Medicinal plants have been important in the nanobiotechnology and medicine due to the benefical secondary metabolites with antioxidant properties. In the medical plants, natural antioxidants that are present in Aractium lappal and Eryngium billardieri, it seams that polyphenols, flavonoids and phenolic compounds responsible for reduction of the disadvantageous consequences of oxidative stress. The antioxidant effect was evaluated using the radical scavenger activity (RSA) method by calculation of DPPH decolourization percentage, the results of which are shown in Fig. 1. As showed in Table 1, DPPH assay showed that the powder methanolic extract of Aractium lappal (34.3 mg/L) could act beter as free radical scavengers than Eryngium billardieri(70.1 mg/L).
Table 1
Comparison of EC50 Methanolic Extract of Aractium lappal and Eryngium billardieri with Synthetic Antioxidants BHT and Gallic Acid.
(mg/L)EC50 DPPH
|
Aractium lappal
|
Eryngium billardieri
|
Galic Acid
|
BHT
|
4.33 ± 34.3
|
4.31 ± 70.1
|
2.61 ± 23.3
|
2.31 ± 9.88
|
Table 2: Mean and standard deviation of FBS on day 1 (A) and last day (B) of diabetic mice treatment.
Descriptives
|
A
|
N
|
Mean
|
Std. Deviation
|
Control
|
5
|
443.8000
|
98.08348
|
Acarbose
|
5
|
455.2000
|
60.20548
|
Aractium lappal methanolic extract
|
5
|
423.6000
|
94.19434
|
B
|
N
|
Mean
|
Std. Deviation
|
Control- Diabetic
|
5
|
442.4000
|
78.24345
|
Acarbose
|
5
|
338.6000
|
61.35389
|
Aractium lappal methanolic extract
|
5
|
236.6000
|
139.59431
|
Control-Non diabetic
|
25
|
365.8800
|
107.46950
|
In this work, the extract of Aractium lappal (methanolic) showed strong inhibitory effect upon pancreatic α-amylase than Eryngium billardieri (Data not shown). In addition, the the effect of methanolic extract Aractium lappal on the α-amylase activity in comparison with acarbose were also determined. Results showed that the IC50 of prepared extract was obtained with 36.5 mg/ml concentration, which is comparable to chemical drug such as acarbose (IC50 37.2 mg/ml) as positive control.
Furthermore, After preparation of diabetic mice from Pasteur Institute of Iran, and in the other part of the study, fasting glucose test was performed using an on-call plus glucometer for 6 weeks based on taking microsamples from each mouse’s tail.
The best control was observed in mice treated with Aractium lappal: Fast Blood Sugar (FBS) levels decreased from 423 ± 94 on the first day of treatment (Table 2A) to 236 ± 139 in the last week (Table 2B). ANOVA Analysis and Tukey’s test also demonstrated that there was a major difference between Aractium lappal treatment with Acarbose and control in the mean FBS on the last day of the trial.
Considering extensive applications of green nanoarticles in several section, however, the synthesis done by some methods which may be harmful to the environment due to the resulting waste material (Abou et al., 2010; Ahamed et al., 2010; Reddy et al., 2014; Mollania et al., 2019; Ramshini et al., 2018). Hereupon, there is a biotechnological increasing penchant to improve more economic, environmentally-friendly, and conveniently reproducible green biosynthesis ways for nanomaterial production such as AgNPs. Since among the two plants (Aractium lappal and Eryngium billardieri) used in this paper, the methanolic extract of Eryngium billardieri synthesized most of the silver nanoparticles, so it was selected to continue.
3.1. Effects of the interactive parameters in the AgNPs biosynthesis
Effect of temperature on the biosynthesis of silver nanoparticles
Temperature has been an important impact on the rate of NPs biosynthesis. Ten different temperatures (20, 25, 35, 45, 55, 65, 75, 85 and 90°C) were studied and it was found that the optimum temperature for the synthesis of silver nanoparticles by extracts of Eryngium billardieri was the 37°C. (Fig. 2A).
3.2. Effect of pH and production time on the biosynthesis of silver nanoparticles
Because the pH parameter has a great effect on the synthesis of nanoparticles (Singh et al., 2009), the pH of extarct was varied and this parameter effect was studied in the AgNPs bio synthesis. Since, experimental solutions color altered from pale yellowto brown, this conversion would be an mark for formation of nanoparticles. Therefore, different observed colors, confirmed thatthe shape and size of the nanoparticles would be controlled by pH (Fig. 2B). The pH of extarct would be a amain affecting arameter in the biosynthesis process and the maximum biosynthesis occurred in the presence of Eryngium billardieri extract with adjusted pH to 7.
To investigate the production time parameter, the methanolic extract was exposed to nanoparticle green biosynthesis at 37°C and pH = 7 at time intervals 12h upto 48 hours. The amount of nanoparticle synthesis was then observed in each of these states (Fig. 2C).
3.3. The produced silver nanoparticles characterization
3.3.1. UV–Vis spectroscopy
The nanoparticle biosynthesis and its stability in colloidal solution after incubation for long time was monitored by UV–Vis spectral analysis that was an useful technique for nanomaterial investigation. As showed in Fig. 3, strong absorption bands with a maximum peak at 426 nm were observed due to formation of AgNPs due to surface plasmon resonances (SPRs). The results are correlating with the reports of other researcher studied in the nanoparticle biosynthesis area (Prakash et al., 2013). The control solution (without silver nitrate salt) shows no evidence of absorption in the mentioned range. Since the optical attributes of AgNPs were reliant on the nanoparticles diameter, therefore, size of the prepared NPs would be estimated by Instrumental methods. For example, by UV-Vis spectroscopy, as the nanoparticle diameter rises, the peak plasmon resonance shifts to the longer wavelengths and a ratio of scattering to total extinction, increases (Haiss et al., 2007). A new broad band appearance after incubation upto 5 months showed agglomeration of Ag-NPs and formation of nanoparticles larger than 85 nm. Interestingly, the produced nanoparticles were stable upto 3 months and during this period, no aggregation was observed (data not shown).
3.3.2. FESEMand EDXmicroanalysis studies
For FESEM analysis, the sample preparation was performed on a carbon coated copper grid by just dropping smallest nanoparticle solution on the grid and then the film were dried by putting it under a mercury lamp. The FESEM showed that formation of well dispersed AgNPs in the suspention (Fig. 3B). Similarly the FESEM images demonstrate the appearance of silver nanoparticles in clusters to individuals in the shape of spherical to irregular and larger size may be due to the reaction conditions are not quite sufficient to get finer particles. In this micrograph, spherical nanoparticles in the size range of 40–79 nm were viewed.
An EDS spectrum of drop coated film with AgNPs was shown in Fig. 3C. The attendance of an optical absorption band at 3 eV revealed the existence of pure metallic AgNPs. EDS showed the occurrence of silver particle in higher amount with trace of magnesium, calcium and chloride. Other elemental signals was recorded due to presence of enzymes or proteins within the extracts.
3.3.3. Dynamic light scattering
Particle-size analysis was done after AgNPs preparation with the culture extarct of Eryngium billardieri. For the DLS evaluation, the suspension of AgNPs was diluted to reduce the noise. The suspention were then filtered and then centrifuged at 12000 rpm (30 min). The particle size histogram of AgNPs showed that the nanoparticle size was almost according to the results obtained with FESEM technique (Fig. 4). The frequency distribution showed that almost 90% of the nanoparticles are in the 45 to 60 nm range.
3.3.4. XRD analysis
The XRD data suggested the crystalline structure for the biosynthetic nanoparticles, which, according to the model, show three extreme peaks in the 2θ range, ranging from 10 to 95°C, with the most obvious peak in the 38°C. this peak is comparable with the standard pattern of silver nanoparticles and it is a certificate to prove the crystal structure of nanoparticles (Fig. 5).
3.3.5. FT-IR spectroscopy
FT-IR was performed to study fabrication of the surface modified AgNPs by using extract secondary metabolites (Fig. 6). The performed FT-IR confirmed the presence of polyphenols, carbohydrates, and alkaloids in the AgNPs solution that biosynthetized by plant extract. The precise position of Ag-O vibration was inconceivable due to a wide range of hydrogen bonding. The broad band observation at ~ 3420 cm− 1 demonstrated coordination of amine ligands to AgNPs at the nanoparticle level and confirms the existence of O–H/N–H stretchings because of protein, peptid or carbohydrate fragments in the extract. In addition, bands at 2920 and 3000 cm− 1 was attributed to the symmetric and assymmetric C-H stretchings, respectively. The band around 1700 cm− 1 is due to the carbonyl functional group upon nanoparticle. The weak asymmetric C = C bendings are also detected at 1472 cm− 1. Other peaks determined at 1210 − 1164, 1084, and 903 cm− 1 due to C-H bending, skeletal C-O and bending C-H, respectively.
3.4. Antibacterial activity
In recent years, due to the increasing resistance of microorganisms to common disinfectants and antibiotics, many studies have been conducted to improve antimicrobial strategies. At present, combination therapy with metal nanoparticle based disinfectants may be one of the probable tactics to overcome bacterial resistance to antibiotics (Takjoo et al., 2017). To test the antibacterial virtues of nanoparticles, these biosynthetized nanodrugs against some pathogenic bacterial strain were used. The antibacterial activity was examined using the agar and broth dilution procedures. Various amounts of AgNPs were placed on the discs and the inhibition zones of the pathogenic growth was assessed. Table 3 showed that the inhibition zone diameter of pathogenic bacteria in the presence of biosynthetized AgNPs.
Table 3. Antibacterial activity in the presence of AgNPs.
Echerishia Coli
|
Pseudomonas aeruginosa
|
Antibiotic
Compounds
|
Sensitive
(20 mm)
|
Intermediate
(19.5 mm)
|
AgNps
(30 mg/L)
|
|
|
Intermediate
(16.1 mm)
|
Resistance
(10.4 mm)
|
FOX 30
|
|
|
Resistance
(10 mm)
|
Intermediate
(16.3 mm)
|
CFM 5
|
|
|
Sensitive
(21.2 mm)
|
Intermediate
(16 mm)
|
Chloramphenicol 30
|
|
|
Resistance
(6.5 mm)
|
Sensitive
(19 mm)
|
Vancomycin 30
|
3.5. Cytotoxicity test
The AgNPs cytotoxicity was determined as a possible property, based on the amount of viability of cell lines exposed to several concentrations of produced nanoparticles. in this part, the common MTT experiment by two cell lines of MCF-7 and MCR-5 were performed. Measuring metabolic activity of cell lines and assessing live/dead cell ratio over a special time was measured the cell viability percentage compared with a control group without any additive (Fig. 7). The results displayed the toxicity of biosynthetized Ag-NPs on both cell lines follow a concentration-affiliate manner. This data is consistent with other results because earlier researcher, in findings considered to this issue that usage of high concentration of some metal nanoparticles might cause the cellular stress induction (Soenen et al., 2009, Ahmad et al., 2015). In addition, by comparing the nanoparticles concentration that caused reduction of viability of two cell lines upto 50% (IC50) it can be understood that the toxicity amount for cancerus cell line of MCF-7 is greater in the compare with MCR-5 normal cell line. According to the results, biosynthetized Ag-NPs can be considered as a suitable sample for drug delivery; moreover, the small cytotoxicity characterization against normal cells is of superior significance in the medical science.