APPLICATION OF PHARMACEUTICAL COMPOSITION IN REGULATION OF FIBROBLAST GROWTH

Abstract

An application of an oral pharmaceutical composition in manufacturing of a medication for regulating fibroblast growth and treating or preventing organ fibrosis. The pharmaceutical composition comprises a homogeneous mixture of an edible oil, beeswax, and -sitosterol, wherein the beeswax forms microcrystals, and on the basis of the total weight of the composition, the content of the beeswax is 0.5-50% and the content of -sitosterol is 0.1%-20%.

Claims

1. A method for regulating fibroblast growth using a pharmaceutical composition, wherein the pharmaceutical composition is a pharmaceutical composition suitable for oral administration comprising a homogenous mixture of edible oil, beeswax and -sitosterol, wherein the beeswax in the composition forms microcrystals, the content of the beeswax is 0.5 to 50% and the content of the -sitosterol is 0.1% to 20% by weight based on the total weight of the composition.

2. A method for treating or preventing organ fibrosis, scar formation and/or tissue aging using a pharmaceutical composition, wherein the pharmaceutical composition is a pharmaceutical composition suitable for oral administration comprising a homogenous mixture of edible oil, beeswax and -sitosterol, wherein the beeswax in the composition forms microcrystals, the content of the beeswax is 0.5 to 50% and the content of the -sitosterol is at least 0.1% to 20% by weight based on the total weight of the composition.

3. The method according to claim 2, wherein the organ comprises heart, liver, lungs, kidneys and bone marrow.

4. The method according to claim 3, wherein the organ is an organ from a mammal, and the mammal is preferably a human.

5. The method according to claim 1, characterized in that the content of the -sitosterol in the pharmaceutical composition is 0.5 to 20% by weight.

6. The method according to claim 1, characterized in that the content of the -sitosterol in the pharmaceutical composition is 1 to 10% by weight.

7. The method according to claim 1, characterized in that the content of the beeswax in the pharmaceutical composition is 3 to 30% by weight.

8. The method according to claim 1, characterized in that the content of the beeswax in the pharmaceutical composition is 5 to 20% by weight.

9. The method according to claim 1, characterized in that the content of the beeswax in the pharmaceutical composition is 6 to 10% by weight.

10. The method according to claim 1, characterized in that the edible oil in the pharmaceutical composition is corn oil, wheat germ oil, soybean oil, rice bran oil, rapeseed oil, sesame oil or fish oil.

11. The method according to claim 1, characterized in that the pharmaceutical composition further comprises propolis, and the content thereof is 0.1 to 30% by weight.

12. The method according to claim 1, characterized in that the pharmaceutical composition comprises water, and the content thereof is less than or equal to 1% by weight.

13. The method according to claim 1, characterized in that the dosage form of the oral pharmaceutical composition is selected from the group consisting of a tablet, pill, capsule, emulsion, gel, syrup and suspension.

14. The method according to claim 1, characterized in that the pharmaceutical composition further comprises Scutellaria baicalensis or the extract of Scutellaria baicalensis, and the content of Scutellaria baicalensis or the extract of Scutellaria baicalensis containing 0.1 to 0.5% of baicalin is 2 to 5% by weight based on the total weight of the composition, the Scutellaria baicalensis is one or more Labiatae plants selected from the group consisting of Scutellaria viscidula bunge, Scutellaria amoena, Scutellaria rehderiana Diels, Scutellaria ikonnikovii Juz, Scutellaria likiangensis and Scutellaria hypericifolia.

15. The method according to claim 1, characterized in that the pharmaceutical composition further comprises Cortex Phellodendri or the extract of Cortex Phellodendri, and the content of Cortex Phellodendri or the extract of Cortex Phellodendri containing 0.1 to 1% of obaculactone is 2 to 5% by weight based on the total weight of the composition, the Cortex Phellodendri is selected from the group consisting of Phellodendron chinense Schneid, Phellodendron amurense, Phellodendron chinense Schneid var. omeiense, Phellodendron Schneid var. yunnanense and Phellodendron chinense Schneid var. falcutum.

16. The method according to claim 1, characterized in that the pharmaceutical composition further comprises 2 to 5% of Coptis chinensis or the extract of Coptis chinensis containing 0.1 to 1% of berberine by weight based on the total weight of the composition.

17. The method according to claim 16, characterized in that the Scutellaria baicalensis extract is a Scutellaria baicalensis extract obtained in sesame oil, the Cortex Phellodendri extract is a Cortex Phellodendri extract obtained in sesame oil, and the Coptis chinensis extract is a Coptis chinensis extract obtained in sesame oil.

18. The method according to claim 1, characterized in that the pharmaceutical composition further comprises 2 to 5% of Scutellaria baicalensis or the extract of Scutellaria baicalensis containing 0.1 to 0.5% of baicalin, 2 to 5% of Cortex Phellodendri or the extract of Cortex Phellodendri containing 0.1 to 1% of obaculactone, 2 to 5% of Coptis chinensis or the extract of Coptis chinensis containing 0.1 to 1% of berberine, 2 to 10% of Pericarpium Papaveris or the extract of Pericarpium Papaveris containing 0.1 to 1% of narcotoline, and 2 to 10% of earthworm or earthworm extract containing amino acid, by weight based on the total weight of the composition.

19. The method according to claim 1, characterized in that the pharmaceutical composition comprises 7% of beeswax, 1% of sterol, 0.5% of obaculactone, 0.3% of baicalin and 0.5% of berberine by weight based on the total weight of the composition.

20. The method according to claim 1, characterized in that the beeswax has microcrystals with a length of 0.1 to 100 microns.

21. The method according to claim 20, characterized in that at least two microcrystals of the beeswax in the pharmaceutical composition are polymerized into a microcrystal complex.

22. The method according to claim 21, characterized in that the microcrystals of the beeswax are sufficiently uniformly dispersed in the edible oil.

Description

DESCRIPTION OF THE DRAWINGS

[0051] FIG. 1: comparison between the pharmaceutical composition well and the control well in the total particle count.

[0052] FIG. 2: comparison between the pharmaceutical composition well and the control well in the total cell count.

[0053] FIG. 3: comparison between the pharmaceutical composition well and the control well in the fibroblast count.

[0054] FIG. 4: comparison between the pharmaceutical composition well and the control well in the viable fibroblast count.

[0055] FIG. 5: comparison of growth profiles between 3 units of the pharmaceutical composition prepared in Example 1 and 4 units of the pharmaceutical composition prepared in Example 1.

[0056] FIG. 6: comparison of growth profiles among 3 units of the pharmaceutical composition prepared in Example 1, 4 units of the pharmaceutical composition prepared in Example 1 and the control group.

[0057] FIG. 7A: in Example 3, there was no fibroblast growth at Day 40 after the culture of the test group.

[0058] FIG. 7B: in Example 3, there was still a lot of fibroblasts growing at Day 40 after the culture of the control group.

[0059] FIG. 8A: in the test group of Example 4, there was no fibroblast proliferation in the myocardial cell.

[0060] FIG. 8B: in the control group of Example 4, myocardial fibrosis was obvious, and fibroblasts proliferated massively.

[0061] FIG. 9A: in the test group of Example 4, there was no fibroblast proliferation in the marrow.

[0062] FIG. 9B: in the control group of Example 4, marrow fibrosis was obvious, and fibroblasts proliferated massively.

[0063] FIG. 10: comparison of growth profiles among 3 units of the pharmaceutical composition prepared in Example 8, 4 units of the pharmaceutical composition prepared in Example 8 and the control group.

[0064] FIG. 11A to 11C: pictures of gastric mucosa taken by SB capsule endoscopy before the Case 1 was administrated with the pharmaceutical composition (FIG. 11A), 9 months after the Case 1 was administrated with the pharmaceutical composition (FIG. 11B), and 4 years after the Case 1 was administrated with the pharmaceutical composition (FIG. 11C) in Example 9.

DETAILED DESCRIPTION OF THE INVENTION

[0065] The present invention is further illustrated by the following examples in combination with the figures, which should not be construed as a limitation to the present invention. Specific materials and sources thereof used in the embodiments of the present invention are provided below. However, it should be understood that these are merely exemplary and are not intended to limit the present invention. Materials that are the same as or similar to the following reagents and instruments in type, model, quality, properties or function can also be used in the embodiment of the present invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials, reagents and the like used in the following examples are commercially available.

Example 1: Preparation of the Pharmaceutical Composition

[0066] The pharmaceutical composition was prepared according to the content disclosed in Examples 1 and 2 of Chinese Patent ZL 02105541.6.

[0067] Briefly, step 1: the refined sesame oil and Scutellaria baicalensis (100 kg: 5 kg) were added to a reaction tank and heated. Heating was stopped when the temperature reached 120 C., and the mixture was kept warm for 50 minutes with stirring. The mixture was filtrated to remove the dregs, the obtained extraction was the medicinal oil I.

[0068] Step 2: the medicinal oil I was added to another reaction tank and heated. When the temperature reached 85 C., the refined beeswax was added at a ratio of 93 kg of medicinal oil I:7 kg of beeswax, and stirred well. Heating was stopped when the temperature reached 120 C., kept stirring the warm mixture for 20 minutes to obtain the medicinal oil II.

[0069] Step 3: the medicinal oil II was grinded using a colloid mill with a pitch of 0.6 to 0.8 mm and an output speed of 15 Kg/15 min. Alternatively, the medicinal oil II could also be homogenized at 402 C. for 15 to 20 minutes using a homogenizer with a rotate speed of 6000 to 10000 rpm. The homogenate was stirred at 100 rpm, vacuumized to below 0.09 MP, cooled to 402 C., and kept warm for 50 minutes. When the temperature decreased to 20 C. and the vacuum degree reached 0.6 to 0.8 MP, the mixture was kept for 20 minutes to obtain the pharmaceutical composition.

[0070] According to Example 2 of Chinese Patent ZL 02105541.6, the active ingredients of the pharmaceutical composition prepared by the above method are shown as follows:

TABLE-US-00001 Ingredients Content per 100 g Natural vitamin E 15 mg~50 mg Total flavone 20 mg~60 mg -sitosterol 0.20 g~1.0 g Linoleic acid 35 g~55 g Oleic acid 25 g~45 g

Example 2: Verification of the Inhibition Effect of the Pharmaceutical Composition on Fibroblast

[0071] 1. Materials and Methods

[0072] 1.1 Instruments, Devices, Materials and Reagents

[0073] Quantitative imaging analytical flow cytometry (AmnisImageStream.sup.xMarkII, Merk Millipore, USA, provided by High-throughput Single Cell Analysis Platform of Phoenix Project of Peking University); ultrapure water system (Milli-Q, Millipore, USA); two-stage reverse osmosis purified water system (Beijing Innogreen Technology Co., Ltd.); electronic scale (AB135-S, Mettler-Toledo, Switzerland); electronic scale (ES-1000HA, Changsha Xiangping Technology Development Co., Ltd.); high-speed refrigerated centrifuge (J20-XP, Beckman-Coulter, USA); desktop high speed refrigerated centrifuge (1-15K, Sigma, Germany); biological clean bench (BCN-1360B, Beijing HDL Apparatus Co., Ltd.); CO.sub.2 incubator (Forma3111, Thermo Fisher Scientific, USA); hybridization oven (Maxi14, Thermo Fisher Scientific, USA); particle ice machine (SIM-F124, Sanyo, Japan); electronic constant temperature water bath (CS501-3C type, Chongqing Sida Experimental Instrument Co., Ltd.), drying oven (Chongqing Sida Experimental Instrument Co., Ltd.); inverted microscope (Nikon TE2000U, Nikon, Japan); microscopic imaging system (Nikon DXM 1200, Nikon, Japan); ordinary optical microscope (BK1201, Chongqing Optical Instrument Factory); micro-sampler (1000 l, 200 l, 20 l, 10 l, Gilson, France); 6-well culture plate; 15 ml centrifuge tube; 1 ml and 0.5 ml Eppendorf centrifuge tube; dripper; sterilized small surgical instrument; sterilized small beaker; sterilized small flask with lid; sterilized culture dish (5 cm, (6 cm); 0.22 m microporous membrane; needle filter; capped triangular flask; capped small test tube; needle; DMEM medium (GIBCO, Invitrogen Corporation, USA, packaged by Beijing Xinjingke Biotechnology Co., Ltd.); 7-AAD staining solution (provided by High-throughput Single Cell Analysis Platform of Phoenix Project of Peking University); super fetal bovine serum (Hangzhou Sijiqing Biological Engineering Material Co., Ltd.); type I mouse tail collagen (Cat. No. C8062, Solarbio Co.); PBS (self-made, stored at 4 C., and used in an ice bath); Hank's balanced salt solution (self-made, stored at 4 C., and used in an ice bath); 75% ethanol (self-made); 8% Na.sub.2S (self-made); penicillin sodium and streptomycin sulfate for injection; trypsin-EDTA cell digest solution (self-made); portable refrigerator.

[0074] 1.2 Method.sup.[6-9]

[0075] 1) Each well of two 6-well plates was coated with 60 l of type I mouse tail collagen. The plate was laid flat, sealed with tape, and placed in a stainless steel box. The box was placed in the Maxi14 hybridization oven at 37 C. overnight, and stored at 4 C.

[0076] 2) A Kunming suckling mouse (5 days old, purchased from Laboratory Animal Breeding Base of Academy of Military Medical Sciences) was placed on a foam board, and wiped with a cotton ball (dipped with 8% Na.sub.2S by tweezers) several times to remove hair, especially trunk hair.

[0077] 3) The mouse was rinsed with deionized water, and killed by breaking its neck.

[0078] 4) The mouse was placed in 75% ethanol for 2 min, 75% ethanol for 5 min, and turned over frequently.

[0079] 5) The suckling mouse was taken out, and placed in a sterile plate after the ethanol was removed completely.

[0080] 6) The skin, especially trunk skin, was cut as much as possible. The collected skin was placed in another sterile plate.

[0081] 7) The sterile skin was spread in the plate with the dermis upward. Tissues such as subcutaneous fat were removed by ophthalmic tweezers.

[0082] 8) The skin was placed in a sterile flask with lid, and added with 10 ml of cold PBS, 60 l of penicillin and 60 l of streptomycin. The skin was turned, stirred and rinsed well.

[0083] 9) The above step was repeated 4 times with a total of 5 times.

[0084] 10) The skin was washed with cold Hank's solution+two antibiotics twice.

[0085] 11) The above suckling mouse skin (washed 7 times) was placed in the sterile flask with lid, added with 10?4) FBS DMEM medium (cold), and cut into skin grafts later.

[0086] 12) The 6-well plate (coated with type I mouse tail collagen on the previous day) was washed with cold PBS 3 times. PBS was removed, and each well was added with 1 ml of 10% FBS DMEM medium. The 6-well plate was shaked and placed still.

[0087] 13) The skin obtained in Step 11 was placed in a sterile plate, and cut into full-thickness skin grafts (about 1 mm.sup.2) by sterile ophthalmic curved scissors (note: the skin grafts were obtained, and moisturized by the culture medium). The plate was covered, and should not let it dry.

[0088] 14) The 10% FBS DMEM medium was removed from the wells of the plate in Step 12 (note: some medium was remained on the bottom of the well as the nutrition for the pre-adherence of graft).

[0089] 15) The skin grafts obtained in Step 13 were averagely placed into each well (A1, A2, A3, B1, B2, B3) of the two 6-well plates. About 10 pieces of skin grafts were placed into one well by straight tweezers under microscope. The skin graft was spread by straight tweezers under microscope, and pressed to adhere closely. Each skin graft was located in the center of the well, and the area of the spread graft in each well was equal (adjustment was carried out if there was a difference). After checking the adherence of each graft, the plate was covered and sealed with tape except for one side (for the entrance of CO.sub.2).

[0090] 16) The freshly formulated 10% FBS DMEM medium (stored in a refrigerator) was incubated in a water bath at 37 C. for 10 min to avoid the expansion and contract of the skin graft, which would cause the skin graft falling from the bottom of the well.

[0091] 17) The plate containing the skin grafts was incubated in an incubator at 37 C., 5% CO.sub.2 for 4 h. 4 ml of the above incubated 10% FBS DMEM medium was slowly added dropwise from the rim to each well. De-adherence of the skin graft was not observed.

[0092] 18) On Day 5 of the experiment, all skin grafts in all wells were well adhered, and recorded by photograph. The test pharmaceutical composition was added to the wells. Specifically, 3 units (about 0.08 g per unit) of pharmaceutical composition, 4 units of pharmaceutical composition or equal amount of medium (as control) was added to the corresponding well, and 3 ml of 10% FBS DMEM medium incubated at 37 C. was added to each well at the same time.

[0093] 19) The medium was changed regularly. Generally, 3 ml of original medium was removed, and an equal amount of 10% FBS DMEM medium incubated at 37 C. was added.

[0094] 20) The growth of fibroblasts was observed and recorded regularly with the Nikon TE2000U inverted microscope during the whole culture process. Photos were recorded with the Nikon DXM1200 micro-camera and saved on a computer. Pictures at certain time points were selected and analyzed with Image-Pro Plus professional image software to draw the growth profile of fibroblast.

[0095] 21) The experiment was stopped after a certain period of cultivation. Cells were collected from one control well, one well with 3 units of pharmaceutical composition and one well with 4 units of pharmaceutical composition. The cells were detected and analyzed with a flow cytometer. The skin grafts and supernatant were removed from the well. The well was rinsed with cold PBS 3 times. 0.5 ml of trypsin and 0.5 ml of EDTA cell digest solution were added to the well, shaked and reacted at room temperature for a certain time. The reaction was stopped when cell morphology change was observed under the inverted microscope. The well was added with 1 ml of 10% FBS DMEM medium to stop the reaction completely, followed by addition of 3 ml of cold Hank's solution. The bottom of the well was piped repeatedly until the cells were completely separated from the bottom of the well. The obtained cell suspension was added to a 15 ml centrifuge tube, and centrifuged at 4 C., 2000 rpm for 5 min. The supernatant was removed, and the volume of the residues was about 0.5 ml. The cells was suspended, added to an Eppendorf tube, and centrifuged at 4 C., 2000 rpm for 5 min. The supernatant was removed, and the volume of the residue was about 25 l. It should be noticed that the above steps were carried out in an ice bath, and the obtained cells were labeled accurately. The cells were placed in the portable refrigerator with ice packs, and immediately tested at Peking University. At the flow cytometer room of Peking University, the cells were piped evenly, added with 10 l of 7-AAD, mixed well and tested immediately. Detection parameters: single laser wavelength of 488 nm, 150 mW; SSC: 0.5 mV (785 nm); 40 objective lens.

[0096] 2. Results

[0097] 2.1 Test Results of Quantitative Imaging Analytical Flow Cytometry

[0098] 2.1.1 Total Particle Count

[0099] All the particles in the cell suspension to be tested were counted to obtain the absolute number of particles. The maximum number of counted particles was set to 300,000. The counted particles included viable cells, dead cells and non-cells. The shape of each particle could be observed clearly through the image recorded by the instrument. Although the total particle count does not accurately reflect the true number of cells, it is of great reference significance. When analyzing the results, the non-cellular components contained therein should be fully considered. It can be seen from the test results that:

[0100] The cells were collected and tested by the flow cytometry to obtain a scatter diagram, which was analyzed by IDEAS software (published by Amnis and matched with the flow cytometer) to obtain the total number of particles. The results are shown in FIG. 1. Compared with the control well, the total number of particles varies greatly, and the pharmaceutical composition well has a significantly decreased total number of particles, indicating that the pharmaceutical composition obviously regulated the growth of fibroblast. The effect may vary in a certain extent according to the dosage of the pharmaceutical composition.

[0101] 2.1.2 Fibroblast Count

[0102] In addition to the total number of particles, the scatter diagram obtained by the flow cytometry was further analyzed by IDEAS software (published by Amnis and matched with the flow cytometer) to obtain the total number of cells, the number of fibroblasts, and the number of viable fibroblasts. In this Example, the parameters were reset, wherein the parameters included two important parameters of aspect ratio and area. The particles that did not meet the cell parameters were excluded to screen the particles that met the cell parameters out of the total particles. The results obtained represented the total number of different types of cells. The results are shown in FIG. 2. Compared with the control well, the total number of cells varies greatly, and the pharmaceutical composition well has a significantly decreased total number of cells, indicating that the pharmaceutical composition obviously regulated the growth of fibroblast. The effect may vary in a certain extent according to the dosage of the pharmaceutical composition.

[0103] The non-fibroblasts were excluded to screen the cells that met the fibroblast parameters out of the total cells and obtain the total number of fibroblasts. The results are shown in FIG. 3. Compared with the control well, the total number of cells varies greatly, and the pharmaceutical composition well has a significantly decreased total number of cells, indicating that the pharmaceutical composition obviously regulated the growth of fibroblast. The effect may vary in a certain extent according to the dosage of the pharmaceutical composition.

[0104] The final step of the flow cytometry test was to detect the number of viable cells among the total number of fibroblasts to reflect the effect of different analytes on fibroblast activity and life span. This was achieved by staining the fibroblasts with 7-AAD staining solution. The results are shown in FIG. 4. Compared with the control well, the number of viable cells varies greatly, and the pharmaceutical composition well has a significantly decreased total number of cells, indicating that the pharmaceutical composition obviously regulated the growth of fibroblast. The effect may vary in a certain extent according to the dosage of the pharmaceutical composition.

[0105] 2.2 Drawing Cell Growth Profile with the Number of Fibroblasts at Different Time Points

[0106] Three areas with high, medium and low cell density were selected from each well of the plate. Three fields of vision with the same area were randomly selected from each area, with a total of 9 fields of vision with the same area. Cells with typical morphology were counted to obtain the total number of cells. Cells were counted by the professional image processing software image-Pro plus Version6.0. Cell count was carried out by clicking the Measure button in the menu, selecting the Measurements . . . item in the drop-down menu, and selecting the Create point feature tool in the Features toolbar in the dialog box. A total of 12 time points (167 h, 186 h, 197 h, 210 h, 222 h, 234 h, 247 h, 258 h, 269 h, 281 h, 291 h and 300 h after the experiment) were selected in the experiment. Fibroblast count results are shown in Table 1:

TABLE-US-00002 TABLE 1 Additive (right) Test time points (below) 3 units 4 units Control 1 27 54 378 2 51 54 432 3 39 34 432 4 57 30 420 5 33 21 369 6 39 15 447 7 24 15 477 8 30 21 396 9 27 12 408 10 12 9 447 11 15 15 459 12 18 12 450

[0107] The fibroblast growth profile is drawn based on the above results. For comparison, the growth profile is expressed as different line charts. The results show that there are a large number of cells in the control well, and the overall trend of the growth profile is upward, indicating that the cells increase over time, and the cell growth is not affected. There are a small number of cells in the pharmaceutical composition well, and the overall trend of the growth profile is downward, indicating that the cells decrease over time. In addition to the obviously inhibited cell growth, the viable cells decrease over time as well, indicating that the cell growth is inhibited. Specific results are shown in FIG. 5 and FIG. 6. It can be seen from the comparison of the growth profiles of 3 units of pharmaceutical composition and 4 units of pharmaceutical composition that the overall trend of the growth profile is obviously downward, and the correlation between the two is good. It can be seen from the comparison of the growth profiles of 3 units of pharmaceutical composition, 4 units of pharmaceutical composition and the control group that the number of cells varies greatly. The overall trend of the first two is downward, while the overall trend of the latter is upward.

[0108] 2.3 Microscopic Examination at Different Time Points

[0109] A total of 14 detection time points for fibroblast growth (48 h, 66 h, 167 h, 186 h, 197 h, 210 h, 222 h, 234 h, 247 h, 258 h, 269 h, 281 h, 291 h and 300 h after the experiment) were selected to visually show the in vitro growth of fibroblast of suckling mouse skin graft under the effect of different biologically active substances. Similar to the results of the previous experiment, it can also be seen under the microscope that the pharmaceutical composition has a significant inhibition effect on fibroblast growth, while the fibroblasts in the control group without the pharmaceutical composition grow vigorously. The results under the microscope show that the pharmaceutical composition has a Very significant inhibition effect on fibroblast. Fibroblasts growing out of the grafts are significantly inhibited by the pharmaceutical composition, and changed in morphology. The cells turn black and shrink until they die and dissolve. The effect of the pharmaceutical composition on fibroblasts that just grow out of the skin graft can also be observed under the microscope. In fact, the cells are inhibited at the early stage of growth without a chance of diffusion and growth. With respect to a large amount of fibroblasts growing in a short period of time, the cells will not be immediately inhibited or die due to the temporary balance of the pharmaceutical composition and fibroblast. After a certain period of time, the cells will eventually be inhibited by the pharmaceutical composition until they die, which is directly related to the amount of the pharmaceutical composition. With respect to fibroblasts that have grown and multiplied before the addition of the pharmaceutical composition, when the pharmaceutical composition is added, it takes time for the pharmaceutical composition to function, and the effect becomes more and more obvious over time. Specific results are shown in Table 2 below:

TABLE-US-00003 TABLE 2 Time points 3 units 4 units Control 1 Fibroblast growth is Fibroblast growth is Fibroblast growth is not found at the edge not found at the edge not found at the edge of the graft of the graft of the graft 2 Fibroblast growth is Fibroblast growth is Fibroblast growth is found at the edge of not found at the edge found at the edge of the graft of the graft the graft 3 There are a few There are very few There are a large fibroblasts growing at fibroblasts growing at number of fibroblasts the edge of the graft the edge of the graft with obvious growth 4 There are a few There are very few Fibroblasts grow fibroblasts at the edge fibroblasts vigorously away from of the graft the graft, and densely arrange with a typical morphology 5 There are fibroblasts There are very few A large number of growing around the fibroblasts fibroblasts gow away edge of the graft from the graft, and densely arrange with a typical morphology 6 There are few There are very few Fibroblasts grow away fibroblasts, which are fibroblasts from the graft, and located around the densely arrange with a graft with a typical typical morphology morphology 7 There are very few There are very few Fibroblasts very fibroblasts with a fibroblasts, which fail densely arrange with a fairly good growth to grow further into the typical morphology, condition, which is distance and grow away from close to the periphery the graft, contact of the graft and failed inhibition is formed to expand farther 8 There are very few There are very few Fibroblasts very fibroblasts, which are viable fibroblasts, densely arrange with a located around the which are located typical morphology graft and failed to around the graft expand farther 9 There are a few viable There are a few viable Fibroblasts densely fibroblasts in an aging fibroblasts in an aging arrange with a typical state with a changed state with a nontypical morphology, and grow morphology, which are morphology, which are away from the graft located around the located around the graft graft 10 There are very few There are very Fibroblasts densely viable fibroblasts, few fibroblasts, arrange with a which are located which are merely typical morphology, around the graft located around the and grow away graft and fail from the graft to expand to the distance 11 There are a few There are a few Fibroblasts very fibroblasts, which are degenerated and dead densely arrange with a located around the fibroblasts, which are typical morphology, graft and fail to expand located around the and glow away from to the distance, the graft and fail to the graft, the growth is growth is significantly expand, the growth is at its best inhibited significantly inhibited 12 There are a few There are very few Fibroblasts densely fibroblasts with a fibroblasts with a arrange with a typical changed morphology, nontypical morphology, and grow which are located morphology, which are away from the graft around the graft, the located around the growth is significantly graft, the growth is inhibited significantly inhibited 13 There are a few There are very few Fibroblasts densely fibroblasts, which are fibroblasts, which are arrange with a typical located around the located around the morphology, and grow graft, the growth is graft, the growth is vigorously away from significantly inhibited significantly inhibited the graft 14 Fibroblasts are located There are very few Fibroblasts densely around the graft, the fibroblasts, which have arrange with a typical morphology is already dead, the morphology, and grow changed, the growth is significantly vigorously away from fibroblasts are dying, inhibited the graft indicating that the cell growth is significantly inhibited

Example 3: Inhibition Effect of the Pharmaceutical Composition on Human Fibroblast

[0110] 1. Materials and Methods

[0111] 1.1 Instruments, Devices, Materials and Reagents

[0112] Ultrapure water system (Milli-Q, Millipore, USA); two-stage reverse osmosis purified water system (Beijing Innogreen Technology Co., Ltd.); electronic scale (AB135-S, Mettler-Toledo, Switzerland); electronic scale (ES-1000HA, Changsha Xiangping Technology Development Co., Ltd.); high-speed refrigerated centrifuge (J20-XP, Beckman-Coulter, USA); desktop high speed refrigerated centrifuge (1-14K, Sigma, Germany); biological clean bench (BCN-1360B, Beijing HDL Apparatus Co., Ltd.); CO.sub.2 incubator (Forma3111, Thermo Fisher Scientific, USA); particle ice machine (SIM-F124, Sanyo. Japan); electronic constant temperature water bath (CS501-3C type, Chongqing Sida. Experimental Instrument Co., Ltd.), drying oven (Chongqing Sida Experimental Instrument Co., Ltd.); inverted microscope (Nikon TE2000U, Nikon, Japan); microscopic imaging system (Nikon DXM 1200, Nikon, Japan); ordinary optical microscope (BK1201, Chongqing Optical Instrument Factory); micro-sampler (1000 l, 200 l, 20 l, 10 l, Gilson, France); 12-well culture plate; 15 ml centrifuge tube; 1 ml and 0.5 ml Eppendorf centrifuge tube; dripper; sterilized small surgical instrument; sterilized small beaker; sterilized small flask with lid; sterilized culture dish (5 cm, (6 cm), 0.22 m microporous membrane; needle filter; capped triangular flask; capped small test tube; needle; DMEM medium and 1640 medium (GIBCO, Invitrogen Corporation, USA, packaged by Beijing Xinjingke Biotechnology Co., Ltd.); fetal bovine serum (FBS, Hangzhou Sijiqing Biological Engineering Material Co., Ltd.); PBS (self-made, stored at 4 C.); 75% ethanol (self-made); trypsin-EDTA cell digest solution (self-made); portable refrigerator; vortex shaker.

[0113] 1.2 Methods and Results

[0114] 1.2.1 Early Human Embryonic Tissue

[0115] Source and transportation of the tissue: with the informed consent of the patient, early human embryonic tissue was obtained from the hospital through artificial abortion. The human embryonic tissue was placed in 10% FBS DMEM complete medium in an ice bath, and immediately processed in the laboratory.

[0116] Graft culture: A small amount of tissue was cut into small pieces. The tissue was washed with PBS once, then washed with a high concentration of double antibiotics-PBS five times. The tissue was rinsed with 15% FBS 1640 medium once, and cut into grafts (1 mm.sup.3). The grafts were cultured in a 12-well plate. Each well comprised several pieces of grafts, and was added with a small amount of 15% FBS1640 medium around. The plate was left to stand in the incubator at 37 C., 5% CO.sub.2 for 2.5 h. Each well was added with 2 ml of 15% FBS1640 medium. The grafts were divided into two groups: test group and control group. The test group was added with two units (0.1 g per unit) of the pharmaceutical composition prepared in Example 1, and the control group was added with the same amount of medium. The plate was incubated in the incubator at 37 C., 5% CO.sub.2. The cell growth in the test group and control group was observed after a certain period of time.

[0117] Results: On Day 70 of the cultivation, no cell growth was observed for the test group, while there was a large number of fibroblasts growth for the control group. Conclusion: The pharmaceutical composition inhibits human fibroblast.

[0118] Single cell culture: A small amount of tissue was cut into small pieces. The tissue was washed with PBS once, then washed with a high concentration of double antibiotics-PBS five times. The tissue was rinsed with 15% FBS 1640 medium once, and cut into grafts (1 mm.sup.3). The grafts were placed in a 50 ml centrifuge tube, and added with a mixed solution of 2 ml of 0.25% trypsin and 2 ml of 0.02% EDTA. The tube was shaked in a constant temperature shaker at 37 C. for 35 min, and vortexed for an appropriate time. The solution was filtrated through a stainless steel filter (80 mesh). The filtrate was added with an appropriate amount of PBS, and centrifuged at 1500 rpm for 7 min. The supernate was removed, and the residues were added with PBS, and centrifuged at 1700 rpm for 5 min. The supernate was removed, and the residues were added with 8 ml of 15% FBS1640 medium. The cells were counted, and cultured in a 12-well plate (2 ml of cell suspension per well). The cells were divided into two groups: test group and control group. The test group was added with two units (0.1 g per unit) of the pharmaceutical composition prepared in Example 1, and the control group was added with the same amount of medium. The plate was incubated in the incubator at 37 C., 5% CO.sub.2. The cell growth in the test group and control group was observed after a certain period of time.

[0119] Results: On Day 70 of the cultivation, no cell growth was observed for the test group, while there was a large number of fibroblasts growth for the control group. Conclusion: The pharmaceutical composition inhibits human fibroblast growth.

[0120] 1.2.2 Gastric Fundus Cancer Tissue

[0121] Source and transportation of the tissue: with the informed consent of the patient, gastric fundus cancer tissue was obtained from the hospital through surgical excision. The gastric fundus cancer tissue was placed in 10% FBS DMEM complete medium in an ice bath, and immediately processed in the laboratory.

[0122] Gastric fundus cancer tissue graft culture: A small amount of cancer core tissue was cut into small pieces. The tissue was washed with PBS once, then washed with a high concentration of double antibiotics-PBS five times. The tissue was rinsed with 15% FBS 1640 medium once, and cut into grafts (1 mm.sup.3). The grafts were cultured in a 12-well plate. Each well comprised several pieces of grafts, and was added with a small amount of 15% FBS1640 medium around. The plate was left to stand in the incubator at 37 C., 5% CO.sub.2 for 2.5 h. Each well was added with 2 ml of 15% FBS1640 medium. The grafts were divided into two groups: test group and control group. The test group was added with two units (0.1 g per unit) of the pharmaceutical composition prepared in Example 1, and the control group was added with the same amount of medium. The plate was incubated in the incubator at 37 C., 5% CO.sub.2. The cell growth in the test group and control group was observed after a certain period of time.

[0123] Results: No fibroblast growth was observed for the test group (FIG. 7A), while there was a large number of fibroblasts growth in the control group (FIG. 7B). Conclusion: The pharmaceutical composition inhibits human fibroblast growth.

Example 4: Inhibition Effect of the Pharmaceutical Composition on Organ Fibrosis

[0124] 1. Materials and Methods

[0125] 1.1 Instruments, Devices, Materials and Reagents

[0126] The laboratory animals were 10-month-old Wistar male rats purchased from Institute of Laboratory Animal Sciences, CAMS. The rats were divided into two groups (59 rats for the test group, and 27 rats for the control group). After adapting the feeding environment for 3 days, the rats were subjected to differentiated feeding. The control group was continuely fed with the general nutrient feed produced by Institute of Laboratory Animal Sciences, CAMS, and the test group was fed with the nutrient feed added with the pharmaceutical composition prepared in Example 1. The first batch of rats (5 rats for the test group, and 5 rats for the control group) which were fed with the nutrient feed comprising the pharmaceutical composition and the normal nutrient feed were euthanized after 526 days of feeding. The second batch of rats (18 rats for the test group, and 10 rats for the control group) which were fed with the nutrient feed comprising the pharmaceutical composition or the normal nutrient feed were euthanized after 623 days of feeding. Samples of various tissues and organs (heart, liver, lungs, kidneys, and bone marrow) were sliced and stained (hematoxylin-eosin staining, HE staining).

[0127] Preparation method of the nutrient feed comprising the pharmaceutical composition: normal nutrient feed:pharmaceutical composition=9:1 (mass ratio). 9 parts of normal nutrient feed were added to a mixer. 1 part of the pharmaceutical composition was properly heated, stirred well, and poured into the mixer. The mixer was covered with a cap and started to run. The normal nutrient feed and the pharmaceutical composition were mixed evenly, and then screened through a fine sieve. The sieve was shaken vigorously to further mix the feed evenly. Finally, the mixture was processed into a biscuit feed with a feed processing machine. The feed was spreaded on a stainless steel plate, and dried in a steam drying room at 80 C. for 4 hours. The feed was cooled naturally after the steam stopped.

[0128] 2. Results

[0129] With regard to myocardial fibrosis of aging rat, there are significant differences between the test group and the control group. Among the 17 samples of the test group, 7 samples have myocardial fibrosis, with an abnormal rate of 41%. Among the 16 samples of the control group, 13 samples have myocardial fibrosis, with an abnormal rate of 81%. The test group is superior to the control group, indicating that the pharmaceutical composition inhibits myocardial fibrosis (see FIG. 8A and FIG. 8B for exemplary tissue pictures).

[0130] With regard to liver fibrosis of aging rat, there are significant differences between the test group and the control group. Among the 18 samples of the test group, 3 samples have fibrosis, with an abnormal rate of 17%. Among the 17 samples of the control group, 10 samples have fibrosis, with an abnormal rate of 59%. The test group is significantly superior to the control group. Bile duct fibrosis is a typical indicator of liver aging. The results indicate that the pharmaceutical composition has a significant effect of inhibiting liver fibrosis and promoting liver regeneration and recovery.

[0131] With regard to lung tissue fibrosis of aging rat, it is determined that the pharmaceutical composition significantly inhibits the lung tissue fibrosis of aging rat. The lung tissue fibrosis rate in the test group is 5/20=25%, while the lung tissue fibrosis rate in the control group is 10/14=71%.

[0132] With regard to renal parenchymal fibrosis of aging rat, there are significant differences between the test group and the control group. Among the 19 samples of the test group, 5 samples have fibrosis, with an abnormal rate of 26%. Among the 16 samples of the control group, 11 samples have fibrosis, with an abnormal rate of 69%. The test group is significantly superior to the control group.

[0133] The pharmaceutical composition inhibits the bone marrow fibrosis of aging rat. None of the 6 samples of the test group have bone marrow fibrosis, while all 6 samples of the control group have fibrosis. In the test group, myeloid cells are densely arranged, and erythroid precursor cells, myeloid (granulocyte) precursor cells and megakaryocytes can be easily found (see FIG. 9A). In the control group, fibroblasts significantly proliferate, and fibrous tissues are generated (see FIG. 9B). The specific results are shown in Table 3 below:

TABLE-US-00004 TABLE 3 The pharmaceutical composition inhibits organ fibrosis Group Fibrosis percentage Organ Test group Control group Difference Heart 41% 81% Significant Liver 17% 59% Significant Lung 25% 71% Significant Kidney 26% 69% Significant Bone marrow 0% 100% Significant

Example 5: Preparation of the Pharmaceutical Composition Comprising Scutellaria baicalensis and Cortex phellodendri as Well as the Effect Thereof on Regulating Fibroblast Growth

[0134] The pharmaceutical composition was prepared according to the method of Example 1, wherein the refined sesame oil, Scutellaria baicalensis and Cortex Phellodendri (100 kg: 5 kg: 4 kg) were added to a reaction tank in step 1, and other steps were the same as in Example 1.

[0135] The pharmaceutical composition obtained in this example was subjected to the test methods of Example 2, 3 and 4. The results are similar to those of the pharmaceutical composition obtained in Example 1, indicating that this pharmaceutical composition can significantly regulate fibroblast growth, and inhibit human fibroblast and organ fibrosis of aging rat.

Example 6: Preparation of the Pharmaceutical Composition Comprising Scutellaria baicalensis and Coptis chinensis as Well as the Effect Thereof on Regulating Fibroblast Growth

[0136] The pharmaceutical composition was prepared according to the method of Example 1, wherein the refined sesame oil, Scutellaria baicalensis and Coptis chinensis (100 kg: 5 kg: 4 kg) were added to a reaction tank in step 1, and other steps were the same as in Example 1.

[0137] The pharmaceutical composition obtained in this example was subjected to the test methods of Example 2, 3 and 4. The results are similar to those of the pharmaceutical composition obtained in Example 1, indicating that this pharmaceutical composition can significantly regulate fibroblast growth, and inhibit human fibroblast and organ fibrosis of aging rat.

Example 7: Preparation of the Pharmaceutical Composition Comprising Scutellaria baicalensis, Cortex Phellodendri and Coptis chinensis as Well as the Effect Thereof on Regulating Fibroblast Growth

[0138] The pharmaceutical composition was prepared according to the method of Example 1, wherein the refined sesame oil, Scutellaria baicalensis and Coptis chinensis (100 kg: 5 kg: 5 kg: 5 kg) were added to a reaction tank in step 1, and other steps were the same as in Example 1.

[0139] The pharmaceutical composition obtained in this example was subjected to the test methods of Example 2, 3 and 4. The results are similar to those of the pharmaceutical composition obtained in Example 1, indicating that this pharmaceutical composition can significantly regulate fibroblast growth, and inhibit human fibroblast and organ fibrosis of aging rat.

Example 8: Preparation of the Pharmaceutical Composition Comprising Scutellaria baicalensis, Coptis chinensis, Cortex phellodendri, Pericarpium Papaveris and Earthworm as Well as the Effect Thereof on Regulating Fibroblast Growth

[0140] The pharmaceutical composition was prepared according to the method of Example 1, wherein the refined sesame oil, Scutellaria baicalensis, Coptis chinensis, Cortex Phellodendri, Pericarpium Papaveris and earthworm (100 kg: 5 kg: 4 kg: 4 kg: 5 kg: 5 kg) were added to a reaction tank in step 1, and other steps were the same as in Example 1.

[0141] The pharmaceutical composition obtained in this example was subjected to the test method of Example 2, and the following results are obtained:

[0142] 1) Drawing cell growth profile with the number of fibroblasts at different time points

[0143] There are a large number of cells in the control well, and the overall trend of the growth profile is upward, indicating that the cells increase over time, and the cell growth is not affected. There are a small number of cells in the M well, and the overall trend of the growth profile is downward, indicating that the cells decrease over time. In addition to the obviously inhibited cell growth, the viable cells decrease over time as well, indicating that the cell growth is inhibited (see FIG. 10). Specific results of fibroblast growth at different time points are shown in Table 4 below:

TABLE-US-00005 TABLE 4 Time point 3 units 4 units Control 1 Fibroblast growth is Fibroblast growth is Fibroblast growth is not found at the edge not found at the edge not found at the edge of the graft of the graft of the graft 2 Fibroblast growth is Fibroblast growth is Fibroblast growth is found at the edge of not found at the edge found at the edge of the graft of the graft the graft 3 There are a few There are a few There are a large fibroblasts growing at fibroblasts growing number of fibroblasts the edge of the graft significantly at the with obvious growth edge of the graft 4 There are a few There are very few Fibroblasts grow scattered fibroblasts at fibroblasts vigorously away from the edge of the graft the graft, and densely arrange with a typical morphology 5 There are a few There are a few A large number of fibroblasts growing fibroblasts with a fibroblasts grow away nontypical from the graft, and morphology, densely arrange with a indicating poor cell typical morphology growth 6 There are a few There are a few Fibroblasts grow away fibroblasts, which are fibroblasts, which are from the graft, and located around the located around the densely arrange with a graft with a typical graft with a poor cell typical morphology morphology, and fail growth, and fail to to expand further expand further 7 There are very few There are very few Fibroblasts very fibroblasts with a viable fibroblasts, and densely arrange with a fairly good growth most of the grown typical morphology, condition, which are fibroblasts have dead and grow away from close to the periphery the graft, contact of the graft and fail to inhibition is formed expand further into the distance 8 There are very few There are a few viable Fibroblasts very fibroblasts with a fibroblasts, which are densely arrange with a normal morphology located around the typical morphology and fairly good growth graft condition, which fail to expand further into the distance 9 There are a few viable There are a few viable Fibroblasts densely fibroblasts in an aging fibroblasts, which are arrange with a typical state with a nontypical located around the morphology, and grow morphology, which are graft and fail to expand away from the graft located around the further into the graft distance 10 The morphology of The morphology of Fibroblasts densely fibroblast is changed, most fibroblasts arrange with a typical the fibroblasts stop to around the graft is morphology, and grow grow and fail to changed, the fibroblast away from the graft expand further into the growth is significantly distance inhibited, and the fibroblast fail to expand further into the distance 11 There are a few There are degenerated Fibroblasts very fibroblasts, which are and dead fibroblasts densely arrange with a located around the and a few viable typical morphology, graft and fail to expand fibroblasts, and the and grow away from to the distance, the growth is significantly the graft, the growth is growth is significantly inhibited at its best inhibited 12 There are a few There are a few Fibroblasts densely fibroblasts, which are fibroblasts, which are arrange with a typical located around the located around the morphology, and grow graft, and the growth is graft, and the growth is away from the graft significantly inhibited significantly inhibited 13 The morphology of There are a few Fibroblasts densely fibroblast is changed fibroblasts with a arrange with a typical obviously, and the changed morphology, morphology, and grow growth is significantly which are located vigorously away from inhibited around the graft, and the graft the growth is significantly inhibited 14 There are very few There are a few Fibroblasts densely fibroblasts, which are fibroblasts, which fail arrange with a typical located around the to expand, and the morphology, and grow graft and fail to growth is significantly vigorously away from expand, and the inhibited the graft growth is significantly inhibited

[0144] The pharmaceutical composition obtained in this example was subjected to the test methods of Example 3 and 4. It is found that the pharmaceutical composition prepared in this example can inhibit human fibroblast and organ fibrosis of aging rat.

Example 9: Clinical Cases of Regulating Fibroblast Growth of the Pharmaceutical Composition

[0145] Case 1: Regeneration and Recovery of Gastric Scar

[0146] Case 1 (male, born on 25 Feb. 1953) has a history of gastric ulcer and chronic erosive gastritis. The patient was administrated with five units each time (0.1 g of the pharmaceutical composition per unit) of the pharmaceutical composition obtained in Example 1 three times a day. Before the administration, SB capsule endoscopy showed obvious scars on the gastric mucosa. After 9 months of administration, it was found that the mucosal scars were significantly reduced. After 4 years of administration, it was found that the mucosal scars were recovered (see FIG. 11A to 11C).

[0147] Case 2: Regeneration and Recovery of Liver Fibrosis

[0148] Case 2 (male, born on 6 Jul. 1950) has a history of chronic hepatitis and liver fibrosis for 30 years, with a spleen 7.4 cm below the ribs. The patient was administrated with five units each time of the pharmaceutical composition obtained in Example 1 three times a day. The patient was recovered to normal after three years. The corresponding biochemical function indexes are normal, which can be seen in Table 5 below:

TABLE-US-00006 TABLE 5 Liver fiberography Before the After 3 years of administration administration Reference value Hyaluronic 44.2 36.0 Less than 40 ng/mL acid Type III 8.0 8.3 Less than 8.6 ng/mL procollagen Type IV 24.9 24.1 Less than 23.8 ng/mL collagen Laminin 125 107 Less than 113 g/L

[0149] Case 3: Regeneration and Recovery of Arteriosclerosis

[0150] Case 3 (female, born on 12 Feb. 1957) has a history of hypertension for 18 years and a history of diabetes for 17 years. The patient was administrated with five units each time of the pharmaceutical composition obtained in Example 1 three times a day. The coronary heart disease and arterial plaque are recovered at present.

[0151] Before the administration, the color B-ultrasound of double carotid arteries showed plaque on the left artery (soft plaque). After one year of administration, the ultrasound showed no obvious abnormalities in bilateral carotid arteries.

[0152] Before the administration, the coronary spiral CT showed coronary artery calcification and stenosis in the proximal segment of the left anterior descending branch (30%). After one year of administration, there were no abnormalities in coronary arteries diagnosed by imaging.

REFERENCES

[0153] [1] Cai L I, Current Research of Organ Fibrosis, Basic and Clinic of Organ Fibrosis [M], Editor-in-Chief: Cai L I, Beijing: People's Medical Publishing House, 2003: 1-15. [0154] [2] Tianshui Y U, Dawei G U A N, Yong M A, Research Progress of Myofibroblast in Fibrotic Disease [J], Chinese Journal of Histochemistry and Cytochemistry, 2013, 22(2): 167-171. [0155] [3] Mitchell R N, Cotran R S, Tissue repair: cellular growth, fibrosis, and wound healing, In: Cotran R S, Kumar V, Collins T (eds.), Robbin's Pathologic Basis of Disease, W.B. Saunders Company, 1999(6th ed); 89-93. [0156] [4] Wei H E, Dermal Fibroblast Biology, Basics of Modern Dermatology, Editor-in-Chief: Xuejun Z H A N G, Weida L I U, Chundi H E, Beijing: People's Medical Publishing House. 2001; 137152. [0157] [5] Jingqu Z H O U, Qinglian P E N G, Inherent Connective Tissue, Histology, Editor-in-Chief: Lingzhong C H E N G, Beijing: People's Medical Publishing House, 1981; 218264. [0158] [6] Translator: Hong Z H O U, Fibroblast Isolation and Culture, Cell Experiment Guide (Volume 1) [M], Translator: Peitang H U A N G et al. Beijing: Science Press, 2001: 27-31. [0159] [7] Zheng E, Culture of Individual Tissue and Cell, Tissue Culture and Molecular Cytology Techniques [M], Editor-in-Chief: Zheng E, Beijing: Beijing Publishing House, 1995: 120-138. [0160] [8] Xiuqin W A N G, Min W U, Culture of Human Skin Fibroblast; Practical Methods and Techniques of Cell Biology [M], Editor-in-Chief: Jingbo Z H A N G, Beijing: United Press of Beijing Medical University and China Union Medical University, 1995: 1-2. [0161] [9] Yuping X I A O, In Vitro Culture of Connective Tissue, Principles and Techniques of In Vitro Culture [M], Editor-in-Chief: Qingshan X U E, Beijing: Science Press, 2001: 442-452.