There exists a balance between the synthesis and degradation of the matrix proteins (collagen, elastin, glycosaminoglycans (GAGs) like hyaluronic acid) in healthy, radiant skin. This is maintained by degradation of the redundant matrix by the corresponding proteinases coupled with its replacement by continual synthesis. However, both chronological as well as photo – aging disrupts this balance by coupling the upregulation of the proteinases, thus enhancing degradation of the proteins, with downregulation of their synthesis. This disparity can be improved by either stimulating the synthesis of the proteins or by altering their breakdown by proteinases.1,2,3,4,5,6,7,8 In addition to maintaining the skin integrity, these proteinases also play a vital role in inflammatory responses to photo – aging.9,10
For this purpose, a variety of skin rejuvenation formulations containing ethanolamines, sodium lauryl sulphates, polypeptides or oligopeptides (copper peptides, palmitoyl pentapeptides), tretinoin etc. are available that are either aimed at improving the synthesis of the matrix proteins (except elastin) or inhibiting the proteinases.11,12,13,14 However, the inadequate efficacies of these formulations due to the variations in skin types coupled with an increase in consumer suspicion about their adverse effects (allergic contact dermatitis, irritant contact dermatitis, phototoxic and photoallergic reactions) eventually led to an incessant search for novel bioactives to replace their synthetic counterparts in the cosmetic formulations.11 The green cosmetic industry therefore, has been for long, leveraging on the bioactive power packs - algae as moisturizing as well as thickening agents.15 The secondary metabolites of microalgae with antioxidant, anticancer, antibacterial, anti-inflammatory, anti – aging, anti-allergic properties16,17 have made them an attractive alternative to their synthetic equivalents for cosmeceutical formulations. According to one of our previous studies, methanolic extracts of Chlorella emersonii KJ725233, an isolate from western Maharashtra, exhibited higher antioxidant capacity as compared to other solvent extracts18 and hence was subjected to an array of anti - aging and toxicity tests.
The anti – aging potential was evaluated for elastase, hyaluronidase, collagenase activity inhibition assays. Radical scavenging potential was determined by 1,1-diphenyl-2-picrylhydrazyl radical scavenging assay along with its cytotoxicity by eye irritation potential and sulforhodamine B assay using 3T3 fibroblast cell line. Being a novel isolate it was also subjected to GC – HRMS analysis so as to identify its possible anti-aging constituents.
MATERIALS AND METHODS
BG – 11, calcium chloride and DPPH were purchased from HiMedia, India. Methanol, dimethyl sulfoxide (DMSO) and aluminium trichloride of AR grade were obtained from S D Fine-chem ltd., India. SAAANA, porcine pancreatic elastase, bovine hyaluronidase, p- dimethyl amino benzaldehyde, bovine collagen, N – [Tris(hydroxymethyl)methyl] – 2 – aminoethanesulfonic acid (TES buffer), ninhydrin, citric acid, sulforhodamine B, tween 20, triton X – 100 and sodium lauryl sulphate of molecular biology grade were procured from Sigma.
All the tests have been performed in triplicate and the values are expressed as mean ± SD.
Culturing of Chlorella emersonii KJ725233 and preparation of its extract
Chlorella emersonii KJ725233 was grown in BG – 11 medium in 12 h: 12 h light: dark conditions with 12 h of aeration at 32±1°C. After 20 d of incubation, the biomass was harvested by centrifugation at 5000 rpm for 15 min and dried at 60°C for 24 h. The dried biomass was suspended in methanol at a concentration of 0.1 mg/mL and sonicated for 30 min. The methanolic suspension was then centrifuged at 5000 rpm for 15 min and the supernatant transferred to a crucible for drying. The mass was subjected thrice to sequential extractions and all the supernatants were pooled. 1 μL of this extract was subjected to for its metabolite composition by GC-HRMS (Agilent Technologies, USA) analysis whereas the remaining extract was allowed to dry at 28± 2°C and then reconstituted in DMSO so as to achieve a concentration of 1 mg/mL.18
Anti – aging activity
The anti – aging activity was determined in terms of its in vitro elastase, hyaluronidase and collagenase inhibition activity.
The anti – elastase activity was determined spectrophotometrically by using SAAANA substrate as described by Thring et al.7 Porcine pancreatic elastase enzyme was incubated with varying concentrations of the extract (100 μg/mL, 500 μg/mL, 1000 μg/mL). After 15 min of incubation the substrate was added and the reaction was further incubated for 30 min at 25°C. The absorbance was read at 410 nm. Epigallocatechin was assayed as a positive control.
Anti - Hyaluronidase activity was spectrophotometrically determined by measuring the amount of N-acetyl glucosamine produced by the action of bovine hyaluronidase on sodium hyaluronate as earlier reported by Lee et al.19 Bovine hyaluronidase was added to varying concentrations of the extract (100 μg/mL, 500 μg/mL, 1000 μg/mL) and incubated at 37°C for 20 min. Hyaluronidase was activated by adding 12.5 mM calcium chloride to the reaction mixture and was further incubated at 37°C for 20 min. After the incubation, 1.2 mg/mL sodium hyaluronate was added and incubated at 100°C in a water bath for 3 min. The reaction mixture was allowed to cool at room temperature, after which 1.5 mL of p-dimethyl amino benzaldehyde was added to it and incubated at 37°C for 20 min. The absorbance was measured at 585 nm. Gallic acid was used as a positive control.
Anti-collagenase activity was determined as earlier described by Mandl et al and Moore et al.20,21 Collagenase was incubated with varying concentrations of extract at 37°C for 30 min. To 25 mg of bovine collagen, 0.05 M TES buffer was added and incubated at 37°C for 15 min. After incubation, 0.1 mL of the treated collagenase was added to the collagen containing mixture and allowed to react. After 5 h, 0.2 mL of this reaction mixture was transferred to tubes containing 1.0 mL of ninhydrincitric acid mixture and was incubated in a boiling water bath for 20 min. After cooling 5 mL of 50% n-propanol was added to it and allowed to stand for 15 min. The absorbance was measured at 600 nm.. Catechin was used as a positive control.
Radical scavenging activity
The radical scavenging potential was determined by using DPPH as described by Bendaoud et al.22 To 150 μL of extract concentrations ranging from 0.2 – 1 mg/mL, an equal volume of 0.2 mM methanolic DPPH was added. The reaction mixture was incubated in the dark at 28±2°C for 30 min.
The absorbance was measured at 517 nm. Percent inhibition was calculated by the following formula:
A graph of percent inhibition versus the extract concentration was plotted and IC50 was determined.
Total flavonoid content
The flavonoid content of the methanolic extract of C.emersonii KJ725233 was evaluated by the aluminium trichloride method.23 To 150 μL of the extract, an equal volume of 2% aluminium trichloride was added and incubated in the dark at 28±2°C for 30 min. The absorbance was read at 470 nm. Quercetin was used as a standard and the total flavonoid content was expressed as mg QE/g dried biomass.
Identification of bioactives by gas chromatography – high resolution mass spectrometry
GC- HRMS analysis of all the methanolic extracts were carried out using GC (Agilent Technologies, USA) equipped with Accutoff MS. Compounds were separated on HP-5 MS capillary column having 5% phenyl polysiloxane as stationary phase, column length 30 m, internal diameter 0.32 mm and film thickness 0.25 μm. 1 μL of the methanolic extracts were injected in the split ratio of 10:1, the injector and transfer line temperature was 250°C and 260°C while the ion source temperature was 200°C. Oven temperature programmed from 80 to 280°C at 10°C min-1; flow rate of carrier gas helium was 1 mL min-1. Compounds were identified by comparing their retention times and mass fragmentation patterns with those of the standards at the NIST library.
Cytotoxicity and Irritation assessment
Cytotoxicity and irritation assessment was determined by the sulforhodamine B assay using 3T3 Swiss albino mouse embryonic fibroblast cell lines. Cells were seeded into a 96 well plate and incubated at 37 °C for 24h in 5% CO2 95% air with 60 – 70% humidity. Varying concentrations of the extracts were added to the cells and incubated for another 24 h. After incubation, the cells were observed under microscope, fixed, washed and dried. The cells were then stained with sulforhodamine B stain, washed and dried. The stain was further solubilized in Tris buffer and the absorbance was read at 530 nm. Tween 20, sodium lauryl sulphate and Triton – X 100 were used as reference standards.24,25
RESULTS AND DISCUSSIONS
Anti – aging activity
Aging leads to a decrease in the synthesis of skin structural proteins like elastin, collagen and hyaluronic acid resulting in the loss of skin elasticity, laxity, integrity eventually leading to the visible signs of aging.1,8 The age dependent downregulation of skin elastic properties is not only attributed to a decreased synthesis of these proteins, but also to an upregulation of the corresponding proteinases.26,27 Elastase, a member of the chymotrypsin family of serine proteinases is mainly responsible for the degradation of elastin.7 Hyaluronic acid is degraded by another serine proteinase – hyaluronidase whereas collagenase a zinc containing matrix metalloproteinase hydrolyzes collagen.7,8 The activity of these proteinases is not only stimulated with chronological aging but also with a prolonged UV – exposure.7 Therefore, the prime mover for anti-aging strategy to achieve healthy, smooth, blemish free and resilient skin is by means of controlling the degradation of the skin structural constituents with regulation of proteinase activity.12,28
With its intrinsically high antioxidant activity, C.emersonii KJ725233 was taken up for assessing its potential as a proteinase inhibitor in the present study. The anti - elastase activity of its methanolic extract was evaluated using SAAANA as a substrate and the percent enzyme inhibition was correlated to the amount of p – nitroaniline formed.7 Significant elastase inhibition of 92.04±2.27% was observed at 1000 μg/mL (Figure 1) with an IC50 of 0.49±0.01 g/mL (Table 1). The IC50 value is inversely proportional to the enzyme inhibition potential. Therefore, a lower IC50 signifies a higher anti – proteinase activity. Although the IC50 value was found to be equivalent to that of C.emersonii 11N i.e 0.42±0.05 mg/mL, it was lower than that of 0.503±0.06 mg/mL for Tetraselmis suecica.28,29
Although the methanolic extract of C.emersonii KJ725233 exhibited a modest anti-hyaluronidase activity of 39.32±0.92% at 1000 μg/mL (Table 1); its IC50 of 1.58±0.06 mg/mL (Figure 1) was fivefold lower than that of Dunaliella tertiolecta (5.542±0.12 mg/mL), Tetraselmis suecica (5.907±0.26 mg/mL) and four fold lower than that of Nannochloropsis species (4.202±0.33 mg/mL) as previously reported.28
The methanolic extract of C.emersonii KJ725233 exhibited a significant anti – elastase and a modest anti – hyaluronidase activity however, its anti – collagenase potential was found to be less significant with a percent inhibition of 20.33±2.90% at 1000 μg/mL (Table 1) with an IC50 of 2.50±0.03 mg/mL (Figure 1). However, the present study is one of the first to report anti-collagenase activity of any microalga, signifying its potential in cosmeceuticals.
Radical scavenging potential and total flavonoid content
Repetitive and prolonged UV exposure leads to an over production of the ROS which induces oxidative cellular stress and promotes genetic alterations thereby affecting matrix proteins structure as well as functions.1 ROS induces the over expression of the matrix metalloproteinases and serine proteinases in the fibroblasts therefore causing the remodelling of skin extracellular matrix, resulting in skin damage.28,29,30 To combat this oxidative stress, there is need to scavenge these free radicals. The methanolic extract of C.emersonii KJ725233 was thus evaluated for its radical scavenging potential by the DPPH method (Figure 2). A graph of percent inhibition versus extract concentration was plotted and the IC50 value was found to be 5.40±0.14 mg/mL. The IC50 of the methanolic extract of C.emersonii KJ725233 was found to be lower than that reported for the commercial Sun chlorella powder of C. pyrenoidosa i.e 9.62 mg/ mL as previously reported.31
DPPH has been widely used for measuring the antioxidant status of phenolic compounds like flavonoids, since they have the ability to donate their hydrogen and thus function as strong free radical scavengers.32,33 Flavonoids are also known to bind metalloenzymes like collagenase via hydrogen as well as hydrophobic interactions, thereby inducing conformational changes and inhibiting it from binding to the substrate.7,34 However, flavonoids not only inhibit collagenase, but also are potent elastase inhibitors.34
The total flavonoid content of the methanolic extract of C.emersonii KJ725233 was determined by the aluminium trichloride method and was found to be 14.26±1.31 mg QE/g dried biomass i.e 158.09±0.23 mg QE/g dried extract which is 20.54% higher as compared to that of 131.15 mg QE/g dried extract of C.vulgaris.35 The coefficient of correlation for DPPH radical scavenging potential and total flavonoid content was found to be 0.9971 signifying the contribution of flavonoids to the radical scavenging potential of the methanolic extract of C.emersonii KJ725233.
Identification of bioactives by GC-HRMS
Since the methanolic extract of C.emersonii KJ725233 exhibited proteinase inhibition as well as radical scavenging potential it was thought worthwhile to identify the bioactives contributing to this activity. The methanolic extract was therefore, subjected to GC-HRMS analysis and the compounds were identified by comparing the mass fragmentation pattern with those in the NIST library as seen in (Table 2).
Phytol - a major contributor of the methanolic extract and its isomer 3,7,11,15 – tetramethyl – 2 – hexadecen – 1 - ol are reported for their antioxidant as well as anti – inflammatory activities.36,37 The hydrocarbon – hexadecane another major contributor of the methanolic extract after phytol is known for its antioxidant as well as anti-inflammatory activities.38,39 9,12 – octadecadienoyl chloride is reported as an anti – inflammatory, cancer preventive, anti – acne, anti – eczema, anti – histaminic etc. whereas 3 – trifluoroacetoxypentadecane is ant oxidative as well as anti-inflammatory in nature.40,41,42
The presence of several anti-inflammatory molecules in the methanolic extract may contribute to the anti-proteinase activity of C.emersonii KJ725233 since the proteinases elastase, hyaluronidase and collagenase are known to be involved in the inflammatory responses.
Cytotoxicity and Irritation assessment
Although the efficiency of bioactives in particular therapeutic approaches is imperative, its safety and absence of adverse cellular side effects is of utmost concern. Hence, along with cytotoxicity, assessment of irritation potential is a vital part of toxicology studies of new materials prior to consumer use.
|Concentration of the extract (μg/mL)||Percent inhibition Elastase||Percent inhibition Hyaluronidase||Percent inhibition Collagenase|
Therefore, to analyze the safety of the methanolic extract of C.emersonii KJ725233 for use as a probable anti-aging agent its cytotoxicity was studied in terms of percent viability of the 3T3 fibroblast cell line (Figure 3) by sulforhodamine B assay.
By plotting a graph of percent viability versus extract concentration the IC50 (Figure 2) was found to be 0.82±0.11 mg/mL. Thus the methanolic extract was found to exhibit cytotoxicity at concentration greater than 0.82±0.11 mg/mL.
The eye irritation potential of the extract was determined in terms of the IC50 values derived from the dose response curves were graded in comparison with those of the reference standards (Figure 4).
As per the Japanese Ministry of Healthcare and welfare guidelines for manufacturing of cosmetics and quasidrugs,25 comparing the IC50 value of the methanolic extract of C.emersonii KJ725233 with those of the reference standards, it can be concluded that it is practically a non – irritant since it has an IC50 higher than all the tested reference samples.
The presence of anti – inflammatory molecules like phytol, its isomer and hydrocarbons, as revealed from the GC-HRMS analysis are known to reduce the proteinases activity thus reducing inflammation of the dermis. The anti – aging properties of Chlorella emersonii KJ725233 thus stem from its ability to inhibit elastase, hyaluronidase, and collagenase and to scavenge free radicals which might aid in supporting skin tissue leading to skin rejuvenation. Due to a plethora of compounds exhibiting anti – aging, antioxidant, anti – inflammatory properties; Chlorella emersonii KJ725233 has a great potential as a source of bioactive for cosmeceuticals.