TWI522127B - Phyla nodiflora extract for skin whitening - Google Patents

Description

Dendrobium extract for skin whitening

The technology relates to a composition formed by the extract of Dendrobium can be added to cosmetics, skin care products or external medicines to exhibit skin whitening function, and can be applied to the pharmaceutical industry and chemical raw materials, and has industrial use.

In recent years, the application of natural extracts has been paid more and more attention by individual industries. Due to the high safety of natural products, it is often used as a raw material for the development of the cosmetics industry, and added to the beauty care products as an improvement of skin texture. .

The scientific name of sarcophagus is Phyla nodiflora (L.) Greene, synonymously called Lippie nodiflora (L.) Michx or Verbena nodiflora (L.), which belongs to the genus Verbena, and is used by the folk. Irregular menstruation, feelings of drinking after drinking, vaginal discharge of women, inflammation of the ear, swelling of the throat, herpes zoster, sputum, gums, dysentery and other diseases.

Dendrobium plants have a variety of biological activities; including anti-atherosclerosis, analgesic, anti-E. coli, inhibition of microbial growth, liver protection and lipid peroxidation, Shukla et al. 2009 Int.J .Pharm.Pharm.Sci. It was found that the crude extracts of methanol and water in the shoot had diuretic effects. In 2010, Dodoala et al . pointed out that Indian J. Nat. Prod. Sci. pointed out that the crude ethanol extract of Dendrobium candidum has the ability to prevent stones; and Turaskar et al. in 2011, Asian J. Pharm. Clin. Res . pointed out that ethanol and The crude extract of chloroform has an inhibitory effect on the central nervous system and can be used in the treatment of anti-caries and anti-anxiety.

The chemical constituents isolated from Dendrobium have been published in the literature, including flavonoids, monoterpenes, sesquiterpenes. (sesquiterpenes), quinols and quinol glucosides, triterpenoids, steroids, volatiles, alkaloids, tannins, Saponins and sugars.

The inventor, in view of Chen Peijun's 2009 master's thesis, pointed out that the methanol extract of the aboveground part of Dendrobium and the known compound such as eupafolin have the activity of inhibiting extracellular mushroom tyrosinase, but the prior art is still Incompletely, after careful experimentation and research, and a spirit of perseverance, I finally conceived the case "Dendrobium extract for skin whitening", which can overcome the shortcomings of the prior art. The following is a brief description of the case.

The present invention is the first to find that the methanol extract of Dendrobium can be safe for cells and inhibits the synthesis of tyrosinase by inhibiting the regulation of microphthalmia transcription factor (MITF) protein expression, and further inhibits melanin production and whitens utility.

In light of the above concept, one of the present techniques provides a cosmetic composition comprising a plant extract of Phyla nodiflora (L.) Greene.

According to the above concept, the cosmetic composition provides discomfort or disease which can be used to treat hyperpigmentation, such as freckle, chloasma, striae of pregnancy, and senile plaque. And melanoma. It also reduces the anti-aging effects of procollagen synthesis, elasticity and collagen content.

In accordance with the above concept, another aspect of the present technology provides a multi-administered method of cosmetic composition comprising a plant extract of Phyla nodiflora (L.) Greene.

According to the above concept, the cosmetic composition can be subjected to multiple combination administration methods to treat discomfort or disease caused by excessive pigmentation, and to reduce anti-aging state such as procollagen synthesis, elasticity and collagen content. Specifically, in the embodiment, the cosmetic composition can be externally applied or administered in accordance with a general cosmetic or pharmaceutical method, and its efficacy can be exhibited alone. An additional cosmetic or pharmaceutical product may also be used, which may be used separately or in combination with the cosmetic composition in accordance with the state of the desired improvement site.

The skin is a complex organ with a variety of cell types and structures that cover the surface of the human body as a protective barrier to isolate internal organs and external environmental stimuli. In recent years, it has been affected by artificial chlorofluorocarbons, which has caused the thickness of the ozone layer to become thinner, resulting in an increase in the amount of ultraviolet radiation. Moderate exposure to sunlight can help the body to synthesize enough vitamin D to maintain healthy bone growth, and also promote the proper amount of melanin production as a protective mechanism against UV damage. However, excessive exposure may lead to an increase in the expression of matrix metalloproteinases (MMPs) in the skin, degradation of extracellular matrix proteins, reduction of procollagen synthesis, reduction in elasticity and collagen content, and photoaging ( Photoaging) phenomena such as rough, dry and wrinkled skin. Even promote melanocyte differentiation to produce more melanin to the surface of the skin, causing abnormal pigmentation, such as freckles, sunburn and chloasma, or senile plaque, melanoma The beauty of the skin increases the risk of skin cancer.

The melanin produced by mammals consists of dark brown eumelanin and red-yellow pheomelanin, which determines the color change of mammalian skin and hair according to the ratio of mixing. In the melanin synthesis pathway, tyrosinase plays an important role as a rate determining step. This is a structure containing copper polyphenol oxidase (polyphenol oxidase, PPO), widely found in microorganisms, animals and plants, is a key enzyme in controlling browning of foods and producing melanin in the skin. Tyrosinase catalyzes the hydroxylation of tyrosine (L-tyrosine) to form L-dopa, which is then catalyzed by tyrosinase to form dopaquinone. The pathway, in the presence of glutathione or cysteine, reacts to form melanoids. Second, dopaquinone produces dopachrome via cyclization and oxidation. Dopachrome is further tyrosinase and dopachrome tautomerase (TRP-2). And 5,6-dihydroxy indole carboxylic acid (DHICA) oxidase (TRP-1) catalyzes the formation of true melanin, and melanin determines the color of skin and hair according to the mixing ratio. In addition, ultraviolet radiation also promotes the formation of reactive oxygen molecules such as nitric oxide, which plays an important role in ultraviolet B (UVB)-induced erythema. Nitric oxide also acts as a melanin production regulator to promote melanin production by regulating the expression of the tyrosinase gene and phosphorylation of melanocyte growth and survival of the small eye malformation transcription factor (MITF). Therefore, if it can effectively inhibit the production of nitric oxide or effectively regulate the expression of the small-eye deformity transcription factor or tyrosinase and its related proteins, it will effectively improve the skin-related diseases derived from melanin transitional hyperplasia.

The scale is taken from the upper part of the sarcophagus. As shown in the extraction flow chart of the first figure, it is first soaked in dichloromethane, soaked in methanol, and the crude extract is concentrated under reduced pressure to obtain crude dichloromethane and methanol. Pumping.

The methylene chloride crude extract was further subjected to gradient elution with hexane and acetone by cerium oxide (SiO ₂ ) column chromatography, and was divided into five divided layers. Purification and separation by normal phase, reverse phase thin layer chromatography, column chromatography, medium pressure liquid chromatography, etc. according to the properties of each division layer, to obtain 3,7,4',5'-tetrahydroxy-3'-3,7,4',5'-tetrahydroxy-3'-methoxyflavone is referred to as Compound 1.

The methanol crude extract was subjected to gradient elution with n-hexane, ethyl acetate and methanol via cerium oxide column chromatography, and was divided into eight divided layers. Purified and separated by normal phase, reverse phase thin layer chromatography, column chromatography, medium pressure liquid chromatography and molecular sieve according to the properties of each division layer, respectively, and obtained nodifloretin called compound 2, 4 '-hydroxywogonin is called compound 3, onopordin is called compound 4, and cirsiliol is called compound 5, 5, 7, 8, 4' -5,7,8,4'-tetrahydroxy-3'-methoxyflavone, referred to as compound 6 and eupafolin, is referred to as compound 7.

The compound including the high-precursor (hispidulin) compound 8 and 3'-methyl myricetin (larycitrin) is referred to as compound 9 and the activity test is carried out. These flavonoid compounds are conventional compounds which are purified and purified from Dendrobium.

Crude extract of Dendrobium for 2,2-diphenyl-l-picrylhydrazyl (DPPH) free radical scavenging test, 50 μl of analyte and 150 μl of DPPH free After mixing the base solution (0.1 mM), after 30 minutes and 60 minutes of reaction, the absorbance was measured at 517 nm and the clearance was calculated, n = 3. The percentage of DPPH free radical scavenging by the analyte is the scavenging rate (%). As shown in Table 1, the longer the dichloromethane crude extract and the methanol crude extract, the higher the concentration and the higher the concentration, the effect of scavenging free radicals. The better, the dose-dependent phenomenon. In contrast to the two layers of crude extracts, the effect of methanol extracts on DPPH removal is better than that of methylene chloride. The dose with a clearance rate of 50% (SD ₅₀ ) was 75.7 ± 2.3 μg/ml for 30 minutes and 67.8 ± 0.7 μg/ml for 60 minutes, compared to 381.9 ± 17.3 and 235.1 ± 12.7 μg/ml for the latter.

Wang et al . , J. Agric. Food Chem. , 1998, pointed out that some compounds, although not having DPPH scavenging ability, are suitable for 2,2'-diazobis(3-ethylbenzothiazoline-6-sulfonic acid). The ammonium salt (2,2'-azinobis-3-ethylbenzthiazoline-6-sulfonic acid, ABTS) showed a scavenging effect on the cationic free radical test. Therefore, the crude extract was also subjected to the ABTS radical scavenging test. After mixing 30 μl of the analyte and 170 μl of the ABTS cationic radical solution, the absorbance was measured at 734 nm after 7 minutes and 20 minutes of reaction. Calculate the clearance rate, n = 3. The percentage of the ABTS cation free radicals removed by the analyte at this concentration is the scavenging rate (%). As shown in Table 2, the methanol crude extract has a good ABTS ^+. The scavenging effect indicates that the crude extract has the ability to provide hydrogen to terminate the chain reaction of free radical malignancy, reduce damage to cellular tissues, and reduce cell death.

In contrast to the two layers of crude extract, the methanol crude extract removes ABTS ^+. The effect is better than the methylene chloride crude extract. The dose with a clearance rate of 50% (SD ₅₀ ) was 81.9 ± 1.1 μg/ml for 7 minutes and 75.2 ± 0.8 μg/ml for 20 minutes, compared to 464.0 ± 19.4 and 384.9 ± 15.7 μg/ml for the latter.

Methanol crude extract has better ABTS ^{. +} Clearing ability, also represents methanol as a good electron supplier, able to stop the chain reaction caused by free radicals, reduce the amount of free radicals to protect human cells, DNA, lipids or proteins. In addition, both layers of crude extract have a tendency to scavenge free radicals with longer and higher concentrations over time, showing dose-dependent and time-dependent.

Tyrosinase is a copper-containing polyphenol oxidase, which is specific to melanocytes and is regulated by tyrosinase in melanin synthesis, which is a rate determining step. Mainly via (1) monophenolase activity: catalyzing the conversion of tyrosine to dopa; (2) diphenolase activity: catalyzing the formation of dopaquinone in dopa, followed by a series of chemical reactions to form melanin. Decreasing the activity of tyrosinase can effectively reduce the production of melanin and prevent pigmentation. After reacting 80 μl of tyrosine (2 mM) and 100 μl of the crude extract for 10 minutes, add 20 μl of tyrosinase solution (1000 U/ml) for 30 minutes and 60 minutes after the reaction. The absorbance was measured at 490 nm and the inhibition rate of tyrosinase inhibition was calculated, n = 3. As a result, as shown in Table 3, as the sample concentration increased, the ability of the methylene chloride crude extract and the methanol crude extract to inhibit the activity of the enzyme also increased, showing a dose-dependent characteristic. The dose (ID ₅₀ ) of the two-layer crude extract with an inhibition rate of 50% was not significantly inhibited in either 30 minutes or 60 minutes compared with the standard arbutin. However, the inhibition rate of Dendrobium extract at 500 μg/ml is similar to that of arbutin at 400 μg/ml, indicating that there is a component that inhibits tyrosinase activity in the crude extract of Dendrobium, because many chemicals in the crude extract The synergistic or antagonistic action of the components does not produce significant tyrosinase inhibition. Therefore, the compound was evaluated for related activity evaluation based on the separation.

Compound tyrosinase inhibition

In the tyrosinase inhibition test, 80 μl of tyrosine (2 mM) and 100 μl of the test compound 1-9 were reacted for 10 minutes, and then 20 μl of tyrosinase solution (1000 U/ml) was added. After a minute and 60 minute reaction, the absorbance was measured at 490 nm and the inhibition was calculated, n = 3. Among them, vitamin C, citric acid and arbutin were used as positive control. These are effective whitening agents commonly found on the market, but they are limited in their application. Vitamin C is unstable, tannic acid is light-sensitive and causes allergies, while arbutin is a hydroquinone derivative that may be metabolized into genotoxic hydroquinone in the body. If the whitening agent developed can effectively solve these problems, the application value of the developed whitening agent can be improved.

The results are shown in Table 4. The ability of onopordin (4) and eupafolin (7) to inhibit tyrosinase has a significant inhibitory effect compared to the standard arbutin, citric acid and vitamin C. The reaction of nodifloretin (2) and cirsiliol (5) in 60 minutes inhibited tyrosinase activity better than vitamin C and tannic acid. Conversely, 4'-hydroxywogonin (3) and 5,7,8,4'-tetrahydroxy-3'-methoxyflavone (5,7,8,4'-tetrahydroxy-3'-methoxyflavone, 6) has no obvious inhibitory effect. As for 3,7,4',5'tetrahydroxy-3'-methoxyflavone (3,7,4',5'-tetrahydroxy-3'-methoxyflavone, 1), high psyllium (hispidulin, 8) And 3'-methyl myricetin (larycitrin, 9) did not detect inhibition because the amount was too small. From the experimental results, it can be found that the inhibition ability of citric acid declines with the increase of reaction time, but the inhibition ability of compounds 2, 3, 5 and 6 increases with the reaction time and the higher the concentration, the better the inhibition effect is presented. Concentration-positive and time-dependent phenomenon.

The mode of action of a compound and an enzyme, depending on the way in which the activity of the enzyme is reduced or inactivated, or the non-covalent binding to the enzyme hinders the entry of the substrate into the active region of the enzyme or changes the conformation of the enzyme to inactivate it. Application of the compound. The mode of action of the compound of onopordin (4) and eupafolin (7) with enzymes inhibiting tyrosinase activity was evaluated by an enzyme kinetic assay. In the enzyme kinetics experiment, a tyrosinase solution (20 U) was reacted with a fixed concentration of the compound dapterin (4) and Zeein lutein (7) and different concentrations of tyrosine solution to A line-average-burk plot was used to determine the type of inhibition of the compound by tyrosinase. The results are shown in the second panel and in the third panel of the comparison graph.

It can be seen from the second figure that the intersection of the big-feather phenol (4) is in the first quadrant, indicating that the pterin is a mixed inhibition mode for tyrosinase, and has the characteristics of both competitive and non-competitive inhibitors. The Km value is the concentration of the matrix at half the maximum catalytic rate and is an indicator of the affinity between the substrate and the enzyme. The larger the Km value, the smaller the affinity. The highest rate ( _Vmax ) is the highest rate of reaction catalyzed by a certain amount of enzyme under a sufficient matrix. The Km values of the compound dapterin were 3.73 (10 μM), 3.66 (60 μM) and 2.77 mM (100 μM), respectively; V _max [optical density (OD) ₄₉₀ / min] was 0.107 (10 μM). ), 0.033 (60 μM) and 0.015 (100 μM). In the 2009 master's thesis, Chen Peijun pointed out that the compound eleufolin (7) shown in the third figure is a competitive inhibition mode.

According to Kubo et al . , Bioorgan. Med. Chem., 2000, the hysteresis effect is typical of the process by which tyrosine is oxidized to form dopa, and when a reducing agent or hydrogen donor is present, especially For o-diphenol hydroxy-substituted compounds, the lag time is shortened or disappeared. The compound dapterin can provide hydrogen or electrons because it has an ortho-diphenolic hydroxyl group on the B ring. Therefore, the fourth graph does not have a delay time, and can shorten the reaction time of converting tyrosine into dopa, and the compound Zeeland Lutein (7) also has the same phenomenon.

In view of this, the present invention can confirm that the extract of Dendrobium methanol does contain an active ingredient which can be developed into a whitening agent. However, this is only the result of extracellular, and it is impossible to completely cover the phenomenon in the cells. In order to make the present invention more complete, the evaluation of intracellular whitening activity of the extract of Dendrobium methanol is carried out. The whitening activity of the extract was evaluated in the B16F10 cell mode.

The cell viability of the methanol extract of Dendrobium sinensis was compared with that of the control group. As shown in the fifth figure, the cell viability of the methanol extract of Dendrobium sinensis was compared with that of the control group, and the extract of Dendrobium sinense was below 200 μg/ml. The effect on cell growth is not statistically significant and can therefore be considered as non-toxic to cells, so concentrations from 12.5 μg/ml to 200 μg/ml will be used for other activity assays.

The inhibitory effect of the melanin production content was evaluated by B16F10 melanocytes at a concentration of 12.5 to 200 μg/ml of Dendrobium extract. After comparison with the control group, as shown in the sixth figure, the methanol extract of Dendrobium candidum showed a dose-dependent inhibition effect at this concentration. Under the experimental concentration conditions, the formation of melanin was inhibited at 50 μg/ml or more. The effect is not cytotoxic, and the best effect at 200 μg/ml can effectively inhibit the melanin content to 52.31%. In addition, At 50 and 100 μg/ml, the melanin content was effectively inhibited by 20.16 and 44.29%, respectively. Below this concentration, such as 12.5 μg/ml and 25 μg/ml, although it has the effect of inhibiting melanin, it is not as effective as the high concentration of the extract of Dendrobium whole grass methanol.

Analysis of the results of inhibition of tyrosinase activity by total extract of Dendrobium

The tyrosinase activity of B16F10 melanocytes was inhibited by the concentration of methanol extract of Dendrobium sinensis from 12.5 to 200 μg/ml. Compared with the control group, as shown in the seventh figure, it was confirmed that the methanol extract of Dendrobium candidum showed a dose-dependent inhibition effect at a series concentration, wherein 200 μg/ml inhibited 30.75% tyrosinase activity and 100 μg/ml. Inhibition of 26.55% tyrosinase activity.

Dendrobium sinensis methanol extract inhibits melanin production pathway-related protein expression

The concentration of methanol extract of Dendrobium candidum from 12.5 to 200 μg/ml showed a dose-dependent effect on melanin production pathway-related proteins in B16F10 cells. As shown in the eighth to eleventh figures, compared with the control group, it was confirmed that the methanol extract of Dendrobium candidum effectively inhibited the expression of the small-eye deformity transcription factor (MITF) at 4 μg/ml, and further inhibited tyramine. The inhibitory rates of acidase (TYR), tyrosinase-related protein-1 (TRP-1) and -2 (TRP-2) were 43.11%, 29.97%, and 49.31%, respectively. At 100 μg/ml, it can effectively inhibit the regulation of small eye deformity transcription factor (MITF) by 29.27%, and further inhibit tyrosinase (TYR), tyrosinase-related protein-1 (TRP-1) and -2 ( TRP-2), which reached 26.11%, 28.41%, and 26.13%, respectively, ultimately inhibited melanin biosynthesis.

In view of this, the Dendrobium methanol extract of the present invention evaluated the whitening activity of the extract in the B16F10 cell mode. According to the results of the invention, the extract of Dendrobium candidum is not toxic to cells, so it is safe; in addition, the extract of Dendrobium can inhibit the regulation of microphthalmia transcription factor protein expression, indicating that the extract has inhibitory tyramine Synthesis of acid enzymes and further inhibition of black The pigment (melanin) is produced to achieve whitening effect. In summary, the extract of Dendrobium can be added to cosmetics, skin care products or external medicines to present skin whitening function.

In addition to the whitening effect of the extract of Dendrobium, the present invention also investigates related molecular biomechanics of ezefalin (7) in the composition, and the results are as shown in the twelfth to eighteenth. As shown in Figure 12, the cell viability of eupafolin (7) compared with the control group, except for 80 μM, affects the survival rate of B16F10 cells. The effect of other concentrations on cell growth is not statistically significant. Meaning, it can be regarded as not toxic to cells, but for more stringent safety considerations, only use the concentration below 10 μM for the following other activity tests.

Zealin lutein inhibited melanin production in B16F10 melanocytes at a concentration of 0.01 to 10 μM. As shown in Fig. 13, after comparison with the control group, it was confirmed that Zeein lutein appeared at this concentration. The dose-dependent inhibition effect, under the experimental concentration conditions, above 0.01 μM, has the effect of inhibiting melanin production, and is not cytotoxic, among which the best effect is 10 μM, which can effectively inhibit the melanin content to 43.33%. In addition, the melanin content was effectively inhibited by 14.5 and 28.77% at 0.1 and 1 μM, respectively.

Zelan Lutein inhibits tyrosinase activity of B16F10 melanocytes at a concentration of 0.01 to 10 μM, as shown in Figure 14, after comparison with the control group, it was confirmed that Zeein lutein was at this concentration. It showed a dose-dependent inhibition effect. Under the experimental concentration conditions, 0.01 μM or more had the effect of inhibiting tyrosinase activity, and it was not cytotoxic, and the best effect was 10 μM, which effectively inhibited tyrosinase. The activity reached 56.04%. In addition, tyrosinase activity was effectively inhibited at 24.47 and 47.77%, respectively, at 0.1 and 1 μM. Zeein lutein showed a dose-dependent effect on melanin production pathway-related proteins in B16F10 cells at concentrations of 0.01 to 10 μM. As shown in Figure 15, after comparison with the control group, it was confirmed that Eupafolin can effectively inhibit the small-eye deformity transcription factor at 10 μM. (MITF) showed a performance of 65.9%, which effectively inhibited the expression of the small-eye deformity transcription factor (MITF) at 4 μM by 48.9%. In addition, the MTF performance was inhibited by 40.6 and 33.0% at 0.1 and 0.01 μM, respectively.

Zeein lutein showed a dose-dependent effect on melanin production pathway-related proteins in B16F10 cells at concentrations of 0.01 to 10 μM. As shown in Figure 16, after comparison with the control group, it was confirmed that Zeein lutein can effectively inhibit the expression of tyrosinase (TYR) at 5 μM, which is 58.6%, and can effectively inhibit tyrosine at 1 μM. The performance of the enzyme (TYR) was 42.6%. In addition, the TYR performance was inhibited by 21.0 and 15.3% at 0.1 and 0.01 μM, respectively.

Zeein lutein showed a dose-dependent effect on melanin production pathway-related proteins in B16F10 cells at concentrations of 0.01 to 10 μM. As shown in Figure 17, after comparison with the control group, it was confirmed that Zeeanthin could effectively inhibit the expression of tyrosinase-related protein-1 (TRP-1) at 10 μM, which was 49.5%, at 1 μM. Effectively inhibited the performance of TRP-1 by 27.2%. In addition, the expression of TRP-1 was inhibited by 0.1 and 7.8% at 0.1 and 0.01 μM, respectively.

Zeein lutein showed a dose-dependent effect on melanin production pathway-related proteins in B16F10 cells at concentrations of 0.01 to 10 μM. As shown in Fig. 18, after comparison with the control group, it was confirmed that Zeeanthin could effectively inhibit the expression of tyrosinase-related protein-2 (TRP-2) at 10 μM, which was 68.6%, at 1 μM. Effectively inhibited the performance of TRP-2 by 51.8%. In addition, TRP-2 expression was inhibited at 27.0 and 12.5%, respectively, at 0.1 and 0.01 μM.

In summary, Zeein lutein can inhibit the expression of tyrosinase and its related proteins by regulating the expression of small eye deformity transcription factors, thereby inhibiting the synthesis of melanin, and further supporting the extract of Dendrobium can be added to Cosmetics, skin care products or topical medicines are used for skin whitening.

A fingerprint print or a fingerprint print chromatogram is used to establish a chemical fingerprint of a Chinese herbal medicine by the analytical ability and reproducibility of high performance liquid chromatography (HPLC). In the fingerprinting method of the invention, the upper part of the stone is firstly weighed, firstly digested with dichloromethane, and then the soaked stone is soaked in methanol, and the crude extract is concentrated under reduced pressure to obtain a crude dichloromethane extract. And methanol crude extract. The two layers of crude extracts were evaluated for activity, and the chemical components were separated according to the active layering, and the compound with the highest content or better activity was used as the index component to establish the fingerprint of the stone sarcophagus.

Choice of mobile phase composition

Different solvent systems can be used, including acetonitrile-0.1% formic acid, methanol-0.1% formic acid and methanol-0.1% acetic acid. As shown in the map of Fig. 19 (a) methanol-0.1% formic acid and the nineteenth (b) acetonitrile-0.1% formic acid, it was found that the extraction effects of the two solvent systems were similar. Among them, acetonitrile-0.1% formic acid is the mobile phase, and a group of compounds will accumulate in 80 to 100 minutes. Because acetonitrile is a regulated drug and expensive in purchase price, methanol-0.1% formic acid is selected based on safety and economic considerations. Mobile phase. 0.1% formic acid was replaced with 0.1% acetic acid as shown in the twentieth diagram. The two maps mainly have the difference of residence time. In the 20th map (a), when the methanol-0.1% acetic acid is used as the mobile phase, the residence time of the peak will move forward, which may cause the original single peak to overlap with other peaks. . In addition, the peaks presented are also obviously tailed. Therefore, the map presented by the twentieth figure (b) methanol-0.1% formic acid as the mobile phase is suitable.

Detection wavelength selection

The methanol extract was scanned by a spectrophotometer, and the change in absorbance was observed in the ultraviolet (200-400 nm) wavelength range. From the map you can find the difference At the wavelengths of 200, 273 and 326 nm, the methanol extract has the highest absorbance, but the maximum absorption range of the methanol solution itself is between 205 and 215 nm. If this wavelength is selected, it may affect the absorbance of the sample. The selected 273 and 326 nm are typical absorption ranges of the flavonoids band I (300~400 nm) and band II (240~280 nm), which are suitable for detecting flavonoids.

Comparing the changes at the spectra at different wavelengths, the spectrum set at 273 nm will have some unknown compounds at 15 to 20 minutes, and the main 40 to 70 minutes signal will be smaller or It disappears and the baseline produces a significant drift, so 326 nm is selected as the detection wavelength. Therefore, the optimized chromatographic method is high pressure liquid chromatography with UV/visible light detector, and is extracted with methanol-0.1% formic acid at a wavelength of 326 nm, and the obtained fingerprint of the crude extract of Dendrobium is as follows. Figure 21 (a) shows that the twenty-first figure (b) is the indicator component of nodifloretin (2), onopordin (4), and eupafolin (7). Corresponds to the map of the crude material. Since nodifloretin(2) is not completely separated from the unknowns and impurities, there will be errors in the quantitative, so the indicator components onopordin(4) and eupafolin(7) can be confirmed, and the onopordin(4) and The content of eupafolin (7).

The technology is prepared as a composition by extracting Phyla nodiflora plants, which can provide skin whitening function. Unless otherwise specified, the crude extract or extract refers to the product of the extraction of the earthworm plant using an organic solvent.

According to the results of the invention, the stone white extract is a safe whitening raw material, which can be added to cosmetics, skin care products or external medicines to present skin whitening functions, and is used in industries such as the pharmaceutical industry and chemical raw materials.

The above excipients, or "pharmaceutically acceptable carriers or excipients", "bioavailable carriers or excipients", include solvents, dispersants, coatings, and antibiotics. Any suitable compound for preparing a dosage form, such as a fungus or an antifungal agent, preserving or delaying an absorbent. Usually such carriers or excipients do not themselves have the activity of treating diseases, and the derivatives disclosed in the present technology, together with pharmaceutically acceptable carriers or excipients, are prepared for administration to animals or humans. Causes adverse reactions, allergies or other inappropriate reactions. Thus, the derivatives disclosed in the present technology, in combination with pharmaceutically acceptable carriers or excipients, are suitable for clinical and human use. "Effective dose" means a dose sufficient to ameliorate or prevent a medical condition or a biological state. The effective dose also means that the dose of the administered compound is sufficient for the diagnosis. Unless otherwise stated in the specification, "active compound" and "pharmaceutically active compound" are used interchangeably herein to refer to a substance having a pharmaceutical, pharmacological or therapeutic effect.

The dosage form of the compound can be administered intravenously, orally, by inhalation or by local or sublingual administration such as nasal, rectal, vaginal, etc., to achieve a therapeutic effect. For patients with different conditions, about 0.1 mg to 100 mg of the active ingredient is administered daily.

The carrier will vary with each dosage form, and the sterile injectable compositions may be solution or suspended in a non-toxic intravenous diluent or solvent such as 1,3-butanediol. A carrier acceptable therebetween may be Mannitol or water. In addition, fixed oils or synthetic single or diglyceride suspension media are common solvents. Fatty acids, such as oleic acid, olive oil or castor oil, and their glyceride derivatives, especially in the form of polyoxyethylation, are useful as injections and are natural pharmaceutically acceptable oils. These oil solutions or suspensions may contain long chain alcohol diluents or dispersants, carboxymethyl cellulose or similar dispersing agents. Other commonly used surfactants such as Tween, Spans or other similar emulsifiers or generally used in the pharmaceutical industry are commercially available solid, liquid or other bioavailable enhancers which are useful in the development of dosage forms.

The composition for oral administration is in any orally acceptable dosage form, and the form thereof includes a capsule, a tablet, a tablet, an emulsifier, a liquid suspension, a dispersing agent, and a solvent. Oral dosage forms are generally used as carriers, and in the case of tablets, lactose, corn starch, and a lubricant such as magnesium stearate are basic additives. The diluent used in the capsules includes lactose and dried corn starch. The liquid suspension or emulsifier dosage form is prepared by suspending or dissolving the active substance in an oily interface combined with an emulsifier or a suspending agent, and adding a moderate sweetener, flavor or pigment as needed.

Nasal gasifying sprays or inhalant compositions can be prepared according to known formulation techniques. For example, the composition is dissolved in physiological saline, benzyl alcohol or other suitable preservative, or an absorbent is added to enhance bioavailability. Compositions of the compounds disclosed in the present technology may also be formulated as a suppository for rectal or vaginal administration.

The compounds disclosed in the present technology may also be administered "intravenous administration", including subcutaneous, intraperitoneal, intravenous, intramuscular, or intra-articular, intracranial, intra-articular, intraspinal injection, aortic injection, intrathoracic injection, diseased parts. Intra-injection, or other suitable drug delivery techniques.

The composition of the present invention can form an appearance such as a cream, an ointment, an emulsion, a lotion, a serum, a paste or a mousse. It may be applied to the skin via an aerosol form as needed, or may be in the form of a solid, such as a stick. It can also be applied to any topical application of the skin care preparations and/or as a cosmetic, in particular an aqueous solution, a water-alcoholic solution or an oily solution, the oil is soluble in water or water soluble in oil or multiple emulsions, aqueous or oily. Gum, liquid, paste or solid anhydrous product. Needless to say, those skilled in the art will be cautious in selecting adjuvants, and/or amounts thereof, which are not considered or substantially not adversely affected by the envisaged additions, in view of the nature of the compounds of the present invention.

The composition of the present invention may also be included in cosmetic and dermatological preparations. Adjuvants, such as hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic active agents, preservatives, antioxidants, solvents, perfumes, fillers, sunscreens, pigments, odor absorbers, and dyes, and the like. The amount of these various adjuvants is a conventional amount in the field of interest, for example, from 0.01 to 20% by weight based on the total weight of the composition. Depending on their nature, these adjuvants can be fed into the fatty phase, the aqueous phase, and the lipid vesicles.

When the composition of the present invention is an emulsion, the ratio of the fat phase may range from 5 to 80% by weight, and preferably from 5 to 50% by weight, based on the total weight of the composition. The oil, emulsifier, co-emulsifier and the like used in the present composition in the form of an emulsion are selected from those conventionally used in the field of consideration. The proportion of the emulsifier and the co-emulsifier present in the composition ranges from 0.3 to 30% by weight, and preferably from 0.5 to 20% by weight, relative to the total weight of the composition.

Oily materials which can be used in the present invention, such as mineral oil (liquid petroleum), vegetable oil (avocado oil, soybean oil), animal oil, lanolin, synthetic oil, perhydrosqualene, eucalyptus oil, cyclomethoxine Cyclomethicone and fluorinated oil, perfluoropolyether. A fatty alcohol (cetyl alcohol), a fatty acid, and a wax (carnauba wax, ozokerite) or the like can also be used as the fatty substance.

As a raw material of an emulsifier and a co-emulsifier which can be used in the present invention, for example, a fatty acid ester of polyethylene glycol such as PEG-20 stearate, and a fatty acid ester of glycerin such as glyceryl stearate. Hydrophilic gelling agent materials, such as: carboxyvinyl polymers, carbomers, acrylic copolymers (such as acrylates/alkyl acrylate copolymers), polypropylene decylamines, polysaccharides, natural gums and Clay, and as a raw material for lipophilic gelling agents, such as modified clay (such as bentonite), metal salts of fatty acids, hydrophobic vermiculite and polyethylene.

Active agents can be used, such as polyols, vitamins, keratolytics and/or descaling tablets, anti-inflammatory agents, calmants and mixtures thereof, depigmenting agents such as citric acid and its derivatives. Lipophilic or hydrophilic can also be used in the composition. UV masking agent, such as benzene-1,4-bis(3-methylene-10-camphorsulfonic acid), 2-ethylhexylα-cyano-β,β-diphenylacrylic acid or cyanide Octocrylen, butyl methoxy benzhydryl methane, octyl methoxycinnamate and/or titanium oxide and zinc oxide.

Cosmetic powders which can be used generally contain fillers, pigments, and pearls. Suitable fillers include vermiculite, surface treated vermiculite, alumina, surface treated alumina, talc and surface treated talc, zinc stearate, mica and surface treated mica, kaolin, nylon (Nylon) powder ( Such as OrgasolTM), polyethylene powder, TeflonTM, starch, boron nitride, lauric acid amide, copolymer microspheres, crosslinked polymethacrylate copolymer, and polyoxyxene microbeads, and the like .

The "Dendrobium extract for skin whitening" proposed in the present application will be fully understood by the following examples, so that those skilled in the art can do so, but the implementation of the present invention is not limited by the following examples. Other embodiments may be derived from the spirit of the disclosed embodiments, and such embodiments are within the scope of the present disclosure. Therefore, the innovative technology revealed in this case has been deeply embedded in industrial value and has been applied for in accordance with the law.

Experimental materials and methods:

Phosphate-buffered saline (PBS)-pH 7.4 potassium dihydrogen phosphate (KH ₂ PO ₄ ): 0.27 g, disodium hydrogen phosphate (Na ₂ HPO ₄ ): 1.42 g, sodium chloride (NaCl) : 8g, potassium chloride (KCl) 0.2g, add about 800mL of deionized water, stir and dissolve, add concentrated hydrochloric acid to adjust the pH to 7.4, and finally set to 1L. Store at room temperature after autoclaving.

Phosphate buffer (PBS-T) containing 0.05% Tween - 300 μl of Tween-20 was added to 100 ml of PBS, and it was mixed and used.

Transfer buffer -30.3 g tris base, 144 g glycine, 20% methanol.

Blocking buffer - 5% skim milk powder was dissolved in phosphate buffer (PBS-T).

Cell safety test of Dendrobium chinensis methanol extract

First, 10,000 mouse melanoma cancer cells (B16F10) were cultured in each well of a 96-well microplate, and cultured in an incubator for 24 hours, then the culture solution was removed, and the culture solution and different concentrations of Dendrobium solanaceous methanol were re-added. Extracts, 12.5, 25, 50, 100 and 200 μg/ml. After the culture solution was removed after 48 hours, 150 μl of a concentration of 5 mg/mL of methyl thiazolil tetracolium (MTT) was added to each well. Thereafter, the cells were cultured in an incubator containing 5% carbon dioxide at 37 ° C for 4 hours, and the culture solution was removed, and 100 μl of Dimethyl sulfoxide (DMSO) was added and reacted for 30 minutes. Finally, the microplate was placed. The cell absorbance was measured at a wavelength of 550 nm by placing it on a microplate spectrophotometer to determine the cell viability of the cell extract of Dendrobium sinensis.

Test of cell melanin production by the methanol extract of Dendrobium

First, 100000 B16F10 cells were cultured in 24-well plates, and cultured in an incubator for 24 hours, the culture solution was removed, and the culture solution and different concentrations of Dendrobium sinensis methanol extract were added again, 12.5, 25, 50, 100. And 200 μg/ml, placed in the incubator for 48 hours, washed twice with Phosphate-buffered saline (PBS) and added with trypsin solution (Trypsin-EDTA) to collect the cells and put them into a microtube. Centrifuge. After the supernatant was removed, 150 μL of 1N sodium hydroxide solution was added and Heat at 95 ° C for 10 minutes. The absorbance of each group was taken from 100 μl to 96 microplates and the absorbance was measured by microplate spectrophotometer at 405 nm. The absorbance was the total melanin content.

Inhibition of intracellular tyrosinase activity by methanol extract of Dendrobium

First, 100000 B16F10 cells were cultured in 24-well plates, and cultured in an incubator for 24 hours, the culture solution was removed, and the culture solution and different concentrations of Dendrobium sinensis methanol extract were added again, 12.5, 25, 50, 100. After 200 hours in the incubator and 200 μg/ml, the cells were washed twice with PBS and Trypsin-EDTA was added to collect the cells and centrifuged in a microtube. After the cells were removed, 100 μL of 1% (v/v) Triton X 100 and 0.1 mM phenlymethylsulfonyl fluoride (PMSF) / 50 mM PBS (pH 6.8) were added to dissolve the cells. It was placed in a microtube and placed at -80 ° C. After 30 minutes, it was removed and placed at 37 ° C for 5 minutes to warm. It was centrifuged at 12,000 rpm for 30 minutes. Take 80 μL of the supernatant and add 20 μL of dopa (L-dopa, 2 mg/mL) in a 96-well microplate, and measure the absorbance at 405 nm on a microplate spectrophotometer to detect intracellular. Tyrase activity.

Experiment on the inhibition of melanin production pathway-related protein expression by Dendrobium extract

The cells were seeded in a 6-well plate and cultured in an incubator until after eight minutes, and different concentrations of Dendrobium sinensis methanol extract were added, 12.5, 25, 50, 100 and 200 μg/ml. After 48 hours, the cells were washed twice with iced PBS, and a fixed amount of cell lysate was added, and then transferred to a microcentrifuge tube, and then heated at 95 ° C for 10 minutes to denature the protein. After completion, 10~15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was placed in the electrophoresis tank and filled with mobile buffer To the solution, 20 μl of the denatured protein sample was injected into each well for electrophoresis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control group.

First, the voltage was 80 V for 30 minutes until the sample was applied to the interface of the stacking gel and the separation gel, and the voltage was adjusted to 100 V for protein electrophoresis separation. After the electrophoresis is finished, the stain is applied. The cut polyvinylidene difluoride (PVDF) was placed in a transfer buffer for use. The SDS-PAGE layer was overlaid on the PVDF membrane, and two 3 mm filter papers were placed on top of each other, and then the sandwich was placed in a transfer buffer, and the current was set at 400 mA, and the stain was applied at 4 ° C for 2 hours.

After completion, the PVDF membrane was added with a blocking buffer for 1 hour at room temperature, and then washed three times with phosphate buffer (PBS-T) containing 0.05% Tween for five minutes each time. Then, dilute the different primary antibodies to cover the PVDF membrane evenly, and shake overnight, such as tyrosinase and its related protein-1 (TRP-1) and -2 (TRP-2), to regulate the small-eye deformity transcription factor ( After completion of MITF), it was washed three times with PBS-T for five minutes each time. Diluted the secondary antibody and poured it evenly on the PVDF membrane for 1 hour, such as a perserase-labeled rabbit anti-goat (HRP-Rabbit anti-Goat) and a peroxidase-labeled sheep. Anti-mouse antibody (HRP-Goat anti-mouse) was washed 3 times with PBS-T. Finally, add the chemical cold light coloring agent (ECL detection buffer) and then take a photo with a cold light camera system to compare the results.

Example 1, the preparation process of the crude extract of Dendrobium

Weigh 4.6 kg of the upper part of the stone, first with dichloromethane After soaking (dichloromethane), the soaked sarcophagus was soaked in methanol, and the crude extract was concentrated under reduced pressure to obtain 101 g of crude dichloromethane extract and 525 g of crude methanol extract.

Example 2, separation and purification of compounds

Dichloromethane crude extract 101 g was subjected to gradient elution with ruthenium dioxide column chromatography (SiO ₂ ) with n-hexane and acetone, and divided into five divided layers (Fr. 1-Fr. 5): division layer 1 (n-hexane/acetone: 100/0, 1.3 g), partition layer 2 (n-hexane/acetone: 90/10, 45.8 g), partition layer 3 (n-hexane/acetone: 80/20, 8.7 g), division Layer 4 (n-hexane/acetone: 65/35, 12.4 g) and layer 5 (n-hexane/acetone: 30/70, 16.0 g). Then use forward or reverse thin layer chromatography or column chromatography, medium pressure liquid chromatography for purification and separation, and obtain 3,7,4',5'-tetrahydroxy-3'-methoxyflavone in Fr.2 (1) (3.9 mg).

650 g of methanol crude extract was taken from 160 g of the column chromatography and eluted with n-hexane, ethyl acetate and methanol, and divided into eight divided layers (Fr. 21-Fr. 28): layer 21 (n-hexane/ethyl acetate/methanol: 100/0/0, 0.3 g), partition layer 22 (hexane/ethyl acetate/methanol: 90/10/0, 0.8 g), partition layer 23 (n-hexane / Ethyl acetate / methanol: 60 / 40 / 0, 1.2 g), partition layer 24 (n-hexane / ethyl acetate / methanol: 40 / 60 / 0, 4.3 g), partition layer 25 (n-hexane / ethyl acetate / Methanol: 7/80/13, 7.7 g), layer 26 (n-hexane/ethyl acetate/methanol: 6/69/25, 33.8 g), partition layer 27 (hexane/ethyl acetate/methanol: 6/) 54/40, 10.5 g) and partition layer 28 (hexane/ethyl acetate/methanol: 5/45/50, 65.6 g). The purification is then carried out by normal phase or reverse phase thin layer chromatography or column chromatography, medium pressure liquid chromatography and molecular sieve. As shown in the first figure, nodifloretin (2) (9.9 mg) is obtained from the division layer 25, respectively. And eupafolin (7) (13.5 mg), dividing layer 26 to get 4'-hydroxywogonin (3) (10.4 mg), onopordin (4) (21.8 mg), cirsiliol (5) (11.8 mg) and 5,7,8,4'-tetrahydroxy-3'-methoxyflavone (6) (10.8 mg), from the sarcophagus Seven pure compounds were isolated.

Example 3, extraction process of Dendrobium whole grass methanol extract

First, 200 g of dried Dendrobium whole grass was weighed and ground into a powder by a grinder, placed in a 2 liter bead flask, and then 1 liter of methanol was added. The triangular conical flask was placed on a heater and placed on a heater, and the temperature was controlled at 100 ° C and heated for 2 hours to conduct a heating reflow test. After 2 hours, the extract was filtered to obtain the first filtrate, and then 1 liter of methanol was again added to the residue of the triangular conical flask for heating and reflux extraction. After a total of 2 cycles, the filtrate was combined and reduced. The concentrate was concentrated and dried in a vacuum freeze dryer to obtain a dry powder.

Example 4, fingerprint preparation

The two layers of crude extracts were evaluated for activity, and the chemical components were separated according to the active layering, and the compound with the highest content or better activity was used as the index component to establish the fingerprint of the stone sarcophagus.

1. Sample and reagent pretreatment

After weighing the sample, it was dissolved in a mobile phase solvent (methanol: 0.1% formic acid = 1:1) to give a final concentration of 1 mg/ml, followed by a 0.45 μm polytetrafluoroethylene (PTFE) filter. Filter to remove impurities from the sample. The solvent used was also filtered through a filter of 0.45 μm polyvinylidene fluoride (PVDF) to remove impurities, and degassed by shaking with an ultrasonic oscillating machine for 15 minutes.

2. Mapping chromatographic conditions

Equipment: ELITE LaChrom with L-2420 UV-Vis detector (detector), L-2200 autosampler (autosampler) and L-2130 push motor (pump)

Column type: Hypersil ^® ODS-C18 column (250 mm × 4.6 mm, 5 μm)

Mobile phase composition: methanol and 0.1% formic acid

Gradient elution: 0-5 minutes, 0-2% methanol; 5-9 minutes, 2-6% methanol; 9-12 minutes, 6-8% methanol; 12-14 minutes, 8-12% methanol; 20 minutes, 12-17% methanol; 20-30 minutes, 17-22% methanol; 30-35 minutes, 22-27% methanol; 35-41 minutes, 27-31% methanol; 41-60 minutes, 31-40 % methanol; 60-75 minutes, 40-43% methanol; 75-80 minutes, 43-45 methanol; 80-120 minutes, 45-80% methanol.

Sample concentration: 1 mg/ml

Flow rate: 0.5 ml/min

Wavelength: 326 nm

Injection volume: 20 μl

III. Indicator components

A compound isolated from the crude extract of Dendrobium sinensis, nodifloretin (2), onopordin (4) and eupafolin (7)

Example 5, cream

Example 6, emulsion

Example 7, whitening cosmetic cream

Other embodiments

An anti-aging composition comprising an extract of Dendrobium.

2. The composition of claim 1, wherein the composition exhibits an effect via a small eye malformation transcription factor-related pathway.

3. The cosmetic composition of claim 1, wherein the HPLC extract of the extract of Dendrobium is selected from the following figures.

4. An anti-aging composition comprising a compound selected from the group consisting of nodifloretin, onopordin, cirsiliol, eupafolin, 5 ,7,8,4'-tetrahydroxy-3'-methoxyflavone, 4'-hydroxywogonin A compound or a mixture thereof.

5. The composition of claim 4, which is characterized by a small eye-shaped transcription factor-related pathway.

6. The composition according to claim 4, wherein the HPLC chart of the extract of Dendrobium is selected from the following figures.

7. The composition of claim 4, which may be a cosmetic or a pharmaceutical.

8. The composition of claim 4, wherein a multi-administered method can be employed.

9. An anti-aging extract comprising a dendrobium extract.

10. The extract of claim 9, which is characterized by a small eye-shaped transcription factor-related pathway.

11. The extract of the extract of Dendrobium extract as described in claim 9 of the patent application, which is selected from the following figures.

12. The extract of claim 9, which comprises a compound selected from the group consisting of nodifloretin, onopordin, cirsiliol, and zealand yellow. Eupafolin, 5,7,8,4'-tetrahydroxy-3'-methoxyflavone, 4'-hydroxyhanurin (4,-hydroxy-hanorin) One of 4'-hydroxywogonin), or a compound thereof.

13. The extract according to claim 9, which may be a cosmetic or a pharmaceutical.

14. The extract according to claim 9 of the patent application, which can be subjected to a multi-administered method.

references:

S. Shukla, R. Patel, R. Kukkar, Study of phytochemical and diuretic potential of methanol and aqueous extracts of aerial parts of Phyla nodiflora Linn., Int. J. Pharm. Pharm. Sci. , 2009, 1 , 85-91 .

S. Dodoala, R. Diviti, B. Koganti, KVSRG Prasad, Effect of ethanolic extract of Phyla nodiflora (Linn.) Greene against calculi producing diet induced urolithiasis, Indian J. Nat. Prod. Sci. , 2010, 1 , 314- 321.

AO Turaskar, SL Bhongade, SM More, AS Dongarwar, VS Shende, VB Pande, Effects of Lippia nodiflora extracts on motor coordination, exploratory behavior pattern, locomotor activity, anxiety and convulsions on albino mice, Asian J. Pharm. Clin. Res. , 2011, 4 , 133-138.

I. Kubo, I. Kinst-Hori, SK Chaudhuri, Y. Kubo, Y. Sánchez, T. Ogura, Flavonols from Heterotheca inuloides : Tyrosinase inhibitory activity and structural criteria, Bioorgan. Med. Chem. , 2000, 8 , 1749- 1755.

The first figure is the extraction flow chart of the whole plant methanol extract of Dendrobium

Figure 2 Kinetics of the tyrosinase enzyme of the compound onion (phenol)

(◆) does not add

( ) Add 10 μM dapterin

(▲) Add 60 μM dapterin

(×) Add 100 μM dapterin

Figure 3 Kinetics of the tyrosinase enzyme of the compound zeain lutein (eupafolin, 7)

(×) does not add

(▲) Add 28 μM Zeein Lutein

(●) Add 56 μM Zeein Lutein

(○) Add 112 μM Zeein Lutein

Figure 4 Inhibition curve of tyrosinase by onopordin (4) with different concentrations of compound

(a◆) 10 μM dapterin

(b ) 60 μM dapterin

(c▲) 100 μM dapterin

Figure 5 Safety analysis of Dendrobium methanol extract on B16F10 melanocytes

Figure 6: Inhibition of melanin production by B16F10 melanocytes

Figure 7: Inhibition of tyrosinase activity by B16F10 melanocytes by Dendrobium methanol extract

Figure 8: Regulating the expression of transcription factors of small eye deformity

A-small eye deformity transcription factor protein

B-glyceraldehyde-3-phosphate dehydrogenase (GADPH)

Figure IX Tyrosinase protein expression

A-tyrosinase

B-glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

Figure 10 shows the performance of tyrosinase-related protein-1

A-tyrosinase-related protein-1 (TRP-1)

B-glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

Figure 11 Tyrosinase-related protein-2

A-tyrosinase-related protein-2 (TRP-2)

B-glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

Figure 12 Cell survival rate of Zeein lutein

Thirteenth image Zeein lutein inhibits melanin production in B16F10 melanocytes

Figure 14 Zela lutein inhibits tyrosinase activity of B16F10 melanocytes

Figure 15 Activity of Zeein Lutein on Pathway-associated Proteins of B16F10 Melanocytes

A-small eye deformity transcription factor protein

B-glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

Figure 16 Diagram of the expression of tyrosinase protein by Zeeland lutein

A-tyrosinase

B-glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

Figure 17 shows the expression of tyrosinase-related protein-1 by Zeeland lutein

A-tyrosinase-related protein-1 (TRP-1)

B-glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

Figure 18 Zeeopsis lutein on tyrosinase-related protein-2

A-tyrosinase-related protein-2 (TRP-2)

B-glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

Figure 19 Fingerprint of different mobile phases

A-methanol-0.1% formic acid

B-acetonitrile-0.1% formic acid

Figure 20 Fingerprint of different mobile phases

A-methanol-0.1% acetic acid

B-methanol-0.1% formic acid

Twenty-first map Shijie crude extract fingerprint

A-HPLC extract of crude extract

B- indicator component crossing the river vine (nodifloretin, 2)

Dapterin (onopordin, 4)

Zeolite lutein (eupafolin, 7)

Claims (8)

A use of a composition for the preparation of a whitening composition comprising a Phyla nodiflora (L.) Greene plant extract. The use of the whitening composition of claim 1, wherein the whitening composition can be a cosmetic or a pharmaceutical. The use according to claim 2, wherein the whitening effect of the whitening composition is for treating freckle, chloasma, striae of pregnancy, and senile plaque ) and discomfort or disease caused by excessive pigmentation of melanoma. The use of the invention of claim 1, wherein the whitening composition can be subjected to a multi-administered method. The use according to claim 1, wherein the extract of Dendrobium plant extract is separated by methanol and separated by HPLC, and the conditions are as follows: equipment: ELITE LaChrom with L-2420 UV-Vis detector, L-2200, autosampler and L-2130 push pump, column type: Hypersil ^® ODS-C18 column (250mm × 4.6mm, 5μm); mobile phase composition: methanol and 0.1% Formic acid; gradient elution: 0-5 minutes, 0-2% methanol; 5-9 minutes, 2-6% methanol; 9-12 minutes, 6-8% methanol; 12-14 minutes, 8-12% methanol; 14-20 minutes, 12-17% methanol; 20-30 minutes, 17-22% methanol; 30-35 minutes, 22-27% methanol; 35-41 minutes, 27-31% methanol; 41-60 minutes, 31 -40% methanol; 60-75 minutes, 40-43% methanol; 75-80 minutes, 43-45 methanol; 80-120 minutes, 45-80% methanol; sample concentration: 1 mg/ml, flow rate: 0.5 ml/min , Wavelength: 326nm, injection volume: 20μl, the index component is the compound isolated from the crude extract of Dendrobium methanol, nodifloretin, onopordin and eupafolin. A use of a composition for preparing a whitening composition comprising a compound selected from the group consisting of nodifloretin, onopordin, cirsiliol, and zebra Eupafolin, 5,7,8,4'-tetrahydroxy-3'-methoxyflavone, 4'-hydroxyhan One of 4'-hydroxywogonin, or a compound thereof. The use of the whitening composition of claim 6, wherein the whitening composition can be a cosmetic or a pharmaceutical. The use according to the invention of claim 7, wherein the whitening effect of the whitening composition is for treating freckle, chloasma, striae of pregnancy, and senile plaque ) and discomfort or disease caused by excessive pigmentation of melanoma.