Antioxidant, Cytotoxic, and Antihemolytic Activity of Greenly Synthesized Selenium Nanoparticles Using Elettaria Cardamomum Extract

INTRODUCTION
Herbal phytomedicines have attracted researchers’ attention because of their potential biological activities [1]. Cardamom (Elettaria cardamomum) of the ginger family (Zingiberaceae) is a spice composed of small pods with dark seeds that possess a good fragrance and used in sweets, spicy food, coffee and tea [2]. It is referred to as small cardamom or green cardamom and is cultivated and grown in some Asian countries, including Nepal, Costa Rica, Guatemala, Indonesia. Sri Lanka, India, Tanzania, and Mexico [3]. It is also recognized as ‘’Hel” in gulf countries like Kuwait, Saudi Arabia, United Arab Emirates, Iran, Iraq, and other regions [3, 4].
The pleasant odor is attributed to the essential oils of cardamom that include pinene, myrcene, methyl eugenol, 1,8-cineole, a-terpinyl acetate, sabinene, phellandrene, terpinene, limonene, p-cymene, linalool, terpinen-4-ol, geraniol, and transnerolidal. Limonene, cineole, linalool, pinene, and borneol were reported to possess antioxidant scavenging activity [5]. This plant has been used for treatment of teeth and throat infections, lung congestion, tuberculosis and digestive problems [6].
They have aromatic, sweet, carminative, deodorant, diuretic, purgative, thirst reliever, and tonic characteristics. Moreover, it is used in asthma, burning sensation, cold, cough, bladder and kidney disorders, maldigestion and scanty urine [7]. Cardamom has exhibited anticancer [8] gastroprotective [9], antihypertensive [10], antiinflammatory [11] and immunomodulatory [12] antifungal and antibacterial properties [13] in numerous experimental studies.
cardamom expressed cancer chemopreventive potential against B(a)P induced forestomach papilloma genesis where it has the ability to inhibit forestomach carcinogenesis at peri-initiation stages of carcinogenesis significantly [14]. Chemo-preventive activity of cardamom has also been shown to regulate colorectal cancer [15]. D-Limonene, one of the bioactive components of cardamom, has been demonstrated to have chemopreventive action against skin, stomach, colon, liver, mammary and lung cancers in rodents [16].
This study aimed to prepare Elettaria cardamomum aqueous extract and to use it in synthesizing selenium nanoparticles in ecofriendly technique. It also aimed to estimate the biological activity of them as antioxidant, hemolytic, and cytotoxic materials.

MATERIALS AND METHODS
Preparation of investigated Elettaria cardamomum extract
The phytochemical components were extracted using 5 grams of Elettaria cardamomum in 100 ml distilled water upon shaking for 20 minutes at 65o C using water bath (Memmert WB14, Germany) then filtered using Whatman no.1 filter paper [17]. 

Preparation of Selenium Nanoparticles
20 mL of 1 mmol Selenium sulfate were added step wise to equal volume of  plant extract upon shaking for 2 hours at 35oC [18]. 
Characterization of the Selenium Nanoparticles
Transmission Electron Microscope (TEM)
Particle’s size, shape, crystal structure, and morphology of the prepared nano selenium were defined using TEM (JEOL TEM-2100) as described by Otunola et al., [19]. 

Zeta Potential technique
This technique was used for characterization of the nature of surface charge and stability of the prepared selenium nanoparticles [20] using Zeta potential analyzer (Malvern Instruments Ltd Zeta Potential Ver. 2.3) [21]. 

Phytochemical Analysis
Total Phenolics 
Phenolics were estimated using Folin-Ciocalteu procedures adopted by Wolfe et al. [22], using gallic acid as a reference compound. Phenolics were determined as milligram gallic acid equivalent / gram dried extract using the standard curve (y = 0.0063x, r2 = 0.997).

Total Flavonoids
Flavonoids were determined by aluminum chloride method adopted by Zhishen et al. [23] using catechin as a reference compound. Flavonoids were determined as milligram catechin equivalents per gram dried extract using the standard curve (y = 0.0031x, r2 = 0.998).

Cytotoxic activity using MTT assay
MTT assay was used for assessing the cytotoxicity of the samples using cell lines of Human prostate cancer (PC3), Hepatocellular carcinoma (HePG-2), Colorectal carcinoma (HCT-116), Epithelioid cervix carcinoma (Hela), Epidermoid larynx carcinoma (HEP2), Mammary gland carcinoma (MCF-7) through estimating the cell growth according to the procedures adopted by Bondock et al., [24]. Doxorubicin was used as standard anticancer drug.

Erythrocyte hemolysis Assay
5ml of whole blood were collected from healthy rats and transferred to tubes containing anticoagulation agent then centrifuged at 2000 rpm for 4 min. The obtained red blood cells were washed by phosphate buffer saline (pH 7.4) several times then resuspended in 0.5% saline [25]. 
0.5ml of different extract and selenium nano solution concentrations was added to 0.5ml of prepared red blood cells, then kept for 30 minutes at 37°C then centrifuged 5 minutes at 4000 rpm. The absorbance was detected at 540 nm. Negative control was prepared by phosphate buffer saline without extract and positive control using distilled water without extract. Erythrocyte hemolytic activity was estimated according to the following equation:

Where: At = tested samples absorbance, An = negative control absorbance, Ac = positive control absorbance.

RESULTS AND DISCUSSIONS
The phytochemical components of Elettaria cardamomum aqueous extract, include a broad variety of diversified secondary constituents that are used in the biosynthesis of nanoparticles [26]. This study illustrated that Elettaria cardamomum extract is rich with polyphenols (142.61± 1.32 milligram gallic acid equivalent/gram plant extract), flavonoids (36.76 milligram catechin equivalent/gram plant extract), and tannins (70.84 milligram gallic acid equivalent/gram plant extract) that could be consumed for reducing and stabilizing selenium ions to produce nanoparticles. phenolics play essential role in the biological reduction of ions into nanoparticles and contribution in their stabilization [29,30]. Flavonoid compounds are stable compounds that are used in the reduction of selenium ions into nanoparticles forming novel compounds that possess very small sizes with large surface area that are chemically and biologically active [27–29]. Polyphenol’s content (35.12 milligram gallic acid equivalent/gram plant extract), flavonoids (9.16 milligram catechin equivalent/ gram plant extract), and tannins (17.53 milligram gallic acid equivalent/ gram plant extract) were markedly decreased in the prepared solution of nano selenium, accordingly the results assure the utilization of these phytochemicals in the formation of nanometals.

Characterization of the synthesized Nano-selenium 
TEM technique
TEM was used for obtaining morphological description of the synthesized selenium particles. Fig. 1 demonstrates the micrographs, size distributions, spherical and tetragonal shapes of the synthesized selenium nanoparticles. The size of the nanoparticles ranged from 38.26 to 68.49 nm. TEM helped to assess agglomeration and/or aggregation of the prepared nano. There was little aggregation of the selenium nanoparticle that provides large surface area so improve efficacy as cytotoxic agents.

Zeta Potential Analysis
 It is used to study the nature of the nano-particles and to ensure their long-term stability. The nanoparticles possess double layer of ions where the electrical potentials at the borders of this layer were described as zeta potential of the nanoparticles with values in the range of +100 mV to −100 mV. Zeta potential of the synthesized selenium nanoparticles using Elettaria cardamomum extract found to be −11 mV (Fig. 2), that was highly stable where zeta potential with values lower than +25 mV or higher than −25 mV described to gain higher degrees of stability [20].

Biological Potentials 
Antioxidant Activity
 The aqueous extract of Elettaria cardamomum expressed an IC50 of 0.023 mg/ml that was comparable to ascorbic acid (IC50 = 0.0225 mg /ml), meanwhile the selenium nanoparticles solution expressed less antioxidant activity with IC50 value of 0.124 mg /ml. The results described the role of polyphenols, flavonoids, and tannins in the very good antioxidant activity of Elettaria cardamomum [30,31]. The decrease in these phytochemicals after synthesis indicates the utilizing of their active hydroxyl groups in the biosynthesis leading to the decrease in antioxidant activity. 

Cytotoxic Activity 
Elettaria cardamomum serve as an antitumor agent that induces less side effects than chemical drugs, such as Cyclophosphamide [8]. It was recorded that selenium nanoparticles and its zero-oxidation state express less toxic effect and more availability as well as it could easily be encapsulated [32,33]. Nano-selenium possess a broad range of applications as antioxidants [34], potential antitumor drugs, and antimicrobial agents [35,36]. Previous studies showed that nano selenium could play a crucial role in cancer chemoprevention [37,38] and as an antitumor drug delivery carrier [39] in addition to its remarkable anticancer activity [40–42]. Treatment with selenium nanoparticles alter the mechanical characteristics of the cancer cells as they clearly diminish the adhesion forces and young’s modulus [43]. In addition to the unique anticancer efficacy of selenium nanoparticles, they possess better selectivity for tumor cells [44]. 
In the present study, cytotoxic activity of the Elettaria cardamomum extract and the synthesized selenium nanoparticles were measured using MTT assay. 6 tumor cells (HepG-2, MCF-7, HCT-116, PC3, HeP2, and HeLa cell lines) and doxorubicin as a standard drug were used. IC50 is a parameter used to reflect the concentrations at which 50% of tumor cells in µg/mL were killed. IC50 is inversely proportional to the efficacy of the sample to stop the growth of tumor cells. The results of in vitro cytotoxicity expressed as percent of inhibition, the cell’s viability or potency of the samples and IC50 values are presented in Fig. 3, Fig. 4, and Fig. 5). The results revealed, that the synthesized selenium nanoparticles possessed cytotoxic potency against the tested cancer cell lines than the main extract. The nanoparticles have large surface area that increased their efficacy to inhibit tumor cells growth. The selenium nanoparticles synthesized using Elettaria cardamomum showed the most potent cytotoxic effect against tumor cell lines of HePG-2 with an IC50 of 23.33 µg/ml followed by HeLa with an IC50 of 27.59 µg/ml, HeP2 with an IC50 of 31.04 µg/ml, HCT-116 of moderate or less potency with an IC50 of 37.36 µg/ml, PC3 of moderate or less potency with an IC50 of 38.68 µg/ml and WI-38 that expressed the lowest activity with an IC50 of 52.91 µg/ml, respectively.   In addition, all the tested samples revealed weak cytotoxicity with the normal lung fibroblast cell line. The results illustrated the applicability of the tested Elettaria cardamomum and the synthesized selenium nanoparticles as antitumor drugs. The results also demonstrated that the prepared selenium nanoparticles had higher efficacy in inhibiting the growth of tumor cells more than E. cardamomum extract.
The results of selenium sulfate solution demonstrated that it displayed lower activities against HePG-2 cell lines (IC50 = 46.15 µg/ml), HeP2 cell lines (IC50 = 54.1 µg/ml), and HeLa cell lines (IC50 = 44.12 µg/ml). Selenium sulfate showed very low cytotoxic effect against MCF-7(IC50 = 58.16 µg/ml), HCT-116(IC50 = 60.14 µg/ml), and PC3 tumor cell lines (IC50 = 64.26 µg/ml). So the salt was not effective against the tested tumor cell lines. 

Erythrocyte hemolytic activity
The in vitro hemolysis assessment estimated the released hemoglobin in the plasma (as an indication for red blood cell lysis) following exposure to the tested agent. According to the US Food and Drug Administration, the in vitro hemolysis study should be implemented at the intended concentration for four administrations to evaluate the hemolytic potential of excipients intended for injectable use [45-47]. In the current study, Elettaria cardamomum extract and its synthesized nano selenium were screened in vitro for their hemolytic activity against human erythrocytes. The samples were prepared in serial dilutions at concentrations of 125, 250, 500, and 1000 μg/ml. The assay was implemented using an amended spectroscopic technique to investigate the hemolytic activity of the desired samples. The percentage of hemolysis was expressed as the hemolytic effect of the investigated samples at four different concentrations. Fig. 6 shows a comparison of the hemolytic activities of the Elettaria cardamomum extract and its synthesized nano selenium at different concentrations.
High concentrations revealed a high percentage of hemolysis in a proportional relationship. All tested samples displayed very low hemolytic effects on human erythrocytes. Based on these results, Elettaria cardamomum synthesized nano selenium solution showed maximum hemolytic activity [48]. The results demonstrated that the investigated samples were potent hemolytic agents. At the same time, the samples were safe for human erythrocytes. Generally, the results of this research established that aqueous extracts from Elettaria cardamomum and its nano-solutions are non/less toxic to human erythrocytes. 

CONCLUSION
This study declares the possibility of formulating selenium nano-composites with aqueous extracts of Elettaria cardamomum to improve the natural biological efficacy of the chemical constituents. These solutions inhibited the growth of various cancer cells while not affecting the growth of normal cells. This study also proved that selenium nano-composites were less efficient than aqueous extracts as antioxidants, which confirms that the formation of selenium nanoparticles decreases the efficiency of nanomaterials to trap DPPH• free radicals. Phytochemical analyses and antioxidant evaluations revealed similar results, showing that decreases in the phenolic and flavonoid contents impacted the DPPH• free radical stabilization. Maximum hemolytic activities were recorded Elettaria cardamomum selenium nano-solutions. Because of the favorable biological properties of Elettaria cardamomum extract, and its selenium nano-solutions, they may be potentially useful for drug development and food processing. 

CONFLICT OF INTEREST
The authors declare that there is no conflict of interests regarding the publication of this manuscript.