CN109730952B

CN109730952B - Water extraction process of lava seawater containing dendrobium officinale

Info

Publication number: CN109730952B
Application number: CN201910008542.6A
Authority: CN
Inventors: 金基洙; 金止焄; 卢勇; 金庚泰; 陈秀玉; 朴素延; 李昭憲; 史永昇; 金藝香
Original assignee: Botaniera Hangzhou Health Technology Co ltd; Hyundai Bioland Jiangsu Co ltd
Current assignee: Botaniera Hangzhou Health Technology Co ltd; Hyundai Bioland Jiangsu Co ltd
Priority date: 2019-01-04
Filing date: 2019-01-04
Publication date: 2021-09-10
Anticipated expiration: 2039-01-04
Also published as: CN109730952A

Abstract

The invention discloses a lava seawater extraction process containing dendrobium officinale, which adopts lava seawater as an extract stock solution, and the lava seawater is sterilized by a sterilizing filter; adding Dendrobium officinale stems into the lava seawater, reflux-extracting, filtering with filter cloth, and filtering with filter to completely remove solvent to obtain the powder of the extract of Dendrobium officinale from lava seawater. The invention adopts the lava seawater dendrobium officinale extract as the effective component, and has the effects of improving wrinkles, inhibiting saccharification, whitening and moisturizing the skin.

Description

Water extraction process of lava seawater containing dendrobium officinale

Technical Field

The invention relates to a lava seawater extraction process for dendrobium officinale.

Background

The skin in the human body is a body organ which is most closely contacted with the external environment, has the function of protecting the interior of the human body, and can be divided into epidermis, dermis and subcutaneous fat. The skin is a complex organ composed of cells with various functions and substances adapted to the cells, wherein the dermis layer is a combination of elastic solid substances (fibers) and viscous liquid substances, and can maintain the inherent elasticity of the skin. The skin therefore responds to tension with unique physical properties, which we call viscoelastic. The skin has such viscoelasticity because proteoglycan includes collagen fibers and elastic fibers, which constitute a unique three-dimensional component. Generally, the skin function is decreased and the skin becomes atrophic due to aging, ultraviolet rays, pollution, stress, and the like, and the number of skin cells is decreased or the thickness of the skin is reduced. For these reasons, the elastic fibers of the skin deform to cause a reduction in the elasticity of the skin or a relaxation of the skin.

Collagen is a main component constituting the skin, and type II collagen accounts for 80% and type III collagen accounts for 15%. Under the long-term exposure to ultraviolet rays, active nutrients in the skin are generated in large amounts, resulting in the promotion of the expression or activity of the collagen-degrading enzyme MMP-1 in the matrix metalloproteinases MMPs, resulting in the reduction of collagen synthesis and thus in the reduction of skin elasticity. Therefore, in order to treat skin and prevent skin aging, we have developed a novel material having the efficacy of expressing MMP-1, inhibiting its activity, and promoting collagen synthesis, which can be a good strategy.

Also, collagen and elastin are susceptible to chemical reactions within the body known as glycation. This is a non-enzymatic reaction that results from the combination of free amino groups containing proteins and reducing sugars, such as glucose. Glucose, which powers cells, may react with proteins such as collagen. This will result in the Advanced Glycation End-products (AGEs) and Reactive Oxygen Species (ROS). One of the AGEs, carboxymethylethylysine (cml), in particular, causes changes in skin elasticity during UV-induced photoaging, gives the skin elasticity and collagen to accumulate AGEs, which negatively affects cross-linking of protein fibers, loss of elasticity, intra-dermal changes, etc.

AGEs generated in the skin act on the receiver site to generate AGE (RAGE) complex. RAGE signaling conveys inflammatory and sequelae-related intracellular processes. These are important as they are the main causes of the inflammatory aging process and the catalytic action of many diseases. For example, diabetic patients have a high glycemic index in their blood and develop complications such as cataract and arteriosclerosis accompanied by the formation of AGEs in their bodies. For these reasons, diabetes is considered to be a disease in which aging is accelerated due to increased inflammation caused by the production of AGEs. This is not limited to diabetes. Physical weakness, heart disease and many brain related diseases are associated with glycation. Scientists believe that reducing glycation means slowing the aging process and disease induction. The generation of AGEs is a mechanism that determines how old the skin is and how the disease is generated in our body, and is a field of most attention.

Pigmentation of skin such as black nevi and freckles is caused by an increase in melanin in the epidermis, and melanin is also an important factor determining skin color. Melanin is produced in pigment granule organs within melanocytes of the basal layer of the epidermis. That is, the mitosis of melanocytes occurs due to ultraviolet rays, and then melanocytes are activated. The synthesis of tyrosinase and tyrosinase is promoted in activated melanocytes, and the production of melanin is enhanced to transport and discharge the melanin to the outside of epidermis.

Naturally occurring melanin is a mixture of alkali-insoluble black melanin (neomelanin) and alkali-soluble yellow to red melanin (pheomelanin). The melanin polymers in general are mostly referred to as neomelanin. In contrast, according to the studies of Prota (g. Prota., j. invest. dermotol., 75, p122, 1980.) and Pavel (s. Pavel et al, Cancer Detection and preservation, 6, p311., 1983.), the new melanin includes not only the dihydroxyindole 5, 6-dihydroindole which we have thought, but also other melanin monomers (5, 6-dihydroindole-2-Carboxylic acid) and melanin produced from various melanin intermediate metabolites. The melanogenesis process in melanosomes is tyrosine oxidation by the enzyme tyrosinase (DOPA) → (dopaquinone) → (5, 6-dihydroindole) → (indole-5,6-quinone), followed by the production of melanin due to the polymerization of (indole-5, 6-quinone). Wherein tyrosinase is intervening until the tyrosine step, and the possibility that several subsequent steps are autoxidized is high. The phenomenon of pigmentation, i.e., blackening, which occurs in sunbathing or the like is caused by an increase in melanin formation due to ultraviolet rays in sunlight.

The processes of melanogenesis and the mechanisms of action for inhibiting melanogenesis in melanocytes, which have been known so far, are roughly classified into two types. The first is to inhibit the production of melanin by cytotoxic action on melanocytes, and the second is to inhibit the production of melanin in melanocytes, thereby inhibiting the production of melanin. According to the recently published papers, not only tyrosinase but also TRP-1 and TRP-2 produce melanin, which is an important enzyme for the intervention of melanin biosynthesis, and if substances capable of inhibiting the production of these enzymes are developed, it is a good method for preventing skin pigmentation.

The human body is mostly composed of water, which is an important component for maintaining a living body. Normal skin contains 70% of water in the body, and the stratum corneum contains 20% to support the elasticity and softness of the skin. When the water content of the horny layer is 10% or less, dry skin is commonly called a cause of skin troubles. The moisture balance of the skin is regulated according to the epidermis. Although the stratum corneum of the epidermis is formed by the accumulation of dead cells, it plays a very important role in maintaining water in the skin. The water content in the horny layer due to regular keratinization is maintained at 10 to 20%. This is a sebum membrane formed by mixing sweat and sebum, and protects the skin from the outside and prevents the evaporation of water from the stratum corneum. The natural moisturizing factor and the dermal lipid capture and maintain moisture. The dermal lipid membrane NMF and dermal lipids act together to maintain moisture. NMF is produced during keratinization, is a hydrophilic, hygroscopic substance, and plays a very important role in skin moisturization. It is composed of amino acids and can lock the moisture of stratum corneum. Also PCA in NMF is of particular importance. As a matrix filling between the respective cells, the layer is formed so as to maintain the moisture inside.

It is reported that the decrease in the amount of hyaluronic acid in human skin with aging is considered to be one of the direct causes of the decrease in skin elasticity and the decrease in moisture content with aging.

Disclosure of Invention

In order to solve the technical problems, the invention provides a lava seawater extraction process containing dendrobium officinale, lava seawater is used as an extract stock solution, and the lava seawater is sterilized by a sterilization filter; adding Dendrobium officinale stems into the lava seawater, reflux-extracting, filtering with filter cloth, and filtering with filter to completely remove solvent to obtain the powder of the extract of Dendrobium officinale from lava seawater.

Further, the cosmetic composition further comprises AQP-3 or HAS-2.

Further, 0.01-10 wt% of the lava seawater dendrobium officinale extract is added based on 100 wt% of the whole.

The invention adopts the lava seawater dendrobium officinale extract as the effective component, and has the effects of improving wrinkles, inhibiting saccharification, whitening and moisturizing the skin.

Drawings

FIG. 1 shows the cytotoxicity results of Fibroplast in example 1 and comparative example 1 on fibroblasts.

FIG. 2 shows the cytotoxicity results of example 1 and comparative example 1 on keratinocytes HaCaT.

FIG. 3 shows the results of comparison of the expression of the Collagen type 1 gene in fibroblasts of example 1 and comparative example 1.

FIG. 4 shows the results of comparing RAGE genetic gene expression of example 1 and comparative example 1 in fibroblasts.

FIG. 5 shows the results of comparing the expression of AQP-3 genes of example 1 and comparative example 1 in keratinocytes.

FIG. 6 shows the results of comparing the expression of HAS-2 gene in keratinocytes in example 1 and comparative example 1.

FIG. 7 shows cytotoxicity results for melanoma (B16-F1) in example 1 and comparative example 1.

FIG. 8 shows the results of comparison of the melanin production per cell in melanoma (B16-F1) in example 1 and comparative example 1.

Detailed Description

Production example 1: water intake of lava seawater

In east China, county, old Leyi-Han, 150m underground, the lava seawater collected at 44.35m of the average surface of the underground sea (sea level as reference) was sterilized by a sterilizing filter. The thus sterilized lava seawater (in a state of not desalted brine) is used for the subsequent production of the dendrobium officinale lava seawater extract. The mineral composition of the sterilized lava seawater is as shown in table 1 below.

[ TABLE 1 ]

Example 1: preparation method of water extract of Dendrobium officinale Kimura et Migo from lava sea

Taking 1kg of dendrobium officinale stem, adding 20 kg of lava seawater, extracting at 90 ℃ under reflux for 3 hours, filtering with 250-mesh filter cloth, and then filtering with a 1 ㎛ filter. The filtrate was completely freed of solvent by a vacuum concentrator (EYELA 사, N-1000, Japan) to obtain 226 g of extract powder of Dendrobium officinale and lava seawater.

The extract of dendrobium candidum in lava seawater is added in an amount of 0.01-10 wt% based on 100 wt% of the whole body, so that the extract has good effects of improving skin wrinkles and inhibiting saccharification, and has whitening and moisturizing effects.

Comparative example 1: preparation of pure water extract of Dendrobium officinale

The extract was prepared from Dendrobium officinale stems under the same conditions as in example 1, i.e., pure water was used instead of lava seawater. 29g of dendrobium officinale pure water extract powder is obtained.

And (3) comparison: cytotoxicity measurements with fibroblasts (fibroplasts) and keratinocytes (HaCaT)

To measure the anti-wrinkle effect, anti-glycation effect, whitening effect and moisturizing effect of the dendrobium officinale kimura sea water extract of example 1 and the dendrobium officinale pure water extract of comparative example 1, toxicity test was performed by MTT assay in human Fibroblast and keratinocyte HaCaT. MTT assay is used for detecting the number of living cells and is generally used for thin cellA method for detecting cell proliferation or cytotoxicity. The living cells are water soluble in mitochondria and the assay utilizes the yellow salt MTT (3- [4, 5-dimethylthiozole-2-yl)]-2, 5-diphenyltetrazolium bromide) is reduced to a water-insoluble blue formazan derivative by succinate dehydrogenase (or mitogenic dehydrogenase). Fibroblast Fibrolast and keratinocyte HaCaT cell were seeded on the bottom of a culture dish, and then cultured in medium containing 5% carbon dioxide at 37 ℃ in IMDM (using Iscove's modified Dulbecco's medium-Fibrolast cell, or DMEM (using Dulbecco's modified Eagle's medium-HaCaT cell) containing penicillin 100U/㎖, streptomycin 100 ㎍/㎖, 10% bovine serum (FBS), human Fibroblast cell and keratinocyte HaCaT cell were cultured in 24-well cell culture plates at 1X 25 10⁵cells/ml, 1.5x10⁵cells/ml were diluted and inoculated with 1 ㎖ cells, respectively, and cultured for 24 hours. After the culture, the culture medium was completely removed, and the culture medium containing no bovine serum was placed, and the extracts of example 1 and comparative example 1 were treated at the concentrations. After culturing at 37 ℃ for 24 hours in a medium containing 5% carbon dioxide, the whole culture medium was purged and replaced with a 10-fold diluted culture medium of 2.5mg/㎖ MTT solution, and the culture medium was cultured for 4 hours. After 4 hours, the supernatant was removed, a DMSO solution as a solvent for dissolving 1 ㎖ was added, and the absorbance of the resulting crystals at 570nm of MTT formazan was measured. In this case, the control group used a dissolution solvent, DMSO. Please refer to table 1 and table 2 for toxicity test results.

[ TABLE 1 ]

[ TABLE 2 ]

The water extract of Dendrobium officinale Kimura et Migo of example 1 and the pure water extract of Dendrobium officinale Kimura et Migo of comparative example 1 both showed no toxicity at concentrations below 1000 ㎍/㎖.

Experimental example 2: investigation of amount of expression of Collagen type I mRNA

To measure the Collagen synthesis-promoting effects of the sea water extract of Dendrobium officinale Kimura et Migo of example 1 and the pure water extract of Dendrobium officinale Kimura et Migo of comparative example 1, Collagen type I genetic gene expression experiments were performed on samples processed on commercially purchased human fibroblasts. Human Fibroblast Fibroplasts purchased at ATCC (American Type Culture Collection) at 1X10 together with IMDM (Iscove's Modified Dulbecco's Media) Culture broth containing 10% bovine serum in 60mm dish⁶cells/well concentration aliquot at 37 ℃ 5% CO₂Incubated under conditions for 24 hours. After 24 hours, the sample was diluted with an IMDM culture medium containing no bovine serum at a certain concentration for 24 hours, and the cells were collected in TRIzol (invitrogen, USA) to isolate RNA. The isolated RNA was used with a Qubit

After quantification of fluoroemeter with RNA BR Assay kit, the gene was amplified using Real-Time PCR 7500 Fast with Power SYBR Green PCR master mix (Applied Biosystems, USA), and the product was quantitatively analyzed. Using Collagen type I and β -Actin primer (Korea) in PCR, primer sequence please refer to Table 3. The control group used solvent-treated DMSO (dimethyl sulfoxide), and the positive control group used well-known antioxidant Vitamin C. See table 4, fig. 3 for experimental results.

[ TABLE 3 ]

[ TABLE 4 ]

The aqueous extract of Dendrobium officinale Kimura et Migo of example 1 and the pure aqueous extract of Dendrobium officinale Kimura et Migo of comparative example 1 confirmed the expression of the Collagen type I genetic gene. Experimental example 1A larger amount of Collagen type I than the control group was expressed, and it was confirmed that it had an anti-wrinkle effect. It was confirmed that the wrinkle preventing effect was more excellent in example 1 than in comparative example 1.

EXAMPLE 3 investigation of expression amount of RAGE mRNA

To examine the anti-glycation effects of the sea water extract of Dendrobium officinale Kimura et Migo of example 1 and the pure water extract of Dendrobium officinale Kimura et Migo of comparative example 1, RAGE genetic gene expression experiments were performed on samples processed on commercially purchased human fibroblasts. Human fibroblasts (fibroplast) were cultured at 1X10 in 60mm dish together with IMDM (Iscove's Modified Dulbesco's Media) medium containing 10% bovine serum⁶Cell/dish concentration aliquots at 37 ℃ with 5% CO₂Incubated under conditions for 24 hours. After 24 hours, the culture medium was removed, HBSS (Hank's Balanced salt solution) 3 ㎖ was added, and UVA 6J/cm was performed²After the treatment, the sample was diluted in a bovine serum-free IMDM culture medium for 48 hours at a certain concentration, and the cells were collected in TRIzol (invitrogen, USA) to isolate RNA. The isolated RNA was used with a Qubit

After quantitative determination of fluorometer with RNA BR Assay kit, cDNA (complementary DNA) was synthesized and subjected to Real-time PCR. cDNA Synthesis Using a cDNA Synthesis Kit (High Capacity RNA to cDNA Kit, Applied Biosystems, USA), experiments were performed according to the method of Kit. The gene was amplified using a Real-Time PCR 7500 Fast with Power SYBR Green PCR master mix (Applied Biosystems, USA) and the product was quantitatively analyzed. RAGE and beta-Actin primer (Korea) used in PCR, primer sequence please refer [ Table 5]. The control group used solvent-treated DMSO (dimethyl sulfoxide), and the UVA-treated group used UVA 6J/cm²And solvent-treated dmso (dimethylsulfoxide). The positive control group used the well known antioxidant Resveratrol. See table 6 and fig. 4 for experimental results.

[ TABLE 5 ]

[ TABLE 6 ]

The aqueous extract of the Dendrobium officinale Kimura et Migo sea of example 1 and the pure aqueous extract of Dendrobium officinale Kimura et Migo of comparative example 1 determined the expression of RAGE genetic genes. Both example 1 and comparative example 1 had a significant inhibitory effect on UVA-induced increased RAGE genetic gene expression. It was confirmed that the wrinkle-preventing effect was more excellent in example 1 than in comparative example 1.

Experimental example 4: AQP-3, HAS-2 mRNA expression survey

To measure the moisturizing effects of the dendrobium officinale kimura sea water extract of example 1 and the dendrobium officinale pure water extract of comparative example 1, samples were treated on human keratinocytes to perform experiments. Keratinocyte HaCaT was cultured in 60mm dish in 1.5X10 DMEM (Dulbecco's modified Eagle's medium) containing 10% bovine serum⁶Cell/dish concentration aliquots at 37 ℃ with 5% CO₂Incubated under conditions for 24 hours. After 24 hours, the sample was diluted at a certain concentration in a DMEM culture solution containing no bovine serum for 24 hours, and the cells were collected in TRIzol (invitrogen, USA) to isolate RNA. The isolated RNA was used with a Qubit

After quantitative determination of fluorometer with RNA BR Assay kit, cDNA (complementary DNA) was synthesized and Real-time PCR was performed. cDNA Synthesis Using a cDNA Synthesis Kit (High Capacity RNA to cDNA Kit, Applied Biosystems, USA), experiments were performed according to the method of Kit. After amplification of the genetic gene using the Real-Time PCR 7500 Fast with Power SYBR Green PCR master mix (Applied Biosystems, USA), the increased product was quantitatively analyzed. AQP-3, HAS-2 and beta-Actin primer (Korea) used in PCR, primer sequence please refer to [ Table 7 ]]. The control group used solvent-treated DMSO (dimethyl sulfoxide), and the positive control group used Retinoic acid with moisturizing effect. Fruit of Chinese wolfberrySee table 8, table 9, fig. 5, and fig. 6 for experimental results.

[ TABLE 7 ]

[ TABLE 8 ]

The expression of AQP-3 genetic genes of the dendrobium officinale kimura sea water extract of example 1 and the dendrobium officinale pure water extract of comparative example 1 was confirmed. It was demonstrated that AQP-3 was expressed in a larger amount than the control group in Experimental example 1, and it was confirmed that it had a moisturizing effect. It was also confirmed that example 1 has a better moisturizing effect than comparative example 1.

[ TABLE 9 ]

The expression of the HAS-2 genetic genes of the dendrobium officinale lava sea water extract of example 1 and the dendrobium officinale pure water extract of comparative example 1 was confirmed. It was shown that experimental example 1 and comparative example 1 expressed a larger amount of HAS-2 than the control group, and it was confirmed that the moisturizing effect was exhibited. It was also confirmed that example 1 has a better moisturizing effect than comparative example 1.

Experimental example 8: detection of cytotoxicity Using melanoma cells (B16-F1)

To measure the toxicity of the water extracts of Dendrobium officinale Kimura et Migo of example 1 and the purified water extract of Dendrobium officinale Kimura et Migo of comparative example 1, samples were treated on commercially available melanoma (B16-F1) cells for experiments. Melanoma (B16-F1) cells were plated at 1X10 in 6 well plates in DMEM (Dulbecco's modified Eagle's medium) with 10% bovine serum⁵Cell/well concentration aliquot at 37 ℃ 5% CO₂Incubated under conditions for 24 hours. After 24 hours, the sample was placed in a medium containing alpha-Melanocyte stimulane hormone (alpha-MSH) and 10% bovine serumThe cells were cultured in fresh DMEM at a diluted concentration for 72 hours. After 72 hours of culture, all the culture medium was removed, and the culture medium was cultured for 4 hours while alternating 10-fold dilution with MTT solution at 2.5mg/㎖. After 4 hours, the supernatant was removed, and 2 ㎖ DMSO (dimethyl sulfoxide) solution was added to detect the absorbance of the MTT formazan crystals at 570 nm. The non-treated group used solvent-treated DMSO (dimethyl sulfoxide), and the alpha-MSH-treated group used alpha-MSH and solvent-treated DMSO (dimethyl sulfoxide). Arbutin (SK Bioland) was used as the positive control group, and the toxicity test results are shown in Table 10 and FIG. 7.

[ TABLE 10 ]

The water extract of Dendrobium officinale Kimura et Migo of example 1 and the pure water extract of Dendrobium officinale Kimura et Migo of comparative example 1 both showed no toxicity at concentrations below 50 ㎍/㎖.

Experimental example 9: melanoma (B16-F1) cell detection for melanin biosynthesis inhibition effect

To measure the whitening effects of the water extracts of Dendrobium officinale Kimura et Migo of example 1 and the purified water extract of Dendrobium officinale Kimura et Migo of comparative example 1, samples were treated on commercially available melanoma B16-F1 cells for experiments. The melanogenesis inhibitory effect of melanoma cells was examined by the method (Fukuda et al, j. soc. Cosmetic chem., 42(361), 1991) and its modification. Melanoma B16-F1 cells were plated at 1X10 in 6 well plates in DMEM (Dulbecco's modified Eagle's medium) with 10% bovine serum⁵Cell/well concentration aliquot at 37 ℃ 5% CO₂Incubated under conditions for 24 hours. After 24 hours, the samples were diluted and cultured in α -Melanocyte potentiate hormon (α -MSH) and fresh DMEM containing 10% bovine serum at the given concentrations for 72 hours. After 72 hours of culture, all the cells from which the culture medium had been removed were washed with PBS (saline) 2 ㎖, treated with 1N NaOH 0.5 ㎖, and the cells were recovered with 1.5 ㎖ tube to obtain melanin in the cells. Will come back toThe collected cells move to a 96 well plate, and the absorbance is detected at 450nm to obtain a certain amount of protein carbohydrate melanin. The non-treated group used solvent-treated DMSO (dimethyl sulfoxide), and the alpha-MSH-treated group used alpha-MSH and solvent-treated DMSO (dimethyl sulfoxide). Arbutin (SK Bioland) was used as the positive control group. See table 11 and fig. 8 for the results of the melanin biosynthesis inhibition assay.

[ TABLE 11 ]

Claims

1. The preparation method of the dendrobium officinale lava sea water extract is characterized by comprising the following steps: adopting lava seawater as an extract stock solution, and sterilizing the lava seawater by using a sterilizing filter; adding Dendrobium officinale stems into the lava seawater, reflux-extracting, filtering with filter cloth, filtering with a filter, and completely removing solvent from the filtrate with a vacuum concentrator to obtain the powder of the lava seawater extract of Dendrobium officinale.