CN113521130B

CN113521130B - Traditional Chinese medicine granular preparation for treating scleroderma and preparation method thereof

Info

Publication number: CN113521130B
Application number: CN202111037267.4A
Authority: CN
Inventors: 王强; 秦万章; 杨春欣; 梁健
Original assignee: Zhongshan Hospital Fudan University
Current assignee: Zhongshan Hospital Fudan University
Priority date: 2021-09-06
Filing date: 2021-09-06
Publication date: 2022-07-29
Anticipated expiration: 2041-09-06
Also published as: CN113521130A

Abstract

The invention discloses a traditional Chinese medicine granular preparation for treating scleroderma and a preparation method thereof. The raw materials of the traditional Chinese medicine granular preparation for treating scleroderma comprise 15-45 parts by weight of salvia miltiorrhiza, 15-45 parts by weight of angelica sinensis, 15-45 parts by weight of astragalus membranaceus, 15-45 parts by weight of caulis spatholobi, 15-45 parts by weight of moutan bark and 15-45 parts by weight of red peony root. The invention also provides a preparation method of the traditional Chinese medicine granules for treating scleroderma. The invention can effectively extract the effective components of six raw material medicines, and the prepared granules have better drug effect than the original Danguihuo blood-activating mixture, more effectively control the quality and the dosage, are more convenient to take and carry, and have longer storage time.

Description

Traditional Chinese medicine granular preparation for treating scleroderma and preparation method thereof

Technical Field

The invention relates to a traditional Chinese medicine granular preparation for treating scleroderma and a preparation method thereof, belonging to the technical field of compound traditional Chinese medicine preparations.

Background

Scleroderma, also known as systemic sclerosis (SSc), is a systemic autoimmune disease characterized by fibrosis or sclerosis, atrophy of the connective tissue of the skin and internal organs, a widespread tissue fibrosis caused by multiple system inflammation, vascular lesions and immune disorders. The first diagnosis is made in dermatology, where the skin is swollen with erythema, followed by firmness and brightness, the grayish yellow is waxy, and the skin, subcutaneous tissue and muscle can be atrophied, often accompanied by Raynaud phenomenon; thereafter, damages of internal organs such as the lung and the kidney occur, and the accompanying pulmonary interstitial disease is one of clinically acute and serious diseases and also is a main cause of hospitalization and death of patients. From the pathogenesis, the following opinions exist in recent times: the theory of immunity; the theory of abnormal collagen synthesis; ③ the theory of blood vessels; and the theory of methylation, etc. These theories are quite similar to the Chinese medicine opinion on the onset of the disease, and belong to the category of arthralgia syndrome in Chinese medicine according to the expression of scleroderma.

In the seventh and eighty years of the last century, according to the fact that scleroderma is related to stagnation of qi and blood flow stasis, the indication of treatment rules of 'activating blood and dissolving stasis' in traditional Chinese medicine application is met, and the scleroderma is treated by blood-activating and blood-dissolving medicines such as 'salvia miltiorrhiza' at first in China. On the basis, according to the 'blood stasis pathogenesis' of scleroderma, a compound preparation of the salvia miltiorrhiza bunge is developed for treating various scleroderma, and in view of the fact that the scleroderma has more blood stasis symptoms, such as blood vessel incoordination and blood flow blocking phenomena such as skin hardening, joint ache, tongue stretching difficulty and the like, treatment rules of 'tonifying qi and activating blood, regulating qi and activating blood, activating blood circulation and removing blood stasis' are adopted for treating 335 cases of scleroderma patients, and the curative effect is over 94.2%; the medicine is best for promoting blood circulation and removing blood stasis, the total effective rate reaches 97.5%, and the significant efficiency is 43.1%. At present, in the active period of the disease, the state of the disease such as the pain of the relevant joints, the pulmonary interstitial disease, the critical occurrence of the kidney and the like is generally advocated, hormone and immunosuppressant are mainly adopted, and Dan Gui Huo Xue Jie is taken as the auxiliary; after the disease condition is stable, the treatment of the Dangui Huoxue mixture is mainly performed, and the dosage of hormone is reduced, so that the hormone is used less or not used at all in common medium and light scleroderma. Local scleroderma and acral scleroderma are preferably treated by using the Dan Gui Huo Xue mixture alone without hormone, and if necessary, hormone is added. From the view of the onset speed, the traditional Chinese medicine takes effect generally for 1 to 2 weeks, and is only second to the 'sub-rapid degree' of 'hormone'; has no obvious side effect and no influence on treatment. The Dangui blood-activating mixture can treat various scleroderma, and has good curative effects on various lupus erythematosus, dermatomyositis/polymyositis, sjogren syndrome, mixed connective tissue diseases, antiphospholipid antibody syndrome and overlap syndrome, rheumatoid arthritis, Behcet disease, vasculitis (called as vasculitis in traditional Chinese medicine, such as erythema nodosum, maculoid vasculitis, anaphylactoid purpura, cutaneous allergic nodular vasculitis, urticaria vasculitis, thrombophlebitis, various arteritis and the like), vitiligo, lichen planus, chronic prurigo nodularis, eczema and other dozens of diseases, has wide adaptation diseases and good feasibility and repeatability, and treats thousands of patients and achieves good effects only in the application condition of a small range in our hospital.

The Dangui Huoxue mixture is prepared with red sage, angelica, spatholobus stem, astragalus root, tree peony bark and red peony root. The salvia miltiorrhiza has the functions of promoting blood circulation to remove blood stasis and tranquilizing and allaying excitement after entering heart and liver channels, and is always regarded as an essential drug for promoting blood circulation and nourishing blood; the angelica can help blood circulation and has the functions of enriching and activating blood; caulis Spatholobi has effects of replenishing blood, promoting blood circulation and regulating menstruation, and contains chicken blood alcohol, and can increase leukocyte count of patient, improve microcirculation, and regulate menstruation; radix astragali has effects in enhancing immunity, invigorating qi, consolidating superficial resistance, arresting sweating, promoting wound healing, and expelling pus; cortex moutan has antiinflammatory, antibacterial, heat clearing away, blood cooling, immunity enhancing, antipyretic, and algefacient effects; radix Paeoniae Rubra has effects in clearing away heat, cooling blood, removing blood stasis, and relieving pain. The formula integrates the advantages of various medicines, supplements each other to achieve the aims of improving the curative effect and reducing the side effect, and the clinical application of the formula for nearly forty years is durable. The preparation method of the Dangui Huoxue mixture is a water decoction ethanol precipitation method, and a preparation prepared by adding a proper amount of sucrose has the advantages of complex components, difficult quality control, short preservation time, difficult carrying and unstable clinical curative effect, so the Dangui Huoxue mixture needs to be reformed.

Disclosure of Invention

The invention aims to provide traditional Chinese medicine granules for treating scleroderma and a preparation method thereof, which can overcome the defects that the quality of the Dangui Huoxue mixture is not easy to control, easy to mildew, difficult to carry and difficult to store, and are superior to the Dangui Huoxue mixture in the aspects of effective component enrichment, quality control, storage time, carrying, curative effect and the like.

In order to achieve the purpose, the preparation process of the Dangui Huoxue mixture is optimized, and the traditional Chinese medicine granular preparation for treating scleroderma is provided, wherein the raw materials comprise 15-45 parts by weight of salvia miltiorrhiza, 15-45 parts by weight of angelica sinensis, 15-45 parts by weight of astragalus membranaceus, 15-45 parts by weight of caulis spatholobi, 15-45 parts by weight of moutan bark and 15-45 parts by weight of red peony root.

The invention also provides a preparation method of the traditional Chinese medicine granular preparation for treating scleroderma, which comprises the following steps:

the first step is as follows: weighing the salvia miltiorrhiza, the angelica, the astragalus, the suberect spatholobus stem, the moutan bark and the red paeony root according to a proportion, mixing, adding 8-16 times of water for decocting twice, each time for 1 hour, combining decoctions, filtering, concentrating the filtrate, cooling, adding 6 times of water by weightFully stirring the 90vol% ethanol, standing for 24 hours, taking supernate, and concentrating the supernate under reduced pressure until the relative density is 1.2-1.3 g/cm ³ Obtaining fluid extract;

the second step is that: adding 0.6-1.8 parts by weight of dextrin, mixing, drying at 80 ℃, preparing dry powder, and filling into a plastic package bag to obtain the traditional Chinese medicine granular preparation for treating scleroderma.

In the first step, the weight ratio of the salvia miltiorrhiza, the angelica, the astragalus, the suberect spatholobus stem, the tree peony bark and the red paeony root is 2.5: 1.25: 2.5: 2.5: 1: 1.

the principle of the invention is as follows:

the following 3 extraction processes are drawn up according to the conventional extraction mode of traditional Chinese medicinal materials, and the specific steps are as follows:

process 1 (preparation method of original Dan Gui Huoxue mixture, namely water decoction and alcohol precipitation method):

taking the components in a weight ratio of 2.5: 1.25: 2.5: 2.5: 1: 1, extracting 6 Chinese medicaments of salvia miltiorrhiza, angelica, astragalus, suberect spatholobus stem, tree peony bark and red paeony root for 2 times, and adding 16/12 times of water respectively. Heating for 1 hr each time, mixing, concentrating, and cooling; adding 95vol% ethanol 2 times the weight of the extract, stirring, standing overnight, and concentrating the supernatant to obtain dry extract.

A process 2 (a partial medicinal material water decoction and alcohol precipitation method and a partial medicinal material ethanol reflux method):

according to the weight ratio of 6 traditional Chinese medicines of salvia miltiorrhiza, angelica, astragalus, suberect spatholobus stem, tree peony bark and red peony root of 2.5: 1.25: 2.5: 2.5: 1: 1, extracting 3 Chinese medicaments of salvia miltiorrhiza, astragalus and suberect spatholobus stem for 2 times, and adding 16/12 times of water respectively. Heating for 1 hour each time, mixing, concentrating, and cooling; adding 95vol% ethanol 2 times the weight of the extract, stirring, standing overnight, collecting supernatant, and recovering under reduced pressure to obtain dry extract (I).

And then 3 traditional Chinese medicines of angelica, moutan bark and red peony root are taken, 5 times of 90% ethanol is added for refluxing for 2 times, the reflux time is 1 hour each time, 2 times of extracts are combined, and the extracts are recovered under reduced pressure to be dry to obtain dry extract (II).

And (3) mixing the dry extract (I) and the dry extract (II).

And (3) a process (an ethanol reflux method of all medicinal materials):

taking the components in a weight ratio of 2.5: 1.25: 2.5: 2.5: 1: 1, adding 5 times of 90vol% ethanol into 6 traditional Chinese medicines of salvia miltiorrhiza, angelica, astragalus, suberect spatholobus stem, tree peony bark and red paeony root, refluxing for 2 times, refluxing for 1 hour each time, combining the extracts obtained by 2 times, and recovering under reduced pressure to be dry to obtain a dry extract.

And (3) evaluating the influence of the Dangui Huoxue granules preparation prepared by 3 different extraction processes on a scleroderma model mouse. The results of 3 extracts of the Danggui blood-activating granule preparation and the Sanchi Tongshu capsule in the scleroderma-improving mice are found as follows:

a first part: changes in the skin:

after the Danggui blood-activating preparation intervenes in a scleroderma model mouse experiment for 12 weeks, the skin is obviously improved, which is shown in the aspects of relevant inflammation and fibrosis of skin tissues:

(1) aspect of skin inflammation: compared with the skin inflammation degree of the scleroderma mouse model group (1.33 +/-0.47) and the normal group (0.44 +/-0.39), the difference is significant (P < 0.05), which indicates that the molding is successful; after 12 weeks of the intervention experiment, the skin inflammation of each intervention group is reduced to different degrees, and the groups with the best improvement degree are AH, AL and CL groups respectively. Wherein, compared with the model group (1.33 +/-0.47), the AH group (0.17 +/-0.24), the AL group (0.38 +/-0.33) and the CL group (0.75 +/-0.25) have statistical significance in difference (p values are all less than 0.05).

(2) The fibrosis aspect of the skin: after 12 weeks of intervention experiment, although the degree of skin fibrosis was decreased, it was found that the difference was not statistically significant (p > 0.05) in the AH group (0.31. + -. 0.11), BL group (0.30. + -. 0.17) and D group (0.27. + -. 0.19) in each intervention group compared with the model group (0.49. + -. 0.21).

(3) Aspect of skin thickness: after 12 weeks of the intervention experiment, the skin thickness of each group is reduced to different degrees, and the groups with the best improvement degree are AH and AL groups respectively. The differences between AH (199.98 + -18.02 μm) and AL (188.59 + -16.46 μm) were statistically significant (p values < 0.05) compared to model (235.68 + -13.31 μm).

(4) Detecting the content of hydroxyproline in the skin: after 12 weeks of the intervention experiment, the hydroxyproline content of each group of skin tissues is reduced to different degrees, and the groups with the best improvement degree are respectively AL, BH and CL. Compared with the model group (4453.23 +/-1099.18 mu g/g), the differences of AL (3024.68 +/-654.15 mu g/g), BH (2948.35 +/-1101.89 mu g/g) and CL (2992.37 +/-560.21 mu g/g) are all statistically significant (p values are all less than 0.05).

A second part: changes in the lung:

after the Danggui blood-activating preparation intervenes in a scleroderma model mouse experiment for 12 weeks, lung tissues are obviously improved, which is shown in the aspects of related inflammation and fibrosis of the lung tissues:

(1) Inflammation of lung tissue: compared with the normal group (0.69 +/-0.35), the difference of the lung tissue inflammation (1.33 +/-0.47) in the scleroderma model group is statistically significant (p = 0.03), which indicates that the molding is successful; after 12 weeks of intervention, the inflammation degree of lung tissue was reduced to different degrees, but the difference between AH group (0.42 + -0.34) and model group (1.33 + -0.47) was statistically significant (p < 0.05).

(2) Fibrosis of lung tissue: after 12 weeks of intervention, pulmonary fibrosis tended to be reduced to different degrees, but the differences between the groups compared with the model group (0.45. + -. 0.20) were not statistically significant (p > 0.05).

(3) Detecting the hydroxyproline content of lung tissues: after 12 weeks of intervention experiment, the hydroxyproline content of each group of lung tissues is reduced to different degrees, and the groups with the best improvement degree are AH, BH, CH and CL in sequence. Compared with the model group (349.22 +/-49.65 mu g/g), the AH group (253.92 +/-114.11 mu g/g), the BH group (212.86 +/-61.24 mu g/g), the CH group (229.05 +/-86.33 mu g/g) and the CL group (284.23 +/-46.23 mu g/g) have statistical significance (p values are all less than 0.05).

Through the chemical and pharmacodynamic researches, the preparation method of the traditional Chinese medicine granular preparation for treating scleroderma is determined:

6 Chinese medicinal materials of salvia miltiorrhiza, angelica, astragalus, suberect spatholobus stem, tree peony bark and red paeony root are extracted for 2 times, and 8-16 times of water is added. Heating for 1 hr each time, mixing, concentrating, and cooling; adding 95vol% ethanol 2 times the weight of the extract,stirring, standing for 24h, taking supernatant, and concentrating under reduced pressure until the relative density is 1.2-1.3 g/cm ³ Adding 0.6-1.8 parts by weight of dextrin, mixing, drying at 80 ℃, preparing dry powder, and filling into a plastic package bag to obtain the traditional Chinese medicine granule preparation for treating scleroderma.

Compared with the prior art, the invention has the beneficial effects that:

1. the preparation method can effectively extract the components with curative effect on scleroderma from the six raw material medicines, and the prepared granules have good drug effect;

2. the granule preparation prepared by the invention can effectively control the quality, is more convenient to take and carry, has accurate dosage and longer storage time.

Drawings

FIG. 1 is a graph of HE staining of pathological tissues of skin of mice in groups AH and AL (Process 1 high and low dose groups);

FIG. 2 is a HE staining pattern of pathological tissues of skin of CH and CL group (Process 3 high and low dose group) mice;

FIG. 3 is a staining graph of skin pathological tissue HE of mice in a positive control group (group D, positive control of SANQITONGSHU Capsule) and a negative control group (group E, SSc model group);

FIG. 4 is a graph showing HE staining of skin pathological tissues of normal mice (group F, blank control group);

FIG. 5 is a Masson staining pattern of pathological skin tissues of mice in AH and AL groups (Process 1 high and low dose group);

FIG. 6 is a Masson staining pattern of skin pathological tissues of BH, BL group (Process 2 high and low dose group) mice;

FIG. 7 is a Masson staining pattern of skin pathology in C H, C L (Process 3 high and low dose) mice;

FIG. 8 is a Masson staining chart of pathological tissues of the skin of mice in a positive control group (group D, positive control of SANQITONGSHU Capsule) and a negative control group (group E, SSc model group);

FIG. 9 is a Masson staining pattern of pathological tissues of the skin of normal mice (group F, blank control group);

FIG. 10 shows comparison of skin tissue inflammation levels (X + -S, N = 6-8) in scleroderma-treated mice;

FIG. 11 shows comparison of collagen fibers in skin tissues of scleroderma-treated mice (X + -S, N = 6-8);

FIG. 12 shows comparison of collagen content in skin tissues of scleroderma mice (X + -S,. mu.g/g, N = 6-8) in each group;

FIG. 13 is a comparison of the thickness of the skin tissue of scleroderma mice in each group (X + -S, μm, N = 6-8);

FIG. 14 is a graph of HE staining of pathological tissues of the lungs of mice in group AH and AL (Process 1 high and low dose);

FIG. 15 is a graph showing HE staining of pathological tissues of lungs of mice in BH and BL groups (Process 2 high and low dose groups);

FIG. 16 is a graph of HE staining of pathological tissues of the lungs of C H, C L (Process 3 high and low dose) mice;

FIG. 17 is a lung pathological tissue HE staining diagram of mice in a positive control group (group D, positive control of Sanchi Tongshu Capsule) and a negative control group (group E, SSc model group);

FIG. 18 is a graph showing HE staining of pathological tissues of lungs of normal mice (group F, blank control group);

FIG. 19 is a Masson staining pattern of pathological lung tissues of mice in AH and AL groups (Process 1 high and low dose group);

FIG. 20 is a graph showing Masson staining of pathological tissues of lung in mice in BH and BL groups (Process 2 high and low dose groups);

FIG. 21 is a Masson staining pattern of pathological lung tissue of C H, C L mice (Process 3 high and low dose group);

FIG. 22 is a Masson staining chart of lung pathological tissue of mice in a positive control group (group D, positive control of Sanchi Tongshu capsule) and a negative control group (group E, SSc model group);

FIG. 23 is a Masson staining pattern of pathological lung tissue of normal mice (group F, blank control group);

FIG. 24 shows the comparison of the degree of inflammation of lung tissues in each group (X + -S, N = 6-8);

FIG. 25 shows comparison of lung tissue collagen fibers (X + -S, N = 6-8) in scleroderma-treated mice;

FIG. 26 shows comparison of collagen content in lung tissue (X. + -. S, N = 6-8) in scleroderma-treated mice.

Detailed Description

In order to make the invention more comprehensible, preferred embodiments accompanied with figures are described in detail below.

Example 1

Preparing a traditional Chinese medicine granular preparation for treating scleroderma:

respectively placing 547.6g, 273.7g, 547.6g, 547.6g, 219.2g and 219.2g (2354.9 g) of 6 identified traditional Chinese medicines of the salvia, the angelica, the astragalus, the suberect spatholobus stem, the tree peony bark and the red paeony root into an extraction tank, mixing, respectively adding 8 times of weight of water for two times, each time for 1h, combining extracting solutions, concentrating, adding 90vol% ethanol (14.1294 kg) with 6 times of weight, standing for 24h, filtering, recovering ethanol from filtrate under reduced pressure, and concentrating until the relative density is 1.287g/cm ³ 411.00g of Dan Gui Huo fluid extract. Adding dextrin as a medicinal auxiliary material which does not interfere with medicinal components, and mixing the components according to the weight ratio of the salvia miltiorrhiza, angelica and the blood activating fluid extract: mass ratio of dextrin = 1: 0.6, drying at 80 ℃, preparing dry powder, and subpackaging in plastic bags, wherein the weight difference of the prepared finished product of the tea bag is small, and the quality of the granules is ensured to meet the regulations of Chinese pharmacopoeia.

Example 2

respectively placing 547.6g, 273.7g, 547.6g, 547.6g, 219.2g and 219.2g (2354.9 g) of 6 identified traditional Chinese medicines of the salvia, the angelica, the astragalus, the suberect spatholobus stem, the tree peony bark and the red paeony root into an extraction tank, mixing, respectively adding 8 times of weight of water for two times, each time for 1h, combining extracting solutions, concentrating, adding 90vol% ethanol (14.1294 kg) with 6 times of weight, standing for 24h, filtering, recovering ethanol from filtrate under reduced pressure, and concentrating until the relative density is 1.293g/cm ³ 519.4g of Danguihuo blood-activating fluid extract is obtained. Adding dextrin as a medicinal auxiliary material which does not interfere with medicinal components, and mixing the components according to the weight ratio of the salvia miltiorrhiza, angelica and the blood activating fluid extract: mass ratio of dextrin = 1: 1.2, drying at 80 ℃, preparing dry powder, and subpackaging in plastic bags, wherein the weight difference of the prepared finished product of the tea bag is small, and the quality of the granules is ensured to meet the regulations of Chinese pharmacopoeia.

Example 3

respectively placing 547.6g, 273.7g, 547.6g, 547.6g, 219.2g and 219.2g (2354.9 g) of 6 identified traditional Chinese medicines of salvia miltiorrhiza, angelica, astragalus, suberect spatholobus stem, tree peony bark and red paeony root into an extraction tank, mixing, respectively adding 8 times of weight of water for two times, each time for 1h, mixing extracting solutions, concentrating, adding 6 times of weight of 90vol% ethanol (14.1294 kg), standing for 24h, filtering, recovering ethanol from filtrate under reduced pressure, and concentrating until the relative density is 1.281g/cm ³ 452.8g of Dan Gui Huo fluid extract. Adding dextrin as a medicinal auxiliary material which does not interfere with medicinal components, and mixing the components according to the weight ratio of the salvia miltiorrhiza, angelica and the blood activating fluid extract: mass ratio of dextrin = 1: 1.8, drying at 80 ℃, preparing dry powder, and subpackaging in plastic bags, wherein the weight difference of the prepared finished product of the tea bag is small, and the quality of the granules is ensured to meet the regulations of Chinese pharmacopoeia.

Example 4

Chemical and pharmacodynamic studies of three extraction processes:

the process 1 comprises the following steps: according to the total weight of the medicinal materials of 2000g, the weight ratio is 2.5: 1.25: 2.5: 2.5: 1: 1, extracting 6 Chinese medicaments of salvia miltiorrhiza, angelica, astragalus, suberect spatholobus stem, tree peony bark and red paeony root for 2 times, and adding 16/12 times of water respectively. Heating for 1 hour each time, mixing, concentrating, and cooling; adding 95vol% ethanol 2 times the weight of the extract, stirring, standing overnight, collecting supernatant, concentrating to obtain dry extract, adding water and diluting to 250mL, which is equivalent to 1mL =8g crude drug.

And (2) a process: according to the total weight of the medicinal materials of 2000g, the weight ratio of 6 Chinese medicaments of salvia miltiorrhiza, angelica, astragalus, suberect spatholobus stem, tree peony bark and red paeony root is 2.5: 1.25: 2.5: 2.5: 1: 1, extracting 3 Chinese medicaments of salvia miltiorrhiza, astragalus and suberect spatholobus stem for 2 times, and adding 16/12 times of water respectively. Heating for 1 hour each time, mixing, concentrating, and cooling; adding 95vol% ethanol 2 times the weight of the extract, stirring, standing overnight, collecting supernatant, and recovering under reduced pressure to obtain dry extract (I).

And (3) combining the dry extract (I) and the dry extract (II), and adding water to dilute to 250mL, which is equivalent to 1mL =8g of crude drug.

And (3) a process: according to the total weight of the medicinal materials of 2000g, the weight ratio is 2.5: 1.25: 2.5: 2.5: 1: 1, adding 5 times of 90vol% ethanol into 6 traditional Chinese medicines of salvia miltiorrhiza, angelica, astragalus, suberect spatholobus stem, tree peony bark and red paeony root, refluxing for 2 times, refluxing for 1 hour each time, combining 2 extracts, decompressing and recovering to be dry to obtain dry extract, adding water to dilute to 250mL, which is equivalent to 1mL =8g crude drugs.

The content of the main effective component danshensu in the extract prepared by 3 processes is measured by adopting a high performance liquid chromatography:

1. chromatographic conditions are as follows:

a chromatographic column: phenomenex Hyper Clore BDS-C18 (4.6 mm' 250mm, 5 mm);

mobile phase: 0.05% trifluoroacetic acid (a) -methanol (B);

gradient elution: 0-15 min, A: 85% → 15%, B: 15% → 85%;

flow rate: 1.0 mL/min ^-1 ；

Column temperature: 35 ℃;

the detection wavelength is 280 nm;

the sample injection amount is 10mL;

2. preparation of control solutions:

accurately weighing 8.5mg of salvianic acid A sodium, adding distilled water to a constant volume of 10mL;

3. preparation of a test solution:

accurately weighing 0.85g of the extracted Dangui blood activating extract, placing the Dangui blood activating extract in a conical flask with a plug, accurately adding 10mL of distilled water, weighing, soaking for 30 minutes, performing ultrasonic treatment for 30 minutes, cooling, weighing again, supplementing the lost weight with distilled water, shaking uniformly, filtering, and taking the subsequent filtrate to obtain the Chinese medicinal preparation.

The results show that: the extraction method of the process 1 has the highest danshensu efficiency, the other two extraction methods have slightly low content and relatively approximate content, and the measurement of the danshensu is not influenced by the angelica, the astragalus, the suberect spatholobus stem, the tree peony bark and the red paeony root.

Preliminary experiments on the pharmacodynamics of the products obtained by three different extraction processes:

the method comprises the following steps:

1. animal grouping:

BALB/c 6 weeks old fed with normal feed under SPF-level closed state, dividing 72 mice into 9 groups according to weight; respectively divided into high dose group (AH group, 0.875g crude drug/10 g) and low dose group (AL group, 0.265g crude drug/10 g) for process 1; process 2 high dose group (BH group, 0.875g crude drug/10 g), low dose group (BL group, 0.265g crude drug/10 g); process 3 high dose group (CH group, 0.875g crude drug/10 g), low dose group (CL group, 0.265g crude drug/10 g); notoginseng Tongshu Capsule control group (group D, 0.124g Capsule/10 g) and model group (group E, physiological saline 0.1mL/10 g). Continuously feeding for 12 weeks, wherein the gavage volume of the mice is 0.1mL/10g each time; it is administered 1 time daily. 8 BALB/c normal control groups (group F); the normal saline is administered.

2. Observation indexes are as follows:

(1) general observations: weighing every 3-5 days after the beginning of the experiment until the end of the experiment; the appetite, hair changes, mobility, etc. of the mice were carefully observed during the administration period.

(2) Observation indexes are as follows: skin and lung tissue were retained and subjected to routine pathology (HE staining, semi-quantitative analysis) and Masson staining (collagen volume fraction method), dermal thickness measurement, Hydroxyproline (HYP) assay (sample alkaline water method).

3. Statistical method

And (3) performing pairwise comparison between groups by adopting an R software (version: 3.6) 'ggsignif' R language package, and adopting t test. P < 0.05 is statistically significant.

The drug effect evaluation of 3 types of salvia miltiorrhiza bunge and angelica sinensis blood-activating extracts with different extraction processes is carried out by adopting a scleroderma model mouse, and the panax notoginseng bunge blood-activating extract capsule with 3 types of different extraction processes are found to have the following results in improving the scleroderma mouse by taking the panax notoginseng bunge and angelica sinensis blood-activating capsule as positive control:

general observation:

before the experiment begins, the weight of each group of mice is not different, but the skin hair is withered and falls off, the state is not good, the activity is reduced and the activity is slow; with the increase of observation time, the diet and activity of each group of mice gradually return to normal, and the outer skin hair is gradually moistened. However, in the course of the observation treatment, the death phenomenon of the mice occurred. Animal mortality 12 weeks after intervention treatment: AH group 2, BH group 1 and CH group 1; these dead mice may be associated with too high a concentration of drug and too viscous of a liquid.

And (II) detecting each group of skin tissues:

the HE staining results of the skin tissues of each group are shown in FIGS. 1 to 4, and the Masson staining results are shown in FIGS. 5 to 9. Wherein, the results of HE staining of the skin of the model-making mouse compared with the control group (fig. 3) show that the dermis layer is remarkably thickened, the number of collagen fiber bundles is increased, the shape is thickened, the arrangement is compact, and the skin is expanded to the deep part to partially replace subcutaneous fat adipose tissues; typical vessel wall thickening fibrosis seen deep in the dermis; there was no significant pathological change in the skin tissue of the control (FIG. 4) mice. The concrete aspects are as follows:

the degree of inflammation of skin tissue:

the inflammation degree of the model group (1.33 +/-0.47) is significantly different from that of the normal group (0.44 +/-0.39) ((P<0.05), indicating that molding was successful; after 12 weeks of drug intervention, the degree of skin inflammation was significantly reduced in the AH group (0.17. + -. 0.24), AL group (0.38. + -. 0.33), CL group (0.75. + -. 0.25) compared to the E group (model group) (1.33. + -. 0.47),Pthe values are 0.0014, 0.0041 and 0.039, respectively, as shown in fig. 10.

② comparison of change of collagen fiber of skin tissue:

after the model animal intervenes for 12 weeks, the skin fibrosis of each intervention group has a reduction trend of different degrees, and the improvement degree groups are AH group (0.31 +/-0.11), BL group (0.30 +/-0.17) and D group (0.27 +/-0.19) in sequence; however, none of them was statistically significant (p > 0.05) compared to the model group (0.49. + -. 0.21), as shown in FIG. 11.

③ change of collagen content of skin tissues of each group:

after 12 weeks of intervention experiments, there was a different degree of reduction in skin fibrosis in each group, with the AL group (3024.68 + -654.15 μ g/g), BH group (2948.35 + -1101.89 μ g/g) and CL group (2992.37 + -560.21 μ g/g) compared to the model group (4453.26 + -1099.18 μ g/g); and the differences in the scleroderma model group compared to the normal control group (2810.11 ± 458.45 μ g/g) (p = 0.019) were statistically significant (p values were all < 0.05), as shown in fig. 12.

Skin thickness variation:

after 12 weeks of intervention, there was a different degree of reduction in skin thickness in each of the intervention groups, where the differences between the AH (199.98. + -. 18.02 μm) and AL (188.59. + -. 16.46 μm) groups were statistically significant (p values were all < 0.05) compared to the model group (235.68. + -. 13.30 μm), as shown in FIG. 13.

And (III) detecting the inflammation degree of lung tissues in each group:

the HE staining results of lung tissues of each group are shown in FIGS. 14-18, the Masson staining results are shown in FIGS. 19-23, and alveolar septal broadening with inflammatory cell exudation and infiltration, alveolar structure destruction and pulmonary tissue vascular hyperplasia and dilatation can be seen in lung tissues of model-making mice (FIG. 17); control mice had normal alveolar structure (FIG. 18).

Detecting the inflammation degree of lung tissues in each group:

compared with the normal group (0.69 +/-0.35), the difference of the model group lung tissue inflammation (1.33 +/-0.47) is statistically significant (p = 0.03), which indicates that the model is successfully made; after 12 weeks of intervention, the lung tissue inflammation degree of each intervention group is reduced to different degrees; the difference between the AH group (0.42. + -. 0.34) and the model group (1.33. + -. 0.47) was statistically significant (p < 0.05), as shown in FIG. 24.

② change detection of collagen fiber of lung tissue of each group:

after 12 weeks of the intervention experiment, pulmonary fibrosis of each intervention group is relieved to different degrees, and the groups with the best improvement degree are AH group (0.32 +/-0.09), AL group (0.31 +/-0.09) and BL group (0.31 +/-0.13) in sequence, but are compared with the model group (0.45 +/-0.20); none of the differences were statistically significant (p > 0.05)), as shown in FIG. 25.

③ change of collagen content of lung tissues of each group:

after 12 weeks of intervention, pulmonary fibrosis was reduced to different degrees in each group, wherein differences between AH (253.92 + -114.11 μ g/g), BH (212.86 + -61.24 μ g/g), CH (229.05 + -86.33 μ g/g), CL (284.23 + -46.23 μ g/g) and model (349.22 + -49.65 μ g/g) were statistically significant (both p values < 0.05) in each group, and between 349.22 + -49.65 μ g/g and normal (229.30 + -65.93 μ g/g), as shown in FIG. 26.

The above-described embodiments are only preferred embodiments of the present invention, and are not intended to limit the present invention in any way and substantially, it should be noted that those skilled in the art may make several modifications and additions without departing from the scope of the present invention, which should also be construed as a protection scope of the present invention.

Claims

1. A preparation method of a traditional Chinese medicine granular preparation for treating scleroderma is characterized in that the traditional Chinese medicine granular preparation is prepared from 15-45 parts by weight of salvia miltiorrhiza, 15-45 parts by weight of angelica sinensis, 15-45 parts by weight of astragalus membranaceus, 15-45 parts by weight of caulis spatholobi, 15-45 parts by weight of moutan bark and 15-45 parts by weight of red peony root, and comprises the following steps:

the first step is as follows: weighing the salvia miltiorrhiza, the angelica, the astragalus, the suberect spatholobus stem, the moutan bark and the red paeony root according to a proportion, mixing, adding 8-16 times of water for decocting twice, 1 hour each time, combining decoction, filtering, concentrating filtrate, cooling, adding 90vol% ethanol with 6 times of weight, fully stirring, standing for 24 hours, taking supernate, and concentrating under reduced pressure until the relative density is 1.2-1.3 g/cm ³ Obtaining fluid extract;

the second step: adding 0.6-1.8 parts by weight of dextrin, mixing, drying at 80 ℃, preparing dry powder, and filling into a plastic package bag to obtain the traditional Chinese medicine granular preparation for treating scleroderma.

2. The method of claim 1, wherein in the first step, the weight ratio of red sage root, angelica, astragalus, spatholobus stem, moutan bark and red peony root is 2.5: 1.25: 2.5: 2.5: 1: 1.