Chamaoshan composition rich in lysimachia capillipes hemsl saponin B and preparation method of Chamaoshan composition

Abstract

The invention discloses a rhodiola rosea composition rich in lysimachia capillipes saponin B. The rhodiola rosea composition is prepared by mixing prepared rehmannia roots and dried rehmannia roots according to the weight part ratio of (1-10): (10-1). Meanwhile, the invention further discloses a preparation method and application of the rhodiola rosea composition rich in lysimachia capillipes hemsl saponin B. The prepared rehmannia root and the raw rehmannia root are combined, so that the traditional Chinese medicine composition has a multi-link and multi-target-point integral regulation effect and has a huge advantage in the aspect of improving the exercise ability of a body; meanwhile, a nematode model is adopted to directly prove that the alcohol extract combined by the radix rehmanniae recen and the prepared rehmannia root has anti-aging biological activity and can be applied to preparation of anti-aging drugs.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and particularly relates to a Manxiangshan composition rich in scutellaria saponin B and a preparation method and application thereof. Background Art

[0002] When facing the elderly and prematurely aged groups, in order to fundamentally improve their quality of life, we must find ways to improve their physical functions and delay aging so that they can age healthily.

[0003] The tonic medicines raw rehmannia and cooked rehmannia are different processed varieties of rehmannia. Raw rehmannia is the dried root of fresh rehmannia, and cooked rehmannia is made by steaming and drying raw rehmannia nine times. Raw rehmannia: sweet, cold in nature, has the effects of clearing heat and cooling blood, nourishing yin and promoting fluid. Cooked rehmannia: sweet, slightly warm in nature, has the effects of nourishing blood and yin, nourishing essence and filling marrow. The combination of raw rehmannia and cooked rehmannia can exert the effects of both raw and cooked, and reduce the side effects of using rehmannia. In terms of its function, it has the effects of replenishing the body's loss caused by disease invasion, improving the body's own activity function, enhancing the ability to resist its own diseases, and eliminating human weakness. Clinical and modern pharmacological studies have shown that raw rehmannia and cooked rehmannia have certain therapeutic effects in treating neurological, metabolic, anti-aging, digestive, and cardiovascular diseases. Raw rehmannia and cooked rehmannia can promote the body's own metabolism, regulate the central nervous system, and enhance the immunity of cells to external diseases, and then be used to treat and prevent Alzheimer's disease, Parkinson's disease, cardiovascular and cerebrovascular diseases and other senile-related diseases. It can be seen that the research on the combined use of raw Rehmannia root and cooked Rehmannia root is of great significance. Using nematodes as a model to study its anti-aging effects may develop more of its functions.

[0004] Caenorhabditis elegans, also known as nematodes, is a type of nematode that lives in the soil and feeds on microorganisms. As long as there are suitable basic conditions such as temperature, humidity, oxygen, and food, nematodes can grow and reproduce well. The adult body of Caenorhabditis elegans is about 1 mm long and transparent throughout. Its morphology and movement trajectory can be clearly observed under a microscope. In the laboratory, nematodes feed on OP50, can grow and reproduce on NGM culture medium, and do not emit a noticeable odor. This provides great possibilities for cultivating nematodes under laboratory conditions and conducting related experimental research. Summary of the invention

[0005] Purpose of the invention: The purpose of the present invention is to provide a Manxiangshan composition rich in scutellaria saponin B in view of the shortcomings of the prior art.

[0006] Another object of the present invention is to provide a method for preparing the Manxiangshan composition rich in scutellaria saponin B.

[0007] The third object of the present invention is to provide an application of the Manxiangshan composition rich in scutellaria saponin B.

[0008] Technical solution: In order to achieve the above-mentioned purpose of the invention, the present invention is specifically implemented as follows: a Manxiangshan composition rich in fine-stemmed vanilla saponin B is prepared by mixing prepared rehmannia root and raw rehmannia root in a weight ratio of 1-10:10-1.

[0009] The method for preparing the above-mentioned Manxiangshan composition rich in scutellaria saponin B comprises the following steps:

[0010] (1) Take cooked rehmannia root and raw rehmannia root according to the formula, grind and sieve;

[0011] (2) Add ethanol or water 8 to 12 times the mass of Rehmannia glutinosa and Rehmannia glutinosa, and reflux for 3 times, with each reflux time controlled at 0.5 to 1 hour;

[0012] (3) The drug solutions extracted from the three reflux extractions were collected and combined, concentrated under reduced pressure, frozen, and freeze-dried in vacuo to obtain powder, which was then stored in a brown bottle at room temperature.

[0013] Wherein, in step (2), when ethanol reflux extraction is adopted, ethanol with a volume fraction of 70% is adopted.

[0014] The Manxiangshan composition rich in scutellaria saponin B can be used as a medicine or health product for improving the body's motor ability.

[0015] Beneficial effects: The present invention uses Rehmannia glutinosa and Radix Rehmanniae glutinosae together, which has a multi-link and multi-target overall regulatory effect, and has great advantages in improving the body's motor ability; at the same time, the present invention uses a nematode model to directly prove that the ethanol extract of Radix Rehmanniae glutinosae and Radix Rehmanniae glutinosae combined has anti-aging biological activity and can be used in the preparation of anti-aging drugs. DETAILED DESCRIPTION

[0016] Example:

[0017] The present invention is applied to experiments on survival and improving movement ability of Caenorhabditis elegans

[0018] 1. Experimental Materials

[0019] 1. Experimental organisms:

[0020] C. elegans strain: Wild-type C. elegans N2, used to evaluate the anti-aging effects of drugs on normal populations.

[0021] Bacterial strain: Uracil mutant Escherichia coli OP50 (E. coli).

[0022] 2. Culture medium and drugs

[0023] LB medium: 5g yeast extract, 10g tryptone, 5g NaCl, add ultrapure water to 1L, autoclave at 121℃ (solid LB medium plus 15-20g agar on this basis)

[0024] M9 buffer: KH2PO4 3 g, Na2HPO4 6 g, NaCl 5 g, MgSO4 1 ml, add ddH2O to 1 L; sterilize at 121℃.

[0025] NGM medium: 3g NaCl, 2.5g tryptone, 17g agar, add ddH2O to 975ml. Sterilize at 121℃. Cool the medium to about 55℃. Add 1ml 1M CaCl2, 1ml 5mg / ml cholesterol ethanol solution, 1ml 1M MgSO4, 25ml1M KH2PO4, and mix well. (Liquid NGM can be made without agar on this basis)

[0026] S-complate Medium: 977ml s-basal 10ml 1M Potassium citrate (PH6.0 sterile) 3ml 1M CaCl2 (sterile) 10ml Trace metal solution (sterile) 3ml 1M MgSO4 (sterile).

[0027] Lysis solution: Bleach: 5M NaOH = 2:1 (Bleach 1ml, 5M NaOH 0.5ml)

[0028] Drugs: Astragalus, ginseng, licorice and cinnamon were purchased from Beijing Tong Ren Tang Nanjing Pharmacy.

[0029] 3. Experimental instruments and materials

[0030] Biochemical incubator Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory

[0031] Refrigerator Freezer Qingdao Haier Special Electric Appliance Co., Ltd. Medical Refrigerator Nanjing Gengchen Scientific Instrument Co., Ltd. High Pressure Sterilizer (American owned) Zhiwei (Xiamen) Instrument Co., Ltd. Ultra-clear workbench SW-CJ-2FD, Sujing Antai Air Technology Co., Ltd. Low speed centrifuge DM0412, Celocy

[0032] Stereo microscope SZX7, olympus

[0033] Analytical balance BSA-124S, Beijing Sartorius Instrument Co., Ltd. Shaker SK-O180-E, Cylo Czech

[0034] 60mm culture dish Corning, Anjieyou Biotechnology

[0035] 96-well culture plate Corning, Anjieyou Biotechnology

[0036] Preparation of the Manxiangshan composition rich in scutellaria saponin B: The ratio of raw rehmannia root: cooked rehmannia root is 1-10:10-1 respectively, and a total of 10 groups (integer ratio) are grinded and sieved. After the medicinal materials are weighed, add 10 times the amount of 70% ethanol / water, and reflux extract 3 times, each time for 1 hour. Collect and combine the 3 times of medicinal liquid, concentrate under reduced pressure, freeze, and vacuum freeze-dry to obtain the crude drug extract powder, which is divided into brown bottles and stored at room temperature for use.

[0037] Activation and plating of E. coli OP50: Turn on the ultraviolet light of the clean bench for 30 minutes, then turn off the ultraviolet light and start aseptic operation. Take a sterile 15ml centrifuge tube, add 10ml LB liquid culture medium with a pipette, and then add 100μl E. coli. Place the centrifuge tube in a 37℃ constant temperature shaker and shake overnight (8-12).

[0038] E. coli OP50 plate: After E. coli OP50 is shaken and cultured overnight, turn on the ultra-clean workbench UV lamp for sterilization before operation. Use a pipette to take 30μl of bacterial solution and add it to the culture medium plate, and gently rotate the plate to spread the bacterial solution. Place the inoculated plate in a 37℃ constant temperature incubator and culture for 8h.

[0039] Inoculate Caenorhabditis elegans in NGM culture plates: Nematodes are subcultured in a 20°C biochemical incubator and sterilely operated on a clean bench. They are grown in NGM culture medium to prevent microbial contamination of the culture medium. When subculturing large quantities of nematodes, prepare fresh culture medium, evenly spread E. coli, and then use a sterilized scalpel to cut a small piece of the old culture medium containing nematodes, invert it and attach it to the surface of the fresh culture medium, and culture it at 20°C.

[0040] Synchronization of Caenorhabditis elegans: Rinse adult Caenorhabditis elegans with liquid NGM into a 10ml centrifuge tube, centrifuge at 1300g for 1min, discard the supernatant, wash twice with sterile water, discard the supernatant, add 6ml liquid NGM and centrifuge and wash, discard the supernatant, retain about 1ml, add 3ml sterile water, 0.5ml 5M NaOH, and 1ml bleach to the centrifuge tube, shake and lyse on a vortex for about 5min, observe the lysis state of the worm under a microscope, centrifuge at 1500g for 1min, aspirate the supernatant, rinse twice with NGM, rinse twice with SC culture medium, add 5ml SC culture medium, incubate in an incubator, and inoculate into a 96-well culture plate. Synchronously growing Caenorhabditis elegans can be obtained by culturing at 20℃.

[0041] Drug preparation and concentration screening: Weigh 0.375g of the extract accurately, dissolve it in an appropriate amount of DMSO, add sterile water to 10ml, prepare a mother solution with a concentration of 37.5mg / ml, and store it at -20℃ for later use. According to relevant literature reports, dilute the mother solution to different concentrations: 18.25mg / ml, 7.5mg / ml, 3.75mg / ml, 1.5mg / ml, 0.75mg / ml, 0.375mg / ml. After the nematodes are synchronized, they are incubated at 20°C until the L1 stage, and then inoculated into a 96-well plate, with 100 μl of worm solution inoculated into each well. They are cultured at 20°C until the L4 stage, and 30 μl of 0.6 mM FUDR is added to each well to inhibit the reproduction of nematodes. 20 μl of drug extract is added, and the final drug concentrations are 5 mg / ml, 2.5 mg / ml, 1 mg / ml, 500 μg / ml, 200 μg / ml, 100 μg / ml, and 50 μg / ml. The drug-treated nematodes are cultured in a 20°C incubator, and the state of the nematodes is observed every 24 hours, and the number of survivors is recorded. The observation is continued for one week to monitor the effect of the drug on the nematodes and further determine the drug dosage.

[0042] Lifespan analysis experimental method: L4 synchronized Caenorhabditis elegans, add 30μl 0.6mM FUDR to each well to inhibit nematode reproduction, add 20μl drug extract, the final drug concentration is 1mg / ml, 500μg / ml, 200μg / ml, 100μg / ml, 50μg / ml, the experimental group is blank group, solvent control group, drug group. Place incubation at 20℃, observe and count the number of survivors and deaths until all nematodes die. Before observation, place the 96-well plate on a shaker and shake gently for 2 minutes. The criterion for nematode death is that the nematode does not bend its body or swing its head after vibration or strong light stimulation, and the body is in a rigid state.

[0043] Movement ability test: Synchronized nematodes were cultured until the L4 stage, and drug treatment was performed for 6 days. Nematodes were pipetted and transferred to a labeled 1.5 ml centrifuge tube and centrifuged at 1300 g for 1 min. The supernatant was removed, M9 buffer was added, and centrifuged at 1300 g for 1 min. Repeated twice. Nematodes were pipetted onto a blank NGM culture dish, 5 per plate. After the nematodes moved freely for 1 min, the number of S-shaped movements of the nematodes within 1 min (i.e., the number of peaks passed within 1 min) was counted. 15 nematodes were counted in each group, and repeated 3 times.

[0044] Head swing test: Synchronized nematodes were cultured until the L4 stage and treated with drugs. After 6 days of drug treatment, the nematodes were pipetted and transferred to a labeled 1.5 ml centrifuge tube and centrifuged at 1300 g for 1 min. The supernatant was removed, M9 buffer was added, and the tube was centrifuged at 1300 g for 1 min. Repeated twice. The nematodes were pipetted onto a blank NGM culture dish, 5 per plate. After the nematodes moved freely for 1 min, the number of head swings within 1 min was recorded under a stereo microscope. The head swing behavior of nematodes was defined as a change in the direction of the head swing, and the change angle was greater than 90 degrees.

[0045] Result analysis: GraphPad Prism5.02 was used for statistical analysis of experimental data, expressed as mean ± standard error, and ANOVA was used for comparison among groups.

[0046] result

[0047] The average lifespan of nematodes in the alcohol / water extract groups of 1 part of raw Rehmannia root and 3 parts of cooked Rehmannia root was significantly prolonged, by 29.91% (100μg / ml group), 24.03% (200μg / ml group) and 18.91% (500μg / ml group), respectively, as shown in Tables 1 and 2.

[0048] Table 1 Statistical table of nematode lifespan analysis under different concentration gradients of alcohol extracts of raw Rehmannia root and cooked Rehmannia root

[0049]

[0050] Table 2 Statistical analysis of nematode lifespan at different concentration gradients of raw and cooked Rehmannia root extracts

[0051]

[0052] Effects of alcohol / water extracts of raw and cooked rehmannia root on nematode reproduction

[0053] Table 3 Effects of alcohol extracts of raw and cooked Rehmannia root on nematode fecundity

[0054]

[0055]

[0056] Table 4 Effects of the combined aqueous extracts of raw and cooked Rehmannia root on nematode fecundity

[0057]

[0058] The alcohol / water extracts of raw Rehmannia root and cooked Rehmannia root can significantly improve the nematode's mobility within a certain dosage range (200, 500 μg / ml, 1 mg / ml), and the number of nematode crawling peaks within 1 minute increased significantly (P<0.05).

[0059] Table 5 Effects of alcohol extracts of raw and cooked Rehmannia root on the motility of nematodes

[0060]

[0061] Table 6 Effects of the combined aqueous extracts of raw and cooked Rehmannia root on the motility of nematodes

[0062]

[0063]

[0064] The alcohol / water extracts of raw Rehmannia root and cooked Rehmannia root can significantly increase the head shaking of nematodes within a certain dosage range (200, 500 μg / ml, 1 mg / ml), and the number of head shaking times of nematodes within 1 minute increased significantly (P<0.05).

[0065] Table 7 Effects of alcohol extracts of raw and cooked Rehmannia root on head swing of nematodes

[0066]

[0067] Table 8 Effects of raw and cooked Rehmannia root aqueous extracts on nematode head swing

[0068]

[0069] In summary, it is suggested that the composition of the present invention has the function of improving the body's motor ability and prolonging lifespan.

Claims

1. A Manxiangshan composition rich in scutellaria saponin B, characterized in that: The prepared drug is prepared by mixing cooked rehmannia root and raw rehmannia root in a weight ratio of 1-10:10-1.

2. A method for preparing the Manxiangshan composition rich in scutellaria saponin B according to claim 1, characterized in that: The following steps are involved: (1) Take cooked Rehmannia root and raw Rehmannia root according to the formula, grind and sieve; (2) Add ethanol or water 8 to 12 times the mass of Rehmannia glutinosa and Rehmannia glutinosa, and reflux for 3 times, with each reflux time controlled at 0.5 to 1 hour; (3) Collect and combine the drug solutions extracted three times by reflux, concentrate under reduced pressure, freeze, and vacuum freeze-dry to obtain powder, which is then stored in a brown bottle at room temperature.

3. The method for preparing a Manxiangshan composition rich in scutellaria saponin B according to claim 2, characterized in that: In the step (2), when ethanol reflux extraction is used, ethanol with a volume fraction of 70% is used.

4. The Manxiangshan composition rich in scutellaria saponin B according to claim 1, characterized in that: Applied to the use of drugs or health products to improve the body's athletic ability.

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