Role of green silver nanoparticles synthesized from Symphytum officinale leaf extract in protection against UVB-induced photoaging
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The present study demonstrated the simple, cheap, eco-friendly synthesis of the silver nanoparticles (S-AgNPs) using Symphytum officinale leaf extract. The biosynthesized S-AgNPs were characterized by UV–Vis, FE-TEM, elemental mapping, EDX, zeta potential, XRD, SAED, and FT-IR. The characterization results revealed the irregular shape and relatively stable nature of synthesized S-AgNPs. The average particle size was determined to be 87.46 nm. The zeta potential shows the negative surface charge (− 25.5 mV) of S-AgNPs. After characterization, we investigated the anti-aging effect of S-AgNPs in HaCaT keratinocyte cells. HaCaT keratinocyte cells were treated with S-AgNPs at concentrations 1, 10, 100 μg mL−1 after UVB or non-UVB irradiation. The S-AgNPs significantly inhibited the production of matrix metalloproteinase-1 and IL-6 but increased the expression of procollagen type 1. The data suggest that S-AgNPs have photoprotective properties and may have potential to be used as an agent against photoaging.
KeywordsSymphytum officinale Silver nanoparticles Photoaging MMP-1 IL-6
Symphytum silver nanoparticles
Field emission transmission electron microscopy
Energy-dispersive X-ray spectroscopy
Selected area electron diffraction
Fourier transform infrared spectroscopy
Transforming growth factor beta
- S. officinale
Dulbecco’s modified eagle’s medium
Fetal bovine serum
Enzyme-linked immune sorbent assay
Reverse transcription polymerase chain reaction
Glyceraldehyde 3-phosphate dehydrogenase
Analysis of variance
Surface plasmon resonance
Dynamic light scattering
Reactive oxygen species
Metal nanoparticles exhibit unique chemical and physical properties including large surface/volume ratio, which is useful in different fields such as electronics, photonics, biomedical, catalysis, etc . Among the various noble metals, silver is the metal of the first choice due to their diverse properties especially high antimicrobial and catalytic nature [2, 3]. Many standard approaches by means of physical and chemical have been used for the preparation of silver nanoparticles (AgNPs) by several researchers. Generally, conventional physical and chemical methods seem to be very expensive and hazardous . Hence, there is always a need for developing an eco-friendly process for the synthesis of nanoparticles which does not use any harmful or toxic agents. The most recent environment-friendly approach is using green chemistry technology . The green synthesis approach provides most advantages over the chemical and physical method as it is fast, cost-effective, eco-friendly and easy to scale up for large-scale synthesis without applying high energy, high pressure, high temperature and toxic chemicals . The green synthesis approach usually employs microorganism or plant parts for the synthesis of nanoparticles. Recently, many researchers have employed different plant parts such as root, stem, bark, leaf, fruit, bud, and latex for the synthesis of AgNPs [7, 8, 9, 10]. Reports have suggested that nanoparticles synthesized from medicinal plants have been found to be pharmacologically active and stable, but no hazardous by-products or toxic chemicals are used in their synthesis . Therefore, as a green approach for the first time, we report the use of Symphytum officinale (Comfrey) leaf extract for the synthesis of AgNPs and further investigating their role in protection against UVB-induced photoaging.
Skin is the primary barrier to the body and protects the body from all kinds of external damage, such as microbial invasion, toxic materials, and ultraviolet (UV) radiation. The solar UV radiation composed of different types of waves depending on the wavelength, UVA (320–400 nm), UVB (280–320 nm) and UVC (100–280 nm). Among all, UVB is responsible for most of this damage . Acute exposure of human skin to UV irradiation causes sunburn, altered pigmentation, inflammation, immune suppression, and dermal connective tissue damage. It is also reported that chronic exposure to UV irradiation over many years disrupts normal architecture of the skin and ultimately causes premature skin aging (photoaging) due to the degradation of collagen and elastin that normally keep skin firm .
Collagen I plays an important role in maintaining skin structure in aged skin cells . The synthesis of collagen I is known to require precursor procollagen type I synthesis by TGF-β/Smad signaling in fibroblasts . Contrarily, collagen I can be degraded by UVB-induced ROS activation through two upregulated pathways using the degrading enzymes of collagen I (Matrix metalloproteinases, MMPs) and a collagen synthesis inhibitor (IL-6), thereby leading to the activation of activator protein 1 (AP-1) . Accordingly, the most common feature associated with UV-induced photoaged skin is increased MMP-1 expression and decreased procollagen type I level. Therefore, compounds that decrease the production of MMP-1 and increase the synthesis of procollagen type I may contribute to the prevention of skin photoaging. Various medicinal plant extracts have already been reported to protect the UVB-induced photoaging [12, 13, 14, 15, 16]. In the present study, we investigated the role of AgNPs synthesized from S. officinale, in protection against UVB-induced photoaging.
Symphytum officinale is a medicinal plant which belongs to family Boraginaceae, commonly known as comfrey. It is a perennial plant found in Asia, Europe, and North America. S. officinale has been commonly used in folk medicine for the treatment of diarrhea, bronchitis, tuberculosis, ulcers, and hemorrhoids. The plant extract was reported to be used as an ointment to promote the wound healing, reduce the inflammation, for the treatment of broken bones, tendon damages, painful joints, and muscles . Although Comfrey also contains dehydropyrrolizidine alkaloids (DHPAs) because of which its internal application is not recommended . Recently, the root extract of comfrey has shown to have anti-oxidant and proliferative activity . However, there is no report of AgNPs synthesized from comfrey on UVB-induced photodamage. Here, we hypothesize that the AgNPs synthesized from Comfrey leaf extract could be used for protection against UVB-induced skin photoaging. To test this hypothesis, markers of skin photoaging, MMP-1, IL-6 and procollagen type 1, were analyzed using PCR with human keratinocyte cells (HaCaT).
Materials and methods
Silver nitrate (AgNO3), crystal violet, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and dimethyl sulphoxide (DMSO) were purchased from Sigma-Aldrich chemicals (St. Louis, MO). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and penicillin/streptomycin were purchased from Gibco BRL (Grand Island, NY, USA). ELISA kits for MMP-1 and IL-6 were purchased from R&D Systems (R&D Systems Inc., Minneapolis, MN, USA). All other chemicals were of reagent grade. Organic solvents were purchased from Samchun chemicals (Korea). Inorganic salts were purchased from Sigma-Aldrich (St. Louis, MO, USA). Unless otherwise mentioned, solvents were purchased from Samchun chemicals (Seoul, Korea). In the present study, a total of seven plant extracts (Table S1) have been used to synthesize AgNPs. The selection of these plants was depending on the medicinal importance and previous study that has been performed. Dried plants were purchased from mountain rose herbs (Eugene, Oregon, USA).
Preparation of aqueous leaf extract
10 g of the dried plants (root, leaf, seeds) was mixed with 100 mL of deionized water and autoclaved for 30 min at 100 °C. The aqueous extract was subsequently centrifuged at 10,000 rpm for 10 min to remove the debris. Finally, the supernatant obtained was filtered through a 0.45 µm PVDF syringe filter (SmartPor, Seoul, Korea). This plant extracts were used for further AgNP synthesis. In the present study, all the plant used for the synthesis of AgNPs are listed in Table S1.
Synthesis of silver nanoparticles
The AgNPs were synthesized as reported previously . Briefly, aqueous plant extracts were diluted in water to 1:5 (v/v), to this solution the final concentration of 1 mM filter-sterilized solution of AgNO3 (Sigma-Aldrich chemicals, St. Louis, MO) has been added. The reaction mixture was kept at 65 °C. The synthesis was monitored for a change in the color, which indicated the synthesis of AgNPs. The AgNPs were collected by high-speed centrifugation at 20,000 rpm for 10 min. The obtained pellet was washed three times with distilled water to remove the unconverted metal ions or any other constituents.
Characterization of silver nanoparticles
UV–Vis spectrophotometer (UV–Vis) (Optizen POP; Mecasys; Daejeon, Korea) was used to confirm the reduction of metal ions and was scanned in the range of 300–800 nm. The transmission electron microscopy (TEM), elemental mapping, energy-dispersive X-ray spectroscopy (EDX) and selected area electron diffraction (SAED) analysis was performed through field emission transmission electron microscopy (FE-TEM), with a JEM-2100F (JEOL, Tokyo, Japan) instrument operated at 200 kV. The sample for FE-TEM was prepared by placing a drop of collected nanoparticles on the carbon-coated copper grid and subsequently drying it at room temperature before transferring it to the microscope. The hydrodynamic size and zeta potential for AgNPs were measured in triplicate using Zetasizer Nano ZS90 (Malvern Instruments, UK), double-distilled water (DDW) was used as a dispersive medium. For DLS and zeta potential analysis, the samples were suspended in water and then used. The X-ray diffraction (XRD) analysis was performed on X-ray diffractometer, D8 Advance, (Bruker), Germany, operated at 40 kV, 40 mA, with CuKα radiation, at a scanning rate of 6° min−1, step size 0.02, over the 2θ range of 20°–80°. The functional groups capped on the surface of AgNPs were identified using a Fourier transform infrared (FT-IR) spectroscopy (Spectrum One System, Perkin-Elmer, Waltham, MA). For XRD and FT-IR, the purified nanoparticles were dried and obtained in powder form then used.
Free-radical-scavenging activity was measured by diphenyl-1-picrylhydrazyl (DPPH) assay as described previously . A 20 µL sample in distilled water was placed in a 96-well plate and 180 µL of 2, 2-diphenyl-1-picrylhydrazyl (0.2 mM) in methanol was also added. After a 30-min reaction in dark conditions, the absorbance was recorded by a microplate reader (Molecular Devices E09090; San Francisco, CA, USA) at the wavelength of 520 nm. AgNPs synthesized from different plant extracts were tested at the different concentration from 100, 500 and 1000 µg mL−1 Arbutin was introduced as a positive control.
Cell culture, UVB irradiation and sample treatment
HaCaT cells originated from human epidermal keratinocytes (Sciencell, Carlsbad, CA, USA) were cultured in DMEM supplemented with 10% heat-inactivated FBS and 1% penicillin–streptomycin at 37 °C in an atmosphere containing 5% CO2. When the cells reached more than 80% confluence, the cells sub-cultured in 35 mm culture dishes (1.0 × 105 cells) and were rinsed twice with phosphate-buffered saline (PBS). Then, the cells were irradiated with UVB (144 mJ cm−2) using UVB radiation machine (Bio-Link BLX-312; Vilber Lourmat GmbH, France). After UVB irradiation, the experimental group cells treated with AgNPs (1, 10 and 100 μg mL−1). The normal cells were treated with the same dose of AgNPs without UVB irradiation. The controls were without the treatment of AgNPs.
Determination of cell viability
To test the effect of AgNPs on the viability of HaCaT cells, MTT assay was carried out. MTT assay was performed as described previously . Briefly, after 24 h of treatment, MTT at a final concentration of 0.1 mg mL−1 was added and further incubated for 2 h. The supernatants were removed and 1 mL dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals. Absorbance was determined on a microplate reader (Molecular Devices Filter Max F5; Sunnyvale, CA, USA) at a wavelength of 570 nm.
Measurement of MMP-1 and IL-6 production
The level of MMP-1 and IL-6 in the medium were determined after 24 h of incubation with AgNPs using ELISA kits according to the manufacturer’s instructions. Each experiment was analyzed in triplicate.
Reverse transcription (RT)-PCR
After 24 h of treatment, total RNA from HaCaT cells was isolated using Trizol reagent according to the manufacturer’s instructions (Invitrogen Life Technologies, Carlsbad, CA). mRNA expression was determined by real-time PCR using SYBR green master mix in a BioRad CFX Connect Real-Time PCR Detection System (BioRad, Hercules, CA). The primer pairs as follows: MMP-1, sense 5′-TGGGAGGCAAGTTGAAAAGC-3′, antisense 5′-CATCTGGGCTGCTTCATCAC-3′; PIP sense 5′-CACAGACAGCTATGACGTG-C-3′ and antisense 5′-TCAGCAGAGAAGACCACCTG-3′. The PCR condition was carried out with an initial denaturation step at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 5 s. The GAPDH gene was used for internal normalization (GAPDH sense 5′-CCAAGGAGTAAGACCCCTGG-3′ and antisense 5′-AGGGGAGATTCAGTGTGGTG-3′). PCR products were separated by 2% agarose gel electrophoresis with ethidium bromide staining.
The data are presented as mean ± standard deviation values of three independent experiments. Statistical analysis was performed using one-way ANOVA test. Statistical significance was set at P < 0.05.
Results and discussion
Synthesis and characterization of AgNPs
EDAX analysis of S-AgNPs
The peaks at 2174 cm−1, 2158 cm−1, 2030 cm−1, 2008 cm−1, 1976 cm−1 and 1722 cm−1 in comfrey extract were lost intensity in the S-AgNPs, which indicated the involvement of these corresponding groups during the reduction of silver to silver nanoparticles.
Effect of AgNPs on DPPH activity
Toxicity analysis of S-AgNPs
Inhibitory effect of S-AgNPs on the production of MMP-1 and IL-6
Interleukin (IL)-6 is a pro-inflammatory cytokine, which results in overexpression of MMPs and eventually photoaging . We next tested the effect of S-AgNPs on the secretion of IL-6. Our data suggested that UVB irradiation also resulted in a severe increase of the pro-inflammatory cytokine IL-6. After, the treatment with S-AgNPs significantly calmed the activation of the IL-6 protein, which showed an inhibition rate of 17.8% and 54.4% at 10 μg mL−1 and 100 μg mL−1 (Fig. 8b).
The present study demonstrated the biological synthesis of irregular-shaped S-AgNPs using S. officinale leaf extract. The method was fast, simple, eco-friendly and cheap. Further, the S-AgNPs prevented UVB-induced photoaging in HaCaT cells. S-AgNPs alleviated the UVB-induced skin damage by suppressing the production of MMP1 and IL-6. On the other hand, S-AgNPs increases the production of procollagen type 1. These findings suggest that S-AgNPs are a promising agent in protection against photoaging, however, further studies including in vivo needs to be done.
This work was conducted under the industrial infrastructure program (Grant no. N0000888) for fundamental technologies which is funded by the Ministry of Trade, Industry, and Energy (MOTIE, Korea).
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