Anti-Aging Skin Care Regimen. Phyto-Stem Cell: Advanced Plant Cell Culture Technology by Bio-FD&C

Transcription

1 Anti-Aging Skin Care Regimen Phyto-Stem Cell: Advanced Plant Cell Culture Technology by Bio-FD&C 01

2 Plant Cell Culture Technology Highly expression of phytochemicals from plant cell culturing process by physical, chemical and biotic stress condition Advantages of Plant Cell Culture in Bioreactors Sustainable eco-friendly No environmental variation in weather, sunlight, soil and water Rare active molecules of interest can be concentrated up Very reproducible biomass and concentration of actives High biological safety of products GMO-free Plant callus, Phytoplacenta cell, Adventitious root, and Plant meristem cell Callus of plant is a mass of undifferentiated cells. Plant callus cells may be made to differentiate into the specialized tissues of a whole plant, with the addition of a number of hormones or enzymes. This is an ability known as totipotency. In this aspect plant callus can be called as plant stem cell like human stem cell. Plasticity Totipotency Callus Adult 02

3 Photoperiod (the day length) Control RF Light Emitting Diodes Ultrasonics Wave Cutting Callus Induction Differentiation Whole body 03

4 Plant part picking Callus Induction 2~3 Weeks 2~3 Weeks (as 20L Scale) 4~6 Weeks (as 1ton Scale) Cultivation in the Bioreactor Test of Efficacy and Safety of the Finished Products 04

5 Plant cell culture technology to accumulate secondary metabolites TOP-C 2 X, SMART-RC 2 TOP-C 2 X Technology of Plant Cell Culture with Elicitor X SMART-RC 2 Secondary Metabolite Accumulated Radiofrequency Technology Re-Controlled Cell Culture Caffeic acid Querceti n Catechi n Vit. C 05

6 Total Polyphenol Content (ppm Gallic Acid Equiv.) What is TOP-C 2 X, SMART-RC 2? Total Polyphenol Content of Rose Callus Control Rose Rose of Sharon (Radiofrequency) TOP-C 2 X Technology of Plant Cell Culture with Elicitor X SMART-RC 2 Secondary Metabolite Accumulated Radiofrequency Technology Re- Controlled Cell Culture 06

7 Macronutrients Micronutrients Plant growth regulators Iron supplement Carbon sources Vitamins & Amino acids Radiofrequency Elicitor 07

8 The seed germination of panax ginseng Cambium Mesophyll Cell Apical Meristem Cutting Callus induction 1~2 Week Bioreactor 08

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10 Cytotoxicity ( % of Contorl) Cytotoxicity Test on Human Skin Cell Panax Ginseng Extract Panax Ginseng Callus Culture Extract Panax Ginseng Callus Culture Extract (RF) Control 1% 2% 5% 1% Vitamin C Method Cytotoxicity of cell was determined by MTT assay, which is based on the reduction of soluble yellow MTT tetrazolium salt [3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] to its blue insoluble MTT formazan products by mitochondrial succinic dehydrogenase. For treatment, primary human dermal fibroblasts ( 1 X 10 4 cell/well) were maintained on culture media without FBS for 24h. After Experimental Compounds treatment, the cells were cultured for 24h. The cells were washed with PBS, 200 μl of MTT (0.05mg/ml) was added to each well, and the cells were incubated for 4h at 37. The supernantant was then removed, and 200 μl of DMSO was added to each well to dissolve the formazan product. Wells without cells were used as blanks. Absorbance was determined at 570 nm, spectrophotometrically, using an ELISA reader. Results The MTT-based cell cytotoxicity assay revealed that Panax Ginseng Extract, Panax Ginseng Callus Culture Extract, Panax Ginseng Callus Culture Extract (RF) were not cytotoxic at 1%, 2% and 5%. 10

11 Collagen Synthesis (% of Control) Panax Ginseng Callus Culture Extract Effect on Collagen Synthesis Panax Ginseng Extract Panax Ginseng Callus Culture Extract Panax Ginseng Callus Culture Extract (RF) Method The levels of type I procollagen protein in cell-free supernatants were determined by ELISA( Procollagen Type I C-Peptide EIA Kit, Takara, Cat No. MK101, Japan). The supernatants of cultured fibroblasts treated with various concentrations of Panax Ginseng Extract, Panax Ginseng Callus Culture Extract, Panax Ginseng Callus Culture Extract (RF) were harvested, and stored at -70. The ELISA was performed according to the manufacturer s instructions accompanying the product. Results 70 Control 1% 2% 5% In this study, the effect of Panax Ginseng Extract on procollagen production in cultured human dermal fibrlblasts was investigated. Cells were treated for 48h with various concentrations of Panax Ginseng Extract, Panax Ginseng Callus Culture Extract, Panax Ginseng Callus Culture Extract (RF), and cell-free supernatants were examined for type I procollagen using a commercially available ELISA kit. Treatment with Panax Ginseng Extract, Panax Ginseng Callus Culture Extract, Panax Ginseng Callus Culture Extract (RF) results in an increase in the synthesis of type I procollagen protein in a concentrationdependent manner. 11

12 Free Radical Scavenging Effect (% of Control) 100 Anti-Oxidant Capacity by Reducing Free-Radicals of DPPH Panax Ginseng Extract Panax Ginseng Callus Culture Extract Panax Ginseng Callus Culture Extract (RF) 20 0 Control 1% 2% 5% 1% Vitamin C Method DPPH Radical Scavenging Activity The 1,1- diphenyl-2-picryl-hydrazyl (DPPH) assay was performed. Various concentrations of Panax Ginseng Extract, Panax Ginseng Callus Culture Extract, Panax Ginseng Callus Culture Extract (RF) were added to 1 ml of the 0.004% methanol solution of DPPH, and the mixtures were vortexed vigorously. The tubes were then incubated at room temperature for 30 min in dark, and the absorbance was taken at 517 nm. Lower absorbance of the reaction mixture indicates higher free radical scavenging activity. Results Exception Panax Ginseng Extract, the Panax Ginseng Callus Culture Extract, Panax Ginseng Callus Culture Extract (RF) exhibited dose dependent inhibition of DPPH activity, and the scavenging activities of the extract and known antioxidants increased with increasing concentration. These results indicated that the Panax Ginseng Callus Culture Extract, Panax Ginseng Callus Culture Extract (RF) had a noticeable effect on scavenging of DPPH free radicals. 12

13 Callus Culture in vitro cultured explants of Neofinetia Falcata Leaf, Stem induction from Callus Callus induction Mesophyll Cell Cutting Neofinetia Falcata 13

14 Scanning electron microscopic(sem) observation of the Callus Neofinetia Falcata Extract Neofinetia Falcata Callus Culture Extract Neofinetia Falcata Callus Culture Extract (RF) Method Cytotoxicity of cell was determined by MTT assay, which is based on the reduction of soluble yellow MTT tetrazolium salt [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] to its blue insoluble MTT formazan products by mitochondrial succinic dehydrogenase. For treatment, primary human dermal fibroblasts ( 1 X 10 4 cell/well) were maintained on culture media without FBS for 24h. After Experimental Compounds treatment, the cells were cultured for 24h. The cells were washed with PBS, 200 μl of MTT (0.05mg/ml) was added to each well, and the cells were incubated for 4h at 37. The supernantant was then removed, and 200 μl of DMSO was added to each well to dissolve the formazan product. Wells without cells were used as blanks. Absorbance was determined at 570 nm, spectrophotometrically, using an ELISA reader. Results The MTT-based cell cytotoxicity assay revealed that Neofinetia Falcata Extract, Neofinetia Falcata Callus Culture Extract, Neofinetia Falcata Callus Culture Extract (RF) were not cytotoxic at 1%, 2% and 5%. 14

15 Human skin fibroblast cells were cultured at a density of cells/well into 24-well plates. The culture medium containing DMEM medium, 10 % fetal bovine serum, 100 IU/ml penicillin and 100 μg /ml streptomycin. At 72 hours after plating, they were washed once with PBS(pH7.4) and exposed to UVB radiation in a thin layer of PBS using three Philips TL 20W/01 lamps, emitting UVB peaking at 311 nm, which were placed 30cm above the flasks. The emitted radiation was checked under the flask lid using a UVR radiometer with a UVB sensor. After irradiation, PBS was replaced by DMEM+10%FBS. The radiation stress was performed twice a day for 5 days. Control cells were kept in the same culture conditions without UVB exposure. Real time RT-PCR At 72 hours after the last stress, total cellular RNA was isolated from CCD986sk after treating Neofinetia Falcata Callus Culture Extract. cdna was synthesized by reverse transcription and quantitation real-time PCR was performed to determine IL-1ß. RNA levels by using Rotor-Gene Q Series software(qiagen, 5-Plex). Gene Name Function GenBank IL-1B Interleukin 1 beta cytokine M

16 Effects of Neofinetia Falcata Callus Culture Extract in Human fibroblast to 10 exposures of UVB at 250 mj/cm 2 per exposure on the mrna level of IL(interleukin)-1ß as inflammatory signal. Control Control-UVB Neofinetia Falcata Callus Culture Extract -UVB Neofinetia Falcata Callus Culture Extract(RF) -UVB Control Control Anti-inflammation Effect 16

17 Method NO 2 - (Nitrite) accumulation was used as an indicator of NO production from cultured cells in the medium. Mouse RAW cells were plated at 5 X 10 5 cells/ml, and stimulated with LPS (200ng/ml) in the presence or absence of callus extract for 24 h. The isolated supernatants were mixed with an equal volume of Griess reagent (1 % sulfanilamide, 0.1 % naphthylethylenediamine dihydrochloride, and 2 % phosphoric acid) and incubated at room temperature for 10 min. NaNO 2 was used to generate a standard curve, and nitrite production was determined by measuring optical density at 570 nm. Results Effect of LPS on NO release. We measured total nitrite content as an indicator of NO released in the absence or presence of LPS for 4h. In the absence of LPS, the total released nitrite content was1 μm. After incubation of Neofinetia Falcata Callus Culture Extract with LPS for 4h, the total nitrite content was significantly in decreased as shown. Anti-inflammation Effect 17

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19 Total Polyphenol Content of Rosa Damascena Callus Culture Extract Rosa Damascena Callus Culture Extract (RF) Rosa Damascena Callus Culture Extract Rosa Damascena Extract Method Total Polyphenol Content (ppm Gallic Acid Equiv.) Extraction of Polyphenols. 0.2g of each sample were weighed in an extraction tube, 5ml of 70% methanol at 70 was added. The extract was mixed and heated at 70 on a vortex for 10 min. After cooling at RT, the extract was centrifuged at 200g for 10 min. The supernatant was decanted in a graduated tube. The extraction step was repeated twice. Both extracts were pooled and the volume adjusted to 10 ml with cold 70% methanol. 1ml of the extract was diluted with water to 100ml. Determination of Total Polyphenol Content(TPC). Total polyphenol content was determined by spectrophotometry, using gallic acid as standard. 1 ml of the diluted sample extract was transferred in duplicated to separate tubes containing 5ml of 1/10 dilution Folin-Ciocalteu s reagent in water. Then, 4 ml of a sodium carbonate solution was added. The tubes were then allowed to stand at RT for 60 min before absorbance at 765 nm was measured. The TPC was expressed as gallic acid equivalents (GAE) in g/100g material. The concentration of polyphenols in samples was derived from a standard curve of gallic acid ranging from 10 to 50 μg/ml Results The total polyphenol content is highest expressed in Rosa Damascena Callus Culture Extract (RF). 19

20 Method DPPH Radical Scavenging Activity The 1,1- diphenyl-2-picryl-hydrazyl (DPPH) assay was performed. Various concentrations of Rosa Damascena Extract, Rosa Damascena Callus Culture Extract, Rosa Damascena Callus Culture Extract (RF) were added to 1 ml of the 0.004% methanol solution of DPPH, and the mixtures were vortexed vigorously. The tubes were then incubated at room temperature for 30 min in dark, and the absorbance was taken at 517 nm. Lower absorbance of the reaction mixture indicates higher free radical scavenging activity. Results Exception Rosa Damascena Extract, Rosa Damascena Callus Culture Extract, Rosa Damascena Callus Culture Extract (RF) exhibited dose dependent inhibition of DPPH activity, and the scavenging activities of the extract and known antioxidants increased with increasing concentration. These results indicated that the Rosa Damascena Callus Culture Extract, Rosa Damascena Callus Culture Extract (RF) had a noticeable effect on scavenging of DPPH free radicals. 20

21 Cosmetics Application - Advanced Plant Stem Cell Cosmetic formulas - Face and Body Care to Protect the Most Valuable Skin Cells - For a vital and healthy-looking skin - Daily Skin Care to Prevent Wrinkle Tea tree Plant INCI NAME : Camellia Sinensis Callus Culture Extract -Effective skin-whitening agent (EGCG ) : Inhibit melanin synthesis and tyrosinase activity - Suitable for oily skin with problems (comedonal acne,pustules) - A plant-based astringent with anti-inflammatory effects. -Proven efficacy on skin cells proliferation Tea tree Callus Active ingredient in Tea Tree [ EGCG ] - EGCG, allatoin, alpha-terpineol, apigenin, ascorbic acid, beta-carotene - beta-sitosterol, caffeic acid, catechin, kaempferol, linoleic acid, - myricetin, polyphenol, quercetin, chlorogenic acid, gallic acid, niacin, - palmitic acid, salicylic acid, thymol, oleic acid 21

22 Chlorogenic acid Accumulation by Radiofrequency Control Chlorogenic acid Chlorogenic acid 22

23 Chlorogenic acid Accumulation by Radiofrequency Control Chlorogenic acid 23