Applied Science and Convergence Technology 2021; 30(3): 81-86
Published online May 31, 2021
Copyright © The Korean Vacuum Society.
Himanshi Mehtaa,†, Megha Guptab,†, Parminder Kaura, Jaspreet Kaura, and Naveen Kaushalb,∗
aUniversity Institute of Engineering and Technology, Panjab University, Chandigarh 160014, India
bDepartment of Biophysics, Panjab University, Chandigarh 160014, India
†Contributed equally to this work as first author.
Correspondence to:E-mail: firstname.lastname@example.org
Biomolecules mediated synthesis of nanoparticles (NPs) has revolutionized the nanotechnology research field due to its eco-friendly and non-toxic nature. The green approaches of NPs synthesis using plant extracts offer an effective and better alternative than other synthesis methods. Metal-based NPs have emerged at the forefront of biomedicine due to their versatility in health and therapeutics. In this context, Selenium (Se), a versatile trace element is known for its critical role in various pathophysiological processes through regulation of cellular redox status and inflammatory pathways besides others. However, these beneficial effects of Se are limited by its narrow physiologically relevant concentration range. Deficiency or excess of Se is associated with numerous pathologies and toxicities. Thus, in current study we have synthesized and characterized Hempseed (Cannabis sativa) based novel biogenic Se nanoparticles (SeNPs) using UV visible spectroscopy, DLS, EDX, FE-SEM, and FT-IR. Further, the phytochemical profiles and antioxidant as well as anti-inflammatory potential of these SeNPs were evaluated to ascertain their physiological benefits. Results indicated the spherical shaped SeNPs of average size 140–150 nm with favourable zeta potential of −45.0 mV. The phytochemical and Se analysis validated the redox modulatory anti-inflammatory potential of these hempseed based green SeNPs.
Keywords: Hempseed, Green synthesis, Selenium nanoparticles, Antioxidant activity, Anti-inflammatory potential
Metal-based nanoparticles (NPs) due to their broad spectrum of applications ranging from optoelectronics, catalysis, biological probes, and drug delivery have emerged as the forefront of therapeutic approaches [1, 2]. In terms of their biological and pathophysiological implications, various metal NPs like gold, silver, copper, etc. have been used due to their enhanced biological activities and lower side effects . Relevant to redox biology, NPs extending up to 200 nm can successfully eliminate free radicals  are considered efficient agents in therapeutics . Gold NPs, for instance, show excellent catalytic properties due to its large surface area in redox reactions , whereas Silver NPs, due to its anti-inflammatory, anti-oxidant and anticancer properties, possess tremendous biomedical applications [6, 7]. Similarly, trace elements-based NPs, due to their diverse properties are generally used as a therapeutic agent.
Previous studies suggested that metal NPs have enhanced bioavail-ability and low toxicity effects on biochemical and hematological assays; thus, they can be considered as a better alternative for the drug delivery system . In this regard, Selenium (Se), an interesting and essential trace element, demonstrates its beneficial antioxidant, anti-inflammatory, antimicrobial, and anti-carcinogenic effects  through incorporation into various selenoproteins . Though at adequate levels, it plays a crucial role in disease resistance and has immune modulatory activity . The beneficial effects of Se are seen at a very narrow concentration range. Any physiological deficiency or excess of Seis associated with pathologies and toxicity of Se respectively [1,12,13]. Thus, to overcome the drawbacks of high dosage of Se and at the same time maintaining its biological effects Se nanoparticles (SeNPs) are taken into consideration . Based on the high catalytic efficiency, absorbing ability, and surface activity  SeNPs are reported to be more effective compared to the Se or selenite . SeNPs regulate thyroid hormones , anti-atherosclerotic activity , anti-leukemia activity , and many others. In terms of the redox regulatory role, SeNPs also play a defensive role against DNA oxidation due to its anti-hydroxyl radicals features .
Despite these plausible benefits, the harsh chemical and expensive physical methods  involved in the NPs synthesis such as UV radiation , evaporation or laser ablation technique , use of harmful solvents, additives, stabilizers, acid decomposition, and reductants limit their use . Whereas the chemical methods of NPs synthesis yield toxic wastes and are not eco-friendly , the physical methods of NPs synthesis require high-cost instrumentation . Apart from this, high temperatures, acidic pH, and other harsh conditions can hinder the use of NPs as therapeutics for biomedical applications . Thus, biological methods using microorganisms, enzymes, and plant extract are emerging as a new way to synthesize NPs [24, 25].
Amongst the biological methods too, the use of microorganisms and enzymes to synthesize NPs have some limitations, as they require aseptic conditions, which are not only difficult to maintain but are also time-consuming . Thus, biosynthesis of NPs by plant extract has the edge over the other biological methods as it is cost-efficient, less time consuming, and does not require any special conditions . Studies over the decade have also demonstrated the enhanced potential benefits of many plant extracts-based metal NPs [24,27–29]. Various reports indicate green synthesized SeNPs using different types of plant derived extract such as Emblica officinalis fruit extract , leave extract of
Considering the exceptional nutritional value and numerous health benefits of hemp (
Plant collection and material:
Preparation of plant extract:
The seeds were rinsed with sterile distilled water to clear contaminants and dried under shade. The desiccated seeds were crushed with a motor and pestle. To prepare the extract, 5 g of powdered seeds were solubilised in 30 ml of deionised water. The solution was heated at 100 °C for 20 min, cooled down to ambient temperature, and then filtered (Whatman no.1). The final extract was obtained by the centrifugation of the filtrate at 8000 rpm for 15 min. The extract was refrigerated at 4 °C.
Biosynthesis of SeNPs:
For the eco-friendly green synthesis of SeNPs, an aqueous solution of sodium selenite (10 mM) was prepared and mixed with fresh plant extract of
Characterization of SeNPs:
Field Emission Scanning Electron Microscopy (
Free radical scavenging antioxidant activity of SeNPs:
The inhibition ratio (%) was calculated as following:
The absorbance of sample was expressed as As and the absorbance at addition of ethanol as Ac.
ABTS assay: To prepare the stock solution of ABTS, 7 mM ABTS aqueous solution was added to 2.4 mM potassium per sulphate and maintained in the dark conditions for 13–14 h at room temperature. The stock solution was diluted in ethanol (about 1:89 v/v) to attain a final absorbance of 0.700 ± 0.02 at 734 nm. 15 µM solution of ascorbic acid was used as standard and ethanol was used as Blank . The percentage inhibition of various dilutions of sample extracts was calculated as :
Selenium estimation by 2,3‐diaminonaphtalene (DAN):
Selenium concentration in NPs was estimated using fluorometric method . For digestion of sample HNO3was added followed by HClO4. After sample hydrolysis with 9 % HCl, the digest was reacted with DAN 2,3‐diaminonaphtalene under acidic condition. The selenodiazole so formed was extracted with cyclohexane. EDTA and hydroxylamine hydrochlorine was used as masking-reducing agent. Subsequently fluorometric Se estimation (PC spectrofluorophotometer) was performed at 376 nm and emission wavelength at 518 nm. Sodium selenite was used as standard.
Anti-inflammatory activity of the SeNPs solution at 0.1 mg/kg concentration were ascertained by the carrageenan-induced hind paw edema model. 16 Balb/C female mice were divided into four groups with four animals per group (
where Vc denotes edema volume in control and treated groups or Indomethacin .
Statistical analysis: The data were analysed using one-way analysis of variance (ANOVA) and multiple posthoc test (Tukey) to compare various treatment groups using the GraphPad Prism 8.0 program (GraphPad Software, San Diego, CA). The significance was set at
Se and Se based compounds have gained substantial attention due to their promising pathophysiological and chemotherapeutic potential. However, oxidative pathologies and toxicity associated with Se at deficient or excess concentration, respectively, restrict its biomedical and therapeutic potential. Chemically synthesized SeNPs due to their low toxicity and excellent bioavailability have a extensive of biomedical applications including chemoprevention, antimicrobial effect, antifungal activity as well as reducing oxidative stress . However, the chemical synthesis of SeNPs uses expensive methods, harsh chemicals, and generates harmful and toxic substances. Thus, recently the usage of plant extracts has become an interest due to its clean and simpler approaches. Different green synthesized SeNPs have been explored for various bio-potential applications such as anti-inflammatory activity  anti-oxidant activity  and anti-cancer activity . SeNPs synthesis employing plant extracts is not only eco-friendly and safe but also has tremendous advantages and wide applicability in the field of nanomedicine [42–46].Considering the unique and balanced nutritional composition as well as therapeutic and pharmacological properties of hempseeds (
Synthesis and characterization of SeNPs: Green synthesis of SeNPs was performed by the reduction of sodium selenite solution (10 mM Na2SeO3) into elemental selenium with the addition of aqueous seed extract of
Metal nanoparticles possess unique optical properties, which change proportionally with the shape and size of nanoparticles. The conversion of selenite (Se+4) to elemental selenium (Se+0) was accompanied by the color change from colorless to orange, indicating the successful formation of SeNPs [Fig. 1(a)]. In accordance with the previous studies, this color change can be attributed to the excitation of the surface plasmon resonance . The synthesis of these biogenic SeNPs was ascertained by UV-Vis spectroscopy. The electronic spectra demonstrated a wide peak around 300 nm representing the formation of SeNPs [Fig. 1(b)]. These results are in concordance with various reported studies concerning the synthesis of SeNPs [11, 50].
The morphological characterization of SeNPs was performed by measuring size, size distribution, and PDI using DLS (dynamic light scattering) analysis. DLS histogram revealed that the mean size of SeNPs is 183.8 nm [Fig. 2(a)]. PDI value is the indicator of the homo-geneity of nanoparticle size. In the current study, the PDI of the SeNPs was 0.032, which indicates a highly monodisperse nanoparticle system with negligible aggregation . Further, the stability of colloidal SeNPs was ascertained by zeta potential analysis (electric potential surrounding the particle). A sharp peak of zeta potential for negatively charged SeNPs was noticed at −45.0 mV [Fig. 2(b)], which confirmed repulsion between particles to prevent agglomeration. The nanoparticles, with a higher magnitude of zeta potential exhibits, increased stability due to greater electrostatic repulsion between nanoparticles . The negative value of zeta potential indicates that the capping of NPs might be responsible for their observed charge and long-term stability .
Further, the size and morphology of the green SeNPs were confirmed by FESEM analysis. The FESEM images of biogenic SeNPs suggest that the particles maintain uniformity and are well dispersed without any aggregation. Synthesized SeNPs were spherical in shape with an average size of 140–150 nm [Fig. 3]. The observed difference between FESEM and DLS size distributions is due to the fact that size measured by DLS is representative of the size of hydrodynamic diameter, which represents the hypothetical sphere that diffuses at the same rate as the particles being measured, while the size measured from
The chemical composition of SeNPs evaluated using EDX revealed the presence of two strong signals of Se at 1.4 and 11.2 keV , indicating the presence of substantial Se concentration in SeNPs. The peak of carbon and oxygen in the sample solution indicates the presence of stabilizers , whereas the peak of sodium suggests the presence of sodium selenite in the solution. The lack of other elemental peaks and strong signals of selenium metal in the spectra confirms the purity of the SeNPs [Fig. 1(c)].
FT-IR spectroscopic analysis was performed to determine the major functional groups present on the surface of phytogenic SeNPs and in the
SeNPs demonstrate enhanced antioxidant properties:
To explore the biological functions of these SeNPs
The phytochemical screening of SeNPs validated these results. Total phenolic and flavonoid content of
Paw edema/anti-inflammatory potential of Se nanoparticles: Upon switching from
Studies indicate that inflammation causes the dilation of blood vessels and contraction of endothelial cells that allows extravasations of nanoparticles (in the size range 20–200 nm) into the tissues in the inflamed areas through enhanced permeability and retention (EPR) effect . In the current study, the anti-inflammatory potential of SeNPs in the edema region may be attributed to enhanced permeability and retention effect. The subcutaneous administration of SeNPs may lead to sitespecific accumulation of SeNPs in the impaired region that eventually resulted in attenuation of inflammation. Reduction in inflammation percentage by nano form of Se proves to be more effective than the inorganic form of Se because of its free radical scavenging activity and lower toxicity . Previous reports demonstrate that plant derived SeNPs possess anti-inflammatory activity . Various studies have shown that phytochemicals prove to be successful in mediating the anti-inflammatory response . Flavonoids have been found to inhibit the release of histamine, cytokine, and prostaglandins [65, 66], and phenolic compounds are effective in treatment against inflammatory disorders . As validated earlier in the FT-IR analysis, the presence of a substantial amount of phenols and flavonoids along with Se present in SeNPs might be responsible for aiding anti-inflammatory action. The obtained results suggest that green synthesized SeNPs provides a better antioxidant alternative in antagonizing the effect of the inflammatory mediators as compared to the sodium selenite.
The green approach towards the synthesis of nanoparticles has been proposed as cost-effective and environment friendly alternative to chemical and physical methods. The current study elucidates the phytofabrication of SeNPs using aqueous seed extract of
NK, PK, JK
HM, MG, NK, PK
NK, PK, JK.
HM, MG, NK, PK
HM, MG, NK, PK
NK, PK, JK
NK, PK, JK
The facilities provided by Central Instrumentation Laboratory; Pan-jab University are acknowledged.
The authors declare that they have no conflict of interest.
All raw and analyzed data as well as the materials are available in this study.
The authors acknowledge the financial support to Pan-jab University (P.U.) and Department of Biophysics, P.U., Chandigarh (India) through DST-PURSE [58-60/RPC] and DST-FIST [SR/FST/LSI-425/2009], UGC-SAP [F.4-1/2015/DSA-1] programs.
PU/45/99/CPCSEA/IAEC/2019374, Panjab University (Himanshi Mehta, Parminder Kaur); PU/45/99/CPCSEA/IAEC/ 2019371, Panjab University (Megha Gupta, Naveen Kaushal)
All authors contributed to the study conception and design. All authors read and approved the final manuscript.