Saussurea involucrata culture as well as preparation method and application thereof

Abstract

The invention discloses a saussurea involucrata culture as well as a preparation method and application thereof, and belongs to the technical field of bioengineering and medicines. The invention relates to a saussurea involucrata culture, in particular to a selenium-rich and chromium-rich saussurea involucrata cell culture with DPP-IV inhibitory activity. The content of selenium in the saussurea involucrata culture reaches 200 mg / Kg dry weight or above, and the content of chromium Cr < 3 + > reaches 20 mg / Kg dry weight or above; in addition, chromium Cr < 6 + > is not detected, and the 70% ethanol extract of the saussurea involucrata cell culture is applied to an in-vitro DPP-IV inhibition test, and the inhibition rate of the 70% ethanol extract of the saussurea involucrata cell culture reaches 99.08%. The selenium-rich and chromium-rich saussurea involucrata culture with the high DPP-IV inhibition rate can be widely applied to preparation of medicines, health foods or functional foods for preventing and / or treating type 2 diabetes, obesity and related metabolic diseases.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of bioengineering and medicine, and relates to a Saussurea culture and a preparation method and application thereof. BACKGROUND

[0002] Saussurea is a commonly used rare ethnic medicine in China, which is recorded in Sheng Mao Gong Mu Shi Yi and Jing Zhu Ben Cao (Crystal Materia Medica) and is derived from the dry aboveground parts or whole plants of Saussurea medusa, Saussurea laniceps and Saussurea involucrata and other plants of the same genus. Saussurea is mainly distributed in the Qinghai-Tibet Plateau and the western region of China and is a rare and endangered medicinal resource that is prohibited from being picked up illegally. Due to the special growth environment, Saussurea takes 4 to 5 years to bloom, has a low natural reproduction rate and grows slowly, so it is difficult to obtain Saussurea medicinal resource through breeding and cultivation technology.

[0003] Plant cell culture (PCC) is a promising biosynthesis platform, which has the advantages of short growth cycle, high cost-effectiveness, no pathogen damage, rich secondary metabolites, and is not affected by geographical environment and seasonal climate, and can efficiently produce natural products with limited accumulation in plants. In the aspect of germplasm resource protection, the application of plant cells reduces the dependence on and damage to wild plants, and there is no problem of transgenic pollution such as release of transgenic organisms, which can effectively solve the contradiction between supply and demand while maintaining ecological diversity and stability.

[0004] Saussurea contains rich natural active substances, which are warm and slightly bitter, have the effects of warming kidney and assisting yang, dispelling wind and dampness, and promoting blood circulation, and have a wide application prospect in the field of biological medicine. The existing research on Saussurea cell culture mainly focuses on flavonoids, polysaccharides, polyphenols and other plant secondary metabolites, but the application research in the fields of diabetes and obesity still needs to be developed.

[0005] In order to solve the above-mentioned defects existing in the prior art, a Saussurea culture and a preparation method and application thereof are developed. SUMMARY

[0006] The purpose of the present application is to provide a Saussurea culture.

[0007] Another purpose of the present application is to provide a preparation method of the Saussurea culture.

[0008] Still another purpose of the present application is to provide an application of the Saussurea culture.

[0009] The present application is realized by the following technical solutions: A saussurea cultivar is a selenium and chromium rich saussurea cell culture with DPP-IV inhibitory activity.

[0010] Further, the selenium content in the saussurea cell culture is above 200 mg / Kg dry weight, and the chromium Cr 3+ content is above 20 mg / Kg dry weight; in addition, the chromium Cr 6+ content is not detected; the 70% ethanol extract of the saussurea cell culture has an inhibition rate of 99.08% in the DPP-IV inhibition test in vitro.

[0011] Further, the preparation method of the saussurea cell culture is obtained by inoculating the explants of saussurea on the MS basic medium containing selenium source and chromium source, and then carrying out plant cell culture.

[0012] The preparation method of the saussurea cell culture comprises the following steps: 1) selecting mature seeds of saussurea or saussurea involucrata, and washing the seeds with water to remove the soil dust on the seeds; 2) soaking the washed seeds with 75% ethanol, washing with sterile water, and rinsing with sodium hypochlorite solution, then absorbing the water, inoculating into the MS medium containing 4% sucrose and 0.8% agar, and culturing for 3-5 days, collecting the sterile seedlings with a length of 2-3 cm as the explants for inducing callus; 3) cutting the hypocotyls, cotyledons and cotyledons of the sterile seedlings into 0.5 cm 2 -1 cm 2 fragments, and placing the fragments into the screening medium for successive screening, and culturing under light at a temperature of 18-25°C to induce loose and high-growth callus; 4) successively screening and amplifying the callus cells to obtain loose and high-growth callus cells and storing the callus cells; 5) culturing the stored callus cells for 15-20 days, collecting the saussurea cell culture, washing and centrifuging with purified water, and then using the boiling drying method to dry the culture at a drying temperature of 40-50°C until the water content of the culture is ≤10%, thereby obtaining the saussurea cell culture.

[0013] The screening medium comprises sodium selenite with a concentration range of 0.1 μM-100 μM, and chromium trichloride with a concentration range of 0.1 μM-100 μM. The MS medium comprises 4% sucrose, 2 mg / L NAA, and 0.5 mg / L 6-BA.

[0014] The storage method of the callus cells is storage by uninterrupted culture at room temperature, storage by low-temperature solid culture, storage by liquid culture, or storage by suspension cell culture. The callus cell culture adopts a culture bottle liquid suspension culture method: the temperature is 18-25 DEG C, the illumination is 2500-3500 lux, 12 hours, the culture period is 15-20 days, and the shaking bed rotating speed is 120-150 r / min.

[0015] The callus cell culture adopts a bioreactor culture method: the temperature is 18-25 DEG C, the illumination is 2500-3500 lux, 12 hours, the culture period is 15-20 days, and the stirring rotating speed is 30-50 r / min.

[0016] The saussurea cell culture can be applied to the preparation of medicines, health foods or functional foods for preventing and / or treating type 2 diabetes, obesity and related metabolic diseases (hyperglycemia, insulin resistance, dyslipidemia).

[0017] Selenium is one of the essential trace elements for human body, which has the functions of antioxidation, immunity, thyroid hormone regulation, detoxification, and male reproductive health protection. Plant cells are good carriers for enriching trace element selenium. The present application uses saussurea cells to enrich selenium, improves the nutritional value and efficacy of cell raw materials, and the enriched selenium can convert inorganic selenium into organic selenium compounds with lower toxicity and stronger function, so that the selenium-enriched saussurea cell culture raw material is more suitable for use as a functional food raw material. Meanwhile, based on the maximum tolerance and recommended intake of selenium per day for an adult, the present application determines how to effectively enrich selenium into saussurea cell raw materials through a large number of tests, and the technical scheme is beneficial to the growth of saussurea cells and the synthesis of existing flavonoids and other secondary metabolites.

[0018] In nature, chromium mainly exists in the forms of Cr(III) and Cr(VI), and trivalent chromium is an essential trace element for human body and has the effect of enhancing insulin. 3+ By forming glucose tolerance factor (GTF), chromium can synergize with insulin to play a physiological role. 3+ When Cr(III) is polymerized with low molecular weight chromium polymer (LMWCr), it can enhance the activity of insulin receptor tyrosine kinase, so that the function of insulin in the body is reduced in the state of chromium deficiency. The present application solves the problems that organic chromium is mainly extracted from chromium-rich yeast with high cost, and the chromium content in ordinary plants is too low to achieve the health care effect by inoculating the explants of saussurea on the MS basic medium containing selenium source and chromium source.

[0019] Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulin polypeptide (GIP) are main members of the gut incretin family, which promote the synthesis and secretion of insulin by stimulating and protecting pancreatic beta cells, and reduce postprandial blood glucose. Dipeptidyl peptidase-IV (DPP-IV) can cleave a variety of peptide hormones including GLP-1 and GIP, and rapidly inactivate them; DPP-IV inhibitors indirectly promote insulin secretion by inhibiting the activity of DPP-IV to increase the level of gut incretins GLP-1 and GIP in vivo, thereby alleviating the symptoms of hyperglycemia in patients with type II diabetes. The selenium and chromium-rich saussurea culture prepared by the above method of the present application can be used for the preparation of a drug for preventing and / or treating type 2 diabetes, obesity and related metabolic diseases (hyperglycemia, insulin resistance, dyslipidemia), a health food or a functional food. The DPP-IV inhibition rate can reach more than 99%, compared with the existing artificially synthesized DPP-IV inhibitors, without side effects and without increasing the burden on the kidneys.

[0020] Compared with the prior art, the present application inoculates the explant of saussurea on the MS basic medium containing selenium source and chromium source, to obtain a selenium and chromium-rich saussurea culture with high DPP-IV inhibition rate, which can be widely used in the preparation of a drug for preventing and / or treating type 2 diabetes, obesity and related metabolic diseases (hyperglycemia, insulin resistance, dyslipidemia), a health food or a functional food. DETAILED DESCRIPTION

[0021] In order to further understand the content of the present application, the present application will be described in detail in conjunction with examples. It should be understood that the examples are only used to explain the present application but not to limit the present application. EXAMPLE

[0022] A preparation method of a saussurea culture, comprising the following steps: 1) Select mature seeds of Saussurea involucrata or Saussurea involucrata granules with full particles and consistent color, place them in a small beaker, cover the beaker opening with degreasing gauze, and wash them under a tap to wash away the dirt on the seeds, then soak the seeds in warm water for 20 min; 2) Soak the washed seeds in 75% ethanol for 30 s, wash them with sterile water, and soak them in 10% calcium hypochlorite solution for 8 min, and continuously stir the seeds with sterile tweezers to make them fully contact with the calcium hypochlorite solution, wash them with sterile water for 3 times, and finally place them on sterile filter paper to absorb the water on the surface of the seeds; inoculate them into MS basic medium containing 3% sucrose and 1% agar, the MS medium contains 3% sucrose, 2 mg / L NAA, and 0.5 mg / L 6-BA; cultivate them at 18-25°C under light for 12 h / d; after 4 days, the seeds will germinate and grow into sterile seedlings, and the germination rate can reach 90%, and the sterile seedlings with a length of 2-3 cm are used as explants for inducing callus.

[0023] 3) Cut the hypocotyl, embryo, and cotyledon of the sterile seedling into 0.5 cm2 ~1 cm 2 The fragments are put into a screening medium for successive screening, and loose and high-yield callus is induced by culture under light at a temperature of 18-25 DEG C. The screening medium comprises sodium selenite with a concentration of 0.1-100 μM and chromium trichloride with a concentration of 0.1-100 μM. 4) The callus cells are subjected to successive screening and amplification to obtain loose and high-yield callus cells, and the callus cells are preserved by liquid culture or suspension cell culture preservation. 5) The preserved callus cells are cultured for 15-20 days. The callus cells are cultured by liquid suspension culture in a culture bottle at a temperature of 18-25 DEG C, light of 2500-3500 lux for 12 hours, a culture period of 15-20 days, and a shaking speed of 120-150 r / min. Then the Saussurea cell culture is collected, washed by purified water, centrifuged, and dried by boiling at a drying temperature of 40-50 DEG C until the moisture content of the culture is less than or equal to 10%.

[0024] DPP-IV inhibitory activity: The detection principle of the method is that DPP-IV catalyzes the hydrolysis of the substrate Gly-Pro-PNA to generate yellow para-nitroaniline under alkaline conditions, and the latter has a characteristic absorption peak at a wavelength of 405 nm. The enzyme activity is reflected by the absorbance measured at 405 nm by an enzyme marker.

[0025] Inhibition rate / %=(OD negative control group-OD blank control group)-(OD Chinese medicine group-OD Chinese medicine blank control group) / (OD negative control group-OD blank control group) x 100%.

[0026]

[0027]

[0028] It is found by experiments that the Saussurea cell culture prepared by the method has the highest DPP-IV inhibition rate, which can reach more than 99%, compared with traditional Saussurea cell culture, selenium-rich Saussurea cell culture obtained by adding selenium, and isatis root Saussurea cell culture obtained by adding isatis root. The DPP-IV inhibition rate is increased with the increase of the concentration of the selenium-rich and chromium-rich Saussurea cell culture. Example

[0029] A preparation method of Saussurea cell culture, comprising the following steps: 1) Select the mature seeds of Tianshan Saussurea or Saussurea involucrata with plump and consistent color, cover the cup opening with a piece of gauze, and wash under a tap to remove the dirt and dust on the seeds. Then, soak the seeds in warm water for 20 minutes; 2) Soak the cleaned seeds in 75% ethanol for 30 seconds, wash with sterile water, and soak in 10% calcium hypochlorite solution for 8 minutes. Stir the seeds with sterile tweezers to ensure that they are fully exposed to the calcium hypochlorite solution. Wash the seeds with sterile water four times, and then place them on sterile filter paper to dry. Inoculate the seeds into MS basic medium containing 3% sucrose and 1% agar. The MS medium contains 3% sucrose, 2 mg / L NAA, and 0.5 mg / L 6-BA. Incubate at 18-25°C with 12 hours of light per day. After 5 days, the seeds will germinate and grow into sterile seedlings with a germination rate of up to 90%. Use the sterile seedlings that have grown to 2-3 cm as explants for callus induction.

[0030] 3) Cut the hypocotyls, cotyledons, and embryos of the sterile seedlings into 0.5 cm 2 ~1 cm 2 pieces and place them in a selection medium for successive selection. Incubate at 18-25°C under light. The selection medium contains sodium selenite at a concentration of 0.1-100 μM and chromium chloride at a concentration of 0.1-100 μM. 4) Perform successive selection and expansion of the callus cells to obtain loose and high-yield callus cells. Store the callus cells at room temperature without interruption. 5) Culture the stored callus cells for 15-20 days. Use a bioreactor for callus cell culture: temperature 18-25°C, light 2500-3500 lux, 12 hours, culture period 15-20 days, stirring speed 30-50 r / min, collect the Saussurea involucrata cell culture, wash and centrifuge with purified water, and then use the boiling drying method at a drying temperature of 40-50°C until the water content of the culture is ≤10%. Example

[0031] A method for preparing Saussurea involucrata culture, comprising the following steps: 1) Select the mature seeds of Tianshan Saussurea or Saussurea involucrata with plump and consistent color, cover the cup opening with a piece of gauze, and wash under a tap to remove the dirt and dust on the seeds. Then, soak the seeds in warm water for 20 minutes; 2) The cleaned seeds are soaked in 75% ethanol for 30 seconds, washed with sterile water and soaked in 10% calcium hypochlorite solution for 8 minutes, and continuously stirred with sterile forceps to ensure that the seeds are fully exposed to the calcium hypochlorite solution. The seeds are washed with sterile water four times and finally placed on sterile filter paper to dry the surface moisture. The seeds are inoculated into MS basic medium containing 3% sucrose and 1% agar. The MS medium contains 3% sucrose, 2 mg / L NAA, and 0.5 mg / L 6-BA. The culture is incubated at 18-25°C under a 12-hour light / dark cycle. After 3 days, the seeds will germinate and grow into sterile seedlings with a germination rate of up to 90%. The sterile seedlings that grow to 2-3 cm in length are used as explants for induction of callus.

[0032] 3) The sterile seedlings are cut into 0.5 cm 2 -1 cm 2 pieces of embryo axis, embryo, and cotyledon, and placed in a screening medium for successive screening. The culture is incubated at 18-25°C under light. The loose and high-yield callus is induced and obtained. 4) The callus cells are subjected to successive screening and amplification to obtain loose and high-yield callus cells, which are stored by low-temperature solid culture. 5) The stored callus cells are cultured for 15-20 days. The callus cells are cultured using a bioreactor culture method. The temperature is 18-25°C, the light is 2500-3500 lux for 12 hours, the culture period is 15-20 days, the stirring speed is 30-50 r / min, and the collected snow lotus cell culture is washed and centrifuged with purified water, then dried by boiling at a drying temperature of 40-50°C until the moisture content of the culture is ≤10%.

Claims

1. A culture of Saussurea lava cultured in vitro, characterized in that, The application discloses a selenium and chromium riched saussurea cell culture with DPP IV inhibiting activity.

2. A saffron culture according to claim 1, characterized in that, The selenium content of the saussurea cell culture is above 200 mg / Kg dry weight, and the chromium Cr3+ content is above 20 mg / Kg dry weight.

3. A method of preparing a culture of Saussurea lava cultured according to claim 1 or 2, characterized in that, The saussurea cell culture is obtained by inoculating saussurea explants on MS basic medium containing a selenium source and a chromium source. The steps are as follows: 1) selecting mature seeds of Saussurea involucrata or Saussurea laniceps, washing the seeds with water to remove soil dust; 2) soaking the washed seeds with 75% ethanol, washing with sterile water and rinsing with sodium hypochlorite solution, drying the seeds, inoculating the seeds into MS medium containing 3-4% sucrose and 0.8-1% agar, and culturing for 3-5 days to collect sterile seedlings with a length of 2-3 cm as explants for inducing callus; 3) Cut the hypocotyl, plumule, and cotyledon of the sterile seedlings into pieces of 0.5 cm x 2–1 cm. 2 The fragments were placed in a selection medium for successive screening, and cultured under light at a temperature of 18℃~25℃ to induce the acquisition of loose callus tissue with high growth rate. 4) screening and amplifying callus cells generation by generation to obtain loose and high-growth callus cells and store the callus cells; 5) culturing the stored callus cells for 15-20 days, collecting saussurea cell culture, washing and centrifuging the cell culture with purified water, and drying the cell culture by boiling at a drying temperature of 40-50 DEG C until the water content of the cell culture is less than or equal to 10%, thereby obtaining the saussurea cell culture.

4. A method of preparing a culture of Saussurea lava divaricata according to claim 3, characterized in that, The screening medium is sodium selenite with a concentration ranging from 0.1 uM to 100 uM. The screening medium is chromium trichloride with a concentration ranging from 0.1 uM to 100 uM. The MS medium contains 3% sucrose, 2 mg / L NAA and 0.5 mg / L 6-BA.

5. A method of preparing a culture of Saussurea lava divaricata according to claim 3, characterized in that, The storage method of the callus cells is normal-temperature uninterrupted culture storage, low-temperature solid culture, liquid culture or suspension cell culture storage.

6. A method of preparing a culture of Saussurea lavaulis according to claim 3, characterized in that, The callus cell culture is carried out by using a culture bottle liquid suspension culture method, the temperature is 18-25 DEG C, the illumination is 2500-3500 lux, the culture period is 12 hours, the culture cycle is 15-20 days, and the shaking bed rotating speed is 120-150 r / min.

7. A method of preparing a culture of Saussurea lavaulis according to claim 3, characterized by, The callus cell culture is carried out by using a bioreactor culture method, the temperature is 18-25 DEG C, the illumination is 2500-3500 lux, the culture period is 12 hours, the culture cycle is 15-20 days, and the stirring rotating speed is 30-50 r / min.

8. The saussurea cell culture of claim 1 is used for preparing medicines for preventing and / or treating type 2 diabetes, obesity, hyperglycemia, insulin resistance and dyslipidemia.

9. The saussurea cell culture of claim 1 is used for preparing health-care food or functional food.

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