CN1176674C - A kind of rehmannia glutinosa extract and its preparation method - Google Patents

Abstract

The invention belongs to the field of traditional Chinese medicine preparations, and in particular relates to an extract of Chinese medicine rehmannia glutinosa and its preparation method and application. The invention prepares the rehmannia glutinosa extract through water extraction, alcohol refining and other methods, and mixes it with various auxiliary materials to make health food and traditional Chinese medicine preparation with clear action mechanism. The invention can be used to prevent and treat obesity and diseases related to obesity such as hyperlipidemia, diabetes, atherosclerosis and other diseases.

Description

A kind of Radix Rehmanniae extract and preparation method thereof

The invention belongs to field of traditional Chinese, be specifically related to Chinese medicine Radix Rehmanniae extract and its production and application.

Obesity is to surpass consumption because energy matter is taken in, and body fat gathers the disease that too much causes.Statistics shows that the fat total number of persons of the U.S. reaches 9,700 ten thousand, in population more than 20 years old, 30% is fat, and China people are along with growth in the living standard, and fat incidence rate also raises year by year, fat number reaches 7,000 ten thousand, and the teenager obesity is influencing nation's quality, has become epidemic diseases.Fat quality of life of not only reducing the patient, and it is closely related with multiple diseases such as hypertension, diabetes, coronary heart disease, hyperlipidemia, sexual dysfunction and even tumors, so, the development of obesity health food or medicine, not only can reduce fat number, improve the people's life, work quality, the relevant disease of obesity also can be prevented and treated and be treated to life-saving.At present, external appetrol act on local link, and more side reaction is arranged, and bounce-back is serious after the drug withdrawal.

The objective of the invention is with the Chinese medicine Radix Rehmanniae is raw material, extracts active component, makes the fat health food of control, and another purpose is that active ingredient is equipped with Chinese medicine preparation and the application in preventing and treating obesity and obesity medicine thereof that adjuvant is made clear and definite mechanism of action.

Radix Rehmanniae Rehmannia glutinosa Libosch is a conventional Chinese medicine, and the beginning is stated from the Sheng Nong's herbal classic, and the row position is top grade, mainly contains iridoids, the function clearing away heat and cooling blood, YIN nourishing and the production of body fluid promoting is used for the calentura crimson tongue, excessive thirst, interior-heat caused by deficiency of YIN, the hectic fever due to YIN-deficiency consumptive fever is spitted blood, and sends out speckle dermexanthesis etc.

The present invention extracts active component by the following method: get Radix Rehmanniae be soaked in water spend the night after, putting 80 ℃ handled 4 hours, contain 1 gram Radix Rehmanniae in the every milliliters of liquid of filtrate simmer down to and promptly get extract Radix Rehmanniae stock solution, the high performance liquid chromatogram test, fine with 2% second is mobile phase, the 210nm scanning wavelength, recording in retention time is the absworption peak that Radix Rehmanniae main component catalpol is arranged about t=9.1min; Radix Rehmanniae stock solution color and luster is brown, news giving off a strong fragrance, mildly bitter flavor, pH meta-acid arranged.Radix Rehmanniae extract of the present invention is equipped with various adjuvants and can be made into capsule, tablet, granule, pill, powder, decoction and compound preparation as active component.

The present invention confirms that through cell in vitro and animal experiment Radix Rehmanniae stock solution can suppress the adipose cell differentiation, suppress the formation of animal body fat tissue, reduce the concentration of plasma cholesterol, triglyceride and blood glucose, and the concentration of increase plasma high density lipoprotein level, as active component, be equipped with various adjuvants, can be made into fat health food of control and the fat Chinese medicine preparation of control.

Explanation by the following examples.

Embodiment 1: Radix Rehmanniae stock solution suppresses the differentiation of adipose cell

The present invention tests with 3T3-L1 mice pre-adipose cell lines and draws the medical college from U.S. John HopKinsUniversity, foundation is induced to differentiate into the model of mature fat cell: cultivate the 3T3-L1 cell and become monolayer in containing the DMEM training liquid of 10% calf serum, add MIX (methyl-isobutyl xanthine) 0.52mM, insulin 1.7uM, dexamethasone 1uM stimulation cultivation 2 days then, change differentiation liquid with an insulin-containing 1.7uM, continue to cultivate, changed differentiation liquid later on every 2 days.Adipose cell changes circle into and greatly, has been full of the adipose cell that fat drips before making fusiformis.

In the test, in irritation cell (Oh) but add the differentiation of Radix Rehmanniae stock solution strong inhibition adipose cell, 12h after stimulation, 24h adds medicine and still has the obvious suppression effect, the Radix Rehmanniae stock solution that adds variable concentrations (0.01%--4%) at Oh, suppressing the effect of differentiation strengthens with the rising of concentration

In order to determine whether that the toxicity owing to medicine suppresses the differentiation of adipose cell, the present invention has carried out the drug toxicity test:

As seen in valid density (0.01%--4%) scope, adipose cell before the 3T3-L1 does not add the differentiation stimulant, only adds Radix Rehmanniae stock solution from the morphology, and cell growth division is vigorous, does not have difference with adipose cell before the 3T3-L1 that does not add Radix Rehmanniae stock solution;

Detect the vigor of cell with mtt assay: the MTT solution of preparation 5mg/ml, black out is preserved; The 3T3-L1 cell reaches in 96 orifice plates, after cell culture is adherent, add and contain 0.25%, effect is 2 days in the Radix Rehmanniae stock solution training liquid of 1%, 4% three kind of concentration, adds MTT solution then, act on 4 hours, remove training liquid, add 200ulDMSO and 25ulSorensen ' sGlysine buffer, analyze its absorptance in the 570nm wavelength period.The result is presented in valid density (0.01%--4%) scope, and medicine does not influence the vigor of cell, is of value to the growth of cell on the contrary.

Inducing differentiation after 3 days, get the normal differentiation cell of contrast and the cell of handling through Radix Rehmanniae stock solution, with the guanidine isothiocyanate method extracting RNA, with DIG labelling C/EBP α and 422/aP2 gene probe (test kit is available from Bao Ling Man), make the nucleic acid dot blot hybridization with cell total rna, (C/EBP α is a transcription regulatory factor important in the adipose cell atomization, can activate the expression of the serial specific proteins that mature fat cell has, as 422/aP2 is the special fatty acid binding protein of adipose cell, adipose cell obtained the ability of synthetic glycerine three esters before the expression of these characteristic proteins made, and formation fat drips the big adipose cell of accumulative circle.) result's demonstration, the cell after Radix Rehmanniae stock solution is handled, C/EBP α gene transcription level reduces by 86.1% than control cells, and 422/aP2 gene transcription level reduces by 37% than control cells.Experiment repeats more than 3 times, and the result is constant.

Inducing differentiation after 3 days, getting the normal differentiation cell of contrast and the noble cells of handling through Radix Rehmanniae stock solution, with the guanidine isothiocyanate method extracting RNA, adopting the RT-PCR method, is internal reference with β-actin, analyzes the influence of medicine to leptin mRNA expression.(leptin is the expression product of ob gene, and expression is the highest in adipose cell, has physiological effect widely such as the energy balance of adjusting, appetite, immunity and reproduction, blood plasma leptin concentration increases, by hypothalamus, appetite is descended, therefore very big dependency is arranged with obesity.Design leptin primer is P1 5 '-ACGCAGTCGGTATCCGCAA-3 ' P2 5 '-GCTCTATGCCAACACAGTGC-3 '; Design β-actin primer is P1 5 '-CTCTATGCCAACACAGTGC-3 ' P2 5 '-GTACTCCTGCTTGCTGATCC-3 '.By total RNA for after the template reverse transcription obtains cDNA, press following scheme pcr amplification: add the Taq enzyme after 94 ℃ of degeneration, through 94 ℃ 30 "; 60 ℃ 1 '; 68 ℃ of 1 ' 30 circulation, 68 ℃ are extended 7 ', finish back 1.2% agarose gel electrophoresis; to Zone electophoresis band gray scale scanning, the result shows that Radix Rehmanniae stock solution can promote that ob gene leptin expresses in the adipose cell with Pharmacia nucleic acid-protein automated imaging system.

In the adipose cell atomization, after Radix Rehmanniae stock solution is handled, adopt the variation of the relevant expressing quantity of Western Blot technology for detection adipose cell differentiation.With 60mM Tris-HCl, 1%SDS solution extracts whole-cell component; After stimulating Oh to add 1% Radix Rehmanniae stock solution, after 1 day, extract whole-cell protein, detect the proteic expression of C/EBP β according to the cultivation of standard differentiation scheme; Cultivated 3 days, 5 days, and detected the expression of C/EBP α; Cultivate and detected 422/aP2, the proteic expression of Glut4 in 5 days.(C/EBP β is the early protein of adipose cell differentiation, may have the C/EBP of promotion alpha expression, and then promotes the effect of differentiation; The characteristic protein that Glut4 also expresses for the adipose cell later stage, relevant with glucose transport.) result shows that these proteic expression all are subjected to inhibition in various degree.

Embodiment 2, and Radix Rehmanniae stock solution prevents the rat diet obesity.

Adopting weight is 12/batches of SD male rats about 200 grams, (buying from laboratory animal portion of Shanghai Medical Univ), the following preparation of its diet program, illumination, two groups of unanimities of environmental factorss such as circadian rhythm.

Sweet whole milk powder 44%

Standard Rodents feedstuff 47%

Semen Maydis oil 8%

Corn starch 1%

Radix Rehmanniae active component 1%

After cultivating for 10 weeks, heavily about 500 grams of control rats apparently higher than the rat of normal diet, illustrate the fat animal model manufacturing success of diet induced.

The SD male rat is divided into 2 groups at random, 12 every group: do not add Radix Rehmanniae stock solution in the control rats high fat diet, Radix Rehmanniae stock solution is closed in the high fat diet of administration group rat.Observe and calculating rat food ration every day, the appetite of administration group and matched group is similar, no statistics difference.

Weigh weekly animal and record obtain the Zhou Shengchang curve of animal, and the result shows that the weight ratio control rats of administration group male rat reduces by 17.6%, P＜0.05.

	Matched group (g)	Administration group (g)
			First week	261.67±27.14	283.33±24.22
Second week	221.67±28.05	235.00±27.02
			The 3rd week	239.33±33.93	229.67±21.81
Around	262.33±28.99	245.67±16.80
			The 5th week	297.00±29.44	269.67±21.96
The 6th week	315.67±38.81	292.33±27.29
			The 7th week	356.67±32.04	309.00±27.76
The 8th week	370.00±38.34	315.67±30.16
			The 9th week	411.00±37.67	338.33±37.28
The tenth week	430.83±42.00	355.00±40.77

Behind the sacrifice of animal, observe intraabdominal adipose tissue, find that administration group rat omentum majus and epididymal adipose all obviously are less than control rats.

Fatty tissue weight in body weight of rat and the rat abdominal cavity, administration group rat significantly is lower than control rats, has statistical significance (p＜0.05).

Radix Rehmanniae stock solution is to the influence of the body weight of rat, Body Mass Index, abdominal cavity fat weight (x ± s)
					Group	The Mus number	Body weight (g)	Body Mass Index	Intraabdominal adipose tissue weight (g)
The administration group	12	349.6±43.11	0.60±0.03	14.55±5.43
					Matched group	12	443.0±33.09	0.70±0.08	18.85±3.18

Get same position and be close to fatty tissue on the epididymis, the form of direct observation rat body fat cell after HE dyeing, the result shows that the adipose cell of administration group rat belongs to little adipose cell (to insulin sensitivity), and control rats is to contain the big adipose cell (to insulin insensitivity) that a large amount of fat drip.

Two groups of serum lipid concentrations have obvious difference: administration group plasma triglyceride, and cholesterol, blood sugar concentration is lower than matched group, and the above two have the statistics difference, and high density lipoprotein is then than matched group height.

	Matched group (n=12)	Administration group (n=12)
			TC	54.3±12..5	44.0±12.0
TG	70.6±41.2	62.4±35.8
			G	143.0±142.8	115.0±60.7
HDL	32.7±5.8	45.2±9.7

TC: T-CHOL, TG: total triglyceride, G: blood glucose, HDL: the variation of leptin protein expression during high density lipoprotein is organized with the detection of original position immunohistochemistry technique, the leptin positive signal is better than matched group in the administration group rat fat tissue.Detect the mRNA expression that the animal body fat is organized leptin with RT-PCR, show the rising of the expression of administration group rat than control rats.With the protein expression level of C/EBP α, 422/aP2, leptin in the Western Blot technology for detection animal adipose tissue, the result shows that the expression of administration group rat C/EBP α, 422/aP2 is lower than matched group, and the expression of leptin is higher than control rats.

Above embodiment explanatorily xanthan liquid has the differentiation of obvious suppression adipose cell, promote leptin to express and the reduction plasma triglyceride, cholesterol and blood glucose, and rising plasma high density lipoprotein level have the effectiveness of the obesity of preventing and prevent and treat atherosclerotic effectiveness.

Embodiment 3: the present invention extracts active ingredient by following distinct methods from Radix Rehmanniae.

Method 1 is got after Radix Rehmanniae shreds, and water is got the decoction effective component extracting after decocting, and temperature is from room temperature to 100 ℃

Method 2 is got after Radix Rehmanniae shreds, and directly uses Diluted Alcohol (methanol) solution to extract

Method 3, extract Radix Rehmanniae stock solution as stated above after, add ethanol (or isopropyl alcohol/methanol) to more than 80%, get its precipitation part.

Description of drawings:

Fig. 1 is the process that adipose cell is divided into adipose cell before the 3T3-L1 mice, and demonstration was fusiformis originally, tall and thin preceding adipose cell changes circle into and greatly, has been full of the big adipose cell of maturation that fat drips.Wherein 1 is differentiation 1 day; 2 are differentiation 2 days; 3 are differentiation 5 days; 4 are 11 days cellular morphology of differentiation.

Fig. 2 is differential stimulus after 5 days for the Radix Rehmanniae stock solution that adds variable concentrations suppresses the situation that adipose cell breaks up, and 1 is matched group, and the sophisticated big adipose cell of volume is arranged; 2 are the cell with the processing of low concentration (0.25%) Radix Rehmanniae stock solution; 3 are the cell with the processing of high concentration (1%) Radix Rehmanniae stock solution, raise with the waste stock solution concentration in the end, and the inhibition effect of adipose cell differentiation is strengthened.

Fig. 3 is the influence of Radix Rehmanniae stock solution to preceding adipose cell growth, and 1 is the Radix Rehmanniae stock solution of adding 1% in the pro-adipose cell culture fluid; 2 is preceding adipose cell contrast, does not have the growth and the division of the preceding adipose cell of influence after the adding Radix Rehmanniae stock solution, and promptly Radix Rehmanniae stock solution does not have direct infringement to the governor of preceding adipose cell, only acts on differentiation.

Fig. 4 when detecting differentiation 3 days and 5 days with the method for dot blot in the adipose cell expression of C/EBP α mRNA and Radix Rehmanniae stock solution to the influence of its expression.A:1,4 is control cells mRNA, 2,3 is the cell mRNA that 1% Radix Rehmanniae stock solution is handled.B: the gray scale scanning value with the hybridization point is vertical coordinate comparative control group and drug treating group cell mRNA, and this figure shows that the expression of C/EBP α mRNA significantly reduces in the adipose cell atomization after Radix Rehmanniae stock solution is handled.

Fig. 5 method of RT-PCR, detect the situation that leptin mRNA expresses in the adipose cell, each group is differentiation 5 days, and A is electrophoretic result, and 1 is the standard molecular weight contrast, 2 are 5 days cellular control unit of differentiation, 3 is to handle through 0.125% Radix Rehmanniae stock solution, and 4 is to handle through 0.25% Radix Rehmanniae stock solution, and 5 is to handle through 1% concentration Radix Rehmanniae stock solution, 6 is β-actin internal reference, and 7 is leptin DNA positive control.B is the influence of the direct comparatively xanthan liquid of vertical coordinate to the adipose cell differentiation with the gray scale scanning ratio of leptin/ β-actin mRNA, shows the rising along with drug level, has to strengthen the effect that leptin mRNA expresses in the adipose cell.

Fig. 6 detects Radix Rehmanniae stock solution to breaking up the influence of C/EBP β protein expression in 1 day the adipose cell with the method for Western, 38KD and 18KD are respectively the proteic two kinds of isomer proteins of C/EBP β, this figure was presented at differentiation in the time of 1 day, and the proteic expression of Radix Rehmanniae stock solution pair cell C/EBP β has the obvious suppression effect.

Fig. 7 detects Radix Rehmanniae stock solution to breaking up the influence of 422/aP2 protein expression in 5 days the adipose cell with the method for Western, A:1,6 usefulness, 4% Radix Rehmanniae stock solution is handled, 2,5 usefulness, 1% Radix Rehmanniae stock solution is handled, and 3,4 is cellular control unit, B: the gray scale scanning value with protein band is the relatively proteic shadow s of variable concentrations Radix Rehmanniae stock solution processing pair cell expression 422/aP2 phase cv of vertical coordinate, shows the rising along with drug level, and its effect that suppresses the 422/aP2 protein expression strengthens.

All body weight change curves of Fig. 8 rat, abscissa is represented all numbers, and vertical coordinate is the body weight of rat, and the rhombus icon is represented the rat of matched group, and square icon is represented the rat of administration group, shows that the body weight of administration group rat is starkly lower than the body weight of control rats.

After Fig. 9 rat fed for 10 weeks, the situation that rat abdominal cavity omentum majus and epididymis fatty tissue distribute, a left side is the rat of matched group, and right is administration group rat, and adipose tissue mass all obviously is less than matched group on the rat omentum majus of demonstration administration group and the epididymis.

Figure 10 Radix Rehmanniae stock solution shows that to the influence of rat fat plasma cholesterol, triglyceride, the blood sugar level of administration group rat obviously reduces, and hdl level then raises.

Figure 11 RT-PCR method detects leptin mRNA expression in the rat fat, and A:1 is the standard molecular weight contrast, and 2,3 is the control rats fatty tissue, 4,5,6,7 is the fatty tissue through administration group rat, and 8 is β-actin internal reference, and 9 is the leptin positive control.B: with the gray level ratio of leptin/ β-actin be vertical coordinate directly comparatively xanthan liquid to the influence of leptin mRNA level in the adipose cell atomization, after the administration in the rat fat tissue leptin mRNA express and increase.

Claims (2)

1. A rehmannia extract, characterized in that the rehmannia extract is prepared according to the following method: soak the raw rehmannia in water overnight, and then treat it at 80°C for 4 hours, or cut the raw rehmannia into pieces and extract it with dilute ethanol or dilute methanol under reflux The active ingredient is obtained by filtering the extract and concentrating the filtrate so that 1 gram of rehmannia root is contained in each milliliter of liquid to obtain the extract rehmannia root stock solution. 2. The rehmannia glutinosa extract according to claim 1, further comprising adding ethanol or isopropanol or methanol to the rehmannia glutinosa extract stock solution obtained by filtering and concentrating the extract, and controlling the alcohol concentration to more than 80%, whichever is used for alcohol precipitation part.

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