J Biomed. In Press(In Press):e6623.

Published online 2016 June 15. Research Article Cytotoxicity of the Methanol Extract of innoxia Petals on MCF-7 and HEK-293 Cell Lines

Hamid Cheshomi,1 Leila Sadat Aldaghi,1 and Hasan Rezaei Seresht2,*

1Cellular and Molecular Research Center, Sabzevar University of Medical Sciences, Sabzevar, IR Iran 2Traditional and Complementary Medicine Research Center, Sabzevar University of Medical Sciences, Sabzevar, IR Iran

*Corresponding author: Hasan Rezaei Seresht, Traditional and Complementary Medicine Research Center, Sabzevar University of Medical Sciences, Sabzevar, IR Iran. Tel: +98-5144445648, Fax: +98-5144445648, E-mail: [email protected]

Received 2016 April 30; Revised 2016 June 01; Accepted 2016 June 11.

Abstract

Background: Medicinal are traditionally used to prevent and treat various diseases, including cancer. Despite the devel- opment of advanced methods of treatment, cancer mortality is still increasing every year. Medicinal plants are one of the most important sources of anticancer agents. Objectives: The present study was conducted to evaluate the cytotoxic effects of the methanolic extracts of Datura innoxia petals, native to central and and distributed in , , , and , on the breast cancer cell line (MCF-7). Materials and Methods: The petals were collected, cleaned, and powdered. The extraction was conducted using them aceration method and then filtered, centrifuged, freeze-dried, and kept at 4°C. The MCF-7 and HEK-293 cell lines wereProof treated with the methano- lic extract of D. innoxia petals at various dilutions. Cell viability was quantitated using the MTT assay after 48 and 72 hours. The methanolic extract of D. innoxia petals showed cytotoxic effects on the MCF-7 cell lines at 48 and 72 hours of incubation but did not affect the non-malignant cell line HEK-293. Conclusions: These findings indicate that the methanolic extracts of D. innoxia petals can act as a possible food supplement with anticancer effects and can be used after complementary tests.

Keywords: Cytotoxicity, MCF-7, HEK-293, Methanolic Extract, Datura innoxia

1. Background soms at night; however, D.metelis known as a wild vari- ety. Its chopped dried roots are used to reduce fever. The poultice from which it is made is also used for the treat- Cancer is the second leading cause of death in the ment of inflammation and bruising (1). Furthermore, the world. Although great advances have been made in in China known as Yangjinhuais known and used the treatment and control of this disease, significant for the treatment of asthma, epilepsy, pain, and rheuma- shortcomings remain to prevent further development. tism (2), while D. stramoniumis mainly used due to its toxic Some unwanted side effects are often observed during effects. Toxication with datura is very common in India. chemotherapy. Hence, natural treatments, such as the use Unintentional poisoning is observed suddenly or when an of plant-derived products, may reduce these harmful side extraction is completed (3). Datura poisoning symptoms, effects. At present, there are few herbal products, which therefore, include delirium, excitement, seizures, dilated have shown anticancer effects in vitro, but are still being pupils, dry mouth, nausea and vomiting, blood pressure, evaluated in humans. Therefore, it is essential that future and coma (4-6). inspecies such as D. stramonium- studies indicate the beneficial effects of these plants for the belongs to the tropane groups (7-9). D. innoxia al- treatment of cancer in humans. kaloids show a similar pattern (10). Datura innoxia is a type of wild grass that belongs to the family. Its name is derived from the word Dhutra meaning divine; therefore, it is named af- 2. Objectives ter its healing properties. Many varieties of Datura are known and are widely used for their medicinal properties and toxicity that is associated with the presence of more The main purpose of this study was to investigate the than 30 different alkaloids. The species such as D. innoxia, cytotoxic effects of plant extracts on the MCF-7 breast can- D.stramoniumUncorrected, and D.wrightiiare cultivated as ornamental cer cell line and the non-malignant cell line, HEK-293, in- plants due to the funnel-shaped forms and fragrant blos- vitro.

Copyright © 2016, Sabzevar University of Medical Sciences. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/) which permits copy and redistribute the material just in noncommercial usages, provided the original work is properly cited. Cheshomi H et al.

3. Materials and Methods MCF-7 and non-malignant cell line HEK-293 was evaluated by MTT assay. Initially, to study the cytotoxic effects of the 3.1. Plant Materials extract on the cell lines, extracts at concentrations between D. innoxia petals were collected from Sabzevar city in 5 µg/mL and 100 µg/mL of this material were prepared at September 2014. In addition, the breast cancer cell line 48 and 72 hours after treatment, where the viability per- MCF-7 and non-malignant cell line HEK-293 were used in centage of the cells was evaluated by the MTT assay. this study and were purchased from the cell bank of the The cytotoxic effects of different extract concentra- Pasteur institute of Iran. tions for each time range was performed for the cell line and then used to determine the most effective concentra- 3.2. Extraction tions and times for these effects. Following this, curves for the extract concentration and cell viability percentage Following the drying of the petals, samples were pow- were drawn, and finally, the IC concentration was calcu- dered in the absence of light. The extraction was com- 50 lated. pleted using the maceration method. For this purpose, the petals were dried (4g) and then incubated for 12 days in the presence of methanol (50 × 2 mL) at room temperature. 4. Results The materials were then filtered and the clear su- pernatant was then concentrated under a reduced pres- 4.1. TPC sure with a vacuum rotary evaporator at 40°C. Finally, The TPC of the methanolic extract of D. innoxia aerial the residue (1.1 g) was used in the following experiments, parts was determined by the Folin-Ciocalteau assay. There- whereby the extract was kept at 4°C. Proof fore, the calibration curve of gallic acid was drawn (Figure 1). The amount of phenol in the methanolic extract was 3.3. Determination of Total Phenolic Content (TPC) 105.684 ± 0.513 mg GAE/g extract. For determining TPC of the extract, Folin-Ciocalteu’s reagent method was applied. In brief, a 0.5 mL of FCR so- 0/8 y = 0/008x + 0/017 lution (10% in distilled water) was added to a test tube con- 0/7 R2 = 0/998 0/6 taining 0.5mL of extract (800µg/mL in methanol) and 1.5 Conc., µg/mL 0/5 Linear (Conc., µg/mL) mL of distilled water. This mixture was thoroughly shaken. 0/4 0/3 After 5 minutes, 2 mL of 10% sodium carbonate solution 0/2 was added and the mixture was shaken for a second time. Absorbance 0/1 0 The mixture was then incubated in the dark for 2 hours at 0 20 40 60 80 100 room temperature. The absorbance was measured at 760 Concentration, µg/mL nm with a UV-VIS spectrophotometer. The analyses were Figure 1. Standard Graph of Galic Acid for TPC Determination conducted in triplicate. Moreover, a gallic acid standard curve was completed (5 - 100µg/mL). TPC was determined in µg of gallic acid equivalents (GAE)/g (11, 12). 4.2. Cytotoxic Effect 3.4. MTT Assay Figures 2,3 show the results of the MTT assay by mea- One of the methods used to evaluate the cytotoxic ef- suring the optical density (OD) based on concentrations fects of different materials was the 3-(4,5-dimethylthiazol- compared with the rate of cellular viability. The percentage 2-yl)-2,5-diphenyltetrazolium bromide (MTT) diagnostic of living cells that had received the extract were calculated test. The basis of this method regards specific metabolic using the following formula (14). reactions in the mitochondria of living cells. On mitochon- Equation 1. drial metabolism, MTT in the mitochondria changed into density Cell Viability, % = Sample optical solid crystals and formed a purple formazan. After the dis- Control optical density solution, different values of crystals in solvents, such as × 100 DMSO, formed varying degrees of a purple color. Measur- (1) ing the absorbance of the solution at a wave length of 545 nm it can be attributed to the optical density of each well The illustrations showed the effects of the different ex- to a number of metabolizing MTT viable cells (13). tract concentrations on cells after 48 and 72 hours, respec- UncorrectedIn this study, the cytotoxic effects of the methanol ex- tively. These findings suggest that the extract will have lit- tract of the petals of D. innoxia on the breast cancer cell line tle effect on non-malignant cells after 48 hours; however, at

2 J Biomed. In Press(In Press):e6623. Cheshomi H et al.

there was significant change in IC50 values. However, with 150 MCF 7 this time change, the toxicity effect on normal cells greatly HEK 293 increased and was derived from the toxicity of the plant. The D. stramonium plant has anticancer effects on nasopha- ryngeal carcinoma at the treatment doses (15). 100 A study was performed on the plant extracts of differ- ent organs and it became clear that D. innoxia contains

Vability, % Vability, compounds such as alkaloids, saponins, flavonoids, essen- 50 tial oils, and phenolic compounds (16). In a study com- pleted on the and stems of the D. metelextracts, it was confirmed that there was anticancer activity against the

0 0 50 100 150 cancer cell line MCF-7. The extract also had more anti- Concentration, ug/mL cancer ability and contained plant compounds such as al-

kaloids, sterols, saponins, phenols, tannins, and flavonoids (17).In another study, the phytochemical screening of D. in- Figure 2. MTT Assay of the Methanolic Extract of Datura innoxia After 48 Hours on MCF-7 Compared to HEK-293. noxiapetals revealed the presence of alkaloids, saponins, flavonoids, coumarins, and tannins. In addition, the re- sults showed that the methanolic extracts of petals of the

60 MCF 7 D. innoxia plant have the potential to bean antioxidant (18). HEK 293 As aforementioned, D. innoxia, containingProof a combination of several compounds, is reported to have anticancer ef- fects for these compounds. Therefore, it can be concluded 40 that the anticancer effects observed in the extract are asso- ciated with the presence of these compounds.

Vability, % Vability, Furthermore, the steroids in this genus have anti- 20 cancer activity against the colon cancer cell line, HCT-116 (19, 20). Many species of the solanaceae family are rich in calystegin, which has glycosidic inhibitory properties 0 against cancer (4). In another study, the anticancer ef- 0 50 100 150 Concentration, ug/mL fects of methanol extracts on the leaves of the D.innoxia in vitro through the induction of apoptosis in cancer cell Figure 3. MTT Assay of the Methanolic Extract of Datura innoxia After 72 Hours on lines, such as Hep-2 and adenocarcinoma of the colon MCF-7 Compared to HEK-293 (HCT-15) was studied. The anticancer effects after 48 hours of incubation, regarding HCT-15 and Hep-2 cell lines with methanolic extract leaves of D. innoxia, have been found concentrations of 50 - 100 µg/mL the extract exhibits a high (21). In another study, which was conducted on the seeds inhibitory effect on tumor cells. Comparing these illus- of the D. innoxia, the anticancer effects of this plant on the trations, 50% of the cell growth inhibitory concentration Hela cell line was established. (IC50) for breast cancer cells was equivalent to28 µg/mL and < 5 µg/mL for 48 and 72 hours, respectively. 5.1. Conclusion According to the results of this study and also in com- 5. Discussion parison with similar studies, it can be concluded that the plant extract in question has anticancer effects for breast The results of this study showed that the D. innoxia cancer treatment. However, further research is warranted extract has cytotoxic effects on the breast cancer cell line to find effective compounds. MCF-7. The methanolic extract of this plant has a severe impact after 72 hours of cell treatment compared to that References at 48 hours of incubation. However, the results indicate that the extract at 48 hours has less cytotoxic effects on nor- 1. DeWolf GP. Notes on cultivated Solanaceae. 2. Datura. Baileya. mal cells. The effect on the cells was dose-dependent and 1956;4(1):13–23. 2. Pan Y, Wang X, Hu X. Cytotoxic withanolides from the flowers of therefore,Uncorrected significantly increased with a rising concentra- . J Nat Prod. 2007;70 (7):1127–32. doi: 10.1021/np070096b. tion. With an increasing time from 48 hours to 72 hours, [PubMed: 17583953].

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