Method for improving active substances of centella asiatica cells and application of method

Abstract

The invention belongs to the technical field of plant cultivation and plant extract application, particularly relates to a method for improving cell active substances of centella asiatica, and further provides application of centella asiatica extract in preparation of an anti-pathogenic bacterium preparation. According to the method for improving the cell active substances of the centella asiatica, on the basis of a traditional centella asiatica cuttage culture system, by optimizing a rooting induction solution and an induction nutrient solution, especially utilizing the synergistic effect of vitamin C, Mg ions and Ca ions which are added in the rooting induction solution and the induction nutrient solution, and especially by adjusting illumination in the culture process, the survival rate of the centella asiatica is increased, and the survival rate of the centella asiatica is increased. The content and the performance of antioxidant active ingredients such as asiaticoside in the centella asiatica are effectively improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of plant cultivation and plant extract application, and particularly relates to a method for improving cell active substances of Centella asiatica (L.) Urb., and further provides use of the Centella asiatica extract for preparing an anti-pathogenic bacteria preparation. BACKGROUND

[0002] Centella asiatica (L.) Urb. is the dry whole plant of Centella asiatica (L.) Urb. of the Umbelliferae family, also known as Luodeda, Bongdabow, Banbianqian, and mainly distributed in the east, south, central south and southwest provinces and regions of China below 2000m of the mountain, roadside, ditch side, field side, fertile and shady places. Relevant data reports that Centella asiatica can be used as a herbal tea and vegetable for eating in folk, has the effects of treating parotitis, pain relief of burns, chronic renal failure and anti-tumor, and can also be used for beauty and skin care. Modern research also shows that Centella asiatica mainly contains triterpenes and their glycosides, polyacetylenes, volatile oils, flavonoids, alkaloids and other substances, among which the triterpenes asiaticoside is most studied and is considered to be the main effective component of Centella asiatica for exerting the related effects.

[0003] At present, the reported natural extraction products of Centella asiatica can be used for atherosclerosis, anti-hypertension, anti-hyperlipidemia, anti-ulcer, anti-depression, antibacterial and antioxidant, and can also be used for treating skin diseases and skin damage, especially in inhibiting scar formation, promoting wound healing and inhibiting melanin production, and has good application prospects in the fields of food, health care products, medicine and cosmetics.

[0004] Therefore, it is expected in the art to develop more application values of the natural extraction products of Centella asiatica. SUMMARY

[0005] The first object of the present application is to provide a method for improving cell active substances of Centella asiatica, which effectively improves the content and performance of antioxidant active ingredients such as asiaticoside in Centella asiatica by optimizing the seedling raising conditions in the cultivation of Centella asiatica; The second object of the present application is to provide an extraction method of active substances of Centella asiatica, which effectively enhances the effective dissolution of active substances based on the method of enzymolysis combined with alcohol extraction; The third object of the present application is to provide a new use of the active substances of Centella asiatica.

[0006] In order to solve the above technical problems, the method for improving cell active substances of Centella asiatica comprises the following steps: (1) selecting healthy Centella asiatica stolons with nodes and leaves, and cutting the lower end to form a notch; (2) preparing rooting induction liquid, and placing the creeping stems of the Centella asiatica in the rooting induction liquid for rooting induction cultivation; wherein, The rooting induction liquid comprises: IBA 100-300 mg / L, honey 1-3 g / L; (3) continuing to place the cuttings of the creeping stems of the Centella asiatica in the seedling raising substrate; wherein, The seedling raising substrate comprises a mixture of peat, vermiculite and wood ash in a mass ratio of 5-8:2-4:1-3; (4) preparing induction nutrient solution for conventional seedling raising cultivation; wherein, The induction nutrient solution comprises the following components in the following mass contents: glucose 0.05-0.08%, vitamin C 0.01-0.03%, MgCl2 0.01-0.03%, CaCl2 0.01-0.03%, urea 0.01-0.02%.

[0007] Specifically, in the method for improving the cell active substance of the Centella asiatica, the rooting induction cultivation in the step (2) is performed for 3-5 days.

[0008] Specifically, in the method for improving the cell active substance of the Centella asiatica, the temperature in the seedling raising cultivation in the step (4) is 20-22℃.

[0009] Specifically, in the method for improving the cell active substance of the Centella asiatica, the seedling raising cultivation in the step (4) further comprises the step of performing ultraviolet light irradiation at an intensity of 1-3 W / m².

[0010] The application further discloses a method for extracting the active substance of the Centella asiatica, which comprises the steps of obtaining the Centella asiatica according to the method and extracting the active substance from the whole plant of the Centella asiatica.

[0011] Specifically, the method for extracting the active substance of the Centella asiatica comprises the following steps. S1, taking the whole plant of the Centella asiatica to be crushed, and mixing with water to obtain a slurry; S2, adding a mixture of cellulase and pectinase to the slurry to perform enzymolysis treatment; S3, collecting the enzymolysis product, adding 40-60% v / v ethanol solution after enzyme inactivation, collecting the extraction liquid through solid-liquid separation, removing the ethanol, and then concentrating and drying to obtain the required active substance of the Centella asiatica.

[0012] Specifically, the method for extracting the active substance of the Centella asiatica comprises the following steps. In the step S1, the water is used in an amount of 2-4 times the mass of the Centella asiatica; The cellulase is used in an amount of 2-5 wt% of the mass of the Yunnan Rehmannia; The pectinase is used in an amount of 1-3 wt% of the mass of the Yunnan Rehmannia in the step S2. The enzymolysis temperature is 45-55℃, and the enzymolysis time is 1-3 hours in the step S2. The amount of the ethanol solution is 5-8 times of the mass of the Yunnan Rehmannia in the step S3.

[0013] The application further discloses a Yunnan Rehmannia active substance extracted by the method.

[0014] The application further discloses a use of the Yunnan Rehmannia active substance for preparing an antioxidant.

[0015] The application further discloses a use of the Yunnan Rehmannia active substance for preparing a Fusarium oxysporum preparation.

[0016] The method for improving the Yunnan Rehmannia active substance improves the content and performance of the antioxidant active components such as Yunnan Rehmannia glycoside in Yunnan Rehmannia by optimizing the rooting induction liquid and the induction nutrient liquid, especially by using the synergistic effect of the added vitamin C, Mg ions and Ca ions, and by adjusting the light in the cultivation process.

[0017] The extraction method of the Yunnan Rehmannia active substance effectively promotes the dissolution of the active components by using the combined action of cellulase and pectinase, and further enhances the effective dissolution of the active substance by using the ethanol extraction method, thereby improving the dissolution rate of the active components.

[0018] The Yunnan Rehmannia active substance extracted by the application not only has the traditional antioxidant effect, but also has a clear inhibitory effect on Fusarium oxysporum which can cause crop root rot, and the inhibitory effect on pathogenic bacteria can be further enhanced by extracting and purifying the Yunnan Rehmannia extract, thereby providing potential and technical support for the development of an anti-plant crop root rot preparation. DETAILED DESCRIPTION

[0019] In order to further provide the effective content of the active components in Yunnan Rehmannia, the application provides a method for improving the Yunnan Rehmannia active substance, which comprises the following steps: (1) selecting healthy Yunnan Rehmannia stolons with nodes and leaves, and cutting the lower end to form a cut; (2) preparing a rooting induction liquid, and placing the Yunnan Rehmannia stolons in the rooting induction liquid for rooting induction cultivation; wherein, The rooting induction solution comprises: IBA 100-300 mg / L, honey 1-3 g / L; (3) continue to place the creeping stem cuttings of the Centella asiatica in the seedling substrate; wherein, The seedling substrate comprises a mixture of peat, vermiculite and wood ash in a mass ratio of 5-8:2-4:1-3; (4) preparing an induction nutrient solution for conventional seedling cultivation; wherein, The induction nutrient solution comprises the following components in the following mass contents: glucose 0.05-0.08%, vitamin C 0.01-0.03%, MgCl2 0.01-0.03%, CaCl2 0.01-0.03%, urea 0.01-0.02%.

[0020] In some specific embodiments, in the step (2), the rooting induction cultivation is performed for 3-5 days.

[0021] In some specific embodiments, in the step (4), the temperature of the seedling cultivation step is 20-22℃.

[0022] In some specific embodiments, in the step (4), the seedling cultivation step further comprises a step of ultraviolet irradiation at an intensity of 1-3 W / m².

[0023] In the following embodiments of the present application, in order to obtain more extracts of active substances, a method for extracting active substances of Centella asiatica is further disclosed, comprising the steps of obtaining Centella asiatica according to the method; and extracting active substances from the whole plant of the Centella asiatica.

[0024] In some specific embodiments, the step of extracting active substances comprises: S1, taking the whole plant of the Centella asiatica and crushing to obtain a slurry by mixing with water; S2, adding a mixture of cellulase and pectinase to the slurry for enzymatic hydrolysis treatment; S3, collecting the enzymatic hydrolysate after enzyme inactivation, adding 40-60% v / v ethanol solution for extraction, collecting the extract liquid after solid-liquid separation, removing ethanol, and then concentrating and drying to obtain the desired active substances of Centella asiatica.

[0025] In some specific embodiments, in the step S1, the amount of water is 2-4 times the mass of the Centella asiatica; In some specific embodiments, in the step S2, the amount of cellulase is 2-5 wt% of the mass of the Centella asiatica; In some specific embodiments, in the step S2, the amount of pectinase is 1-3 wt% of the mass of the Centella asiatica; In some embodiments, the enzymolysis temperature in step S2 is 45-55°C, and the enzymolysis time is 1-3 hours. In some embodiments, the amount of the ethanol solution used in step S3 is 5-8 times the mass of the Centella asiatica.

[0026] The use of the Centella asiatica active substance for preparing an antioxidant is also disclosed in the following examples of the present application.

[0027] The use of the Centella asiatica active substance for preparing an anti-Fusarium oxysporum preparation is also disclosed in the following examples of the present application.

[0028] Example 1 The method for improving the cell activity of Centella asiatica in the present embodiment comprises the following steps: (1) Select healthy Centella asiatica stolon with nodes and leaves (3-5 leaves are enough), and cut the lower end to form a cut; (2) Prepare a rooting induction liquid by dissolving IBA and honey in water at a concentration of IBA 200 mg / L and honey 2 g / L, and immerse the cut end of the Centella asiatica stolon in the rooting induction liquid for rooting induction. The Centella asiatica stolon system is placed at room temperature for rooting induction, and roots can be observed after about 3-5 days; (3) Mix peat, vermiculite and wood ash at a mass ratio of 6:3:2 to obtain a seedling substrate, and evenly place the rooting-induced Centella asiatica stolon in the seedling substrate; (4) Prepare the required induction nutrient solution according to the following ingredient contents: glucose 0.06%, vitamin C 0.02%, MgCl2 0.02%, CaCl2 0.02%, and urea 0.015%; and apply the nutrient solution during the seedling growth of the Centella asiatica, once a day. In addition, during the seedling process, the Centella asiatica can be irradiated with ultraviolet light at an intensity of 2W / m2 for about 30 minutes per day, and the growth of the Centella asiatica can be observed at any time.

[0029] Example 2 The method for improving the cell activity of Centella asiatica in the present embodiment comprises the following steps: (1) Select healthy Centella asiatica stolon with nodes and leaves (3-5 leaves are enough), and cut the lower end to form a cut; (2) according to the concentration of IBA 100mg / L, honey 3g / L, take IBA and honey with water to fully dissolve, prepared to get rooting induction liquid, and the creeping stems of the Centella asiatica are placed in the rooting induction liquid for rooting induction cultivation, it should be noted that the cut position must be fully immersed in the rooting induction liquid, the creeping stems of the Centella asiatica are placed at room temperature for rooting induction, and roots can be obviously seen after about 3-5 days; (3) according to the mass ratio of 5:4:1, take peat, vermiculite and wood ash to fully mix to get seedling substrate, which is uniformly placed in the cultivation container, and the creeping stems of the Centella asiatica after rooting induction are cut and placed in the seedling substrate; (4) the required induction nutrient solution is prepared according to the following ingredient content: glucose 0.05%, vitamin C 0.03%, MgCl2 0.01%, CaCl2 0.03%, urea 0.01%; and the nutrient solution is applied during the seedling growth of the Centella asiatica, once a day, in addition, during the seedling process, the Centella asiatica can be irradiated with ultraviolet light at an intensity of 1W / m² for about 30min, and the growth status of the Centella asiatica is observed at any time.

[0030] Example 3 The method for improving the cell activity substance of the Centella asiatica in this embodiment comprises the following steps: (1) select healthy creeping stems of the Centella asiatica with nodes and leaves (3-5 leaves are enough), and cut the lower end to form a cut; (2) according to the concentration of IBA 300mg / L, honey 1g / L, take IBA and honey with water to fully dissolve, prepared to get rooting induction liquid, and the creeping stems of the Centella asiatica are placed in the rooting induction liquid for rooting induction cultivation, it should be noted that the cut position must be fully immersed in the rooting induction liquid, the creeping stems of the Centella asiatica are placed at room temperature for rooting induction, and roots can be obviously seen after about 3-5 days; (3) according to the mass ratio of 8:2:3, take peat, vermiculite and wood ash to fully mix to get seedling substrate, which is uniformly placed in the cultivation container, and the creeping stems of the Centella asiatica after rooting induction are cut and placed in the seedling substrate; (4) the required induction nutrient solution is prepared according to the following ingredient content: glucose 0.08%, vitamin C 0.01%, MgCl2 0.03%, CaCl2 0.01%, urea 0.02%; and the nutrient solution is applied during the seedling growth of the Centella asiatica, once a day, in addition, during the seedling process, the Centella asiatica can be irradiated with ultraviolet light at an intensity of 1-3W / m² for about 30min, and the growth status of the Centella asiatica is observed at any time.

[0031] Example 4 This embodiment is based on the extraction preparation of active ingredients of the Centella asiatica obtained in Example 1, specifically comprising the following steps: S1, taking the whole plant of the Centella asiatica after washing, drying and fully crushing, adding 3 times the amount of water of the mass of the Centella asiatica to mix, to obtain a slurry; S2, adding 3wt% of cellulase and 2wt% of pectinase of the mass of the Centella asiatica to the slurry to fully mix, and performing enzymatic hydrolysis treatment at 50℃ for 1-3h; S3, collecting the enzymatic hydrolysate after high-temperature enzyme inactivation at 90℃, continuously adding 6 times the amount of 50% v / v ethanol solution of the mass of the Centella asiatica to extract for 2h, collecting the extract liquid after solid-liquid separation, removing ethanol, and then concentrating, drying, to obtain the desired active substance of the Centella asiatica.

[0032] Example 5 This embodiment is based on the extraction preparation of active ingredients of the Centella asiatica obtained in Example 1, specifically comprising the following steps: S1, taking the whole plant of the Centella asiatica after washing, drying and fully crushing, adding 2 times the amount of water of the mass of the Centella asiatica to mix, to obtain a slurry; S2, adding 2wt% of cellulase and 3wt% of pectinase of the mass of the Centella asiatica to the slurry to fully mix, and performing enzymatic hydrolysis treatment at 45℃ for 3h; S3, collecting the enzymatic hydrolysate after high-temperature enzyme inactivation at 90℃, continuously adding 5 times the amount of 60% v / v ethanol solution of the mass of the Centella asiatica to extract for 2h, collecting the extract liquid after solid-liquid separation, removing ethanol, and then concentrating, drying, to obtain the desired active substance of the Centella asiatica.

[0033] Example 6 This embodiment is based on the extraction preparation of active ingredients of the Centella asiatica obtained in Example 1, specifically comprising the following steps: S1, taking the whole plant of the Centella asiatica after washing, drying and fully crushing, adding 2-4 times the amount of water of the mass of the Centella asiatica to mix, to obtain a slurry; S2, adding 5wt% of cellulase and 1wt% of pectinase of the mass of the Centella asiatica to the slurry to fully mix, and performing enzymatic hydrolysis treatment at 55℃ for 1h; S3, collecting the enzymatic hydrolysate after high-temperature enzyme inactivation at 90℃, continuously adding 8 times the amount of 40% v / v ethanol solution of the mass of the Centella asiatica to extract for 2h, collecting the extract liquid after solid-liquid separation, removing ethanol, and then concentrating, drying, to obtain the desired active substance of the Centella asiatica.

[0034] Experimental Example 1. Antioxidant performance In this experimental example, the antioxidant performance of the Centella asiatica extract prepared in the foregoing Example 4 was verified.

[0035] In this experimental example, the verification method of the antioxidant capacity was referred to the determination method of the PPH free radical scavenging capacity and the hydroxyl radical (·OH) scavenging capacity in the prior art.

[0036] In the experimental example scheme as follows, the sample solution of the Centella asiatica extract was prepared into an aqueous solution with a mass concentration of 20 wt%.

[0037] (1) Determination of DPPH free radical scavenging capacity: The experimental group was added with 1 ml of sample solution + 1 ml of 0.2 mmol / L DPPH.

[0038] The control group was added with 1 ml of anhydrous ethanol + 1 ml of sample solution.

[0039] The blank group was added with 1 ml of anhydrous ethanol + 1 ml of 0.2 mmol / L DPPH.

[0040] The above groups were mixed and placed at room temperature (25℃) for 30 min. After completion, 300 ul of reaction solution was taken at 517 nm, the absorbance of the experimental group was determined and recorded as Ai, the absorbance of the control group was determined and recorded as Aj, and the blank group was recorded as A0. Each sample was measured in triplicate.

[0041] The DPPH scavenging rate (%) was calculated as [1-(Ai-Aj) / A0]×100%.

[0042] (2) Determination of hydroxyl radical (·OH) scavenging capacity: In this experimental example, the hydroxyl radical (·OH) scavenging test method was referred to the Fenton reaction method. In a test tube, 2 mL of ferrous sulfate solution (6 mmol / L), 2 mL of hydrogen peroxide solution (6 mmol / L) and 2 mL of sample were sequentially added and then placed for 10 min. Then, 2 mL of salicylic acid solution (6 mmol / L) was added, and the absorbance A0 was measured at 510 nm after being placed for 30 min. Distilled water was used to replace the sample solution and was treated in the same way to measure the absorbance Ax.

[0043] The hydroxyl radical (·OH) scavenging rate (%) was calculated as (1-A0 / Ax)×100%.

[0044] In this experimental example, the DPPH free radical scavenging rate and the hydroxyl radical (·OH) scavenging rate are shown in Table 1 below.

[0045]

[0046] It can be seen that the Centella asiatica active substance extracted by the application has good antioxidant performance.

[0047] 2. Pathogenic fungi inhibition performance The preserved pathogenic fungi Fusarium oxysporum was inoculated into solid PDA medium and cultured in a 25℃ incubator for 2 days in the dark to obtain pathogenic fungi blocks.

[0048] In addition, PDA solid medium was prepared, and the active substance extracted in Example 4 was added in a heated liquid state to control the concentration to 5 mg / mL to obtain the experimental group PDA medium, which was poured into a culture dish and cooled. Under the same operation conditions and parameters, the PDA plate medium added with an equal volume of anhydrous ethanol was used as the control group.

[0049] The preserved pathogenic fungi Fusarium oxysporum blocks were inoculated into the plates of the experimental group and the control group, with the mycelium facing upwards, and the size of the blocks in each plate was kept consistent, and each was set up in triplicate.

[0050] The inoculated plates were placed in a 25℃ incubator and cultured in the dark for 4 days, and the colony size was measured by the cross method. The inhibition rate was calculated according to the following formula.

[0051] Inhibition rate (%) = (control group colony diameter - treatment group colony diameter) / (control group colony diameter - fungus cake diameter) x 100%.

[0052] In the experimental example, the inhibition rate of the Centella asiatica extract on the pathogenic fungi Fusarium oxysporum reached 61.36%, indicating that it has a significant inhibitory effect on pathogenic fungi. Subsequent analysis of active ingredients and improvement of purity can further provide its ability to inhibit pathogenic fungi.

[0053] In summary, the active substance of the Centella asiatica extracted by the present application not only has the traditional antioxidant effect, but also exhibits an inhibitory effect on pathogenic fungi, especially on Fusarium oxysporum which can cause crop root rot. Subsequent extraction and purification of the Centella asiatica extract can enhance its inhibitory effect on pathogenic fungi, and also provide potential and technical support for the development of formulations for resisting crop root rot in the future.

[0054] The embodiments of the present application are described in detail above, and specific examples are applied in this paper to describe the principles and implementation methods of the present application. The above description of the embodiments is only used to help understand the method of the present application and its core idea; at the same time, for those skilled in the art, according to the idea of the present application, the specific implementation method and application range will be changed; in summary, the content of the specification should not be understood as a limitation of the present application.

Claims

1. A method for enhancing the cell activity of Centella asiatica, characterized in that, Includes the following steps: (1) Select healthy Centella asiatica stolons with nodes and leaves, and make a cut at the lower end; (2) Prepare a rooting induction solution and place the Centella asiatica stolons in the rooting induction solution for rooting induction culture; wherein, The rooting induction solution includes: IBA 100-300 mg / L and honey 1-3 g / L; (3) Continue to place the Centella asiatica stolons into the seedling substrate; wherein, The seedling substrate comprises a mixture of peat, vermiculite, and wood ash in a mass ratio of 5-8:2-4:1-3. (4) Prepare induction nutrient solution for routine seedling cultivation; among which, The induced nutrient solution comprises the following components in the indicated mass percentages: glucose 0.05-0.08%, vitamin C 0.01-0.03%, MgCl2 0.01-0.03%, CaCl2 0.01-0.03%, and urea 0.01-0.02%.

2. The method for enhancing the cell activity of Centella asiatica according to claim 1, characterized in that, In step (2), the rooting induction culture time is 3-5 days.

3. The method for enhancing the cell activity of Centella asiatica according to claim 2, characterized in that, In step (4), the temperature of the seedling cultivation step is 20-22℃.

4. The method for enhancing the cell activity of Centella asiatica according to claim 3, characterized in that, In step (4), the seedling cultivation step also includes a step of irradiating the seedlings with ultraviolet light at an intensity of 1-3 W / m².

5. A method for extracting active substances from Centella asiatica, characterized in that, The method includes the step of obtaining Centella asiatica according to any one of claims 1-4; and the step of extracting active substances from the whole plant of Centella asiatica.

6. The method for extracting active substances from Centella asiatica according to claim 5, characterized in that, The step of extracting the active substance includes: S1, take the whole herb of Centella asiatica, crush it, add water and mix to obtain a slurry; S2, add a mixture of cellulase and pectinase to the slurry for enzymatic hydrolysis; S3. After collecting the enzymatic hydrolysate and inactivating the enzyme, add 40-60% v / v ethanol solution for extraction. Collect the extract after solid-liquid separation, remove the ethanol, concentrate and dry to obtain the desired Centella asiatica active substance.

7. The method for extracting active substances from Centella asiatica according to claim 6, characterized in that: In step S1, the amount of water used is 2-4 times the mass of the Centella asiatica. In step S2, the amount of cellulase used is 2-5 wt% of the mass of the Centella asiatica. In step S2, the amount of pectinase used is 1-3 wt% of the mass of the Centella asiatica. In step S2, the temperature of the enzymatic hydrolysis step is 45-55℃, and the enzymatic hydrolysis time is 1-3 hours. In step S3, the amount of ethanol solution used is 5-8 times the mass of the Centella asiatica.

8. An active substance of Centella asiatica obtained by the method described in claim 6 or 7.

9. Use of the Centella asiatica active substance of claim 8 in the preparation of an antioxidant.

10. Use of the Centella asiatica active ingredient of claim 8 in the preparation of an anti-Fusarium oxysporum preparation.

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