Abstract
Context: Cancer chemopreventive action of walnut [Juglans regia L. (Juglandaceae)] has been explored.
Objective: This study evaluated antiproliferative and antioxidant activities of walnut.
Materials and methods: Various fractions of walnut extract have been screened for antiproliferative activity against human cancer cell lines using the MTT assay. All these fractions have also been evaluated for total phenolic content, antioxidant activity, and reducing power capacity.
Results and discussion: Chloroform and ethyl acetate fractions exhibited a high level of antiproliferation against HepG-2, liver cancer cell line (IC50 = 9 and 15 µg/mL, respectively).
Conclusion: Exhibiting high phenolic content, antioxidant activity, and potent antiproliferative activity, walnut may act as a cancer chemopreventive agent.
Introduction
Walnut tree [Juglans regia L. (Juglandaceae)] is traditionally an important and widely distributed deciduous tree. It is cultivated in various temperate regions of Asia, Europe, South Africa, United States, and so on. In India, it exists in lower temperate to temperate zone. The plant possesses antibacterial (CitationPereira et al., 2007) and antioxidant properties (CitationLabuckas et al., 2008). Leaves are astringent, anthelmintic and are useful in herpes, eczema, scrofula, and syphilis (CitationWarrier et al., 1995). Its fruits are thermogenic, aphrodisiac, carminative and are useful in rheumatism (CitationWarrier et al., 1995). Expressed oil of fruit is useful against tapeworm (CitationWarrier et al., 1995). Walnut is a very nutritious fruit, rich in unsaturated fatty acids and phenolic content. Its slight astringent flavor has been associated with the presence of phenolic compounds (CitationColaric et al., 2005). Epidemiological evidence shows that foods rich in phenolic content are associated with a lower risk of cancer and coronary heart disease, as well as cataract, brain, immune dysfunction, and stroke (CitationLattanzio, 2003).
The phenolic content-rich fruit is considered to be cancer protective but the antiproliferative activity of its extracts has not been reported so far. In the present study, we investigated antiproliferative activity of various extracts of raw walnut fruit and its total phenolic content, reducing power, and antioxidant properties to validate the cancer preventive action.
Materials and methods
Plant material
The fresh mature raw fruits were collected in June 2007, from Nishani village (altitude = 1800 m), Pauri Garhwal district, Uttarakhand, India. It is situated in the Shivalik region of Himalaya. The voucher specimen (Voucher no. 12471) was authenticated by Dr. S.C. Singh and preserved in the Botany and Pharmacognosy Division, CIMAP, Lucknow.
Extract
The fresh fruits were crushed and extracted with methanol (200 g, 600 mL, 34 h) and 20% aqueous methanol (200 g, 600 mL, 34 h) separately to get methanol (A) and aqueous methanol extracts (B) at room temperature (24–36°C). Solvents were filtered and evaporated through rotavapor to get crude residues A (3.92 g) and B (4.86 g). The methanol extract (B, 3.74 g) was further partitioned into hexane (0.62 g), chloroform (0.53 g), ethyl acetate (0.67 g), methanol-soluble fraction (1.26 g), and methanol-insoluble fraction (0.37 g).
Quantitative analysis by reverse phase HPLC
All the extracts and fractions were analyzed by Waters analytical HPLC system using photodiode array detector (PDA) for gallic acid and ellagic acid contents. The system was controlled with the Waters Empower software. Waters HPLC column SS (Symmetry, 4.6 × 150 mm, C18, 5 µm) was used. The mobile phase for the determination of gallic acid was methanol:water (1% acetic acid) = 20:80 at the flow rate of 0.5 mL/min. The data acquisition was done at 272 nm. The retention time for gallic acid was 5.4 min. While the mobile phase for ellagic acid determination was methanol:water (1% acetic acid) = 50:50 at the flow rate of 1 mL/min. The data were recorded at 254 nm. The retention time for ellagic acid was 5.2 min.
Evaluation of biological activity
Cell lines and culture media
Human cancer cell lines, such as MCF-7 (estrogen receptor positive breast adenocarcinoma), KB (oral and mouth), HepG-2 (liver), Caco2 (colon), and WRL-68 (liver) (all American Type of Culture Collection, ATCC) were obtained from NCCS, Pune, India. Cells were cultured in DMEM with 25 mM HEPES, 0.22% NaHCO3, and 10% fetal bovine serum (FBS) (CitationWoerdenbag et al., 1993).
Anticancer activity using MTT assay
In vitro cytotoxicity testing was performed as per reported method (CitationSaxena et al., 2007). Two thousand cells per well were incubated in the 5% CO2 incubator for 24 h to enable them to adhere properly to the 96-well polystyrene microplate (Greiner, Germany). Test compound dissolved in dimethyl sulfoxide (DMSO; Merck, Germany), in at least five concentrations, was added into the wells and left for 4 h. After the incubation, the compound and media were replaced with fresh media and the cells were incubated for another 48 h in the CO2 incubator at 37°C. The concentration of DMSO was always kept below 1.25%, which was found to be nontoxic to cells. Then, 10 µL MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] was added to each well and plates were incubated at 37°C for 4 h. One hundred microliters of DMSO were added to all wells and mixed thoroughly to dissolve the dark blue crystals. The plates were read on SpectraMax 190 Microplate reader (Molecular Devices Inc., Sunnyvale, CA) at 570 nm within 1 h of DMSO addition.
Paclitaxel (Taxol), podophyllotoxin, and tamoxifen were used as standard cytotoxic molecules. IC50 values below 20 µg/mL were considered as potent activity, at 20–200 µg/mL as good activity, at 200–500 µg/mL as poorly active, while IC50 values higher than 500 µg/mL were considered as inactive. Four different concentrations of 1, 10, 100, and 500 µg/mL were used against WRL-68, HepG-2, KB, Caco2 cell lines, whereas the concentrations against MCF-7 were used as 1, 10, 100, 500, and 1000 µg/mL.
Total phenolic content, antioxidant activity, and reducing power capacity assays
Total phenolic content in plant extracts was determined by a reported method (CitationSingleton & Rossi, 1965) using Folin-Ciocalteu reagent. Total phenolic content was determined in terms of gallic acid equivalence. Total antioxidant capacity of plant extract was measured using the standard method (CitationPrieto et al., 1999) with l-ascorbic acid as standard. Total antioxidant capacity of the plant extract was estimated in terms of ascorbic acid equivalence. The reducing power capacity of plants extracts was estimated by using the standard method (CitationYen & Chen, 1995).
Data analysis
The experiment was done in triplicate and the inhibitory concentration (IC) values were calculated from dose–response curve. IC50 is the concentration in µg/mL required for 50% inhibition of cell growth as compared with that of untreated control. IC50 was calculated based on dose–response curves of at least five concentrations repeated three times ().
Figure 1. Dose-dependent graphs of antiproliferative activity of various fractions of Juglans regia extract (AKH-1 to AKH-7) against various human cancer cell lines.
Results and discussion
The phenolic compounds from walnut fruits have a positive influence on human health such as a decrease of coronary heart disease, anti-inflammatory and antimutagenic activities (CitationAnderson et al., 2001). Walnut extracts containing ellagic acid monomers, polymeric tannins, and other phenolic contents effectively inhibited human plasma and low-density lipoprotein (LDL) oxidation in vitro (CitationAnderson et al., 2001). Gallic acid, ellagic acid, chlorogenic acid, caffeic acid, vanillic acid, ferulic acid, protocatechic acid, genistein, synapic acid, salicylic acid, catechin, epicatechin, and juglone are the reported walnut phenolic contents (CitationRastogi & Mehrotra, 1998).
Chloroform and ethyl acetate fractions possessed very good antiproliferative activity against HepG-2, KB, WRL, and Caco2 cancer cell lines. Aqueous methanol extracts also showed good activity against HepG-2 and WRL-68 cell lines (). Chloroform fraction that possessed better antiproliferative activity also showed high antioxidant activity.
Table 1. Antiproliferative activity of extracts of Juglans regia fruits against various human cancer cell lines by MTT assay.a
In our study, the extracts and fractions of walnut showed very good antioxidant activity and reducing power capacity, which may be due to high content of phenolic compounds (). HPLC analysis showed that methanol extract had the highest contents of both gallic acid (1.96%) and ellagic acid (0.32%). In all these extracts and fractions, concentration of gallic acid was much higher than that of the ellagic acid. Both hexane and chloroform fractions were devoid of gallic and ellagic acids. Nevertheless, both the fractions possessed high antioxidant activity, which might be due to its oil content that has been reported to possess antioxidant activity. The total phenolic content was found highest in methanol extract and aqueous methanol extracts. But the antiproliferative activity was not very much significant. Earlier, it was reported (CitationAlamprese & Pompei, 2005) that the total phenolic content in liquor decreased with ripening of the walnut fruits. The liquor that was made from more ripened fruits showed a lower antioxidant power. An alcoholic liquor of walnuts with green husk just before the hardening of endocarp is considered a good health drink (CitationAlamprese & Pompei, 2005; Stampar et al., 2006).
Figure 2. Total phenolic content, antioxidant activity, and reducing power capacity of Juglans regia fruits extracts/fractions (at 200 µg) (results present the average and standard deviation of three independent measurements).
Conclusion
In conclusion, chloroform and ethyl acetate fractions of walnut possessed potent antiproliferative activity against various human cancer cell lines. Further, bioactivity-guided isolation is required to reach the bioactive molecule. The extracts and fractions also showed high antioxidant activity and reducing power capacity. Hence, it may be considered as cancer chemopreventive too.
Acknowledgements
The financial support from Council of Scientific and Industrial Research (CSIR), India is duly acknowledged. S.L. is recipient of DST-FAST Track Young Scientist scheme.
Declaration of interest
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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