CN112451519A - Application of butenyl phthalide and method for preparing pharmaceutical composition from butenyl phthalide - Google Patents
Application of butenyl phthalide and method for preparing pharmaceutical composition from butenyl phthalide Download PDFInfo
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Abstract
The invention discloses an application of butenyl phthalide, analogues thereof or a combination of the above components in preparing a pharmaceutical composition for treating or preventing neurodegenerative diseases, and a method for preparing the pharmaceutical composition. The butenyl phthalide has the capability of reducing the concentration of beta amyloid protein in cells, and can achieve the effect of treating or preventing neurodegenerative diseases. Therefore, according to the preparation method of the pharmaceutical composition disclosed by the invention, the macromolecular substance is used as a drug carrier, and the butenyl phthalide which is used as an active ingredient is coated in the macromolecular substance so as to slow down or reduce the cytotoxicity of the butenyl phthalide, thereby achieving the effects of improving the safety of the pharmaceutical composition and treating or preventing neurodegenerative diseases.
Description
The application is divisional application of an invention patent application with the application number of 201410362632.2, the application date of 2014 is 7/28, and the invention name is 'application of butenyl phthalide and a method for preparing the butenyl phthalide into a pharmaceutical composition'.
Technical Field
The invention relates to application of a compound, in particular to application of butenyl phthalide and a method for preparing the butenyl phthalide into a pharmaceutical composition.
Background
Alzheimer's disease is the most common neurodegenerative disease worldwide and is characterized primarily pathologically by the deposition of excess amyloid beta protein (Abeta) in the brain, forming Abeta plaques (plaque) (Glenner and Wong 1984; Masters, C.L.et al, 1985) and Neurofibrillary (NFT) tangles within the cells (Grundke-Iqbal, I.et al, 1986; Goedert, M.et al, 1988). The A β plaque is generated by Amyloid Precursor Protein (APP) after BACE enzyme (Hussain, I.et al, 1999; Vassar, R.et al, 1999) and gamma secretase (gamma-secretase) cleavage (Wolfe, M.S.et al, 1999; Yu, G.et al, 2000), mainly including two forms of A β 40 and A β 42 (Jarrett, J.T.et al, 1993). When amyloid beta is accumulated in large amounts inside and outside cells, it becomes a major cause of neuronal cell death.
In the Down's disease patient, when the 21 st chromosome is meiotic, the chromosome is not uniformly separated, and the cell is provided with one more chromosome. Since the amyloid precursor protein gene is located on chromosome 21 (Rumble, b.et al, 1989; Selkoe, d.j.,1996), it is believed that overexpression of amyloid precursor protein causes symptoms of early-onset cognitive impairment in down's patients (Burger, p.c. and f.s.vogel, 1973). Many studies currently show that plaques of a β appear in the brain of down's patients (Masters, c.l. et al, 1985; Beyreuther, k.et al, 1992; Gyure, k.a. et al, 2001; Mori, c.et al, 2002). In addition, studies have shown that nerve cells generated from Induced Pluripotent Stem Cells (iPSCs) from patients with down's disease can be used to reproduce pathological features typical of alzheimer's disease, such as accumulation of a β 42 and a β 40, highly phosphorylated Tau protein, and the like. Therefore, the induced universal stem cell differentiation system of the Down's disease patient can be used as a drug platform for screening and treating or preventing the neurodegenerative diseases related to the beta amyloid protein.
Currently, clinical drugs for treating alzheimer disease include cholinesterase inhibitors (Birks, j.,2006) and NMDA receptor antagonists (NMDA receptor antagonists) (McShane, r.et al.,2006), both of which are suitable for improving cognitive functions of alzheimer patients, however, for diseases derived from alzheimer disease, such as depression, insomnia, etc., treatment by other suitable drugs is required (Tariot, p.n.et al., 2004; Feldman, h.et al., 2006; Howard, r.et al.,2012), and both of which can only be used for improving symptoms and cannot achieve efficacy of curing alzheimer disease (Farlow, m.r.et al., 2010). In addition, many studies are directed to designing new drugs for treating alzheimer's disease (Hong-Qi, y.et al.,2012) for reducing the accumulation of a β in the brain, specifically, drugs comprising BACE inhibitors (BACE inhibitors), such as MK-8931 and ACI-91(Mullard a.,2012), or gamma secretase inhibitors, such as LY450139(Siemers, e.et al.,2005) and BMS-708163(Tong, g.et al.,2012), or antibodies that antagonize a β in an immune pathway, etc.
Therefore, neurodegenerative diseases such as alzheimer's disease do pose a serious threat to human health, and since there is no clinically effective drug at present, it is an important subject of scientists to develop pharmaceutical compositions for treating or preventing neurodegenerative diseases.
Disclosure of Invention
The main object of the present invention is to provide the use of butenyl phthalide for the preparation of a pharmaceutical composition for the treatment or prevention of neurodegenerative diseases.
To achieve the above objects, the present invention discloses the use of butenyl phthalide, its analogs or a combination of the above components for preparing a pharmaceutical composition for treating or preventing neurodegenerative diseases.
Preferably, the neurodegenerative disease is a condition in which there is an accumulation of excess amyloid-beta protein in the brain.
Preferably, the neurodegenerative disease is alzheimer's disease.
Preferably, the butenyl phthalide can be extracted from natural plants, such as Umbelliferae plants, Compositae plants, etc., wherein the extraction technique used is known to those skilled in the art.
Preferably, the butenyl phthalide can be prepared by chemical synthesis techniques, wherein the chemical synthesis techniques used are chemical synthesis methods well known to those skilled in the art.
Another objective of the present invention is to provide a method for preparing the above-mentioned pharmaceutical composition, which is used to reduce the cytotoxicity of the active ingredient in the pharmaceutical composition, thereby achieving the effects of improving the safety of the pharmaceutical composition and reducing the side effects.
In order to achieve the purpose, the invention discloses a preparation method of a pharmaceutical composition, which comprises the following steps of mixing butenyl phthalide and a high molecular substance in a weight ratio of 1: 1-1: 2 mixing, adding a polar organic solvent and water, making butenyl phthalide and high molecular substance adsorb in water phase by intermolecular attraction to coat butenyl phthalide on the high molecular substance, and removing the polar organic solvent.
Preferably, the polar organic solvent is a heterocyclic ether-like compound.
Preferably, the polar organic solvent is tetrahydrofuran (tetrahydrofuran).
Preferably, the high molecular substance is an F127 high molecular substance.
Preferably, the method for removing the polar organic solvent is a heating method.
The invention has the beneficial effects that:
the butenyl phthalide disclosed by the invention can obviously reduce the concentration of A beta 40 in nerve cells, has the effect similar to the effect of administering a gamma secretagogue inhibitor DAPT, and has the capability of improving or slowing down the accumulation of excessive beta amyloid protein in cells. The preparation method of the pharmaceutical composition disclosed by the invention can use the high molecular substance as a drug carrier, slow down or reduce the cytotoxicity of the butenyl phthalide, and can actually and effectively improve the safety of the pharmaceutical composition. The pharmaceutical composition prepared by the method of the present invention has the function of reducing the concentration of beta amyloid protein in cells, and can achieve the effect of treating or preventing neurodegenerative diseases by administering an effective amount of the pharmaceutical composition to a living body.
Drawings
FIG. 1 shows the results of cytotoxicity tests using butenyl phthalide coated with a high molecular substance F127 and butenyl phthalide uncoated with a high molecular substance F127.
FIG. 2 is a flow chart of neural differentiation culture of T21-induced pluripotent stem cells.
FIG. 3 shows the result of N-cadherin expression observed by immunofluorescence staining analysis after neural differentiation culture of T21-induced pluripotent stem cells, wherein red is immunofluorescent-stained N-cadherin, and blue is DAPI-stained cell nucleus.
FIG. 4 shows the results of immunofluorescent staining analysis of nestin expression in T21-induced pluripotent stem cells after neural differentiation culture, wherein green is immunofluorescent stained nestin and blue is DAPI stained nuclei.
FIG. 5 shows the results of immunofluorescent staining analysis of expression of Pax-6 protein in T21-induced pluripotent stem cells after neural differentiation culture, wherein red is immunofluorescent-stained Pax-6 protein and blue is DAPI-stained nuclei.
FIG. 6 shows the results of immunofluorescent staining analysis of beta III tubulin expression and neurite outgrowth of T21-induced pluripotent stem cells after neural differentiation culture, where green is immunofluorescent stained beta III tubulin and blue is DAPI stained nuclei.
FIG. 7 shows the results of statistical analysis of the expression level of Abeta 40 in neural cells differentiated from different cells by ELISA.
FIG. 8 shows the results of analysis of the expression level of Abeta 40 in each cell by enzyme-linked immunosorbent assay after the neural cells differentiated from T21-induced pluripotent stem cells were cultured under different conditions.
Detailed Description
The invention discloses application of butenyl phthalide (Bdph) and a method for preparing the butenyl phthalide into a pharmaceutical composition. Since the butenyl phthalide can reduce the content of beta amyloid protein in nerve cells, and excessive accumulation of beta amyloid protein in cells can cause neurodegenerative diseases such as Alzheimer's disease, the effect of preventing or treating neurodegenerative diseases can be achieved by administering an effective amount of butenyl phthalide to an organism. Furthermore, the pharmaceutical composition is prepared by organic synthesis reaction, and covalent bonding is generated between the macromolecular substance such as F127 and the butenyl phthalide, so that the macromolecular substance coats the butenyl phthalide, and the effect of reducing the cytotoxicity of the pharmaceutical composition on organisms is achieved.
Unless defined otherwise, all technical and scientific terms used herein in the specification and claims have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present disclosure will control.
The structural formula of the butenyl phthalide disclosed by the invention is shown as the formula (I):
the manner of making or obtaining it is within the ordinary knowledge of a person of ordinary skill in the art, and is not a feature of the invention,therefore, the present invention will not be described in detail. For example, chinese patent application No. 200910066666 discloses that butenyl phthalide is extracted from plants of the family of umbelliferae or Compositae by using an organic solvent; chinese patent publication No. 1041725C discloses that butenyl phthalide is synthesized by chemical synthesis.
The high molecular substance F127 disclosed by the invention is a triblock polymer of polyoxyethylene-polyoxypropylene-polyoxyethylene, and has a chemical formula shown as a formula (II):
The term "intermolecular force" as used herein includes, but is not limited to, van der Waals forces, coulomb forces, hydrogen bonding, and hydrophobic bonding forces.
The term "pharmaceutical composition" as used herein includes an effective amount of a desired compound or active ingredient to produce a particular effect, and at least one pharmaceutically acceptable carrier. As will be appreciated by those skilled in the art, the form of the pharmaceutical composition may vary with the mode of administration desired to produce a particular result, such as tablets, powders, injections, etc., and the carrier may be solid, semi-solid, or liquid depending on the form of the pharmaceutical composition. For example, carriers include, but are not limited to, gelatin, emulsifiers, hydrocarbon mixtures, water, glycerol, physiological saline, buffered physiological saline, lanolin, paraffin, beeswax, dimethicone, ethanol.
The term "effective amount" as used herein refers to the amount of active ingredient or compound in a pharmaceutical composition that is intended to produce a particular desired effect, usually expressed as a percentage by weight of the active ingredient or compound in the pharmaceutical composition. As will be appreciated by those of ordinary skill in the art, such effective amounts will vary depending on the manner of administration desired to bring about the particular effect. Generally, the active ingredient or compound may be present in the composition in an amount of from about 1% to about 100%, preferably from about 30% to about 100%, by weight of the composition.
The term "analog" as used herein includes salts of the compounds, esters thereof, structural isomers thereof, such as Z-type structures or E-type structures, or structurally modified products thereof.
The term "polymeric substance" as used herein refers to a polymer formed by polymerization to form a macromolecule having a relatively high molecular weight, and generally speaking, polymeric substances are all organic molecules.
In the following, in order to further illustrate the efficacy of the present invention, several embodiments are described in detail, which are illustrative examples, and any terms used therein should not be construed to limit the scope and meaning of the present invention.
Example 1: synthesis of aqueous phase butenyl phthalide
Butenyl phthalide (n-butylidenephthalide, Bdph, W333301) and F127 polymer (Pluronic F127, P2443) were obtained from Sigma-Aldrich group, USA. Mixing 10 mg of butenyl phthalide and F127 high molecular substance in a weight ratio of 1: 1 or 1: 2, dissolved in 2 ml of tetrahydrofuran (tetrahydrofuran), added to 10 ml of water, rapidly heated to remove tetrahydrofuran, and freeze-dried. Then, the solution is re-dissolved in water, the size of the butenyl phthalide particles emulsified in the solution is about 30-200 nm, and the polydispersity index (polydispersity index) is 0.2-0.5.
Example 2: cell culture
Culturing human universal stem cells in serum-free culture medium Essential 8TM(Life Technology, USA) and in matrigelTMThe culture was performed by attaching matrigel (Becton-Dickinson, USA). Removing the culture medium by suction, washing the cells with phosphate buffer for 2 times, and adding AccutaseTM(MerckMillipore, usa) for 2-5 minutes, and the cells are washed off and broken into small clumps, centrifuged at 1000rpm for 2 minutes, the supernatant is aspirated, placed in a new culture dish for culture for about 3-5 days, subcultured, and the culture medium is changed every day during the culture.
Example 3: neural cell differentiation
Culturing human universal stem cells to 8-9 min, washing the cells with phosphate buffer solution for 2 times, and adding AccutaseTMAfter 2-5 minutes of action, adding culture solution to dilute enzyme, washing the cells, scattering the cells to a proper size, centrifuging the cells at the speed of 800rpm for about 2 minutes, placing the cells in a DMEM-F12 culture medium containing 20% serum substitute (KSR for short, Life Technology, USA), and performing suspension culture on the cells in a petri dish for 2 days to obtain an embryo-like body (Embryonic body) suspension.
Placing the suspended embryoid bodies in a centrifuge tube, naturally settling, removing supernatant, and performing suspension culture in a nerve induction culture medium containing a BiSF small molecule drug for 2 days, wherein the suspended embryoid bodies have a circular epithelial cell structure, and the small molecule drug comprises 0.5 μ M BIO (Sigma-Aldrich company, USA), 10ng/mL fibroblast growth factor-2 (FGF-2, Peprotech company, USA) and 10 μ M SB431542(Sigma-Aldrich company, USA); the composition of the neural induction culture solution is shown in table 1 below.
TABLE 1 composition of the neural inducing culture solution
| Composition (I) | Content (ml) |
| DMEM medium (Life technologies, 11965- | 326 |
| F12 Medium (Life technologies, 11765- | 163 |
| N2 additive (Life technologies, 17502- | 5 |
| Non-essential amino acids (Life technologies, 11140- | 5 |
| Heparin (1mg/mL) | 1 |
Then, the embryoid bodies were settled and cultured in a neural basal medium containing 10ng/mL fibroblast growth factor-2, wherein the composition of the neural basal medium is shown in Table 2 below. Suspending for 2 days, and precipitating embryoid body, and applying external force or enzyme AccutaseTMScattering the mixture into small blocks, and placing the small blocks on a coated 1% matrigelTMCell attachment was performed in a petri dish with matrigel for more than 1 hour. Cells attach, grow and grow neural structures about 2-7 days. And performing cell separation when the cells grow to 7-8 min, wherein the cells are washed for 1 time by using a phosphate buffer solution and then washed by using the phosphate buffer solution and the enzyme AccutaseTMThe enzyme solution mixed in equal proportion acts for about 2-5 minutes, then the cell is diluted by a nerve basic culture medium, then a cell spatula is used for scraping the cells and breaking the cells into small blocks or single cells by mechanical force, the centrifugation is carried out for about 2-5 minutes at 800-1000 rpm, then the supernatant is sucked and removed for disk sticking, and 10 mu M Y27632(Stemgent company, USA) is added for 1 day when the disk sticking is carried out to help the cell sticking.
TABLE 2 composition of the neural basal Medium
Example 4: cytotoxicity assays
Human embryonic stem cell TW1 was cultured in Essential 8TMIn the culture medium, and in three groups in subculture, 1.2X10 was placed in each 6-well plate5The amount of cells of (a) is cultured under the following conditions, wherein the first group is cultured solely with a culture medium; the second group adds butenyl phthalide without coating F127 high molecular substance in the culture medium, the concentration is 10 μ M; third group to the culture medium was added butenyl phthalide coated with F127 high molecular substance prepared in example 1 at a concentration of 10. mu.M. The cell morphology of each group was observed during the culture, and the cell count was performed for 4 to 5 days of culture, and the results were repeated 1 to 3 times for each group, as shown in FIG. 1, wherein the results of FIG. 1 were obtained by single-factor variance analysis (one-way ANOVA) and Multiple comparative assay (Tukey's Multiple Comparison Test analysis), and the P value was less than 0.05, 95% confidence level, and the notation is below the significance level of 0.05 and the notation is below the significance level of 0.01.
As can be seen from the results of fig. 1, the number of cells of the second group was significantly less than that of the first or third group after 5 days of culture. Therefore, the butenyl phthalide has high cytotoxicity to cells, and the cytotoxicity of the butenyl phthalide can be effectively reduced by coating the high molecular substance F127.
Example 5: neural cell differentiation culture of T21 induced pluripotent stem cells
Referring to example 2 and fig. 2, T21-induced pluripotent stem cells (T21-iPSCs) were neural differentiated as described in example 2 and attached on day 8. Immunofluorescent staining analysis was performed on day 12 after cell attachment to observe the expression of neural stem cells containing N-cadherin, Nestin and Pax-6 proteins, as shown in FIGS. 3 to 5. The growth of neurites after neural differentiation culture of T21-induced pluripotent stem cells was observed, and the expression of a mature neural marker such as β III tubulin (tubulin) was observed by immunofluorescence staining analysis on day 27 of culture and Tuj-1 antibody, and the results are shown in FIG. 6.
The operation flow of the immunofluorescence staining analysis is as follows: when cells are cultured in 4-well culture dishes of a cover glass and stained, a cell culture solution is aspirated, washed with a phosphate buffer solution for 2 times, then 4% Paraformaldehyde (PFA) is added to act on ice for 5 minutes to fix the cells, then the cells are removed, washed with the phosphate buffer solution for 3 times, then 0.3% triton (pbst) is added to act on ice for 10 minutes to punch holes, washed with the phosphate buffer solution for 3 times, then 5% horse serum is added to block (blocking) the holes for 1 hour, the solution is aspirated, a primary antibody configured to 3% horse serum is added, and the cells are reacted at room temperature for 4 hours or at 4 ℃ for 16 hours. Thereafter, the primary antibody was aspirated and washed 3 times with PBST wash solutions for 5 minutes each. The secondary antibody is configured in phosphate buffer, after PBST is absorbed, the secondary antibody is added, the light shielding effect is carried out for 1 hour, then PBST washing liquid is used for washing for 3 times, cell nucleus dye DAPI with the concentration of 1 mu g/mL is added, the light shielding effect is carried out for 10 minutes at room temperature, then PBST washing liquid is used for washing for 2 times, solution which is mixed by glycerol and phosphate buffer solution in equal proportion is soaked for 200 mu L, then the piece picking is carried out, and the fluorescence is observed under an upright fluorescence microscope after the piece sealing.
From the results shown in FIGS. 3 to 5, it is clear that T21-induced pluripotent stem cells express N-cadherin, Nestin and Pax-6 neural stem cell specific proteins on day 12 of neural cell differentiation culture, and that T21-induced pluripotent stem cells can be successfully differentiated into T21 neural cells after being cultured in the above-mentioned medium. Furthermore, from the results of fig. 6, it was found that T21-induced universal stem cells had a large number of neurites after 27 days of culture and differentiation, and were able to express β iii tubulin, which is a target of mature nerves, and that T21-induced universal stem cells were differentiated and cultured into mature T21 nerve cells after 27 days of culture.
Example 6: a beta 40 expression of nerve cells
After counting the number of cells of the neural cells differentiated from the T21-induced pluripotent stem cells in the patch culture, the cells were plated on a 4-well culture dish, the culture medium was replaced every 48 hours, the culture medium collected every 48 hours was stored at-20 ℃, the expression level of a β 40 was analyzed by enzyme-linked immunosorbent assay (ELISA), and the neural cells differentiated from the human embryonic stem cells TW1 having normal karyotype were used as a control group, and the experiment was repeated 2 or 3 times for each group. The analysis results are shown in FIG. 7.
The operation procedure of the enzyme-linked immunosorbent assay is as follows: the standard a β protein of the ELISA kit was sequence diluted with culture buffer (workginubationbuffer) to serve as a standard curve. Mixing the collected cell culture solution with the culture buffer solution in a ratio of 1: 2 for dilution. 100. mu.L of the standard solution and the sample were placed in a 96-well plate and left at 4 ℃ for 16 hours, respectively. After aspirating off the liquid, the well plates were rinsed with 300 μ L and air dried. Adding 200 mu L of TMB substrate, reacting for about 40-45 minutes at room temperature in a dark place, and reading the light absorption value at 620 nm.
As can be seen from the results of fig. 7, expression of a β 40 in the neural cells differentiated from T21-induced pluripotent stem cells was higher than that of the neural cells differentiated from human embryonic stem cells TW1 at the 20 th day of differentiation, and a difference was observed at the 25 th day of differentiation. In detail, the expression level of A.beta.40 in the neural cells differentiated from T21-induced pluripotent stem cells was 105.38pg/mL on day 25 of differentiation, 155.68pg/mL on day 30 of differentiation, and 264.47pg/mL on day 42 of differentiation.
Accordingly, the expression level of a β 40 in the neural cells differentiated from T21-induced pluripotent stem cells was significantly increased with the increase in differentiation time, and thus it was found that the expression level could be used as a platform for screening for the treatment or prevention of neurodegenerative diseases.
Example 7: drug screening
The T21-induced pluripotent stem cells were divided into three groups by neural differentiation culture until day 39, and after being subjected to different culture conditions, the cells were cultured for 3 days, that is, at day 42, the concentrations of a β 40 in each group were analyzed by enzyme-linked immunosorbent assay, and the results are shown in fig. 8, in comparison with T21-induced pluripotent stem cells that were not subjected to neural differentiation culture, in the first group, T21-induced pluripotent stem cells that were not subjected to neural differentiation culture; the second to fourth groups were T21-induced pluripotent stem cells subjected to neural differentiation culture, the second group was not supplemented with any drug during the culture, the third group was supplemented with the gamma secretagogue inhibitor DAPT at day 39 of culture, and the fourth group was supplemented with 10 μ M of F127-coated high-molecular-weight butenyl phthalide at day 39 of culture. Each set of tests is carried out 2-3 times, and the presence or absence of significant differences in the analytical values is determined by single factor variance analysis and multiple comparisons, wherein the P value is less than 0.05, the 95% confidence level, the notation A indicates a significance level below 0.05 and the notation A indicates a significance level below 0.01.
As can be seen from the results of fig. 8, the concentration of a β 40 in the first group was significantly lower than that in the second group, and the concentrations of a β 40 in the third group and the fourth group were respectively lower than that in the second group. Therefore, the butenyl phthalide of the present invention can significantly reduce the concentration of a β 40 in nerve cells, and the effect is similar to that of DAPT, which is an inhibitor of γ secretagogues. Therefore, the butenyl phthalide disclosed by the invention has the capability of improving or slowing the accumulation of excessive beta amyloid protein in cells, and can achieve the effect of treating or preventing neurodegenerative diseases.
Through the description of the above embodiments, the preparation method of the pharmaceutical composition disclosed by the invention can use the high molecular substance as the drug carrier, slow down or reduce the cytotoxicity of the butenyl phthalide, and indeed can effectively improve the safety of the pharmaceutical composition. The pharmaceutical composition prepared by the above method indeed has the function of reducing the intracellular amyloid beta concentration, so that the efficacy of treating or preventing neurodegenerative diseases can be achieved by administering an effective amount of the pharmaceutical composition to a living body.
The present invention has been described in detail with reference to the embodiments, and it is to be understood that any simple modification or variation of the embodiments described in the specification may be made by those skilled in the art without departing from the spirit of the present invention.
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Claims (6)
1. The use of butenyl phthalide in the preparation of an inhibitor of amyloid-beta protein in nerve cells, characterized in that: the concentration of amyloid-beta protein in the nerve cells is higher than 200 pg/ml.
2. Use according to claim 1, characterized in that: the butenyl phthalide is extracted from Umbelliferae plant.
3. Use according to claim 1, characterized in that: the butenyl phthalide is extracted from Compositae plant.
4. Use according to claim 1, characterized in that: the butenyl phthalide or its analogue is prepared by chemical synthesis technology.
5. Use according to claim 1, characterized in that: the beta amyloid protein inhibitor in nerve cells is prepared by coating butenyl phthalide with F127 high molecular substance.
6. Use according to claim 5, characterized in that: the weight ratio of the butenyl phthalide to the F127 high molecular substance is 1: 1-1: 2.
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| CN103169694A (en) * | 2011-12-20 | 2013-06-26 | 东华大学 | Application of n-butenyl phthalide in preparing medicine for treating liver injury and improving liver function and pharmaceutical composition |
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