CN111588762B

CN111588762B - Medicine for treating thromboangiitis obliterans, preparation method and content determination method

Info

Publication number: CN111588762B
Application number: CN202010392759.4A
Authority: CN
Inventors: 唐宏; 宋哲
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-05-11
Filing date: 2020-05-11
Publication date: 2021-11-19
Anticipated expiration: 2040-05-11
Also published as: CN111588762A

Abstract

The invention relates to the field of traditional Chinese medicines, in particular to a medicine for treating thromboangiitis obliterans, a preparation method and a content determination method. The commodity name is as follows: capsule for treating blood arthralgia. The medicinal effective components are prepared from the following raw material medicines in parts by weight: 25-35 parts of astragalus membranaceus, 12-18 parts of salvia miltiorrhiza, 15-25 parts of red paeony root, 12-18 parts of centipede, 8-12 parts of earthworm, 12-18 parts of ground beeltle, 15-25 parts of Chinese starjasmine stem, 12-18 parts of red tangerine peel and 12-18 parts of uncaria. The preparation method has the advantages that the optimal extraction process parameters are 80% ethanol solution, the material-liquid ratio is 1:8, the extraction is carried out for 2 times, each time is 1h, the extraction process is feasible, and the effect is stable. The determination of the content of the effective components is simple and easy, and the quality of the medicine can be objectively, truly and comprehensively monitored.

Description

Medicine for treating thromboangiitis obliterans, preparation method and content determination method

Technical Field

The invention relates to the field of traditional Chinese medicines, in particular to a medicine for treating thromboangiitis obliterans, a preparation method and a content determination method. The commodity name is as follows: capsule for treating blood arthralgia.

Background

In the prior art, thromboangiitis obliterans (TAO), also known as Buerger disease, is a common peripheral vascular disease, the etiology of which is complex and diverse and has no definite theory, and related factors can be classified into two aspects, namely intrinsic factors and extrinsic factors. The external factors mainly comprise cold and damp living environment, smoking, infection, chronic injury and the like, and the internal factors mainly comprise autoimmune system disorder, prostatic hormone disorder, heredity and the like. Endothelial dysfunction, i.e., endothelium-dependent vasodilation, is damaged by various factors, and the pathogenic factors induce the release of inflammatory factors, which results in the adhesion of immune cells and endothelial cells, and finally the formation of intravascular thrombosis and vasculitis. Thromboangiitis obliterans is clinically manifested as bluish purple and cold limbs, numbness, spasm and pain. The main causes of thromboangiitis obliterans are not yet clearly defined. From the perspective of traditional Chinese medicine, the causes of the disease are: feeling exogenous pathogens: invasion of cold tends to cause coagulation of qi, blood and body fluids in the meridians. Cold nature leading to coagulation and cold pathogen hurting human body leads to qi and blood obstruction, unsmooth channels and collaterals, and causes swelling and pain and even gangrene. Ming Xuehe (Xue Yi, pivotal theory of surgery) records that the swelling, burning, pain and redness of toes are caused by the deficiency of three yang meridians and the domination of exogenous pathogenic factors. ② damp-heat toxin accumulation: the dampness is tangible and water-like, and pertains to yin in nature, so the invasion of damp pathogen firstly causes the disease in the lower part, and the invasion of damp pathogen usually causes the disease in the lower part with retention. The dampness is heavy and turbid, the pathogenic factors tend to adhere to each other, the heat-toxicity is endogenous, and the damp-heat goes downward to cause the disease. ③ deficiency of vital qi: qi is the commander of blood and can produce, move and control blood. Blood is the mother of qi and can nourish qi and carry gas. Qi and blood deficiency failing to flow through the meridians and collaterals, failing to nourish the internal organs and the external extremities, resulting in black-burnt and necrotic toes or even loss of toes. Phlegm stasis trauma: spleen governs transportation and transformation, spleen and stomach disharmony leads to water and grain failure, phlegm stasis is generated internally, stasis becomes pathogenic factor for a long time, qi and blood can not reach limbs and bones due to obstruction of veins and collaterals to cause gangrene, or trauma and qi and blood stasis cause qi and blood to fail to reach limbs and bones, and necrosis and abscission are caused without blood supply (Liyu satisfaction, Liu hong Yan, Chen Gui et al. It will be a future development to treat this disease by improving hemorheology, anti-thrombosis, thrombolysis, inhibition of endothelial cell proliferation and vascular wall inflammatory response. The Chinese herbs have various kinds and different curative effects.

Disclosure of Invention

The invention aims to provide a medicine for treating thromboangiitis obliterans, a preparation method and a content determination method aiming at the defects, and the medicine has better effects on protecting and treating oxidative stress injury of cells.

The technical solution of the invention is as follows: the medicine for treating thromboangiitis obliterans is characterized in that the medicinal effective components are prepared by the following raw material medicines in parts by weight: 25-35 parts of astragalus membranaceus, 12-18 parts of salvia miltiorrhiza, 15-25 parts of red paeony root, 12-18 parts of centipede, 8-12 parts of earthworm, 12-18 parts of ground beeltle, 15-25 parts of Chinese starjasmine stem, 12-18 parts of red tangerine peel and 12-18 parts of uncaria. The production is proportionally expanded.

Preferably, the medicament for treating thromboangiitis obliterans is characterized in that the medicament is prepared into capsules by the following raw material medicaments in gram by weight: 30g of astragalus membranaceus, 15g of salvia miltiorrhiza, 20g of red paeony root, 15g of centipede, 10g of earthworm, 15g of ground beetle, 20g of Chinese starjasmine stem, 15g of red tangerine peel and 15g of uncaria. And (5) mixing the extract powder with dextrin at a ratio of 6:4, and encapsulating.

The preparation method of the medicine for treating thromboangiitis obliterans is characterized by comprising the following steps: extracting medicinal materials with 80% ethanol solution at a material-to-liquid ratio of 1:8 for 2 times, extracting for 1 hr each time, cooling, evaporating the extractive solution with rotary evaporator until the extractive solution is viscous but not wall-hung, transferring the extractive solution into evaporating dish, evaporating in 75 deg.C water bath until the extractive solution is pasty, and drying the evaporating dish in vacuum drying oven at 55 deg.C to obtain powder.

The content determination method of the medicine for treating thromboangiitis obliterans is characterized in that the method comprises any one or a combination of the following steps:

(1) determination of astragaloside content

Chromatographic column InertSustain C18 (5 μm, 4.6X 250 mm) using octadecylsilane chemically bonded silica as filler, mobile phase selected from acetonitrile-water (35: 65), evaporative light scattering detector, flow rate of 1.0 mL/min, column temperature of 40 deg.C, drift tube temperature of 40 deg.C. Taking the sample amount (mug) of the astragaloside IV standard substance as a horizontal coordinate, and taking a peak area as a vertical coordinate to draw a standard curve; accurately weighing astragaloside IV standard, adding methanol to prepare standard solution with concentration of 0.05 μ g/μ L, 0.10 μ g/μ L, 0.25 μ g/μ L, 0.50 μ g/μ L, and 0.75 μ g/μ L, sequentially injecting 10 μ L, and obtaining data via high performance liquid phase; obtaining the linear regression equation y =2157223.918x-609361.5306 of astragaloside IV and the correlation coefficient R²=0.9956, which shows that the linear relation is good between 0.5-7.5 mug (figure 2), and the corresponding peak areas of three batches (20190413, 20190428 and 20190511) of samples to be tested are brought in, so that the content of the astragaloside in each 1g of extract powder is 96.172 mug, 98.557 mug and 97.265 mug respectively; the astragaloside obtained by three tests has similar content, and the extraction process is proved to be feasible and stable in effect.

(2) Determination of tanshinone IIA content

Using chromatographic column InertSustain C18 (5 μm, 4.6 x 250 mm) using octadecylsilane chemically bonded silica as filler, selecting acetonitrile and 0.02% phosphoric acid solution as mobile phase, gradient eluting, detecting wavelength of 270 nm, flow rate of 1.0 mL/min, column temperature of 20 deg.C; accurately weighing tanshinone IIA standard substance, adding methanol solution to prepare standard substance solutions with concentrations of 0.05 μ g/μ L, 0.10 μ g/μ L, 0.25 μ g/μ L, 0.50 μ g/μ L and 1.00 μ g/μ L, sequentially injecting 10 μ L, and obtaining data through high performance liquid chromatography. Taking the sample amount mu g of tanshinone IIA as a horizontal coordinate, and taking the peak area as a vertical coordinate to draw a standard curve; obtaining a tanshinone IIA linear regression equation y =42276x +4832, and a correlation coefficient R2=0.9986, which shows that the linear relation of the tanshinone IIA linear regression equation y =42276x +4832 and the correlation coefficient R2=0.9986 is good, and the tanshinone IIA content is 2.968mg, 2.979mg and 2.894mg in each 1g of Xuebitong extract powder by bringing the linear relation into corresponding peak areas of three batches of samples to be tested; the tanshinone IIA content measured by three tests is similar, and the effect of extracting the effective component tanshinone IIA from the Xuebitong extract powder is stable.

(3) And (3) measuring the content of paeoniflorin:

using chromatographic column InertSustain C18 (5 μm, 4.6 x 250 mm) using octadecylsilane chemically bonded silica as filler, selecting methanol and 0.05mol/L potassium dihydrogen phosphate solution (40: 60) as mobile phase, detecting wavelength of 230nm, flow rate of 1.0 mL/min, and column temperature of 40 deg.C; accurately weighing paeoniflorin standard, adding methanol solution to prepare standard solutions with concentrations of 1.00 μ g/μ L, 2.50 μ g/μ L, 5.00 μ g/μ L, 10.10 μ g/μ L and 20.00 μ g/μ L, sequentially injecting 10 μ L, and obtaining data through high performance liquid chromatography. Taking the sampling amount mu g of paeoniflorin as a horizontal coordinate, and taking a peak area as a vertical coordinate to draw a standard curve; the linear regression equation y =24914x +11078 of paeoniflorin and the correlation coefficient R2=0.9996 show that the linear relation between 10.0-200.0 mug is good, the linear relation is brought into the corresponding peak areas of three batches of samples to be measured, and the paeoniflorin content in each 1g of Xuebitong extract powder is 39.650 mg, 40.121 mg and 39.244 mg; the content of paeoniflorin measured by three tests is similar, and the effect of extracting the effective component paeoniflorin from the Xuebitong extract powder is stable.

(4) And (3) measuring the content of the trachelospermin:

chromatographic column InertSustain C18 (5 μm, 4.6 × 250 mm) using octadecylsilane chemically bonded silica as filler, mobile phase selected from acetonitrile and water (30: 70), detection wavelength of 280nm, flow rate of 1.0 mL/min, and column temperature of 40 deg.C; precisely weighing a standard substance of the trachelospermin, adding methanol to prepare standard substance solutions with the concentrations of 0.50 mu g/mu l, 1.00 mu g/mu l, 2.50 mu g/mu l, 5.10 mu g/mu l and 10.00 mu g/mu l, sequentially injecting 10 mu l, and obtaining data through a high performance liquid phase. Taking the sampling amount mu g of paeoniflorin as a horizontal coordinate, and taking a peak area as a vertical coordinate to draw a standard curve; the linear regression equation y =36362x-4853.4 and the correlation coefficient R2=0.9818 show that the linear relation between 5.0-100.0 mug is good, the corresponding peak areas of three batches of samples to be measured are taken, and the contents of the trachelospermin in each 1g of extract powder are 2.750 mg, 2.631 mg and 2.668 mg. The contents of the trachelospermi glycosides measured by three tests are similar, and the extraction effect of the trachelospermi glycosides in the Xuebi Tong extract powder for treating blood arthralgia is stable.

Astragalus root: invigorating qi, consolidating superficial resistance, inducing diuresis, removing toxic substances, expelling pus, healing wound, and promoting granulation. Red sage root: dispel stasis and alleviate pain, cool blood and cure abscess. Red peony root: clear heat and cool blood, activate blood. Centipede: extinguish wind and stop spasm, dredge collaterals and alleviate pain. Earthworm: pacify liver, stop dyspnea and dredge meridians. Ground beetle: break blood and remove stasis, reunion of fractured tendons and bones. Caulis trachelospermi: dispel wind, dredge collaterals, cool blood and relieve swelling. Orange peel: to regulate qi, relax middle energizer, dry dampness and resolve phlegm. Uncaria: clear heat and pacify liver, extinguish wind and stop spasm.

Square solution: monarch drug: radix astragali. Ministerial drugs: red sage root, red peony root, centipede, earthworm, ground beeltle, Chinese starjasmine stem and rhizome. Adjuvant drugs: and (4) exocarpium citri grandis and uncaria. Thromboangiitis obliterans belongs to the category of gangrene of finger in traditional Chinese medicine, and is mainly caused by that the qi and blood can not reach the extremities due to cold-dampness, trauma, blood stasis and other blood stasis blocking veins, and the extremities lose the nourishment of qi and blood. The traditional Chinese medicine considers that: invasion of cold-dampness, stagnation of the vessels; trauma and blood stasis blocking the veins; emotional disorder, qi stagnation and blood stasis, all of which block the meridians, make qi and blood flow unsmooth and fail to reach the extremities, and the extremities lose the warm nourishment of qi and blood, causing pain, ulceration and even necrosis, which are the main pathogenesis of gangrene. Astragalus root, radix astragali has the effects of invigorating qi, expelling pus, healing sore and promoting granulation. The red sage root and the red peony root have the functions of promoting blood circulation and cooling blood. The insect medicine breaks blood and removes stasis, the Chinese starjasmine stem dispels wind and unblocks collaterals, and the uncaria extinguishes wind and stops spasm, so that the whole formula is embodied, and the purposes of nourishing qi and activating blood, cooling blood and dredging channels and collaterals are achieved, and the vasculitis is treated.

The efficacy is as follows: dispel wind, dredge collaterals, break blood, expel blood stasis, extinguish wind and stop convulsion.

The dosage form is not limited, and the above medicinal raw materials and pharmaceutically-acceptable carriers, such as excipient or adjuvant, can be mixed to make into tablet, capsule (soft capsule), granule, powder, pill or other conventional oral preparations.

The invention has the advantages that: 1. experiments prove that the blood bi-tong achieves the treatment purpose by improving the inflammatory environment of the organism, recovering the vasodilation capacity and inhibiting the proliferation of vascular smooth muscle. 2. The Chinese medicinal preparation, XUEBITONG Capsule, has effects in resisting blood platelet aggregation, resisting blood platelet adhesion and resisting thrombosis. The action mechanism of the blood bi tong capsule on the thromboangitis obliterans is related to the regulation of vasodilation and the inhibition of vascular smooth muscle proliferation. Has certain therapeutic effect on thromboangiitis obliterans. 3. The optimal extraction process parameter is 80% ethanol solution with a material-liquid ratio of 1:8, the extraction is carried out for 2 times, each time for 1h, the extraction process is feasible, and the effect is stable. 4. The method for measuring the content of the effective components has the advantages of simplicity, feasibility and capability of objectively, truly and comprehensively monitoring the quality of the medicine. 5. The clinical observation of 50 patients with thromboangiitis obliterans is accumulated by the medicine: the cure rate is 72%, the significant efficiency is 92%, the effective rate is 98%, and the ineffective rate is 2%. Has obvious curative effect.

Embodiments of the present invention will be described in further detail with reference to examples.

Drawings

FIG. 1 shows isolated rat femoral artery with the proximal end of the femoral artery occluded.

FIG. 2 astragaloside IV standard curve.

FIG. 3 shows Thin Layer Chromatography (TLC) chromatogram of radix astragali under ultraviolet lamp (365 nm) in the preparation.

Fig. 4 tanshinone IIA standard curve.

FIG. 5 paeoniflorin standard curve.

FIG. 6A standard curve for trachelospermin.

FIG. 7: adding 800 mu M H in each group except for the normal saline group₂O₂The damage is caused to the patient,^#，P＜0.05，^##，P< 0.01 in comparison to saline group;^*，P＜0.05，^**，P< 0.01 and H₂O₂And (4) comparing the damage groups.

FIG. 8: adding 800 mu M H in each group except for the normal saline group₂O₂The damage is caused to the patient,^#，P＜0.05，^##，P< 0.01 in comparison to saline group;^*，P＜0.05，^**，P< 0.01 and H₂O₂And (4) comparing the damage groups.

FIG. 9: adding 800 mu M H in each group except for the normal saline group₂O₂The damage is caused to the patient,^#，P＜0.05，^##，P< 0.01 in comparison to saline group;^*，P＜0.05，^**，P< 0.01 and H₂O₂And (4) comparing the damage groups.

FIG. 10: adding 800 mu M H in each group except for the normal saline group₂O₂The damage is caused to the patient,^#，P＜0.05，^##，P< 0.01 in comparison to saline group;^*，P＜0.05，^**，P< 0.01 and H₂O₂And (4) comparing the damage groups.

FIG. 11: adding 800 mu M H in each group except for the normal saline group₂O₂The damage is caused to the patient,^#，P＜0.05，^##，P< 0.01 in comparison to saline group;^*，P＜0.05，^**，P< 0.01 and H₂O₂And (4) comparing the damage groups.

FIG. 12: adding 800 mu M H in each group except for the normal saline group₂O₂The damage is caused to the patient,^#，P＜0.05，^##，P< 0.01 and physiologicalComparing saline groups;^*，P＜0.05，^**，P< 0.01 and H₂O₂And (4) comparing the damage groups.

FIG. 13: adding 800 mu M H in each group except for the normal saline group₂O₂The damage is caused to the patient,^#，P＜0.05，^##，P< 0.01 in comparison to saline group;^*，P＜0.05，^**，P< 0.01 and H₂O₂And (4) comparing the damage groups.

Detailed Description

Example 1

The medicine (Xuebaitong) for treating thromboangiitis obliterans is prepared into capsules by the following raw material medicines by weight: 30g of astragalus membranaceus, 15g of salvia miltiorrhiza, 20g of red paeony root, 15g of centipede, 10g of earthworm, 15g of ground beetle, 20g of Chinese starjasmine stem, 15g of red tangerine peel and 15g of uncaria.

Experimental example 1

Clinical report of Xuebaitong capsule

First, 50 cases of observation of blood Bi Tong capsule for treating thromboangiitis obliterans

And (4) conclusion: the clinical observation of 50 patients with thromboangiitis obliterans is accumulated by the medicine: the cure rate is 72%, the significant efficiency is 92%, the effective rate is 98%, and the ineffective rate is 2%. Has obvious curative effect.

II, diagnosis standard of thromboangiitis obliterans:

1. in men, 20-40 years old 2, the symptoms of chronic limb ischemia, such as numbness, fear of cold, intermittent claudication, malnutrition and the like, usually affect the lower limbs and the upper limbs rarely. 3. 40-60% have a history or signs of migratory superficial thrombotic phlebotomy. 4. Various examinations prove that the limb artery occlusion and stenosis are mostly positioned in the popliteal artery and the distal artery (frequently involving the middle and small arteries) 5, the smoking history or the cold exposure history 6, the limb arteriosclerosis obliterans, diabetic gangrene, Takayasu arteritis, limb artery embolism, Raynaud union, traumatic artery occlusion, connective tissue disease angiopathy, allergic vasculitis and the like. 7. The diagnosis is facilitated by immunoglobulin increase and anti-arterial antibody positive, and the skin temperature measurement, limb segment manometry, impedance blood flow diagram examination, percutaneous oxygen partial pressure measurement, limb infrared thermography and other examinations are helpful for judging the artery occlusion part and the lesion degree 8, and Doppler ultrasonic examination and artery angiography can determine the lesion part and degree (the artery angiography can aggravate the ischemia of the affected limb, and the diagnosis should be carried out with caution). Arteriography, but we do not claim to make an invasive examination of the vessels. The popliteal artery and the distal artery are mostly affected, the artery is blocked and narrowed in sections, the artery between the blocked sections and the proximal artery are mostly normal, and the proximal and distal occluded arteries are mostly tree-root-shaped collateral circulation arteries. 9. Positive in the limb position test (Buerger sign).

Thirdly, the treatment standard of thromboangiitis obliterans is as follows:

(one) curing: intermittent claudication disappears, so that the patient can walk normally and the pulse of the dorsum of foot is normal.

(II) effect display: intermittent claudication disappears, the patient can normally walk for 1000 meters, and the pulse of the dorsum of the foot is normal.

(III) effective: intermittent claudication is improved, the patient can normally walk for about 100 meters, and the pulse of the dorsum of the foot is weakened.

(IV) invalidation: the disease nature is progressively worsened, and the dorsal pulse of the foot disappears.

(V) color ultrasound examination

Fourth, case report

Case 1: xuezhi, male, age 54

The patient has no obvious inducement to numbness of the right lower limb and cold feet in one year, and is accompanied with intermittent claudication, and the diagnosis is carried out in a hospital in the city: thromboangiitis obliterans. And (3) carrying out four-dimensional color Doppler ultrasound prompting in 2019, 3 and 27 months: double lower limb arterial plaque formation, deep vein thrombosis. And (3) clinical diagnosis: thromboangiitis obliterans, deep vein thrombosis. The blood circulation activating capsule is orally taken, 55 capsules/time and 3 capsules/day, after the oral administration for 13 days, the pain of the lower limbs disappears, and the pulse of the dorsum of the foot is obvious. Color Doppler ultrasound display in 2019, 4, 9 and 9 months: double lower limb arteriosclerosis (local luminal inflammatory infiltration), deep vein intimal thickening and clinical healing.

Case 2: chua is a certain woman, 70 years old.

The patient has no obvious inducement before one year, has cold feet and numbness and pain of legs, is accompanied by intermittent claudication, and has color ultrasonic examination prompt in 6 months and 1 day in 2019: double lower limb arteriosclerosis. Physical examination: the right foot dorsal artery pulsation disappears, the left lower limb pain disappears after the capsule for treating blood numbness is orally taken for 5 grains/time and 3 times/day for one month, the dorsal artery pulsation is normal, and the color ultrasonography is carried out for 7 months and 3 days in 2019 to prompt the clinical cure of the arteriosclerosis of the two lower limbs.

Experimental example 2

Preparation method, content determination method and curative effect verification of Xuebitong

2.1 materials of the experiment

2.1.1 cells and animals

Cell line: human Umbilical Vein Endothelial Cells (HUVEC).

Animals: clean grade SD rats, male, weighing 180-. And (5) adaptively feeding for one week. Are all purchased from Liaoning Biotechnology Ltd.

Experimental apparatus, see Table 1.1

TABLE 1.1 Experimental instruments

2.1.3 test reagents, see Table 1.2

TABLE 1.2 Experimental reagents

2.1.4 medicinal materials

The experimental medicinal material standards are all according to the medicinal material items in the 'Chinese pharmacopoeia' 2015 edition.

Liquid phase conditions

Astragaloside IV: chromatographic column InertSustain C18 (5 μm, 4.6X 250 mm) using octadecylsilane chemically bonded silica as filler, mobile phase selected from acetonitrile-water (35: 65), evaporative light scattering detector, flow rate of 1.0 mL/min, column temperature of 40 deg.C, drift tube temperature of 40 deg.C.

Tanshinone IIA: using chromatographic column InertSustain C18 (5 μm, 4.6 × 250 mm) using octadecylsilane chemically bonded silica as filler, selecting acetonitrile and 0.02% phosphoric acid solution as mobile phase, gradient eluting, detecting wavelength of 270 nm, flow rate of 1.0 mL/min, and column temperature of 20 deg.C.

Paeoniflorin: chromatographic column InertSustain C18 (5 μm, 4.6X 250 mm) using octadecylsilane chemically bonded silica as filler, mobile phase selected from methanol and 0.05mol/L potassium dihydrogen phosphate solution (40: 60), detection wavelength 230nm, flow rate 1.0 mL/min, and column temperature 40 deg.C.

And (3) trachelospermiside: chromatographic column InertSustain C18 (5 μm, 4.6 x 250 mm) using octadecylsilane chemically bonded silica as filler, mobile phase selected from acetonitrile and water (30: 70), detection wavelength of 280nm, flow rate of 1.0 mL/min, and column temperature of 40 deg.C.

Method

2.2.1 preparation process of Xuebitong extract powder

2.2.1.1 heated reflux extraction reagent study: weighing the medicinal materials according to the prescription of Xuebitong, soaking the medicinal materials in 10 times of water and 80% ethanol solution for 12h, heating and refluxing for 2h, cooling, evaporating the extract by using a rotary evaporator until the extract is viscous but not wall-hung, transferring the extract into an evaporation dish, evaporating the extract in a 75 ℃ water bath until the extract is pasty, and drying the evaporation dish in a reduced pressure drying oven at 55 ℃ to prepare powder.

According to an astragaloside extraction method under the item of astragalus root in the 'Chinese pharmacopoeia' 2015 edition, 1g of extract powder for blood impediment is accurately weighed, the extract powder is soaked in 25 mL of methanol for 12h, heated and refluxed for extraction for 4h, the extract is evaporated by a rotary evaporator until the extract is completely dried, 10 mL of distilled water is added, the extract is completely dissolved by ultrasonic treatment, water saturated n-butanol is used for extraction for 4 times, 25 mL of water saturated n-butanol is added each time, the lower water saturated n-butanol extract is combined and recovered, the extract is sufficiently shaken and washed for 2 times by an ammonia reagent, 25 mL of the ammonia reagent is added each time, the upper ammonia solution is discarded, the water saturated n-butanol layer is recovered and suspended, 5mL of distilled water is added for ultrasonic dissolution, separation is carried out by a D101 type macroporous adsorption resin column (the inner diameter is 1.5 cm, and the column height is 35 cm), eluent is firstly eluted by 150 mL of distilled water, the eluent is discarded, then 120 mL of 40% ethanol solution is used for elution, the eluent is discarded, finally, 250 mL of 70% ethanol solution is used for elution, the eluent is recovered and is suspended by a rotary evaporator, and 1mL of methanol solution is added for ultrasonic dissolution. Taking a proper amount of astragaloside IV sample, and determining astragaloside IV content by High Performance Liquid Chromatography (HPLC) method. The result shows that the content of astragaloside in the 80% ethanol extract is about 1.52 times of that of water under the same dosage, so the 80% ethanol solution is adopted as the blood impediment extraction reagent.

2.2.1.2 study of the Xuebitong ethanol heating reflux extraction process: designing an orthogonal test, weighing the medicinal materials according to the formula, horizontally heating and refluxing for extraction according to corresponding factors shown in table 2.1, combining the extracting solutions, cooling and filtering, evaporating the extracting solution by using a rotary evaporator until the extracting solution is viscous and does not hang on the wall, transferring the extracting solution into an evaporating dish, evaporating the extracting solution to a non-flowing paste in a water bath at 75 ℃, putting the evaporating dish into a reduced-pressure drying oven, and drying at 55 ℃ under reduced pressure to obtain a powder. Dissolving appropriate amount of extract powder, sampling to prepare astragaloside IV extract, and measuring astragaloside IV content by HPLC.

2.2.1.3 verification of the optimal extraction process: weighing the medicinal materials according to the formula, extracting the blood Bi Tong according to the optimal extraction process, and preparing 3 batches of test samples. Accurately weighing astragaloside IV standard, adding methanol to prepare standard solution with concentration of 0.05 μ g/μ L, 0.10 μ g/μ L, 0.25 μ g/μ L, 0.50 μ g/μ L, and 0.75 μ g/μ L, sequentially injecting 10 μ L, and obtaining data by high performance liquid chromatography.

Thin-layer chromatography for identifying medicinal materials

2.2.2.1 thin-layer identification of radix astragali (other components are identified slightly): taking 1g of Xuebaitong extract powder, adding 15 mL of ethanol solution, heating and refluxing for extraction for 20min, cooling, filtering, suspending, adding 7.5mL of 0.3% sodium hydroxide solution, and dissolving by ultrasound. The solution was filtered, the pH was adjusted to 5-6 with dilute hydrochloric acid, and 7.5mL of ethyl acetate was added and extracted with shaking. And taking the ethyl acetate layer, filtering the ethyl acetate layer by using filter paper paved with a proper amount of anhydrous sodium sulfate, suspending the filtrate, adding 1mL of ethyl acetate, and ultrasonically dissolving to prepare a sample solution to be detected. The control solution and the negative control solution are prepared by the same method. Sucking 10 μ L of the above solutions, respectively dropping on the same silica gel G thin layer plate, uniformly spreading all test points with chloroform and methanol (10: 1) as developing agent, taking out the silica gel plate, air drying, fumigating the silica gel plate in ammonia vapor, and inspecting under ultraviolet lamp (365 nm).

Determination of content of active ingredient

2.2.3.1 determination of astragaloside IV: chromatographic column InertSustain C18 (5 μm, 4.6X 250 mm) using octadecylsilane chemically bonded silica as filler, mobile phase selected from acetonitrile-water (35: 65), evaporative light scattering detector, flow rate of 1.0 mL/min, column temperature of 40 deg.C, drift tube temperature of 40 deg.C. Taking the sample amount (mug) of the astragaloside IV standard substance as a horizontal coordinate, and taking a peak area as a vertical coordinate to draw a standard curve; accurately weighing astragaloside IV standard, adding methanol to prepare standard solution with concentration of 0.05 μ g/μ L, 0.10 μ g/μ L, 0.25 μ g/μ L, 0.50 μ g/μ L, and 0.75 μ g/μ L, sequentially injecting 10 μ L, and obtaining data via high performance liquid phase; obtaining the linear regression equation y =2157223.918x-609361.5306 of astragaloside IV and the correlation coefficient R²=0.9956, which shows that the linear relation is good between 0.5-7.5 mug (figure 2), and the corresponding peak areas of three batches (20190413, 20190428 and 20190511) of samples to be tested are brought in, so that the content of the astragaloside in each 1g of extract powder is 96.172 mug, 98.557 mug and 97.265 mug respectively; the astragaloside obtained by three tests has similar content, and the extraction process is proved to be feasible and stable in effect.

2.2.3.2 determination of tanshinone IIA content: accurately weighing tanshinone IIA standard substance, adding methanol solution to prepare standard substance solutions with concentrations of 0.05 μ g/μ L, 0.10 μ g/μ L, 0.25 μ g/μ L, 0.50 μ g/μ L and 1.00 μ g/μ L, sequentially injecting 10 μ L, and obtaining data through high performance liquid chromatography.

2.2.3.3 paeoniflorin content determination: accurately weighing paeoniflorin standard, adding methanol solution to prepare standard solutions with concentrations of 1.00 μ g/μ L, 2.50 μ g/μ L, 5.00 μ g/μ L, 10.10 μ g/μ L and 20.00 μ g/μ L, sequentially injecting 10 μ L, and obtaining data through high performance liquid chromatography.

2.2.3.4 determination of trachelospermin content: precisely weighing a standard substance of the trachelospermin, adding methanol to prepare standard substance solutions with the concentrations of 0.50 mu g/mu l, 1.00 mu g/mu l, 2.50 mu g/mu l, 5.10 mu g/mu l and 10.00 mu g/mu l, sequentially injecting 10 mu l, and obtaining data through a high performance liquid phase.

Research on granulation and filling processes

2.2.4.1 research on granulation adjuvants comprises weighing 5g of XUEBI extract powder, mixing with 5g of adjuvants (sucrose, lactose, dextrin, and starch) at a ratio of 1:1, spraying 85% ethanol, kneading, mechanically pressing, sieving with 20 mesh sieve, and drying at 40 deg.C. And sieving with a 40-mesh sieve to remove dust to obtain the blood arthralgia removing granules.

2.2.4.2 research on the ratio of the extract powder to adjuvants, wherein dextrin is selected as adjuvant, and the ratio of Xuebitong extract powder is increased to increase the content of medicinal components in the final granule and reduce the dosage.

2.2.4.3 bulk density determination: three batches (20190413, 20190428, 20190511) of samples were weighed accurately, the contents pellets were placed in a 25 mL measuring cylinder, and the cylinder was vibrated up and down 5 times to measure the pellet volume.

In-vitro experimental method for treating thromboangiitis obliterans by blood obstruction

2.2.6.1 extraction of serum: 30 clean SD rats were randomly divided into 3 groups of 10 rats each, and divided into a Xuebitong administration group (2.56 g/kg), a positive drug group (0.052 g/kg), and a blank control group (equivalent amount of physiological saline). After adaptive feeding for 7 days, beginning intragastric administration, performing intragastric administration for 7 days according to the dose, performing intragastric injection for 2 hours on the 7 th day, performing intraperitoneal injection on a rat anesthetization by 10% chloral hydrate (0.33 mL/kg), fixing the rat by using a rat plate when the muscle of the rat is free of tension, taking blood from the abdominal aorta, and placing the taken whole blood into a refrigerator at 4 ℃ for standing for 2 hours; taking out whole blood, centrifuging at 3000rpm for 10min, centrifuging, collecting supernatant, subpackaging, inactivating in water bath at 56 deg.C for 30min, filtering with 0.45 μm filter membrane, subpackaging, and storing in-20 deg.C refrigerator.

2.2.6.2 cell culture

2.2.6.2.1 cell recovery: taking out the frozen HUVEC cells in liquid nitrogen, quickly putting into a water bath kettle at 37 ℃ for melting, transferring into a container after meltingPut into a centrifuge tube of 15 mL and centrifuge at 1000 rpm for 5 min. Taking out 15 mL centrifuge tube, discarding supernatant, adding 1mL DMEM medium containing 10% BS (calf serum), resuspending, transferring into culture dish containing 7mL prepared medium, observing under the mirror, standing at 37 deg.C, and 5% CO₂Culturing in a saturated humidity incubator.

2.2.6.2.2 cell passage: culturing cells with DMEM medium containing 10% BS, removing culture medium when cell surface area is estimated to be about 80% of total plate area in 2-3 days, washing residual culture medium on cell surface with PBS, adding pancreatin, adding 5% CO at 37 deg.C₂Digesting for 2 min in a saturated humidity incubator, adding a prepared culture medium to terminate digestion after cell morphology becomes round, repeatedly blowing gently to blow off adherent cells, transferring the cell-containing culture medium into a 15 mL centrifuge tube, placing the centrifuge tube into a centrifuge, centrifuging for 5min at 1000 rpm, taking out the 15 mL centrifuge tube from the centrifuge, discarding supernatant, resuspending with 3 mL culture medium, sucking 1mL cell suspension after blowing uniformly, adding the cell suspension into a flat dish, placing the flat dish into a container at 37 ℃, and adding 5% CO₂Culturing in a saturated humidity incubator.

2.2.6.3 determination of H₂O₂Optimal injury concentration to HUVEC cells: HUVEC were digested and plated into 96-well plates at 6000 cells per well. The experiment was divided into 6 groups of 4 multiple wells, each group being: (ii) a blank control group (15% blank control group serum); ② H₂O₂Lesion group (15% placebo serum); ③ Xubi Tong high dose group (15% Xubi Tong administration group serum); fourthly, a blood arthralgia removing middle dose group (10 percent of blood arthralgia removing administration group serum and 5 percent of blank control group serum); low dosage group for blood arthralgia syndrome (5% blood arthralgia syndrome administration group serum +10% blank control group serum); sixthly, the positive control group of the compound salvia tablet (10 percent of positive medicine group serum and 5 percent of blank control group serum). Culturing in normal culture medium for 12h, removing the upper layer culture medium after cell adherence, adding 100 μ L prepared serum-containing culture medium into each well, removing the upper layer culture medium after culturing for 24 h, and adding 100 μ L medium containing 1200 μm H into each well except blank control group₂O₂The serum-containing medium (2) was cultured for 12 hours, and then the medium was discarded, and 100. mu.L of the prepared CCK8 reagent (800. mu.L of the medium + 160. mu.LC) was added to each wellCK8 reagent) was incubated for 2-4h and absorbance was measured at 490 nm. The above experiment was repeated with setting H₂O₂The lesion concentration gradient was 1200 μm, 1000 μm, 800 μm, 600 μm. Finally, 800 μm H was selected₂O₂Damage was the optimal damage concentration.

2.2.6.4 CCK8 cell viability: repeating the above experimental steps, selecting 800 μm H₂O₂To assess the concentration, cell viability was measured.

2.2.6.5ROS (reactive oxygen species) measurement of intracellular reactive oxygen species concentration: HUVEC were digested and then expressed at 2X 10⁵Each cell was plated in 6-well plates, and the experiments were divided into 6 groups, each: (ii) a blank control group (15% blank control group serum); ② H₂O₂Lesion group (15% placebo serum); ③ Xubi Tong high dose group (15% Xubi Tong administration group serum); fourthly, a blood arthralgia removing middle dose group (10 percent of blood arthralgia removing administration group serum and 5 percent of blank control group serum); low dosage group for blood arthralgia syndrome (5% blood arthralgia syndrome administration group serum +10% blank control group serum); sixthly, the positive control group of the compound salvia tablet (10 percent of positive medicine group serum and 5 percent of blank control group serum). Culturing in normal culture medium for 12h, removing the upper layer culture medium after cell adherence, adding 1mL prepared serum-containing medium into each well, removing the upper layer culture medium after culturing for 24 h, and adding 1mL culture medium containing 800 μm H into each well except blank control group₂O₂The serum-containing medium of (1), after 12h of injury, the medium was discarded by aspiration, 1mL of PBS (phosphate-buffered saline) was added to each well to wash the medium, 500. mu.L of pancreatin was added to each well until cell morphology rounding was observed, 500. mu.L of medium was added to each well to stop digestion, the cells were blown off, separately aspirated into 1.5mL of EP tubes, centrifuged at 170G for 5min, the supernatant was removed and discarded, 100. mu.L of ROS reagent (DMEM: ROS =1000: 1) was added to each tube, the cells were resuspended, placed at 37 ℃ and 5% CO₂Incubating in a saturated humidity incubator for 30min, taking out every 5min, and mixing uniformly. Taking out after 30min, placing in centrifuge, centrifuging for 5min at 170G, discarding supernatant, resuspending with 500 μ L PBS to wash ROS reagent to avoid dark background during detection, repeatedly washing for 3 times, resuspending with 200 μ L PBS for the last time, sucking 8 μ L, adding into counting plate, counting with 10 wells⁴Individual cellThe cells were plated in a 96-well plate and 4 wells were prepared, with an excitation light of 488nm and absorbance at 525 nm.

2.2.6.6 analysis of H under protection of blood impediment by Western felt method₂O₂Changes in the level of endogenous protein expression in damaged cells

2.2.6.6.1 Total protein extraction: the above cultured cell assay procedure was repeated, and after 12 hours of injury, the upper medium was discarded, and 100. mu.L of cell lysate (1% Triton X-100, 50mM HEPES, 50mM sodium pyrophosphate, 100 mM NaF, 10 mM EDTA (pH 8.0), 10 mM sodium vanadate (pH 10.0), 100. mu.g/mL aprotinin, 5. mu.g/mL leupeptin, 4. mu.g/mL pepstatin) was added to each well to lyse the cells, repeatedly scraping and grinding cells in the holes on ice by using a scraper to fully crack the cells, subpackaging the cells into 1.5mL EP tubes, repeatedly freezing and thawing for 3 times, 30min for each time, uniformly mixing after the third freezing and thawing, centrifuging at 4 ℃ and 12000 rpm for 30min, taking out the EP tubes, sucking supernatant, labeling names, quantifying protein by using a BCA method, preparing standard curve concentration, and adding 100 mu L of BCA working solution into each hole (A: B =50: 1). Incubating at 37 ℃ for 30min, and detecting the ultraviolet absorbance at 562 nm. And finally calculating the concentration of the target protein according to a standard curve, adding a proper amount of lysate and protein buffer solution, uniformly mixing, putting into a boiling water bath, boiling for 5min, and storing in a refrigerator at the temperature of-20 ℃.

2.2.6.6.2 Western blotting: the sample was removed from the-20 ℃ freezer, vortexed, centrifuged until the sample was completely thawed, and the sample was added to the lane, followed by 5 μ L marker in the last lane. And after the sample completely runs out, preparing a membrane transferring solution, methanol, a rubber cutting plate, filter paper and a PVDF membrane. Cutting a PVDF membrane according to the size of a rubber block, putting the PVDF membrane into methanol for activation, then soaking the PVDF membrane into a membrane transferring solution, placing the PVDF membrane, the rubber, the filter paper, the foam and the negative electrode in sequence, placing the PVDF membrane, the rubber, the filter paper, the foam and the negative electrode in a membrane transferring groove after clamping, injecting the membrane transferring solution, and performing constant flow of 250 mA. And after the membrane is transferred, taking out the membrane, putting the membrane into lithospermum, shaking the membrane for 10min by a shaking table, fully dyeing, separating samples according to the molecular weight of the protein, and labeling the protein name. Washing membrane with TBST, cleaning in shaking table for 10min for 3 times, cleaning excess membrane transfer solution and dye, preparing 5% confining liquid, and sealing on shaking table for 1 hr. The blocking solution was washed with TBST, primary antibody was added, and the mixture was placed on a 4 ℃ freezer overnight in a shaker. After being taken out, the mixture is washed for 3 times of 10min by TBST. Preparing sealing liquid, and sealing in a shaking table for 1 h. And adding a secondary antibody after the sealing is finished, and putting the mixture into a shaking table for incubation for 1 h. After being taken out, the mixture is washed for 3 times of 10min by TBST.

2.2.6.7GSH (glutathione) kit for detecting the content of glutathione: extracting protein by the same method at the early stage, preparing each reagent of GSH according to the instruction, adding an enzyme label plate according to table 2.2 to prepare double-hole, adjusting the optical path to zero at 420 nm by 1 cm, measuring the OD value of each hole, and calculating the content of GSH according to the OD value, the protein concentration and a formula.

2.2.6.8LDH (lactate dehydrogenase) kit for detecting lactate dehydrogenase content: treating cells by the same method at the early stage, adding 500 mu L of culture medium when the cells are damaged in order to achieve the lowest detection concentration, culturing for 12h, then sucking the supernatant to be detected, preparing a detection reagent according to the instruction, adding the detection reagent according to the table 2.3, making double-hole wells, standing for 5min at room temperature, measuring an OD value by using an enzyme labeling instrument at 450 nm, and calculating the LDH content according to the OD value and a formula.

2.2.6.9 MDA kit for detecting malondialdehyde concentration comprises extracting protein with the same method at the earlier stage, preparing MDA reagent according to the instruction, adding into 5mL EP tube according to Table 2.4, mixing, tightening the tube with preservative film, pricking with needle, decocting 5mL EP tube in 95 deg.C water bath for 40 min, taking out, cooling with flowing water, and centrifuging at 4000 rpm for 10min to obtain complete precipitate. Adding 100 μ L of each well into an enzyme label plate, making double wells, adjusting the light path at 532 nm by 1 cm, adjusting to zero with distilled water, measuring the OD value of each well, and calculating the MDA content according to the OD value, the protein concentration and a formula.

2.2.6.10 NO kit for detecting the concentration of nitric oxide: extracting protein by the same method at the early stage, preparing an NO reagent according to the specification, sequentially adding the NO reagent into an enzyme label plate according to the table 2.5, standing for 10min at 550 nm, adjusting the optical path to 0.5 cm, adjusting the distilled water to zero, measuring the OD value of each hole, and calculating the NO content according to the OD value, the protein concentration and a formula.

2.2.6.11 SOD (superoxide dismutase) kit for detecting the concentration of nitric oxide: extracting protein by the same method at the earlier stage, preparing SOD reagent according to the instruction, sequentially adding into enzyme label plate according to table 2.6, incubating in incubator at 37 deg.C for 20min, measuring OD at 450 nm, and calculating SOD content according to OD value, protein concentration and formula.

2.2.7 in vivo Experimental method for treating thromboangiitis obliterans by Xuebitong

2.2.7.1 modeling of experimental animal: preparing 10% chloral hydrate anesthetic and 10 mg/mL sodium laurate solution, wherein the sodium laurate solution needs to be filtered and sterilized by a 0.45 mu m filter membrane after being prepared. Injecting 10% chloral hydrate anesthetic into the abdominal cavity of SD rat at the dose of 0.33 mL/kg, fixing the rat with rat plate when the rat loses muscle tension, depilating with appropriate amount of depilatory cream, and sterilizing with iodophor for 3 times. Cutting an incision of about 1 cm at the midpoint of a groin, separating subcutaneous tissues and muscle tissues and dissociating a femoral artery sheath, completely separating the femoral artery, clamping the femoral artery at the proximal end by using a blood vessel clamp, blocking blood flow, injecting 0.1 mL of prepared 10 mg/mL sodium laurate solution to the distal end (the femoral artery clamp is shown in figure 1), observing the whitening of the blood vessel at the distal end of the femoral artery by naked eyes, rapidly pulling out a needle head and pressing for hemostasis, suturing muscle layers and epithelial layers when no blood flows out, and injecting 40 ten thousand units of penicillin into the abdominal cavity to prevent infection. After the operation is finished, the rat is placed under an electric oven in a supine position to slowly revive, and is placed in a mouse cage after the rat revives. FIG. 1 shows the femoral artery of a free rat, the proximal end of the femoral artery is clamped and prepared for injection of 0.1 mL of a 10 mg/mL sodium laurate solution.

2.2.7.2 grouping and administration of experimental animals: successfully molded rats were randomly divided into 5 groups: firstly, a blood arthralgia removing high dose group (the medicine feeding dose is 2.56 g/kg/d), secondly, a blood arthralgia removing low dose group (the medicine feeding dose is 1.28 g/kg/d), thirdly, a compound red sage root tablet positive medicine group (the medicine feeding dose is 0.052 g/kg/d) and fourthly, a pseudo-operation group (the same dose of normal saline is fed every day); model group (feeding normal saline with same dosage every day). Sequentially perfusing into stomach for 7 days according to the dosage.

2.2.7.3 evaluation of lesion rating in rats: fasting is carried out on the intragastric administration day 6 and water is not forbidden, the lesion grade of the rats after the model building is observed by naked eyes before the intragastric administration day 7, and the lesion grade of the rats is as follows: stage I: lesions are localized to the toenail; and II, stage: lesions are localized to the toe; grade III: lesions confined to the paw portion; IV stage: lesions are confined below the knee joint; and V stage: the lesions developed above the knee joint.

2.2.7.4 abdominal aorta blood was taken and rat femoral artery was intercepted: the rats were injected with 10% chloral hydrate in the abdominal cavity, and when the rats lost muscle tension, the rats were fixed with a rat plate and tested by taking blood from abdominal aorta using an anticoagulant blood collection tube containing 3.8% sodium citrate. Cutting the groin along the midpoint of the groin at the operation side, dissociating the femoral artery sheath, separating femoral vein and femoral artery, taking the femoral artery below the injection site, fixing with 10% formalin for 3 days, and performing examination to obtain slices.

2.2.7.5 anti-platelet aggregation assay: rat whole blood was taken in an amount of 1mL, transferred into a 1.5mL EP tube, and centrifuged at 1500rpm for 5min to obtain a supernatant of Platelet-rich plasma (PRP). And sucking 200 mu L of PRP, injecting the PRP into a cuvette provided with a stirrer, and placing the cuvette in a rear constant temperature hole for later use. The remaining plasma was centrifuged at 3000rpm for 10min, and the resulting supernatant was Platelet-poor plasma (PPP). And (3) sucking 200 mu L of supernatant, injecting the supernatant into another cuvette, placing the cuvette into a detection hole, automatically detecting and zeroing, taking out a PPP cup, inserting the PPP cup into a PRP cup, sucking 10 mu L of prepared adenosine diphosphate (ADP 75 mu mol/L) by using a microsyringe, quickly pushing the prepared adenosine diphosphate into a sample to be detected, and measuring the platelet aggregation rate and the maximum aggregation rate for 1, 3 and 5 min.

2.2.7.6 platelet adhesion experiments: rat whole blood was taken 1mL and centrifuged at 1000 rpm for 5 min. And (3) adding 380 mu L of platelet diluent into 20 mu L of centrifuged plasma for dilution, and uniformly mixing. And adding 10 mu L of diluted plasma into a cell counting plate, and standing for 3 min. Platelet counts were performed using light microscopy. Adding the residual plasma into a spherical glass bottle of an LBY-5F platelet adhesion instrument, rotating at 3.7 rpm for 15 min, taking 10 mu L of the adhered plasma, adding the plasma into a cell counting plate, standing for 3 min, counting platelets of the adhered plasma by using an optical microscope, and calculating the platelet adhesion rate.

Platelet adhesion rate (%) = (number of platelets before adhesion-number of platelets after adhesion)/number of platelets before adhesion × 100%

2.2.7.7 in vitro thrombosis test: 1.5mL of whole blood was taken, and the whole blood was added to a siliconized thrombus tube, and the tube was rotated clockwise at 37 ℃ for 15 min by using an in vitro thrombometer at 3.7 rpm. Taking off the sleeve, pouring out the thrombus on the filter paper which is weighed to be net weight, putting the filter paper and the thrombus into a constant-temperature drying box at 37 ℃, drying for 30min, weighing the weight (mg) of the thrombus, and calculating the thrombus inhibition rate.

Thrombus inhibition ratio (%) = (control thrombus dry weight-administered group thrombus dry weight)/control thrombus dry weight × 100%

2.2.7.8TXB₂(thromboxane B)₂)、6-k-PGF_1α(6-keto prostaglandin F)_1α) ET-1, IL-6, CRP content determination: whole blood is taken and kept stand for 4h at room temperature, then centrifuged for 20min at 3000rpm, and the supernatant is taken and used as an experimental sample.

2.2.7.8.1TXB₂、6-k-PGF_1αThe determination of (1): after 50. mu.L of the standard working solution (sample) was added to the corresponding plate well, 50. mu.L of the biotinylated antibody working solution was immediately added thereto, and the mixture was incubated in a constant temperature incubator at 37 ℃ for 45 min. The well plate was removed, the liquid in the plate was discarded, and the well plate was washed 3 times repeatedly. After washing, 100 mu L of HRP enzyme conjugate working solution is added into each hole, and the mixture is placed into a constant temperature incubator and incubated at 37 ℃ for 30 min. The well plate was removed and the liquid in the plate was discarded, and the well plate was washed repeatedly 5 times. Adding 90 μ L of substrate solution into each well after washing, placing into a constant temperature incubator, incubating at 37 deg.C for 15 min, and adding 50 μ L into each wellStopping, immediately detecting OD value at 450 nm, and calculating TXB according to the standard curve and the OD value₂、6-k-PGF_1αThe content of (a).

2.2.7.8.2 ET-1, IL-6 and CRP content determination: add 100. mu.L of the standard working solution (sample) to the corresponding well plate, put into a constant temperature incubator, and incubate at 37 ℃ for 90 min. Taking out the pore plate, discarding the liquid in the plate, immediately adding 100 μ L of biotinylated antibody working solution, placing in a constant temperature incubator, and incubating at 37 deg.C for 60 min. The plate was removed and the liquid was discarded and washed 3 times. After washing, 100 mu L of HRP enzyme conjugate working solution is added into each hole, and the mixture is placed into a constant temperature incubator and incubated at 37 ℃ for 30 min. The well plate was removed and the liquid in the plate was discarded, and the well plate was washed repeatedly 5 times. And after washing, adding 90 mu L of substrate solution into each hole, putting the substrate solution into a constant-temperature incubator, incubating for 15 min at 37 ℃, adding 50 mu L of termination solution into each hole, immediately detecting the OD value at the wavelength of 450 nm, and calculating the contents of ET-1, IL-6 and CRP according to the standard curve and the OD value.

Statistical analysis

Data are expressed as Mean. + -. standard deviation (Mean. + -. SEM) andP<0.05 judged that the difference was statistically significant.

Preparation process of blood arthralgia treating extract powder

3.1.1 Xuebitong ethanol heating reflux extraction process research result

On the premise of confirming that the extraction efficiency of ethanol is higher than that of water, in order to explore the optimal extraction process of blood Bi-syndrome, the application designs an orthogonal test astragalus, the extraction efficiency is reflected by detecting the content of astragaloside under each extraction method (table 3.1), and the influence degree on the extraction efficiency of astragaloside is sequentially from large to small as compared with the deviation in the visual analysis (table 3.2): the ethanol concentration is more than the solvent amount is more than the extraction times is more than the extraction time, factors such as production cost, time consumption and the like are comprehensively considered, and the final optimal extraction conditions are determined as follows: extracting with 80% ethanol solution at a ratio of 1:8 for 2 times, each for 1 hr.

3.1.2 validation of the optimal extraction Process

In order to verify the authenticity of the optimal extraction process, a standard curve is drawn by taking the sample volume (mug) of the astragaloside standard as a horizontal coordinate and taking the peak area as a vertical coordinate. Obtaining the linear regression equation y =2157223.918x-609361.5306 of astragaloside IV and the correlation coefficient R²=0.9956, which shows that the linear relationship is good between 0.5-7.5 μ g (fig. 2), and the corresponding peak areas of three batches (20190413, 20190428, 20190511) of samples to be tested are substituted to obtain the contents of astragaloside 96.172 μ g, 98.557 μ g and 97.265 μ g in each 1g of extract powder. The astragaloside obtained by three tests has similar content, and the extraction process is proved to be feasible and stable in effect. FIG. 2 astragaloside IV standard curve.

Thin layer identification results

3.2.1 thin layer identification of Astragalus membranaceus

According to an identification method under the item of astragalus root in the 2015 edition of Chinese pharmacopoeia, the thin-layer chromatography of the astragalus root to be detected in the blood bi-tong is basically consistent with that of the standard astragalus root (figure 3), and the effective astragalus root ingredients in the blood bi-tong are determined. FIG. 3 shows TLC chromatogram of radix astragali in the preparation under ultraviolet lamp (365 nm). (1-3 radix astragali solution to be tested, 4 radix astragali standard medicinal materials, 5 negative control solution)

3.3 measurement results of effective Components

3.3.1 measurement results of tanshinone IIA content

In order to determine the content of tanshinone IIA in the Xuebitong extract powder, a standard curve is drawn by taking the sample amount (mu g) of tanshinone IIA as a horizontal coordinate and taking a peak area as a vertical coordinate. Obtaining tanshinone IIA linear regression equation y =42276x +4832 and correlation coefficient R²=0.9986, which shows that the linear relation is good between 0.5-10.1 μ g (fig. 4), and the corresponding peak areas of three batches (20190413, 20190428, 20190511) of samples to be tested are brought in, so as to obtain the tanshinone IIA content of 2.968mg, 2.979mg and 2.894mg in each 1g of Xuebaitong extract powder. The tanshinone IIA content measured by three tests is similar, which proves that the blood arthralgia is relievedThe extract powder has stable extraction effect on effective component tanshinone IIA. See fig. 4 tanshinone IIA standard curve.

Determination result of paeoniflorin content

In order to determine the paeoniflorin content in the Xuebitong extract powder, a standard curve is drawn by taking the sampling amount (mu g) of the paeoniflorin as a horizontal coordinate and taking a peak area as a vertical coordinate. Obtaining paeoniflorin linear regression equation y =24914x +11078 and correlation coefficient R²=0.9996, which shows that the linear relation is good between 10.0-200.0 μ g (fig. 5), and the corresponding peak areas of three batches (20190413, 20190428, 20190511) of samples to be tested are brought in, and the paeoniflorin content in each 1g of Xuebitong extract powder is 39.650 mg, 40.121 mg, 39.244 mg. The paeoniflorin content measured by the three tests is similar, and the result proves that the effect of extracting the effective component paeoniflorin in the Xuebitong extract powder is stable. FIG. 5 paeoniflorin standard curve.

Determination result of Trachelospermin content

In order to determine the ludwigitrin content in the blood impediment extract powder, a standard curve is drawn by taking the paeoniflorin sample amount (mug) as a horizontal coordinate and taking a peak area as a vertical coordinate. Obtaining a linear regression equation y =36362x-4853.4 of the trachelospermin and a correlation coefficient R²=0.9818, which shows that the linear relation is good between 5.0-100.0 μ g (fig. 6), and the corresponding peak areas of three batches (20190413, 20190428, 20190511) of samples to be tested are brought in, so as to obtain the contents of the ludwigite glycoside in 1g of extract powder, which are 2.750 mg, 2.631 mg and 2.668 mg. The contents of the trachelospermi glycosides measured by three tests are similar, and the stable extraction effect of the trachelospermi glycosides in the Xuebitong extract powder is proved. FIG. 6A standard curve for trachelospermin.

Research results of granulation filling

3.4.1 granulation excipients

Comparing the influence of different auxiliary materials on granulation (table 3.3), the fluidity requirement of capsule production can be met when the repose angle is less than 40 degrees, so that three auxiliary materials of sucrose, lactose and dextrin can be selected, and the dextrin is selected as the auxiliary material in consideration of the difficulty and the cost of granulation.

3.4.2 research result on the mixture ratio of extract powder and auxiliary materials

Experiments prove that the ratio of the extract powder to the auxiliary materials has influence on granulation (Table 3.4), factors such as angle of repose, yield, production cost and the like are comprehensively considered, and the ratio of the extract powder to dextrin is 6:4 as the production ratio.

3.4.3 bulk Density measurement results

Knowing the mass m and volume V of the particles, the bulk density of the particles was calculated according to the formula ρ = m/V, giving an average bulk density of the particles of 0.4714 g/mL (table 3.5), which can be filled using a size 0 capsule.

3.6 in vitro test results of therapeutic action of Xuebitong on thromboangium obliterans

3.6.1 results of CCK8 assay for cellular Activity

Through detecting the activity of the cells, the following results are found: and H₂O₂The injured group had some protection against HUVEC compared to the hebitong administration group and increased with increasing administration dose (fig. 7). FIG. 7: adding 800 mu M H in each group except for the normal saline group₂O₂The damage is caused to the patient,^#，P＜0.05，^##，P< 0.01 in comparison to saline group;^*，P＜0.05，^**，P< 0.01 and H₂O₂And (4) comparing the damage groups.

Results of detecting cellular Activity

Detection Using ROS reagent, discovery with H₂O₂The injured group was effective in reducing intracellular reactive oxygen species concentration compared to the blood Bi-Tong administration group, and the effect was enhanced as the administration dose was increased (FIG. 8). FIG. 8: adding 800 mu M H in each group except for the normal saline group₂O₂The damage is caused to the patient,^#，P＜0.05，^##，P< 0.01 andcomparing the normal saline group;^*，P＜0.05，^**，P< 0.01 and H₂O₂And (4) comparing the damage groups.

Detection result of related protein by protein immunoblotting method

3.6.4GSH kit for detecting intracellular glutathione concentration result

Detecting GSH value, finding and H₂O₂The damaged group was able to effectively maintain glutathione concentration in the body and increased with increasing dose compared to the Xuebitong administration group (fig. 9). FIG. 9: adding 800 mu M H in each group except for the normal saline group₂O₂The damage is caused to the patient,^#，P＜0.05，^##，P< 0.01 in comparison to saline group;^*，P＜0.05，^**，P< 0.01 and H₂O₂And (4) comparing the damage groups.

Result of kit for detecting concentration of lactate dehydrogenase in cell culture solution

Detection of LDH values, discovery and H₂O₂The damaged group was effective in reducing the concentration of lactate dehydrogenase in cell culture compared to the hebetong administration group, and was boosted with increasing administration dose (fig. 10). FIG. 10: adding 800 mu M H in each group except for the normal saline group₂O₂The damage is caused to the patient,^#，P＜0.05，^##，P< 0.01 in comparison to saline group;^*，P＜0.05，^**，P< 0.01 and H₂O₂And (4) comparing the damage groups.

Malondialdehyde) kit for detecting malondialdehyde concentration result

Detecting MDA value, finding and H₂O₂The damaged group was effective in reducing intracellular malondialdehyde concentration compared to the hebeth group and increased with increasing dose (fig. 11). FIG. 11: adding 800 mu M H in each group except for the normal saline group₂O₂The damage is caused to the patient,^#，P＜0.05，^##，P< 0.01 in comparison to saline group;^*，P＜0.05，^**，P< 0.01 and H₂O₂And (4) comparing the damage groups.

Nitric oxide) kit for determining concentration result of vascular endothelial relaxation factor

Detecting NO value, finding and H₂O₂The damaged group was able to effectively maintain the concentration of the intracellular vasodilator in comparison with the administered group, and was enhanced with the increase of the administered dose (fig. 12). FIG. 12: adding 800 mu M H in each group except for the normal saline group₂O₂The damage is caused to the patient,^#，P＜0.05，^##，P< 0.01 in comparison to saline group;^*，P＜0.05，^**，P< 0.01 and H₂O₂And (4) comparing the damage groups.

SOD kit for detecting concentration result of intracellular superoxide dismutase

Detecting SOD value, finding and H₂O₂The damaged group was able to effectively maintain the concentration of superoxide dismutase in the body compared to the Xuebitong administered group (fig. 13). FIG. 13: adding 800 mu M H in each group except for the normal saline group₂O₂The damage is caused to the patient,^#，P＜0.05，^##，P< 0.01 in comparison to saline group;^*，P＜0.05，^**，P< 0.01 and H₂O₂And (4) comparing the damage groups.

In vivo experimental result of therapeutic effect of Xuebaitong on thromboangiitis obliterans

3.7.1 animal model building result of thromboangiitis obliterans

After about 2 hours of operation, the operative limb rachitis and swelling of the rat are accompanied by the phenomenon of stress relief, and the operative limb of the rat falls in white body surface temperature and even turns purple and black on the next day of operation, and the operative limb is dragged during crawling, so that the model building is successful. And the model is successfully assembled and molded on the seventh day.

Evaluation of lesion grade in rats

The results show that the pathological change grades of rats in the administration group are obviously reduced compared with those in the model group, the pathological change grades are reduced along with the increase of the administration dose, and the pathological change grades of the rats in the high-dose blood arthralgia removing group are also obviously lower than those in the positive medicament group of the compound salvia tablets (Table 3.6).

3.7.3 inhibition of ADP-induced platelet aggregation in rats by Xuebitong

Grading the blood arthralgia removing and platelet aggregation resisting effects by a turbidimetric method, wherein the maximum aggregation rate of a model group is (66.88 +/-7.69)%, and compared with the model group, the blood arthralgia removing high dose group obviously reduces the maximum aggregation rate of platelets (the)P< 0.01). The platelet aggregation inhibition rate is increased with the increase of the administration dose, and the blood impediment high dose group and the blood impediment low dose group are respectively 10.44% and 25.45%. The results are shown in Table 3.7.

3.7.4 inhibition of platelet adhesion in rats by Xuebitong

The effect of blood blockage on platelet adhesion was evaluated by the rotating glass sphere method. Compared with model group (44.92 +/-8.36)% of the blood Bi-syndrome treating drug, the high and low dosage groups of the blood Bi-syndrome treating drug remarkably reduce the platelet adhesion rate (P< 0.05), the platelet adhesion inhibition rate increased with the increase of the administered concentration, 39.49% and 32.70%, respectively. The results are shown in Table 3.8.

3.7.5 inhibiting effect of Xuebitong on thrombosis

The inhibition of thrombosis by blood obstruction was reviewed by in vitro thrombosis experiments. Significantly reduced thrombus dry weight in the blood Bi Tong administration group compared to the model group (P< 0.01) and clearly shows a decrease in the dry weight of the thrombus with increasing dose, with thrombus inhibition rates of 26.56% and 39.44%, respectively. The results are shown in Table 3.9.

3.7.6 rat affected limb femoral artery section

The vascular intima of the rat in the sham operation group is normal in shape, the phenomenon of stripping or shedding does not occur, endothelial cells are tightly adhered to the vascular endothelium, the lumen is normal, and blood cells can be seen. The rats in the model group can see that endothelial cells are seriously exfoliated, the lumen is narrowed, and thrombosis is generated. Through the blood bi-tong treatment, the pathological change phenomenon of the rats in the blood bi-tong administration group is obviously improved compared with that in the model group, and the endothelial cell shedding degree, the lumen stenosis degree and the thrombosis degree are in a regression trend and are in dose dependence compared with the model group.

3.7.7 TXB₂、6-keto-PGF_1αDetermination of the content

Compared with the model group, the high-dose group and the low-dose group for treating the blood impediment can effectively reduce TXB₂Content and TXB₂、6-keto-PGF_1αRatio of two (PLess than 0.05) and effectively increases 6-keto-PGF_1αContent of (A)P< 0.05) (Table 3.10).

3.7.8 ET-1 (endothelin-1), IL-6 (leukocyte meso-6) and CRP (C reactive protein) content determination

Compared with the model group, the high-dose group and the low-dose group of the blood impediment can effectively reduce the contents of ET-1, IL-6 and CRP (the contents of ET-1, IL-6 and CRP)P< 0.05) (Table 3.11).

The foregoing description is only exemplary of the invention and is not intended to limit the spirit of the invention.

Claims

1. The medicine for treating thromboangiitis obliterans is characterized in that the medicinal effective components are prepared by the following raw material medicines in parts by weight: 25-35 parts of astragalus membranaceus, 12-18 parts of salvia miltiorrhiza, 15-25 parts of red paeony root, 12-18 parts of centipede, 8-12 parts of earthworm, 12-18 parts of ground beeltle, 15-25 parts of Chinese starjasmine stem, 12-18 parts of red tangerine peel and 12-18 parts of uncaria.

2. The medicament for treating thromboangiitis obliterans according to claim 1, wherein the capsule is prepared from the following raw material medicines in grams by weight: 30g of astragalus membranaceus, 15g of salvia miltiorrhiza, 20g of red paeony root, 15g of centipede, 10g of earthworm, 15g of ground beetle, 20g of Chinese starjasmine stem, 15g of red tangerine peel and 15g of uncaria.

3. A process for the preparation of a medicament according to claim 1 or 2 for the treatment of thromboangiitis obliterans, characterized by the following steps: extracting medicinal materials with 80% ethanol solution at a material-to-liquid ratio of 1:8 for 2 times, extracting for 1 hr each time, cooling, evaporating the extractive solution with rotary evaporator until the extractive solution is viscous but not wall-hung, transferring the extractive solution into evaporating dish, evaporating in 75 deg.C water bath until the extractive solution is pasty, and drying the evaporating dish in vacuum drying oven at 55 deg.C to obtain powder.

4. A method for measuring the content of a drug for the treatment of thromboangiitis obliterans according to claim 1 or 2, wherein the method comprises any one or a combination of the following:

(1) determination of astragaloside content

Using octadecylsilane chemically bonded silica as a chromatographic column InertSustain C18 with a mobile phase of acetonitrile-water =35:65, and an evaporative light scattering detector with a flow rate of 1.0 mL/min, a column temperature of 40 ℃ and a drift tube temperature of 40 ℃; taking the sample amount of the astragaloside IV standard substance mu g as a horizontal coordinate, and taking a peak area as a vertical coordinate to draw a standard curve; accurately weighing astragaloside IV standard, adding methanol to prepare standard solution with concentration of 0.05 μ g/μ L, 0.10 μ g/μ L, 0.25 μ g/μ L, 0.50 μ g/μ L, and 0.75 μ g/μ L, sequentially injecting 10 μ L, and obtaining data via high performance liquid phase; obtaining the linear regression equation y =2157223.918x-609361.5306 of astragaloside IV and the correlation coefficient R²=0.9956, which shows that the linear relation is good between 0.5-7.5 mug, and the corresponding peak areas of three batches of samples to be measured are brought in, so that the contents of the astragaloside in each 1g of extract powder are 96.172 mug, 98.557 mug and 97.265 mug respectively; the astragaloside obtained by three tests has similar content, and the extraction process is proved to be feasible and stable in effect;

(2) determination of tanshinone IIA content

Using octadecylsilane chemically bonded silica as a chromatographic column InertSustain C18 column of a filler, selecting acetonitrile and 0.02% phosphoric acid solution as a mobile phase, carrying out gradient elution, wherein the detection wavelength is 270 nm, the flow rate is 1.0 mL/min, and the column temperature is 20 ℃; accurately weighing tanshinone IIA standard substance, adding methanol solution to prepare standard substance solution with concentration of 0.05 μ g/μ L, 0.10 μ g/μ L, 0.25 μ g/μ L, 0.50 μ g/μ L, 1.00 μ g/μ L, sequentially injecting 10 μ L, and obtaining data by high performance liquid chromatography; taking the sample amount mu g of tanshinone IIA as a horizontal coordinate, and taking the peak area as a vertical coordinate to draw a standard curve; obtaining a tanshinone IIA linear regression equation y =42276x +4832, and a correlation coefficient R2=0.9986, which shows that the linear relation of the tanshinone IIA linear regression equation y =42276x +4832 and the correlation coefficient R2=0.9986 is good, and the tanshinone IIA content is 2.968mg, 2.979mg and 2.894mg in each 1g of Xuebitong extract powder by bringing the linear relation into corresponding peak areas of three batches of samples to be tested; the tanshinone IIA content measured by the three tests is similar, and the effect of extracting the effective component tanshinone IIA in the Xuebitong extract powder is stable;

(3) and (3) measuring the content of paeoniflorin:

using octadecylsilane chemically bonded silica as a chromatographic column InertSustain C18 column of a filler, selecting methanol and 0.05mol/L potassium dihydrogen phosphate solution as a mobile phase =40:60, detecting the wavelength at 230nm, the flow rate at 1.0 mL/min and the column temperature at 40 ℃; accurately weighing paeoniflorin standard substance, adding methanol solution to prepare standard substance solutions with concentrations of 1.00 μ g/μ L, 2.50 μ g/μ L, 5.00 μ g/μ L, 10.10 μ g/μ L and 20.00 μ g/μ L, sequentially injecting 10 μ L, and obtaining data through high performance liquid phase; taking the sampling amount mu g of paeoniflorin as a horizontal coordinate, and taking a peak area as a vertical coordinate to draw a standard curve; the linear regression equation y =24914x +11078 of paeoniflorin and the correlation coefficient R2=0.9996 show that the linear relation between 10.0-200.0 mug is good, the linear relation is brought into the corresponding peak areas of three batches of samples to be measured, and the paeoniflorin content in each 1g of Xuebitong extract powder is 39.650 mg, 40.121 mg and 39.244 mg; the paeoniflorin content measured by the three tests is similar, and the effect of extracting the effective component paeoniflorin in the Xuebitong extract powder is stable;

(4) and (3) measuring the content of the trachelospermin:

using octadecylsilane chemically bonded silica as a chromatographic column InertSustain C18 column of a filler, selecting acetonitrile and water as a mobile phase with a ratio of 30:70, a detection wavelength of 280nm, a flow rate of 1.0 mL/min and a column temperature of 40 ℃; precisely weighing a standard substance of the trachelospermin, adding methanol to prepare standard substance solutions with the concentrations of 0.50 mu g/mu l, 1.00 mu g/mu l, 2.50 mu g/mu l, 5.10 mu g/mu l and 10.00 mu g/mu l, sequentially injecting 10 mu l, and obtaining data through a high performance liquid phase; taking the sampling quantity mu g of the trachelospermin as a horizontal coordinate, and taking a peak area as a vertical coordinate to draw a standard curve; obtaining a trachelospermide linear regression equation y =36362x-4853.4, and a correlation coefficient R2=0.9818, which shows that the linear relation of the trachelospermide linear regression equation y =36362x-4853.4 is good, and the trachelospermide linear regression equation is brought into corresponding peak areas of three batches of samples to be measured, so that the trachelospermide content of each 1g of extract powder is 2.750 mg, 2.631 mg and 2.668 mg; the contents of the trachelospermi glycosides measured by three tests are similar, and the extraction effect of the trachelospermi glycosides in the Xuebi Tong extract powder for treating blood arthralgia is stable.