Compound I and II, their application and preparation method in preparing medicines for preventing formation of melanins
A technology for inhibiting melanin and compounds, applied in the field of application and preparation of compounds I and II and their preparations to treat and inhibit melanin formation
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Embodiment 1
[0031] Embodiment 1 Experimental material
[0032] Cinnamomum subavenium, a Lauraceae plant, grows in mountains at an altitude of 500-1500 meters. It is an evergreen tree native to China. The bark is fragrant and can be used as a spice. Extracted leaf oil can be used in food, cigarette flavor or antibacterial agent.
[0033] Extraction and purification steps
[0034] Two compounds, linderanolide B and subamolide A, are extracted from the stems of Cinnamomum laxifolius.
[0035] The specific steps are as follows: 8 kg of air-dried cinnamon stems were extracted 6 times with 80 L of methanol under normal temperature and low pressure conditions to obtain about 200 g of extract; the extract was resuspended in 1 L of water, and a total of 2 L of chloroform was added in 5 times (CHCl 3 ); after static separation, the chloroform layer was collected and dried to obtain about 120 g of the extract; the extract was placed on a liquid chromatography column (800 g, 70-230 mesh), and mi...
Embodiment 2
[0044] Example 2 In vitro experiment
[0045] In this experiment, human melanocytes were used to test the effect of the compounds isolated in the previous step on inhibiting tyrosinase activity.
[0046] Will 5x10 4 Primary cultured human epidermal melanocytes (HEMn-MP) derived from neonatal foreskin were seeded in 96-well plates at 37 °C, 5% CO 2 cultivated under conditions. After 24 h, different concentrations (0.01 uM, 0.1 uM, 1 uM, 5 uM, 10 uM) of compound I and II, as well as kojic acid (100 uM), phenylthiourea (N-phenylthiourea, 100 uM ) and α-melanocyte-stimulating hormone (MSH, 0.1 uM) were added to each culture well, and cultured for 72 h under the same conditions. Cell viability was detected using MTS assay (CellTiter 96? Aq ueous One Solution Cell Proliferation Assay, Promega). Add CellTiter 96? Aq to each culture well ueous One Solution Reagent, incubate at 37°C for 4 h. The cell viability of each well was obtained by measuring the absorbance of each wel...
Embodiment 3
[0051] Embodiment 3 in vitro experiments
[0052] Zebrafish were used in vitro experiments to determine the whitening effect of the two compounds involved in this patent, as well as the dosage and safety of the compounds used, the results are shown in Figure 5 .
[0053] The day before egg collection, the female and male fish were separated, and on the second day after the light started, the female and male fish were collected together in the tank, and the fertilized eggs were washed with water, and the embryos were cultured in a 90 mm Petri dish with Holfreter solution, placed in In a light incubator, culture at 28.5 ?C until the desired development period, and stage the fertilized eggs according to the description of Kimmel et al.
[0054] The zebrafish embryos were collected within 30 min after fertilization, washed and placed in a 90 mm Petri dish, and the embryos with normal development were selected under a stereoscope for use in experiments.
[0055] Different conc...
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