2009. 8:755-758.
    CrossRef
  57. Satapathy AK, Gunasekaran G, Sahoo SC, Amit K, Satapathy AK, Rodrigues PV. Corrosion inhibition by Justicia gendarussa plant extract in hydrochloric acid solution. Corros Sci. 2009. 51:2848-2856.
    CrossRef
  58. Senyilmaz-Tiebe D, Pfaff DH, Virtue S, Schwarz KV, Fleming T, Altamura S, et al. Dietary stearic acid regulates mitochondria in vivo in humans. Nat Commun. 2018. 9:3129. https://doi.org/10.1038/s41467-018-05614-6.
    Pubmed KoreaMed CrossRef
  59. Shamsa F, Monsef H, Ghamooshi R, Verdian-rizi M. Spectrophotometric determination of total alkaloids in some Iranian medicinal plants. Thai J Pharm Sci. 2008. 32:17-20.
    CrossRef
  60. Takhatajah A. Angiosperms (the flowering plants). 15th ed. In: Swanson C, Benton W, Hutchins RM, editors. Encyclopedia Britannica: Macropaedia. Encyclopædia Britannica Editorial, Chicago, IL, USA. 1992. p 596-610.
  61. Temple VJ, Ojobe TO, Kapu MM. Chemical analysis of tiger nut (Cyperus esculentis). J Sci Food Agric. 1990. 50:261-263.
    CrossRef
  62. Tomasetti C, Li L, Vogelstein B. Stem cell divisions, somatic mutations, cancer etiology, and cancer prevention. Science. 2017. 355:1330-1334.
    Pubmed KoreaMed CrossRef
  63. Trott O, Olson AJ. AutoDock Vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem. 2010. 31:455-461.
    Pubmed KoreaMed CrossRef
  64. Uriu-Adams JY, Keen CL. Copper, oxidative stress, and human health. Mol Aspects Med. 2005. 26:268-298.
    Pubmed CrossRef
  65. Van Buren JP, Robinson WB. Formation of complexes between protein and tannic acid. J Agric Food Chem. 1969. 17:772-777.
    CrossRef
  66. Varsha KK, Devendra L, Shilpa G, Priya S, Pandey A, Nampoothiri KM. 2,4-Di-tert-butyl phenol as the antifungal, antioxidant bioactive purified from a newly isolated Lactococcus sp. Int J Food Microbiol. 2015. 211:44-50.
    Pubmed CrossRef
  67. Wiedemann G, van Gessel N, Köchl F, Hunn L, Schulze K, Maloukh L, et al. RecQ helicases function in development, DNA repair, and gene targeting in Physcomitrella patens. Plant Cell. 2018. 30:717-736.
    Pubmed KoreaMed CrossRef
  68. Willis S, Jackson C, Verghese M. Effects of processing on antioxidant capacity and metabolizing enzyme inhibition of tiger nut tubers. Food Nutr Sci. 2019. 10:1132-1141.
    CrossRef
  69. Zhao L, Chen XJ, Zhu J, Xi YB, Yang X, Hu LD, et al. Lanosterol reverses protein aggregation in cataracts. Nature. 2015. 523:607-611.
    Pubmed CrossRef