US20190269609A1 - A multi-component injection - Google Patents
A multi-component injection Download PDFInfo
- Publication number
- US20190269609A1 US20190269609A1 US15/737,808 US201715737808A US2019269609A1 US 20190269609 A1 US20190269609 A1 US 20190269609A1 US 201715737808 A US201715737808 A US 201715737808A US 2019269609 A1 US2019269609 A1 US 2019269609A1
- Authority
- US
- United States
- Prior art keywords
- ethanol
- parts
- concentrate
- filtrate
- cool
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 238000002347 injection Methods 0.000 title claims abstract description 192
- 239000007924 injection Substances 0.000 title claims abstract description 192
- 239000003814 drug Substances 0.000 claims abstract description 101
- 235000006484 Paeonia officinalis Nutrition 0.000 claims abstract description 97
- 244000020518 Carthamus tinctorius Species 0.000 claims abstract description 76
- 206010040047 Sepsis Diseases 0.000 claims abstract description 49
- 239000004615 ingredient Substances 0.000 claims abstract description 22
- 241000382455 Angelica sinensis Species 0.000 claims abstract description 17
- 241000112528 Ligusticum striatum Species 0.000 claims abstract description 17
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 880
- 239000012141 concentrate Substances 0.000 claims description 240
- 239000007788 liquid Substances 0.000 claims description 201
- 239000000706 filtrate Substances 0.000 claims description 168
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical class CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 137
- 238000001914 filtration Methods 0.000 claims description 100
- 241001106477 Paeoniaceae Species 0.000 claims description 96
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 96
- 229910001868 water Inorganic materials 0.000 claims description 86
- 238000000034 method Methods 0.000 claims description 85
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 81
- 239000000284 extract Substances 0.000 claims description 80
- 238000001816 cooling Methods 0.000 claims description 72
- 238000009835 boiling Methods 0.000 claims description 64
- 230000008569 process Effects 0.000 claims description 54
- 239000000243 solution Substances 0.000 claims description 52
- 239000000463 material Substances 0.000 claims description 50
- 108010010803 Gelatin Proteins 0.000 claims description 48
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- 239000008273 gelatin Substances 0.000 claims description 48
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- 235000011852 gelatine desserts Nutrition 0.000 claims description 48
- 230000003381 solubilizing effect Effects 0.000 claims description 48
- 239000000203 mixture Substances 0.000 claims description 32
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Definitions
- the present invention relates to the technical field of medical technology, and particularly relates to a multi-component injection and preparation processes and applications thereof.
- Sepsis is a fatal organ dysfunction caused by the reaction disorder of the infected hosts, with a hospital mortality of more than 10%, which has posed a huge threat to human health, becoming a focus to which the medical field pays close attention [see JAMA. 2016 Feb. 23; 315(8):801-10].
- Sepsis is not only a process of systemic inflammatory reaction or immune disorder, but also a complex chronic critical disease combining sustained inflammation, immunosuppression and catabolism. [See J Trauma Acute Care Surg. 2012 June; 72(6): 1491-501, BMJ. 2016 May 23; 353: i1585 and Medicine (Baltimore). 2015 December; 94(50):e2044.].
- Xuebijing injection is an intravenous injection developed on the basis of “Xuefu Zhuyu Decoction” recorded in Wang Qingren's “Corrections on the Errors of Medical Works” in Qing Dynasty in accordance with the syndrome differentiation principle of “Three Syndromes and Three Methods” under the theoretical guidance of “Treatment for bacteria, endotoxin and inflammatory media together”.
- the “Xuebijing injection” is prepared from five medicinal materials, i.e., safflower carthamus, Red Paeony Root, Ligusticum wallichii , radix salviae miltiorrnhizae and Chinese angelica , via extraction, refining, drying, blending and other modern techniques.
- the “Xuebijing injection” belongs to the traditional Chinese medicine for dispersing blood stasis and detoxifying, which is applied for warm heat diseases, such as fever, dyspnea, tachypnea, palpitation, dysphoria and other syndromes of intermingling of blood statsis and toxin, and is suitable for the systemic inflammatory reaction syndrome induced by infection.
- the Xuebijing injection can also be used during the period of organ function lesion for treatment of multi-organ disfunction syndrome.
- the “Xuebijing injection” has the pharmacological actions of antagonizing bacterial endotoxin [See Chinese critical care medicine, 2006, 18 (11): 643-4], inhibiting inflammatory factors from being released excessively [See Chinese critical care medicine, 2006, 18 (11): 643-4, Evid Based Complement Alternat Med. 2015; 2015:860259 and Chin J Integr Med.
- the invention provides a multi-component injection, which is characterized by comprising the following active ingredients, by weight: 8.66-35.26 parts of albiflorin std, 1000.0-1700.0 parts of paeoniflorin, 11.13-68.07 parts of Oxypaeoniflorin, 12.20-52.98 parts of Benzoylpaeoniflorin, 0.667-1.617 parts of Benzoyloxypaeoniflorin, 1.915-3.202 parts of Mudanpioside J, 10.63-20.13 parts of galloylpaeoniflorin, 0.804-1.338 parts of Mudanpioside C, 10.0-200.0 parts of benzoic acid, 0.21-15.88 parts of gallic acid, 0.2396-0.6860 parts of ethyl gallate, and 1.31-12.60 parts of catechin.
- the present invention further provides a multi-component injection, which is characterized by comprising the following active ingredients, by weight: 8.66-35.26 parts of albiflorin std, 1000.0-1700.0 parts of paeoniflorin, 11.13-68.07 parts of Oxypaeoniflorin, 12.20-52.98 parts of Benzoylpaeoniflorin, 0.667-1.617 parts of Benzoyloxypaeoniflorin, 1.915-3.202 parts of Mudanpioside J, 10.63-20.13 parts of galloylpaeoniflorin, 0.804-1.338 parts of Mudanpioside C, 10.0-200.0 parts of benzoic acid, 0.21-15.88 parts of gallic acid, 0.2396-0.6860 parts of ethyl gallate, 1.31-12.60 parts of catechin, 1.232-3.547 parts of Kaempferol-3-0-gluco
- the present invention further provides a multi-component injection, which is characterized by comprising the following active ingredients, by weight: 8.66-35.26 parts of albiflorin std, 1000.0-1700.0 parts of paeoniflorin, 11.13-68.07 parts of Oxypaeoniflorin, 12.20-52.98 parts of Benzoylpaeoniflorin, 0.667-1.617 parts of Benzoyloxypaeoniflorin, 1.915-3.202 parts of Mudanpioside J, 10.63-20.13 parts of galloylpaeoniflorin, 0.804-1.338 parts of Mudanpioside C, 10.0-200.0 parts of benzoic acid, 0.21-15.88 parts of gallic acid, 0.2396-0.6860 parts of ethyl gallate, 1.31-12.60 parts of catechin, 1.232-3.547 parts of Kaempferol-3-0-gluco
- the present invention further provides a multi-component injection, which is characterized by comprising the following active ingredients, by weight: 8.66-35.26 parts of albiflorin std, 1000.0-1700.0 parts of paeoniflorin, 11.13-68.07 parts of Oxypaeoniflorin, 12.20-52.98 parts of Benzoylpaeoniflorin, 0.667-1.617 parts of Benzoyloxypaeoniflorin, 1.915-3.202 parts of Mudanpioside J, 10.63-20.13 parts of galloylpaeoniflorin, 0.804-1.338 parts of Mudanpioside C, 10.0-200.0 parts of benzoic acid, 0.21-15.88 parts of gallic acid, 0.2396-0.6860 parts of ethyl gallate, 1.31-12.60 parts of catechin, 1.232-3.547 parts of Kaempferol-3-0-glu
- Any one of multi-component injections of the invention is characterized in that the administration way thereof is injection administration.
- Any one of multi-component injections of the invention is used for preparing medicines for treating Sepsis.
- the preparation method for the multi-component injections of the invention also comprises the steps: mixing the above active ingredients which are purchased or prepared, and preparing the injection via the conventional technology of pharmaceutics.
- the technical solution of the invention patent is further described via the following experiments:
- the multi-component injection of the present invention has therapeutic effects on the Sepsis cell model and the Sepsis mouse model, indicating that the multi-component injection of the present invention has therapeutic effects on the Sepsis of mammals, thereby achieving therapeutic effects on the Sepsis of human bodies.
- mice purchased from Laboratory Animal Center of Peking Union Medical College
- cleaning level 6-8 weeks
- weight 19-23 g
- the medicines of the invention are prepared in accordance with the embodiments 2-5, and endotoxin is purchased from U.S. Sigma Company.
- mice are divided into a normal group, a model group, experimental groups 1-12 randomly, and there are 20 mice in each group. The mice are prohibited from being fed for 12 h before the experiment, weighed and subjected to grouping in accordance with the table of random numbers.
- the model group only the modeling of counteracting toxic substances by endotoxin is carried out; in experimental groups 1-12, 12 batches of multi-component injections in embodiments 2-5 are received respectively for medicine intervention with administration by injection (the first batch in the embodiment 2 to the third batch of the embodiment 5 are assigned to the experiment groups 1-12 successively) after the modeling of counteracting toxic substances by endotoxin has been carried out; and the normal group is not subjected to additional intervention.
- mice purchased from Laboratory Animal Center of Peking Union Medical College
- cleaning level 6-8 weeks
- weight 19-23 g
- the medicines of the invention are prepared in accordance with the embodiments 2-5.
- mice are divided into a control group, a model group, experimental groups 1-12 randomly, and there are 20 mice in each group.
- the mice are prohibited from being fed for 12 h before the experiment, weighed and subjected to grouping in accordance with the table of random numbers.
- the control group only the same operation process is carried out, but neither ligation nor puncture is carried out on caecum; in the model group, the cecal ligation and puncture (CLP) is carried out; for experimental groupsl-12, 12 batches of multi-component injections in embodiments 2-5 are injected respectively (the first batch in the embodiment 2 to the third batch of the embodiment 5 are administered to the experimental groups 1-12 successively) for medicine intervention after CLP modeling.
- CLP cecal ligation and puncture
- the cecum is returned to the enterocoelia, and the incision of abdominal wall is sutured.
- the postoperation skin behind the neck is subjected to conventional disinfection, 1 mL normal saline is subcutaneous injected for anabiosis, and after coming round from anesthesia, each of the mice is raised in a single cage.
- 12 batches of multi-component injections in embodiments 2-5 are respectively injected from the auricular vein in the experimental groupsl-12, at 0.5 h, 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, 84 h and 96 h after the operation, with dosage of 4 mL/kg; and in the model group and the control group, 4 ml/kg (by weight) of normal saline is injected from the auricular vein at corresponding time.
- the death rate of the Sepsis model group is 70%, within 7 days after CLP operation, whereas the death rates of the experimental groups 1-12 are shown as the table below. Where, the death rates of the experimental groups 7-12 are obviously lower than that of the Sepsis model group (p ⁇ 0.05). It indicates that in the embodiment 4 (experiment groups 7-9) and embodiment 5 (experiment groups 10-12), a plurality of batches of multi-component injections have the protective effects on the Sepsis mice caused by endotoxin.
- Wistar male rats purchased from Experimental Animal Research Institute of Chinese Academy of Medical Sciences
- cleaning level weight of 180-220 g
- the medicines of the present invention are prepared in accordance with the embodiments 2-5; the kit for high-mobility family protein B1 (HMGB) enzyme linked immunosorbent assay (ELISA) (Japanese Shino-Test Company).
- HMGB high-mobility family protein B1
- ELISA enzyme linked immunosorbent assay
- rats 140 rats are divided into a control group, a model group, experimental groups 1-12 randomly, and there are 10 rats in each group. The rats are prohibited from being fed for 12 h before the experiment, weighed and subjected to grouping in accordance with the table of random numbers.
- control group laparotomy is carried out to expose the cecum, the skin is sutured, and then, 10 ml of normal saline is subcutaneous injected for anabiosis; in the Sepsis model group, the cecal ligation and puncture (CLP) is carried out for modeling; and in experimental groups 1-12, 12 batches of multi-component injections in embodiments 2-5 are injected respectively (the first batch in the embodiment 2 to the third batch of the embodiment 5 are administered to the experiment groups 1-12 successively) for medicine intervention after CLP modeling.
- CLP cecal ligation and puncture
- the mixed solution of ketamine injection+sumianxin II injection of 2:1 is subjected to intramuscular injection to anesthetize rats, and the Sepsis animal model is prepared using CLP.
- the No. 18 syringe needle penetrates through the cecum for 2 times to form intestinal fistula, 2 drainage strips (0.5 cm ⁇ 2.0 cm) are indwelled to prevent needle hole from healing, then, skin is sutured layer by layer, and 10 mL of normal saline is immediately subcutaneously injected for anabiosis after the operation.
- Animals of each group which are anesthetized, are subjected to sterile blood sampling with 3 mL at abdominal aorta at 2 h, 8 h, 24 h, 48 h and 72 h after CLP, and plasma HMGB1 content is detected via the enzyme linked immunosorbent assay (ELISA).
- ELISA enzyme linked immunosorbent assay
- Wistar male rats purchased from Experimental Animal Research Institute of Chinese Academy of Medical Sciences
- cleaning level weight of 180-220 g
- the medicines of the invention are prepared in accordance with the embodiments 2-5; the kit for tissue factor (TF) enzyme linked immunosorbent assay (ELISA) (USA USCN Company); fluorescein isothiocyanate (FITC) labelled anti-rat monocyte protease activated receptor-1 (PAR-1) antibody (USA Santa Cruz Company).
- TF tissue factor
- ELISA enzyme linked immunosorbent assay
- FITC fluorescein isothiocyanate
- PAR-1 anti-rat monocyte protease activated receptor-1
- rats 140 rats are divided into a control group, a model group, experimental groups 1-12 randomly, and there are 10 rats in each group. The rats are prohibited from being fed for 12 h before the experiment, weighed and subjected to grouping in accordance with the table of random numbers.
- laparotomy is carried out to expose the cecum, after that, the skin is sutured, and then, 10 mL of normal saline is injected subcutaneously for anabiosis; in the Sepsis model group, the cecal ligation and puncture (CLP) is carried out for modeling; and in experiment groups 1-12, 12 batches of multi-component injections in embodiments 2-5 are injected respectively (the first batch in the embodiment 2 to the third batch of the embodiment 5 are administered to the experiment groups 1-12 successively) for medicine intervention after CLP modeling.
- CLP cecal ligation and puncture
- the No. 18 syringe needle penetrates through the cecum for 2 times to form intestinal fistula, 2 drainage strips (0.5 cm ⁇ 2.0 cm) are indwelled to prevent needle hole from healing, then, skin is sutured layer by layer, and 10 mL of normal saline is immediately subcutaneously injected for anabiosis after the operation.
- 12 batches of multi-component injections in embodiments 2-5 are respectively injected from the dorsal vein of penis, with a dose of 4 mL/kg at 2 h, 12 h, 24 h, 36 h, 48 h and 60 h, after the operation; and in the model group and the control group, 4 mL/kg (by weight) of normal saline is injected from the dorsal vein of penis at the corresponding time.
- Animals of each group, which are anesthetized, are subjected to sterile blood sampling with 3 mL at aorta abdominalis at 12 h, 24 h, 48 h, 48 h and 72 h after CLP.
- the expression of the plasma monocyte protease activated receptor-1 (PAR-1) is detected by using the flow immumofluorescence method; and the content of plasma tissue factors (TF) are detected by using the enzyme linked immunosorbent assay (ELISA).
- Detection of PAR-1 expression taking 2 mL of heparin for anticoagulant, and separating mono karyocyte by using rat lymphocyte separating medium; resuspendeding mono karyocyte by using 10% of the full cell culture fluid of fetal calf serum-RPMI 1640, adjusting cell density to be 4 ⁇ 106/mL, and washing a cell culture plate using the cell culture fluid after incubating for 2 h; adding 0.25% trypsin-EDTA digestive juice for digesting 3 min, after majority of cells exfoliating, adding 2 mL of full cell culture fluid of 10% fetal calf serum-RPMI 1640, and terminating digesting; washing using phosphate buffer (PBS) for 3 times to acquire peripheral blood mononuclear cells; adding 1 ⁇ g/1 ⁇ 106 cell fluorescein isothiocyanate (FITC) into cell suspension to mark the anti-rat PAR-1 antibody, incubating away from light at 4° C. for 15 min, adding 2 mL of PBS to wash for
- the average fluorescence intensities of PAR-1 at 24 h and 48 h in the experiment groups 1-3 are lower than that of the model group, with no significant difference; and the fluorescence intensities of the experiment groups at 72 h are significantly lower than that of the model group (p ⁇ 0.05).
- the average fluorescence intensities of PAR-1 of nine intervention groups of the experiment groups 4-12 at 24 h, 48 h and 72 h are significantly reduced (p ⁇ 0.05) by comparison to that of the model group.
- the mononuclear cells TF in the control group have a certain expression; the expression of TF at 12 h, 24 h, 48 h and 72 h after the operation in the model group are significantly higher than that of the control group (p ⁇ 0.05), which is gradually increased with the extension of postoperation time, and reach a peak at 48 h.
- the expression of TF at 12 h and 72 h in the experiment groups 1-3 are lower than that of the model group, with no significant difference; and the expression intensities of the experiment groups at 24 h and 48 h are significantly higher than that of the model group (p ⁇ 0.05).
- the expression of TF of nine intervention groups of the experiment groups 4-12 at 24 h, 48 h and 72 h are significantly reduced (p ⁇ 0.05) by comparison to that of the model group.
- Wistar male rats purchased from Experimental Animal Research Institute of Chinese Academy of Medical Sciences
- cleaning level weight of 180-220 g
- the medicines of the invention are prepared in accordance with the embodiments 2-5; phycoerythrin (PE)-anti-rat CD25, fluorescein isothiocyanate (FITC) labeled anti-rat CD4, allophycocyanin (APC) labeled Annexin V apoptosis reagent kit (USA BD Company); rat anti-PE reagent kit, CD4 magnetic beads, MiniMACS magnetic separation apparatus and separation column (Germany MiltenyiBiotec Company); PE labeled forkhead winged helix transcription factor (Foxp3) reagent kit, PE labeled T lymphocytotoxicity related antigen 4 (CTLA-4), anti-rat CD3 monoclonal antibody, anti-rat CD28 monoclonal antibody (USA eBioscience Company); interferon (IFN)-7, IL-4, IL-10 enzyme linked immunosorbent assay (ELISA) reagent kits (USA Biosource Company); IL-17 ELISA reagent kit (USA Usenlife Science Company); Trypan Blue
- rats 140 rats are divided into a control group, a model group, experimental groups 1-12 randomly, and there are 10 rats in each group. The rats are prohibited from being fed for 12 h before the experiment, weighed and subjected to grouping in accordance with the table of random numbers.
- laparotomy is carried out to expose the cecum, after that, the skin is sutured, and then, 10 mL of normal saline is injected subcutaneously for anabiosis; in the Sepsis model group, the cecal ligation and puncture (CLP) is carried out for modeling; and in experiment groups 1-12, 12 batches of multi-component injections in embodiments 2-5 are injected respectively (the first batch in the embodiment 2 to the third batch of the embodiment 5 are administered to the experiment groups 1-12 successively) for medicine intervention after CLP modeling.
- CLP cecal ligation and puncture
- the mixed solution of ketamine injection:sumianxin II injection of 2:1 is intramuscular injected to anesthetize rats, and the Sepsis animal model is prepared by using CLP.
- the No. 18 syringe needle penetrates through the cecum for 2 times to form intestinal fistula, 2 drainage strips (0.5 cm ⁇ 2.0 cm) are indwelled to prevent needle hole from healing, then, skin is sutured layer by layer, and 10 mL of normal saline is immediately injected subcutaneously for anabiosis after the operation.
- CD25+T cell dissociation reagent After carrying out dissociation by CD25+T cell dissociation reagent, carry out positive selection on negatively selected cells using FITC-anti-CD4 and CD4 magnetic beads to obtain CD4+CD25+Treg; and carry out positive selection on CD25-T cells using FITC-anti-CD4 and CD4 magnetic beads to obtain CD4+CD25-T cells. Test the purity of double positive cells via the flow cytometry. Dye CD4+CD25+Treg using 0.4% (mass fraction) of Trypan Blue, and observe cell survival rate. Put RPM11640 culture solution containing 20% (volume fraction) fetal bovine serum in a 48-pore culture plate, and culture in a CO 2 incubator.
- CD4+CD25+Treg in each group at the 3rd day for culturing for 12 h, adjust the concentration of CD4+CD25+Treg and CD4+CD25-T cells by using the culture solution to be 1:1 for culturing, stimulate using sword bean A (Con A, 5 mg/L), culture in a CO 2 incubator at 37° C. for 68 h, centrifuge to get a supernate which is frozen at ⁇ 70° C. for testing.
- sword bean A Con A, 5 mg/L
- Treg apoptosis rate culturing the separated cells for 12 h, washing the suspending CD4+CD25+Treg (1 ⁇ 10 9 /L) 2 times with phosphate buffer (PBS), adding 100 L of binding buffer and 10 L of APC labeled Annexin V (20 mg/L), keeping in the dark place at room temperature for 30 min, then, adding 10 L of actinomycin D (7-AAD), keeping in the dark place for reaction for 5 min, adding 400 ⁇ L of binding buffer, selecting 7-AAD negative Annexin V positive cells as apoptotic cells by the flow cytometry, and testing the cell apoptosis rate.
- PBS phosphate buffer
- APC labeled Annexin V 20 mg/L
- actinomycin D 7-AAD
- IL-10 secreted by Treg including main inhibitory cell factor IL-10 secreted by Treg, IFN- ⁇ secreted by Th1, and IL-4 secreted by Th2.
- the IL-10 specimen is from supernate collected after the separated Treg is cultured for 12 h in each group, IFN- ⁇ and IL-4 specimens are from supernate collected after CD4+CD25+Treg and CD4+CD25-T cells are subjected to co-culture for 68 h. Tests are performed by using the ELISA reagent kit in accordance with the specification, the standard curve and the regression equation are calculated respectively, the absorbancy of the samples is substituted into the standard curve, and the protein concentration of cell factors in the samples are calculated.
- the expression of Foxp3 and CTLA-4 in the model group is obviously higher than that of the control group (p ⁇ 0.05), the expression of the experimental groups 4-12 are significantly lower than that of the model group (p ⁇ 0.05), and the trends of the experimental groups 1-3 are close, without significant difference.
- the secretion level of IL-10 in the model group is obviously higher than that of the control group (p ⁇ 0.05), the secretion level of the experimental groups 4-12 are significantly lower than that of the model group (p ⁇ 0.05), and the trends of the experimental groups 1-3 are close, without significant difference.
- the levels of IFN- ⁇ and IL-4 in the model group are substantially increased compared with the control group, and the levels of IFN- ⁇ and IL-4 in the experimental groups 4-12 are further increased. Compared with the model group, there are statistical significance for difference (p ⁇ 0.05).
- the trends of experimental groups 1-3 are close, without significant difference.
- the multi-component injections of the present invention have the characteristics of stable quality, simple production process, convenience for production, safety, effectiveness, etc.
- FIG. 1 Comparison of survival rates of endotoxin induced Sepsis mice among groups Note: compared with the model group, the experimental groups 1-12 have the significant statistical difference (p ⁇ 0.05).
- FIG. 2 Comparison of the survival rates of cecal ligation and puncture mediated Sepsis mice among groups
- Chromatographic column Waters HSS T3 UPLC C18 Chromatographic column (100 mm ⁇ 2.1 mm; 1.8 ⁇ M, Waters, USA);
- Mobile phase A: H 2 O (containing 25 mM HCOOH, B: MeOH (containing 25 mM HCOOH);
- Chromatographic column Waters HSS T3 UPLC C18 Chromatographic column (100 mm ⁇ 2.1 mm; 1.8 ⁇ M, Waters, USA);
- Chromatographic column Waters HSS T3 UPLC C18 Chromatographic column (100 mm ⁇ 2.1 mm; 1.8 ⁇ M, Waters, USA);
- Mobile phase A: MeOH—H 2 O (v/v, 1:99), including 1 mM HCOOH and 25 ⁇ M, CH 3 COOLi; B: MeOH—H 2 O (v/v, 99:1), including 1 mM HCOOH and 25 ⁇ M, CH3COOLi; Gradient elute is shown in Table 8; flow velocity: 0.35 mL/min; injection volume: 5 ⁇ L; analysis time: 8 min.
Abstract
Description
- The present invention relates to the technical field of medical technology, and particularly relates to a multi-component injection and preparation processes and applications thereof.
- Sepsis is a fatal organ dysfunction caused by the reaction disorder of the infected hosts, with a hospital mortality of more than 10%, which has posed a huge threat to human health, becoming a focus to which the medical field pays close attention [see JAMA. 2016 Feb. 23; 315(8):801-10]. Under the big data retrospective analysis in combination with the microarray technology and analysis of leukocyte gene expression, people gradually realize that Sepsis is not only a process of systemic inflammatory reaction or immune disorder, but also a complex chronic critical disease combining sustained inflammation, immunosuppression and catabolism. [See J Trauma Acute Care Surg. 2012 June; 72(6): 1491-501, BMJ. 2016 May 23; 353: i1585 and Medicine (Baltimore). 2015 December; 94(50):e2044.].
- For many years, antibiotics, antiviral medicines, vasopressor medicines, etc. are used for the traditional treatment of Sepsis. However, there are not enough specific medicines put into clinical practice aiming at the pathogenesis of Sepsis. [See Crit Care Med. 2013 February; 41(2):580-637]. How to timely correct the systemic inflammatory reaction, coagulation disorders and immune dysfunction in the development process of Sepsis, restore proinflammatory-anti-inflammatory dynamic balance of organisms as early as possible, and effectively improve the prognosis of patients have become the important issues to be solved in the research and development of Sepsis treatment medicines.
- In 1970s, Professor Jinda Wang, a Chinese famous first-aid medical expert, presented the rules of treatment by traditional Chinese medicine for treating acute and critical diseases: the method of heat-clearing and detoxicating remedy is used for treating toxic-heat syndrome, the method for promoting blood circulation and removing blood-stasis is used for treating syndrome of blood stasis, and the method for supporting the healthy energy is used for treating acute asthenic symptoms, i.e., “Three Syndromes and Three Methods”, which are used as the basic methods of clinical practice for treating Sepsis [See China first-aid medicine of critical diseases, 2006, 18 (11): 643-4]. In 1975, he firstly confirmed that endotoxemia was the initial etiology of infectious multiple system organ failure, and proposed a new treatment strategy of “Treatment for both germs andendotoxin”. As he realized that the harm of endotoxin to organisms included inducing the production of in-vivo inflammatory media and then showing toxic effects, he further proposed treatment for bacteria, endotoxin and inflammatory media together, i.e., “Treatment for bacteria, endotoxin and inflammatory media together” [See Chinese Journal of Surgery of Integrated Traditional and Western Medicine, 2012, 18 (6): 553-554].
- “Xuebijing injection” is an intravenous injection developed on the basis of “Xuefu Zhuyu Decoction” recorded in Wang Qingren's “Corrections on the Errors of Medical Works” in Qing Dynasty in accordance with the syndrome differentiation principle of “Three Syndromes and Three Methods” under the theoretical guidance of “Treatment for bacteria, endotoxin and inflammatory media together”. The “Xuebijing injection” is prepared from five medicinal materials, i.e., safflower carthamus, Red Paeony Root, Ligusticum wallichii, radix salviae miltiorrnhizae and Chinese angelica, via extraction, refining, drying, blending and other modern techniques. The “Xuebijing injection” belongs to the traditional Chinese medicine for dispersing blood stasis and detoxifying, which is applied for warm heat diseases, such as fever, dyspnea, tachypnea, palpitation, dysphoria and other syndromes of intermingling of blood statsis and toxin, and is suitable for the systemic inflammatory reaction syndrome induced by infection. The Xuebijing injection can also be used during the period of organ function lesion for treatment of multi-organ disfunction syndrome. The “Xuebijing injection” has the pharmacological actions of antagonizing bacterial endotoxin [See Chinese critical care medicine, 2006, 18 (11): 643-4], inhibiting inflammatory factors from being released excessively [See Chinese critical care medicine, 2006, 18 (11): 643-4, Evid Based Complement Alternat Med. 2015; 2015:860259 and Chin J Integr Med. 2009; 15(1): 13-5], overcoming coagulation disorders [See Chinese Journal of Experimental Surgery, 2010, 27 (1): 32-4 and Chinese Journal of Integrated traditional and Western Medicine in Critical Care, 2009, 16 (4): 218-22], protecting vascular endothelial cells [See Chinese Journal of Integrated traditional and Western Medicine in Critical Care, 2009, 16 (4): 218-22], improving the microcirculation of tissue [See J Surg Res. 2016 May 1; 202(1):147-54], and improving immune dysfunction [see Chinese Journal of Surgery, 2009, 47 (1): 58-61 and Evid Based Complement Alternat Med. 2015; 2015:352642], which fully embodies the integrated regulatory effects of having multiple components, multiple links, multiple channels and multiple target points of traditional Chinese medicine. The results of the multi-centric clinical study with large sample sizes and meta analysis (Meta) indicate that it can reduce the 28 d case fatality rate and incidence rate of complication of Sepsis patients, shorten the average hospital stay, effectively improve the clinical indexes including systemic inflammatory response, coagulation function of patients, and the scoring of the scoring system II for acute physiology and chronic health condition (APACHE II), protect organ function, and significantly improve the clinical effect on the basis of the conventional comprehensive treatment in combination with the use of the “Xuebijing injection” [See Chinese critical care medicine 2015, 27(6): 465-76, Chinese Journal of Emergency Medicine, 2013, 22 (2): 130-5 and Medical Journal of Chinese People's Liberation Army, 2010, 35 (1): 9-12].
- In 2003, Chinese patent 03104977.X disclosed “a Chinese medicinal preparation for treating Sepsis and a preparation method thereof”, i.e., a preparation method for the “Xuebijing injection”, and its preparation process has been very matured. With the progress and innovation of science and technology, we carry out optimization on the process and elaboration of technological parameters, i.e., changing the removal of the heat source by 1% activated carbon in the original preparation process into ultrafiltration, thereby ensuring the safety of finished products and uniform and stable quality. The inventors, by means of the advanced research concept and instrument and equipment, have achieved the full-ingredient testing of such injection. We acquire a bran-new multi-component injection via the study on the material basis of the Xuebijing injection, in which the effective ingredients of the original “Xuebijing injection” are reserved.
- The invention provides a multi-component injection, which is characterized by comprising the following active ingredients, by weight: 8.66-35.26 parts of albiflorin std, 1000.0-1700.0 parts of paeoniflorin, 11.13-68.07 parts of Oxypaeoniflorin, 12.20-52.98 parts of Benzoylpaeoniflorin, 0.667-1.617 parts of Benzoyloxypaeoniflorin, 1.915-3.202 parts of Mudanpioside J, 10.63-20.13 parts of galloylpaeoniflorin, 0.804-1.338 parts of Mudanpioside C, 10.0-200.0 parts of benzoic acid, 0.21-15.88 parts of gallic acid, 0.2396-0.6860 parts of ethyl gallate, and 1.31-12.60 parts of catechin.
- The above-mentioned multi-component injection is characterized in that the preparation method thereof comprises the following steps:
- providing 100 g of Red Paeony Root decoction pieces; heating and boiling the Red Paeony Root decoction pieces with process water of 10 times the weight of the Red Paeony Root decoction pieces to obtain a first decoction after 2 hours slight boiling; filtering the first decoction to obtain filtrate I and a first dreg; boiling the first dreg with process water of 8 times the weight of the first dreg to obtain a second decoction after 1 hour slight boiling; filtering the second decoction to obtain filtrate II and a second dreg; mixing filtrate I and filtrate II to obtain a mixture; concentrating the mixture to obtain a first concentrate of 100 ml; adding a proper amount of gelatin solution to the first concentrate under stirring to obtain a gelatin-containing first concentrate; adding 95% ethanol to the gelatin-containing first concentrate to obtain an ethanol-containing first concentrate having an ethanol volume content of 70%; storing the ethanol-containing first concentrate under cooling condition for 24 hours to obtain a cool ethanol-containing first concentrate; filtering and concentrating the cool ethanol-containing first concentrate to obtain a second concentrate of 100 ml; extracting the second concentrate with water-saturated n-butanol for 4 times and using water-saturated n-butanol of 50 ml for each time; combining extract liquors of the 4 times extraction to obtain a combined extract liquor; recycling n-butanol from the combined extract liquor to obtain a n-butanol-reduced extract liquor without alcohol taste; and vacuum drying the n-butanol-reduced extract liquor to obtain a Red Paeony Root dry paste;
- providing a proper amount of the Red Paeony Root dry paste; dissolving the Red Paeony Root dry paste in injection water to obtain a dilute of 200 ml; storing the dilute under cooling condition to obtain a first cool liquid; adding glucosum anhydricum of an amount in accordance with 4.5% mass fraction of the multi-component injection and injection water to the first cool liquid to obtain a 1000 ml liquid; adjusting the pH value of the 1000 ml liquid to 5.5-7.0 with a sodium hydroxide solution of 10% mass fraction to obtain a pH adjusted liquid; storing the pH adjusted liquid under cooling condition to obtain a second cool liquid; subjecting the second cool liquid to ultrafiltration to obtain an ultrafiltrate; adding a proper amount of solubilizing auxiliary materials which are dissolved in a proper amount of injection water into the ultrafiltrate to obtain a solubilizing auxiliary materials-containing ultrafiltrate; adjusting the pH value of the solubilizing auxiliary materials-containing ultrafiltrate to 5.5-7.0 using the sodium hydroxide solution of 10% mass fraction to obtain a pH adjusted ultrafiltrate; filtering the pH adjusted ultrafiltrate to obtain a filtrate; encapsulating and sterilizing the filtrate to obtain the multi-component injection.
- On the basis of the above-mentioned multi-component injection, the present invention further provides a multi-component injection, which is characterized by comprising the following active ingredients, by weight: 8.66-35.26 parts of albiflorin std, 1000.0-1700.0 parts of paeoniflorin, 11.13-68.07 parts of Oxypaeoniflorin, 12.20-52.98 parts of Benzoylpaeoniflorin, 0.667-1.617 parts of Benzoyloxypaeoniflorin, 1.915-3.202 parts of Mudanpioside J, 10.63-20.13 parts of galloylpaeoniflorin, 0.804-1.338 parts of Mudanpioside C, 10.0-200.0 parts of benzoic acid, 0.21-15.88 parts of gallic acid, 0.2396-0.6860 parts of ethyl gallate, 1.31-12.60 parts of catechin, 1.232-3.547 parts of Kaempferol-3-0-glucoside, 0.0500-0.4184 parts of scutellarin, 0.755-2.570 parts of quercetin-3-0-glucoside, 8.42-29.40 parts of Kaempferol-3-0-rutinoside, 4.036-7.695 parts of Kaempferol-3-0-sophoroside, 1.517-5.598 parts of quercetin-3-0-rutinoside, 200.0-500.0 parts of Hydroxysafflor yellow A, 0.316-0.774 parts of uracil, 13.77-30.56 parts of adenine, 20.50-44.99 parts of phenylalanine, 11.44-27.13 parts of uridine, 5.07-12.63 parts of adenosine, 8.00-24.11 parts of guanosine, 4.96-16.86 parts of butanedioic acid, 2.384-5.404 parts of p-hydroxybenzoic acid, 3.00-17.98 parts of p-coumaric acid, 4.837-7.806 parts of caffeic acid and 3.83-8.59 parts of chlorogenic acid.
- The above-mentioned multi-component injection is characterized in that the preparation method thereof comprises the following steps:
- providing 100 g of safflower carthamus decoction pieces; leaching the safflower carthamus decoction pieces with 30% ethanol of 8 times the weight of the safflower carthamus decoction pieces for 8 hours to obtain a leach liquor; filtering the leach liquor to obtain a liquid medicine of 4-6 times the weight of the safflower carthamus decoction pieces; adding 95% ethanol to the liquid medicine to obtain an ethanol-containing liquid medicine having an ethanol volume content of 70%; storing the ethanol-containing liquid medicine under cooling condition for 48 hours to obtain a cool ethanol-containing liquid medicine; filtering the cool ethanol-containing liquid medicine to obtain a first filtrate; concentrating the first filtrate under reduced pressure to obtain a concentrate of 100 ml; adding 95% ethanol to the concentrate to obtain an ethanol-containing concentrate having an ethanol volume content of 80%; storing the ethanol-containing concentrate under cooling condition for 48 hours to obtain a cool ethanol-containing concentrate; filtering the cool ethanol-containing concentrate to obtain a second filtrate; recycling ethanol from the second filtrate to obtain an ethanol-reduced filtrate; concentrating and vacuum drying the ethanol-reduced filtrate to obtain a safflower carthamus dry paste;
- providing 100 g of Red Paeony Root decoction pieces; heating and boiling the Red Paeony Root decoction pieces with process water of 10 times the weight of the Red Paeony Root decoction pieces to obtain a first decoction after 2 hours slight boiling; filtering the first decoction to obtain filtrate I and a first dreg; boiling the first dreg with process water of 8 times the weight of the first dreg to obtain a second decoction after 1 hour slight boiling; filtering the second decoction to obtain filtrate II and a second dreg; mixing filtrate I and filtrate II to obtain a mixture; concentrating the mixture to obtain a first concentrate of 100 ml; adding a proper amount of gelatin solution to the first concentrate under stirring to obtain a gelatin-containing first concentrate; adding 95% ethanol to the gelatin-containing first concentrate to obtain an ethanol-containing first concentrate having an ethanol volume content of 70%; storing the ethanol-containing first concentrate under cooling condition for 24 hours to obtain a cool ethanol-containing first concentrate; filtering and concentrating the cool ethanol-containing first concentrate to obtain a second concentrate of 100 ml; extracting the second concentrate with water-saturated n-butanol for 4 times and using 50 ml water-saturated n-butanol for each time; combining extract liquors of the 4 times extraction to obtain a combined extract liquor; recycling n-butanol from the combined extract liquor to obtain a n-butanol-reduced extract liquor without alcohol taste; and vacuum drying the n-butanol-reduced extract liquor to obtain a Red Paeony Root dry paste;
- providing the safflower carthamus dry paste and the Red Paeony Root dry paste each of a proper amount; dissolving the two dry pastes in injection water to obtain a dilute of 200 ml; storing the dilute under cooling condition to obtain a first cool liquid; adding glucosum anhydricum of an amount in accordance with 4.5% mass fraction of the multi-component injection and injection water to the first cool liquid to obtain a 1000 ml liquid; adjusting the pH value of the 1000 ml liquid to 5.5-7.0 with a sodium hydroxide solution of 10% mass fraction to obtain a pH adjusted liquid; storing the pH adjusted liquid under cooling condition to obtain a second cool liquid; subjecting the second cool liquid to ultrafiltration to obtain an ultrafiltrate; adding a proper amount of solubilizing auxiliary materials which are dissolved in a proper amount of injection water into the ultrafiltrate to obtain a solubilizing auxiliary materials-containing ultrafiltrate; adjusting the pH value of the solubilizing auxiliary materials-containing ultrafiltrate to 5.5-7.0 using the sodium hydroxide solution of 10% mass fraction to obtain a pH adjusted ultrafiltrate; filtering the pH adjusted ultrafiltrate to obtain a filtrate; encapsulating and sterilizing the filtrate to obtain the multi-component injection.
- On the basis of the above-mentioned multi-component injection, the present invention further provides a multi-component injection, which is characterized by comprising the following active ingredients, by weight: 8.66-35.26 parts of albiflorin std, 1000.0-1700.0 parts of paeoniflorin, 11.13-68.07 parts of Oxypaeoniflorin, 12.20-52.98 parts of Benzoylpaeoniflorin, 0.667-1.617 parts of Benzoyloxypaeoniflorin, 1.915-3.202 parts of Mudanpioside J, 10.63-20.13 parts of galloylpaeoniflorin, 0.804-1.338 parts of Mudanpioside C, 10.0-200.0 parts of benzoic acid, 0.21-15.88 parts of gallic acid, 0.2396-0.6860 parts of ethyl gallate, 1.31-12.60 parts of catechin, 1.232-3.547 parts of Kaempferol-3-0-glucoside, 0.0500-0.4184 parts of scutellarin, 0.755-2.570 parts of quercetin-3-0-glucoside, 8.42-29.40 parts of Kaempferol-3-0-rutinoside, 4.036-7.695 parts of Kaempferol-3-0-sophoroside, 1.517-5.598 parts of quercetin-3-0-rutinoside, 200.0-500.0 parts of Hydroxysafflor yellow A, 0.316-0.774 parts of uracil, 13.77-30.56 parts of adenine, 20.50-44.99 parts of phenylalanine, 11.44-27.13 parts of uridine, 5.07-12.63 parts of adenosine, 8.00-24.11 parts of guanosine, 4.96-16.86 parts of butanedioic acid, 2.384-5.404 parts of p-hydroxybenzoic acid, 3.00-17.98 parts of p-coumaric acid, 4.837-7.806 parts of caffeic acid, 3.83-8.59 parts of chlorogenic acid, 6.51-69.39 parts of senkyunolide I (42), 1.55-18.27 parts of senkyunolide H, 4.30-14.22 parts of senkyunolide N, 2.460-5.648 parts of open-loop senkyunolide I, 1.55-10.74 parts of senkyunolide G, 1.43-8.67 parts of 3-hydroxyl-3-butylphthalide, 0.10-0.961 parts of senkyunolide A, and 7.66-47.15 parts of ferulaic acid.
- The above-mentioned multi-component injection is characterized in that the preparation method thereof comprises the following steps:
- taking 100 g of Ligusticum wallichii and Angelica sinensis decoction pieces respectively, treating in accordance with the Red Paeony Root process, keeping 300 ml of concentrated solution every time, and extracting with 150 ml of water saturated normal butanol each time to prepare dry paste;
- providing 100 g of safflower carthamus decoction pieces; leaching the safflower carthamus decoction pieces with 30% ethanol of 8 times the weight of the safflower carthamus decoction pieces for 8 hours to obtain a leach liquor; filtering the leach liquor to obtain a liquid medicine of 4-6 times the weight of the safflower carthamus decoction pieces; adding 95% ethanol to the liquid medicine to obtain an ethanol-containing liquid medicine having an ethanol volume content of 70%; storing the ethanol-containing liquid medicine under cooling condition for 48 hours to obtain a cool ethanol-containing liquid medicine; filtering the cool ethanol-containing liquid medicine to obtain a first filtrate; concentrating the first filtrate under reduced pressure to obtain a concentrate of 100 ml; adding 95% ethanol to the concentrate to obtain an ethanol-containing concentrate having an ethanol volume content of 80%; storing the ethanol-containing concentrate under cooling condition for 48 hours to obtain a cool ethanol-containing concentrate; filtering the cool ethanol-containing concentrate to obtain a second filtrate; recycling ethanol from the second filtrate to obtain an ethanol-reduced filtrate; concentrating and vacuum drying the ethanol-reduced filtrate to obtain a safflower carthamus dry paste;
- providing 100 g of Red Paeony Root decoction pieces; heating and boiling the Red Paeony Root decoction pieces with process water of 10 times the weight of the Red Paeony Root decoction pieces to obtain a first decoction after 2 hours slight boiling; filtering the first decoction to obtain filtrate I and a first dreg; boiling the first dreg with process water of 8 times the weight of the first dreg to obtain a second decoction after 1 hour slight boiling; filtering the second decoction to obtain filtrate II and a second dreg; mixing filtrate I and filtrate II to obtain a mixture; concentrating the mixture to obtain a first concentrate of 100 ml; adding a proper amount of gelatin solution to the first concentrate under stirring to obtain a gelatin-containing first concentrate; adding 95% ethanol to the gelatin-containing first concentrate to obtain an ethanol-containing first concentrate having an ethanol volume content of 70%; storing the ethanol-containing first concentrate under cooling condition for 24 hours to obtain a cool ethanol-containing first concentrate; filtering and concentrating the cool ethanol-containing first concentrate to obtain a second concentrate of 100 ml; extracting the second concentrate with water-saturated n-butanol for 4 times and using 50 ml water-saturated n-butanol for each time; combining extract liquors of the 4 times extraction to obtain a combined extract liquor; recycling n-butanol from the combined extract liquor to obtain a n-butanol-reduced extract liquor without alcohol taste; and vacuum drying the n-butanol-reduced extract liquor to obtain a Red Paeony Root dry paste;
- providing 100 g of Ligusticum wallichii decoction pieces and 100 g of Angelica sinensis decoction pieces; treating the Ligusticum wallichii decoction pieces and Angelica sinensis decoction pieces with a same treatment process as the Red Paeony Root decoction pieces except for keeping 300 ml of concentrate every time and extracting with 150 ml of water-saturated n-butanol each time, to obtain a third dry paste;
- providing the safflower carthamus dry paste, the Red Paeony Root dry paste and the third dry paste each of a proper amount; dissolving the three dry pastes in injection water to obtain a dilute of 200 ml; storing the dilute under cooling condition to obtain a first cool liquid; adding glucosum anhydricum of an amount in accordance with 4.5% mass fraction of the multi-component injection and injection water to the first cool liquid to obtain a 1000 ml liquid; adjusting the pH value of the 1000 ml liquid to 5.5-7.0 with a sodium hydroxide solution of 10% mass fraction to obtain a pH adjusted liquid; storing the pH adjusted liquid under cooling condition to obtain a second cool liquid; subjecting the second cool liquid to ultrafiltration to obtain an ultrafiltrate; adding a proper amount of solubilizing auxiliary materials which are dissolved in a proper amount of injection water into the ultrafiltrate to obtain a solubilizing auxiliary materials-containing ultrafiltrate; adjusting the pH value of the solubilizing auxiliary materials-containing ultrafiltrate to 5.5-7.0 using the sodium hydroxide solution of 10% mass fraction to obtain a pH adjusted ultrafiltrate; filtering the pH adjusted ultrafiltrate to obtain a filtrate; encapsulating and sterilizing the filtrate to obtain the multi-component injection.
- On the basis of the above-mentioned multi-component injections, the present invention further provides a multi-component injection, which is characterized by comprising the following active ingredients, by weight: 8.66-35.26 parts of albiflorin std, 1000.0-1700.0 parts of paeoniflorin, 11.13-68.07 parts of Oxypaeoniflorin, 12.20-52.98 parts of Benzoylpaeoniflorin, 0.667-1.617 parts of Benzoyloxypaeoniflorin, 1.915-3.202 parts of Mudanpioside J, 10.63-20.13 parts of galloylpaeoniflorin, 0.804-1.338 parts of Mudanpioside C, 10.0-200.0 parts of benzoic acid, 0.21-15.88 parts of gallic acid, 0.2396-0.6860 parts of ethyl gallate, 1.31-12.60 parts of catechin, 1.232-3.547 parts of Kaempferol-3-0-glucoside, 0.0500-0.4184 parts of scutellarin, 0.755-2.570 parts of quercetin-3-0-glucoside, 8.42-29.40 parts of Kaempferol-3-0-rutinoside, 4.036-7.695 parts of Kaempferol-3-0-sophoroside, 1.517-5.598 parts of quercetin-3-0-rutinoside, 200.0-500.0 parts of Hydroxysafflor yellow A, 0.316-0.774 parts of uracil, 13.77-30.56 parts of adenine, 20.50-44.99 parts of phenylalanine, 11.44-27.13 parts of uridine, 5.07-12.63 parts of adenosine, 8.00-24.11 parts of guanosine, 4.96-16.86 parts of butanedioic acid, 2.384-5.404 parts of p-hydroxybenzoic acid, 3.00-17.98 parts of p-coumaric acid, 4.837-7.806 parts of caffeic acid, 3.83-8.59 parts of chlorogenic acid, 6.51-69.39 parts of senkyunolide I (42), 1.55-18.27 parts of senkyunolide H, 4.30-14.22 parts of senkyunolide N, 2.460-5.648 parts of open-loop senkyunolide I, 1.55-10.74 parts of senkyunolide G, 1.43-8.67 parts of 3-hydroxyl-3-butylphthalide, 0.10-0.961 parts of senkyunolide A, 7.66-47.15 parts of ferulaic acid, 1.43-17.25 parts of protocatechualdehyde, 2.361-4.030 parts of protocatechuic acid, 2.776-6.845 parts of tanshinol, 5.07-12.78 parts of rosmarinic acid, 0.0697-0.4005 parts of salvianolic acid D, 1.123-4.732 parts of salvianolic acid C, 0.366-2.505 parts of salvianolic acid A, 0.429-0.945 parts of alkannic acid, and 4.00-11.00 parts of salvianolic acid B.
- The above-mentioned multi-component injection is characterized in that the preparation method thereof comprises the following steps:
- providing 100 g of safflower carthamus decoction pieces; leaching the safflower carthamus decoction pieces with 30% ethanol of 8 times the weight of the safflower carthamus decoction pieces for 8 hours to obtain a leach liquor; filtering the leach liquor to obtain a liquid medicine of 4-6 times the weight of the safflower carthamus decoction pieces; adding 95% ethanol to the liquid medicine to obtain an ethanol-containing liquid medicine having an ethanol volume content of 70%; storing the ethanol-containing liquid medicine under cooling condition for 48 hours to obtain a cool ethanol-containing liquid medicine; filtering the cool ethanol-containing liquid medicine to obtain a first filtrate; concentrating the first filtrate under reduced pressure to obtain a concentrate of 100 ml; adding 95% ethanol to the concentrate to obtain an ethanol-containing concentrate having an ethanol volume content of 80%; storing the ethanol-containing concentrate under cooling condition for 48 hours to obtain a cool ethanol-containing concentrate; filtering the cool ethanol-containing concentrate to obtain a second filtrate; recycling ethanol from the second filtrate to obtain an ethanol-reduced filtrate; concentrating and vacuum drying the ethanol-reduced filtrate to obtain a safflower carthamus dry paste;
- providing 100 g of Red Paeony Root decoction pieces; heating and boiling the Red Paeony Root decoction pieces with process water of 10 times the weight of the Red Paeony Root decoction pieces to obtain a first decoction after 2 hours slight boiling; filtering the first decoction to obtain filtrate I and a first dreg; boiling the first dreg with process water of 8 times the weight of the first dreg to obtain a second decoction after 1 hour slight boiling; filtering the second decoction to obtain filtrate II and a second dreg; mixing filtrate I and filtrate II to obtain a mixture; concentrating the mixture to obtain a first concentrate of 100 ml; adding a proper amount of gelatin solution to the first concentrate under stirring to obtain a gelatin-containing first concentrate; adding 95% ethanol to the gelatin-containing first concentrate to obtain an ethanol-containing first concentrate having an ethanol volume content of 70%; storing the ethanol-containing first concentrate under cooling condition for 24 hours to obtain a cool ethanol-containing first concentrate; filtering and concentrating the cool ethanol-containing first concentrate to obtain a second concentrate of 100 ml; extracting the second concentrate with water-saturated n-butanol for 4 times and using 50 ml water-saturated n-butanol for each time; combining extract liquors of the 4 times extraction to obtain a combined extract liquor; recycling n-butanol from the combined extract liquor to obtain a n-butanol-reduced extract liquor without alcohol taste; and vacuum drying the n-butanol-reduced extract liquor to obtain a Red Paeony Root dry paste;
- providing 100 g of Ligusticum wallichii decoction pieces, 100 g of radix salviae miltiorrhizae and 100 g of Angelica sinensis decoction pieces; treating the three decoction pieces with a same treatment process as the Red Paeony Root decoction pieces except for keeping 300 ml of concentrate every time and extracting with 150 ml of water-saturated n-butanol each time, to obtain a third dry paste; providing the safflower carthamus dry paste, the Red Paeony Root dry paste and the third dry paste each of a proper amount; dissolving the three dry pastes in injection water to obtain a dilute of 200 ml; storing the dilute under cooling condition to obtain a first cool liquid; adding glucosum anhydricum of an amount in accordance with 4.5% mass fraction of the multi-component injection and injection water to the first cool liquid to obtain a 1000 ml liquid; adjusting the pH value of the 1000 ml liquid to 5.5-7.0 with a sodium hydroxide solution of 10% mass fraction to obtain a pH adjusted liquid; storing the pH adjusted liquid under cooling condition to obtain a second cool liquid; subjecting the second cool liquid to ultrafiltration to obtain an ultrafiltrate; adding a proper amount of solubilizing auxiliary materials which are dissolved in a proper amount of injection water into the ultrafiltrate to obtain a solubilizing auxiliary materials-containing ultrafiltrate; adjusting the pH value of the solubilizing auxiliary materials-containing ultrafiltrate to 5.5-7.0 using the sodium hydroxide solution of 10% mass fraction to obtain a pH adjusted ultrafiltrate; filtering the pH adjusted ultrafiltrate to obtain a filtrate; encapsulating and sterilizing the filtrate to obtain the multi-component injection.
- Any one of multi-component injections of the invention is characterized in that the administration way thereof is injection administration.
- Any one of multi-component injections of the invention is used for preparing medicines for treating Sepsis.
- As the active ingredients in the multi-component injections of the invention are known substances, which can be acquired via the known methods, the preparation method for the multi-component injections of the invention also comprises the steps: mixing the above active ingredients which are purchased or prepared, and preparing the injection via the conventional technology of pharmaceutics.
- The technical solution of the invention patent is further described via the following experiments: The multi-component injection of the present invention has therapeutic effects on the Sepsis cell model and the Sepsis mouse model, indicating that the multi-component injection of the present invention has therapeutic effects on the Sepsis of mammals, thereby achieving therapeutic effects on the Sepsis of human bodies.
- The advantages of the present invention will be further detailed via the following experiment data:
- Experiment Animals
- Balb/C male mice (purchased from Laboratory Animal Center of Peking Union Medical College), cleaning level, 6-8 weeks, weight of 19-23 g, adaptive feeding for 3 days.
- Medicine and Reagent
- The medicines of the invention are prepared in accordance with the embodiments 2-5, and endotoxin is purchased from U.S. Sigma Company.
- Experiment Method
- 3.1 Grouping and Intervention
- 280 mice are divided into a normal group, a model group, experimental groups 1-12 randomly, and there are 20 mice in each group. The mice are prohibited from being fed for 12 h before the experiment, weighed and subjected to grouping in accordance with the table of random numbers. In the model group, only the modeling of counteracting toxic substances by endotoxin is carried out; in experimental groups 1-12, 12 batches of multi-component injections in embodiments 2-5 are received respectively for medicine intervention with administration by injection (the first batch in the
embodiment 2 to the third batch of theembodiment 5 are assigned to the experiment groups 1-12 successively) after the modeling of counteracting toxic substances by endotoxin has been carried out; and the normal group is not subjected to additional intervention. The modeling process of counteracting toxic substances by endotoxin is as below: the mixed solution of ketamine hydrochloride:sumianxin II:PBS (phosphate buffer)=1:1.5:2.5 is selected as anaesthetic, and intramuscular injection is carried out for anesthesia in a dose of 2 mL/kg. After anesthesia is satisfied, the mice are fixed on the operating table, and 10 mg/kg of endotoxin is injected to the enterocoelia of the mice. In experimental groups 1-12, 12 batches of multi-component injections in embodiments 2-5 are injected at the auricular vein with a dose of 4 mL/kg (by weight) at 30 min and 60 min before counteracting toxic substances by endotoxin and 30 min after counteracting toxic substances by endotoxin respectively; in the model group, normal saline are injected at auricular vein with 4 mL/kg (by weight) at 30 min and 60 min before counteracting toxic substances by endotoxin and 30 min after counteracting toxic substances by endotoxin. - 3.2 Observation and Statistics of Death Rate
- Dead mice are observed and counted every 24 h. Statistic analysis is carried out by Kaplan-Merer method (statistics software is SPSS 16.0), and p<0.05 indicates significant difference.
- Experiment Results
- The results indicate that the death rate of the Sepsis model group is 100%, within 7 days after the injection of endotoxin, whereas the death rates of the experimental groups 1-12 are shown as the table below, which are obviously lower than that of the Sepsis model group (p<0.05).
- The above results prompt that in embodiments 2-5, 12 batches of multi-component injections have the protective effects on Sepsis mice caused by endotoxin.
-
TABLE 1 The effects of 12 batches of multi-component injections in embodiments 2-5 on the death of Sepsis mice caused by endotoxin. Death number of the 7th day after being attacked Group Quantity by endotoxin Death rate (%) Normal group 20 0 0 Model group 20 20 100 Experimental group 120 7 35* Experimental group 220 8 40* Experimental group 320 9 45* Experimental group 420 8 40* Experimental group 520 8 40* Experimental group 620 9 45* Experimental group 720 8 40* Experimental group 820 8 40* Experimental group 920 7 35* Experimental group 1020 7 35* Experimental group 1120 7 35* Experimental group 1220 7 35* Note: *indicates p < 0.05 compared with the model group. - Experiment Animals
- Balb/C male mice (purchased from Laboratory Animal Center of Peking Union Medical College), cleaning level, 6-8 weeks, weight of 19-23 g, adaptive feeding for 3 days.
- Medicine and Reagent
- The medicines of the invention are prepared in accordance with the embodiments 2-5.
- Experiment Method
- 3.1 Grouping and Intervention
- 280 mice are divided into a control group, a model group, experimental groups 1-12 randomly, and there are 20 mice in each group. The mice are prohibited from being fed for 12 h before the experiment, weighed and subjected to grouping in accordance with the table of random numbers. In the control group, only the same operation process is carried out, but neither ligation nor puncture is carried out on caecum; in the model group, the cecal ligation and puncture (CLP) is carried out; for experimental groupsl-12, 12 batches of multi-component injections in embodiments 2-5 are injected respectively (the first batch in the
embodiment 2 to the third batch of theembodiment 5 are administered to the experimental groups 1-12 successively) for medicine intervention after CLP modeling. The CLP modeling process is as below: the mixed solution of ketamine hydrochloride:sumianxin II:PBS=1:1.5:2.5 is selected as anaesthetic, and intramuscular injection is carried out for anesthesia at a dose of 2 mL/kg. After anesthesia is satisfied, the mice are fixed on the operating table, thorax and abdomen of the mice are subjected to conventional disinfection for draping, the median abdominal incision is carried out at aposition 1 cm below the xiphoid, with an incision length of 1.5 cm. The cecum is exposed and is subjected to ligation at ½ position, and a 21 G syringe needle penetrates through ligated cecum and aviods blood vessels. After it is confirmed that there is no active bleeding, the cecum is returned to the enterocoelia, and the incision of abdominal wall is sutured. The postoperation skin behind the neck is subjected to conventional disinfection, 1 mL normal saline is subcutaneous injected for anabiosis, and after coming round from anesthesia, each of the mice is raised in a single cage. After the mice are subjected to the CLP operation, 12 batches of multi-component injections in embodiments 2-5 are respectively injected from the auricular vein in the experimental groupsl-12, at 0.5 h, 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, 84 h and 96 h after the operation, with dosage of 4 mL/kg; and in the model group and the control group, 4 ml/kg (by weight) of normal saline is injected from the auricular vein at corresponding time. - 3.2 Observation and Statistics of Death Rate
- Dead mice are observed and counted every 24 h. Statistic analysis is carried out via the Kaplan-Merer method (statistics software is SPSS 16.0), and p<0.05 indicates significant difference.
- Experiment Results
- The results indicate that the death rate of the Sepsis model group is 70%, within 7 days after CLP operation, whereas the death rates of the experimental groups 1-12 are shown as the table below. Where, the death rates of the experimental groups 7-12 are obviously lower than that of the Sepsis model group (p<0.05). It indicates that in the embodiment 4 (experiment groups 7-9) and embodiment 5 (experiment groups 10-12), a plurality of batches of multi-component injections have the protective effects on the Sepsis mice caused by endotoxin.
-
TABLE 2 The effects of 12 batches of multi-component injections in embodiments 2-5 on the death of CLP mediated Sepsis mice. Death number of the 7th day after CLP Group Quantity operation Death rate (%) Control group 20 0 0 Model group 20 14 70 Experimental group 120 10 50 Experimental group 220 11 55 Experimental group 320 10 50 Experimental group 420 8 40 Experimental group 520 8 40 Experimental group 620 8 40 Experimental group 720 5 25* Experimental group 820 6 30* Experimental group 920 6 30* Experimental group 1020 5 25* Experimental group 1120 5 25* Experimental group 1220 5 25* Note: *indicates P < 0.05 compared with the Sepsis model group. - Experiment Animals
- Wistar male rats (purchased from Experimental Animal Research Institute of Chinese Academy of Medical Sciences), cleaning level, weight of 180-220 g, adaptive feeding for 1 week.
- Medicine and Reagent
- The medicines of the present invention are prepared in accordance with the embodiments 2-5; the kit for high-mobility family protein B1 (HMGB) enzyme linked immunosorbent assay (ELISA) (Japanese Shino-Test Company).
- Experiment Method
- 3.1 Grouping and Intervention
- 140 rats are divided into a control group, a model group, experimental groups 1-12 randomly, and there are 10 rats in each group. The rats are prohibited from being fed for 12 h before the experiment, weighed and subjected to grouping in accordance with the table of random numbers. In the control group, laparotomy is carried out to expose the cecum, the skin is sutured, and then, 10 ml of normal saline is subcutaneous injected for anabiosis; in the Sepsis model group, the cecal ligation and puncture (CLP) is carried out for modeling; and in experimental groups 1-12, 12 batches of multi-component injections in embodiments 2-5 are injected respectively (the first batch in the
embodiment 2 to the third batch of theembodiment 5 are administered to the experiment groups 1-12 successively) for medicine intervention after CLP modeling. In the CLP modeling process, the mixed solution of ketamine injection+sumianxin II injection of 2:1 is subjected to intramuscular injection to anesthetize rats, and the Sepsis animal model is prepared using CLP. At the joint of the ligated cecum and the ileum, the No. 18 syringe needle penetrates through the cecum for 2 times to form intestinal fistula, 2 drainage strips (0.5 cm×2.0 cm) are indwelled to prevent needle hole from healing, then, skin is sutured layer by layer, and 10 mL of normal saline is immediately subcutaneously injected for anabiosis after the operation. After the rats are subjected to the CLP operation, in the experiment groupsl-12, at 2 h, 12 h, 24 h, 36 h, 48 h and 60 h, after the operation, 12 batches of multi-component injections in embodiments 2-5 are respectively injected from the dorsal vein of penis, with a dose of 4 mL/kg; and in the model group and the control group, 4 mL/kg (by weight) of normal saline is injected from the dorsal vein of penis at the corresponding time. - 3.2 Blood Sampling and Testing
- Animals of each group, which are anesthetized, are subjected to sterile blood sampling with 3 mL at abdominal aorta at 2 h, 8 h, 24 h, 48 h and 72 h after CLP, and plasma HMGB1 content is detected via the enzyme linked immunosorbent assay (ELISA).
- Experiment Results
- The results indicate that in the control group, plasma contains a little amount of HMGB1; in the early stage after the CLP operation, HMGB1 content in the model group is obviously increased, the HMGB1 level at 8 h is further improved, and is gradually reduced after 24 h, but is still higher than that of the control group at 72 h after the operation. There is statistical significance between the two groups (p<0.05). While in the experiment groups 1-12 with intervention treatment via 12 batches of multi-component injection in embodiments 2-5, the plasma content at 2 h after the operation is obviously lower than that of the model group (p<0.05), HMGB1 is reduced more obviously (p<0.01) after 24 h, which is close to the level of the control group. The results prompt that 12 batches of multi-component injections in embodiments 2-5 have the inhibiting effects on the expression of HMGB1 during Sepsis.
-
TABLE 3 The effects of 12 batches of multi-component injections in embodiments 2-5 on the HMGB1 level of Sepsis rat plasma ( x ± s, n = 10) Unit: μg/L2 h after 8 h after 24 h after 48 h after 72 h after Group operation operation operation operation operation Control group 8.2 ± 2.8 8.3 ± 2.5 8.0 ± 2.3 8.1 ± 1.6 8.2 ± 2.2 Model group 28.8 ± 10.8# 31.7 ± 7.2# 23.2 ± 6.3# 15.3 ± 1.3# 17.3 ± 1.1# Experimental group 1 15.3 ± 5.2* 17.4 ± 3.3* 8.2 ± 1.7* 8.0 ± 2.2* 8.1 ± 1.3* Experimental group 2 17.2 ± 5.7* 19.2 ± 7.8* 8.4 ± 2.0* 6.8 ± 1.5* 10.5 ± 1.4* Experimental group 3 17.0 ± 5.3* 19.5 ± 6.1* 9.0 ± 2.7* 7.1 ± 2.2* 10.7 ± 2.5* Experimental group 4 16.3 ± 5.5* 19.0 ± 3.9* 8.4 ± 2.7* 7.9 ± 2.0* 8.9 ± 1.8* Experimental group 5 15.9 ± 5.4* 19.4 ± 3.9* 8.5 ± 2.4* 7.2 ± 1.9* 10.1 ± 1.4* Experimental group 6 17.0 ± 5.5* 18.7 ± 3.9* 8.5 ± 1.7* 7.9 ± 1.9* 10.4 ± 1.9* Experimental group 7 15.3 ± 5.6* 19.2 ± 5.6* 9.0 ± 2.2* 6.9 ± 2.0* 10.4 ± 1.7* Experimental group 8 17.0 ± 5.3* 18.0 ± 6.6* 8.9 ± 2.4* 7.4 ± 2.0* 8.3 ± 1.6* Experimental group 9 17.2 ± 5.6* 18.9 ± 7.5* 8.8 ± 2.6* 7.7 ± 2.0* 9.4 ± 2.5* Experimental group 10 16.4 ± 5.3* 19.2 ± 5.4* 8.9 ± 2.5* 7.6 ± 1.5* 9.6 ± 1.6* Experimental group 11 16.3 ± 5.3* 18.5 ± 6.0* 8.4 ± 2.5* 7.7 ± 2.1* 9.7 ± 1.4* Experimental group 12 16.2 ± 5.5* 17.6 ± 6.4* 8.5 ± 2.5* 7.4 ± 1.5* 10.1 ± 2.4* Note: #indicates that p < 0.05 compared with the control group; *indicates p < 0.05 compared with the model group. - Experiment Animals
- Wistar male rats (purchased from Experimental Animal Research Institute of Chinese Academy of Medical Sciences), cleaning level, weight of 180-220 g, adaptive feeding for 1 week.
- Medicine and Reagent
- The medicines of the invention are prepared in accordance with the embodiments 2-5; the kit for tissue factor (TF) enzyme linked immunosorbent assay (ELISA) (USA USCN Company); fluorescein isothiocyanate (FITC) labelled anti-rat monocyte protease activated receptor-1 (PAR-1) antibody (USA Santa Cruz Company).
- Experiment Method
- 3.1 Grouping and Intervention
- 140 rats are divided into a control group, a model group, experimental groups 1-12 randomly, and there are 10 rats in each group. The rats are prohibited from being fed for 12 h before the experiment, weighed and subjected to grouping in accordance with the table of random numbers. In the control group, laparotomy is carried out to expose the cecum, after that, the skin is sutured, and then, 10 mL of normal saline is injected subcutaneously for anabiosis; in the Sepsis model group, the cecal ligation and puncture (CLP) is carried out for modeling; and in experiment groups 1-12, 12 batches of multi-component injections in embodiments 2-5 are injected respectively (the first batch in the
embodiment 2 to the third batch of theembodiment 5 are administered to the experiment groups 1-12 successively) for medicine intervention after CLP modeling. In the CLP modeling process, the mixed solution of ketamine injection:sumianxin II injection=2:1 is used to intramuscular injection to anesthetize rats, and the Sepsis animal model is prepared using CLP. At the joint of the ligated cecum and the ileum, the No. 18 syringe needle penetrates through the cecum for 2 times to form intestinal fistula, 2 drainage strips (0.5 cm×2.0 cm) are indwelled to prevent needle hole from healing, then, skin is sutured layer by layer, and 10 mL of normal saline is immediately subcutaneously injected for anabiosis after the operation. After the rats are subjected to the CLP operation, in the experimental groups 1-12, 12 batches of multi-component injections in embodiments 2-5 are respectively injected from the dorsal vein of penis, with a dose of 4 mL/kg at 2 h, 12 h, 24 h, 36 h, 48 h and 60 h, after the operation; and in the model group and the control group, 4 mL/kg (by weight) of normal saline is injected from the dorsal vein of penis at the corresponding time. - 3.2 Blood Sampling and Testing
- Animals of each group, which are anesthetized, are subjected to sterile blood sampling with 3 mL at aorta abdominalis at 12 h, 24 h, 48 h, 48 h and 72 h after CLP. The expression of the plasma monocyte protease activated receptor-1 (PAR-1) is detected by using the flow immumofluorescence method; and the content of plasma tissue factors (TF) are detected by using the enzyme linked immunosorbent assay (ELISA).
- Detection of PAR-1 expression: taking 2 mL of heparin for anticoagulant, and separating mono karyocyte by using rat lymphocyte separating medium; resuspendeding mono karyocyte by using 10% of the full cell culture fluid of fetal calf serum-RPMI 1640, adjusting cell density to be 4×106/mL, and washing a cell culture plate using the cell culture fluid after incubating for 2 h; adding 0.25% trypsin-EDTA digestive juice for digesting 3 min, after majority of cells exfoliating, adding 2 mL of full cell culture fluid of 10% fetal calf serum-RPMI 1640, and terminating digesting; washing using phosphate buffer (PBS) for 3 times to acquire peripheral blood mononuclear cells; adding 1 μg/1×106 cell fluorescein isothiocyanate (FITC) into cell suspension to mark the anti-rat PAR-1 antibody, incubating away from light at 4° C. for 15 min, adding 2 mL of PBS to wash for 1 time, adding 0.4 mL of PBS, and detecting its average fluorescence intensity on the flow cytometry.
- Experiment Results
- The results indicate that the mononuclear cells PAR-1 in the control group have a certain expression; there is no statistical significance for difference of the expression quantity of PAR-1 at 12 h after the operation between the model group and the control group upon comparison, the average fluorescence intensity is significantly higher than that of the control group at 24 h, 48 h and 72 h after the operation (p<0.05), and gradually increased with the extension of postoperation time. The average fluorescence intensities of PAR-1 at 24 h and 48 h in the experiment groups 1-3 are lower than that of the model group, with no significant difference; and the fluorescence intensities of the experiment groups at 72 h are significantly lower than that of the model group (p<0.05). The average fluorescence intensities of PAR-1 of nine intervention groups of the experiment groups 4-12 at 24 h, 48 h and 72 h are significantly reduced (p<0.05) by comparison to that of the model group.
- The mononuclear cells TF in the control group have a certain expression; the expression of TF at 12 h, 24 h, 48 h and 72 h after the operation in the model group are significantly higher than that of the control group (p<0.05), which is gradually increased with the extension of postoperation time, and reach a peak at 48 h. The expression of TF at 12 h and 72 h in the experiment groups 1-3 are lower than that of the model group, with no significant difference; and the expression intensities of the experiment groups at 24 h and 48 h are significantly higher than that of the model group (p<0.05). The expression of TF of nine intervention groups of the experiment groups 4-12 at 24 h, 48 h and 72 h are significantly reduced (p<0.05) by comparison to that of the model group.
- The results indicate that the 9 batches of multi-component injections in embodiments 3-5 can reduce the expression of Sepsis induced monocyte PAR-1, thereby obviously reducing the secretion of TF, and improving the monocyte mediated coagulation dysfunction.
-
TABLE 4-1 The effects of multi-component injections on the expression of Sepsis rats PAR-1 ( x ± s, n = 10)12 h after 24 h after 48 h after 72 h after Group operation operation operation operation Control group 20.0 ± 0.7 17.6 ± 2.0 15.7 ± 0.2 16.8 ± 2.9 Model group 19.8 ± 1.1 21.9 ± 1.1# 24.2 ± 0.7# 29.7 ± 0.4# Experimental 19.6 ± 1.9 20.2 ± 2.3 22.9 ± 2.0 22.6 ± 2.3* group 1 Experimental 20.8 ± 2.1 20.4 ± 1.7 23.1 ± 1.8 21.6 ± 2.2* group 2 Experimental 20.7 ± 3.3 20.9 ± 1.6 22.7 ± 2.7 22.8 ± 1.5* group 3 Experimental 20.0 ± 3.2 18.6 ± 1.7* 20.7 ± 2.2* 18.0 ± 2.1* group 4 Experimental 20.2 ± 2.9 18.8 ± 1.7* 18.6 ± 2.6* 18.3 ± 1.7* group 5 Experimental 20.3 ± 2.3 19.2 ± 1.8* 18.3 ± 2.5* 18.1 ± 2.2* group 6 Experimental 20.2 ± 2.7 19.1 ± 1.9* 20.6 ± 2.0* 17.9 ± 2.1* group 7 Experimental 20.7 ± 2.2 18.7 ± 1.7* 21.4 ± 2.3* 17.9 ± 1.5* group 8 Experimental 20.0 ± 2.5 18.4 ± 2.0* 18.7 ± 2.1* 17.7 ± 1.7* group 9 Experimental 19.9 ± 2.4 18.4 ± 1.6* 18.0 ± 2.2* 17.9 ± 2.3* group 10 Experimental 20.3 ± 2.5 18.7 ± 1.9* 21.5 ± 2.1* 17.8 ± 2.1* group 11 Experimental 20.7 ± 2.6 18.5 ± 2.3* 18.1 ± 1.9* 17.7 ± 2.2* group 12 Note: #indicates that p < 0.05 compared with the control group; *indicates p < 0.05 compared with the model group. -
TABLE 4-2 The effects of multi-component injections on the expression of TF of Sepsis rats ( x ± s,n = 10) Unit: ng/L 12 h after 24 h after 48 h after 72 h after Groups operation operation operation operation Control group 238.3 ± 15.6 221.8 ± 40.7 259.0 ± 27.57 240.5 ± 34.4 Model group 324.0 ± 18.9# 505.2 ± 82.8# 695.4 ± 138.9# 342.9 ± 36.3# Experimental group 1 299.3 ± 29.6 370.0 ± 22.3* 377.8 ± 26.1* 319.7 ± 28.3 Experimental group 2 296.4 ± 32.3 366.7 ± 19.3* 380.7 ± 41.3* 313.9 ± 25.3 Experimental group 3 297.7 ± 33.3 374.3 ± 21.3* 381.0 ± 19.7* 320.0 ± 31.2 Experimental group 4 283.5 ± 24.6* 273.2 ± 20.8* 281.4 ± 39.2* 252.5 ± 23.8* Experimental group 5 285.3 ± 29.1* 273.7 ± 21.4* 282.6 ± 31.3* 255.8 ± 19.4* Experimental group 6 279.7 ± 26.5* 268.6 ± 21.1* 284.9 ± 39.6* 257.1 ± 20.0* Experimental group 7 283.3 ± 31.9* 270.5 ± 20.7* 284.0 ± 20.0* 251.7 ± 25.4* Experimental group 8 284.2 ± 30.7* 273.0 ± 21.2* 280.2 ± 31.5* 254.8 ± 29.7* Experimental group 9 281.9 ± 29.7* 273.4 ± 20.8* 280.5 ± 40.0* 252.1 ± 25.2* Experimental group 10 284.5 ± 23.9* 271.1 ± 19.5* 281.8 ± 30.3* 250.8 ± 26.3* Experimental group 11 283.9 ± 31.2* 270.1 ± 19.9* 286.1 ± 21.4* 250.1 ± 27.5* Experimental group 12 282.7 ± 20.3* 268.5 ± 19.9* 283.7 ± 39.4* 252.8 ± 19.8* Note: #indicates that p < 0.05 compared with the control group; *indicates p < 0.05 compared with the model group. - Experiment Animals
- Wistar male rats (purchased from Experimental Animal Research Institute of Chinese Academy of Medical Sciences), cleaning level, weight of 180-220 g, adaptive feeding for 1 week.
- Medicine and Reagent
- The medicines of the invention are prepared in accordance with the embodiments 2-5; phycoerythrin (PE)-anti-rat CD25, fluorescein isothiocyanate (FITC) labeled anti-rat CD4, allophycocyanin (APC) labeled Annexin V apoptosis reagent kit (USA BD Company); rat anti-PE reagent kit, CD4 magnetic beads, MiniMACS magnetic separation apparatus and separation column (Germany MiltenyiBiotec Company); PE labeled forkhead winged helix transcription factor (Foxp3) reagent kit, PE labeled T lymphocytotoxicity related antigen 4 (CTLA-4), anti-rat CD3 monoclonal antibody, anti-rat CD28 monoclonal antibody (USA eBioscience Company); interferon (IFN)-7, IL-4, IL-10 enzyme linked immunosorbent assay (ELISA) reagent kits (USA Biosource Company); IL-17 ELISA reagent kit (USA Usenlife Science Company); Trypan Blue (USA Sigma Company).
- Experiment Method
- 3.1 Grouping and Intervention
- 140 rats are divided into a control group, a model group, experimental groups 1-12 randomly, and there are 10 rats in each group. The rats are prohibited from being fed for 12 h before the experiment, weighed and subjected to grouping in accordance with the table of random numbers. In the control group, laparotomy is carried out to expose the cecum, after that, the skin is sutured, and then, 10 mL of normal saline is injected subcutaneously for anabiosis; in the Sepsis model group, the cecal ligation and puncture (CLP) is carried out for modeling; and in experiment groups 1-12, 12 batches of multi-component injections in embodiments 2-5 are injected respectively (the first batch in the
embodiment 2 to the third batch of theembodiment 5 are administered to the experiment groups 1-12 successively) for medicine intervention after CLP modeling. In the CLP modeling process, the mixed solution of ketamine injection:sumianxin II injection of 2:1 is intramuscular injected to anesthetize rats, and the Sepsis animal model is prepared by using CLP. At the joint of the ligated cecum and the ileum, the No. 18 syringe needle penetrates through the cecum for 2 times to form intestinal fistula, 2 drainage strips (0.5 cm×2.0 cm) are indwelled to prevent needle hole from healing, then, skin is sutured layer by layer, and 10 mL of normal saline is immediately injected subcutaneously for anabiosis after the operation. After the rats are subjected to the CLP operation, in the experiment groupsl-12, after the operation, 12 batches of multi-component injections in embodiments 2-5 are respectively injected from the dorsal vein of penis, with a dose of 4 mL/kg; and in the model group and the control group, 4 mL/kg (by weight) of normal saline is injected from the dorsal vein of penis at the corresponding time. - 3.2 Cell Separation and Culture
- Take spleen from each group of rats after breaking necks to death under the sterile condition, grinding the spleen, sieve it with a 400-mesh sieve, centrifuge cell suspension, add lymphocyte separating medium for centrifuging, and obtain white cells in the middle layer. Add PE-anti-CD25 and PE magnetic beads, carry out positive selection (positive) to obtain CD25+T cells, and carry out negative selection (negative) to obtain CD25-T cell. After carrying out dissociation by CD25+T cell dissociation reagent, carry out positive selection on negatively selected cells using FITC-anti-CD4 and CD4 magnetic beads to obtain CD4+CD25+Treg; and carry out positive selection on CD25-T cells using FITC-anti-CD4 and CD4 magnetic beads to obtain CD4+CD25-T cells. Test the purity of double positive cells via the flow cytometry. Dye CD4+CD25+Treg using 0.4% (mass fraction) of Trypan Blue, and observe cell survival rate. Put RPM11640 culture solution containing 20% (volume fraction) fetal bovine serum in a 48-pore culture plate, and culture in a CO2 incubator. Separate CD4+CD25+Treg in each group at the 3rd day for culturing for 12 h, adjust the concentration of CD4+CD25+Treg and CD4+CD25-T cells by using the culture solution to be 1:1 for culturing, stimulate using sword bean A (Con A, 5 mg/L), culture in a CO2 incubator at 37° C. for 68 h, centrifuge to get a supernate which is frozen at −70° C. for testing.
- 3.3 Detection and Analysis
- Testing of Treg apoptosis rate: culturing the separated cells for 12 h, washing the suspending CD4+CD25+Treg (1×109/L) 2 times with phosphate buffer (PBS), adding 100 L of binding buffer and 10 L of APC labeled Annexin V (20 mg/L), keeping in the dark place at room temperature for 30 min, then, adding 10 L of actinomycin D (7-AAD), keeping in the dark place for reaction for 5 min, adding 400 μL of binding buffer, selecting 7-AAD negative Annexin V positive cells as apoptotic cells by the flow cytometry, and testing the cell apoptosis rate.
- Testing of Foxp3 and CTLA-4 expression: Adding 1 ml of newly prepared film-breaking liquid into 100 μL prepared CD4+CD25+Treg (1×109/L), keeping in the dark place at 4° C. for incubating for 2 h, and washing with 2 mL of film-breaking buffer; adding PE-anti-Foxp3, incubating in the dark place at 4° C. for 30 min, washing with 2 mL of film-breaking buffer, centrifuging and discarding supernate, adding 0.5 mL of PBS, and testing the average fluorescence intensity of Foxp3 by the flow cytometry. Directly adding PE-CTLA-4 into the resuspended cells (1×109/L), keeping in the dark place at 4° C. for incubating for 30 min, and testing the average fluorescence intensity of CTLA-4.
- Testing of cell factors: including main inhibitory cell factor IL-10 secreted by Treg, IFN-γ secreted by Th1, and IL-4 secreted by Th2. The IL-10 specimen is from supernate collected after the separated Treg is cultured for 12 h in each group, IFN-γ and IL-4 specimens are from supernate collected after CD4+CD25+Treg and CD4+CD25-T cells are subjected to co-culture for 68 h. Tests are performed by using the ELISA reagent kit in accordance with the specification, the standard curve and the regression equation are calculated respectively, the absorbancy of the samples is substituted into the standard curve, and the protein concentration of cell factors in the samples are calculated.
- Experiment Results
- The results indicate that the apoptosis rate of Treg in the model group is obviously lower than that of the control group (p<0.05), the apoptosis rates of the experimental groups 4-12 are significantly higher than that of the model group (p<0.05), and the trends of the experimental groups 1-3 are close, without significant difference. The expression of Foxp3 and CTLA-4 in the model group is obviously higher than that of the control group (p<0.05), the expression of the experimental groups 4-12 are significantly lower than that of the model group (p<0.05), and the trends of the experimental groups 1-3 are close, without significant difference. The secretion level of IL-10 in the model group is obviously higher than that of the control group (p<0.05), the secretion level of the experimental groups 4-12 are significantly lower than that of the model group (p<0.05), and the trends of the experimental groups 1-3 are close, without significant difference. The levels of IFN-γ and IL-4 in the model group are substantially increased compared with the control group, and the levels of IFN-γ and IL-4 in the experimental groups 4-12 are further increased. Compared with the model group, there are statistical significance for difference (p<0.05). The trends of experimental groups 1-3 are close, without significant difference.
- The results indicate that 9 batches of multi-component injections in embodiments 3-5 can promote the apoptosis of Sepsis Treg and improve the secretion of cell factors of Treg and effector T cells, and are conducive to correcting the inhibitory state of cellular immunity of organisms.
-
TABLE 5-1 The effects of 12 batches of multi-component injections in embodiments 2-5 on the apoptosis rate of Treg and the expression of Foxp3 and CTLA-4 of Sepsis rats ( x ± s, n = 10) Unit: %Group Apoptosis rate Foxp3 CTLA Control group 9.48 ± 2.2 126.5 ± 12.2 119.1 ± 6.4 Model group 5.9 ± 1.5# 175.0 ± 19.6# 172.0 ± 10.1# Experimental group 1 7.1 ± 3.0 159.8 ± 13.3 159.7 ± 14.1 Experimental group 2 7.3 ± 2.5 159.7 ± 16.8 161.4 ± 19.9 Experimental group 3 7.0 ± 2.3 158.5 ± 15.1 160.0 ± 20.7 Experimental group 4 17.4 ± 2.5* 118.9 ± 14.4* 118.3 ± 15.2* Experimental group 5 17.9 ± 3.0* 118.8 ± 14.1* 116.2 ± 18.3* Experimental group 6 17.9 ± 2.9* 119.6 ± 14.1* 116.4 ± 15.6* Experimental group 7 28.0 ± 2.9* 59.7 ± 4.2* 55.5 ± 6.5* Experimental group 8 27.3 ± 2.4* 58.9 ± 5.5* 55.6 ± 6.3* Experimental group 9 27.4 ± 2.8* 58.6 ± 6.3* 56.3 ± 6.6* Experimental group 10 27.9 ± 2.9* 59.0 ± 3.5* 58.6 ± 5.8* Experimental group 11 27.8 ± 2.5* 59.5 ± 5.8* 56.5 ± 7.4* Experimental group 12 27.2 ± 3.0* 59.5 ± 3.3* 56.8 ± 4.2* Note: #indicates that p < 0.05 compared with the control group; *indicates p < 0.05 compared with the model group. -
TABLE 5-2 The effects of 12 batches of multi-component injections in embodiments 2-5 on the level of secretory cell factors of Treg and effector T cells of Sepsis rats ( x ± s, n = 10) Unit: ng/LGroup IL-10 IFN-γ IL-4 Control group 133.7 ± 25.7 19.0 ± 6.7 5.4 ± 0.7 Model group 318.1 ± 28.3# 254.7 ± 44.9# 8.8 ± 0.6# Experimental group 1 299.3 ± 22.3 311.6 ± 75.2 9.3 ± 0.9 Experimental group 2 300.6 ± 22.1 321.5 ± 84.3 9.5 ± 1.2 Experimental group 3 302.6 ± 22.5 322.8 ± 88.9 9.3 ± 1.1 Experimental group 4 245.2 ± 22.5* 414.7 ± 82.2* 10.4 ± 0.8* Experimental group 5 251.5 ± 22.2* 413.2 ± 78.2* 10.3 ± 1.0* Experimental group 6 251.6 ± 19.3* 408.8 ± 84.1* 10.5 ± 1.3* Experimental group 7 51.2 ± 2.5* 498.7 ± 81.7* 10.5 ± 1.2* Experimental group 8 49.6 ± 2.2* 494.2 ± 85.3* 10.7 ± 1.1* Experimental group 9 52.2 ± 2.3* 495.6 ± 81.9* 10.6 ± 0.9* Experimental group 10 50.3 ± 2.1* 499.4 ± 85.6* 10.8 ± 1.4* Experimental group 11 49.3 ± 2.3* 501.4 ± 87.6* 10.7 ± 1.1* Experimental group 12 51.7 ± 2.1* 494.5 ± 81.0* 10.8 ± 1.3* Note: #indicates that p < 0.05 compared with the control group; *indicates p < 0.05 compared with the model group. - After related testing, we find that the multi-component injections of the present invention have the characteristics of stable quality, simple production process, convenience for production, safety, effectiveness, etc.
- The summary of relevant compounds presented in the present application:
-
Name CAS Molecular formula Structural formula Kaempferol-3-0-glucoside 480-10-4 C21H20O11 Scutellarin 27740-01-8 C21H18O12 Quercetin-3-0-glucoside 482-35-9 C21H20O12 Kaempferol-3-0-rutinoside 17650-84-9 C27H30O15 Kaempferol-3-0-sophoroside 19895-95-5 C27H30O16 Quercetin-3-0-rutinoside 949926-49-2 C27H30O16 Hydroxysafflor yellowA 78281-02-4 C27H32O16 Uracil 66-22-8 C4H4N2O2 Adenine 73-24-5 C5H5N5 Phenylalanine 167088-01-9 C9H11NO2 Uridine 58-96-8 C9H12N2O6 Adenosine 58-61-7 C10H13N5O4 Guanosine 118-00-3 C10H13N5O5 Butanedioic acid 110-15-6 C4H6O4 p-hydroxybenzoic acid 99-96-7 C7H6O3 p-coumaric acid 501-98-4 C9H8O3 Caffeic acid 331-39-5 C9H8O4 Chlorogenic acid 327-97-9 C16H18O9 Albiflorin std 39011-90-0 C23H28O11 Paeoniflorin 23180-57-6 C23H28O11 Oxypaeoniflorin 39011-91-1 C23H28O12 Benzoylpaeoniflorin 38642-49-8 C30H32O12 Benzoyloxy paeoniflorin 72896-40-3 C30H32O13 Mudanpioside J 262350-52-7 C31H34O14 Galloylpaeoniflorin 122965-41-7 C30H32O15 Mudanpioside C 172760-03-1 C30H32O13 Benzoic acid 65-85-0 C7H6O2 Gallic acid 149-91-7 C7H6O5 Ethyl gallate 831-61-8 C9H10O5 Catechin 154-23-4 C15H14O6 Protocatechualdehyde 139-85-5 C7H6O3 Protocatechuic acid 99-50-3 C7H6O4 Tanshinol 76822-21-4 C9H10O5 Rosmarinic acid 537-15-5 C18H16O8 Salvianolic acid D 142998-47-8 C20H18O10 Salvianolic acid C 115841-09-3 C26H20O10 Salvianolic acid A 96574-01-5 C26H22O10 Alkannic acid 28831-65-4 C27H22O12 Salvianolic acid B 121521-90-2 C36H30O16 Senkyunolide I 94596-28-8 C12H16O4 Senkyunolide H 94596-27-7 C12H16O4 Senkyunolide N 140694-58-2 C12H18O4 Open-loop senkyunolide I 1809299-03-3 C12H18O5 Senkyunolide G 94530-85-5 C12H16O3 3-hydroxy-3-Butylphthalide 162050-42-2 C12H14O3 Senkyunolide A 62006-39-7 C12H16O2 Ferulaic acid 1135-24-6 C10H10O4 -
FIG. 1 Comparison of survival rates of endotoxin induced Sepsis mice among groups Note: compared with the model group, the experimental groups 1-12 have the significant statistical difference (p<0.05). -
FIG. 2 Comparison of the survival rates of cecal ligation and puncture mediated Sepsis mice among groups - Note: compared with the model group, the experimental groups 7-12 have the significant statistical difference (p<0.05).
- There is a further description to the invention via the following specific embodiments, which is not used as limits.
- Method 1: Chromatographic Separation Conditions for Analyzing Ingredients of Safflower Carthamus and Red Paeony Root
- Chromatographic column: Waters HSS T3 UPLC C18 Chromatographic column (100 mm×2.1 mm; 1.8 μM, Waters, USA);
- Column temperature: 45° C.;
- Mobile phase: A: H2O (containing 25 mM HCOOH, B: MeOH (containing 25 mM HCOOH);
- Gradient elute is shown in Table 6; flow velocity: 0.35 mL/min; injection volume: 5 μL; analysis time: 13 min.
-
TABLE 6 Liquid-phase gradient elute conditions of ingredients of safflower carthamus and Red Paeony Root Time (min) Solvent A Solvent B 0.0 98% 2% 8.0 30% 70% 11.0 2% 98% 13.0 98% 2% - Method 2: Chromatographic Separation Conditions for Analyzing Ingredients of Radix Salviae Miltiorrhizae
- Chromatographic column: Waters HSS T3 UPLC C18 Chromatographic column (100 mm×2.1 mm; 1.8 μM, Waters, USA);
- Column temperature: 45° C.;
- Mobile phase: A: H2O (containing 25 mM HCOOH), B: MeOH (containing 25 mM HCOOH);
- Gradient elute is shown in Table 7; flow velocity: 0.35 ml/min; injection volume: 5 μL; analysis time: 20 min.
-
TABLE 7 Liquid-phase gradient elute conditions of ingredients of radix salviae miltiorrhizae Time (min) Solvent A Solvent B 0.0 98% 2% 1.0 98% 2% 15.0 30% 70% 17.0 2% 98% 20.0 98% 2% - Method 3: Chromatographic Separation Conditions for Analyzing Ingredients of Ligusticum wallichii and Angelica sinensis, Adenine and Adenosine
- Chromatographic column: Waters HSS T3 UPLC C18 Chromatographic column (100 mm×2.1 mm; 1.8 μM, Waters, USA);
- Column temperature: 45° C.;
- Mobile phase: A: MeOH—H2O (v/v, 1:99), including 1 mM HCOOH and 25 μM, CH3COOLi; B: MeOH—H2O (v/v, 99:1), including 1 mM HCOOH and 25 μM, CH3COOLi; Gradient elute is shown in Table 8; flow velocity: 0.35 mL/min; injection volume: 5 μL; analysis time: 8 min.
-
TABLE 8 Liquid-phase gradient elute conditions of ingredients of Ligusticum wallichii and Angelica sinensis, adenine and adenosine Time (min) Solvent A Solvent B 0.0 94% 6% 7.0 5% 95% 8.0 94% 6% - providing 100 g of Red Paeony Root decoction pieces; heating and boiling the Red Paeony Root decoction pieces with process water of 10 times the weight of the Red Paeony Root decoction pieces to obtain a first decoction after 2 hours slight boiling; filtering the first decoction to obtain filtrate I and a first dreg; boiling the first dreg with process water of 8 times the weight of the first dreg to obtain a second decoction after 1 hour slight boiling; filtering the second decoction to obtain filtrate II and a second dreg; mixing filtrate I and filtrate II to obtain a mixture; concentrating the mixture to obtain a first concentrate of 100 ml; adding a proper amount of gelatin solution to the first concentrate under stirring to obtain a gelatin-containing first concentrate; adding 95% ethanol to the gelatin-containing first concentrate to obtain an ethanol-containing first concentrate having an ethanol volume content of 70%; storing the ethanol-containing first concentrate under cooling condition for 24 hours to obtain a cool ethanol-containing first concentrate; filtering and concentrating the cool ethanol-containing first concentrate to obtain a second concentrate of 100 ml; extracting the second concentrate with water-saturated n-butanol for 4 times and using water-saturated n-butanol of 50 ml for each time; combining extract liquors of the 4 times extraction to obtain a combined extract liquor; recycling n-butanol from the combined extract liquor to obtain a n-butanol-reduced extract liquor without alcohol taste; and vacuum drying the n-butanol-reduced extract liquor to obtain a Red Paeony Root dry paste;
- providing a proper amount of the Red Paeony Root dry paste; dissolving the Red Paeony Root dry paste in injection water to obtain a dilute of 200 ml; storing the dilute under cooling condition to obtain a first cool liquid; adding glucosum anhydricum of an amount in accordance with 4.5% mass fraction of the multi-component injection and injection water to the first cool liquid to obtain a 1000 ml liquid; adjusting the pH value of the 1000 ml liquid to 5.5-7.0 with a sodium hydroxide solution of 10% mass fraction to obtain a pH adjusted liquid; storing the pH adjusted liquid under cooling condition to obtain a second cool liquid; subjecting the second cool liquid to ultrafiltration to obtain an ultrafiltrate; adding a proper amount of solubilizing auxiliary materials which are dissolved in a proper amount of injection water into the ultrafiltrate to obtain a solubilizing auxiliary materials-containing ultrafiltrate; adjusting the pH value of the solubilizing auxiliary materials-containing ultrafiltrate to 5.5-7.0 using the sodium hydroxide solution of 10% mass fraction to obtain a pH adjusted ultrafiltrate; filtering the pH adjusted ultrafiltrate to obtain a filtrate; encapsulating and sterilizing the filtrate to obtain the multi-component injection.
- Three batches of injections are prepared in accordance with the process, and the testing results of the effective ingredients are as below:
-
Effective The First The ingredient (Unit ug/ml) batch Second batch The Third batch Albiflorin std 8.66 9.51 11.98 Paeoniflorin 1000.0 1010.7 1051.6 Oxypaeoniflorin 11.13 14.43 14.45 Benzoylpaeoniflorin 12.20 15.48 18.09 Benzoyloxypaeoniflorin 0.667 0.711 0.753 Mudanpioside J 1.915 1.934 1.960 Galloylpaeoniflorin 10.63 11.03 11.15 Mudanpioside C 0.804 0.805 0.941 Benzoic acid 10.0 20.4 30.1 Gallic acid 0.21 0.73 0.77 Ethyl gallate 0.2396 0.2428 0.2526 Catechin 1.31 1.91 1.60 - providing 100 g of safflower carthamus decoction pieces; leaching the safflower carthamus decoction pieces with 30% ethanol of 8 times the weight of the safflower carthamus decoction pieces for 8 hours to obtain a leach liquor; filtering the leach liquor to obtain a liquid medicine of 4-6 times the weight of the safflower carthamus decoction pieces; adding 95% ethanol to the liquid medicine to obtain an ethanol-containing liquid medicine having an ethanol volume content of 70%; storing the ethanol-containing liquid medicine under cooling condition for 48 hours to obtain a cool ethanol-containing liquid medicine; filtering the cool ethanol-containing liquid medicine to obtain a first filtrate; concentrating the first filtrate under reduced pressure to obtain a concentrate of 100 ml; adding 95% ethanol to the concentrate to obtain an ethanol-containing concentrate having an ethanol volume content of 80%; storing the ethanol-containing concentrate under cooling condition for 48 hours to obtain a cool ethanol-containing concentrate; filtering the cool ethanol-containing concentrate to obtain a second filtrate; recycling ethanol from the second filtrate to obtain an ethanol-reduced filtrate; concentrating and vacuum drying the ethanol-reduced filtrate to obtain a safflower carthamus dry paste;
- providing 100 g of Red Paeony Root decoction pieces; heating and boiling the Red Paeony Root decoction pieces with process water of 10 times the weight of the Red Paeony Root decoction pieces to obtain a first decoction after 2 hours slight boiling; filtering the first decoction to obtain filtrate I and a first dreg; boiling the first dreg with process water of 8 times the weight of the first dreg to obtain a second decoction after 1 hour slight boiling; filtering the second decoction to obtain filtrate II and a second dreg; mixing filtrate I and filtrate II to obtain a mixture; concentrating the mixture to obtain a first concentrate of 100 ml; adding a proper amount of gelatin solution to the first concentrate under stirring to obtain a gelatin-containing first concentrate; adding 95% ethanol to the gelatin-containing first concentrate to obtain an ethanol-containing first concentrate having an ethanol volume content of 70%; storing the ethanol-containing first concentrate under cooling condition for 24 hours to obtain a cool ethanol-containing first concentrate; filtering and concentrating the cool ethanol-containing first concentrate to obtain a second concentrate of 100 ml; extracting the second concentrate with water-saturated n-butanol for 4 times and using 50 ml water-saturated n-butanol for each time; combining extract liquors of the 4 times extraction to obtain a combined extract liquor; recycling n-butanol from the combined extract liquor to obtain a n-butanol-reduced extract liquor without alcohol taste; and vacuum drying the n-butanol-reduced extract liquor to obtain a Red Paeony Root dry paste;
- providing the safflower carthamus dry paste and the Red Paeony Root dry paste each of a proper amount; dissolving the two dry pastes in injection water to obtain a dilute of 200 ml; storing the dilute under cooling condition to obtain a first cool liquid; adding glucosum anhydricum of an amount in accordance with 4.5% mass fraction of the multi-component injection and injection water to the first cool liquid to obtain a 1000 ml liquid; adjusting the pH value of the 1000 ml liquid to 5.5-7.0 with a sodium hydroxide solution of 10% mass fraction to obtain a pH adjusted liquid; storing the pH adjusted liquid under cooling condition to obtain a second cool liquid; subjecting the second cool liquid to ultrafiltration to obtain an ultrafiltrate; adding a proper amount of solubilizing auxiliary materials which are dissolved in a proper amount of injection water into the ultrafiltrate to obtain a solubilizing auxiliary materials-containing ultrafiltrate; adjusting the pH value of the solubilizing auxiliary materials-containing ultrafiltrate to 5.5-7.0 using the sodium hydroxide solution of 10% mass fraction to obtain a pH adjusted ultrafiltrate; filtering the pH adjusted ultrafiltrate to obtain a filtrate; encapsulating and sterilizing the filtrate to obtain the multi-component injection.
- Three batches of injections are prepared in accordance with the above process, and the testing results of the effective ingredients are as below:
-
Effective The First The Second The ingredient (Unit ug/ml) batch batch Third batch Albiflorin std 13.58 15.46 15.01 Paeoniflorin 1119.7 1139.7 1209.5 Oxypaeoniflorin 21.45 24.17 24.24 Benzoylpaeoniflorin 21.20 21.55 24.19 Benzoyloxypaeoniflorin 0.820 0.844 0.904 Mudanpioside J 2.083 2.463 2.480 Galloylpaeoniflorin 11.48 11.81 13.96 Mudanpioside C 0.942 1.031 1.048 Benzoic acid 84.8 85.8 100.2 Gallic acid 6.68 5.72 8.41 Ethyl gallate 0.3083 0.3967 0.4453 Catechin 4.01 5.86 5.97 Kaempferol-3-0-glucoside 1.232 1.440 1.506 Scutellarin 0.0500 0.0570 0.1033 Quercetin-3-0-glucoside 0.755 0.859 0.973 Kaempferol-3-0-rutinoside 8.42 11.02 12.59 Kaempferol-3-0-sophoroside 4.036 4.369 4.954 Quercetin-3-0-rutinoside 1.517 1.529 1.774 Hydroxysafflor yellow A 200.0 225.5 229.8 Uracil 0.316 0.338 0.424 Adenine 13.77 14.31 16.45 Phenylalanine 20.50 24.22 36.02 Uridine 11.44 13.25 14.26 Adenosine 5.07 5.59 5.70 Guanosine 8.00 9.06 10.02 Butanedioic acid 4.96 5.67 8.23 p-hydroxybenzoic acid 2.384 2.997 3.040 p-coumaric acid 3.00 3.23 3.79 Caffeic acid 4.837 5.298 5.584 Chlorogenic acid 3.83 3.86 4.28 - providing 100 g of safflower carthamus decoction pieces; leaching the safflower carthamus decoction pieces with 30% ethanol of 8 times the weight of the safflower carthamus decoction pieces for 8 hours to obtain a leach liquor; filtering the leach liquor to obtain a liquid medicine of 4-6 times the weight of the safflower carthamus decoction pieces; adding 95% ethanol to the liquid medicine to obtain an ethanol-containing liquid medicine having an ethanol volume content of 70%; storing the ethanol-containing liquid medicine under cooling condition for 48 hours to obtain a cool ethanol-containing liquid medicine; filtering the cool ethanol-containing liquid medicine to obtain a first filtrate; concentrating the first filtrate under reduced pressure to obtain a concentrate of 100 ml; adding 95% ethanol to the concentrate to obtain an ethanol-containing concentrate having an ethanol volume content of 80%; storing the ethanol-containing concentrate under cooling condition for 48 hours to obtain a cool ethanol-containing concentrate; filtering the cool ethanol-containing concentrate to obtain a second filtrate; recycling ethanol from the second filtrate to obtain an ethanol-reduced filtrate; concentrating and vacuum drying the ethanol-reduced filtrate to obtain a safflower carthamus dry paste;
- providing 100 g of Red Paeony Root decoction pieces; heating and boiling the Red Paeony Root decoction pieces with process water of 10 times the weight of the Red Paeony Root decoction pieces to obtain a first decoction after 2 hours slight boiling; filtering the first decoction to obtain filtrate I and a first dreg; boiling the first dreg with process water of 8 times the weight of the first dreg to obtain a second decoction after 1 hour slight boiling; filtering the second decoction to obtain filtrate II and a second dreg; mixing filtrate I and filtrate II to obtain a mixture; concentrating the mixture to obtain a first concentrate of 100 ml; adding a proper amount of gelatin solution to the first concentrate under stirring to obtain a gelatin-containing first concentrate; adding 95% ethanol to the gelatin-containing first concentrate to obtain an ethanol-containing first concentrate having an ethanol volume content of 70%; storing the ethanol-containing first concentrate under cooling condition for 24 hours to obtain a cool ethanol-containing first concentrate; filtering and concentrating the cool ethanol-containing first concentrate to obtain a second concentrate of 100 ml; extracting the second concentrate with water-saturated n-butanol for 4 times and using 50 ml water-saturated n-butanol for each time; combining extract liquors of the 4 times extraction to obtain a combined extract liquor; recycling n-butanol from the combined extract liquor to obtain a n-butanol-reduced extract liquor without alcohol taste; and vacuum drying the n-butanol-reduced extract liquor to obtain a Red Paeony Root dry paste;
- providing 100 g of Ligusticum wallichii decoction pieces and 100 g of Angelica sinensis decoction pieces; treating the Ligusticum wallichii decoction pieces and Angelica sinensis decoction pieces with a same treatment process as the Red Paeony Root decoction pieces except for keeping 300 ml of concentrate every time and extracting with 150 ml of water-saturated n-butanol each time, to obtain a third dry paste;
- providing the safflower carthamus dry paste, the Red Paeony Root dry paste and the third dry paste each of a proper amount; dissolving the three dry pastes in injection water to obtain a dilute of 200 ml; storing the dilute under cooling condition to obtain a first cool liquid; adding glucosum anhydricum of an amount in accordance with 4.5% mass fraction of the multi-component injection and injection water to the first cool liquid to obtain a 1000 ml liquid; adjusting the pH value of the 1000 ml liquid to 5.5-7.0 with a sodium hydroxide solution of 10% mass fraction to obtain a pH adjusted liquid; storing the pH adjusted liquid under cooling condition to obtain a second cool liquid; subjecting the second cool liquid to ultrafiltration to obtain an ultrafiltrate; adding a proper amount of solubilizing auxiliary materials which are dissolved in a proper amount of injection water into the ultrafiltrate to obtain a solubilizing auxiliary materials-containing ultrafiltrate; adjusting the pH value of the solubilizing auxiliary materials-containing ultrafiltrate to 5.5-7.0 using the sodium hydroxide solution of 10% mass fraction to obtain a pH adjusted ultrafiltrate; filtering the pH adjusted ultrafiltrate to obtain a filtrate; encapsulating and sterilizing the filtrate to obtain the multi-component injection.
- Three batches of injections are prepared in accordance with the process, and the testing results of the effective ingredients are as below:
-
Effective The First The Second The ingredient (Unit ug/ml) batch batch Third batch Albiflorin std 19.06 22.10 22.27 Paeoniflorin 1227.3 1263.2 1286.0 Oxypaeoniflorin 40.38 43.28 43.69 Benzoylpaeoniflorin 32.04 34.49 35.72 Benzoyloxypaeoniflorin 1.364 1.396 1.436 Mudanpioside J 2.516 2.729 2.773 Galloylpaeoniflorin 15.07 15.71 16.16 Mudanpioside C 1.060 1.074 1.170 Benzoic acid 153.4 165.4 176.3 Gallic acid 8.80 10.12 13.70 Ethyl gallate 0.5517 0.5527 0.5863 Catechin 9.95 10.37 10.45 Kaempferol-3-0-glucoside 1.646 1.723 1.898 Scutellarin 0.1236 0.1307 0.1766 Quercetin-3-0-glucoside 1.482 1.775 1.918 Kaempferol-3-0-rutinoside 13.87 14.82 14.93 Kaempferol-3-0-sophoroside 5.321 5.542 5.619 Quercetin-3-0-rutinoside 2.669 2.788 3.150 Hydroxysafflor yellow A 250.4 311.5 329.9 Uracil 0.440 0.476 0.477 Adenine 16.56 17.27 19.82 Phenylalanine 31.17 31.85 32.93 Uridine 14.78 15.23 19.32 Adenosine 6.66 6.72 7.24 Guanosine 10.07 10.81 11.95 Butanedioic acid 9.51 9.78 10.04 p-hydroxybenzoic acid 3.054 3.447 3.712 p-coumaric acid 10.61 12.46 12.55 Caffeic acid 5.627 5.971 6.212 Chlorogenic acid 4.32 4.53 4.68 Senkyunolide I 6.51 7.72 8.06 Senkyunolide H 1.55 2.93 2.51 Senkyunolide N 4.30 4.86 6.17 Open-loop senkyunolide I 2.460 3.037 3.132 Senkyunolide G 1.55 2.06 2.12 3-hydroxy-3-Butylphthalide 1.43 1.7 2.38 Senkyunolide A 0.10 0.183 0.203 Ferulaic acid 7.66 8.41 10.23 - providing 100 g of safflower carthamus decoction pieces; leaching the safflower carthamus decoction pieces with 30% ethanol of 8 times the weight of the safflower carthamus decoction pieces for 8 hours to obtain a leach liquor; filtering the leach liquor to obtain a liquid medicine of 4-6 times the weight of the safflower carthamus decoction pieces; adding 95% ethanol to the liquid medicine to obtain an ethanol-containing liquid medicine having an ethanol volume content of 70%; storing the ethanol-containing liquid medicine under cooling condition for 48 hours to obtain a cool ethanol-containing liquid medicine; filtering the cool ethanol-containing liquid medicine to obtain a first filtrate; concentrating the first filtrate under reduced pressure to obtain a concentrate of 100 ml; adding 95% ethanol to the concentrate to obtain an ethanol-containing concentrate having an ethanol volume content of 80%; storing the ethanol-containing concentrate under cooling condition for 48 hours to obtain a cool ethanol-containing concentrate; filtering the cool ethanol-containing concentrate to obtain a second filtrate; recycling ethanol from the second filtrate to obtain an ethanol-reduced filtrate; concentrating and vacuum drying the ethanol-reduced filtrate to obtain a safflower carthamus dry paste;
- providing 100 g of Red Paeony Root decoction pieces; heating and boiling the Red Paeony Root decoction pieces with process water of 10 times the weight of the Red Paeony Root decoction pieces to obtain a first decoction after 2 hours slight boiling; filtering the first decoction to obtain filtrate I and a first dreg; boiling the first dreg with process water of 8 times the weight of the first dreg to obtain a second decoction after 1 hour slight boiling; filtering the second decoction to obtain filtrate II and a second dreg; mixing filtrate I and filtrate II to obtain a mixture; concentrating the mixture to obtain a first concentrate of 100 ml; adding a proper amount of gelatin solution to the first concentrate under stirring to obtain a gelatin-containing first concentrate; adding 95% ethanol to the gelatin-containing first concentrate to obtain an ethanol-containing first concentrate having an ethanol volume content of 70%; storing the ethanol-containing first concentrate under cooling condition for 24 hours to obtain a cool ethanol-containing first concentrate; filtering and concentrating the cool ethanol-containing first concentrate to obtain a second concentrate of 100 ml; extracting the second concentrate with water-saturated n-butanol for 4 times and using 50 ml water-saturated n-butanol for each time; combining extract liquors of the 4 times extraction to obtain a combined extract liquor; recycling n-butanol from the combined extract liquor to obtain a n-butanol-reduced extract liquor without alcohol taste; and vacuum drying the n-butanol-reduced extract liquor to obtain a Red Paeony Root dry paste;
- providing 100 g of Ligusticum wallichii decoction pieces, 100 g of radix salviae miltiorrhizae and 100 g of Angelica sinensis decoction pieces; treating the three decoction pieces with a same treatment process as the Red Paeony Root decoction pieces except for keeping 300 ml of concentrate every time and extracting with 150 ml of water-saturated n-butanol each time, to obtain a third dry paste; providing the safflower carthamus dry paste, the Red Paeony Root dry paste and the third dry paste each of a proper amount; dissolving the three dry pastes in injection water to obtain a dilute of 200 ml; storing the dilute under cooling condition to obtain a first cool liquid; adding glucosum anhydricum of an amount in accordance with 4.5% mass fraction of the multi-component injection and injection water to the first cool liquid to obtain a 1000 ml liquid; adjusting the pH value of the 1000 ml liquid to 5.5-7.0 with a sodium hydroxide solution of 10% mass fraction to obtain a pH adjusted liquid; storing the pH adjusted liquid under cooling condition to obtain a second cool liquid; subjecting the second cool liquid to ultrafiltration to obtain an ultrafiltrate; adding a proper amount of solubilizing auxiliary materials which are dissolved in a proper amount of injection water into the ultrafiltrate to obtain a solubilizing auxiliary materials-containing ultrafiltrate; adjusting the pH value of the solubilizing auxiliary materials-containing ultrafiltrate to 5.5-7.0 using the sodium hydroxide solution of 10% mass fraction to obtain a pH adjusted ultrafiltrate; filtering the pH adjusted ultrafiltrate to obtain a filtrate; encapsulating and sterilizing the filtrate to obtain the multi-component injection.
- Three batches of injections are prepared in accordance with the process, and the testing results of the effective ingredients are as below:
-
Effective The First The Second The ingredient (Unit ug/ml) batch batch Third batch Albiflorin std 34.94 32.30 35.26 Paeoniflorin 1588.2 1677.0 1700.0 Oxypaeoniflorin 59.42 64.52 68.07 Benzoylpaeoniflorin 47.98 48.27 52.98 Benzoyloxypaeoniflorin 1.452 1.551 1.617 Mudanpioside J 2.775 3.030 3.202 Galloylpaeoniflorin 18.37 19.71 20.13 Mudanpioside C 1.225 1.277 1.338 Benzoic acid 186.2 191.9 200.0 Gallic acid 13.85 15.40 15.88 Ethyl gallate 0.6277 0.6633 0.6860 Catechin 11.48 11.94 12.60 Kaempferol-3-0-glucoside 2.859 3.328 3.547 Scutellarin 0.2824 0.3620 0.4184 Quercetin-3-0-glucoside 2.295 2.263 2.570 Kaempferol-3-0-rutinoside 22.90 26.66 29.40 Kaempferol-3-0-sophoroside 7.384 7.667 7.695 Quercetin-3-0-rutinoside 4.420 5.288 5.598 Hydroxysafflor yellow A 422.4 476.9 500.0 Uracil 0.723 0.758 0.774 Adenine 24.44 28.49 30.56 Phenylalanine 38.44 40.78 44.99 Uridine 23.22 26.86 27.13 Adenosine 10.34 12.28 12.63 Guanosine 20.68 22.95 24.11 Butanedioic acid 15.29 15.44 16.86 p-hydroxybenzoic acid 4.690 5.363 5.404 p-coumaric acid 15.63 17.92 17.98 Caffeic acid 6.909 7.774 7.806 Chlorogenic acid 7.74 8.48 8.59 Senkyunolide I 60.32 63.21 69.39 Senkyunolide H 15.76 17.29 18.27 Senkyunolide N 13.36 14.18 14.22 Open-loop senkyunolide I 5.126 5.190 5.648 Senkyunolide G 10.31 10.68 10.74 3-hydroxy-3-Butylphthalide 7.02 7.08 8.67 Senkyunolide A 0.656 0.938 0.961 Ferulaic acid 35.62 42.68 47.15 Protocatechualdehyde 1.435 8.511 17.25 Protocatechuic acid 2.361 2.469 4.030 Tanshinol 2.776 4.150 6.845 Rosmarinic acid 5.07 9.57 12.78 Salvianolic acid D 0.0697 0.2892 0.4005 Salvianolic acid C 1.123 2.185 4.732 Salvianolic acid A 0.366 1.508 2.505 Alkannic acid 0.429 0.480 0.945 Salvianolic acid B 4.00 8.96 11.00
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CN106727649A (en) * | 2016-12-28 | 2017-05-31 | 天津红日药业股份有限公司 | A kind of multicomponent parenteral solution |
CN107589191B (en) * | 2017-09-26 | 2020-03-17 | 山东宏济堂制药集团股份有限公司 | Method for establishing and detecting HPLC (high Performance liquid chromatography) fingerprint spectrum of golden scallop oral liquid |
CN108743600B (en) * | 2018-07-04 | 2021-03-09 | 天津中医药大学 | Natural medicine composition, traditional Chinese medicine composition containing natural medicine and application of natural medicine composition |
CN109394750A (en) * | 2018-10-19 | 2019-03-01 | 天津红日药业股份有限公司 | Application of the hydroxyl radical carthamin yellow carthamus A in preparation treatment medication for treating pyemia |
CN109260214A (en) * | 2018-10-19 | 2019-01-25 | 天津红日药业股份有限公司 | Application of the paeoniflorin compound in preparation treatment medication for treating pyemia |
CN109709244A (en) * | 2019-02-22 | 2019-05-03 | 天津红日药业股份有限公司 | A kind of 'Xuebijing ' injection phthalide lactone component content measuring method |
CN111481535B (en) * | 2020-04-09 | 2023-01-10 | 西北大学 | Application of IDHP in preparation of anti-septicemia and myocardial damage drug induced by IDHP |
Citations (1)
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CN105748782A (en) * | 2016-02-25 | 2016-07-13 | 太和县中医院 | Traditional Chinese medicine oral liquid for treating cervical spondylosis |
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CN1190223C (en) * | 2003-03-04 | 2005-02-23 | 天津红日药业股份有限公司 | Recipe of Chinese medicine with treating and health care functions |
CN101007072B (en) * | 2007-01-18 | 2010-09-29 | 天津红日药业股份有限公司 | Quality-control method of a traditional Chinese medicine 'Xuebijing' injection |
CN101396441B (en) * | 2007-09-26 | 2011-11-30 | 首都医科大学附属北京友谊医院 | Traditional Chinese medicine composition for treating blood stasis syndrome of multi viscera organs incompetence syndrome |
CN101683318A (en) * | 2008-09-28 | 2010-03-31 | 南京中医药大学 | Method for removing pyrogen from injections |
CN104297375B (en) * | 2014-10-23 | 2016-03-30 | 天津红日药业股份有限公司 | The detection method of content of polyoxyethylene sorbitan monoleate in a kind of Chinese medicine 'Xuebijing ' injection |
CN106727649A (en) * | 2016-12-28 | 2017-05-31 | 天津红日药业股份有限公司 | A kind of multicomponent parenteral solution |
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2016
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CN105748782A (en) * | 2016-02-25 | 2016-07-13 | 太和县中医院 | Traditional Chinese medicine oral liquid for treating cervical spondylosis |
Cited By (2)
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CN112704681A (en) * | 2020-12-17 | 2021-04-27 | 天津中医药大学 | Composition, traditional Chinese medicine composition containing composition and application of traditional Chinese medicine composition |
CN112891362A (en) * | 2021-03-01 | 2021-06-04 | 天津红日药业股份有限公司 | Pharmaceutical composition for treating sepsis and application thereof |
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US20210346281A1 (en) | 2021-11-11 |
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