Journal of Engineering Research xxx () xxx–xxx

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Journal of Engineering Research

journal homepage: www.journals.elsevier.com/journal-of-engineering-research

Formulation of a modern anti-human breast cancer drug from silver

nanoparticles green-synthesized using Allium saralicum
Xin Xua, Sara Amirpour Amraiib, Reza Toushmalanic, Maryam Almasid,
⁎

a
Department of Thyroid Gland and Breast Vascular Surgery, Xijing Hospital, The First Affiliated Hospital of Air Force Medical University, Xi'an 710032, Shaanxi Province,
China
b
Department of Microbiology, School of Biology, College of Science, University of Tehran, Tehran, Iran
c
College of Skills and Entrepreneurship, Rasht Branch, Islamic Azad University, Rasht, Iran
d
Department of Clinical Sciences, Faculty of Veterinary Medicine, Razi University, Kermanshah, Iran

A R T I C L E I N F O A B S T R A C T

Keywords: Nanotechnology has immense potential for the development of novel and necessary products that benefit human
Chemotherapeutic supplement health, the environment, and industries. Silver nanoparticles and their nanocomposites have gained tremendous
Formulation, Human breast cancer importance in the field of nanotechnology owing to their widespread usage in various realms of science and
technology, including electronics, biomedical, environmental protection, textile industry, cosmetics, ther­
apeutics, photonics, agriculture, and so on. As the characteristic properties of silver nanostructures, like excellent
SERS/SPR, surface properties, diversity in shape, surface charge, dissolution rate, controlled silver ion release for
mediating the antimicrobial toxicity and cytotoxicity towards cancer cells, and efficient biocompatibility, which
render them to be potential antimicrobial, anticancer and diagnostic agents. Recently, Allium saralicum extract
has been used for green-synthesizing the silver nanoparticles and investigation of its biomedical effects. In the
present research, we formulated a modern chemotherapeutic drug by silver nanoparticles (AgNPs) containing
Allium saralicum aqueous extract for the treatment of breast cancer. Characterization of AgNPs was done by UV-
Vis, FT‐IR, TEM, and FE‐SEM. The bands at 488 and 539 cm−1 (belonging to the Ag-O bond) approves the
production of silver nanoparticles. The spherical morphology with a range size of 13.9–55.2 nm is obtained for
synthesized nanoparticles. The band at 434 nm approves the AgNPs formation. To survey the cytotoxicity and
anti-human breast cancer effects of AgNO3, Allium saralicum, and AgNPs, MTT assay was used on the SK-BR-3,
MDA-MB-231, AU565 [AU-565], and Hs 281. T cell lines. Silver nanoparticles had very low cell viability and
anticancer properties dose-dependently against cancer cell lines without any cytotoxicity on the normal cell line.
The best result of anticancer properties of AgNPs against the above cell lines was seen in the case of the MDA-
MB-231 cell line. The IC50 of nanoparticles were 208, 250, 200, and 188 µg/ml against SK-BR-3, MDA-MB-231,
AU565 [AU-565], and Hs 281. T cell lines, respectively.

Introduction sometimes abnormal proliferation of body cells. Cancer can be induced

through various agricultural toxins, environmental pollutants, che­
The use of chemotherapy drugs and synthetic additives has dis­ motherapy drugs and food additives [1–3]. In general, many chemical
rupted the functioning of healthy cells. Studies conducted on cell drugs and food additives cause changes in the process of cell division
toxicity have shown that these synthetic substances cause severe and disrupt the reproduction and differentiation of healthy cells [2,3].
apoptosis and DNA breakage. Due to the increasing concerns about the The main drawback of chemotherapy drugs and synthetic food ad­
safety of chemical drugs and additives, the demand for the use of ditives is that they harm the healthy cells of a person. Therefore, sci­
bioactive has attracted the most attention in recent years [1,2]. The entists are trying to use natural bioactive with low toxic effects to re­
search for natural biological compounds led to the introduction of plant place these chemical drugs and synthetic additives [4–6].
nanoparticles as a bioactive compound in the pharmaceutical and food Scientific evidence shows that the medicinal and biological effects of
industries [2,3]. Today, cancer means the growth, proliferation and Allium saralicum (garlic) are due to the large amount of sulfurous

⁎
Corresponding author.
E-mail address: [email protected] (M. Almasi).

https://doi.org/10.1016/j.jer.2023.100136
Received 21 April 2023; Received in revised form 15 June 2023; Accepted 25 June 2023
2307-1877/© 2023 Published by Elsevier B.V. on behalf of Kuwait University. This is an open access article under the CC BY-NC-ND license (http://creative­
commons.org/licenses/by-nc-nd/4.0/).

Please cite this article as: X. Xu, S.A. Amraii, R. Toushmalani et al., Formulation of a modern anti-human breast cancer drug from silver
nanoparticles green-synthesized using Allium saralicum, Journal of Engineering Research, https://doi.org/10.1016/j.jer.2023.100136i
X. Xu, S.A. Amraii, R. Toushmalani et al. Journal of Engineering Research xxx () xxx–xxx

compounds of this plant [7]. These ingredients create the special taste
of garlic. Flavonoids are not found in garlic. The biological properties of
garlic constituents such as lectin, prostaglandin, fructan, pectin, ade­
nosine, vitamins CB B2, B and E, nicotinic acid, glycolide fatty acid,
phospholipid, and essential amino acids have been studied for decades
[8]. So far, biological importance and medicinal properties such as anti-
fungal, anti-bacterial, anti-tumor, anti-thrombosis and hypocholester­
olemic properties of saponins such as B chlorogenin are known. Allicin
and organoselenium compounds can be mentioned among other che­
mical substances in garlic [7–9]. These substances have biological
properties such as reducing cholesterol, preventing cancer, and also
have a citergestive effect with organosulfur compounds. Garlic products
in international markets are classified into four groups: garlic essential
oil, garlic extract oil, garlic powder (Carisin) and garlic extract (Kyolic),
which is known as Age garlic extract (AGE) [9]. Products containing
garlic, which are safe, very effective, stable and odorless, are considered
valuable food supplements. Based on many studies, AGE has a greater
effect in terms of consistency and stability compared to raw garlic,
dehydrated garlic powder and other products. Research shows that the
main and medicinal properties of garlic depend on the compounds
present in it [10]. One of the products derived from garlic is Age Garlic
Extract (AGE), which has more antioxidant activity than fresh garlic Fig. 1. FT-IR Spectrum of silver NPs.
and other commercial garlic supplements [10,11]. In vivo and in vitro
studies of researchers show that thiosulfate extracted from garlic pre­
vents the growth of cancer cells. Laboratory and epidemiological stu­
dies also proved that garlic reduces the risk of carcinoma in different
tissues and organs. The course of anticarcinogenic action is carried out
by different mechanisms, such as increasing the cycle of carcinogenic
metabolism and the easy secretion of the anti-toxin enzyme system,
which increase the production of anticarcinogens from the body [11].
Also, this plant prevents procarcinogen activity by the effect of garlic on
cytochrome P450, which leads to antioxidant action; On the other
hand, garlic prevents cell proliferation, cell division, chromosomal
damage, lipogenesis and cyclooxygenase activities (anti-inflammatory
effects) [12]. Many evidences show that water and lipid-soluble com­
pounds, sulfur, organic sulfur compounds, Eruboside B, a steroid sa­
ponin, and organic selenium compounds play an important role in the
anti-cancer activities of garlic. Garlic has a greater anti-cancer role
compared to other plants due to its high selenium content. This prop­
erty is due to the replacement of sulfur with selenium in garlic [13]. In
addition to the studies conducted on the anti-cancer activities of garlic
compounds, some recent researches have also focused on their anti-
mutation activities [13,14]. The results of the research of researchers of
the American National Cancer Institute showed that garlic has great Fig. 2. AgNPs FE-SEM image.
power in preventing cancer [24]. In addition to the mentioned biolo­
gical activities, the absorption of AGE from the small intestine can and can provide solutions for technological and environmental chal­
protect patients against the side effects of antitumor drugs such as vo­ lenges. These materials have unique properties due to their particle size
miting, nausea, stomach inflammation, stomach ulcers, bleeding and used in several science fields such as medicine, engineering and tech­
intestinal ulcers. Consumption of garlic can stimulate the digestive nology (including electronics, environmental measurement, Raman
process and increase the speed of food absorption and decrease the time spectroscopy of bacterial surface, antibacterial agent, drug delivery and
of food passing through the digestive tube [13–16]. The prebiotic ac­ water purification) [4–6]. The application of nanomaterials in medi­
tivity of garlic is related to the amount of soluble fibers, especially the cine, especially in drug delivery, has increased in the last decade.
fructo-oligosaccharides starch of this plant, which are absorbed in the However, some nanomaterials have harmful effects, the most important
large intestine. Because there are Lactobacilli and Bifidobacteria in the of which is the poisoning of humans and animals [20–25]. For example,
large intestine, which have a positive effect on people's health. Starch zinc oxide nanoparticles are liver function disorder and silver nano­
and fructo-oligosaccharides in garlic can be used to enrich foods toxicity in zebrafish embryos. In addition, this toxicity can directly
without having a negative effect on the taste of foods [16]. harm human health, which hurts reproduction and fetal development.
Recently, plants have been used for synthesizing metallic NPs due to These factors have been researched to develop non-toxic nanomaterial
their high therapeutic activities. The pollution created during the NPs production methods [20–22]. Therefore, to minimize waste products
biosynthesis by plant extract is almost zero. As a result, the herbal use and maximize the chemical processes efficiency, the basic principles
nanoparticles have very low environmental effects. But, the quantity of green chemistry should be adopted. Therefore, any chemical process
and quality effects of metallic NPs depend on many factors such as the or synthetic pathway must meet these principles with environmentally
plant extract nature, extract concentration, temperature, metal salt non-toxic chemicals and benign solvents [4–6]. Nanoparticles enter the
concentration, pH, and reaction time [17–19]. Nanotechnology is a body in different ways and can be transferred to the vital organs of the
branch of material science, recognized as one of the key technologies of body through the bloodstream and cause cell damage [17–19]. In
the future. Nanomaterials are used in many different parts of human life general, the size of nanoparticles is between 1 and 100 mm, and these

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X. Xu, S.A. Amraii, R. Toushmalani et al. Journal of Engineering Research xxx () xxx–xxx

Fig. 5. The cell viability of AgNPs against normal (HUVEC) cell line.

Fig. 3. TEM image of AgNPs. Experimental

Green formulation

To synthesize the silver nanoparticles, AgNO3 stock with a con­

centration of 600 mg in 60 ml of distilled water was first prepared.
Then, this stock was used to prepare 50 ml of 1 mM AgNO3 solution.
5 ml of extract of the plant was added to this solution and placed on a
shaker at 100 rpm at room temperature. To separate nanoparticles from
the solution, a centrifuge was used with a speed of 12000 rpm and a
duration of 30 min. To confirm producing silver nanoparticles, a
spectrophotometer (200–800 nm) was used, and a FE-SEM was applied
to measure the size and shape. Also, the FTIR method was used to de­
termine the plant extract possible organic compounds that are involved
in the NPs synthesis.

Anticancer activities

Synthesized silver nanoparticles cytotoxic properties on breast

cancer cells i.e. SK-BR-3, MDA-MB-231, AU565 [AU-565], and Hs 281.
T were assessed using an MTT colorimetric method. 10% RPMI culture
medium was used for the cultivation of cancer cells. To prepare the
Fig. 4. UV-Vis pattern of AgNPs. complete culture medium, 10% RPMI was added to 90 cc of prepared
RPMI culture medium in the amount of 10 ml of FBS and 1 ml of
Table 1 streptomycin and penicillin. The vial containing cancer cells was re­
The IC50 of AgNPs in the anticancer test. moved from the incubator and then transferred to Bain-Marie. Then the
contents of the vial were mixed with about 5 cc of the culture medium
HUVEC SK-BR-3 MDA- AU565 Hs 28
in the Falcon and the contents of the Falcon were centrifuged. The
MB-231
supernatant and 1 ml culture medium were added to the cell plate.
IC50 (µg/ - 208 250 200 188 Then, the cells were transferred to a flask containing 5 cc of culture
ml) medium, and the flasks were placed in a CO2 incubator with appro­
priate humidity and temperature. At regular intervals, the cells were
passaged and transferred to a fresh culture medium. An inverted mi­
metal nanoparticles have various physiochemical properties than the
croscope was applied to estimate the number of live cells and check any
metal itself [20–23]. Nanoparticles are applied in all life dimensions
type of contamination. Then the liquid solution in the flasks was re­
such as the fight against microbes, electrical systems, diseases treat­
moved. After that, the bottom of the flask was washed with PBS. To
ment and diagnosis [21–25]. One of the most important nanoparticles
separate the cells from the plate bottom, trypsin enzyme was added.
in medicine is silver NPs, so we used them to treat breast cancer.
The cells were counted with trypan blue dye and finally after trans­
The characteristics of silver nanoparticles green-formulated by
ferring the cells to a plate, MTT test was done and placed in an in­
Allium saralicum extract against human breast cancer cells i.e. SK-BR-3,
cubator. The basis of the work is that living cells have the succinate
MDA-MB-231, AU565 [AU-565], and Hs 281. T were assessed in the
dehydrogenase enzyme in their mitochondrial membrane, which reacts
recent study.
with tetrazolium and creates purple crystals, and since dead cells do not

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X. Xu, S.A. Amraii, R. Toushmalani et al. Journal of Engineering Research xxx () xxx–xxx

Fig. 6. The anticancer properties of AgNPs against SK-BR-3 (A), MDA-MB-231 (B), AU565 (C), and Hs 281.T (D) cell lines.

have this enzyme, curmazan is not formed and remains colorless. The obtained for synthesized nanoparticles. The images show an aggrega­
working method is that cancer cells prepared from the cell bank were tion of silver nanoparticles which is one of the common properties of
cultured in a 96-well plate in an RPMI culture medium. After 24 h, green synthesized metallic nanoparticles [25–30]. So far, silver nano­
treatment with nanoparticles synthesized in several dilations particles, which have green synthesized using plant extracts, are formed
(1–1000 µg/ml) was done. Finally, 20 µl MTT solution was added to in a wide range size of 23–54 nm. This reveals a resemblance with the
wells and the plates were placed in an incubator at a temperature of AgNPs particle size [31–34].
37 °C for about 4 h [24]: Using UV Vis spectroscopy is very common to find the information
about the plasmonic resonance of nanoparticles. Confirmation of silver
Sample A.
Cell viability(%) = x100 nano-particle formation and plasmonic resonance can be found by
Control A.
analyzing the absorbance data of UV-VIs spectroscopy. It can also
roughly estimate the size of a particle [25–30]. In the recent study,
Statistical analysis Fig. 4 exhibits the silver NPs UV–Vis. spectrum. The band at 434 nm
approves the AgNPs formation.
One-way ANOVA in SPSS version 22 software was used to calculate It seems that the silver nanoparticles anti-cancer effect against
the HUVEC and cancer cells viability percentage of the synthesized NPs breast cancer cells is because of their antioxidant effects.
and to test for significant differences (p ≤ 0.01). The viability of cancer cell lines was reduced by AgNPs (Table 1 and
Figs. 5 and 6).
Results and discussion The AgNPs IC50 was 208, 250, 200, and 188 µg/ml against SK-BR-3,
MDA-MB-231, AU565 [AU-565], and Hs 281. T, respectively (Table 1
Fig. 1 reveals the silver nanoparticles FT-IR spectrum. The bands at and Figs. 5,6).
488 and 539 cm−1 (belonging to the Ag-O bond) approves the pro­ The numbers indicate the cell viability percentages in the con­
duction of silver nanoparticles. Furthermore, there is a similarity be­ centrations of 0–1000 μg/ml of AgNPs against several cancer cell lines.
tween silver nanoparticles FT-IR spectrum and green synthetic nano­ Today, the use of nanomaterials in combination with anticancer
particles using extract [24]. The peaks at other regions including 3419 drugs is increasing widely, and the use of these compounds has many
and 2917, 1384–1622, and 1045 belong to the extract organic com­ applications in medicine. One of the practical aspects of nanomaterials
pounds bonds such as triterpenes, flavonoid, and phenolic, which were is to combine them with anticancer drugs so that the drug can be de­
reported previously [5,6]. livered to the cell with a higher effect, also, its toxicity on healthy cells
Figs. 2 and 3 exhibit the silver nanoparticles TEM and FE-SEM can be decreased [5,6]. Due to the rising death prevalence caused by
images. The spherical morphology with a range size of 13.9–55.2 nm is cancers and defects in chemotherapy and radiotherapy methods in

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X. Xu, S.A. Amraii, R. Toushmalani et al. Journal of Engineering Research xxx () xxx–xxx

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