Platycladus orientalis extract for inhibiting activity of emasculation-related enzyme as well as extraction method and application thereof

Abstract

The invention relates to the field of cosmetics, in particular to a platycladus orientalis extract for inhibiting the activity of emasculation-related enzymes as well as an extraction method and application of the platycladus orientalis extract, and the extraction method comprises the following steps: A, taking, cleaning and drying platycladus orientalis leaves to obtain a platycladus orientalis raw material; b, adding the platycladus orientalis raw material into water, and heating and extracting to obtain a first crude extract; c, sieving the first crude extract to obtain a second crude extract; d, adding a preservative into the second crude extract to obtain a third crude extract; e, adding a pH regulator into the third crude extract, and regulating the pH to be acidic to obtain a fourth crude extract; and F, carrying out solid-liquid separation on the fourth crude extract to obtain the platycladus orientalis extract. According to the invention, the post-treatment process flow of the extract is greatly simplified, the cosmetic application is met, and the prepared extract also has the effects of inhibiting 5a-reductase activity and preventing alopecia; meanwhile, no organic solvent is adopted for extraction, environment friendliness is achieved, and the danger coefficient is low.

Description

Technical Field

[0001] The invention relates to the field of cosmetics, and in particular to a Platycladus orientalis extract capable of inhibiting the activity of androgenetic alopecia-related enzymes, and an extraction method and application thereof. Background Art

[0002] Platycladus orientalis leaf is a traditional Chinese herbal medicine, and is also a widely used and widely recognized anti-hair loss Chinese medicine in the cosmetics industry. Therefore, its extract is widely used in anti-hair loss cosmetics. The preparation process of its extract is mainly solvent extraction methods such as water extraction and alcohol extraction, and the extract is obtained after multiple steps of impurity removal. This extraction method generally has more processes, which affects the timeliness, and the organic solvents such as ethanol used have high risk factors and are not environmentally friendly.

[0003] Androgenic alopecia (AGA) is the most common type of hair loss. Its pathogenic mechanism has been widely studied. The widely accepted mechanism is that androgen (DT) is transported to hair follicles through the blood and reduced to more active DHT by 5α-reductase in hair papilla cells. DHT binds to hair papilla cell receptors and regulates the expression of genes related to hair growth, ultimately leading to gradual atrophy of hair follicles and hair loss. Inhibiting 5α-reductase activity can inhibit the conversion of DT to DHT, thereby preventing hair loss. Oxidative stress is also one of the factors affecting hair loss. Oxidative stress caused by endogenous or exogenous factors affects the normal physiological cycle of hair. Providing a certain degree of antioxidant activity may also be one of the strategies to prevent hair loss. Summary of the invention

[0004] The purpose of the present invention is to disclose an extraction method and application of a Platycladus orientalis extract for inhibiting the activity of enzymes related to androgenetic alopecia, simplify the extraction process of Platycladus orientalis leaves, and the obtained extract has good stability, has antioxidant properties, and inhibits 5a-reductase activity, and can be used as a raw material for the formulation of anti-hair loss shampoo and hair care products.

[0005] The process of the present invention does not contain organic solvents, is environmentally friendly and has a low risk factor. Water extraction is less irritating than alcohol extraction, thus avoiding the eye irritation problem caused by the extract after application.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions: A method for extracting a Platycladus orientalis extract for inhibiting the activity of androgenetic related enzymes comprises the following steps: A. Take Platycladus orientalis leaves, wash and dry them to obtain Platycladus orientalis raw materials; B. adding the Platycladus orientalis raw material into water and performing heating extraction to obtain a first crude extract; C. sieving the first crude extract to obtain a second crude extract; D. adding a preservative to the second crude extract to obtain a third crude extract; E. adding a pH regulator to the third crude extract to adjust the pH to acidic to obtain a fourth crude extract; F. Performing solid-liquid separation on the fourth crude extract to obtain a Platycladus orientalis extract.

[0007] Preferably, in step D, the preservative includes one or more of phenoxyethanol and sodium benzoate; in step E, the pH adjuster includes citric acid.

[0008] Preferably, in step D, in the preservative, the mass ratio of the phenoxyethanol to the sodium benzoate is 7-8:2-3.

[0009] The specific ratio of phenoxyethanol + sodium benzoate is compounded, and the antiseptic performance is better than that of a single ingredient with the same content.

[0010] Preferably, in step B, the extraction temperature of the heating extraction includes 93° C., and the extraction time includes 5 hours; in step F, the solid-liquid separation includes centrifugal separation.

[0011] Preferably, in step F, the separation speed of the centrifugal separation is 3000 rpm, and the centrifugal time is 30 min; the centrifugal separation is performed in a centrifuge.

[0012] The purification process after extraction of the present invention only uses centrifugation for treatment, which greatly simplifies the post-processing process of the extract, is suitable for cosmetic applications, and the prepared extract also has the function of inhibiting 5a-reductase activity and having the effect of preventing hair loss; at the same time, no organic solvent is used for extraction, which is environmentally friendly and has a low risk factor.

[0013] Preferably, in step B, the mass ratio of the Platycladus orientalis raw material to water is 1:8~20; in step D, the amount of the preservative added is 1% of the mass of the second crude extract; in step E, the pH of the fourth crude extract is 5.5; in step F, the solid content of the Platycladus orientalis extract is 1~3%.

[0014] Preferably, in step A, the drying method includes oven drying; in step B, the heating extraction is performed in a steam generator; in step C, the sieving screen includes 300 mesh.

[0015] A Platycladus orientalis extract obtained by the extraction method of the Platycladus orientalis extract for inhibiting the activity of androgenetic related enzymes.

[0016] An application of the above-mentioned Platycladus orientalis extract is used to prepare one or more of a preparation for inhibiting the activity of androgenetic atrophy-related enzymes and a preparation for scavenging free radicals.

[0017] Preferably, the androgenetic degeneration-related enzyme includes 5α-reductase; and the free radical includes DPPH free radical.

[0018] Compared with the prior art, the present invention has the following beneficial effects: 1. Simplify the extraction process of Platycladus orientalis leaf extract, improve the timeliness, and keep it stable without precipitation.

[0019] 2. It adopts water extraction method, which is environmentally friendly and has low risk factor. The 100% concentration extract has no eye irritation.

[0020] 3. The prepared extract has the ability to inhibit 5α-reductase activity and has the effect of improving androgenic alopecia.

[0021] 4. The extract has the effect of scavenging DPPH free radicals, with a free radical scavenging rate of 45.5%, and has certain antioxidant properties.

[0022] 5. The extract can be used in the formulation of mild, non-eye-irritating hair care products, which can prevent consumers from losing their hair. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of the extract without centrifugation.

[0024] Figure 2 Schematic diagram of the centrifuged extract after 3 months.

[0025] Figure 3 This is a graph showing the average peak results of each group in the 5α-reductase activity inhibition test.

[0026] Figure 4 This is the chicken embryo irritation picture of 100% concentration of Platycladus orientalis leaf water extract.

[0027] Figure 5 This is the chicken embryo irritation picture of 50% concentration of Platycladus orientalis leaf alcohol extract. DETAILED DESCRIPTION

[0028] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings.

[0029] Example 1 Extraction process of Platycladus orientalis leaf extract: (1) Wash the raw leaves, dry them, and weigh 86g.

[0030] (2) Add 688 g of water (feed-liquid ratio = 1:8) into the transparent emulsifying pot and control the steam generator to extract at 93 °C for 5 hours.

[0031] (3) Pass through a 300-mesh sieve and extract 497.4 g of extract.

[0032] (4) Add 1% preservative (phenoxyethanol: sodium benzoate = 0.8:0.2).

[0033] (5) Add appropriate amount of citric acid to adjust the pH to 5.5.

[0034] (6) Centrifuge (3000 rpm, 30 min) to obtain a solid content of 2.32% to obtain Platycladus orientalis leaf extract. No precipitation occurred after long-term storage.

[0035] Example 2 Extraction process of Platycladus orientalis leaf extract: (1) Wash the raw leaves, dry them, and weigh 86g.

[0036] (2) Add 860 g of water (feed-liquid ratio = 1:10) into the transparent emulsifying pot and control the steam generator to extract at 93 °C for 5 hours.

[0037] (3) Pass through a 300-mesh sieve and extract 647 g of extract.

[0038] (4) Add 1% preservative (phenoxyethanol: sodium benzoate = 0.8:0.2).

[0039] (5) Add appropriate amount of citric acid to adjust the pH to 5.5.

[0040] (6) Centrifuge (3000 rpm, 30 min) to obtain a solid content of 1.95%; Platycladus orientalis leaf extract was obtained without precipitation after long-term storage.

[0041] Example 3 Extraction process of Platycladus orientalis leaf extract: (1) Wash the raw leaves, dry them, and weigh 86g.

[0042] (2) Add 1290 g of water (feed-liquid ratio = 1:15) into the transparent emulsifying pot and control the steam generator to extract at 93 °C for 5 hours.

[0043] (3) Pass through a 300-mesh sieve and extract 1105 g of extract.

[0044] (4) Add 1% preservative (phenoxyethanol: sodium benzoate = 0.8:0.2).

[0045] (5) Add appropriate amount of citric acid to adjust the pH to 5.5.

[0046] (6) Centrifuge (3000 rpm, 30 min) to obtain a solid content of 1.31% Platycladus orientalis leaf extract, which has no precipitation after long-term storage.

[0047] Example 4 Extraction process of Platycladus orientalis leaf extract: (1) Wash the raw leaves, dry them, and weigh 86g.

[0048] (2) Add 1720 g of water (feed-liquid ratio = 1:20) into the transparent emulsifying pot and control the steam generator to extract at 93 °C for 5 hours.

[0049] (3) Pass through a 300-mesh sieve and extract 1512 g of extract.

[0050] (4) Add 1% preservative (phenoxyethanol: sodium benzoate = 0.8:0.2).

[0051] (5) Add appropriate amount of citric acid to adjust the pH to 5.5.

[0052] (6) Centrifuge (3000 rpm, 30 min) to obtain a solid content of 1.02% Platycladus orientalis leaf extract, which has no precipitation after long-term storage.

[0053] Comparative Example 1 Extraction process of Platycladus orientalis leaf extract: (1) Wash the raw leaves, dry them, and weigh 86g.

[0054] (2) Add 516 g of water (feed-liquid ratio = 1:6) into the transparent emulsifying pot and control the steam generator to extract at 93 °C for 5 hours.

[0055] (3) Pass through a 300-mesh sieve and extract 313 g of extract.

[0056] (4) Add 1% preservative (phenoxyethanol: sodium benzoate = 0.8:0.2).

[0057] (5) Add appropriate amount of citric acid to adjust the pH to 5.5.

[0058] (6) Centrifuge (3000 rpm, 30 min) to obtain a solid content of 2.58%. Platycladus orientalis leaf extract was obtained. After being left overnight, precipitation was visible.

[0059] Effect Example 1 Taking Example 1 as an example, the extract after completing step (5) of Example 1 was taken for observation. After being left overnight, Figure 1 shown.

[0060] At the same time, the extract after completing step (6) of Example 1 was taken for observation. After being placed under the same conditions for three months, Figure 2 shown.

[0061] from Figure 1 , Figure 2From the comparison, it can be seen that the extract that has not been centrifuged will precipitate after being placed, while the extract that has been centrifuged will still not precipitate after being placed for up to 3 months, and has better stability.

[0062] Effect Example 2 Taking Example 1 as an example, the Platycladus orientalis leaf extract obtained in Example 1 was used to conduct an antiseptic challenge experiment.

[0063] The theoretical inoculation amount of bacteria is 5.0*106 cfu / g; the theoretical inoculation amount of mold is 1.2*10 5 .

[0064] The results are shown in Table 1.

[0065] Table 1

[0066] The results show that the Platycladus orientalis leaf extract obtained by the present invention can pass the antiseptic challenge test.

[0067] Effect Example 3 Taking Example 1 as an example, a test on the inhibition of 5α-reductase activity was performed.

[0068] Test steps: (1) Preparation of rat 5α-reductase: Take 5 male SD rats, remove the prostate gland and cut it into pieces on an ice table. Use pre-cooled buffer to make a homogenate in a glass homogenizer at 0℃, centrifuge, take the supernatant and add buffer to make the volume to 20 mL, and dispense into EP tubes, 1 mL per tube, which is the enzyme extract, and store in a -80℃ refrigerator.

[0069] (2) 5α-reductase activity assay: A 2 mL reaction system contained: phosphate buffer L, enzyme extract, testosterone solution, NADPH solution (10% ethanol in the complete reaction group, finasteride in the positive control group, and sample solution in the sample group). In the control group, the enzyme extract was replaced with phosphate buffer and dichloromethane was added to terminate the reaction; the complete reaction group, the positive group, and the sample group were reacted at 37°C for 30 min. After the reaction, dichloromethane was added to stop the reaction, and the reaction was shaken for 1 min and then centrifuged. The upper aqueous phase was removed, the organic phase was removed and evaporated to dryness, the residue was dissolved in methanol, and the testosterone peak area (T) was determined by high performance liquid chromatography. Three parallel tubes were set up for each reaction.

[0070] (3) Inhibition rate = (T 样品管 -T 反应管 ) / (T 空白管 -T 反应管 )*100% Experimental results: The average testosterone peak area and inhibition rate of each group are shown in Table 2 and Figure 3 shown.

[0071] Table 2

[0072] *: The difference was statistically significant compared with the complete response group (P<0.05).

[0073] From Table 2 and Figure 3 It can be seen from the statistical analysis that compared with the complete reaction group, the average peak area of ​​testosterone (T) in the positive group increased, and the inhibition rate of type II 5α-reductase was 77.73%, which was statistically significant (P<0.05); the average peak area of ​​testosterone (T) in the sample "Platycladus orientalis leaf extract" increased at concentrations of 100%, 50% (v / v), and 5% (v / v), and the inhibition rates of type II 5α-reductase were 73.76%, 67.57%, and 35.20%, respectively, which were statistically significant (P<0.05). Under the conditions of this experiment, this sample "Platycladus orientalis leaf extract" had an anti-hair loss effect at 100%, 50% (v / v), and 5% (v / v).

[0074] Effect Example 4 The Platycladus orientalis leaf water extract obtained by the method of the present invention (taking Example 1 as an example) and the Platycladus orientalis leaf alcohol extract obtained by conventional alcohol extraction were compared and tested as follows: 1. Test Purpose Prediction of conjunctival eye irritation of test samples by CAM membrane model 2. The test steps are shown in Table 3.

[0075] Table 3

[0076] 3. Test parameters 1. Stimulus Rating (IS) Result judgment: IS<1: non-irritating; 1≤IS<5: mildly irritating; 5≤IS<9: moderately irritating; IS≥9: strongly irritating / corrosive.

[0077] 4. The test data are shown in Table 4 and Table 5.

[0078] Table 4 CAM membrane eye irritation score of 100% concentration of Platycladus orientalis leaf water extract (scoring method: IS)

[0079] 1. 100% concentration of Platycladus orientalis leaf water extract IS=0, and is classified as non-irritating to the eyes; 2. The IS score of the positive control 1% SLS benchmark substance is 6.65, which is moderately irritating.

[0080] Table 5 Eye irritation score of CAM membrane of 50% concentration of Platycladus orientalis leaf alcohol extract (scoring method: IS)

[0081] 1. IS of 50% concentration of Platycladus orientalis leaf alcohol extract is 9.43, and its eye irritation is classified as strong irritation; 2. The IS score of the positive control 1% SLS benchmark substance is 5.98, which is moderately irritating; 5. Test pictures The Platycladus orientalis leaf water extract of test sample 1 acted on the CAM membrane for 5 minutes, and a chicken embryo irritation picture was taken for one group as an example, as shown in FIG. Figure 4 As shown, the chicken embryo irritation status of the other groups of test sample 1 was similar.

[0082] The alcohol extract of Platycladus orientalis leaves of test sample 2 acted on the CAM membrane for 5 minutes, and a chicken embryo irritation picture was taken for one group as an example, as shown in FIG. Figure 5 As shown, the chicken embryo irritation status of the other groups of test sample 2 was similar.

[0083] 6. Test Conclusion The comprehensive test results are summarized and shown in Table 6.

[0084] Table 6

[0085] Effect Example 5 Different Platycladus orientalis leaf extracts were compared and tested, and the results are shown in Table 7.

[0086] Table 7

[0087] It can be seen from the irritation score and antiseptic performance comparison table in Table 7 that, on the one hand, the irritation of the Platycladus orientalis leaf water extract of the present invention is significantly reduced compared with the Platycladus orientalis leaf alcohol extract. On the other hand, when the preservative of the present invention is mixed with phenoxyethanol: sodium benzoate = 0.8:0.2, the average antibacterial rate of bacteria can be improved to a certain extent compared with the single use while maintaining low irritation.

[0088] Effect Example 6 The Platycladus orientalis leaf extract obtained in Example 1 was used to prepare an essence, which was then tested and evaluated. The results are shown in Table 8.

[0089] Table 8

[0090] It can be seen from Table 8 that the essence prepared by using the Platycladus orientalis leaf extract of the present invention can effectively reduce the occurrence of hair loss, especially when the added amount reaches 10%, the hair loss condition can be significantly improved.

[0091] The above disclosure is only the preferred embodiment of the present invention, which certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. A method for extracting Platycladus orientalis extract for inhibiting the activity of androgenetic related enzymes, characterized in that: The following steps are involved: A. Take Platycladus orientalis leaves, wash and dry them to obtain Platycladus orientalis raw materials; B. adding the Platycladus orientalis raw material into water and performing heating extraction to obtain a first crude extract; C. sieving the first crude extract to obtain a second crude extract; D. adding a preservative to the second crude extract to obtain a third crude extract; E. adding a pH regulator to the third crude extract to adjust the pH to acidic to obtain a fourth crude extract; F. Performing solid-liquid separation on the fourth crude extract to obtain a Platycladus orientalis extract.

2. The method for extracting the Platycladus orientalis extract for inhibiting the activity of androgenetic related enzymes according to claim 1, characterized in that: In step D, the preservative includes one or more of phenoxyethanol and sodium benzoate; in step E, the pH adjuster includes citric acid.

3. The method for extracting the Platycladus orientalis extract for inhibiting the activity of androgenetic related enzymes according to claim 2, characterized in that: In step D, in the preservative, the mass ratio of the phenoxyethanol to the sodium benzoate is 7-8:2-3.

4. The method for extracting the Platycladus orientalis extract for inhibiting the activity of androgenetic related enzymes according to claim 1, characterized in that: In step B, the extraction temperature of the heating extraction includes 93° C., and the extraction time includes 5 hours; in step F, the solid-liquid separation includes centrifugal separation.

5. The method for extracting the Platycladus orientalis extract for inhibiting the activity of androgenetic related enzymes according to claim 4, characterized in that: In step F, the separation speed of the centrifugal separation includes 3000 rpm / min, and the centrifugal time includes 30 min; the centrifugal separation is performed in a centrifuge.

6. The method for extracting the Platycladus orientalis extract for inhibiting the activity of androgenetic related enzymes according to claim 1, characterized in that: In step B, the mass ratio of the Platycladus orientalis raw material to water is 1:8~20; in step D, the amount of the preservative added is 1% of the mass of the second crude extract; in step E, the pH of the fourth crude extract is 5.5; in step F, the solid content of the Platycladus orientalis extract is 1~3%.

7. The method for extracting the Platycladus orientalis extract for inhibiting the activity of androgenetic related enzymes according to claim 1, characterized in that: In step A, the drying method includes oven drying; in step B, the heating extraction is performed in a steam generator; in step C, the sieving screen includes 300 meshes.

8. A Platycladus orientalis extract obtained according to the method for extracting the Platycladus orientalis extract for inhibiting the activity of androgenetic apoptosis-related enzymes according to claim 1.

9. An application of the Platycladus orientalis extract according to claim 8, characterized in that: Used to prepare one or more of the preparations for inhibiting the activity of androgenetic degeneration-related enzymes and the preparations for scavenging free radicals.

10. The use of the Platycladus orientalis extract according to claim 9, characterized in that: The male degeneration-related enzyme includes 5α-reductase; the free radical includes DPPH free radical.

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