IN VITRO CYTOTOXICITY OF AG (SILVER) NANOPARTICLES SYNTHESIZED FROM (AEGLE MARMELOS(L)

1,*Mahanthesh Kumar

1,2,3Department of Biotechnology, Sahyadri Science College, Shivamogga

Received 25th March 2020

ABSTRACT

The Ag nanoparticles synthesized from the plant sources are ecopresent work was to evaluate In vitro cytotoxic activity of Ag nanoparticles green synthesized from Aegle marmelos exMCF-7 Cell Lines (breast cancer). The aqueous extract is prepared by cold extraction (maceration) using ethanol as a solvent. Phytochemical analysis was done by using the standard procedures. Aqueous extract of Aegle marmelos was used for synnanoparticles. The nanoparticles were characterized by UVTransform Infra-red Spectroscopy (FTIR) techniques. In vitro cytotoxicity studies of Ag nanoparticles were done by MTT asMCF-7 cell lines. The preliminary phytochemical results revealed that the aqueous extract of A. marmelos contains broad spectrum secondary metabolites like Tannins, Saponins, Glycosides, Flavonoids, Anthraquinones, Terpenoids and Steroids. Thespectrophotometric analysis of Ag nanoparticles displayed maximum absorption at 300 nm and scanning AFM and FTIR studies showed that the nanoparticles size ranges from 50potent cytotoxicity against MCF-7 cell lines with IC50 value of 45.82 µg/ml. Thus, the present study concludes that Ag nanoparticles can be used as a potent drug in alternative therapy for treating the breast cancer patients.

Keywords: Aegle marmelos, Ag nanoparticles, MTT assay, Breast cancer, Cytotoxicity.

INTRODUCTION Nan particles are minute particles with ranges from in 1large, whose size is comparable to the Biological molecules with its new and enhanced size-dependent properties compared to its bulk material, they penetrate more than larger substances and ideally interact with cellular structures .These nanoparticlesbe synthesized by chemical processes like pyrolysis, hydrothermal method, chemical precipitation chemical processes cause pollution and are costly practices. Synthesis of metal oxide nanoparticles can be done using biological materials such as microbes and plants. Synthesis of nano particles using microorganism involves lengthy process maintaining microbial cultures, intracellular synthesis andmultiple purification steps. However, using “green” methods in the synthesis of Silver nanoparticles has increasingly become a topic of interest. Nan particles in recent days have revolutionizethe modern medicinal practices because of their potential activities in treatment of various diseases. Plants have been used from centuries to treat various human diseases. Herbal drugs as compared to synthetic drugs have no or lesser are less expensive. Since plant mediated synthesis is easy and safe with one-step protocol and don’t involve the use of harsh solvents or surfactant as the reducing agents, studies have

*Corresponding author: Mahanthesh Kumar.G.T,

Department of Biotechnology, Sahyadri Science College, Shivamogga

International Journal of Current Science and EngineeringVol. 02, Issue, 0

Available online at http://www.journalijcse

R

IN VITRO CYTOTOXICITY OF AG (SILVER) NANOPARTICLES SYNTHESIZED FROM (AEGLE MARMELOS(L) CORREA) AGAINST MCF-7 CELL LINES.

Mahanthesh Kumar.G.T, 2C.K.Ramesh, 3K.Pradeepa

of Biotechnology, Sahyadri Science College, Shivamogga-577203

2020; Accepted 20th April 2020; Published Online 30th May 2020

The Ag nanoparticles synthesized from the plant sources are eco-friendly and are potent anticancer agents. The objective of the present work was to evaluate In vitro cytotoxic activity of Ag nanoparticles green synthesized from Aegle marmelos ex

7 Cell Lines (breast cancer). The aqueous extract is prepared by cold extraction (maceration) using ethanol as a solvent. Phytochemical analysis was done by using the standard procedures. Aqueous extract of Aegle marmelos was used for synnanoparticles. The nanoparticles were characterized by UV-Visible spectrometry, Atomic Force Microscopy (AFM) and Fourier

red Spectroscopy (FTIR) techniques. In vitro cytotoxicity studies of Ag nanoparticles were done by MTT as7 cell lines. The preliminary phytochemical results revealed that the aqueous extract of A. marmelos contains broad spectrum

secondary metabolites like Tannins, Saponins, Glycosides, Flavonoids, Anthraquinones, Terpenoids and Steroids. Thespectrophotometric analysis of Ag nanoparticles displayed maximum absorption at 300 nm and scanning AFM and FTIR studies showed that the nanoparticles size ranges from 50-500 nm. The MTT assay results revealed that the of Ag nanoparticles exhibits

7 cell lines with IC50 value of 45.82 µg/ml. Thus, the present study concludes that Ag nanoparticles can be used as a potent drug in alternative therapy for treating the breast cancer patients.

nanoparticles, MTT assay, Breast cancer, Cytotoxicity.

Nan particles are minute particles with ranges from in 1-100 nm Biological molecules with properties compared to its

they penetrate more than larger substances and ideally interact with cellular structures .These nanoparticles can be synthesized by chemical processes like pyrolysis, hydrothermal method, chemical precipitation etc. But these chemical processes cause pollution and are costly practices.

nanoparticles can be done using biological materials such as microbes and plants. Synthesis of

using microorganism involves lengthy process of maintaining microbial cultures, intracellular synthesis and multiple purification steps. However, using “green” methods in

increasingly become a topic of interest. Nan particles in recent days have revolutionized

practices because of their potential activities in treatment of various diseases. Plants have been used

centuries to treat various human diseases. Herbal drugs as side effects and

e less expensive. Since plant mediated synthesis is easy and and don’t involve the use of harsh

solvents or surfactant as the reducing agents, studies have

Biotechnology, Sahyadri Science College, Shivamogga-577203

suggested their use to be more ideal and compatible for their use in nano medicine because of their stability in variousmedia. The bimolecular present in plants mediates the synthesiof nano particles and also stabilize the nanowith desired size and shape as well as ions to the nano size, and in the capping of nanonumerous factors such as temperature, pH, concentration ofextracts, concentration of raw material, etc. influence the reduction process of metal ions into the metalCancer is a class of disease where abnormal cells proliferate uncontrollably, producing malignant tumors that invade surrounding healthy tissue. Breast cancer is the most common type of cancer and the second leading cause of death. This disease is the primary cause of mortality among women aged55 years, and is the second leading cause of cancerdeath. The incidence of breast cancer isrequiring complete tissue removal, chemotherapy, radiotherapy, and hormone therapy most of the time. Due to serious side effect so currently available cancer treatments chemotherapy andradiation therapy, death rates are high.plant derived nano particles can be used toin the near future with minimum side effects. The Plant based Ag nano particles were used because of their inbuilt ability to penetrate tissue and cells and interacThis property of Ag-nano particles can be further engineered by fictionalization with target proteins or chemicalrendering them benign to normal cells while retaining their cancer targeting and killing properties. St

International Journal of Current Science and Engineering , Issue, 05, pp.289-0295, May, 2020

le online at http://www.journalijcse.com

Research Article

IN VITRO CYTOTOXICITY OF AG (SILVER) NANOPARTICLES SYNTHESIZED FROM (AEGLE 7 CELL LINES.

577203

2020

friendly and are potent anticancer agents. The objective of the present work was to evaluate In vitro cytotoxic activity of Ag nanoparticles green synthesized from Aegle marmelos extract against

7 Cell Lines (breast cancer). The aqueous extract is prepared by cold extraction (maceration) using ethanol as a solvent. Phytochemical analysis was done by using the standard procedures. Aqueous extract of Aegle marmelos was used for synthesis of Ag

Visible spectrometry, Atomic Force Microscopy (AFM) and Fourier red Spectroscopy (FTIR) techniques. In vitro cytotoxicity studies of Ag nanoparticles were done by MTT assay using

7 cell lines. The preliminary phytochemical results revealed that the aqueous extract of A. marmelos contains broad spectrum of secondary metabolites like Tannins, Saponins, Glycosides, Flavonoids, Anthraquinones, Terpenoids and Steroids. The U.V spectrophotometric analysis of Ag nanoparticles displayed maximum absorption at 300 nm and scanning AFM and FTIR studies

500 nm. The MTT assay results revealed that the of Ag nanoparticles exhibits 7 cell lines with IC50 value of 45.82 µg/ml. Thus, the present study concludes that Ag nanoparticles

use to be more ideal and compatible for their use in nano medicine because of their stability in various biological

present in plants mediates the synthesis stabilize the nano particles formed

with desired size and shape as well as play a role in reducing the the nano size, and in the capping of nano particles

numerous factors such as temperature, pH, concentration of extracts, concentration of raw material, etc. influence the reduction process of metal ions into the metal nano particles. Cancer is a class of disease where abnormal cells proliferate

malignant tumors that invade thy tissue. Breast cancer is the most common and the second leading cause of death. This

disease is the primary cause of mortality among women aged 45–55 years, and is the second leading cause of cancer-induced

cancer is almost 1in8 women, requiring complete tissue removal, chemotherapy, radiotherapy,

most of the time. Due to serious side effect so currently available cancer treatments chemotherapy and radiation therapy, death rates are high. Studies have showed that

particles can be used to treat cancer patients in the near future with minimum side effects. The Plant based Ag

used because of their inbuilt ability to penetrate tissue and cells and interact with the cancerous cells.

particles can be further engineered by with target proteins or chemical groups and

rendering them benign to normal cells while retaining their properties. Studies demonstrate that

cytotoxic property of Ag nanoparticles depends on their size i.e. smaller nanoparticles exhibit greater toxicity. The surface charge nature of Silver nanoparticles is typically due to neutral hydroxyl groups attached to their surface. Also, at lower pH Ag-nanoparticles gain positive charge from the environment, which then interact with negatively charged phospholipids on the membrane of cancer cells, there by promoting cellular uptake, phagocytosis and cytotoxicity. Studies have showed that Ag nanoparticles exposure at particular concentration induces the production of various proinflammatory cytokines that elucidates Th1-mediated immune response which interexchange tumor cell killing through production of TNF-α (Tumor Necrosis Factor). A. marmelos generally known as Bael belongs to the family rutacea. It is spread throughout number of tropical and subtropical areas including India. Almost all the parts of A. marmelos are used as medicine traditionally for the treatment of various ailments such as chest infection, urethritis, strangury, hematuria, leprosy, ulcer, toothache, inflammation of bladder, piles, laxative, in chronic cystitis, gleet and gonorrhea, arthritis, seizures and in liver protection. There are limited numbers of reports available on the cytotoxic effect of Ag nanoparticles against breast cancer, so the present study aims at profiling the same. Materials and method: Collection of plant materials The leaves of Aegle marmelos were collected from the Lingaraj Nagar, Hubballi, Karnataka during June, 2018. The plant were identified by Dr.Joy Hoskeri, Assistant professor.Dept,of Biotechnology, Karnataka State Akka Mahadevi Women’s University, Vijaya Pura, Karnataka. Preparation of leaf extract The leaf materials of the Aegle marmelos were rinsed in running tap water and shade dried at room temperature for 6-7 days. The shade dried leaves were powdered and stored in a well closed vessel or a container for further extraction process. Preliminary photochemical analysis Photochemical analysis was performed using standard procedures by Harborne JB, 1998 Synthesis of silver nanoparticles by cold Maceration method The plant leaf materials were subjected to cold extraction with methanol (cold maceration method). Maceration was carried out in a two closed conical flask for 48 hours. In both case 5g powdered Aegle marmelos leaf sample and 80% methanol were used. This mixture was kept in dark for 7 days at room temperature with intermittent shaking. After incubation, the whole extracts were filtered through What man grade 41 filter paper, dried and stored for further analysis. An aliquot (5ml) of aqueous plant extract sample was added to 50ml of 1mM aqueous AgNO3. To drive nanoparticle formation the reaction mixtures were kept in dark. Colour change of the reaction mixtures were monitored to determine nanoparticle formation, which is indicated by a dark brown colour. Once colour intensities of the solutions reached maximum, the vessels were removed and stored in dark at room temperature to prevent

agglomeration of the nanoparticles. A 50ml aliquot of AgNO3 containing 5ml distilled water was processed as described above and used as a negative control. Characterization of Ag Nanoparticles The Ag nanoparticles synthesized were observed by change of white to brown color precipitate, the precipitate was dried and used for UV spectrophotometer analysis. Graph 1 shows the U.V spectrophotometer result with maximum absorption at 300nm. Atomic Force Microscopy (AFM) The Atomic Force Microscopy (AFM) analysis was used to determine a wide distribution of particles size and shapes, depending on the conditions of the irradiating laser. Fourier Transform Infra-red Spectroscopy (FTIR) The nanoparticles were distinguished using a Fourier Transform Infrared Spectrophotometer (FTIR Thermo-scientific iS5). 2mg of the sample was mixed with 100 mg potassium bromide (KBr). Then, condensed to prepare a salt disc approximately 3 mm in diameter and the disc were directly kept in the sample holder. FTIR spectra were verified in the absorption range between 400 and 4000 cm1 and it used for identifying the phytochemical compound present in the silver nanoparticles. Cell line and culture medium MCF-7(Breast cancer) cell lines were procured from National Centre for Cell Sciences (NCCS), Pune, India. Stock cells were cultured in DMEM supplemented with 10% inactivated Fetal Bovine Serum (FBS), Penicillin (100IU/ml), streptomycin (100µg/ml) and amphotericin B (5 µg/ml) in humidified atmosphere of 5% CO2 at 37°C until confluent. The cells were dissociated with DMSO solution (0.2%trypsin, 0.02% EDTA, 0.05% glucose in PBS). The stock cultures were grown in 25 cm2 culture flasks and all experiments were carried out in 96 microtiterplates (Tar sons India Pvt. Ltd., Kolkata, India). Preparation of test solution For Cytotoxicity studies, each weighed test drugs were separately dissolved in DMSO and volume was made up with DMEM supplemented with 2% inactivated FBS to obtain a stock solution of 1 mg/ml concentration and sterilized by filtration. Serial twofold dilutions were prepared from this for carrying out cytotoxic studies.

Determination of cell viability by MTT Assay

The monolayer cell culture was tyrosinazed and the cell count was adjusted to1.0 × 105 cells/ml using DMEM containing 10% FBS. To each well of the 96 well microtiter plate,0.1ml of the diluted cell suspension (approximately 10,000 cells) were added. After 24 h, when a partial monolayer was formed, the supernatant was flicked off, washed the monolayer once with medium and 100 µl of different test concentrations of test drugs were added on to the partial monolayer in microtiter plates. The plates were then incubated at 37 0C for 3 days in 5% CO2 atmosphere, and microscopic examination was carried out and

International Journal of Current Science and Engineering 290

observations were noted every 24 h interval. After 72 h, the drug solutions in the wells were discarded and 50 µl of MTT inwas added to each well. The plates were gently shaken and incubated for 3 h at 37 0C in 5% CO2 atmosphere. The supernatant was removed and 100 µl of propanol was added and the plates were gently shaken to solubilize the formed formazan. The absorbance was measured using a microplate reader at awavelength of 540 nm. the percentage growth inhibition was calculated using the following formula and concentration of test drug needed to inhibit cell growth by 50% (ICgenerated from the dose response curves for each cell line.

% Growth Inhibition = 100 –Mean OD of individual test groupMean OD of control group ×100

The Ag nanoparticles were characterized by using a UVspectrometer, the nanoparticle suspension wasdeionized water the sample was analyzed from 200 nm to 800 nm range in UV-Vis spectrophotometer. (UV-2450 Agilent) Result and discussion

Aegle marmelosis an important medicinal plant used traditionally to treat many human ailments. In thethe preliminary phytochemical analysis of Aegle marmelos revealed the presence of broad range of phytoconstituents like flavonoids, tannins, saponins, anthraquinones, terpenoids, glycosides and steroids (Table 1).

Table 1: Preliminary phytochemical analysis of aqueous extract of Aegle Marmelos

These phytoconstituents are used as potent drugs in treating diabetes, cancer, inflammation and as potential antioxidants. The plant phytochemicals with anti-oxidant or reducingusually responsible for reduction of metal compounds into their respective nanoparticles. The Ag nanoparticles were synthesized

SL NO. TEST INFERENCE

1 Reducing Sugar +

2 Tannin +

3 Alkaloid -

4 Saponin +

5 Flavonoids +

6 Terpenoids +

7 Glycosides +

8 Anthraquinones +

9 Steroid +

Table 2: MTTassayresultsshowingcellviability(%)based onconcentration

MCF-7 Cell line Vs

Sample Am

Blank Untreated

Reading 1 0.017 0.774

Reading 2 0.014 0.776

Mean OD 0.0155 0.775

Mean OD-Mean Blank 0.7595

Standard deviation 0.0014142

Standard error 0.001

% Standard error 0.1316656

% Viability 100

% Growth Inhibition = 100 – Mean OD of individual test group

Mean OD of control group

International Journal of Current Science and Engineering

noted every 24 h interval. After 72 h, the drug solutions in the wells were discarded and 50 µl of MTT in PBS was added to each well. The plates were gently shaken and

atmosphere. The supernatant was removed and 100 µl of propanol was added and

shaken to solubilize the formed formazan. The absorbance was measured using a microplate reader at a

rcentage growth inhibition was concentration of test

drug needed to inhibit cell growth by 50% (IC 50) values is response curves for each cell line.

Mean OD of individual test group

The Ag nanoparticles were characterized by using a UV-Vis spectrometer, the nanoparticle suspension was prepared in deionized water the sample was analyzed from 200 nm to 800

2450 Agilent)

Aegle marmelosis an important medicinal plant used traditionally to treat many human ailments. In the present study the preliminary phytochemical analysis of Aegle marmelos

phytoconstituents like flavonoids, tannins, saponins, anthraquinones, terpenoids,

Table 1: Preliminary phytochemical analysis of aqueous extract of

These phytoconstituents are used as potent drugs in treating inflammation and as potential antioxidants. The

oxidant or reducing properties are usually responsible for reduction of metal compounds into their

Ag nanoparticles were synthesized

using silver nitrate as precursor with the plant extracts. These Agnanoparticles when subjected to UVthe maximum absorption at 300 nm

Graph 1: Absorption maximum of Ag nanoparticles

Graph 2: Graph showing the concentration of sample against the cell Viability

TEM analysis revealed that the nanoparticles were spherical in shape and found as aggregates in the range of 50(Figure1and 2). When these Ag nanoparticles subjected to cytotoxicity assay exhibited potential cytotoxicity against MCFcelllines with IC 50 value of 42.82µg/ml (Graph 2). Thecytotoxicity was found to be in a dose dependent manner (Graph3). These Ag nano particles may selectively

INFERENCE

MTTassayresultsshowingcellviability(%)based onconcentration

Test concentrations (in µg/ml)

Cisplatin

15 µg/ml

10 20 40

0.144 0.754 0.733 0.701

0.161 0.761 0.77 0.759

0.1525 0.7575 0.7515 0.73

0.137 0.742 0.736 0.7145

0.012020815 0.00495 0.026163 0.041012

0.0085 0.0035 0.0185 0.029

1.11915734 0.460829 2.435813 3.818302

18.03818302 97.69585 96.90586 94.07505

Mean OD of individual test group

Mean OD of control group ×100

ational Journal of Current Science and Engineering

using silver nitrate as precursor with the plant extracts. These Ag nanoparticles when subjected to UV-Vis characterization showed

(Graph 1).

Graph 1: Absorption maximum of Ag nanoparticles

Graph 2: Graph showing the concentration of sample against

TEM analysis revealed that the nanoparticles were spherical in in the range of 50-500 nm

(Figure1and 2). When these Ag nanoparticles subjected to exhibited potential cytotoxicity against MCF-7

50 value of 42.82µg/ml (Graph 2). The cytotoxicity was found to be in a dose dependent manner (Graph3). These Ag nano particles may selectively kill the

Test concentrations (in µg/ml)

80 160

0.412 0.017

0.425 0.02

0.4185 0.0185

0.403 0.003

0.009192 0.002121

0.0065 0.0015

0.855826 0.197498

53.06122 0.394997

291

cancer cells by apoptosis induced by the production of reactive oxygen species (ROS) viap 53pathway.This ability of the Ag nanoparticles to inhibit the growth of cancer cell can be used in the treatment of breast cancer. (Figure 3, 4 and 5)

Figure 1:TEM image of Ag nanoparticle 1500 X

Figure 2: TEM image of Ag nanoparticle 3000 X

10µg

Figure 3:The figure shows untreated and 10µg drug treated cell viability untreated cisplatin

40µg

Figure 4:The figure shows 20µg to 40µg drug treated cell viability 20µg

80µg

160µg

Figure 5:The figure shows 80µg to 160µg drug treated cell viability

CONCLUSION

The Ag nanoparticles synthesized using Aegle marmelos, showed positive results for cytotoxic activity with an IC50 value of 42.82 µg/ml. These positive results confirmed the cytotoxic potential of the silver nanoparticles against breast cancer. thus,

International Journal of Current Science and Engineering 292

silver nanoparticles can be used as a potent drug in alternative therapy for treating the breast cancer patients in the near future. Further potential of cytotoxicity against cancer can be enhanced by In vivo studies in animal models and human volunteers. Acknowledgement Authors are thankful to Department of Biotechnology, Sahyadri Science College Kuvempu University for providing the necessary infra structure for carrying out this work.

REFERENCES 1. Jae YS, Beom SK. Rapid biological synthesis of silver nanoparticles using plant leaf extracts. Bioprocess Biosyst Eng. 2008;32(1):79-84.

2. Cheon J, Horace G. Inorganic nanoparticles for biological sensing, imaging and therapeutics. J Mater Chem. 2009; 19:6249-50. 3. Jitendra K, Meenakshi K C, Maheshwari M. Nanobiotechnology: Application of nanotechnology in diagnosis, drug discovery and drug development. Asian Journal of Pharmaceutical and Clinical Research. 2011;4(1):23-8. 4. Sharmila RD, Gayathri R. Green Synthesis of Zinc Oxide Nanoparticles by using Hibiscus rosa-sinensis. International Journal of Current Engineering and Technology. 2014;4(4): 2444-46 5. Kavitha KS, Syed B, Rakshith D, Kavitha HU, Yash wantha Rao HC, Harini BP. et al. Plants as Green Source towards Synthesis of Nanoparticles. International Research Journal of Biological Sciences.2013;2(6):66-76. 6. Baker S, Satish S. Endophytes: Toward a Vision in Synthesis of Nanoparticles for Future Therapeutic Agents. Int J Bio-Inorg Hybd Nanomat. 2012;1:1-11.

7. Hanley C, Layne J, Punnoose A, Reddy KM, Coombs I, Coombs A, et al. Preferential killing of cancer cells and activated human T cells using silver nitrate nanoparticles. Nanotechnology. 2008;19(29):295103- 13.

8. Hanley C, Thurber A, Hanna C, Punnoose A, Zhang J, Wingett DG . The influences of cell and Ag nanoparticle size and immune cell cytotoxicity and cytokine induction. Nanoscale Res Lett.2009;4(12):1409-20. 9. Nagao M. Physiosorption of water on zinc oxide surface. J Phys Chem. 1971; 75(25):3822-28.10. Horie M, Nishio K, Fujita K, Endoh S, Miyauchi A, Saito Y et al. Protein adsorption of ultrafine metal oxide and its influence on cytotoxicity toward cultured cells. Chem Res Toxicol. 2009;22(3):543-53. 11. Grabarek Z, Gergely J. Zero-length crosslinking procedure with the use of active esters. Anal Biochem. 1990;185(1):131-5.

12. Nair S, Sasidharan A, Divya Rani V V, Menon D, Nair S, Manzoor K, et al. Role of size scale of Zn nanoparticles and microparticles on toxicity toward bacteria and osteoblast cancer cells. J Mater Sci Mater Med. 2009;20(1):235-41.

13. John WR, Ezequiel M, Panagiota L, Denise G, Wingett. Zinc Oxide Nanoparticles for Selective Destruction of Tumor Cells and Potential for Drug Delivery Applications. Expert Opin Drug Deliv. 2010;7(9):1063-77.

14. Qu F, Morais PC. Energy levels in metal oxide semiconductor quantum dots in water-based colloids. J of Chem Physics. 1999;111(18):8588-94. 15. Qu F, Morais PC. The pH dependence of the surface charge density in oxidebased semiconductor nanoparticles immersed in aqueous solution. IEEE Transactions on Magnetics. 2001;37(4):2654-6.

16. Abercrombie M, Ambrose EJ. The surface properties of cancer cells: a review. Cancer Res.1962;22(5):525-48.

17. Degen A, Kosec M. Effect of pH and impurities on the surface charge of zinc oxide in aqueous solution. J Europena Ceramic Society. 2000;20:667-73. 18. Papo N, Shahar M, Eisenbach L, Shai Y. A novel lytic peptide composed of DLamino acids selectively kills cancer cells in culture and in mice. J Biol Chem. 2003;278(23):2101823. 19. Lappin MB, Campbell JD. The Th1-Th2 classification of cellular immune responses: concepts, current thinking and applications in hematological malignancy. Blood Rev. 2000;14(4):228-39. 20. Sayes CM, Reed KL, Warheit DB. Assessing toxicity of fine and nanoparticles: comparing in vitro measurements to in vivo pulmonary toxicity profiles. Toxicol Sci. 2007;97(1):163-80. 21. Croft M. The role of TNF super family members in T-cell function and diseases. Nat Rev Immune. 2009; 9(4):271-85.

22. Archna S, Sharma RA, Hemlata S, Saharia RL. Phytochemical and Pharmacological Profile of Abutilon indicum L. Sweet : A Review. Int J Pharm Sci Rev Res. 2013;20(1):1207. 23. Mohite MS, Shelar PA, Raje VN, Babar SJ, Sapkal RK. Review on Pharmacological Properties of Abutilon indicum. Asian J Pharm Res. 2012;2(4):156-60.

24. Harborne JB. In: Phytochemical methods, In: A guide to modern techniques of plant analysis. 3rd ed. Chapman and Hall, London; 1998 P. 235.

25. Gnanasangeetha D, Sarala D. Thambavani. Biogenic Production of Ag Nanoparticles Using Acalypha indica. An International Peer Review E-3 Journal of Sciences. 2014;4(1):238-46.

26. Saraniya DJ, B. Valentin Bhimba. Anticancer Activity of Silver Nan particles Synthesized by the Seaweed Ulva lactuca In vitro. Open Access Scientific Reports. 2012; 1(4):242.

27. Akhtar MJ, Ahamed M, Kumar S, Khan MM, Ahmad J, Alrokayan SA. Zinc oxide nanoparticles selectively induce

International Journal of Current Science and Engineering 293

apoptosis in human cancer cells through reactive oxygen species. Int J Nanomedicine. 2012;7:845-57. 28. A guide for journalists on breast cancer and its treatment-Roche https://www.roche.com/dam/jcr:5260dc48-ffc1-4991-9f3f-0bdae3e42128/en/med-breastcancer.pdf 29. Patel P, Mohammed S, Asdaq B (2010). “Immunomodulatory activity of methanolic fruit extract of Aeglemarmelos in experimental animals” Saudi Pharmaceutical Journal.; 18(3):161-165. 30. A guide for journalists on breast cancer and its treatment-Roche., American Cancer Society, Treating Breast Cancer., American Cancer Society, Treating Breast Cancer 31. A review on aetio-pathogenesis of breast cancer. J. Genet. Syndr. Gene Ther. 4, 1–5. 32. Alley, M. C., Scudiere, D. A., Monks, A., Czerwinski, M., Shoemaker, R. II., and Boyd, M. R. Validation of an automated micro culture tetrazolium assay (MTA) to assess growth and drug sensitivity of human tumor cell lines. Proc. Am. Assoc. Cancer Res., 27: 389, 1986 33. American Cancer Society, Treating Breast Cancer. https://www.cancer.org/cancer/breastcancer/treatment.html Breastcancer.org, Treatments and side effects of breast cancer. http://www.breastcancer.org/treatment 34. Arul V, Miyazaki S, Dhananjayan R. (2005). “Studies on the anti-inflammatory, antipyretic and analgesic properties of the leaves of Aegle marmelos Corr.” Journal of Ethnopharmacology, 96: 159-163. 35. Atkinson, H.G., 2003. Alcohol’s ‘‘darker side.” A drink a day may raise a woman’s risk of breast cancer. Health News 9, 1–4. 36. Barreto, A.M., Schwartz, G.G., Woodruff, R., Cramer, S.D., 2000. 25-Hydroxyvitamin D3, the prohormone of 1, 25-dihydroxyvitaminD3, inhibits the proliferation of primary prostatic epithelial cells. Cancer Epidemiol. Biomarkers Prev. 9, 265–270. 37. Breastcancer.org, Stages of breast cancer.,Mitsuk Anzhelika, Breast cancer information for young women, Turku University of Applied Science 38. Breastcancer.org, Stages of breast cancer., Chun Christina, What are the sign and symptoms of Breast Cancer, Health line., Swelling of all or part of the breast. 39. Xi-Feng Zhang 1 ,Zhi-Guo Liu 1 , Wei Shen 2 and SangiliyandiGurunathan 3, Int J Mol Sci. 2016 Sep 13;17(9). pii: E1534. doi: 10.3390/ijms17091534 40. Breastcancer.org,Treatments and side effects of breast cancer. http://www.breastcancer.org/treatment

41. Brooks, J.D., Ward, W.E., Lewis, J.E., Hilditch, J., Nickell, L., Wong,E., Thompson, L.U., 2004. Supplementation with flaxseed alters estrogen metabolism in postmenopausal women to a greater extentthan does supplementation with an equal amount of soy. Am. J.Clin. Nutr. 79, 318–325.

42. Brown, P., Allen, A.R., 2002. Obesity linked to some forms of cancer. W. Va. Med. J. 98 (6), 271–272.

43. Cancer Res. 48: 589-601, 1988. Mosmann, T. J. Immunol. Methods 65: 55-63, 1983.

44. Chen, J., Stavro, P.M., Thompson, L.U., 2002. Dietary flaxseed inhibits human breast cancer growth and metastasis and down regulates expression of insulin-like growth factor and epidermal

45. Chen, J., Tan, K.P., Ward, W.E., Thompson, L.U., 2003. Exposure to flaxseed or its purified lignin during suckling inhibits chemically induced rat mammary tumorigenesis. Exp. Biol. Med. (Maywood)228, 951–958.

46. Kuklev, D.V. V.M. Dembitsky, Prog. Lipid Res College of Biological and Pharmaceutical Engineering, Wuhan Polytechnic University, Wuhan 430023, China; zhangxf9465@163.com (X.-F.Z.); zhiguo_l@126.com (Z.-G.L.). Department of Stem Cell and Regenerative Biotechnology, Konkuk University, Seoul 143- 701, Korea * Correspondence: gsangiliyandi@yahoo.com; Tel.: +82-2-450-0457; Fax: +82-2-544-4645 18. 56, 67–91(2014) 19 47. Binning,G.C,F,Quate,Gerber,Eweibel.phys.Rev.Lett.49,57-61(1982) 48. Gerlier D., and N. Thomasset. J. Alley, M.C., et al Immunol. Methods 94: 57-63, 1986.. 49. Growth factor receptor. Nutr. Cancer 43, 187–192. 50. Jaswant, Akilandeswari, V. Loganathan, S. Manimaran and Ruckmani, (2001). “Wound healing activity of Aeglemarmelos”. Indian J. Pharm. Sci, 63(1), 41-44. 51. Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ. Cancer statistics, 2009. CA Cancer J Clin. 2009-(54-28). 52. Key Laboratory of Animal Reproduction and Germplasm Enhancement in Universities of Shandong, College of Animal Science and Technology, Qingdao Agricultural University, Qingdao 266109, China; shenwei427@126.com

53. Lampronti I, Martello D, Bianchi N, Borgatti M, Lambertini E, Piva R, (2003). “In vitro antiproliferative effects on human tumor cell lines of extracts from the Bangladeshi medicinal plant Aeglemarmelos Correa.” Phytomedicine, 10: 300-308.

54. Maity P, Hansda D, Bandyopadhyay and Mishra DK 2009. Biological Activity of crude extracts and chemical constituents of bael, Aeglemarmelos (L.) Corr.#. Ind J Experimental Biology 47: 849-861.

55. MTT Cell Proliferation Assay Instruction Guide – ATCC, VA, USA www.atcc.org

56. Patel P, Mohammed S, Asdaq B (2010). “Immunomodulatory activity of methanolic fruit extract of Aeglemarmelos in experimental animals” Saudi Pharmaceutical Journal.; 18(3):161-165.

International Journal of Current Science and Engineering 294

57. Rai M, Gupta PN, Dewedi R (1991). Variability in Baelgermplasm. Indian J. Plant Genet. Resour. 40:86-91.

58. Rana B. K,.Singh U. P and Taneja V., (1997). “Anti-fungal activity and kinectics of inhibition by essential oil isolated from leaves of Aeglemarmelos”, J Ethnopharmacology. 57, Page No.29-34

59. S.Vearasilp, S. Trakhtenberg, S. Gorinstein, Food Res.Int. 44, 1671–1701 (2011)Abdulkareem, I.H., 2013.

60. Sahare KN, Anandhraman V, Meshram VG, Meshram SU, Reddy MV, Tumane PM, ( 2008) “Antimicrofilarial activity of methanolic extract of Vitexnegundo and Aeglemarmelos and their phytochemical analysis.” Indian Journal of Experimental Biology.; 46(9022):128-131.

61. Shankarananth V, Balakrishnan N, Suresh D, Sureshpandian G, Edwin E, Sheeja E. (2007). “ Analgesic activity of methanol extract of Aeglemarmelos leaves.” Biological Trace Element Research, 78: 258 - 259.

62. Dembitsky V.M., Chem. Biodivers. 1, 673–781 (2004)

63. Dembitsky V.M., Eur. J. Med. Chem. 43, 223–251 (2008) 64. Dembitsky V.M., Phytomedicine 21, 1559–1581 (2014) 65. Dembitsky V.M., S. Poovarodom, H. Leontowicz, M. Leontowicz, 39. Xi-Feng Zhang 1,Zhi-Guo Liu 1 , Wei Shen 2 and SangiliyandiGurunathan 3, Int J Mol Sci. 2016 Sep13;17(9). pii: E1534. doi: 10.3390/ijms17091534

Abbreviation Used

Ag: Silver; MCF-7: Michigan Cancer Foundation-7; AFM: Atomic Force Microscopy; FTIR: Fourier Transform Infra-red Spectroscopy UV-Vis: Ultraviolet Visible; MTT:3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; nm: Nanometre; Th1: TNF-α: Tumour Necrosis Factor; hrs: Hours; ml: Millilitre; M: Molar; gm: Gram; min: Minutes; KV: Kilovolts; DMEM:

Dulbecco’s Modified Eagle’s Medium; EDTA: Ethylene Diamine Tetra Acetic acid; DMSO: Dimethyl Sulfoxide; FBS: Fetal Bovine Serum; IU/ml: International Units/millilitres; mg: Microgram; TPVG: Trypsin Phosphate Versene Glucose; cm:

centimetre; mg: milligram; ml: microliter; OD: optical density; IC: Inhibitory Concentration.

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