Sirius Red Method Research Articles

In vitro anti-ageing activities of ethanolic extracts from Pink rambutan (Nephelium lappaceum Linn.) for skin applications

Skin ageing is characterized by features such as wrinkles, loss of elasticity, laxity, rough-textured appearance, melasma and freckles. Several researches have focused for preventing, and treating skin ageing by many natural ingredients. This study aimed to assess the anti-ageing activities for anti-skin ageing of the ethanolic extracts of Pink rambutan (PR) (Nephelium lappaceum Linn.) from leaves (L), branches (B), seeds (S), and peels from ripe (R) and young (Y) fruits. The extraction yields of all Pink Rambutan (PR) extracted by the Maceration (M) and the Soxhlet extraction (Sox) using 95% ethanol as a solvent, ranged from 10.62% to 30.63%. Flavonoids were found as the main phytochemicals in almost all the PR extracts. The PR-Y-M and PR-Y-Sox extracts gave the highest total phenolic contents by the Folin-Ciocalteu assay of 67.60 ± 4.38 mgGAE/g, and total flavonoid contents by the modified aluminum chloride colorimetric assay of 678.72 ± 23.59 mgQE/g, respectively. The PR-L-M extracts showed the highest three anti-oxidative activities; the free radical scavenging (SC50 of 0.320 ± 0.070 mg/mL), the lipid peroxidation inhibition (LC50 of 0.274 ± 0.029 mg/mL), and the metal chelation activity (MC50 of 0.203 ± 0.021 mg/mL). All the PR extracts at 0.01 and 0.1 mg/mL showed no cytotoxicity on B16F10 cells, and human skin fibroblasts, respectively. Likewise, the PR-R-Sox extract exhibited the highest anti-melanogenesis on B16F10 cells (52.7 ± 0.9%) and, the mushroom tyrosinase inhibition activity (IC50 of 0.04 ± 0.02 mg/mL), which was significantly comparable to kojic acid (p < 0.05). The PR-Y-Sox extract showed the collagen biosynthesis by the Sirius Red method, and the stimulation of anti-ageing genes (Sirt1 and Foxo1) on human skin fibroblasts by the RT-PCR method, which were similar to standards ʟ-ascorbic acid and resveratrol, respectively. This study suggests that the PR-R-Sox and PR-Y-Sox extracts can be further developed as natural anti-ageing agents for whitening and anti-wrinkle in the cosmetics, cosmeceutical, and pharmaceutical industries.

A new method for determining the denaturation temperature of collagen

The denaturation temperature of collagen has been determined using several methods, such as circular dichroism, fourier transform infrared spectroscopy, and differential scanning calorimetry, etc. Such methods need specific equipment or mass samples (more than 75 mg samples), which means higher inspection costs. In this study, Sirius red was employed to determine the Tds of collagen from calf tendon, silver carp skin, frog and salmon skins (38.2 °C, 32.6 °C, 33.8 °C, and 15.6 °C, respectively) to explore a new method that does not require special equipment and only needs 2–3 mg sample for one measurement. This method was suitable for the determination of the denaturation temperature of collagen from terrestrial, aquatic and amphibian animals. Analysis of variance and t-test revealed that no significant difference was found between Sirius Red and viscosity methods. However, the Sirius Red method needs simpler equipment and less sample than viscosity and other methods. So it could be used as a convenient approach to determine the denaturation temperature of collagen instead of the viscosity method.

Antifibrotic Effect of Ethanolic Extract of Nerium indicum Mill. Standardized 5α-Oleandrin on Keloid Fibroblasts Cells

Keloid is a skin lesion caused by an abnormal proliferation of fibroblasts and the deposition of collagen in the wound healing process. Therapy for keloids is relatively limited and mostly has side effects. Recent studies show that 5α-oleandrin from N. indicum Mill. (N. indicum) has anti-keloid effect by inhibiting (proliferation, migration activity and TGF-β1 expression) keloid fibroblast. Further studies with N. indicum extract standardized 5α-oleandrin should be conducted to develop it as an anti-keloid agent by performing at proliferative effects, collagen accumulation, and keloid fibroblast migration. The antifibrotic activity of N. indicum extract on the proliferation of keloid fibroblasts was measured by MTT assay, whereas collagen deposits were observed by Sirius Red method. Observation of keloid fibroblast migration by scratch assay is according to Liang et al. (2009). Ethanol extract of N. indicum standardized 5α-oleandrin can inhibit the proliferation of keloid fibroblas with IC50 0.458 μg/mL, also inhibited collagen deposits with IC50 0.055 μg/mL at incubation for 72 hours. In incubation 48 hours after treatment with ethanol extract of 1.0 μg/mL occurred inhibition of migration significantly compared with control.

Experimental study on the expression of α-SMA and HMGB1 in silicotic fibrosismodel rats interfered by lumbricus

Objective: To observe the expression of alpha smooth muscle actin (α-SMA) and high mo-bility group protein B1 (HMGB1) in silicosis model rats interfered by lumbricus. Methods: 45 rats were ran-domly divided into the control group, model group and group interfered by lumbricus. The silicosis model rats were established. The group interfered by lumbricus were intragastric administered with lumbricus decoction by the 4 ml/kg dose. The control group and model group were ig administered with the equal amount of normal saline. Each group were killed 5 rats on the 7(th), 14(th) and 28(th) day. The lung tissues were stained with HE and Sirius red methods. The mRNA expressions of α-SMA and HMGB1 were determined with RT-PCR; The pro-tein levels of α-SMA and HMGB1 were determined with Western blotting. Results: Compared with the control group, the expression levels of α-SMA and HMGB1mRNA and protein in lung tissue of model group were grad-ually increased in the 7(th), 14(th) and 28(th) days, the difference was statistically significant (P< 0.01) . Compared with model group, the levels of α-SMA and HMGB1mRNA and protein in lung tissue of group interfered by lumbricus were gradually lowered in the 7th, 14th and 28th days, the difference was statistically significant (P<0.05, P<0.01) . Conclusion: Lumbricus inhibits the collagen deposition and the formation of silicosis pulmo-nary fibrosis, which may be related to the inhibition of HMGB1 expression and activation of α-SMA in lung tis-sue.

Certification procedures for sirius red F3B (CI 35780, Direct red 80)

Sirius red F3B (CI 35780, Direct red 80) is a polyazo dye used principally in staining methods for collagen and amyloid. For certification by the Biological Stain Commission, a sample of the dye must exhibit an absorption spectrum of characteristic shape with a maximum at 528-529 nm, a small shoulder near 500 nm and narrow peaks at 372, 281-282 and 230-235 nm. Spot tests (color changes with addition of concentrated H2SO4 or HCl and subsequent dilution or neutralization) also are applied. The dye must perform satisfactorily in the picro-sirius red method for collagen by providing red staining of all types of collagen with yellow and green birefringence of fibers. Llewellyn's alkaline sirius red method applied to tissue known to contain amyloid must show red coloration of the products with green birefringence. Dye content, which does not influence significantly the staining properties of sirius red F3B, is not assayed.

Morphological aspects of the myocarditis and myositis in Calomys callosus experimentally infected with Trypanosoma cruzi: fibrogenesis and spontaneous regression of fibrosis.

Calomys callosus a wild rodent, is a natural host of Trypanosoma cruzi. Twelve C. callosus were infected with 10(5) trypomastigotes of the F strain (a myotropic strain) of T. cruzi. Parasitemia decreased on the 21st day becoming negative around the 40th day of infection. All animals survived but had positive parasitological tests, until the end of the experiment. The infected animals developed severe inflammation in the myocardium and skeletal muscle. This process was pronounced from the 26th to the 30th day and gradually subsided from the 50th day becoming absent or residual on the 64th day after infection. Collagen was identified by the picro Sirius red method. Fibrogenesis developed early, but regression of fibrosis occurred between the 50th and 64th day. Ultrastructural study disclosed a predominance of macrophages and fibroblasts in the inflammatory infiltrates, with small numbers of lymphocytes. Macrophages had active phagocytosis and showed points of contact with altered muscle cells. Different degrees of matrix expansion were present, with granular and fibrillar deposits and collagen bundles. These alterations subsided by the 64th days. Macrophages seem to be the main immune effector cell in the C. callosus model of infection with T. cruzi. The mechanisms involved in the rapid fibrogenesis and its regression deserve further investigation.