CN105106303B - Medicinal uses of Prunella vulgaris water extract - Google Patents

Abstract

The invention relates to the field of medicine, in particular to the medicinal use of an aqueous extract of Prunella vulgaris, wherein the water extract of Prunella vulgaris contains 0.1-20 wt% of rosmarinic acid. The water extract of Prunella vulgaris of the present invention can be used for treating diabetic retinopathy.

Description

Medicinal uses of Prunella vulgaris water extract

technical field

The invention relates to the field of medicine and health care products, in particular to the medicinal use of a traditional Chinese medicine plant extract.

Background technique

Diabetic retinopathy (DR) is one of the most important manifestations of diabetic microangiopathy, a fundus disease with specific changes, and one of the serious complications of diabetes. Clinically, according to whether there is retinal neovascularization as a marker, diabetic retinopathy without retinal neovascularization, but with leakage of the blood-retinal barrier (BRB) and retinal inflammatory damage is called nonproliferative diabetic retinopathy (nonproliferative diabetic retinopathy). diabetic retinopathy (NPDR) (or simple type or background type), and diabetic retinopathy with retinal neovascularization is called proliferative diabetic retinopathy (PDR). The former is mainly manifested as scattered retinal microangiomas, spotting hemorrhages, rigid exudates, macular edema, etc.; the latter is manifested as retinal or optic papilla neovascularization, which grows toward the vitreous, and is prone to vitreous hemorrhage and traction retinal detachment.

Prunella vulgaris L, also known as Prunella vulgaris L, alias Prunella vulgaris, Prunella vulgaris (Yunnan Congshu), Prunella vulgaris, Prunella vulgaris (Materia Medica in southern Yunnan), Xiju, etc., is a perennial of the Lamiaceae Wild Sesame subfamily herb. Dried fruit ears are used as medicine, with strong adaptability and are produced all over the country. Its medicinal properties are pungent, bitter, and cold, and it returns to the liver and gallbladder meridians. It is mostly used for the treatment of red eyes, swelling and pain, headache, dizziness, night pain in the eyeballs, scrofula, gall tumors, and breast carbuncle swelling and pain. The rosmarinic acid (C ₁₈ H ₁₆ O ₈ ) contained in the Prunella vulgaris extract is an index component for the identification and content determination of Prunella vulgaris in the Pharmacopoeia.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a new application of Prunella vulgaris water extract.

Specifically, the first aspect of the present invention is to provide an application of a Prunella vulgaris water extract in the preparation of a medicine or food for preventing or treating diabetic retinopathy, wherein the Prunella vulgaris water extract contains 0.1-20wt% of marinic acid.

In a preferred example, the water extract of Prunella vulgaris contains 1.98wt% rosmarinic acid

In another preferred embodiment, the diabetic retinopathy is non-proliferative diabetic retinopathy.

The details of various aspects of the invention will be described in detail in subsequent sections. The features, objects and advantages of the present invention will become more apparent from the description below and in the claims.

Description of drawings

n＝16)^###P<0.001vs.空白对照；Figure 1 Effects of Prunella vulgaris water extract (PV) on body weight and blood sugar of diabetic mice (A) body weight; (B) blood sugar; (

n=16) ^### P<0.001 vs. blank control;

n＝6)^###P<0.001vs.空白对照；***P<0.001vs.糖尿病组；Fig. 2 Effects of Prunella vulgaris water extract (PV) on BRB leakage in diabetic mice (

n=6) ^### P<0.001vs. blank control; ***P<0.001vs. diabetes group;

n＝10)^#P<0.05vs.空白对照；*P<0.05,**P<0.01,***P<0.001vs.糖尿病组；Figure 3 Prunella vulgaris water extract (PV) reduces the elevated levels of IL-1β and TNF-α in the serum of diabetic mice (

n=10) ^# P<0.05vs. blank control; *P<0.05, **P<0.01, ***P<0.001vs. diabetes group;

n＝8)^###P<0.001vs.空白对照；***P<0.001vs.糖尿病组；Figure 4 The effect of Prunella vulgaris water extract (PV) on the expression of Iba-1 in the retina of diabetic mice (

n=8) ^### P<0.001vs. blank control; ***P<0.001vs. diabetes group;

n＝8)^###P<0.001vs.空白对照；*P<0.05,***P<0.001vs.糖尿病组；Figure 5 Effects of Prunella vulgaris water extract (PV) on phosphorylation and nuclear translocation of NF-κB p65 subunit in retina of diabetic mice (

n=8) ^### P<0.001vs. blank control; *P<0.05, ***P<0.001vs. diabetes group;

Fig. 6 The effect of different batches of Prunella vulgaris water extract on the leakage of BRB in diabetic mice ( n=6) ^### P<0.001 vs. blank control; ***P<0.001 vs. diabetes group.

Detailed ways

The advent of the present invention is partly based on the unexpected discovery that the aqueous extract of Prunella vulgaris, a traditional Chinese medicine, can significantly improve the inflammatory damage and BRB leakage of STZ-induced diabetic retinopathy in mice. Therefore, the water extract of Prunella vulgaris may be expected to be developed as a drug for the prevention or treatment of diabetic retinopathy

Furthermore, the first aspect of the present invention provides the application of the water extract of Prunella vulgaris in the preparation of medicine or food for preventing or treating diabetic retinopathy, the water extract of Prunella vulgaris contains 0.1-20wt% rosmarinic acid.

More preferably, the water extract of Prunella vulgaris contains 1.98wt% rosmarinic acid

More preferably, the diabetic retinopathy is non-proliferative diabetic retinopathy.

As known to those skilled in the art, as the identification of Prunella vulgaris in the pharmacopoeia and the index component of content determination, rosmarinic acid (Rosmarinic acid) has the following structural formula:

The water extract of Prunella vulgaris of the present invention can be purchased commercially from Nanjing Zelang Pharmaceutical Technology Co., Ltd. Concentrated.

The water extract of Prunella vulgaris of the present invention can be used alone or in the form of a pharmaceutical composition. The pharmaceutical composition includes the Prunella vulgaris water extract of the present invention as an active ingredient and a pharmaceutically acceptable carrier. Preferably, the pharmaceutical composition of the present invention contains 0.1-99.9% by weight of the water extract of Prunella vulgaris of the present invention as an active ingredient. "Pharmaceutically acceptable carrier" will not destroy the pharmaceutical activity of the water extract of Prunella vulgaris of the present invention, and at the same time, its effective amount, that is, the amount that can play the role of drug carrier, is nontoxic to human body.

The pharmaceutically acceptable carriers include, but are not limited to: soft phospholipids, aluminum stearate, alumina, ion exchange materials, self-emulsifying drug delivery systems, Tween or other surfactants, serum proteins, buffer substances such as phosphates, amino acids Acetic acid, sorbic acid, water, salt, electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salt, magnesium silicate, mixture of saturated fatty acid partial glycerides, etc.

Other commonly used pharmaceutical excipients such as binders (such as microcrystalline cellulose), fillers (such as starch, glucose, anhydrous lactose and lactose beads), disintegrants (such as cross-linked PVP, cross-linked sodium carboxymethyl starch) , croscarmellose sodium, low-substituted hydroxypropyl cellulose), lubricants (such as magnesium stearate) and absorption enhancers, adsorption carriers, flavors, sweeteners, excipients, diluents, Wetting agents, etc.

The water extract of Prunella vulgaris and its pharmaceutical composition of the present invention can be prepared according to conventional methods in the art and can be administered through enteral or parenteral or topical routes. Oral preparations include capsules, tablets, oral liquids, granules, pills, powders, pills, ointments, etc.; parenteral preparations include injections, etc.; topical preparations include creams, patches, ointments, sprays, etc. Oral formulations are preferred.

The administration route of the water extract of Prunella vulgaris and its pharmaceutical composition of the present invention can be oral, sublingual, transdermal, intramuscular or subcutaneous, mucocutaneous, intravenous, urethral, vaginal and the like.

In addition to being prepared as a medicine, various food additives such as antioxidants, pigments, and enzyme preparations can also be added to the water extract of Prunella vulgaris of the present invention, and the health food can be prepared according to the conventional methods in the art.

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. In the following examples, the experimental methods without specific conditions are usually in accordance with conventional conditions or in accordance with the conditions suggested by the manufacturer. All percentages, ratios, ratios, or parts are by weight unless otherwise indicated.

Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be used in the methods of the present invention. Methods and materials for preferred embodiments described herein are provided for illustrative purposes only.

The above features mentioned in the present invention or the features mentioned in the embodiments can be combined arbitrarily. All features disclosed in this patent specification may be used in combination with any composition, and each feature disclosed in the specification may be replaced by any alternative feature serving the same, equivalent or similar purpose. Therefore, unless otherwise stated, the disclosed features are only general examples of equivalent or similar features.

Embodiment 1 Determination of rosmarinic acid (RA) content in the water extract of Prunella vulgaris

1.1 Experimental materials and methods

Preparation of water extract of Prunella vulgaris: weigh 100g of Prunella vulgaris from different batches, powder or cut into granules and fragments suitable for drug extraction, add ten times of deionized water to soak for 1 hour, boil in boiling water for 1.5 hours, and collect the decoction. , the medicinal materials were decocted again, extracted three times in total, combined the decoction, concentrated under reduced pressure and evaporated to dryness to obtain solid water extracts of Prunella vulgaris in different batches, which were stored in a refrigerator at 4°C for later use.

Preparation of reference substance solution: take an appropriate amount of rosmarinic acid reference substance, accurately weigh it, put it in a brown volumetric flask, add 50% methanol to make a reference substance solution containing 100 μg per 1ml, that is (store below 10°C).

Preparation of the test solution: Precisely weigh the water extract of Prunella vulgaris into a volumetric flask, add 50% methanol, dissolve by ultrasonic, and pass through a 0.22 μm microporous membrane to obtain (stored below 10°C).

Determination of rosmarinic acid (RA) content: the chromatographic column is Phenomenex C18 column (4.6×250mm, 5μm); the mobile phase condition is methanol-1% formic acid (40:60, v/v), the flow rate is 1.0mL/min, Isocratic elution; the injection volume was 5 μL; the column temperature was 25°C; the detection wavelength was 330 nm.

1.2 Experimental results

Table 1 The percentage content of rosmarinic acid in different batches of Prunella vulgaris water extract

Example 2: Pharmacodynamic activity and mechanism of Prunella vulgaris water extract (PV) in improving diabetic retinopathy

2.1 Experimental materials and methods

2.1.1 Animals: SPF grade C57 mice, male, weighing 18-24 g, purchased from Shanghai Experimental Animal Center, Chinese Academy of Sciences. Animal certificate number: SCXK (Shanghai) 2012-0002. The animals were kept in the SPF animal room of Shanghai University of Traditional Chinese Medicine and used after one week of rearing. The rearing conditions were: temperature 22±1°C, humidity 55±5%, 12-hour light-dark cycle, free intake of feed and water after disinfection. The experiments were carried out in strict accordance with the national and Shanghai University of Traditional Chinese Medicine Animal Center Animal Use Management Regulations.

2.1.2 Preparation of medicines: Dissolve an appropriate amount of Prunella vulgaris water extract (PV) extract in physiological saline, and administer by gavage, the concentrations are 100 mg/kg and 200 mg/kg.

2.1.3 Animal experiments:

(1) Mix 0.1M citric acid solution and trisodium citrate solution at a ratio of 14:11, adjust the pH of the mixture to 4.3-4.5, and set aside. Weigh an appropriate amount of STZ powder, dissolve it in the dark to make it into a solution of 5.5mg/ml, and immediately give it to mice who have fasted for 12h by intraperitoneal injection, 0.1ml/10g, the final dose of STZ is 55mg/kg, continuous administration 5 days.

(2) One week after the last administration, the blood glucose concentration of the mice was measured by the tail cutting method, and the blood glucose value ≥ 16.7 mmol/L (250 mg/dl) was regarded as a successful model. The successfully modeled mice were randomly divided into 3 groups: DR model group (DR model group), Prunella vulgaris water extract 100 mg/kg group, and Prunella vulgaris water extract 200 mg/kg group, with 16 mice in each group. In addition, 16 normal C57 mice without i.p STZ were used as the normal group (Control group). Changes in body weight and blood sugar were measured every two weeks.

(3) The drug administration (0.1ml/10g) was started 1 month after the successful modeling. The 100 mg/kg Prunella vulgaris water extract group and the Prunella vulgaris water extract 200 mg/kg group were respectively given different concentrations of Prunella vulgaris aqueous extract solutions , the final doses were: 100mg/kg, 200mg/kg, the Model group was given the same volume of vehicle control, after 1 month of administration, 6 mice in each group were taken for Evans blue experiment, and the remaining 10 mice were treated with pentamethylene The patients were anesthetized with sodium barbital (30 mg·kg-1, intraperitoneal), blood was collected from the abdominal aorta, and the eyeballs were removed.

2.1.4 Evans blue leakage test: dissolve Evans blue in PBS to prepare a solution with a concentration of 0.2 g·L ^-1 . The Evans blue solution prepared by intraperitoneal injection (10ul·g ^-1 ) was injected into the mice, and the left ventricle of the mice was perfused with PBS 2 h after the injection to completely remove the residual Evans blue dye in the blood vessels, and then the eyeballs were quickly removed and the retinal tissue was centrifuged. In the tube, the retinal tissue was freeze-dried and weighed. Next, the retinal tissue was incubated with 120ul of formamide at 70°C for 18h to extract the Evans blue dye, and the extract was centrifuged twice (10000g, 4°C), carefully pipetted and combined. The supernatant was measured for absorbance at 620 nm using a spectrophotometer. The amount of Evans blue dye in the formamide extract was calculated from a pre-made standard curve and expressed as Evans blue content divided by dry retinal weight.

2.1.5 ELISA experiment: After the whole blood was left standing at room temperature for 2 hours, centrifuged at 4°C and 4000g for 15 minutes, the upper serum was separated, and the contents of TNF-α and IL-1β were detected according to the kit instructions.

2.1.6 Extraction of cytoplasmic and nuclear proteins: According to the instructions of the kit, use the cytoplasmic/nuclear protein extraction kit to extract the proteins in the cytoplasmic and nuclear parts of the retinal tissue, and use the BCA protein quantification kit to measure the samples of protein concentration, all samples were unified to equal protein concentration.

2.1.7 Protein electrophoresis experiment: The prepared protein samples were electrophoresed on SDS-PAGE gel, and the protein on the gel was transferred to PVDF membrane, blocked with TBST solution containing 5% skim milk for 1 h, added with primary antibody, and incubated at 4°C overnight. After washing off the primary antibody, it was incubated with the secondary antibody for 1 h at room temperature. After washing off the excess antibody with TBST, chemiluminescence solution was added for imaging, and the protein bands were quantified with GeneTools image analysis software.

)表示，采用SPSS16.0统计软件进行分析，以One-Way ANOVA方式进行方差分析，两两比较采用LSD法，P<0.05表示差异具有显著性2.1.8 Statistical analysis: the experimental data are used as mean ± standard error (

) means that SPSS16.0 statistical software was used for analysis, One-Way ANOVA was used for variance analysis, and LSD method was used for pairwise comparison, and P<0.05 indicated that the difference was significant

2.2 Experimental results

2.2.1 Effects of Prunella vulgaris water extract (PV) on blood sugar and body weight in diabetic mice

It can be seen from Figure 1 that each dose group of Prunella vulgaris water extract could not improve the weight loss of diabetic mice, nor could it reduce the elevated blood sugar. It indicated that the water extract of Prunella vulgaris did not significantly improve the pharmacodynamic activity of diabetes.

2.2.2 Effects of Prunella vulgaris water extract (PV) on retinal BRB leakage in diabetic mice

It can be seen from Figure 2 that the leakage value of Evan's blue in the retina of STZ-induced diabetic mice was significantly increased (P<0.001), and PV (100, 200 mg·kg ^-1 ) both significantly improved this (P<0.001). 0.001). Damage to the blood-retinal barrier (BRB) is a feature of non-proliferative diabetic retinopathy. Our study shows that the water extract of Prunella vulgaris can alleviate the leakage caused by BRB damage, so it has the potential to improve diabetic retinopathy, especially non-proliferative diabetic retinopathy. pharmacological activity.

2.2.3 Prunella vulgaris water extract (PV) reduces the elevated levels of IL-1β and TNF-α in the serum of diabetic mice

As shown in Figure 3, the serum levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in STZ-induced diabetic mice were significantly higher than those in the normal group (P<0.05). In contrast, the levels of IL-1β and TNF-α in serum were decreased to different degrees after PV (100,200 mg·kg ^-1 ) administration (P<0.01, P<0.05, P<0.001). Our research shows that: Prunella vulgaris water extract improves diabetic retinopathy by inhibiting the release of pro-inflammatory factors, resisting retinal inflammatory damage.

2.2.4 Effects of Prunella vulgaris water extract (PV) on Iba-1 expression, NF-κB p65 phosphorylation and nuclear translocation in the retina of diabetic mice

As can be seen in Figures 4A and 4B, the expression of Iba-1 protein in retina tissue of STZ-induced diabetic mice was significantly up-regulated (P<0.001). In contrast, PV (100, 200 mg·kg ^-1 ) significantly reduced the elevated expression of Iba-1 in retinal tissue (P<0.001). Iba1 is a marker of microglia activation. Studies have found that the activation of microglia and the inflammatory damage mediated by them play an important role in the occurrence and development of diabetic retinopathy. Our study shows that: Prunella vulgaris water extract inhibits retinal inflammatory damage by inhibiting the activation of microglia.

As shown in Figure 5A-C, compared with the normal group, the expression of phosphorylated NF-κB p65 protein in the retina tissue of STZ-induced diabetic mice was significantly increased (P<0.001), and the expression of NF-κB p65 protein in the nucleus was also significantly increased. increased (P<0.001). In contrast, PV (100, 200 mg·kg ^-1 ) could reduce the expression of elevated phosphorylated NF-κB p65 protein (P<0.05, P<0.001), and also reduced the increase in the nuclear the expression of NF-κB p65 protein (P<0.001), but had no significant effect on the expression of p65 protein in the cytoplasm. NF-κB is an important regulator of inflammatory damage in the body. Our research shows that the water extract of Prunella vulgaris inhibits the inflammatory damage of the retina in the process of diabetic retinopathy by inhibiting the NF-κB signaling pathway.

Example 3: Pharmacodynamic activity of different batches of Prunella vulgaris water extracts in improving diabetic retinopathy

3.1 Experimental materials and methods

3.1.1 Animals: SPF grade C57 mice, male, weighing 18-24 g, purchased from Shanghai Experimental Animal Center, Chinese Academy of Sciences. Animal certificate number: SCXK (Shanghai) 2012-0002. The animals were kept in the SPF animal room of Shanghai University of Traditional Chinese Medicine and used after one week of rearing. The rearing conditions were: temperature 22±1°C, humidity 55±5%, 12-hour light-dark cycle, free intake of feed and water after disinfection. The experiments were carried out in strict accordance with the national and Shanghai University of Traditional Chinese Medicine Animal Center Animal Use Management Regulations.

3.1.2 Preparation of medicine: Take appropriate amount of different batches of water extract of Prunella vulgaris (prepared according to Example 1), dissolve it in physiological saline, and administer it by gavage at a concentration of 100 mg/kg.

3.1.3 Animal experiments:

(1) Mix 0.1M citric acid solution and trisodium citrate solution at a ratio of 14:11, adjust the pH of the mixture to 4.3-4.5, and set aside. Weigh an appropriate amount of STZ powder, dissolve it in the dark to make it into a solution of 5.5mg/ml, and immediately give it to mice who have fasted for 12h by intraperitoneal injection, 0.1ml/10g, the final dose of STZ is 55mg/kg, continuous administration 5 days.

(2) One week after the last administration, the blood glucose concentration of the mice was measured by the tail cutting method, and the blood glucose value ≥ 16.7mmol/L (250mg/dl) was regarded as a successful model. The successfully modeled mice were randomly divided into 5 groups: DR model group (DR model group), Prunella vulgaris water extract (PV) group, Prunella vulgaris water extract-1 (PV-1) group, Prunella vulgaris water extract group. Material-2 (PV-2) group and Prunella vulgaris water extract-3 (PV-3) group, 6 animals in each group. In addition, 6 normal C57 mice without i.p STZ served as normal group (Control group).

(3) The drug administration (0.1ml/10g) was started 1 month after the successful modeling, and different batches of Prunella vulgaris water extract solutions were given respectively, with a dose of 100 mg/kg. The Model group was given the same volume of vehicle control, and the Evan's blue test was performed to detect the leakage of the blood retinal barrier (BRB) 1 month after the drug.

3.1.4 Evans blue leakage test: dissolve Evans blue in PBS to prepare a solution with a concentration of 0.2 g·L ^-1 . The Evans blue solution prepared by intraperitoneal injection (10ul·g ^-1 ) was injected into the mice, and the left ventricle of the mice was perfused with PBS 2 h after the injection to completely remove the residual Evans blue dye in the blood vessels, and then the eyeballs were quickly removed and the retinal tissue was centrifuged. In the tube, the retinal tissue was freeze-dried and weighed. Next, the retinal tissue was incubated with 120ul of formamide at 70°C for 18h to extract the Evans blue dye, and the extract was centrifuged twice (10000g, 4°C), carefully pipetted and combined. The supernatant was measured for absorbance at 620 nm using a spectrophotometer. The amount of Evans blue dye in the formamide extract was calculated from a pre-made standard curve and expressed as Evans blue content divided by dry retinal weight.

3.2 Experimental results

It can be seen from Figure 6 that the leakage value of Evans blue in the retina of STZ-induced diabetic mice was significantly increased (P<0.001), while PV (100 mg·kg ^-1 ), PV-1 (100 mg·kg ^-1 ), PV -2 (100 mg·kg ^-1 ) and PV-3 (100 mg·kg ^-1 ) both had significant improvement effects (P<0.001). Damage to the blood retinal barrier (BRB) is a feature of non-proliferative diabetic retinopathy. Our study showed that: All batches of Prunella vulgaris water extract can alleviate the leakage of Evans blue caused by BRB damage, so it can improve diabetes Pharmacodynamic activity in retinopathy, especially nonproliferative diabetic retinopathy.

The various aspects involved in the present invention have been described above. However, it should be understood that equivalent changes and modifications may be made thereto by those skilled in the art without departing from the spirit of the present invention, which also fall within the scope of coverage of the appended claims of the present application.

Claims (2)

1. Application of Prunella vulgaris water extract in preparing medicine or food for preventing or treating diabetic retinopathy, said Prunella vulgaris water extract contains 0.1-20wt% rosmarinic acid, and said diabetic retinopathy is non-proliferative Diabetic retinopathy. 2. The application of the water extract of Prunella vulgaris as claimed in claim 1, wherein the water extract of Prunella vulgaris contains 1.98wt% rosmarinic acid.

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