CN101732440B - Raspberry extract, raspberry element separated from raspberry extract and application of raspberry element - Google Patents

Abstract

本发明覆盆子提取物及覆盆子素具有防治慢性肾炎、肾阳虚和骨质疏松的活性，因而可以用于制备治疗和预防慢性肾炎、肾阳虚或骨质疏松的药物。The present invention relates to the field of medical technology, and is a raspberry extract extracted from Rubus chingii Hu and raspberry extract isolated therefrom, as well as their use in preparing drugs for treating and preventing chronic nephritis, kidney yang deficiency and osteoporosis. The chemical structure of raspberry extract is as follows:

The raspberry extract and raspberry element of the invention have the activity of preventing and treating chronic nephritis, kidney yang deficiency and osteoporosis, and can be used for preparing medicine for treating and preventing chronic nephritis, kidney yang deficiency or osteoporosis.

Description

Raspberry extract and raspberry extract isolated therefrom and uses thereof

technical field

The invention relates to the technical field of medicine, and relates to raspberry extract extracted from Rubus chingii Hu and raspberry extract isolated therefrom and their preparation for treating and preventing chronic nephritis, kidney yang deficiency or osteoporosis application in medicine.

Background technique

Raspberry (Fructus rubi) is the dry near-ripe fruit of Rubus chingii Hu in the family Rosaceae. Sweet, sour, warm. Return kidney, urinary bladder channel. Tonifying the kidney, strengthening essence, reducing urination. For enuresis due to kidney deficiency, frequent urination, impotence and premature ejaculation, spermatorrhea and spermatorrhea embolism. Raspberry is rich in resources in my country, mainly distributed in Zhejiang, Jiangsu, Anhui, Jiangxi, Fujian, Guangxi and other places, and it has been recorded in "Chinese Pharmacopoeia". In recent years, there has been some research on raspberry at home and abroad, and it was reported to the German army that raspberry extract has a certain effect of warming the kidney and helping yang (Journal of Guangdong Pharmaceutical University 2002.18(3): 217-218), but did not mention its active ingredients . So far, there is no report that raspberry extract can prevent and treat chronic nephritis, kidney yang deficiency or osteoporosis.

Contents of the invention

The invention provides a raspberry extract with the activity of preventing and treating chronic nephritis and osteoporosis and a new compound-raspberry element isolated from the raspberry extract. The chemical structural formula of raspberry element is:

The research of the present invention shows that the raspberry element is the active ingredient of the raspberry extract, and has the activity of preventing and treating chronic nephritis, osteoporosis and kidney-yang deficiency.

The preparation method of raspberry extract of the present invention and raspberry element is as follows:

1. Preparation of Raspberry Extract

Extract the dried fruit of raspberry palmate with 0-95% ethanol aqueous solution according to conventional percolation or soaking extraction, preferably with 50-80% ethanol aqueous solution, most preferably with 70% ethanol aqueous solution, and concentrate the extract under reduced pressure until it has no alcohol smell The extract, raspberry extract;

2. Preparation of crude raspberry extract

Dilute the above-mentioned raspberry extract with water, then extract with petroleum ether and dichloromethane in sequence, discard the extract, then extract with saturated n-butanol with water, concentrate the n-butanol extract under reduced pressure to obtain a crude raspberry element;

3. Separation and extraction of raspberry element

Suspend the above crude product of raspberry extract with water, apply it to macroporous resin D101, and then elute with water, 30%, 60% and 95% ethanol aqueous solution respectively, collect the 30% ethanol eluate, concentrate under reduced pressure into extract and use Dissolve in methanol, mix the sample with silica gel H (type 60, Qingdao Ocean Chemical Group Co., Ltd.), put it on a silica gel H chromatography column, and elute with chloroform-methanol (30:1-1:1) solvent system to collect chloroform-methanol 5: 1, and concentrated, washed with methanol to obtain a yellow powder, that is, raspberry element.

According to animal experiments, the raspberry extract and raspberry element of the present invention have the activity of preventing and treating chronic nephritis, deficiency of kidney yang and osteoporosis, so they can be used to prepare medicines for preventing and treating chronic nephritis, deficiency of kidney yang and osteoporosis.

The preparation method of the extract and the raspberry element compound of the invention is simple and low in cost, and the invention provides a new drug source for preventing and treating chronic nephritis, kidney-yang deficiency and osteoporosis.

Detailed ways:

The present invention is described in detail in conjunction with embodiment now.

The raspberry palmate used was collected in September 2007 in Hangzhou, Zhejiang.

Example 1. Preparation of raspberry extract

50 kg of dried fruit of raspberry palmate was extracted by percolation with an aqueous solution as usual, and the extract was concentrated under reduced pressure to a non-alcoholic extract to obtain 12.6 kg of raspberry extract.

Example 2. Preparation of raspberry extract

50 kg of dried fruit of raspberry palmate was extracted by percolation with a 95% solution as usual, and the extract was concentrated under reduced pressure to an alcohol-free extract to obtain 2.4 kg of raspberry extract.

Example 3. Preparation of raspberry extract

50 kg of dried fruit of raspberry palmate was extracted by percolation with 70% ethanol aqueous solution as usual, and the extract was concentrated under reduced pressure to a non-alcoholic extract to obtain 6.5 kg of raspberry extract.

Embodiment 4. prepare raspberry element with raspberry extract

1. Preparation of crude raspberry extract

The raspberry extract prepared in Example 3 was diluted with water to a volume of 50L, first extracted 3 times with 50L petroleum ether, discarded the extract, then extracted 3 times with 50L dichloromethane, discarded the extract, then saturated with n-butyl ether Alcohol extraction 3 times, 50 L each time, combined n-butanol extracts, concentrated under reduced pressure to obtain crude raspberry element.

2. Separation and extraction of raspberry element

Suspend the above-mentioned raspberry extract crude product with water, apply it to macroporous resin D101, elute with water, 30%, 60% and 95% ethanol aqueous solution in sequence, collect the 30% ethanol eluate, concentrate under reduced pressure into an extract and wash with methanol Dissolve, mix the sample with silica gel H (type 60, Qingdao Ocean Chemical Group Co., Ltd.), put it on a silica gel H chromatography column, elute with chloroform-methanol (30:1-1:1) solvent system, collect chloroform-methanol 5:1 The eluate was concentrated and washed with methanol to obtain a yellow powder, that is, raspberry element.

Structure Identification:

Yellow powder, mp: 267-269°C, ESI-MS: m/z 247[MH] ^- , the molecular weight of the compound is determined to be 248, and the molecular formula shown by HR-MS is: C ₁₂ H ₈ O ₆ . UV (EtOH): λmax (ε) = 364 (10143), 299 (28317), 200 (17918). IR(KBr): v _OH = 3404 cm ^-1 , v _{C = O} = 1684 cm ^-1 , v _{C = C} = 1599 cm ^-1 , v _CH2 = 1392 cm ^-1 , v _CO = 1305 cm ^-1 .

In ¹ H-NMR (400MHz, δ, pyridine-d5), ¹ HNMR signal δ=2.5(2H, m), δ=3.5(2H, m), showed -CH ₂ -CH ₂ -, δ=8.0( 1H, s), shown as H on the double bond. Signals δ=195.5 and δ=160.5 in ¹³ C-NMR indicated the presence of an α,β-unsaturated ketone and α,β-unsaturated ester. From ¹³ C-NMR spectrum and DEPT spectrum, it can be seen that the compound contains 9 quaternary C, 1 -CH, 2 -CH ₂ , 110-150 in ¹³ C-NMR has 6 peaks, and DEPT spectrum shows 6 peaks The peaks are all quaternary C, indicating the presence of 6-substituted benzene rings.

Based on the above information, it can be deduced that the compound is a benzene ring, one side is connected with a six-membered lactone ring, the other side is connected with a five-membered ketone ring, and the other two Cs are replaced by OH.

According to the HMQC spectrum, the hydrocarbons of the compound can be further assigned.

In the HMQC spectrum, the secondary C of δ = 23.8 corresponds to the H of δ = 3.5 (2H, m), the secondary C of δ = 33.0 corresponds to the H of δ = 2.5 (2H, m), and the quaternary C of δ = 108.0 corresponds to δ = 8.0 H of (1H, s). In HMBC, H of δ=8.0 is related to C of δ=160.5, 140.1, 144.2, 149.3, and 113.2, indicating that the double bonds are connected to ester bonds and benzene rings respectively, H of δ=2.5 is related to C of δ=141.1, 195.5, and 23.8 C is related, indicating that this CH ₂ is connected to the ketone carbonyl, and H of δ=3.5 is related to C of δ=141.1, 145.0, 195.5, and 23.8, indicating that two CH ₂ are connected, and the latter is connected to the benzene ring. The assignment of each model is shown in Table 1.

Based on the above analysis, the complete structure of the compound is determined, named as raspberry element, and its structural formula is:

Table 1. ¹ H, ¹³ C-NMR data of raspberry (400MHz, δ, pyridine-d5)

Location C DEPT h HMBC 1 2 160.5 3 140.1 4 108.0 CH 8.0(1H, s) 2, 3, 5, 9, 10 5 144.2 6 115.5 7 141.1 8 145.0 9 149.3 10 113.2 11 195.5 12 33.0 CH ₂ 2.5(2H, m) 7, 11, 13 13 23.8 CH ₂ 3.5(2H, m) 7, 8, 11

Pharmacology, efficacy experiment

Raspberry extract of the present invention is basically the same as the pharmacology or pharmacodynamics experimental method of raspberry element, now detailed as follows:

1. Prevention and treatment effect of raspberry extract and raspberry element on passive Heymann nephritis in rats

Passive Heymarm nephritis is a classic model for studying human membranous nephropathy, which is characterized by massive proteinuria [Chinese Journal of Nephrology. 1999.15(3): 158]. Therefore, the present invention uses the Heymann nephritis model to study the prevention and treatment effect of raspberry extract and raspberry element on nephritis.

1. Test material

1.1. Experimental Instruments

BCA protein detection kit (Pierce company, the United States), ReverTra DashTM kit (Toyobo company, Japan).

1.2. Samples for testing:

Raspberry extract (prepared in Example 3)

Raspberry element (prepared in embodiment 4, the purity is not less than 93.2%, the same below);

Dexamethasone (Hebei Jiheng Pharmaceutical Co., Ltd.)

Before use, the raspberry extract, raspberry element and dexamethasone were mixed with 0.5% carboxymethylcellulose sodium (CMCNa) respectively to prepare corresponding concentrations.

2. Test animals

Strain: Wistar rat

Gender: male

Weight: 175-200 grams

Source: Shanghai Animal Center, Chinese Academy of Sciences (the same below)

Animal certificate: Shanghai Donghe Zhengzi No. 005

Breeding: constant temperature, purified and ventilated animal room, free access to food and water, 25±2°C.

3. Test method

3.1 Preparation of anti-FX1A serum

1) Preparation of FX1A antigen

For normal male Wistar rats, disinfect the skin after ether anesthesia, cut open the chest and abdomen cavity, lavage the kidney with sterile cold saline, then separate the renal cortex and weigh it, grind the cortex into a homogenate, add appropriate amount of saline, and use Filter through 80 mesh, 100 mesh and 220 mesh sieves, collect the final filtrate, centrifuge the filtrate at 1600r/min for 20min, take the supernatant, repeat the centrifugation once, take the supernatant, then centrifuge at 10000r/min for 30min, discard the supernatant, and put The precipitate was washed 3 times with double distilled water, and the obtained precipitate was FXA1A antigen.

2) Preparation of anti-FX1A serum

After fully emulsifying the above-prepared FXA1A antigen and an equal volume of complete Freund's adjuvant, large-eared white rabbits were subcutaneously immunized at intervals of 2 weeks, and the serum was collected after 6 times of immunization. The titer was determined by two-way immunodiffusion and indirect immunofluorescence, and the titer was 1:32 and 1:2000 respectively. Blood was collected from the carotid artery, and serum was separated, which was anti-FX1A serum, kept at -20°C for use, and inactivated before use.

3.2. Establishment of passive Heymann nephritis model and animal grouping

Seventy-five male Wistar rats were taken, and their 24-h urine protein was less than 10 mg. Randomly divided into 5 groups on average: positive control group, raspberry extract group, raspberry element group, model control group (abbreviated model group, the same below) and blank control group (abbreviated blank group, the same below). The blank group was injected with normal saline 5ml/kg into the tail vein at one time; the rats in the other groups were injected with 5ml/kg of anti-FX1A serum through the tail vein at one time to establish the model. Calculation per kilogram of body weight), totally 4 weeks, administration dosage is respectively positive control group (dexamethasone, 0.1mg/kg/d); Raspberry extract group (30g/kg/d); Raspberry element group ( 10 mg/kg/d); the model group and the blank group were orally perfused with 0.5% carboxymethylcellulose sodium (CMCNa) suspension 5 mg/kg/d every day.

3.3. Observation indicators

Urinary protein was quantified using the BCA (bicinchoninnacid) method. All five groups were placed in metabolic cages at the end of the second and fourth weeks of modeling, and urine was collected for 24 hours. The measurement data are expressed as mean ± standard deviation (x±SD). Each group was compared with the model group, and the two-sided two-sample t test was used for statistical analysis. The results are shown in Table 2.

Table 2. Statistical table of urine protein in rats

Compared with the model group: **P<0.01

Visible by table 2, raspberry extract and raspberry element urine protein amount are obviously lower than model group (P＜0.05), close to positive control group, illustrate that raspberry extract of the present invention and raspberry element have positive effect on passive Heymann's nephritis. Significant curative effect.

2. Prevention and treatment effect of raspberry extract and raspberry element on hydrocortisone-induced kidney-yang deficiency in mice

The present invention selects the mouse model of kidney-yang deficiency induced by hydrocortisone, which is a classic and commonly used model of kidney-yang deficiency.

1. Test material

1.1. Drugs for modeling

Name: Hydrocortisone

Source: Tianjin Biochemical Pharmaceutical Factory

Content: 50mg/bottle

Properties: powder for injection

Preparation: use physiological saline to make 3.57mg/ml.

1.2. Samples for testing:

Raspberry extract and raspberry element are the same as above; Methyltestosterone Tablets (Shanghai Xinyi Kangjie Pharmaceutical Co., Ltd.); Jinkuishenqi Pills (Beijing Tongrentang Science and Technology Development Co., Ltd. Pharmaceutical Factory).

Solution preparation: before use, dissolve raspberry extract and raspberry element in 1% tragacanth gum to make a suspension of corresponding concentration;

Methyltestosterone: use 1% tragacanth gum to make a 30mg/ml suspension;

Jinkuishenqi Pills: use 1% tragacanth gum to make a 150mg/kg suspension.

2. Test animals

Strain: ICR mice

Age: 6 weeks old

Level: clean level

Gender: Male

Weight: 25g±2g

Source: Shanghai Slack Experimental Animal Co., Ltd.

Animal certificate number: SCXK (Shanghai) 2007-0005

Breeding: clean animal room, free access to food and water, temperature: 25±2°C, humidity: 40-60%, artificial lighting to simulate diurnal changes.

3. Test method

Sixty-four male ICR mice were adapted to the experimental conditions for 3 days. Randomly grouped, 8 in each group. Except for the blank group, each mouse was intraperitoneally injected with 0.2ml of normal saline every day for 10 days, and each mouse of the other groups was injected with 0.2ml of hydrocortisone solution every day to establish a model, and at the same time, the above-mentioned prepared medicinal solution was administrated into the stomach respectively. Dosage: raspberry extract low dose group 10g/kg; raspberry extract high dose group 30g/kg; raspberry extract low dose group 5mg/kg; raspberry extract high dose group 10mg/kg; methyltestosterone group 0.65 g/kg; Jinkuishenqi pill group 6g/kg; model group and blank group were given 0.5ml 1% tragacanth gum and injected hydrocortisone solution continuously for 10 days. On the 13th day after the administration, the mice in each group were weighed, blood was collected from the orbit, and then the mice were sacrificed. The testes and kidneys of the mice were removed and weighed. After the mouse blood was left for 2 hours, it was centrifuged, and the serum was collected and placed in a -20°C refrigerator for testing.

There was no difference in the weights of the kidneys and testes of the mice in each group. The testosterone chemiluminescence kit was used for serum samples, and the serum testosterone concentration was determined according to the instructions. Calculate the mean and standard deviation (n=8) of each group of mouse body weight and testosterone concentration, the experimental data is represented by x ± SD, each group is compared with the model group respectively, adopts two-sided two-sample t test to carry out statistical analysis, the results are shown in table 3.

Table 3 Statistical table of body weight and testosterone concentration level of mice

Compared with the model group: *P<0.05, **P<0.01

It can be seen from Table 3 that the body weight of the mice in the model group was significantly lower than that of the blank group, and the blood testosterone level was significantly different from that of the blank group (P<0.01). Raspberry extract and raspberry element group of the present invention, body weight is close to blank group and methyltestosterone and Jinkuishenqi pill group; blood testosterone level is close to methyltestosterone and Jinkuishenqi pill group, especially raspberry extract and raspberry The level of blood testosterone in the high-dose group of Penzisu was basically the same as that of the blank group, methyltestosterone and Jinkuishenqiwan group, and had a significant difference compared with the model group (P<0.01). The results show that the raspberry extract and raspberry element of the present invention have obvious prevention and treatment effects on mice with kidney-yang deficiency.

3. Therapeutic effect of raspberry extract and raspberry element on retinoic acid-induced osteoporosis in mice

In order to study the anti-osteoporosis effect of raspberry extract and raspberry element, this experiment adopts the animal model of retinoic acid-induced osteoporosis, because all-trans retinoic acid is an immunosuppressant, which can inhibit bone synthesis, etc. Therefore, it is often used to establish animal models of osteoporosis.

1. Test material

1.1. Experimental Instruments

AE240 electronic balance (Mettler-Toledo Instruments).

1.2. Samples for testing:

Raspberry extract and raspberry element are the same as above; all-trans retinoic acid (Henan Zhongtian Industrial Co., Ltd.); alendronate sodium (North China Pharmaceutical Company).

Preparation: before use, the raspberry extract, raspberry element, all-trans retinoic acid and sodium alendronate were respectively dissolved in 0.5% carboxymethylcellulose sodium (CMCNa) suspension for use.

Hydroxyproline (HOP) detection kit (Nanjing Jiancheng Bioengineering Institute).

2. Test animals

Strain: Kunming mouse

Gender: male

Weight: 33±4 grams

Source: Shanghai Animal Center, Chinese Academy of Sciences

Animal certificate: Shanghai Donghe Zhengzi No. 005

Breeding: constant temperature, purified and ventilated animal room, free access to food and water, 25±2°C.

3. Test method

40 Kunming mice were randomly divided into 4 groups, 10 in each group: blank group, model group, raspberry extract group (30g/kg), and raspberry element group (10mg/kg). Except for the blank group, all the other groups were modeled with all-trans retinoic acid (105 mg/kg). Retinoic acid was intragastrically administered every morning, and mice were given corresponding doses of drugs by intragastric administration in the afternoon. The model group and the blank group were administered respectively. Stomach 0.5% carboxymethylcellulose sodium (CMCNa) suspension for 14 days. During the experiment, the body weight was weighed once every weekend, and the animals were sacrificed on the 15th day, and the right femur was taken for measurement.

After the mouse femur was dissected, the attached soft tissue was removed, dried at 80°C to constant weight, and measured as follows:

(1) backbone weight measurement: weigh with an electronic balance to get the backbone weight;

(2) Measurement of bone length and transverse diameter of the base bone: measure the length and transverse diameter of the femur with a vernier caliper;

The experimental results were expressed as mean ± standard deviation (x ± SD), and the measurement data were compared between groups by t test.

The results are shown in Table 4.

Table 4 Statistical table of body weight and bone index of mice

Compared with the model group: *P<0.05, **P<0.01; compared with the model group: #P<0.05, ##P<0.01

As can be seen from Table 4, the dry weight of the right femur and the transverse short diameter of the right femur in the model group were significantly smaller than the blank group, while the raspberry extract or raspberry element group were significantly higher than the model group (P<0.05, P<0.01) . It shows that raspberry extract or raspberry element has preventive effect on osteoporosis.

Claims (2)

1. The application of raspberry extract as the only active ingredient in the preparation of drugs for the prevention and treatment of chronic nephritis or osteoporosis, the preparation method of the raspberry extract is: using 0-95% ethanol aqueous solution to dry fruit of raspberry palmate According to conventional percolation extraction or immersion extraction, the extract is concentrated under reduced pressure to a non-alcoholic extract to obtain raspberry extract. 2. The application of raspberry element in the preparation and prevention of chronic nephritis, kidney yang deficiency or osteoporosis medicine, the chemical structural formula of said raspberry element is: