CN1569017A - Total epimeddium flavone tablet and its preparation method - Google Patents

Abstract

The invention discloses a total barrenwort flavones tablet, wherein the tablet comprises total barrenwort flavones and medicinal auxiliary material, which has actions of reducing side effect of hormones application and undesired reaction, and delaying immunity ageing. The invention also provides the preparing process.

Description

Herba Epimedii total flavones tablet and preparation method

Technical field:

The invention belongs to the Chinese medicine preparation technical field.Be specifically related to a kind of Herba Epimedii total flavones sheet and preparation method.

Background technology:

The complication and the side effect of using hormone and causing can be divided three classes: the first kind is the outward manifestation that causes hypercortisolism owing to hormonosis, as obesity, moon face, buffalo hump, hirsutism, purple striae, excitement be insomnia, easy hungry polyphagia etc.; Second class is that hormonosis causes metabolism disorder, as osteoporosis, hyperglycemia, hyperlipidemia, hypertension, peptic ulcer in addition hemorrhage, suppress immunologic function and infect easily; The 3rd class is to cause adrenal cortex to be suppressed after the hormone prolonged application and be disuse atrophy, this type of patient often is dependent status to hormone and is difficult for removing, and run into big stress, as operation or severe infections, the insufficient crisis of acute adrenal cortex function can take place at any time and life-threatening.Therefore the side effect and the complication that how to alleviate hormone are clinical difficult problems that presses for solution.On the chemical constitution that changes hormone, many work have been done abroad, be intended to improve antiinflammatory, anti-allergic effects and reduce the side effect of salt retention, but this mainly acts on still to fail to accomplish neither to influence its antiinflammatory, can correct its side effect and complication again more comprehensively, and himself adrenal gland is not suppressed, and help hormone and remove, domestic like this too.

In " kidney essence " research, it is low to observe insufficiency of kidney-YANG patient hypophysis-adrenal cortex s function the beginning of the sixties, uses the warming and recuperating the kidney-YANG medicine and can make it to improve.Zoopery proof warming and recuperating the kidney-YANG medical instrument has similar hormone-like effect, and can protect the adrenal gland to avoid exogenous hormones and suppress and avoid atrophy.

The inventor can help hormone to successively decrease smoothly the obstinate asthma patient of 10 examples long-term (average 46 months) dependence hormone with the warming and recuperating the kidney-YANG medicine, make 7 examples remove hormone and succeed, and hypophysis-adrenal cortex function also recovers normal.Studies show that of kidney essence, syndrome of deficiency of kidney-YANG patient and old people have the neuroendocrine immunity (Neuroendocrine and immune, NEI) network function are low, and the warming and recuperating the kidney-YANG compound recipe can improve lowered NEI network function.At the kidney warming compound recipe of intending certainly in order to delay or to improve the NEI network function, as warming YANG sheet, tonifying kidney and prolonging life sheet, longevity and health sheet and from intending that Herba Epimedii is all arranged in the Yougui Yin, the kidney-Yang-Reinkforcing Decotion, these four the kidney warming compound recipes all show and can obviously improve its NEI network function in syndrome of deficiency of kidney-YANG and aged person's clinical and zoopery.

Summary of the invention:

Technical problem to be solved by this invention is the warming and recuperating the kidney-YANG preparation of development and Design single Herba Epimedii.

The invention provides a kind of Herba Epimedii total flavones sheet, this tablet comprises what Herba Epimedii total flavones extract and pharmaceutic adjuvant were formed, and wherein the content of Herba Epimedii total flavones extract is 60%～65%.

The drug efficacy study of the tablet of Herba Epimedii total flavones of the present invention (Herba Epimedii) is as follows:

One, the comparison of single Herba Epimedii (Herba Epimedii) and warming and recuperating the kidney-YANG compound recipe

On dexamethasone Mus (simulation insufficiency of kidney-YANG) animal model, observe the influence of the kidney invigorating compound recipe and single Herba Epimedii to the external secretory function of adrenal cortical cell.As a result in the adrenal cortical cell culture fluid of the kidney invigorating compound recipe group and Herba Epimedii group corticosterone content under Cortrosyn (synthetic ACTH) stimulates all between Dexamethasone group and matched group.See Table 1:

Table 1. kidney-reinforcing drug is subjected to press down the influence that the external Cortrosyn of rat adrenal cortical cell stimulates to the adrenal cortex axle

Culture fluid corticosterone concentration (nmol/L, x ± SD)

After stimulating before the group number of animals stimulates

Contrast 12 259.91 ± 21.52 381.64 ± 121.84 ^{△ △}

Dexamethasone 10 7.09 ± 6.81* 91.89 ± 105.38

Hormone compound recipe 10 17.02 ± 10.72* 182.04 ± 134.60 ^△

Hormone Herba Epimedii 10 4.44 ± 6.46* 231.63 ± 245.02 ^△

Annotate: compare before stimulating with matched group ^*P＜0.01; Stimulate the back relatively with Dexamethasone group ^△P＜0.05; Stimulate the back relatively with Dexamethasone group ^{△ △}P＜0.01

Two, the clinical and zoopery of single Herba Epimedii

Selected to need to use patient's 65 examples of hormone in 1998, the sick kind has nephrotic syndrome, lupus erythematosus, thrombocytopenic purpura, myasthenia gravis.Be divided into compound recipe prednisone group (prednisone adds Herba Epimedii) and prednisone group at random and compare, the hypophysis-adrenal cortex of compound recipe prednisone group and immunologic function are all than prednisone group obviously rise (P＜0.05) as a result.See Table 2:

Blood plasma cortisol, ACTH and the comparison of pouring commentaries on classics measured value before and after table 2.3 group is observed (x ± s)

Group example number hydrocortisone (ug/L) ACTH (pg/ml) drenches changes (cpm)

Contrast 15 observes preceding 120.4 ± 50.2 32.9 ± 8.2 120335 ± 12231

Observe back 110.6 ± 50.0 33.0 ± 7.5 124673 ± 10981

Prednisone 25 medications preceding 110.8 ± 40.9 30.5 ± 7.0 119082 ± 10965

50.2 ± 20.1*, 12.9 ± 6.2*, 52409 ± 4587* after the medication

Compound recipe 40 medications preceding 110.5 ± 40.6 31.2 ± 7.4 115896 ± 11245

After the prednisone medication 80.1 ± 20.8 ^△20.2 ± 6.9 ^△98336 ± 5495 ^△

Annotate: with matched group comparison same period * P＜0.05; With comparison after the medication of prednisone group ^△The corresponding zoopery in P＜0.05 also obtains identical result.See Table 3, table 4:

Table 3.3 group rat pituitary-adrenal gland, thymic weight be (mg, x ± s) relatively

Group example number hypophysis adrenal gland thymus

Contrast 10 8.55 ± 1.10 24.02 ± 3.84 393.61 ± 65.01

Model 10 6.90 ± 0.98* 11.06 ± 2.04** 204.52 ± 38.49**

Herba Epimedii 10 7.96 ± 1.17 ^△20.81 ± 5.35 ^{△ △}250.32 ± 35.94 ^{△ △}

Annotate: compare * P＜0.05, * * P＜0.01 with matched group; Compare with the corticosterone group ^△P＜0.05, ^{△ △}P＜0.01

Table 4. Herba Epimedii suppresses the influence of rat model cellular immunization and cytokine and antiinflammatory action to the HPAT axle

(x±s)

LPR NKCC IL-2 γ-IFN granuloma induced by implantation of cotton pellets weight

(%) (u/ml) (mg) for group Mus number (cpm)

Contrast 10 119716 ± 24,856 61.8 ± 8.2 81.20 ± 8.90 85.2 ± 6.1 92.5 ± 6.8

Model 10 65187 ± 22802* 15.6 ± 7.8* 37.47 ± 5.34* 40.5 ± 5.2* 68.7 ± 4.5*

Herba Epimedii 10 101290 ± 29580 ^△48.5 ± 9.2 ^△62.92 ± 12.89 ^△63.3 ± 5.8 ^△70.1 ± 5.8 ^△

Annotate: compare * P＜0.05 with matched group; Compare with the corticosterone group ^△P＜0.05

Illustrate that the single Herba Epimedii can resist the inhibitory action of exogenous hormones to hypophysis-adrenal gland-thymus axle effectively.

Three, the corticosterone rat model of simulation " insufficiency of kidney-YANG " is adopted in another experiment, and with Herba Epimedii and extractive total flavone (EF) and polysaccharide (EPS), why not observe it respectively has same-action to neuroendocrine and immune system.See Table 5, table 6:

Table 5. Herba Epimedii and extract thereof are to the influence of rat model plasma CO RT, ACTH (x ± s)

Group example number CORT (μ g/ml) ACTH (pg/ml)

Normal 10 10.10 ± 4.47 99.33 ± 12.92

Model 10 4.08 ± 2.15** 59.50 ± 35.91*

Herba Epimedii 10 5.97 ± 1.88 ^△79.80 ± 24.46 ^△

EF 10 7.59±1.88 ^△△ 113.0±32.43 ^△△

EPS 10 4.28±1.67 69.42±24.57

Annotate: compare * P＜0.05, * * P＜0.01 with normal group; Compare with model group ^△P＜0.05, ^{△ △}P＜0.005

Table 6 Herba Epimedii and extract thereof are to the influence of rat model lymphproliferation response, NKC activity and γ-IFN level (x ± s)

Group example number drenches and changes (cpm) NKC activity (%) γ-IFN (U/ml)

Contrast 10 119716 ± 24,856 61.80 ± 8.20 85.20 ± 6.10

Model 10 72854 ± 28485* 15.60 ± 7.80** 41.69 ± 8.69**

Herba Epimedii 10 104307 ± 29695 ^△46.60 ± 8.30 ^{△ △}59.93 ± 11.78 ^{△ △}

EF 10 60401±22884 15.25±8.40 54.68±15.25 ^△

EPS 10 126211±40657 ^△ 48.62±10.20 ^△△ 51.83±8.64 ^△

Annotate: compare * P＜0.05, * * P＜0.001 with normal group; Compare with model group ^△P＜0.05, ^{△ △}P＜0.005

The result shows that HPA (HPA) axle and the cellular immune function of model group are subjected to obvious inhibition simultaneously.The Herba Epimedii water decoction all has clear improvement to hpa axis and cellular immunization, and EF has clear improvement to hpa axis, and the pair cell immunity also has improvement effect to a certain degree; And EPS only the pair cell immunity improvement effect is arranged, point out the contained different active components of Herba Epimedii to have the regulating action that NEI system difference is stressed thus.

Four, Herba Epimedii total flavones and the kidney invigorating compound recipe are to the comparative study of corticosterone rat t cell proliferation related gene group regulation and control:

The Herba Epimedii that this research is adopted is that Herba Epimedii (Epimedium KereanumNakai) is produced in Liaoning, the EF. the kidney invigorating compound recipe that extracts is selected Yougui Yin, the kidney-Yang-Reinkforcing Decotion and BUSHEN YISHOU JIAONANG for use, and Yougui Yin, the kidney-Yang-Reinkforcing Decotion consists of Radix Aconiti Lateralis Preparata, Cortex Cinnamomi, Radix Rehmanniae Preparata, Fructus Corni, Rhizoma Dioscoreae, Fructus Lycii, Herba Epimedii, Radix Glycyrrhizae Preparata; BUSHEN YISHOU JIAONANG is formed (group provides by Sichuan Tai Ji) by baked polygonum capitatum, Rhizoma Polygonati, Radix Ginseng, Fructus Lycii, Herba Epimedii, Radix Ginseng, Ganoderma etc., and making concentration is the 1.2g/100ml working solution; The spleen invigorating compound recipe adopts decoction of four noble drugs.60 of 4 monthly age male SD rats are divided into 6 groups at random and experimentize.The modeling medicine is a corticosterone, the results are shown in Table 7-9:

Table 7. is respectively organized rat t cell proliferation percentage rate (x ± s)

Group example number t cell proliferation percentage rate

％

Normal group 10 34.25 ± 6.47

Model group 10 45.87 ± 7.22*

Decoction of four noble drugs group 10 36.92 ± 11.82

Yougui Yin, the kidney-Yang-Reinkforcing Decotion group 10 35.90 ± 7.39 ^{△ △}

Tonifying kidney and prolonging life group 10 36.20 ± 9.14 ^△

EF organizes 10 36.81 ± 10.33 ^△

* compare P＜0.01 with normal group; ^△Compare P＜0.05 with model group; ^{△ △}Compare P＜0.01 with model group

Table 8. is respectively organized rat t cell proliferation related gene expression (x ± s)

Group example number Fas/GAPDH FasL/GAPDH TNFR1/GAPDH TNFR2/GAPD Bcl-2/GAPDH Bax/GAPDH

Normal group 8 1.572 ± 0.063 1.553 ± 0.077 1.725 ± 0.127 1.328 ± 0.082 1.294 ± 0.134 1.516 ± 0.193

Model group 8 1.422 ± 0.077 ^*1.413 ± 0.050 ^*1.390 ± 0.163 ^*1.525 ± 0.232 ^*1.573 ± 0.223 ^*1.332 ± 0.120 ^*

Decoction of four noble drugs group 8 1.427 ± 0.062 1.464 ± 0.060 1.519 ± 0.055 1.356 ± 0.087 1.367 ± 0.028 ^△1.362 ± 0.125

Yougui organizes 8 1.486 ± 0.080 1.525 ± 0.067 ^{△ △}1.603 ± 0.046 ^{△ △}1.375 ± 0.131 1.350 ± 0.163 ^△1.489 ± 0.177

Tonifying kidney and prolonging life group 8 1.454 ± 0.048 1.512 ± 0.078 ^{△ △}1.541 ± 0.070 ^△1.348 ± 0.050 1.376 ± 0.046 ^△1.369 ± 0.138

EF organizes 8 1.430 ± 0.080 1.506 ± 0.089 ^△1.543 ± 0.054 ^△1.384 ± 0.043 1.369 ± 0.057 ^△1.374 ± 0.095

* compared significant difference (P＜0.05) with normal group; * has compared utmost point significant difference (P＜0.01) with normal group

^△Compared significant difference (P＜0.05) with model group; ^{△ △}Compare the utmost point with model group significant difference (P＜0.01) is arranged

Table 9. is respectively organized (x ± s) of rat caspase8, caspase3 specific activity

Group example number caspase8 caspase3

Normal group 8 0.125 ± 0.030 0.269 ± 0.094

Model group 8 0.190 ± 0.066* 0.491 ± 0.157**

Decoction of four noble drugs group 8 0.158 ± 0.032 0.366 ± 0.079

Yougui organizes 8 0.132 ± 0.031 ^△0.299 ± 0.126 ^△

Tonifying kidney and prolonging life group 8 0.134 ± 0.025 ^△0.354 ± 0.027 ^△

EF organizes 8 0.134 ± 0.023 ^△0.326 ± 0.131 ^△

* compared significant difference (P＜0.05) with normal group; * has compared utmost point significant difference (P＜0.01) with normal group

^△Compared significant difference (P＜0.05) with model group.

In view of two the kidney invigorating compound recipes (Yougui Yin, the kidney-Yang-Reinkforcing Decotion and BUSHEN YISHOU JIAONANG) only Herba Epimedii, establish EF and two the kidney invigorating compound recipes and spleen invigorating compound recipe and compare so this research is model with the corticosterone rat for total.The result shows that EF and two the kidney invigorating compound recipes all have obvious downward modulation effect for corticosterone rat T cell transition apoptosis, and the mRNA expression of urging apoptosis FasL, TNFR1 gene in downward modulation, the mRNA that raises anti-apoptosis Bcl-2 gene expresses, and the active aspect of reduction apoptosis startup enzyme Caspase8, execution enzyme Caspase3 all has identical regulative mode.See that thus Herba Epimedii total flavones is in t cell proliferation and gene regulation process thereof; can represent the kidney invigorating compound recipe neuroprotective endocrine-immune system antagonism exogenous hormones to the T cell inhibiting, the untoward reaction of removing hormone or alleviating hormone is had its good prospects for application.

5, the protective effect that suppressed model of Herba Epimedii total flavones HPAT axle that exogenous glucocorticoid is caused is studied:

Material and method

Medication preparation: Herba Epimedii total flavones is extracted by Shanghai City medical industry academy, and content is 60%.

Animal grouping, modeling: 55 of male SD rats, at 4 months monthly ages, body weight 230-250g is provided by zoopery portion of Fudan University.The modeling medicine is that (Sigma USA), is dissolved in during use in the sterilization olive oil corticosterone, and working concentration is 20mg/ml.Rat is divided into 4 groups at random, every group each 11, each organizes the rat body weight no difference of science of statistics, be respectively normal control group, corticosterone model group, Herba Epimedii total flavones treatment group (EF group) and the kidney invigorating compound treatment group, model group and each treatment group are all according to 10mg/kg subcutaneous injection corticosterone, the normal control group replaces corticosterone with equal-volume sterilization olive oil, every day 1 time, continuous 14 days, each treatment group is irritated stomach according to body weight with corresponding medicine respectively, normal group and model group replace medicine to irritate stomach, every day 1 time, continuous 14 days with distilled water.The 15th day, behind all rat etherizations, weigh respectively, heart blood sampling, aseptic separating spleen, peel off thymus, adrenal gland.Measure level of plasma cortisol, thymus index, adrenal gland's index, lymhocyte transformation rate.

The result

1. respectively organize rat body weight relatively: the results are shown in Table 1.Experiment shows that model group compares with normal group, and body weight significantly alleviates, P＜0.01; Herba Epimedii total flavones treatment group body weight and model group relatively increase, P＜0.01, and the kidney invigorating compound recipe group body weight does not have significant change.Show that Herba Epimedii total flavones can improve rat body weight, offset the consumption effect of hormone each tissue such as whole skin, connective tissue, muscle.

Table 10 is respectively organized rat body weight (x ± SD)

Group example number body weight

Normal 11 308.36 ± 11.93 ^*

Model 11 266.55 ± 14.94

EF organizes 11 297.09 ± 9.31 ^*

Compound recipe group 11 278.91 ± 15.06

^*Compare P＜0.01 with model group

2. respectively organize the exponential comparison of rat chest gland: the results are shown in Table 2.Experiment shows that model group compares with normal group, and thymic weight significantly alleviates, P＜0.01; Herba Epimedii total flavones treatment group, the kidney invigorating compound recipe group are compared with model group all has utmost point significant difference, P＜0.01.Show that Herba Epimedii total flavones and the kidney invigorating compound recipe all can improve rat chest gland weight, offset the inhibition of hormone the central lymphoid organ.

Table 11 is respectively organized rat chest gland index (x ± SD)

Group example number thymus index

Normal 11 151.01 ± 26.33 ^*

Model 11 91.52 ± 28.23

EF organizes 11 146.60 ± 23.49 ^*

Compound recipe group 11 128.40 ± 21.572 ^*

^*Compare P＜0.01 with model group

3. respectively organize rat adrenal gland index relatively: the results are shown in Table 3.Experiment shows that model group compares with normal group, and adrenal gland's weight significantly alleviates, and P＜0.01 shows that hormone is inhibited to the adrenal gland; Herba Epimedii total flavones and the kidney invigorating compound recipe and model group relatively have utmost point significant difference, and P＜0.01 shows that Herba Epimedii total flavones and the kidney invigorating compound recipe all can improve rat adrenal gland weight, suppress adrenal effect to hormone and have antagonistic effect.

Table 12 is respectively organized rat adrenal gland index (x ± SD)

Group example number adrenal gland index

Normal 11 8.22 ± 2.99 ^*

Model 11 3.76 ± 0.21

EF organizes 11 7.94 ± 2.15 ^*

Compound recipe group 11 9.16 ± 1.98 ^*

^*Compare P＜0.01 with model group

4. respectively organize the comparison of rat lymphocyte conversion ratio (LPR): the results are shown in Table 4.Experiment shows that model group compares with normal group, and lymhocyte transformation rate significantly reduces, and P＜0.05 shows that hormone is inhibited to periphery lymphocyte; Herba Epimedii total flavones and the kidney invigorating compound recipe all can improve LPR, compare Herba Epimedii total flavones group P＜0.01 with model group, and the kidney invigorating compound recipe group P＜0.05 shows that Herba Epimedii total flavones has the effect that the external all lymphocyte immunities of hormone antagonist are suppressed.

Table 13 is respectively organized rat LPR (x ± SD)

Group example number LPR

Normal 11 1.30 ± 0.23 ^*

Model 11 0.96 ± 0.12

EF organizes 11 1.31 ± 0.13 ^*

Compound recipe group 11 1.21 ± 0.14 ^*

^*Compare P＜0.05 with model group;

5. respectively organize rat plasma hydrocortisone content relatively: the results are shown in Table 5.Experiment shows that model group compares with normal group, and blood plasma cortisol content significantly reduces, and P＜0.05 shows that adrenal cortex function obviously goes down; The kidney invigorating compound recipe group blood plasma cortisol content is compared nothing with model group and is significantly increased P＞0.05; Herba Epimedii total flavones group blood plasma cortisol content is compared with model group and is increased, and has significant difference, and P＜0.05 shows that Herba Epimedii total flavones can improve and protect the rat adrenal cortex function, and the effect that hormone is suppressed adrenal cortex function has antagonistic effect.

Table 14 is respectively organized the rat plasma corticosterone content and is measured

Group example number plasma corticosterone

Normal group 11 3.68 ± 2.30 ^*

Model group 11 1.94 ± 1.27

EF organizes 11 4.06 ± 3.06 ^*

Compound recipe group 11 4.58 ± 3.40

^*Compare P＜0.05 with model group; ^*Compare P＜0.01 with model group

More than research prompting; Herba Epimedii total flavones and the kidney invigorating compound recipe are relatively; have the exogenous glucocorticoid of comprehensive protection and cause the downtrod effect of rat HPAT axle; in conjunction with the series of studies result in early stage, show that fully Herba Epimedii total flavones has its good prospects for application to the untoward reaction of removing hormone or alleviating hormone.

This research intend to the senile rat lymphocyte at once apoptosis (spontaneous apoptosis) carry out quantitative analysis, and adopt high-throughout biochip technology to analyze its gene expression profile.Further observe Herba Epimedii total flavones (Epimedium Flavonoids simultaneously, EF) to the influence of Lymphocyte Apoptosis and gene expression profile thereof, purpose is to understand the gene regulation pattern that Herba Epimedii total flavones is rebuild old and feeble immune stable state, to illustrate the molecular mechanism that Herba Epimedii total flavones is rebuild old and feeble immune stable state.

Materials and methods

1 material

1.1 animal grouping: experiment is provided by the department of the Chinese Academy of Sciences of animals and plants section of Fudan University with cleaning level SD rat, 30 of 23 months old rats, and male and female half and half are divided into 2 groups at random, i.e. aged control (OC group), Herba Epimedii total flavones group (EF group); The EF group gives EF and irritates stomach, and the OC group gives distilled water simultaneously and irritates stomach, and irritating the stomach time is 3 months.Other establishes 7 young matched groups of conduct of 4 monthly age SD rats (YC group).All animal etherizations, the aseptic spleen of getting, separating spleen lymphocyte.

1.2 medication preparation: this research adopts Liaoning to produce Herba Epimedii (EpimediumKereanum Nakai), extracts Herba Epimedii total flavones by Shanghai City medical industry academy mesothecium.Be formulated as the solution of 3g/100ml during experiment with distilled water, irritate stomach according to 0.06g/kgBW/d.

1.3 reagent: lymphocyte separation medium be Lympholyte-Rat (CL5045, CEDARLANE, CANADA); ANNEXIN V-FITC APOPTOSISDETECTION KIT I (BD Bioscience, U.S.A); TRI _ZOLReagent (GIBCOBRL, Life technologies, U.S.A); Rneasy Mini Kit (Qiagen, Germany); Adopt synthetic cDNA first chain of GeneChip T7-oligo (dT) Promoter Primer Kit and SuperScript II RT; Adopt Enzo BioArray HighYield RNA TranscripLabeling Kit ³(Affymetrix, U.S.A) cRNA of synthesizing biotinylated labelling; Adopt the cRNA of the plain labelling of GeneChip Sample Cleanup Module purifying biological; Adopt Test3 Array (Affymetrix) to carry out preliminary experiment, to determine Biotin-labeled cRNA quality.After Test Chip detects, adopt Rat Genome U34A Array (Affymetrix), each group cRNA sample is detected.

2 methods

2.1 flow cytometer detects percentage of cell apoptosis: PBS washing lymphocyte 2 times, adjusting cell concentration with 1 * binding buffer is 1 * 10 ⁶Cells/ml; Get 100 μ l samples, add 5 μ l AnnexinV and 5 μ l PI; Slight mixing cell, 15min is hatched in 25 ℃ of darkrooms; Add 400 μ l, 1 * binding buffer in every duplicate samples; The cell percentage of flow cytometer check and analysis AnnexinV (+), PI (-).

2.2 gene chip detects the gene of respectively organizing rat lymphocyte gene expression profile and comparative analysis aged control and young matched group, Herba Epimedii total flavones group and aged control differential expression.Concrete grammar is as follows: after total RNA extracting, purification, evaluation reach quantitatively in the cell, and synthetic cDNA first chain, second chain.Behind the double-stranded cDNA product purification, adopt the cRNA. of EnzoBioArray HighYield RNA Transcrip Labeling Kit synthesizing biotinylated labelling further the cRNA product to be carried out quantitatively and after identifying, carry out the segmentization of cRNA product, the cRNA after the segmentization promptly can be used for hybridization.Hybridization carries out in HybridizationOven640 (Affymetrix).Further wash and dye, be reflected among the FluidicsStation 400 (Affymetrix) and carry out, adopt Washing Buffer A, WashingBuffer B to wash; Adopt Streptavidin Phycoerythrin Stain solution to dye.Adopt Scanner (Affymetrix) to scan, scanning process is carried out under the control of AffymetrixMicroarray Suite5.0.

3 date processing

3.1 the apoptotic cell percentage rate adopts mean ± standard deviation to represent, adopts SPSS11.0 forwindows software kit to carry out statistical procedures, p＜0.05 is for there being statistical significance.

3.2 gene chip detects The data Affymetrix Microarray Suite5.0 software and analyzes.Data analysis adopts Statistical Algorithm. under the precursor to single sample expression analysis, and promptly according to Detection p-value,＜0.04 is Present; 0.04-0.06 be Marginal;＞0.06 is Absent, further carries out comparing between two groups, judges according to Signal LogRatio (SLR).Multiple＞2 (are promptly expressed in SLR＞1 ¹) be up-regulated, statistical significance is arranged; Multiple＜2 (are promptly expressed in SLR＜-1 ^-1), be down-regulated expression, statistical significance is arranged.

The result

1, Herba Epimedii total flavones is to the apoptotic influence of senile rat lymphocyte:

We adopt flow cytometer in conjunction with the two dyeing of FITC-Annexin V-PI each group rat splenocyte apoptosis percentage rate to be carried out quantitative analysis.The result shows: senile rat splenocyte apoptosis percentage rate is significantly higher than young rats splenocyte apoptosis percentage rate, and there is excessive apoptosis in prompting senile rat body endolymph cell; Splenocyte apoptosis percentage rate after the Herba Epimedii total flavones intervention is compared remarkable reduction with the senile rat matched group, the prompting Herba Epimedii total flavones can significantly be intervened the unbalance excessive apoptosis of important mechanisms-lymphocyte of senile rat immunity stable state, has the effect that senile rat is rebuild immune stable state.See Table 15:

Table 15 is respectively organized rat splenocyte apoptosis percentage rate (%) x ± SD

Group example number percentage of cell apoptosis (%)

YC 7 10.91±6.70

OC 7 25.81±9.11 ^△

EF 7 11.95±4.38 ^☆

Annotate: ^△OC compares with YC, p＜0.01; ^☆EF compares with OC, p＜0.01

2, senile rat lymphocyte gene expression profile is compared with youth: senile rat is compared with young rats, and the gene of up-regulated has 116, and the gene of down-regulated expression has 215.Analyze in conjunction with our background of research, the distinctive gene expression pattern of senile rat lymphocyte has following characteristics: (1) has and promotes the gene expression of apoptosis effect significantly to raise, as: the Caspasel, the Caspase3 that participate in control of apoptosis generative center and effect link; Calpain II; Dnase gamma; PKC delta; Bnip31; P47 etc.Gene such as Cathepsin B, Mtal etc. with anti-apoptotic effect express significantly downward modulation.(2) gene expression with antiproliferative effect is significantly raised, as Rb, Anti-proliferative factor etc.Gene expression with promotion cel l proliferation is significantly reduced, as c-myc, c-fos, Jun, A-raf; Rgc32, Cyclin B1, Cyclin G-associated kinase etc.(3) participate in the element of baseband signal paths such as lymphocyte activation propagation and immunological effect, the expression of a plurality of signal of interest molecules is significantly reduced, as the related receptor signal approach of enzyme signal of interest molecule Serine/Threonine protein kinase, cAMP dependent protein kinase, Ca ²⁺/ calmodulin-dependent protein kinase 2, Spleen tyrosine kinase, inositol trisphosphate receptor subtype 3 (IP3R-3), RAC protein kinasegamma,, guanylate cyclase, protein-tyrosine-phospatase etc.(4) the basic regulatory factor abnormal expression of immunocyte effect and function significantly raises as MHC I class antigen presentation, and MHC II class antigen presentation is significantly reduced; IFN-γ and CD24 express significantly downward modulation etc.

3, Herba Epimedii total flavones is to the influence of senile rat lymphocyte gene expression: the senile rat lymphocyte gene profile after the Herba Epimedii total flavones intervention is compared with aged control lymphocyte gene expression profile, the up-regulated gene of expressing has 447, and the gene of down-regulated expression has 456.The research background of binding immunoassay aging the analysis showed that further the binding mode that Herba Epimedii total flavones is regulated and control the gene expression of senile rat lymphocyte has following characteristics: the expression of (1) Herba Epimedii total flavones scalable gene participating in apoptosis-remarkable rise anti-apoptotic genes expression transcribed when significantly transcribing of apoptogene urged in downward modulation.As have gene C aspase1, Caspase2, Caspase3, a Caspase6 that promotes the apoptosis effect; Calpains; CathepsinD, CathepsinH, CathepsinS; Dnase gamma; PKC delta etc. significantly reduces in the expression of Herba Epimedii total flavones group.Meanwhile, the gene expression with anti-apoptotic effect of Cathepsin B, Mta1, NF-kappa B, Notch is significantly raised.(2) the Herba Epimedii total flavones scalable cell proliferation related because of expression, be in particular in: significantly raise c-Jun, PCNA, A-raf, Rgc32, isogenic the transcribing of Cyclin G-associated kinase that promotes cells survival and propagation, simultaneously significantly downward modulation antiproliferative Rb is isogenic transcribes.(3) Herba Epimedii total flavones significantly raises the expression of a plurality of signaling molecules in the relevant baseband signal conduction element of lymphocyte activation and immunological effect, as Serine/Threonine protein kinase, Serine/Threonine protein kinase (pim3), Serine/Threonine protein kinase (tpl2); CAMP dependent protein kinase; PI3K, inositol trisphosphatereceptor subtype 3 (IP3R-3), PI3K Receptor, protein-tyrosine-phospatase, isogenic the transcribing in the Herba Epimedii total flavones group of protein kinase C-eta, protein kinase are significantly raised.(4) Herba Epimedii total flavones significantly raises the expression of the basic regulatory factor of immunocyte effect and function, significantly raises CD2, CD3, CD5, TCR as Herba Epimedii total flavones; IL-2, IL-2R (CD25), the isogenic expression of CD28, CTLA4.

Therefore, transcribing of apoptogene urged in Herba Epimedii total flavones downward modulation when the rise anti-apoptotic genes expression is transcribed; Downward modulation antiproliferative gene transcription when the short amplified gene of rise is transcribed; Raise and promote the conduction of lymphocyte activation signal, isogenic the transcribing of immunological effect regulation and control.

Discuss

Herba Epimedii total flavones can significantly reduce the lymphocytic excessive apoptosis of senile rat, points out out it to delay the effect of immunosenescence.A plurality of short apoptogenes, anti-apoptotic genes expression, the pattern of a kind of collaborative, antagonism of the function embodiment of short amplified gene, antiproliferative gene.Keeping of immunity stable state must be expressed as prerequisite with the balance of the gene " antithetical phrase " of these functions reversed.The unbalance important gene background of old and feeble immune stable state is exactly that the expression stable state of these genes " antithetical phrase " is broken.When Herba Epimedii total flavones raises the anti-apoptotic genes expression expression, the expression of the short apoptogene of downward modulation; Raise when urging the amplified gene expression downward modulation antiproliferative expression of gene.The general trend of its effect is to readjust the contrast of decision cells survival and dead both sides' strength, lymphocytic gene expression trend presents a kind of pattern that helps cells survival, propagation and normal function performance under the aging state thereby make, and it rebuilds the molecular mechanism place of old and feeble immune stable state just for this.

The bulk information that full genome analysis is provided is pointed out out the rule of Herba Epimedii total flavones regulator gene imbalance of expression, be short apoptogene and anti-apoptotic genes expression, short amplified gene and the antiproliferative gene of reversing function opposition in old and feeble lymphocyte, express unusually, reinvent the benign balance of gene expression, make the gene expression after the integration embody anti-apoptosis, rebuild the biological effect of immune stable state.This result of study fully points out out Herba Epimedii total flavones to have the effect that delays immunosenescence, and illustrated the molecular mechanism of action that Herba Epimedii total flavones delays immunosenescence comparatively all sidedly.

Another object of the present invention has provided the preparation method of above-mentioned a kind of Herba Epimedii total flavones tablet, and this method comprises the following steps:

(1) preparation of Herba Epimedii total flavones extract: the Herba Epimedii leaf decocts with water secondary, and each water consumption is 16 times of medical material weight.For the first time decocting time keeps little boiling 2 hours for boiling the back, boils the back for the second time and keeps little boiling 1.5 hours, and decoction liquor is with 4 layers of filtered through gauze at every turn, and merging filtrate fully is cooled to room temperature.Last macroporous adsorbent resin, resin demand are 1Kg Herba Epimedii leaf with 1 liter of resin, and flow velocity is 1.5L/ minute.Behind the end of the sample, with the deionized water eluting, flow velocity is the same, the deionized water consumption is 500 liters, and washing rises eluting with 15%EtOH200 after finishing, flow velocity is the same, rises eluting with 40%EtOH500 again, and flow velocity is constant, add 50%EtOH300 and rise eluting, flow velocity is the same, contains the alcohol amount with alcohol hydrometer mensuration eluent and reaches at 45% o'clock, begin to collect eluent to add 50%EtOH eluting end, be evaporated to do after, pulverize, promptly get the Herba Epimedii total flavones extract.

(2) Herba Epimedii total flavones extract that step (1) is made and pharmaceutic adjuvant promptly get the Herba Epimedii total flavones tablet by the film-making of tablet conventional method.

Tablet content scope of the present invention: Herba Epimedii total flavones content is 60%-65%.

Dosage form and adjuvant: tablet, adopt starch (making 10% slurry), magnesium stearate as adjuvant.

Another object of the present invention has provided above-mentioned a kind of Herba Epimedii total flavones and has alleviated the application of using exogenous glucocorticoid side effect and delaying the immunosenescence medicament in preparation.

Herba Epimedii total flavones tablet of the present invention has following effect characteristics:

1, alleviates and use exogenous glucocorticoid side effect: mainly show as Antiglucocorticoid thymus, adrenal gland and lymphocytic inhibitory action, improve body weight, thymus index, adrenal gland's index, lymhocyte transformation rate and serum corticosterone etc. that hormone is used the back rat.Alleviate the effect that hormone is used side effect and untoward reaction thereby have.

2, delay immunosenescence: the important symbol-lymphocytic excessive apoptosis that mainly shows as immunosenescence has significant downward modulation effect, and polygenic expression such as the old and feeble Lymphocyte Apoptosis of scalable, propagation, signal conduction and immunizing potency.Help to lower the generation of relevant disease of immunosenescence such as infection, tumor, autoimmune disease, improve life quality, life-saving.

The specific embodiment:

Example: Herba Epimedii total flavones sheet

(prescription) Herba Epimedii total flavones 1.00kg, starch (making 10% slurry) 0.25g, magnesium stearate 0.01g make 10000 altogether

(method for making) gets the Powdered extract of Herba Epimedii total flavones, adds 10% starch slurry, fully stirs, and makes soft material behind the mix homogeneously, cross 14 mesh sieves and granulate, 50-70 ℃ is dried to the dry granular water content and is about 6%, granulate, add the magnesium stearate mix homogeneously, tabletting contains Herba Epimedii total flavones 0.1g in every.

(application) alleviates the exogenous glucocorticoid side effect of application; Delay immunosenescence.

(usage) is oral, a 0.1g, 0.3g on the one.

Claims (3)

1, a kind of Herba Epimedii total flavones tablet, its special sheet are that this tablet comprises Herba Epimedii total flavones extract and pharmaceutic adjuvant composition, and wherein the Herba Epimedii total flavones extractive content is 60%～65%.

2, a kind of preparation method of a kind of Herba Epimedii total flavones tablet as claimed in claim 1 is characterized in that this method comprises the following steps:

(1) preparation of Herba Epimedii total flavones extract: the Herba Epimedii leaf decocts with water secondary, each water consumption is 16 times of medical material weight, decocting time keeps little boiling 2 hours for boiling the back for the first time, boil for the second time the back and keep little boiling 1.5 hours, each decoction liquor is with 4 layers of filtered through gauze, and merging filtrate fully is cooled to room temperature, last macroporous adsorbent resin, resin demand is a 1Kg Herba Epimedii leaf with 1 liter of resin, and flow velocity is 1.5L/ minute, behind the end of the sample, with the deionized water eluting, flow velocity is the same, and the deionized water consumption is 500 liters, after washing finishes, rise eluting with 15%EtOH200, flow velocity is the same, rises eluting with 40%EtOH500 again, and flow velocity is constant, add 50%EtOH300 and rise eluting, flow velocity is the same, contains the alcohol amount with alcohol hydrometer mensuration eluent and reaches at 45% o'clock, begins to collect eluent to institute and adds the end of 50%EtOH eluting, be evaporated to do after, pulverize, promptly get the Herba Epimedii total flavones extract

(2) Herba Epimedii total flavones extract that step (1) is made and pharmaceutic adjuvant promptly get the Herba Epimedii total flavones tablet by the film-making of tablet conventional method.

3, a kind of Herba Epimedii total flavones extract alleviates in preparation and uses exogenous glucocorticoid side effect and delay application in the immunosenescence medicament.