CHEMICAL PROFILE OF TWO JASMINUM SAMBAC L. (AIT) CULTIVARS CULTIVATED IN EGYPT–THEIR MEDIATED SILVER NANOPARTICLES SYNTHESIS AND SELECTIVE CYTOTOXICITY

Objective: Evaluation of two Jasminum sambac L. (Ait) cultivars; Arabian Nights (JSA) and Grand Duke of Tuscany (JSG) ethanolic leaves extracts as reducing agents for the green synthesis of silver nanoparticles (AgNPs) and evaluation of their cytotoxicity against MCF-7 breast cancer and 5637 bladder cancer cell lines and chemical profiling of the two cultivars. 
Methods: The synthesis of silver nanoparticles (AgNPs) by the two cultivars and characterization of AgNPs by ultraviolet (UV)–visible spectroscopy, Transmission electron microscopy (TEM) and Fourier Transform Infrared Spectroscopy (FTIR). Additionally, the use of The high-performance liquid chromatography coupled with photodiode array-mass-mass-spectroscopy (HPLC-PDA-MS/MS) for chemical profiling of both cultivars and evaluation of total leaves extracts and corresponding nanoparticles towards MCF-7 and 5637 cell lines compared to aneuploidy immortal keratinocyte (Ha Cat) normal cells by neutral cell assay. 
Results: The green synthesized AgNPs (of an average size range of 8.83 and 11.24 nm for JSA and JSG, respectively) exhibited cytotoxicity against MCF-7 and 5637 cell lines. The IC50 was determined for each total extract JSA (15.29±2.16 μg/ml) and JSG (20.28±1.20 μg/ml) and corresponding AgNPs 17.32±2.22 μg/ml and 6.32±1.01μg/ml for JSA and JSG, respectively. The IC50 of JSA and JSG against 5637 bladder cancer cell line were 13.76±1.11 μg/ml and 50.69±3.75 μg/ml, while the corresponding AgNPs showed IC50 of 5.54±0.88 μg/ml and 27.89±2.84 μg/ml, respectively. The HPLC-PDA-MS/MS allowed the identification of 59 compounds; 10 simple phenols, 17 flavonoids; quercetin and kaempferol glycosides, 2 lignans, and 30 secoiridoids; oleuropein, molihauside, and sambacoside. 
Conclusion: This study proved that JSA is an excellent source for the synthesis of AgNPs with optimum characters and enhanced activities toward MCF-7 and 5637 cell lines in correlation to identified compounds.


INTRODUCTION
Cancer is the second leading death cause 9.6 million death cases worldwide in 2018, most death cases are in low-and-middle-income countries. The most common cancers are breast cancer around 2.09 million cases [1]. Precancerous lesions convert normal cells to malignant tumors due to several factors as exposure to ultraviolet (UV) and infrared (IR) radiation, chemical carcinogens such as tobacco smoke, aflatoxin and arsenic and biological carcinogens due to infectious diseases: viruses, parasites and bacteria and may be due to genetic causes Bladder cancer is the second prominent cancer in males, this type of cancer is resistant for most medical treatments, with the highest incidence in developing countries, most cases of bladder tumor are subjected to tumor recurrence after radical cystectomy [2]. In the early stages, medication therapies are involved in the form of hormonal therapy, targeted therapy [3] or chemotherapy. Several studies have been devoted to the discoveries of new natural therapies that can fight the cancerous cell progression with limited effect on normal cells to achieve the maximum healing properties of breast cancer that can overcome the side effects of previous treatment protocols [4].
Nanoparticle sciences involve recently considerable interest from both academic and industrial fields and spreading of their application practically in medicinal [5][6][7], electrical [8], agriculture, environment [9] and aquaculture fields due to spontaneous discoveries of their diverse and interesting properties. Development of biologically based and inspired processes for the optimization of nanoparticles characters to target specific diseases or drug delivery pathway is an important branch of nanoscience and nanotechnology. In recent tendency, silver nanoparticles are introduced in medical researches as antimicrobial [10], antifungal, antiviral and cytotoxicity against many cell lines as NIH 3T3 cells and Hela cells [11].
Synthesis of nanoparticles is achieved using chemical, thermal or biological synthesis using bacteria or natural plant extracts [12,13]. The biological green pathway involves using of natural plants extracts as reducing agents is more favorable for the development of nanoparticles of optimized characters excluding the effects of chemicals which could alter the nanoparticles characters, toxicity and biological activity [14].
Jasminum sambac L. (Ait), Oleaceae is also known as Arabian Jasmine is native to Middle east and Asia. The two cultivars Jasminum sambac L." Arabian Nights"; (JSA) and Jasminum sambac L."Grand duke of Tuscany"; (JSG) are cultivated in Egypt for thousands of years [15]. Both cultivars are characterized by the high scent aroma of the shiny white composite flowers. They differ from each other by the shape of leaves and the structure of the corolla. They are extensively used in the perfume industry and as a flavoring agent in jasmine tea and aromatherapy [16]. Considerable attention has been gained to Jasminum sambac cultivars and their pharmacological activity [17]. Several studies were performed on its antidiabetic [18], antiinflammatory [19], vasodilator activity [20] and effect on morphine withdrawal symptoms [21]. Additionally, extracts of the flowers were reported to exhibit cytotoxic activity towards brine shrimp Artemia [22], Hep-G2 [23] and Dalton's ascites lymphoma [24].
High-performance liquid chromatography coupled to photodiode array-mass spectroscopy-mass spectroscopy (HPLC-PDA-MS/MS) is a type of liquid chromatography-mass spectroscopy used for tentative identification and profiling of the chemical composition and the fragmentation pattern of base peaks of selected plant species [25][26][27]. This chromatographic technique is used for complete qualitative profiling of secondary metabolites with link to the library for tentative identification.
The current research aims to evaluate the silver nanoparticles (AgNPs) synthesized by the JGA and JSG total ethanolic extracts towards MCF-7 breast cancer cell line and 5637 bladder cancer cell line in comparison to the leaves total extracts. Also, the identification of the chemical composition of the total extracts of leaves was performed using HPLC-PDA-MS/MS technique.

Plant material
The

Preparation of extracts
The dry leaves (100) g of each of JSA and JSG were extracted with 99% ethanol by percolation (4 x 1 L), filtered through Whatmann No.1 filter paper (pore size 0.6 m), each filtrate was concentrated under vacuum using rotary evaporator at 45° C and lyophilized to yield 24% and 20% dry extract, respectively. One hundred mg of each lyophilized extract was used for HPLC-PDA-MS/MS analysis, nanoparticles synthesis, characterization, and cytotoxicity study.

Green synthesis of silver nanoparticles (AgNPs)
AgNPs were synthesized as in the following protocol: 1 Mm aqueous solution of silver nitrate (AgNO3) was prepared and kept in a cool and dark place to use in the synthesis of 1 mmol aqueous solution of silver nitrate (AgNO3) was prepared and used for the synthesis of silver nanoparticles. 10 ml of each ethanolic extract of leaves of JSA and JSG added separately into 90 ml of an aqueous solution of 1 mmol silver nitrate for reduction of Ag+ions and incubated overnight at room temperature in dark place. The resultant yellowish brown solutions were the indication for the formation of silver nanoparticles. The formed solutions were used directly for TEM and UV quantifications [12,13]. Centrifugation at 4000 rpm for 30 min. followed by a series of washing by dist. H2O, filtration to obtain pure AgNPs. The pure AgNPs were used for cytotoxicity study.

Characterization of AgNPs
The UV-Vis spectroscopy of AgNPs were monitored as a function of time in 10 mm optical path-length-quartz-cuvettes with UV-Vis range 3600 spectrophotometer (Shimadzu, Japan). Samples were diluted 5 times with distilled water before being measured. The morphology of the particles (shape and dimensions) was examined by Transmission electron microscope (TEM). (JEOL-JEM-1011, Japan). Sample for TEM analysis were prepared by placing 3 ml of the sample on the copper grid and kept for drying at room temperature for 15 min. The different functional groups of the prepared nanomaterials in the range of 4000-400 cm_1 were measured by Fourier transform infrared spectroscopy (FTIR) 6100 spectrometer (Jasco, Japan).

Cytotoxicity assay
Cells of breast cancer cell line (MCF-7) and colon cancer cell line (5637) were obtained from the CLS Cell Lines Service (Eppelheim, Germany). Cells were cultured in RPMI 1640 medium (BioWhittaker, Lonza, Belgium) supplemented with 8 % fetal bovine serum (Sigma Aldrich, Germany) and antibiotics (100 U/ml penicillin/100 µg/ml streptomycin; Sigma Aldrich, Germany) at 95% humidity, 5% CO2 and 37.5 •C. MCF-7 and 5637 cells were sub-cultured twice a week and regularly tested for mycoplasma contamination. Cytotoxicity of test samples investigated cell line using the neutral red uptake (NRU) assay [28]. Statistical analysis of the data was expressed as mean±SD for triplicate trials of each measurement.

Therapeutic index
The therapeutic index is calculated as the ratio between the IC50 of the extract or nanoparticles on the normal keratinocyte cells and the IC50 on the cancer cell line. The drug or extract is considered effective with low cellular toxicity if the therapeutic index (TI) is high [29]. TI = IC50 on normal cells / IC50 on cancer cells

HPLC-PDA-MS/MS
HPLC-PDA-MS/MS using A Thermo Finnigan LC system (Thermo Electron Corporation, Austin, TX, USA). A Zorbax Eclipse XDB-C18; Rapid resolution, 4.6 × 150 mm, 3.5 µm column was used (Agilent, Santa Clara, CA, USA). A gradient consisting of water, 0.1% formic acid and acetonitrile, acetonitrile was increased to 30% within 60 min with a flow rate1 ml/min and a 1:1 split before the ESI source [30]. The sample was injected using autosampler. LCQ-Duo ion trap having a Thermo Quest ESI source was used for MS analysis. Xcalibur software (Xcalibur™ 2.0.7, Thermo Scientific, Waltham, MA, USA) was used to control the system. MS operating parameters in the negative mode were used as described in [31].

UV-vis spectroscopy
UV-vis spectroscopy is a reliable, accurate, simple, selective technique for monitoring the synthesis and stability of AgNPs. AgNPs have unique optical properties, which make them strongly interact with specific wavelengths of light. The conduction band and valence band lie close to each other in which electrons move freely. These free electrons give rise to a surface plasmon resonance (SPR) absorption band due to the collective oscillation of electrons of AgNPs [32]. The absorption of AgNPs depends on the dielectric medium, and chemical surroundings, particles dimensions, and particle size. Observation UV measurements of the formed nanoparticles showed absorbance at 443 nm for JSA AgNPs and 447 nm for JSG AgNPs.

Transmission electron microscopy (TEM)
TEM photography fig. (1) Showed biosynthesized AgNPs were predominantly spherical in shape with an average size ranging of 8.83 and 11.24 nm for JSA AgNPs and JSG AgNPs, respectively.

Fourier transform infrared spectroscopy (FTIR)
The FTIR spectroscopy analysis was performed to investigate plant metabolites acting as reducing agents for the metal ions to form nanoparticles and supporting their subsequent stability [33,34]. For JSA and JSG FIG (2A, 2B) The peaks near 3280, 2942 and 1648 cm −1 could be due to the O-H, aliphatic C-H and C=O stretching vibration of flavonoids and phenolic groups. The peak 1408 cm −1 corresponds for polyphenol OH and confirms the presence of an aromatic group, while the absorption peaks at 1012 cm −1 were assigned for C-O-C and secondary OH group. (In fig. 2 C, D), there is a deviation at 3280 and 1648 cm −1 of peak observed for JSA AgNPs and JSG AgNPs fig. (2 C, D). It suggests that the O-H and C=O groups were adsorbed on the surface of AgNPs have a deterministic role in the reduction of silver nitrate for AgNPs formation. These functional groups are attributed to flavonoids and secoiridoids the main components of the extracts.  fig. (2, 3). Values are expressed as mean±SD (N=3) Indicating the effectiveness of JSA AgNPs toward the two cell lines with very low cellular toxicity.

HPLC-PDA-MS/MS
Phytoconstituents of the two Jasminum sambac cultivars were identified via HPLC-PDA-MS/MS, a total of 59 compounds were identified as listed in table (2) and fig. (4), assignments were done by comparing retention times data and UV-vis spectral data for the screening and qualitative determination of phenolic acids, secoiridoids glycosides and flavonoids in plants has been illustrated with the ethanolic leaves extracts of Jasminum sambac (Ait.) cultivars. In this paper, it has been shown that parent ion scan and base peaks are powerful tools to identify the presence of certain compounds often occurring in genus Jasminum, interpretation of HPLC-PDA-MS/MS of JSA and JSG showed some variations among these 2 cultivars, JSA result in tentative identification of 42 compounds the main class is the secoiridoid glycosides 23 compounds and 2 lignans in addition to simple phenols and flavonoids. While in JSG cultivar a total of 26 compounds were tentatively identified composed of 9 secoiridoids, 7 phenolic acids derivatives, and 9 flavonoid glycosides and one lignan. Table (2), fig. 4

Identification of simple phenols and phenolic acids
Simple phenols i.e. free hydroxytyrosol (2) and hydroxytyrosol hexoside (1) with molecular ion peaks at [M-H] -of m/z 315, 153 were identified in JSA, while not identified in JSG. Phenolic acids and derivatives; caftaric acid, caftaric acid rhamnoside, ethyl cinnamate, syringic acid, and salvianolic acid were determined in JSG, and coumaroyl hexoside has been detected in JSA only. Protocatechualdehyde and sinapoyl hexoside were detected in both cultivars.   µg/ml. These results showed that a plant with higher cytotoxic results produces silver nanoparticles with higher characteristics (less particle size and better cytotoxic activities toward the same cell lines). The chemical profile of the JSA cultivar differs from the JSG cultivar. HPLC-PDA-MS/MS of JSA showed the abundance of secoiridoids and secoiridoids glycosides different from JSG like oleoside methyl ester, oleoside dimethyl ester, jasmolactone B, polyanoside, jaspolyanoside, polyanthoside, oleuropein, oleuropein hexoside, ligstroside, and ligstroside hexoside, while common secoiridoids in the two cultivars is sambacoside. Both cultivars showed secoiridoid as a major metabolite, but JSA cultivar possessed a higher abundance of secoiridoid derivatives than JSG.
Flavonoid glycosides were derivatives of kaempferol and quercetin in both cultivars with slight differences among them. From the above fig 3 and table 2, the main components of JSA were; deacyl sambacoside, sambacoside A, quercetin dihexoside, jaspolyanoside, and elonolic acid hexoside, while major compounds in JSG were sambacoside A, sambacolignoside, jasnudifloside H, molihauside A, kaempferol hexoside, kaempferol rutinoside, and oleoside pentoside. These results are in accordance with a previous report on Chinese Jasminum sambac flowers which identified molihauside A, sambacoside A and quercetin hexosides as major constituents [78].
The silver nanoparticles green synthesis with the optimum characters by JSA and their selective cytotoxicity may be attributed to the presence of secoiridoids in this cultivar. Additionally, the chemical profile could be used to distinguish the two cultivars of Egyptian Jasminum sambac (Arabian Nights and Grand Duke of Tuscany)

CONCLUSION
Jasminum sambac (Arabian Nights) cultivar is an excellent source for the synthesis of green biofriendly silver nanoparticles with selectivity to MCF-7 breast cancer and 5637 bladder cancer cell lines and limited toxicity towards the normal cells, thus offering a high safety margin when used as a cytotoxic agent.

FUNDING
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.