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ORIGINAL ARTICLE
Year : 2015  |  Volume : 5  |  Issue : 2  |  Page : 82-88

Mutagenic evaluation and spectroscopic characterization of flavonoidal fraction of Apium leptophyllum (Pers.) fruit


1 Department of Pharmaceutical Sciences, NIMS Institute of Pharmacy, Jaipur, Rajasthan, India
2 Department of Pharma Analysis and Quality Assurance, Institute of Pharmacy and Technology, Cuttack, Odisha, India
3 Department of Pharmacognosy and Phytochemistry, Vedica College of Pharmacy, Bhopal, Madhya Pradesh, India
4 Department of Pharmacology, NIMS Medical College, NIMS University, Jaipur, Rajasthan, India

Date of Submission15-Dec-2014
Date of Acceptance17-Feb-2015
Date of Web Publication23-Mar-2015

Correspondence Address:
Himanshu Bhusan Sahoo
NIMS Institute of Pharmacy, NIMS University, Jaipur - 303 121, Rajasthan
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2231-0738.153798

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   Abstract 

Objective: The present study was aimed to evaluate the antimutagenic potential of flavonoidal fraction of Apium leptophyllum fruits (FFALF) and isolate the bioactive phytoconstituents from that fraction along with their subsequent characterization. Materials and Methods: For mutagenicity study, a single application of FFALF at doses of 5 mg/kg, 10 mg/kg, and 20 mg/kg of body weight were orally administered to Swiss mice, 24 h prior the i.p. administration of cyclophosphamide (50 mg/kg). After 24 h of administration of cyclophosphamide, the rodents were sacrificed and the aberrant chromosomal cells as well as micronuclei formations from the bone marrow cells were compared with the positive control. Then, the phytoconstituents were isolated from FFALF using column chromatography and characterized by spectroscopic evidences of Fourier transform infrared spectroscopy (FTIR), mass spectroscopy (MS), hydrogen-1 nuclear magnetic resonance ( 1 H NMR), and carbon-13 nuclear magnetic resonance ( 13 C NMR). Results: The pretreatment of FFALF significantly (P < 0.01) reduced the frequency of micronuclei formation and the incidence of chromosomal aberration in a dose-dependent manner from the bone marrow cells of mice as compared to the cyclophosphamide induced group. The cumulative spectral data showed the presence of two flavonoids isolated from FFALF, i.e., apigenin and quercetin. Conclusion: In the present study, apigenin and quercetin have been successfully isolated from FFALF. The isolation of the above characterized flavonoids exerts antimutagenic activity against cyclophosphamide-induced genetic abnormalities in Swiss mice. In the future, it may be useful to prepare plant-based pharmaceutical preparation to treat various genetic or immunological disorders.

Keywords: Apigenin, Apium leptophyllum, chromosomal aberrations, cyclophosphamide, micronucleus, mutagenicity, spectroscopy, quercetin
Abbreviations: FFALF-Flavonoidal fractions from Apium leptophyllum fruits, i.p.-Intraperitoneal, PO-Per Oral, CP- Cyclophosphamide, IR-Infrared, MS-Mass Spectroscopy, 1 H- and 13 C- NMR-Proton and 13 C nuclear magnetic resonance.


How to cite this article:
Sahoo HB, Patro SK, Sagar R, Santani DD. Mutagenic evaluation and spectroscopic characterization of flavonoidal fraction of Apium leptophyllum (Pers.) fruit. Int J Nutr Pharmacol Neurol Dis 2015;5:82-8

How to cite this URL:
Sahoo HB, Patro SK, Sagar R, Santani DD. Mutagenic evaluation and spectroscopic characterization of flavonoidal fraction of Apium leptophyllum (Pers.) fruit. Int J Nutr Pharmacol Neurol Dis [serial online] 2015 [cited 2019 Nov 19];5:82-8. Available from: http://www.ijnpnd.com/text.asp?2015/5/2/82/153798


   Introduction Top


Nature has provided us with a storehouse of remedies to cure all ailments of mankind. Today, many of the modern drugs for clinical use are of natural origin. In developing countries, many people have deep faith in herbal folk medicines for primary health treatments. So, the value of these herbal medicines is increasing in arithmetic progression, but is limited due to only some constituents present in a medicinal plant that have the potential to counter toxicities like mutagenic, carcinogenic, teratogenic, etc. [1] These potential toxicities of herbal drugs have not been recognized by the general public or by the traditional practitioners, which affect the safety, efficacy, and quality of herbal drugs. Presently, both the laymen and the medical professionals are updated toward the safety and efficacy of drugs prior to their use. Therefore, evaluating the toxicological effects of any herbal extract intended to be used in humans is of utmost importance. [2] Among these toxicological parameters, genetic damage is a very important one, which could affect the present as well as future generations. These genetic alterations lead to mutagenesis, carcinogenesis, or other chronic degenerative diseases like atherosclerosis and heart diseases, which are the leading causes of death in the human population at present. [3]

Apium leptophyllum (A. leptophyllum) Pers. (family: Umbelliferae), popularly known as Ajmuda is distributed in India, Sri Lanka, Pakistan, South America, Queensland (Australia), and some tropical areas. In India, it is cultivated in Andhra Pradesh, Gujarat, Madhya Pradesh, Jammu and Kashmir, and Karnataka. [4] In the traditional system of Ayurvedic medicine, the fruit is widely used as an antinephritic, antirheumatic, and carminative agent, and is also beneficial for the prevention of tumor, anorexia, vomiting, and colic pain. [5],[6] In Ethiopian traditional medicine, the leaves are used for the treatment of Mitchell's disease (a disease associated with inflammation, sweating, and loss of appetite). [7] The volatile oil from the leaves possesses antimicrobial activity and in vitro radical scavenging activity. [8] Earlier scientific investigation showed that the fruit of this plant possesses good antioxidant property and chemopreventive activity in skin carcinogenic cases. [9],[10] Phytochemically, it contains volatile oils, coumarin derivatives, terpene hydrocarbons, phenols, and alkaloids, and is a rich source of flavonoids. [10],[11] No previous work has been reported on the isolation of flavonoids from the fruit. So, the present study focuses on the isolation of flavonoids and antimutagenic potential from A. leptophyllum fruits.


   Materials and methods Top


Chemicals and reagents

Cyclophosphamide, colchicine, Giemsa stain, and fetal bovine serum (FBS) were purchased from Sigma-Aldrich Chemicals Pvt. Ltd., Bengaluru, Karnataka, India. The solvents such as methanol, ethanol, petroleum ether, diethyl ether, chloroform, ethyl acetate, n-butanol, benzene, ammonia, and formic acid were purchased from Merck India Ltd., Mumbai, India and the other chemicals used in this study were of analytical grade.

Extract preparation from plant material

The fruits of A. leptophyllum were collected from Bhopal District, Madhya Pradesh, India. The collected fruits were cleaned, washed with distilled water, and dried in shade for 4-6 days. The fine powder (≈60 mesh size) was obtained from the dried fruits by using a blender and then the powdered material (50 g) was defatted with petroleum ether and exhaustively extracted with 80% methanol into the Soxhlet assembly for 48 h. The extract was separated by filtration through Whatman Grade No.1 filter paper, concentrated on vacuum evaporator. The extract (yield-16.4% w/w) was filled in a plastic bottle and stored at 4°C until used.

Preliminary phytochemical screening

The preliminary phytochemical tests were performed on the methanolic extract of A. leptophyllum for the presence of various phytoconstituents as per the described methods. [12]

Separation of flavonoid fraction from the methanolic extract

The crude methanolic extract (10 g) was subjected to column chromatography (silica gel 120 mesh, 500 g) using solvents such as diethyl ether, chloroform, ethyl acetate, and n-butanol. The collected fractions were subjected to the Shinoda test, followed by thin-layer chromatography (TLC) using benzene:methanol:ammonia (9:1:0.1) solvent system. The spot was visualized by spraying with ammonia, a reagent specific for flavonoids. The fractions showing positive response for flavonoid were pooled together and considered as the total flavonoid fraction. The total flavonoid fraction was concentrated and subjected to further studies.

Experimental animals

Healthy male Swiss mice (6-8 weeks old, 25-30 g) were randomly selected and were maintained in an ambient room temperature (25 ± 2°C), relative humidity (50 ± 5%), and 12:12 h dark-light cycle. The mice were housed in a polypropylene cage and fed pellet diet with water ad libitum throughout the experiment. They were allowed to stabilize for 1 week prior to the commencement of experiment. The protocols were approved by the Institutional Animal Ethics Committee and carried out under strict compliance with the Committee for the Purpose of Control and Supervision of Experiments on Animals, Ministry of Environment, Forest and Climate Change, Government of India.

Treatment schedule

Male Swiss mice were divided into six groups (n = 6) in the laboratory cage. They were fasted overnight, with free access to only water.

Group I: Vehicle control - Received only distilled water

Group II: FFALF control - Received with FFALF alone (5 mg/kg) orally

Group III: Positive control - Received single i.p. injection of 50 mg/kg cyclophosphamide in 0.9% w/v saline

Groups IV-VI: Test groups - received single dose of 5 mg/kg, 10 mg/kg, and 20 mg/kg body weight per oral (PO) of FFALF, respectively, before 24 h treatment of cyclophosphamide (50 mg/kg, i.p.)

Mutagenicity evaluation

Mutagenic study in the bone marrow cells was evaluated by chromosomal aberrations and micronuclei formation by cyclophosphamide induction. [13]

Chromosomal aberrations analysis

The mice were treated with colchicine (4 mg/kg i.p.) 2 h before to arrest metaphase. Then, they were sacrificed by cervical dislocation and the femur bones were isolated. The bone marrow was flushed out from both the femurs using 0.56% (w/v) KCl solution and incubated at 37°C. The bone marrow suspension was centrifuged for 10 min at 1000 revolutions per minute (rpm) and the supernatant was discarded. The collected pellet was resuspended in freshly prepared Carnoy's fixative (3:1 mixture of ethanol and glacial acetic acid) on cyclomixer till white pellet free of debris were obtained. The suspension was dropped on the ice-cold slides (previously kept in 90% alcohol in freezer) by the air drop method using pasteur pipette and the slides were immediately flamed for a few seconds and allowed to dry at room temperature. After drying, the slides were stained with phosphate-buffered 5% Giemsa solution for 15 min. A total of 100 well-spread metaphase plates were analyzed (total 600 metaphases in each group) for chromosomal aberrations at a magnification of 1000 X (100 × 10 X) for each group. Different types of chromosomal aberrations such as chromatid breaks, fragments, gaps, ring formation, centromeric association, etc. were scored and expressed as the percentage of chromosomal aberrations. [14]

Micronucleus analysis

After sacrificing, the bone marrow cells were flushed with 1 mL FBS from both the femurs. The collected bone marrow cells were allowed to incubate at 25°C and centrifuged at 1500 rpm for 10 min. The supernatant was discarded and the pellets were resuspended again with 100 μL of FBS. Two to three drops of cell suspension were spread on precleaned slides, air dried, and fixed in absolute methanol. The slides were stained with acridine orange (0.003%). The prepared slides were coded and examined under a fluorescent microscope. To evaluate bone marrow toxicity, the ratio of polychromatic erythrocyte (PCE) to total erythrocytes [normochromatic erythrocyte (NCE)], i.e., PCE/(PCE+NCE) was calculated by counting a total of 1,000 erythrocytes per mouse using these slides. To determine the frequency of micronucleated polychromatic erythrocytes (MNPCE), all together 2,000 PCEs per mouse were examined for the presence of micronuclei, which means that 12,000 PCEs were scored per dose group. [15]

Isolation and spectroscopic characterization of phytoconstituents from FFALF

The FFALF mixture was allowed to run in column chromatography for separation of flavonoids. The 45 cm length and 3 cm width of the column were used and is filled with the slurry of silica gel-H of mesh size 60-120 μm to 1/3rd portion using n-hexane. The column was set with the solvent of n-hexane and special care was taken to avoid air bubble formation during column packing. Then, 10 g of total flavonoidal fraction was bound with silica gel and loaded on the top of the column. The column was eluted with the gradient solvent system of n-butanol-water-acetic acid system in the ratio of 12:2:1 v/v/v until the color of the column was colorless.

Each layered fraction were collected carefully and and dried at 45-50°C. The isolated compounds were subjected to infrared (IR), hydrogen-1 nuclear magnetic resonance ( 1 H NMR), carbon-13 nuclear magnetic resonance ( 13 C NMR), and mass spectroscopy (MS) studies to obtain the spectral data for the detection of functional group, number of protons, carbons, and molecular mass of the compound, respectively. [16] All these spectral evaluations were conducted in the Sophisticated Analytical Instrumentation Facility, CIL and UCIM, Punjab University, Chandigarh, India.

MS (LC-MS)

The samples were analyzed on the Micromass Q-ToF Mass Spectrometer equipped with electrospray ionization (ESI) and atmospheric pressure chemical ionization (APcI) sources having mass range 4000 amu in quadruple and 20000 amu in time of flight (ToF). The sample was introduced into the ion source with detector voltage 1.25 kV, detector temperature 300°C, ion source temperature 225°C, and ionization energy 70 eV. The sample was dissolved in methanol, sonicated for 10 min, and diluted further to obtain a concentration of 10 μL/mL. Then, 5 μL of sample was introduced into a mass spectrophotometer using an autosampler.

NMR spectroscopy

Each isolate was dissolved in 0.6 mL of dimethyl sulfoxide-d6 (DMSO-d6 ) containing tetramethylsilane (TMS) and were performed on a Bruker Avance II 400 NMR spectrometer with a z-gradient unit at 295.3 K with 400 MHz for 1 H NMR (10 mg of sample) and 100 MHz for 13 C NMR (50 mg of sample), employing the manufacturer's pulse programs. The instrument is equipped with a cryomagnet of field strength 9.4 T.

The sample of 10 mg and 50 mg were taken for 1 H NMR and 13 C NMR, respectively. The chemical shift values were reported in ppm (d), relative to TMS as the internal standard.

IR spectroscopy

The IR spectra were recorded in the solid state (10 mg of sample) as KBr pellets using PerkinElmer Spectrum RX I FTIR Spectrophotometer (PerkinElmer Inc., USA). It has a resolution of 1 cm -1 and a scan range 4000 cm -1 -250 cm -1 .

Statistical analysis

The results were expressed as mean ± standard deviation (SD) and analyzed using one-way analysis of variance (one-way ANOVA) followed by the Student's t-test using the software SYSTAT 7.0, to find out the level of significance. The data were considered statistically significant at the minimum level of P < 0.01 and P < 0.05.


   Results Top


Preliminary phytochemical screening

The methanolic extract of A. leptophyllum showed the positive test for carbohydrate, flavonoids, terpenoids, glycosides, tannins, volatile oils, and proteins.

Isolation of flavonoid fraction from A. leptophyllum extract

A total of 39 fractions were eluted from the methanolic extract of A. leptophyllum. The Shinoda test was carried out for confirming the presence of flavonoids and TLC studies were carried out using chloroform:ethyl acetate (6:4) and iodine vapor as the detecting agent for all the fractions. The eluates chloroform:ethyl acetate (25:75), ethyl acetate (100), ethyl acetate:n-butanol (50:50), and n-butanol (100) gave positive response for flavonoids producing yellowish color in the Shinoda test. The fractions 1-9, 13-16, 30-31, and 35-35 did not exhibit any spots. The fractions 10-12 and 17-20 exhibited spots but flavonoids were absent. However, in between the 21-24, 25-29, 32-34, and 36-39 the fractions exhibited flavonoidal spots. The fractions with similar spots and positive test for flavonoids were pooled together and concentrated. The concentrated fractions after evaporation revealed yellowish-brownish crystalline powder.

Effects of FFALF against cyclophosphamide induced chromosomal aberration

In chromosomal aberration study, cyclophosphamide (50 mg/kg/i.p.) administration showed a significant (P < 0.01) increase in the incidence of chromosomal aberrations in the form of breaks, fragments, gap, ring, and association as compared to the untreated group [Table 1]. But the pretreatment of FFALF with cyclophosphamide showed statistical significance (P < 0.01) and dose-dependently decreased the incidence of chromosomal aberrations [Figure 1]. Also, treatment of FFALF alone showed decrease in the percentage of aberrant cells (P > 0.05) as compared to the untreated group. The dose-dependent protection in chromosomal aberrations of FFALF with the cyclophosphamide treated groups showed a better genoprotective effect [Figure 2].
Figure 1: Effect of FFALF on chromosomal aberrations in bone marrow cells of mouse. (a) CP (50 mg/kg/i.p.) and (b) FFALF alone (5 mg/kg/PO)

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Figure 2: Effect of FFALF on percentage of chromosomal protection in bone marrow cells of mouse **P < 0.01 as compared with cyclophosphamide control by one-way ANOVA, followed by the Dunnett's test. CP- Cyclophosphamide

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Table 1: Effect of FFALF on chromosomal aberration in mouse bone marrow cells

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Effects of FFALF against cyclophosphamide induced micronuclei formation

The results of FFALF showed modulatory effects on cyclophosphamide-induced bone marrow clastogenicity [Table 2]. In positive control, cyclophosphamide showed significant (P < 0.01) increase in micronuclei formation and reduction of positive/negative (P/N) ratio as compared to the untreated control. But all the doses of FFALF with the posttreatment of cyclophosphamide showed dose-dependently and significantly decreased the nu of micronuclei formation (P < 0.01) and significantly increased in P/N ratio (P < 0.05). Further, treatment of FFALF alone did not show any micronuclei formation [Figure 3].
Figure 3: Effect of FFALF on micronucleus formation in bone marrow cells of mouse. (a) CP (50 mg/kg/i.p.) and (b) FFALF alone (5 mg/kg/PO)

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Table 2: Effect of FFALF on micronucleus formation in mouse bone marrow cells

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Identification of isolated flavonoids from FFALF

Two compounds were isolated from the flavonoidal fraction of A. leptophyllum and purified. The structural elucidation of each compound was carried out by MS, FTIR, 1 H NMR, and 13 C NMR spectroscopy and the structure of these compounds were analyzed [Figure 4].
Figure 4: Chemical structure of isolated flavonoids from FFALF

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Spectral analysis of sample-I

Molecular formula: C 15 H 10 O 5 ; melting point (m.p.) 347-350°C; FTIR (KBr) cm -1 : 3285 (OH str of phenols), 3093, 3016, 2924 (Ar-Hydrogen), 1653 (C = O str), 1607, 1557, 1501 (C = C str), 1244 (C-O-C), 829, 806 (Ar-CH def); fast atom bombardment (FAB)-MS: 270 [M] + , EI-MS: 269 [M + -1]; 1 H NMR (400 MHz, DMSO-d6 ) d: 12.9670 (1H, s), 10.8237 (1H, s), 10.3596 (1H, s), 7.8726-7.9016 (2H, d), 6.8972-6.9332 (2H, d), 6.4605-6.4656 (1H, d), 6.1845 (1H, d); 13 C NMR (100 MHz, DMSO-d6 ) d: 181.67 (C-4), 164.05 (C-2), 163.65 (C-7), 161.41 (C-9), 161.01 (C-4'), 157.24 (C-5), 128.36 (C-6', C-2'), 121.14 (C-1'), 115.89 (C-3', C-5'), 103.66 (C-10), 102.75 (C-3), 98.78 (C-6), 93.90 (C-8).

Spectral analysis of sample- II

Molecular formula: C 15 H 10 O 7 ; m.p. 300-305˚C; FTIR (KBr) cm -1 : 3408, 3322 (OH str of phenols), 2854, 2714 (Ar-CH str), 1665 (C=O of aryl ketonic str), 1611, 1561, 1522 (C=C str of aromatic ring), 1262 (C-O str of aryl ether), 824, 796 (-CH def of aromatic hydrocarbon); MS: 302 [M] + , 301 [M+-1]; 1 H NMR (400 MHz, DMSO-d6 ) d, ppm: 12.2990 (1H, s), 9.3506-9.5853 (3H, m), 7.3649-7.5188 (3H, d), 6.7306 (1H, s), 6.0106-6.2359 (2H, d); 13 C NMR (100 MHz, DMSO-d6 ) d:175.76 (C-4) , 163.54 (C-7), 160.66 (C-5), 156.10 (C-9), 147.61 (C-2), 146.71 (C-4'), 144.98 (C-3'), 135.68 (C-3), 121.97 (C-1'), 119.99 (C-6'), 115.57 (C-5'), 115.04 (C-2'), 102.97 (C-10), 98.17 (C-6), 93.14 (C-8).


   Discussion Top


Genotoxicants constitute the leading cause of death in the human population by acting as carcinogen and mutagen with strong interference of somatic cells and germline cells. So, investigation on the genotoxicants is of growing interest in fresh vegetables or herbal drugs in the present era, because these may affect our future generations. The chemicals that interfere with DNA repair or mutagen metabolism can be accepted as effective antimutagens. [17] Chromosomal aberration and micronucleus assays are considered as highly reliable methods to investigate the cytogenetic potential of any herbal treatment. [18],[19] Micronucleus assay is a well characterized biomarker of structural and numerical chromosomal damages, which arise from the acentric chromosome fragments or the lagging whole chromosome(s) that fail to incorporate into the daughter nuclei after nuclear division. Chromosomal aberration involves the blockage of DNA synthesis or spindle formation during cell division. [20],[21] These spindle formation is mediated by polymerization or depolymerization of the microtubules and by premature anaphase onset in cell division. Chromosomal aberrations include chromatid break, ring shaped chromosome, exchange, deletion, minute or multiple breaks, fragmentation, pulverisation, etc. Again, micronucleus formation is mainly due to the incorporation of the daughter nuclei in the mother cell during cell division, or lack of centromere or centomeric damage and defect in the cytokinesis, etc. [22]

The doses of FFALF with cyclophosphamide treatment showed significant reduction in chromosomal aberration as compared to positive control and the percentage of chromosomal protection followed in a dose-dependent manner against cyclophosphamide [Figure 1] and [Figure 2]. In a similar study, all the doses of FFALF-treated groups with posttreatment of cyclophosphamide reduced the frequency of micronucleus formation in a dose-dependent manner as compared to the positive control group. While administration of FFALF alone (5 mg/kg) showed reduction of micronuclei formation as compared to the untreated control, it suggested that FFALF is genoprotective in mice [Figure 3]. At the same time, the ratio of PCE and NCE in FFALF-treated groups was not statistically different from the positive control group, showing that FFALF did not induce erythropoietic cell toxicity.

Flavonoids have a great role in mutagen inactivation. [13],[23] Furthermore, various scientific studies conclude that the genotoxicity or chromosomal instability depends on oxidative stress or radical mechanisms. [24],[25] These oxidative stresses generated by reactive oxygen or nitrogen molecules, which affect cell physiology by damaging cell membrane and DNA, resulted in cancer or other degenerative diseases. [26] So, there is a constant requirement for maintaining equilibrium between these oxidative species and the antioxidant defense system of the body; otherwise, it may lead to various pathological disturbances like arthritis, asthma, carcinogenesis, or mutagenesis. A. leptophyllum has been previously reported to have free radical scavenging property, which might be responsible for genoprotective action against cyclophosphamide induction in mice. [10]

Identification of the flavonoids was done from the molecular ion peak and the fragmentation pattern of mass spectra. It was supported and confirmed by the bands observed in the IR spectra and the signals recorded in 1 H and 13 C NMR spectra [Figure 4]. Two bioactive flavonoids have been successfully isolated from FFALF in the present study. On the basis of spectral data, the compounds were identified as apigenin and quercetin. Apigenin comes under the flavones class of flavonoid while quercetin comes under the flavonol class. It is well known that both the flavonoids have antioxidant and anticarcinogenic activities and can reduce high blood pressure. [27] So, the fruit of this plant can be added to food as a kind of functional ingredient, which has many beneficial effects on human health.


   Conclusion Top


In conclusion, FFALF treatment alone of mice or pretreatment with cyclophosphamide reduced the cytogenetic damage by inhibiting the frequency of micronuclei formation and chromosomal aberration. So our data suggest that FFALF possesses significant antimutagenic activity, which may be due to the antioxidative potential and the presence of flavonoids such as apigenin and quercetin. Due to both antioxidative and antimutagenic roles, FFALF may be used as a weapon for treatment of various types of carcinogenesis or other chronic inflammatory diseases.


   Acknowledgements Top


I express my sincere thanks to the Principal of Vedica College of Pharmacy, Bhopal, Madhya Pradesh, India and the Dean of NIMS Institute of Pharmacy, NIMS University, Jaipur, Rajasthan, India for guiding and permitting the lab facility for my research work.

 
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    Figures

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