CN106967132A - Reactive compound and its preparation method and application in Shenzhou City's honey peach - Google Patents

Abstract

The present invention provides reactive compound and its preparation method and application in Shenzhou City's honey peach.After Shenzhou City's honey peach fruit is crushed first, it is placed in methanol and extracts, filtering and concentrating, and extract is carried out to isolate and purify two sequiterpene glycoside reactive compounds of acquisition, one of them is novel compound, is named as SZMT01.Two compounds that the present invention is extracted from Shenzhou City's honey peach can significantly extend the replicability life-span of yeast cells, with anti-senescence function.Find that SZMT01 is the expression by adjusting sod gene by the analysis to SZMT01 anti-aging mechanism, improve the oxidation resistance of yeast, so as to reach the purpose in extension yeast cells replicability life-span.Oxidative stress description of test SZMT01 can improve the survival rate of yeast under conditions of hydrogen peroxide presence.The present invention being studied in terms of anti-aging and treatment senile disease for Shenzhou City's honey peach reactive compound, has important practical significance to new drug development.

Description

Active compounds in Shenzhou Peach and its preparation method and application

technical field

The invention relates to the technical field of medicines, in particular to two active compounds in Shenzhou honey peach and its preparation method and application.

Background technique

According to the latest statistics, the number of people aged 60 and above in the world reached 901 million in 2014, and this number is expected to exceed 2 billion by 2050. The world has entered an aging society, and the accompanying diseases related to aging have become increasingly significant problems. Neurodegenerative diseases including Alzheimer's disease and Parkinson's disease are one of the worldwide medical problems. In China alone, the number of patients with Alzheimer's disease has increased to 5.69 million in 2010. In the field of medicine, finding medicines for preventing and treating senile diseases has become a top priority.

After long-term research on the mechanism of aging, a variety of aging theories have been formed in this field, including the theory of programmed aging, somatic cell mutation, error disaster, free radical theory, neuroendocrine theory, immune aging theory and so on. It can be seen that aging is a very complicated process. Even the same type of anti-aging drugs have different ways of exerting their effects, and there is no necessary connection between them.

Shenzhou peaches come from Shenzhou City, Hebei Province. This place has a unique geographical environment and soil conditions. It is rich in peaches and has distinctive characteristics. Shenzhou City was also named "Hometown of Chinese Peach" by the State Forestry Administration. Shenzhou peaches are huge in size, unique in shape, gorgeous in color, protruding at the top of the fruit, deep in suture, thin in skin and thin in flesh, sweet as honey in juice, and rich in fragrance. They are known as the king of peaches, also known as Kui Tao. After more than two thousand years, it has been used as a tribute to the royal family. It is a unique and precious variety, known as "tribute peach" in history.

Contents of the invention

The object of the present invention is to provide a kind of active compound 1 (compound SZMT01) and compound 2 in Shenzhou honey peach, the active compound in described Shenzhou honey peach is sesquiterpene glycoside compound, and wherein compound 1 is sesquiterpene A new structure compound of terpene glycosides, two active compounds have strong anti-aging ability. Its structural formula is as follows:

The physical and chemical properties of the above Shenzhou peach active compound 1 are: colorless powder; (c 0.16, MeOH); molecular formula is C ₂₁ H ₃₄ O ₈ ; high resolution mass spectrum ESI-TOF-MS m/z 437.2151[M+Na] ⁺ , theoretical value: C ₂₁ H ₃₄ O ₈ Na ⁺ 437.2151; ¹ H and ¹³ C NMR are shown in Table 1.

The physical and chemical properties of active compound 2 are: colorless powder; (c 0.2, MeOH); molecular formula is C ₂₁ H ₃₆ O ₇ ; high resolution mass spectrum ESI-TOF-MS m/z 423.2368[M+Na] ⁺ , theoretical value: C ₂₁ H ₃₆ O ₇ Na ⁺ 423.2359; ¹³ CNMR (125MHz, CD ₃ CD) data are as follows: δ _C 14.1(C-13), 16.0(C-14), 23.7(C-8), 27.4(C-4), 27.6(C-15), 40.3( C-5), 43.4(C-9), 62.8(C-6′), 71.8(C-4′), 73.9(C-10), 75.1(C-2′), 76.0(C-1), 77.9 (C-5′), 78.2 (C-3′), 102.6 (C-1′), 112.0 (C-12), 126.0 (C-7), 129.9 (C-3), 132.9 (C-2 ), 135.7 (C-6), 146.4 (C-11).

Another object of the present invention is to provide the preparation method of two active compounds 1 (compound SZMT01) and compound 2 in Shenzhou honey peach, realize through the following steps:

(1) Take the Shenzhou peach fruit, crush it, place it in methanol for extraction, filter and concentrate to obtain the extract;

(2) Separating and purifying the extract to obtain the Shenzhou peach active compound 1 and compound 2.

Preferably, in step (1), the temperature of the leaching is 25-30° C., and the time is 0.5-24 h. More preferably, the temperature of the leaching is 25° C. at room temperature, and the time is 24 hours.

Further, in step (2), the process of said separation and purification includes:

(a) using methanol: water solvent system as eluent, the extract is separated for the first time to obtain target fraction I;

(b) using methanol: water solvent system as eluent, the target fraction I is separated for the second time to obtain active fraction II and active fraction III;

(c) Using methanol aqueous solution as mobile phase, utilize reversed-phase HPLC to separate active fraction II and active fraction III for the third time to obtain the Shenzhou peach active compound 1 and compound 2;

Wherein: in step (a), an octadecyl bonded silica gel open column is used for separation; methanol: water solvent system is eluted sequentially at a volume ratio of 30:70, 50:50, 70:30, and 100:0, and the volume ratio is 70: The 30 eluted fraction is the target fraction I.

In step (b), use octadecyl bonded silica gel open column for separation; methanol: water solvent system according to the volume ratio of 50:50,55:45,60:40,65:35,70:30,80:20,100 :0 eluted in turn, the volume ratio 60:40 and 65:35 eluted fractions were active fraction II and active fraction III.

In step (c), the mobile phase is 63% methanol aqueous solution; during the separation process, the fraction with a retention time of 13.9 minutes is taken to obtain Shenzhou Peach active compound 1.

In the step (c), the mobile phase is 65% methanol aqueous solution; during the separation process, the fraction with a retention time of 14.5 minutes is taken to obtain Shenzhou Peach active compound 2.

Another object of the present invention is to provide the application of the active compound of Shenzhou peach in the preparation of anti-aging medicine or health products, for example, making Shenzhou peach into an anti-aging peach drink. Made from deep state peach active compounds and pharmaceutically acceptable carriers. Studies have shown that both compound SZMT01 and compound 2 can significantly prolong the replicative lifespan of yeast cells in the in vitro screening model of anti-aging compounds.

The pharmaceutically acceptable carrier refers to conventional drug carriers in the pharmaceutical field, such as fillers, adhesives, wetting agents, absorption promoters, surfactants and the like.

The filler can adopt starch, sucrose or microcrystalline cellulose; the binder can adopt starch slurry, hydroxypropyl cellulose, gelatin or polyethylene glycol; the wetting agent can adopt magnesium stearate, micropowder silica gel Or polyethylene glycols; the absorption enhancer can use polysorbate or lecithin; the surfactant can use boloxamer, fatty acid sorbitan or polysorbate. In addition, other adjuvants such as flavoring agents and sweetening agents can also be added.

The dosage form of described anti-aging medicine or health product can be tablet, pill, powder, dispersible tablet, sachet, elixir, suspension, emulsion, solution, syrup, aerosol, soft capsule, hard capsule Capsules, sterile injectable solutions, liniments or suppositories; available in conventional, immediate-release, sustained-release or delayed-release formulations.

The anti-aging medicine of the present invention can be administered through various routes, including oral administration, nasal cavity injection, intramuscular injection, subcutaneous injection, intravenous injection and the like.

In order to further explore the anti-aging mechanism of compound SZMT01, we conducted a preliminary study on the anti-aging mechanism of compound SZMT01 through oxidative stress experiments, and found that the compound SZMT01 can improve the survival rate of yeast in the presence of hydrogen peroxide, and also found that The compound improves the antioxidant capacity of yeast by regulating the expression of SOD gene, thereby prolonging the replicative lifespan of yeast cells.

According to the oxidative free radical theory, oxidative stress is the main cause of aging. Even under normal conditions, mitochondria can produce harmful metabolites—reactive oxygen species (ROS), which can damage cell membranes and biomacromolecules, such as proteins and nucleic acids, thereby disrupting cell functions, causing aging and death.

SOD is a gene associated with resistance to oxidative stress. The compound SZMT01 could not prolong the replicative lifespan of the SOD-knockout yeast, which further proves that SZMT01 prolongs the replicative lifespan of yeast cells by regulating the expression of the SOD gene.

Compared with the prior art, the present invention has the following beneficial effects:

(1) In the yeast aging model K6001 Saccharomyces cerevisiae cells, the compounds SZMT01 and 2 extracted from Shenzhou peach in the present invention can significantly prolong the replicative lifespan of yeast cells and have strong anti-aging ability.

(2) The Shenzhou Peach active compound of the present invention has important practical significance for basic research on the research and development of new drugs for delaying aging and treating senile diseases.

Description of drawings

Fig. 1 is the effect result of Shenzhou peach active compound 1 (A) and compound 2 (B) obtained in embodiment 2 on yeast K6001 replicative lifespan; Wherein, Control represents negative control; Res (Resveratrol) is positive control Resveratrol (10 μM); 1 7.5 μM indicates compound 1 at a concentration of 7.5 μM, 1 25 μM indicates compound 1 at a concentration of 25 μM; 27.5 μM indicates compound 2 at a concentration of 7.5 μM, 2 25 μM indicates compound 2 at a concentration of 25 μM .

Fig. 2 is the result of the effect of active compound 1 on the replicative lifespan of yeast mutant strains Δsod1 (A) and Δsod2 (B) in Example 3; wherein, Control (K6001) represents the K6001 yeast without adding active compound 1; Control (Δsod1) and Control(Δsod2) respectively represent K6001 yeast mutant strains Δsod1 and Δsod2 without adding active compound 1; .

Fig. 3 is the oxidative stress test result of active compound 1 in embodiment 3; Wherein, Control (K6001) represents the K6001 yeast that does not add active compound 1; Control represents negative control; Res (Resveratrol) is positive control resveratrol (10 μ M ); 1 7.5 μM indicates compound 1 at a concentration of 7.5 μM, and 1 25 μM indicates compound 1 at a concentration of 25 μM.

detailed description

The present invention is further described in conjunction with drawings and embodiments.

Example 1

1. The preparation of active compound 1 and compound 2 in Shenzhou honey peach, the specific steps are as follows:

(1) Put 3.0 kg of Shenzhou peaches in methanol (industrial grade) and extract at room temperature for 24 hours (shaking); after concentrated by suction filtration, 100.0 g of methanol extracts were obtained.

(2) Separating the methanol extract with an octadecyl bonded silica gel open column, using methanol:water solvent system as eluent, according to the volume ratio of methanol:water=30:70,50:50,70: 30,100:0, eluted in sequence, and 0.7g of the fraction eluted with a volume ratio of 70:30 was taken.

(3) Purify the fraction obtained in step (2) with an open column of octadecyl bonded silica gel, using methanol: water solvent system as eluent, according to the volume ratio of methanol: water = 50:50, 55:45 , 60:40, 65:35, 70:30, 80:20, and 100:0 were eluted in sequence, and 3.7 mg and 2.3 mg of fractions eluted at volume ratios of 60:40 and 65:35 were taken.

(4) Get the cut that step (3) takes, purify with reverse HPLC, chromatographic condition: chromatographic column ODS-HG-5 (10/250mm), flow rate 3ml/min, detection wavelength 210nm, mobile phase is 63% aqueous methanol , to obtain 1.1 mg of active compound 1 (retention time of 13.9 min); the mobile phase was 65% aqueous methanol, and 1.8 mg of active compound 2 was obtained (retention time of 14.5 min). 2. Physical and chemical characteristics and chemical structure analysis of active compound 1 and compound 2

After ¹³ C NMR, ¹ H NMR, HRMS, HSQC, HMBC, NOESY analysis, the results are as follows:

Physical and chemical properties of compound 1: colorless powder, molecular formula C ₂₁ H ₃₄ O ₈ , high-resolution mass spectrum ESI-TOF-MS m/z 437.2151[M+Na] ⁺ . ¹ H NMR and ¹³ C NMR data are shown in Table 1 below.

Table 1 1 ¹ H NMR and ¹³ C NMR data (CD ₃ OD; δ/ppm, J/Hz).

^a 500MHz, ^b 125MHz

Physicochemical properties of compound 2: colorless powder, molecular formula C ₂₁ H ₃₆ O ₇ ; high-resolution mass spectrum ESI-TOF-MS m/z 423.2368[M+Na] ⁺ . ¹³ C NMR (125MHz, CD ₃ CD) data are as follows: δC 14.1( _C -13), 16.0(C-14), 23.7(C-8), 27.4(C-4), 27.6(C-15), 40.3 (C-5), 43.4 (C-9), 62.8 (C-6′), 71.8 (C-4′), 73.9 (C-10), 75.1 (C-2′), 76.0 (C-1 ), 77.9 (C-5′), 78.2 (C-3′), 102.6 (C-1′), 112.0 (C-12), 126.0 (C-7), 129.9 (C-3), 132.9 (C -2), 135.7(C-6), 146.4(C-11).

The structural formulas of compound 1 and compound 2 are as follows;

Anti-aging activity analysis of active compound 1 (SZMT01) and compound 2 in Shenzhou honey peach in embodiment 2

At present, the biological models used for anti-aging research mainly include mice, nematodes, fruit flies and yeast. In this example, Saccharomyces cerevisiae was selected as the active system for anti-aging research; because yeast is a single-celled eukaryote with a short life cycle, its complete genome data has been obtained, and it is currently a commonly used aging model organism.

At the same time, resveratrol is used as a positive control, which is a well-known small molecular compound that shows anti-aging effects in various animal models.

The steps of the analysis method are as follows:

(1) Take out the K6001 yeast strain from the refrigerator at -30°C, wash it three times with PBS, 5ml each time, and remove the glycerol therein; then add 1ml PBS, pipette to suspend it, and then add it to 5ml liquid medium (1% yeast powder , 2% peptone, 3% galactose); cultured at 28° C. for 24 hours with shaking (160 r/min).

(2) After the cultivation, wash three times with 5ml PBS, remove the liquid medium therein, count with a hemocytometer, and calculate the concentration of yeast.

(3) Using absolute ethanol as a solvent, prepare 7.5 μM, 25 μM 1 and 2, and 10 μM resveratrol for later use.

(4) Add 5ml of solid medium (1% yeast powder, 2% peptone, 2% glucose, 2% agar powder) to the sterilized petri dish. After the medium solidifies, add For the sample prepared in step (3), 4,000 yeasts were added after the solvent evaporated, spread evenly with a spreader, and incubated at a constant temperature of 28°C for 48 hours.

(5) Randomly count the number of daughter cells produced by 40 mother cells in each plate under the microscope, record, draw and analyze. The results are shown in Figure 1.

As can be seen from Figure 1, the average lifespan of the negative control (no sample added, Control) is 7.10 ± 0.34, and that of the positive control (10 μM resveratrol, Resverstrol) is 8.40 ± 0.47*; 7.5 μM and 25 μM concentrations of 1 are 8.87±0.53**, 8.37±0.51*; 7.5μM, 25μM concentrations of 2 were 8.40±0.54*, 8.20±0.41*, respectively. (*p<0.05,**p<0.01).

Therefore, Compound 1 and Compound 2 can prolong the replicative lifespan of yeast.

Example 3 Anti-aging Mechanism Analysis of Active Compound 1 in Shenzhou Peach

1. To test whether 1 can prolong the replicative lifespan of K6001 yeast mutant strains Δsod1 and Δsod2 with the SOD gene knocked out at an active concentration of 7.5 μM.

The steps of the analysis method are as follows:

(1) Take out the K6001 yeast strain and the K6001 yeast mutant strains Δsod1 and Δsod2 from the -30°C refrigerator, wash with PBS three times, 5ml each time, and remove the glycerol; then add 1ml of PBS, pipette to suspend, and then add to 5ml of liquid culture medium (1% yeast powder, 2% peptone, 3% galactose); cultured at 28° C. with shaking (160 r/min) for 24 hours.

(2) After the cultivation, wash three times with 5ml PBS, remove the liquid medium therein, count with a hemocytometer, and calculate the concentration of yeast.

(3) Using absolute ethanol as a solvent, prepare 7.5 μM of 1 and set aside.

(4) Add 5ml of solid medium (1% yeast powder, 2% peptone, 2% glucose, 2% agar powder) to the sterilized petri dish. After the medium solidifies, add The sample prepared in step (3), after the solvent evaporates. Add 4000 yeasts (K6001 yeast strain or K6001 yeast mutant strains Δsod1 and Δsod2, spread evenly with a spreader, and incubate at a constant temperature of 28°C for 48 hours.

(5) Randomly count the number of daughter cells produced by 40 mother cells in each plate under the microscope, record, draw and analyze. The results are shown in Figure 2.

It can be seen from Figure 2 that the average lifespan of the negative control (no sample added, Control) on the K6001 strain was 6.85±0.42, and the positive control (10 μM resveratrol, Resverstrol) was 8.70±0.48**, 7.5 μM concentration of 1 is 8.40±0.48*; the average lifespan of the negative control (no sample added, Control) on the K6001 yeast mutant strain Δsod1 is 6.55±0.33, and the positive control (10 μM resveratrol, Resverstrol) is 6.95±0.32, 7.5 μM concentration 1 is 7.05±0.37; the average lifespan of the negative control (no sample added, Control) on the K6001 yeast mutant strain Δsod2 is 6.65±0.36, and the positive control (10 μM resveratrol, Resverstrol) is 7.17±0.46, 7.5 μM The concentration of 1 is 7.25 ± 0.32. (*p<0.05,**p<0.01)

Therefore, Compound 1 was unable to prolong the replicative lifespan of K6001 yeast mutant strains Δsod1 and Δsod2 in which the SOD gene was knocked out at an active concentration of 7.5 μM.

Embodiment 5 SZMT01 improves the activity analysis of yeast viability

To test whether SZMT01 can improve the survival rate of yeast, the test method is as follows:

(1) Wash the wild-type yeast BY4741 stored at -30°C with 5ml PBS three times to remove the glycerol; add 1ml sterile water, blow it to suspend it, add it to 5ml glucose medium (1% yeast powder, 2% peptone, 2% glucose); put it into a shaker, and shake (160r/min) at 28°C for 24 hours to restore its growth ability.

(2) Inoculate BY4741 into 25 mL of new glucose medium, adjust the OD ₆₀₀ value to 0.1, and then incubate with 7.5 μM, 25 μM cholesterol and positive control substance (10 μM resveratrol, Resverstrol) for 12 hours.

(3) 5 μL of culture solution containing the same yeast cells in each group was dropped on glucose solid medium (1% yeast powder, 2% peptone, 2% glucose, 2% agar) containing 9mM H ₂ O ₂ ; After culturing at 28°C for 3 days, the growth status of the yeast was observed and photographed, and the observation results are shown in Figure 3.

It can be seen from Figure 3 that compared with the negative control, 7.5 μM and 25 μM SZMT01 significantly increased the survival rate of yeast on the glucose solid medium containing 9mM H ₂ O ₂ .

Claims (10)

1. reactive compound in a kind of Shenzhou City's honey peach, it is characterised in that Shenzhou City's honey peach reactive compound is referred to as compound 1, Its structural formula is：

2. the preparation method of reactive compound in a kind of Shenzhou City's honey peach, it is characterised in that the compound is respectively the He of compound 1 Compound 2, is realized by following steps：

(1) Shenzhou City's honey peach fruit is taken, after crushing, is placed in methanol and shakes extraction, filtering and concentrating obtains extract；

(2) extract is isolated and purified, obtains described Shenzhou City's honey peach reactive compound；

The structural formula of compound 1 and compound 2 is as follows：

3. preparation method according to claim 2, it is characterised in that in step (1), the temperature of the extraction is room temperature, Time is 24h.

4. preparation method according to claim 2, it is characterised in that described to isolate and purify by following step in step (2) It is rapid to realize：

(a) with methanol:Aqueous solvent system makees eluant, eluent, and first time separation is carried out to extract, obtains target fraction I；

(b) with methanol:Aqueous solvent system makees eluant, eluent, and carrying out second to the target fraction I separates, and obtains active fractions II And III；

(c) using methanol aqueous solution as mobile phase, third time separation is carried out to active fractions II and III using reversed-phase HPLC, respectively Obtain Shenzhou City's honey peach reactive compound 1 and compound 2.

5. preparation method according to claim 4, it is characterised in that in step (a), methanol：Aqueous solvent system presses volume Than 30:70,50:50,70:30,100:0 elutes successively, volume ratio 70:The cut of 30 elutions is target fraction I.

6. preparation method according to claim 4, it is characterised in that in step (b), methanol：Aqueous solvent system is according to body Product compares 50:50,55:45,60:40,65:35,70:30,80:20,100:0 elutes successively, volume ratio 60:40 and 65:35 elutions Cut be active fractions II and active fractions III.

7. preparation method according to claim 4, it is characterised in that in step (c), the mobile phase is 63% methanol-water Solution；In separation process, the cut for taking retention time to be 13.9 minutes obtains Shenzhou City's honey peach reactive compound 1.

8. preparation method according to claim 4, it is characterised in that in step (c), the mobile phase is 65% methanol-water Solution；In separation process, the cut for taking retention time to be 14.5 minutes obtains Shenzhou City's honey peach reactive compound 2.

9. Shenzhou City's honey peach reactive compound that method is prepared according to claim 2 is preparing antiaging agent or health care Application in product, the compound is compound 1 and compound 2.

10. application according to claim 9, it is characterised in that the medicine or health products are as the depth described in claim 1 Two reactive compounds of state honey peach and pharmaceutically acceptable carrier are made.