CN107198708B - A kind of sunflower disc extract and its preparation method and use - Google Patents

Abstract

The invention relates to the field of medicine, in particular to a sunflower disc extract and a preparation method and application thereof. The sunflower disc extract of the present invention contains 17.1±0.2wt% of total flavonoids and 6.7±0.1wt% of chlorogenic acid. The sunflower disc extract of the present invention can be used for preparing vasodilators.

Description

A kind of sunflower disc extract and its preparation method and use

technical field

The present invention relates to the field of medicine, in particular to a plant extract and its preparation method and use.

Background technique

The sunflower plate is the sunflower (Helianthus annuus L.) with the sunflower seeds removed. The sunflower belongs to the Asteraceae Helianthus genus and is an annual herb, also known as sunflower. Sunflower cultivation in my country has a history of at least four hundred years. In the past, people would burn sunflower trays with sunflower seeds removed as waste. In recent years, sunflower trays have been made into feed or used for extraction due to the fact that they contain a variety of nutrients. pectin. In fact, sunflower plate is also a blind Chinese medicinal material. Sunflower plate is sweet, cold in nature, non-toxic, and belongs to the liver meridian. Functions Dispel wind, calm liver, clear dampness and heat, disperse stagnant qi, nourish qi and invigorate kidney. "Chinese Materia Medica" records that it has the effect of treating high blood pressure, headache, dizziness, tinnitus and other symptoms.

As an important oil crop in my country, sunflower is widely planted, and its seeds are used for oil extraction, while the flower plate has not been developed and utilized, but is only used as a medicine in folk prescriptions. Studies have shown that sunflower disc has the effect of lowering blood pressure, but there is no relevant report on its vasodilatory effect.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a sunflower disc extract and its preparation method and vasodilation effect.

Specifically, the first aspect of the present invention provides a sunflower disc extract, the sunflower disc extract contains 16.9-17.3 wt % of total flavonoids and 6.6-6.8 wt % of chlorogenic acid.

In a preferred example, the sunflower disc extract is prepared by the following method: pulverizing the sunflower disc and extracting it with 75% ethanol through a reflux device; rotating the extract and centrifuging to remove the precipitation; petroleum ether-ethyl acetate-normal The butanol is extracted in turn, the petroleum ether and ethyl acetate extraction step retains the lower layer liquid, and finally n-butanol is added, and after standing, the layers are separated, and the upper layer extract is retained; n-Butanol phase extract.

The second aspect of the present invention provides a method for preparing the above-mentioned sunflower disc extract, the method comprising: pulverizing the sunflower disc, extracting with 75% ethanol through a reflux device; rotating the extract and centrifuging to remove the precipitation; Petroleum ether-ethyl acetate-n-butanol are extracted in turn, the petroleum ether and ethyl acetate extraction step retains the lower layer liquid, and finally n-butanol is added, and after standing, the layers are separated, and the upper layer extract is retained; the obtained upper layer extract is Rotary evaporation and centrifugation to remove the precipitate to obtain n-butanol phase extract.

The present invention also provides the use of the above sunflower disc extract in preparing a vasodilator drug.

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

The rutin standard curve of Fig. 1 sunflower disc n-butanol phase extract of the present invention illustrates that the rutin concentration and the absorbance linearity are good in the range of 0～640ug/ml;

Fig. 2 is the chlorogenic acid standard curve of the n-butanol phase extract of sunflower disc of the present invention, illustrating that the chlorogenic acid concentration and the absorbance linearity are good in the range of 0～128ug/ml;

Fig. 3 Sunflower disc n-butanol phase extract of the present invention has relaxation effect on rat thoracic aorta, and its vasodilation effect is concentration-dependent, and the relaxation effect is stronger than rutin and chlorogenic acid;

Fig. 4 The vasodilation of the n-butanol phase extract of the sunflower disc of the present invention is mainly through the NO pathway.

Detailed ways

Example 1 Extraction and quantitative analysis of n-butanol phase of sunflower plate

1. Materials and methods

1.1 Materials

Sunflower plate, purchased from Guoan, Hebei, was identified as Asteraceae Helianthus annuus L. by Professor Ke Yan of the Teaching and Experiment Center of Shanghai University of Traditional Chinese Medicine; rutin and chlorogenic acid standard (Shanghai Yuanye Biological Technology Co., Ltd.); 75% ethanol, n-butanol (Sinopharm Group Chemical Reagent Co., Ltd.); 722 visible light spectrophotometer (Shanghai Precision Instrument Co., Ltd.); Blast drying oven (Shanghai Jinghong Experimental Equipment Co., Ltd.).

1.2 Methods

1.2.1 Extraction of n-butanol phase of sunflower plate

The collected fresh sunflower discs were dried at 60°C in a blast drying oven, and directly pulverized by a pulverizer; 1 kg of sunflower disc powder was taken, the sunflower discs were pulverized, and extracted with 75% ethanol through a reflux device; the extract was spun and centrifuged to remove Precipitation; petroleum ether-ethyl acetate-n-butanol is extracted successively, and petroleum ether and ethyl acetate are extracted to retain the lower layer liquid, and finally n-butanol is added, and the layers are separated after standing, and the upper layer extract is retained; the obtained upper layer is extracted The liquid was rotary evaporated and centrifuged to remove the precipitation to obtain 1.024 g of the n-butanol phase extract, which was formulated into 1 mg/mL for quantitative analysis of total flavonoids and chlorogenic acid in the n-butanol phase extract of sunflower disc.

1.2.2 Quantitative analysis of total flavonoids in the n-butanol phase extract of sunflower disc

1.2.2.1 Preparation of rutin standard curve

Accurately weigh 10 mg of rutin standard, dissolve it with 90% ethanol and dilute to 10 mL, and store it in a brown bottle at 4°C for future use (1000 μg/mL). Pipette 0.1, 0.2, 0.4, 0.8, 0.16, 0.32, 0.64 mL of standard solution into a 10 mL volumetric flask, add 0.3 mL of 5% NaNO ₂ solution, mix well, add 0.3 mL of 10% Al(NO3) ₃ after 6 minutes, and mix After standing for 6 minutes, add 4 mL of 4% NaOH solution, and after standing for 15 minutes, dilute to 10 mL with 90% ethanol solution, mix well, and measure the absorbance at 510 nm after 15 minutes. A linear regression analysis was performed on the absorbance (Y) with the concentration of rutin (X), and the standard curve of rutin was drawn.

1.2.2.2 Quantitative analysis of total flavonoids in the n-butanol phase extract of sunflower disc

After diluting the extract in 1.2.1 above, referring to the preparation method of the standard curve, add 5% NaNO ₂ solution, 10% Al(NO 3 ) ₃ and 4% NaOH solution according to the ratio, make the sample solution at a constant volume, at 510nm Measure its absorbance. The extraction rate calculation formula is:

W=C×V×n/m

Among them: C - the concentration of the sample, calculated according to the standard curve; V - the volume of the sample after dilution;

n-dilution factor; m-sample mass.

1.2.3 Quantitative analysis of chlorogenic acid in the n-butanol phase extract of sunflower disc

1.2.3.1 Preparation of chlorogenic acid standard curve

Precisely weigh 5.12 mg of chlorogenic acid reference substance into a 10 ml brown volumetric flask, add 50% methanol solution to the mark, and prepare a chlorogenic acid reference substance solution with a concentration of 512 μg/ml (stored below 10°C). Take 0.039ml, 0.078ml, 0.156ml, 0.313ml, 0.625ml, 1.25ml, 2.5m, and 15ml of chlorogenic acid standard solution in a 10ml brown volumetric flask, add 50% methanol to the volume, and prepare a concentration of 2, 4, 8, 16, 32, 64, 128, 256 μg/ml of chlorogenic acid standard solution, 50% methanol as blank control. Measure the absorbance values of different concentrations of standard solution at 327nm wavelength, perform linear regression analysis on the absorbance (Y) of the chlorogenic acid concentration (X), and draw the chlorogenic acid standard curve.

1.2.3.2 Quantitative analysis of chlorogenic acid in the n-butanol phase extract of sunflower disc

After diluting the extract in 1.2.1 above, accurately weigh 5 mg of the n-butanol phase extract sample of sunflower disc and place it in a 10 ml brown volumetric flask, add 50% methanol solution to the mark, and prepare a sample with a concentration of 500 μg/ml solution. Its absorbance was measured at 327 nm. The extraction rate calculation formula is:

W=C×V×n/m

Among them: C - the concentration of the sample, calculated according to the standard curve; V - the volume of the sample after dilution;

n-dilution factor; m-sample mass.

2. Results

2.1 The rutin concentration has a good linearity with the absorbance in the range of 0-640ug/ml. The standard curve is y=0.0013x+0.0429. See Table 1 and Figure 1.

Table 1 Standard curve of rutin

2.2 According to the standard curve in 2.1, substitute the total flavonoid content in the n-butanol phase extract of sunflower disc=17.1±0.2wt%.

2.3 The concentration of chlorogenic acid has a good linearity with absorbance in the range of 0～128ug/ml. The standard curve is y=0.0177x+0.1827. See Table 2 and Figure 2.

Table 2 Standard curve of chlorogenic acid

2.4 According to the standard curve in 2.3, substitute the chlorogenic acid content in the n-butanol phase extract of sunflower disc=6.7±0.1wt%.

Example 2 Study on the vasodilatory effect of n-butanol phase extract of sunflower disc

1. Materials and methods

1.1 Materials

Acetylcholine (ACh), phenylephrine (Phenylephrine, PE), Sodium Nitroprusside (SNP), etc. were purchased from Sigma Company, and inorganic reagents such as NaCl were of domestic analytical grade. Kreb-Henseleit (K-H) solution (mmol·L-1): NaCl 118, KCl 4.7, KH2PO4 1.2, MgCl2 1.2, NaHCO3 25, CaCl2 2.5, Glucose 11, PH=7.4). Animals: 200±20g, 30 rats, purchased from Shanghai Slack Laboratory Animal Co., Ltd. and raised in the Animal Experimental Center of Shanghai University of Traditional Chinese Medicine. Separation funnel, dissection plate, tack, dissection scissors, forceps, cotton ball (absorbent cotton), petri dish, etc. PowerLab eight-channel multifunction physiological recorder (AD Instruments, Australia).

1.2 Methods

1.2.1 Preparation of thoracic aortic rings

The rats were sacrificed by anesthesia, and the thoracic aorta was quickly taken out and placed in KH solution at 4°C, and the surrounding connective tissue was stripped to make a 2-3mm vascular ring. The vascular ring was suspended in a bath of 4 mL KH solution at a constant temperature of 37°C, and a mixture of 95% O ₂ +5% CO ₂ was continuously fed.

Each vascular ring was given a pulling force of 2.0g, KH solution was replaced every 15min, and it was balanced for 60min. Pre-contract twice with 60 mmol·L ^-1 KCl to induce the maximum shrinkage. After eluting with KH solution, after the tensile force is stable, add 10 ^-6 mol·L ^-1 PE to shrink. After the tensile force reaches the plateau value, add 10 ^-5 mol/L Ach, if the vasodilation is more than 80%, the endothelium can be considered intact and can be used in the next experiment.

1.2.2 The effect of sunflower disc n-butanol phase extract on endothelium-dependent relaxation of rat thoracic aorta

Rat thoracic aortic vascular rings with intact endothelium were prepared, and equal doses of PE were used to contract, and different concentrations of sunflower disc n-butanol phase extract (prepared according to Example 1) were added to observe the effect of different concentration gradient groups on thoracic aortic endothelium-dependent relaxation. effect. Repeat the experiment more than 3 times according to the above experimental steps.

1.2.3 Mechanism of n-butanol phase extract of sunflower disc on endothelium-dependent relaxation of rat thoracic aorta

Rat thoracic aortic vascular rings with intact endothelium were prepared and pretreated with L-NAME (0.1mmol·L ^-1 ), indomethacin (0.1mmol·L ^-1 ) and TEA (5mmol·L ^-1 ), respectively 20min, pre-shrink with PE, add the n-butanol phase extract of sunflower disc (prepared according to Example 1) to observe its effect, and add the same dose of KH solution to the control group.

2 results

2.1 Sunflower disc n-butanol phase extract of the present invention has diastolic effect on rat thoracic aorta, and its vasodilatation effect is concentration-dependent, and the effect is better than the rutin and chlorogenic acid it contains (see Table 3 and image 3).

Table 3 Effects of different concentrations of sunflower disc n-butanol phase extract on vasodilation

^** P<0.01 compared to 0.1mg/μL sunflower dish n-butanol

2.2 The relaxation effect of n-butanol extraction from sunflower disc relative to rat thoracic aorta is mainly through the NO pathway (see Table 4 and Figure 4).

Table 4 The effect of different concentrations of sunflower disc n-butanol phase extract on vasodilation

^** P<0.01 compared to 0.1mg/μL sunflower dish n-butanol

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 (3)

1. The blood vessel dilating sunflower disc extract is characterized by comprising 16.9-17.3 wt% of total flavonoids and 6.6-6.8 wt% of chlorogenic acid, and is prepared by the following method: pulverizing receptaculum Helianthi, and extracting with 75% ethanol under reflux; steaming the extract, and centrifuging to remove precipitate; sequentially extracting petroleum ether, ethyl acetate and n-butyl alcohol, wherein the lower layer liquid is reserved in the petroleum ether and ethyl acetate extraction step, the n-butyl alcohol is added finally, the mixture is layered after standing, and the upper layer extracting solution is reserved; and (4) carrying out rotary evaporation and centrifugation on the obtained upper-layer extracting solution to remove precipitates to obtain the n-butanol phase extract.

2. The method of preparing the sunflower disc extract of claim 1, comprising: pulverizing receptaculum Helianthi, and extracting with 75% ethanol under reflux; steaming the extract, and centrifuging to remove precipitate; sequentially extracting petroleum ether, ethyl acetate and n-butyl alcohol, wherein the lower layer liquid is reserved in the petroleum ether and ethyl acetate extraction step, the n-butyl alcohol is added finally, the mixture is layered after standing, and the upper layer extracting solution is reserved; and (4) carrying out rotary evaporation and centrifugation on the obtained upper-layer extracting solution to remove precipitates to obtain the n-butanol phase extract.

3. The use of the sunflower disc extract of claim 1 in the preparation of a vasodilator.

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