Anti-elastase Activity Research Articles

Characterization of Novel Missense Variants of SERPINA1 Gene Causing Alpha-1 Antitrypsin Deficiency.

The SERPINA1 gene is highly polymorphic, with more than 100 variants described in databases. SERPINA1 encodes the alpha-1 antitrypsin (AAT) protein, and severe deficiency of AAT is a major contributor to pulmonary emphysema and liver diseases. In Spanish patients with AAT deficiency, we identified seven new variants of the SERPINA1 gene involving amino acid substitutions in different exons: PiSDonosti (S+Ser14Phe), PiTijarafe (Ile50Asn), PiSevilla (Ala58Asp), PiCadiz (Glu151Lys), PiTarragona (Phe227Cys), PiPuerto Real (Thr249Ala), and PiValencia (Lys328Glu). We examined the characteristics of these variants and the putative association with the disease. Mutant proteins were overexpressed in HEK293T cells, and AAT expression, polymerization, degradation, and secretion, as well as antielastase activity, were analyzed by periodic acid-Schiff staining, Western blotting, pulse-chase, and elastase inhibition assays. When overexpressed, S+S14F, I50N, A58D, F227C, and T249A variants formed intracellular polymers and did not secrete AAT protein. Both the E151K and K328E variants secreted AAT protein and did not form polymers, although K328E showed intracellular retention and reduced antielastase activity. We conclude that deficient variants may be more frequent than previously thought and that their discovery is possible only by the complete sequencing of the gene and subsequent functional characterization. Better knowledge of SERPINA1 variants would improve diagnosis and management of individuals with AAT deficiency.

Déficit de Alfa-1-antitripsina y su tratamiento

Alpha-1 antitrypsin deficiency (AATD) is a genetic disorder caused by a mutation in the codifying gene for the alpha-1 antitrypsin (AAT) protein, which has anti-elastase activity. While there is extensive genetic variability, the most common genotypes associated with AATD are PI*Z y PI*S. Most clinical manifestations are pulmonary (i.e. emphysema) and hepatic, though other pathologies, such as panniculitis and Wegener’s granulomatosis, can also present with this disorder. To date, there is no cure for AATD, and treatment options are focused on reducing the frequency of exacerbations and relieving symptoms; among these, augmentation therapy,
 which raises AAT levels through serum injections of the protein to correct the deficit, has been the most effective. Novel findings of this therapy are discussed, along with research efforts that explore new treatment options based on gene therapy or the use of protein chaperones, for instance. In Chile, no epidemiological studies on AATD have been conducted, so it is unknown exactly how many individuals are affected or if they receive adequate treatment.

Role of the P2 residue of human alpha 1-antitrypsin in determining target protease specificity.

Alpha 1-antitrypsin (A1AT) is a serine protease inhibitor that mainly inhibits neutrophil elastase in the lungs. A variant of A1AT at the P1 position with methionine 358 to arginine (A1AT-Pittsburgh) is a rapid inhibitor of thrombin with greatly diminished anti-elastase activity. The P2 residue (position 357) of A1AT-Pittsburgh has been shown to play an important role in interactions with thrombin and kallikrein, but the role of P2 residue in wild-type A1AT has largely been unraveled. Here, we investigated the effects of P2 proline substitutions in wild-type A1AT on interactions with porcine pancreatic elastase (PPE) and human neutrophil elastase (HNE). The mutant A1AT proteins (P357A, P357D, P357K, P357L, P357N, P357S, and P357W) were less efficient than the wild-type A1AT at inhibiting PPE and HNE. Among the mutants, P357D did not form a complex with PPE, whereas P357L, P357N, and P357W showed significantly reduced complex formation with PPE. Surprisingly, mass spectrometry analysis revealed that P357D had two cleavage sites after the P9 alanine and the P3 isoleucine residues. Our results indicate that the size and negative charge of the R group of the P2 residue influence the interaction with elastases. Specifically, the negative charge at the P2 residue is disfavored and the resulting conformational changes in the reactive center loop upon interaction with PPE lead to cleavage at new sites. Overall, the results of this study demonstrate a previously unknown role for P2 residue in determining inhibitory specificity of A1AT.

Inhibitory effect of burdock leaves on elastase and tyrosinase activity.

Burdock (Arctium lappa L.) leaves generate a considerable amount of waste following burdock root harvest in Taiwan. To increase the use of burdock leaves, the present study investigated the optimal methods for producing burdock leaf extract (BLE) with high antioxidant polyphenolic content, including drying methods and solvent extraction concentration. In addition, the elastase and tyrosinase inhibitory activity of BLE was examined. Burdock leaves were dried by four methods: Shadow drying, oven drying, sun drying and freeze-drying. The extract solution was then subjected to total polyphenol content analysis and the method that produced BLE with the highest amount of total antioxidant components was taken forward for further analysis. The 1,1-diphenyl-2-pycrylhydrazyl scavenging, antielastase and antityrosinase activity of the BLE were measured to enable the evaluation of the antioxidant and skin aging-associated enzyme inhibitory activities of BLE. The results indicated that the total polyphenolic content following extraction with ethanol (EtOH) was highest using the freeze-drying method, followed by the oven drying, shadow drying and sun drying methods. BLE yielded a higher polyphenol content and stronger antioxidant activity as the ratio of the aqueous content of the extraction solvent used increased. BLE possesses marked tyrosinase and elastase inhibitory activities, with its antielastase activity notably stronger compared with its antityrosinase activity. These results indicate that the concentration of the extraction solvent was associated with the antioxidant and skin aging-associated enzyme inhibitory activity of BLE. The reactive oxygen species scavenging theory of skin aging may explain the tyrosinase and elastase inhibitory activity of BLE. In conclusion, the optimal method for obtaining BLE with a high antioxidant polyphenolic content was freeze-drying followed by 30-50% EtOH extraction. In addition, the antielastase and antityrosinase activities of the BLE produced may be aid in the development of skincare products with antiwrinkle and skin-evening properties.

ANTI-ELASTASE, ANTI-COLLAGENASE AND ANTIMICROBIAL ACTIVITIES OF THE UNDERUTILIZED RED PITAYA PEEL: AN IN VITRO STUDY FOR ANTI-AGING APPLICATIONS

Objective: To investigate the in vitro anti-elastase, anti-collagenase, and antimicrobial activities of the red pitaya peel extract for cosmetic application focusing on skin aging.Methods: Extraction was performed by the reflux method for 103 minutes at 56°C with 82% aqueous ethanol solution and the red pitaya peel extract was evaporated using a rotary evaporator. Anti-elastase and anti-collagenase properties were evaluated using the drug discovery kits (neutrophil elastase colorimetric and matrix metalloproteinase-1 colorimetric, respectively). The antimicrobial potential was analyzed using agar well diffusion method against 10 selected microorganisms, and the presence or absence of the inhibition zones was identified.Results: The red pitaya peel extract exhibited remarkable inhibition percentage 87.62±0.05% and 96.92±0.02% for anti-elastase and anti-collagenase activities, respectively. Red pitaya peel extract showed significant inhibition against the Gram-positive Bacillus subtilis B29 with an inhibition zone diameter of 8.0±0.3 mm.Conclusion: The excellent anti-aging properties displayed by the underutilized red pitaya peel extract highlighted its potential as a natural source of anti-aging agent for cosmetic formulations.

English

Collagen is the main structural component of connective tissues in vertebral and invertebral animal tissues and organs. The collagen isolated from the body wall of sea cucumber (Holothuria lecospilota) can be used in the pharmaceutical field. The research aimed at isolating collagen from the sea cucumber body wall and testing the anti-tyrosinase and anti-elastase activity. This was done by isolation of collagen by soaking the body wall of sea cucumber using Tris-HCl-EDTA 0.1 M and dialyzing using 0.5 M acetic acid. The step was followed by characterization of collagen using FT-IR spectrophotometer and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The resulting collagen was tested for anti-tyrosinase and anti-elastase in vitro. The research showed that the resulting collagen was of the amide group with molecular weight of 166.43 and 138.35 kDa, showing α1 and α2 chains. Anti-tyrosinase activity test showed an IC50 value of 1.20 mg/mL, while anti-elastase activity test showed an IC50 value of 125 µg/mL. The collagen isolated from the sea cucumber has weak anti-tyrosinase and moderate anti-elastase activity. Key words: Crude collagen, anti-tyrosinase, anti-elastase, sea cucumber (Holothuria leucospilota).

Antioxidant, Anti-tyrosinase, and Anti-elastase Activities of Alternative Tea Materials (Mugwort, Bower Actinidia, Arrowroot) by Harvest Time and Region: A Comparative In Vitro Study

Antioxidant, Anti-tyrosinase, and Anti-elastase Activities of Alternative Tea Materials (Mugwort, Bower Actinidia, Arrowroot) by Harvest Time and Region: A Comparative In Vitro Study

The anti-dermatophyte activity of Zataria multiflora essential oils

Dermtophytes are a group of pathogenic fungi and the major cause of dermatophytosis in humans and animals. Fighting dermatophytes by natural essential oils is one important issue in new researches. In this investigation, we evaluated the anti-dermatophyte activities of three samples of Z.multiflora essential oils against dermatophytes along with analysis of chemical compositions of the essential oils and their anti-elastase activities on elastase production in dermatophytes. Carvacrol (1.5-34.4%), thymol (25.8-41.2%), carvacrol methyl ether (1.9-28.3%) and p-cymene (2.3-8.3%) were the main components of Z.multiflora essential oils. Z.multiflora essential oils (100ppm) inhibited the mycelium growth of dermatophytes (6±1.7-47.0±1.4%) and had the minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) values of 0.03-0.25μl/ml against dermatophytes. Essential oils inhibited elastase produced in dermatophytes and pure porcine elastase. Z.multiflora essential oils can be used as natural anti-dermatophyte agent for fighting dermatophytes in further preclinical and clinical studies.

Greek flora as a source of new anti-oxidant, anti-elastase, anti-collagenase and anti-hyaluronidase natural agents

In order to investigate new cosmetic ingredients of natural origin, 50 plant extracts obtained from Greek flora were screened for their capacity to scavenge free radicals and inhibit enzymes related to skin ageing. Degradation of extracellular matrix (ECM) has directly been linked to skin ageing and is correlated to increase in activity of enzymes such as elastase, collagenase and hyaluronidase. Elastin, collagen and hyaluronic acid decrease, leading to a loss of strength and flexibility in skin and formation of deep wrinkles. Also free radicals induce the activity of these enzymes [1,2]. Discovery of bioactive molecules which show both anti-oxidant and anti-ageing properties is the target of the present study. Anti-elastase, anti-collagenase and anti-hyaluronidase activities of 50 methanolic and ethyl acetate extracts were determined using spectrophotometric methods. Radical scavenging activity was determined by the ability of the extracts to scavenge DPPH and ABTS radicals. The majority of the samples especially the methanolic extracts showed high anti-oxidant capacity, e.g. Rosa damascena extract exhibited IC50 value at 47.6 µg/ml. In anti-elastase assay 8 extracts showed more than 50% inhibition at 100 µg/ml, in anti-collagenase assay 15 extracts showed more than 60% inhibiton at 100 µg/ml and in anti-hyaluronidase assay 8 extracts showed more than 80% inhibition at 300 µg/ml. The methanolic extracts of Sedum sediforme and Umbilicus horizontalis aerial parts inhibited elastase and hyaluronidase by more than 85%. Rosa damascena inhibited 80% of collagenase activity at 100 µg/ml. Antioxidant and enzyme inhibitory activities of the plant extracts suggest that they can restore skin elasticity and delay wrinkling process. Bioguided isolation process was performed in order to reveal the active secondary metabolites. In the case of Rosa damascena quercetin-3-O-rhamnoside, kaempferol-3-glucopyranoside, phenylethanol were proved to exhibit high anti-collagenase activity.

Viburnum mullaha D. DON fruit (Indian cranberry): A potential source of polyphenol with rich antioxidant, anti-elastase, anti-collagenase, and anti-tyrosinase activities

ABSTRACTViburnum mullaha (Buch.-Ham. Ex D. Don), is an underexplored wild edible fruit of Indian Himalayan region, analyzed for total polyphenol contents, antioxidant, anti-elastase, anti-collagenase, and anti-tyrosinase activities using in vitro assays. High values of total phenolic contents of 1257 ± 40 mg gallic acid equivalents/100 g fruit weight and total flavonoid contents of 3501 ± 203 mg catechin equivalents/100 g fruit weight were observed. V. mullaha fruit extracts showed outstanding antioxidant activities (ABTS+, 1,1-diphenyl-2-picrylhydrazyl, superoxide anion, linoleate peroxyl radicals scavenging, and ferric reducing activities) and remarkable anti-elastase, anti-collagenase, and anti-tyrosinase activities. High resolution liquid chromatography–mass spectroscopy analysis revealed presence of 15 phenolic compounds, namely, chlorogenic acid, acetyl salicylic acid, dihydrorobinetin, dihydromyricetin, 2-isoprenylemodin, rutin, cosmosiin hexaacetate, pectolinarin, dihydroquercetin, eriodictyol, iriginol hexaacetate, theaflavin, epicatechin-pentaacetate, lomatin, and peucenin in fruit extracts. This study recommends utilization of V. mullaha fruit as functional food with prospective pharmaceutical, nutraceutical, and cosmeceutical properties.

115 - Plant-Based Production, Purification, and Characterization of Oxidation-Resistant Alpha-1 Antitrypsin

Alpha-1 antitrypsin (AAT) is an acute phase protease inhibitor. Currently, the only available treatment for patients with COPD caused by AAT deficiency is replacement therapy, requiring weekly infusions of human plasma-derived AAT. While AAT has shown potential when inhaled to treat both emphysema and cystic fibrosis, the oxidation susceptibility of methionine residues at positions 351 and 358 in the reactive site loop have led to difficulties in maintaining AAT activity and stability when aerosolized. Using a novel cucumber mosaic virus-based inducible transient expression system, we have produced a biobetter plant recombinant AAT (prAAT) in Nicotiana benthamiana . This variant replaces the oxidation-susceptible methionine residue at position 358 with a valine residue to increase resistance to oxidation. N. benthamiana plants were vacuum infiltrated as detached leaves with two strains of Agrobacterium tumefaciens : one carrying the biobetter AAT gene and one carrying a gene for the tomato bushy stunt virus p19 viral RNA silencing suppressor driven by a constitutive cauliflower mosaic virus 35S promoter. Leaves were incubated in humidity chambers at 20°C in the dark for 6 days before flash freezing in liquid nitrogen, grinding, and protein extraction in a 20 mM Tris, 150 mM NaCl, and 0.01% (v/v) Tween-80 AAT stability buffer. Purification was achieved using Alpha-1 Antitrypsin Select Affinity Chromatography (GE Healthcare) with a 58.1±8.1% yield of active AAT. Activity of purified AAT was confirmed via a band shift in a Western blot after incubation with excess porcine pancreatic elastase, however, degradation of prAAT was also observed in all purified extracts. Purified prAAT was tested against an analytical standard (Calbiochem) and Prolastin-C (Griffols), a common therapeutic formulation of human AAT, to determine its activity and oxidation resistance. 41±11% of purified prAAT and 71.9±5.5% of Prolastin-C showed activity against porcine pancreatic elastase as determined by a residual elastase activity inhibitory assay. Under oxidation with 48.9 mM H 2 O 2 for 60 minutes at room temperature, prAAT was found to retain 102.8±20.4% of its original anti-elastase activity, while Prolastin-C retained 13.8±5.0% anti-elastase activity and analytical standard AAT retained 11.6±7.5% anti-elastase activity. Future work will include determinations of oxidation resistance to OCl – /HOCl and development of more optimal isolation and purification processes.

Biological activities of triterpenoids from Poraqueiba sericea stems

Eleven compounds were isolated from Poraqueiba sericea stems and identified as niga-ichigoside-F1 (1), trachelosperoside B1 (2), 4-epi-niga-ichigoside (7), 19α-hydroxyasiatic acid (3), myrianthic acid (4), hyptatic acid (5), trachelosperogenin B (6), arjunolic acid (8), and trachelosperogenin E (9), secologanoside (10) and secoxyloganin (11). Compounds 1–11 were tested for their antileishmanial activities against Leishmania infantum promastigotes, 1–6 and 8–11 were tested for their cytotoxic activities on fibroblasts, 1–3, 5–6, 8–11 were evaluated for their anti-elastase and anti-acetylcholinesterase assays activities by a spectrophotometric method and 1–2, 5 and 7–10 were tested using bioautography for their β-glucosidase. No antileishmanial activity was detected; compounds 1, 2 and 11 showed a moderate cytotoxic activity with IC50 17.7, 20.5 and 10.9 μg/mL, respectively; compounds 2, 8, 9 and 10 gave a percentage of inhibition ranging from 13 to 16% (at 50 μg/mL) and compounds 1 and 2 showed an inhibition zone on β-glucosidase and anti-acetylcholinesterase assays.

Residual biomasses and protein hydrolysates of three green microalgae species exhibit antioxidant and anti-aging activity

It is widely recognized that microalgae represent a promising strategy in the development of next-generation biofuels; however, their economic viability remains questionable. Several studies have shown microalgal biomass is a relatively unexplored source of natural components with nutraceutical activities; indeed, some authors state that the remaining residue after oil extraction from the microalgae biomass, known as microalgae residual biomass (MRB), could have outstanding biological activities. Therefore, in the present study, we analyzed the antioxidant capacity and anti-aging potential of MRB and protein hydrolysates of three green microalgae species: Dunaliella tertiolecta, Tetraselmis suecica, and Nannochloropsis sp. Antioxidant capacity, determined through oxygen radical absorbance capacity (ORAC), radical cation activity (ABTS), and radical scavenging activity (DPPH), of the three MRBs was good overall, with D. tertiolecta and Nannochloropsis having the highest antioxidant capacity. Interestingly, their protein hydrolysates showed markedly higher antioxidant capacities than their MRBs at the concentrations tested. Anti-aging potential, determined by the inhibition of the activity of two extracellular matrix-degrading enzymes, elastase and hyaluronidase, was found to be higher in D. tertiolecta and Nannochloropsis biomasses for anti-elastase activity; however, no significant differences between microalgae species were found in anti-hyaluronidase activity. For the protein hydrolysates, Nannochloropsis had the highest anti-elastase and anti-hyaluronidase activity. In conclusion, selected MRBs and their protein hydrolysates exhibited a good antioxidant capacity and/or anti-aging potential overall, indicating the economical importance of MRBs in the nutraceutical and nutricosmetic industries. Overall, this study contributes to convert microalgae biofuel production into a more sustainable and economically feasible process, through the comprehensive utilization of residual biomass.

Results from in vitro and ex vivo skin aging models assessing the antiglycation and anti-elastase MMP-12 potential of glycylglycine oleamide.

BackgroundGlycation is an aging reaction of naturally occurring sugars with dermal proteins. Type I collagen and elastin are most affected by glycation during intrinsic chronological aging.AimTo study the in vitro and ex vivo assays in human skin cells and explants and the antiaging effects of glycylglycine oleamide (GGO).Materials and methodsThe antiglycation effect of GGO was assessed in a noncellular in vitro study on collagen and, ex vivo, by immunohistochemical staining on human skin explants (elastin network glycation). The ability of GGO to contract fibroblasts was assessed in a functional assay, and its anti-elastase (MMP-12) activity was compared to that of oleic acid alone, glycylglycine (GG) alone, and oleic acid associated with GG.ResultsIn vitro, GGO reduced the glycation of type I collagen. Ex vivo, GGO restored the expression of fibrillin-1 inhibited by glycation. Furthermore, GGO induced a tissue retraction of almost 30%. Moreover, the MMP-12 activity was inhibited by up to 60%.ConclusionUnder the present in vitro and ex vivo conditions, GGO prevents glycation of the major structural proteins of the dermis, helping to reduce the risk of rigidification. By maintaining the elastic function of the skin, GGO may be a promising sparring partner for other topical antiaging agents.

Antioxidant, anti-collagenase and anti-elastase activities of Phyllanthus emblica, Manilkara zapota and silymarin: an in vitro comparative study for anti-aging applications

Context Phyllanthus emblica L. (Euphorbiaceae) (amla), Manilkara zapota L.P. Royen (Sapotaceae) (sapota) and silymarin are reported to contain antioxidant effects. However, information on other biological activities relating to the anti-aging properties is limited.Objective To compare in vitro antioxidants, anti-collagenase (MMP-1 and MMP-2) and anti-elastase properties as well as the phenolic and flavonoid contents of amla, sapota and silymarin as potential anti-aging ingredients.Materials and methods The ethanol amla and sapota fruit extracts were prepared by three cycles of maceration with 24 h duration each. The total phenolic (TPC) and flavonoid (TFC) contents were determined. The antioxidant capacity was evaluated by DPPH and ABTS assays. The effects of MMP-1, MMP-2 and elastase inhibitions were determined by using the EnzChek® assay kits (Molecular-Probes, Eugene, OR).Results Amla exhibited the highest in TPC (362.43 ± 11.2 mg GAE/g) while silymarin showed the highest in TFC (21.04 ± 0.67 mg QE/g). Results of antioxidant activity by DPPH and ABTS methods showed that amla possessed the most potent capacity with IC50 values of 1.70 ± 0.07 and 4.45 ± 0.10 μg/mL, respectively. Highest inhibitions against MMP-1, MMP-2 and elastase were detected for sapota with IC50 values of 89.61 ± 0.96, 86.47 ± 3.04 and 35.73 ± 0.61 μg/mL, respectively.Discussion and conclusion Test extracts offered anti-aging properties in different mechanisms. Amla showed the highest phenolic content and antioxidant property with moderate anti-collagenase. Silymarin exhibited measurable flavonoid content with anti-elastase effect. Sapota showed the highest collagenase and elastase inhibitions with moderate antioxidant effect. Thus, extracts might be added as a mixture to gain the overall anti-aging effects.

Fruits of selected wild and cultivated Andean plants as sources of potential compounds with antioxidant and anti-aging activity

It is known that most of the species growing in high-elevation environments in tropical mountains remain largely unexplored. In particular, little is known from a chemical and biological standpoint. The aim of this study was to conduct a preliminary screening of the effects of selected wild and cultivated Andean plants characterized by small and colorful fruits on collagenase, elastase, hyaluronidase and tyrosinase, along with antioxidant capacity, to identify possible sources of potential anti-aging substances for the development of natural cosmetic products. Sixty-five samples consisting of methanol extracts of the fruits of thirty-five Andean plants at different stages of growth were used in the study. The anti-collagenase, anti-elastase, anti-hyaluronidase and anti-tyrosinase activities were determined by spectrophotometric and fluorometric assays. The antioxidant capacity was measured by oxygen radical absorbance capacity (ORAC) and trolox equivalent anti-oxidant capacity (TEAC) assays. The total phenolic content was determined by the Folin–Ciocalteu method. Liquid–liquid partitions of extracts of interest were performed, and each fraction was tested. The inhibitory activity against skin aging-related enzymes and antioxidant properties has provided evidence for the potential utility of Alchornea triplinervia, Gaultheria erecta, Rubus compactus and Ugni myricoides, which were selected as plants of interest. In addition, the maturity state of fruits alters the properties of extracts in a statistically significant manner. In the vast majority of cases, the immature fruits presented a higher antioxidant capacity and enzymatic inhibitory activity. The extracts, fractions and sub-fractions of G. erecta and U. myricoides fruits showed effective inhibitory activity against skin aging-related enzymes and antioxidant properties, which supports the need to more extensively examine extracts from this plant material. The results of this study illuminated possible ways in which to add value to fruits of thirty-five wild and cultivated Andean plants based on their potential use as anti-aging ingredients in cosmetic formulations. After additional studies of the security and isolation of the active compounds, new cosmetic ingredients useful for anti-aging formulations could be obtained.

Evaluation of Skin Anti-aging Potential of Citrus reticulata Blanco Peel.

Background:The peel of Citrus reticulata Blanco is traditionally used as tonic, stomachic, astringent, and carminative. It is also useful in skin care.Objective:To study the anti-aging potential of alcoholic extracts of C. reticulata Blanco peel using in vitro antioxidant and anti-enzyme assays.Materials and Methods:Plant extracts were obtained by Soxhlation (CR HAE- Hot Alcoholic Extract of Citrus reticulata) and maceration method (CR CAE- Cold Alcoholic Extract of Citrus reticulata). Qualitative and quantitative phytochemical analysis was performed. Further, in vitro antioxidant, anti-enzyme, and gas chromatography-mass spectrometry (GC-MS) analyses were performed.Results:Total phenolic and flavonoid contents of CR HAE were found to be higher than CR CAE. EC50 value of CR HAE and CR CAE for 1,1-Diphenyl-2-picrylhydrazyl, Superoxide anion, and 2, 2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) assays were 250.33 ± 40.16 μg/ml and 254.73 ± 15.78 μg/ml, 221.27 ± 11.25 μg/ml and 354.20 ± 23.79 μg/ml, and 59.16 ± 2.17 μg/ml and 59.12 ± 6.21 μg/ml, respectively. Oxygen radical absorbance capacity values for CR HAE and CR CAE were found to be 1243 and 1063 μmoles 6-hydroxy-2,5,7,8-tetra methylchromane-2-carboxylic acid equivalent/g of substance, respectively. Anti-collagenase and anti-elastase activities were evaluated for both CR HAE and CR CAE. EC50 values of CR HAE and CR CAE for anti-collagenase and anti-elastase were 329.33 ± 6.38 μg/ml, 466.93 ± 8.04 μg/ml and 3.22 ± 0.24 mg/ml, 5.09 ± 0.30 mg/ml, respectively. CR HAE exhibited stronger anti-collagenase and anti-elastase activity than CR CAE. GC-MS analysis of CR HAE was carried out because CR HAE exhibited higher antioxidant and anti-enzyme potential than CR CAE.Conclusion:C. reticulata peel can be utilized in anti-wrinkle skin care formulations.SUMMARY Skin anti-aging potential of Citrus reticulata Blanco peel was evaluated throughIn vitro antioxidant and anti-enzyme assaysTwo types of extraction were performed and extracts were subjected to qualitative and quantitative phytochemical analysis. Extract obtained by Soxhlation (CR HAE) showed higher total phenolic and flavonoid contents than extract obtained by maceration (CR CAE)CR HAE demonstrated strong DPPH and Superoxide free radical scavenging activity whereas, ABTS scavenging activity of both the extracts were found to be similar. Oxygen Radical Absorbance Capacity (ORAC) of CR HAE was found to be more; indicating its strong antioxidant potentialIn vitro collagenase and elastase enzyme inhibition activities were evaluated for both the extracts and CR HAE showed strong anti-collagenase and antielastase potential indicating its anti-aging abilityGC-MS analysis of CR HAE revealed the presence of various compounds mainly including Polymethoxyflavones. CR HAE exhibited promising antioxidant and anti-enzymatic activity and can be used as a potent antiwrinkle agent in anti-aging skin care formulations. Abbreviation Used: ECM: Extracellular matrix, UV: Ultra violet, ROS: Reactive Oxygen Species, MMP: Matrix metalloproteinase, Chc: Clostridium histolyticum collagenase, DPPH: 2, 2-diphenyl-1-picrylhydrazyl, GC-MS: Gas Chromatography-Mass Spectroscopy, RT: Room Temperature, μg GAE/ mg: Microgram Gallic acid equivalent / milligram, W/V: Weight by Volume, μg QE/ mg: Microgram Quercetin equivalent / milligram, CR HAE: Hot Alcoholic Extract of Citrus reticulata Blanco, CR CAE: Cold Alcoholic Extract of Citrus reticulata Blanco, EC50: Half Maximal Effective Concentration, PMS NADH: Phenazine methosulfate nicotinamide adenine dinucleotide, NBT: Nitroblue tetrazolium, DMSO: Dimethyl sulfoxide, APS: Ammonium Persulphate, AAPH: 2,2 -azobis(2-amidino-propane) dihydrochloride, TROLOX: (±) 6-hydroxy-2,5,7,8-tetramethyl chromane-2-carboxylic acid, ORAC: Oxygen Radical Absorbance Capacity, FALGPA: N-[3-(2-Furyl) acryloyl)]-Leu-Gly-Pro-Ala, SANA: Succinyl-Ala-Ala-Ala-p-nitroanilide, Rf: Retardation Factor, MSD: Mass Selective Detector

Evaluation and comparison of polyphenols and bioactivities of wild edible fruits of North-West Himalaya, India

ABSTRACT Objective To evaluate and compare the polyphenol contents, antioxidant, anti-elastase, anti-collagenase, anti-tyrosinase and anti-inflammatory activities of 13 wild edible fruits [ Pyracantha crenulata, Berberis asiatica ( B. asiatica ) , Ficus subincisa ( F. subincisa ) , Morus serrata, Ziziphus nummularia, Leea asiatica ( L. asiatica ) , Dendrobenthamia capitata, Ziziphus mauritiana, Prunus cerasoides, Ampelocissus latifolia ( A. latifolia ) , Vitis jacquemontii, Morus alba and Grewia optiva ] of North-West Himalayan Region of India. Methods Fruits extracts were prepared with 80% aqueous acetone and evaluated for total phenolic contents (TPC) and total flavonoid contents (TFC). Free radical scavenging activities [against 1,1-diphenyl-2-picryl-hydrazyl, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), linoleate hydroperoxyl and superoxide radicals], ferric reducing ability, ferrous metal chelating capacity, anti-elastase, anti-collagenase, anti-tyrosinase and anti-inflammatory activities were determined by using various in vitro assays. Results TPC varied from 58.83 to 4 496.39 mg gallic acid equivalents/100 g fruit weight (FW), being highest in A. latifolia and lowest in F. subincisa . TFC ranged from 108.00 to 1 963.75 mg catechin equivalents/100 g FW, standing highest in L. asiatica and lowest in Prunus cerasoides. A. latifolia and L. asiatica possessed the highest antioxidant activities while B. asiatica and L. asiatica owned uppermost anti-elastase and anti-collagenase activities, respectively. B. asiatica revealed the highest anti-tyrosinase activity and F. subincisa demonstrated the highest anti-inflammatory activity. The present study revealed differential contribution of TPC and TFC in various antioxidant activities. However, no obvious relationship was visible between anti-elastase/anti-collagenase/anti-tyrosinase/anti-inflammatory activities and TPC/TFC, suggesting the role of individual or combination of specific phenolics and flavonoids. Conclusions The abilities of Himalayan wild edible fruits to scavenge a variety of free radicals, inhibit enzymes causing skin-aging and skin-darkening are highly appreciable, suggesting their possible utilization for the development of effective formulations for general health maintenance and anti-aging, skin-whitening cosmetics.

Development and Evaluation of a Novel Delivery System Containing Phytophospholipid Complex for Skin Aging.

Citrus auranticum and Glycyrrhiza glabra are rich in anti-oxidant polyphenols helpful in prevention of skin aging. Polyphenols have high polarity and lower skin penetration resulting in lower cutaneous delivery. The present work is attempted to develop a novel polyherbal phospholipid complex cream to improve cutaneous delivery of polyphenols for sustained anti-oxidant action. Phytochemical and in vitro anti-oxidant evaluation was done on methanolic extracts of orange peel and liquorice powder. Total phenolic content, total flavonoid content, and anti-oxidant assays were done on different ratios of orange peel and liquorice extract. Ratio 1:2 gave highest total phenolic content (TPC) (530.00 ± 1.56mg gallic acid equivalent (GAE) g(-1) extract), total flavonoid content (TFC) (246.25 ± 1.03mg rutin equivalent (RUE) g(-1) extract), 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity (87.99 ± 0.64%), and H2O2 scavenging activity (72.47 ± 0.86%) and hence was used for formulation. Solvent evaporation method using methanol with 1:1 extract to phospholipid ratio was found to have entrapment efficiency of 93.22 ± 0.26%. Evaluation parameters like scanning electron microscopy (SEM), Fourier transform infrared spectrophotometry (FT-IR), and differential scanning calorimetry (DSC) confirmed formation of complex. The complex was formulated as oil-in-water cream and evaluated for various parameters. The optimized cream containing 1% complex was non-irritant and was found to be stable for 3-month period under conditions of stability study. Ex vivo diffusion studies showed that extract phospholipid complex cream had better retention of polyphenols in the skin when compared to conventional extract cream giving prolonged and stronger topical action. The cream had an anti-elastase activity of 28.02 ± 0.95% at concentration of 3000μgml(-1) (w/v). Thus, the developed safe and stable polyherbal phytophospholipid complex cream exhibited good potential as anti-aging cosmeceutical.

Stalked sea squirt (Styela clava) tunic waste as a valuable bioresource: Cosmetic and antioxidant activities

The aim of this study was to assess the skin aging related-enzyme inhibitory effects and antioxidant effects of different solvent extracts of the tunics of the solitary ascidian tunicate Styela clava, which are regarded as fishery waste. All extracts dose-dependently inhibited the enzyme activities of collagenase, elastase and hyaluronidase. The anticollagenase activities (IC50) of hot water (5.058mg/ml) and n-butanol (5.760mg/ml) extracts were comparable to that of ascorbic acid (3.838mg/ml), but lower than that of EDTA (10.856mg/ml). The antielastase activities (IC50) of ethanol (0.356mg/ml), acetone (1.668mg/ml), and n-butanol (2.040mg/ml) extracts were much lower than that of ursolic acid (4.073mg/ml), a representative elastase inhibitor, and the antihyaluronidase activities of ethyl acetate (0.210mg/ml) and hexane (0.596mg/ml) extracts were lower than that of ketoprofen, a well-known hyaluronidase inhibitor. Hot water extract scavenged ABTS radicals with an IC50 value of 0.192mg/ml, which was comparable to that of ascorbic acid (0.052mg/ml). These results suggest that S. clava tunics (SCTs) are a potential candidate for wrinkle-care cosmetics and for the treatment of inflammation-related disorders. As far as we are aware, this is the first report to describe the potential antiaging activity of SCT.