A method for fermenting indigo with trichoderma, a product obtained and application thereof

Abstract

The application discloses a kind of trichoderma fermentation processing indigo blue method, the product obtained and application.The method includes: preparation trichoderma seed liquid;After primary indigo blue sterilization, access seed liquid, fermentation 5-9 days under 25-30 DEG C, 120-180 rpm, avoid light, obtain secondary processing indigo blue.The application first utilizes trichoderma to carry out biological transformation to indigo blue, can significantly improve the content of its key anti-tumor component indigo carmine with specificity, and the increase is more than 150%.In vitro anti-tumor experiment shows that the inhibitory activity of secondary processing indigo blue prepared by the application to human leukemia K562 cells and human colon adenocarcinoma cell SW480 cell is significantly better than ordinary indigo blue.The product of the application can be used for preparing medicine for preventing and / or treating leukemia, colon adenocarcinoma, process controllable, suitable for industrialized production, and provides a new way for developing efficient indigo blue anticancer new drug.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine processing technology, specifically to a method for processing Indigo Naturalis using Trichoderma fermentation, the resulting product and its application, and more specifically to a new method for secondary processing of Indigo Naturalis using Trichoderma fermentation, the secondary processed Indigo Naturalis product obtained by this method, and the application of this product in the preparation of drugs for treating leukemia and colon adenocarcinoma. Background Technology

[0002] Indigofera tinctoria is a plant of the Acanthaceae family, specifically the stork tree (Ind Baphicacanthus cusia (Nees) Bremek. ), Polygonaceae plant Polygonum indica ( Polygonum tinctorium Ait. ) or Isatis indigotica, a plant of the Brassicaceae family ( Isatis indigotica Fort Indigofera tinctoria is a dried powder, lumps, or granules obtained from the processed leaves or stems of the plant. It is cold in nature, salty in taste, and enters the liver meridian. It has the effects of clearing heat and detoxifying, cooling the blood and eliminating spots, purging fire and calming the nerves. Modern research shows that the main active ingredients of Indigofera tinctoria include indigo, indirubin, and tryptanthrin, among which indirubin has been proven to have significant anti-tumor activity, especially with good efficacy against chronic myeloid leukemia.

[0003] However, the content of indigo in natural indigo is generally low (the 2025 edition of the Pharmacopoeia requires it to be no less than 0.20%), and its water solubility and bioavailability are poor, which seriously limits its clinical efficacy and formulation development. Traditional processing methods such as water levigation are mainly aimed at purification and removal of impurities, but cannot significantly change the proportion of its internal components and bioavailability.

[0004] Microbial fermentation, as a traditional Chinese medicine processing and modification technique, has received widespread attention in recent years. Utilizing the powerful enzyme systems of microorganisms to biotransform components of Chinese medicine can produce new active substances, increase the content of original components, or reduce their toxicity, thereby enhancing efficacy and expanding the scope of clinical applications. Currently, there are no reports of using Trichoderma to conduct targeted fermentation of Indigo Naturalis to specifically increase the content of Indirubin and enhance its anti-leukemia and colon adenocarcinoma activity. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a new method for secondary processing of indigo naturalis using Trichoderma. This method can significantly increase the content of indigo carmine in indigo naturalis and enhance its anti-tumor activity.

[0006] Another object of the present invention is to provide a secondary processed indigo product with high indigo content prepared by the above method.

[0007] Another object of the present invention is to provide the application of the above-mentioned secondary processed indigo product in the preparation of antitumor drugs.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides a method for processing indigo naturalis using Trichoderma fermentation, comprising the following steps:

[0010] (1) Preparation of Trichoderma seed culture;

[0011] (2) Prepare the primary indigo naturalis into a suspension and sterilize it;

[0012] (3) The seed liquid prepared in step (1) is inoculated into the sterilized indigo suspension at an inoculation amount of 5% to 15% by volume (v / v), and fermented in the dark at 25 to 30°C and 120 to 180 rpm for 5 to 9 days. After fermentation, the precipitate is centrifuged, collected, and dried to obtain the secondary processed indigo.

[0013] Preferably, the Trichoderma fungus mentioned in step (1) is Trichoderma longifolia ( Trichoderma longibrachiatum Trichoderma harzianum ( Trichodermaharzianum ), dark green Trichoderma ( Trichoderma atroviride The preparation method of the fungal seed liquid includes: inoculating Trichoderma strains onto PDA plate medium for activation, culturing at 28-37℃ for 48-72 hours; then picking up the activated colonies with an inoculation loop and inoculating them into PDB liquid medium, and culturing them in the dark with shaking at 25-30℃ and 120-180 rpm for 2-4 days.

[0014] Preferably, the primary indigo described in step (2) is commercially available indigo or indigo obtained by soaking raw material from Isatis indigotica, adding lime milk to make indigo, and filtering.

[0015] Preferably, the concentration of the suspension is 0.2 to 3.33 mg / ml.

[0016] Preferably, the sterilization process in step (2) is high-pressure steam sterilization, with conditions of 121°C for 15–20 min.

[0017] Preferably, the bacterial concentration of the seed culture in step (3) is OD. 600 Between 0.5 and 0.7.

[0018] Preferably, the fermentation culture time in step (3) is 7 days.

[0019] Secondly, the present invention provides a secondary processed indigo naturalis prepared by any of the above methods.

[0020] Preferably, the content of the active ingredient indirubin in the secondary processed indigo is ≥0.33%.

[0021] In a third aspect, the present application provides use of the twice processed indigo caeruleum in preparation of an anti-tumor drug.

[0022] Preferably, the tumor is leukemia or colon adenocarcinoma.

[0023] In a fourth aspect, the present application provides a pharmaceutical composition comprising the twice processed indigo caeruleum and one or more pharmaceutically acceptable carriers or excipients.

[0024] Preferably, the pharmaceutically acceptable carriers or excipients include any one of or a combination of sustained-release agents, excipients, fillers, binders, humectants, disintegrants, absorption promoters, surfactants, lubricants, and the like, such as a combination of binders and excipients, a combination of binders and flavorings, a combination of binders and fillers, and the like, and any other combination can be selected, which will not be described herein.

[0025] Preferably, the dosage form of the pharmaceutical composition can be a conventional dosage form such as tablets, capsules, granules, powders, or oral liquids.

[0026] Compared with the prior art, the present application has the following beneficial effects:

[0027] Specific synergistic effect: the present application first uses Trichoderma to ferment and process indigo caeruleum, and the strain and its metabolic enzyme system can specifically and directionally transform certain components in indigo caeruleum, significantly increasing the content of key anti-tumor active ingredient indigo, with an increase of more than 150%, which cannot be achieved by traditional physical processing methods.

[0028] Significant increase in activity: the indigo caeruleum processed by the method of the present application has significantly better in-vitro anti-human chronic myelocytic leukemia K562 cell and human colon adenocarcinoma SW480 cell activities than ordinary indigo caeruleum, with a significantly reduced IC 50 value, indicating a significant improvement in drug efficacy.

[0029] Controllable process, easy to promote: the fermentation process provided by the present application has clear conditions and controllable parameters, good reproducibility, and is suitable for industrial large-scale production, providing high-quality raw materials for the development of efficient indigo caeruleum anti-cancer drugs.

[0030] High safety: the fermentation process is carried out in a closed condition and through pre-sterilization treatment, effectively controlling bacterial contamination, and the microbial limit of the final product meets the requirements, ensuring the safety of drug use. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 Comparison of HPLC chromatograms of the twice processed indigo caeruleum and the once processed indigo caeruleum of the present application;

[0032] Figure 2The figure is a comparison of the inhibitory effects of secondary processed indigo and primary processed indigo on K562 and SW480 cell proliferation. In the figure, secondary processed product 1 is the sample prepared in Example 2, secondary processed product 2 is the sample prepared in Example 3, and primary processed product is the sample prepared in Example 1. DETAILED DESCRIPTION

[0033] The application will be further described in detail below with reference to the accompanying drawings and examples, but in no way limits the application, any transformation or improvement based on the teaching of the application falls within the protection scope of the application.

[0034] The process, conditions, reagents, experimental methods, etc. for implementing the application, except for the following specifically mentioned contents, are the general knowledge and common sense in the art, and the application has no special limitations. The experimental methods not specified in the examples are usually performed according to the conventional conditions or the conditions recommended by the manufacturers.

[0035] Unless otherwise specified, the meanings of all professional terms and scientific terms used in this specification are the same as those generally understood by the skilled person in the technical field to which the application belongs. However, if there is a conflict, the specification containing the definition shall prevail.

[0036] The following are the sources of some main raw materials in the examples:

[0037] Trichoderma longibrachiatum Trichoderma longibrachiatum was purchased from China General Microbiological Culture Collection Center, with the preservation number of CGMCC No. 3.8877. Trichoderma atroviride Trichoderma atroviride was purchased from China General Microbiological Culture Collection Center, with the preservation number of CGMCC No. 3.12963; Trichoderma harzianum Trichoderma harzianum was purchased from China General Microbiological Culture Collection Center, with the preservation number of CGMCC No. 3.12069.

[0038] The south radix isopophammi is the south radix isopophammi planted in Yuan Yang, Yunnan.

[0039] Example 1 Preparation of primary indigo

[0040] 80 g of a mixture of dried south radix isopophammi branches and leaves was weighed and placed in a 2000 mL beaker, 1000 mL of tap water was added, and it was soaked at room temperature, with stirring every 5 hours. After 3 days, it was filtered with gauze, the branches were discarded, and the filtrate was collected. 20 mL of lime milk solution containing 5 g of lime was added to the filtrate, and it was stirred uniformly. Then it was stirred at high speed using a stirrer (‘beating indigo’) for 10 times, a large amount of blue foam was generated, and the foam was collected to obtain indigo flower. After standing and precipitation, the supernatant was discarded, the bottom precipitate was collected, filtered, and dried at 55°C to obtain primary indigo.

[0041] Example 2 Secondary processing of indigo

[0042] (1) Strain activation: In the clean bench, the frozen Trichoderma longibrachiatum powder was picked up with an inoculation loop and streaked on PDA plate medium. The plate was inverted in a 28°C constant temperature incubator and cultured for 72 hours.

[0043] (2) Seed liquid preparation: The mycelium growing vigorously was picked up from the activated plate with an inoculation loop and inoculated into a 500 mL triangular flask containing 100 mL PDB liquid medium. It was placed in a shaker and cultured for 3 days at 28°C, 150 rpm, and in the dark, to obtain Trichoderma longibrachiatum seed liquid (OD 600 between 0.6).

[0044] (3) 1 g of the primary indigo prepared in Example 1 was weighed and placed in a 1000 mL triangular flask, 300 mL of purified water was added to prepare a suspension. The triangular flask was wrapped and sterilized in a 121°C high-pressure steam sterilization pot for 15 minutes, and then cooled to room temperature. In the clean bench, 10 mL of Trichoderma longibrachiatum seed liquid prepared in step (2) was inoculated at a 10% inoculation amount (v / v). The triangular flask was placed in a shaker and cultured for 7 days at 28°C, 150 rpm, and in the dark. After the fermentation was completed, the fermentation liquid was centrifuged (4000 rpm, 10 min) to remove the mycelium, the supernatant was collected, 20 mL of lime milk was added, and after the indigo was beaten, the precipitate was vacuum dried at 55°C, ground, and passed through a 100-mesh sieve to obtain the secondary processed indigo of the application.

[0045] Example 3 Secondary processing of indigo

[0046] (1) Strain activation: In the clean bench, the frozen Trichoderma longibrachiatum powder was picked up with an inoculation loop and streaked on PDA plate medium. The plate was inverted in a 28°C constant temperature incubator and cultured for 72 hours.

[0047] (2) Seed liquid preparation: The mycelium growing vigorously was picked up from the activated plate with an inoculation loop and inoculated into a 500 mL triangular flask containing 100 mL PDB liquid medium. It was placed in a shaker and cultured for 3 days at 28°C, 150 rpm, and in the dark, to obtain Trichoderma longibrachiatum seed liquid (OD 600 between 0.6).

[0048] (3) Weigh 50 mg of the primary indigo naturalis prepared in Example 1 and place it in a 500 mL Erlenmeyer flask. Add 100 mL of purified water to prepare a suspension. Wrap the Erlenmeyer flask and sterilize it in a high-pressure steam sterilizer at 121℃ for 20 minutes. Remove and cool to room temperature. In a clean bench, inoculate 5 mL of the Trichoderma longifolia seed solution prepared in step (2) at a 5% inoculation rate (v / v). Place the Erlenmeyer flask in a shaker and ferment it in the dark at 25℃ and 120 rpm for 9 days. After fermentation, centrifuge the fermentation broth (8000 rpm, 10 min), collect the precipitate, vacuum dry it at 55℃, grind it, and pass it through a 100-mesh sieve to obtain the secondary processed indigo naturalis described in this invention.

[0049] Example 4: Secondary processing of indigo

[0050] (1) Activation of strain: In a clean bench, use an inoculation loop to pick up frozen Trichoderma harzianum powder and streak it onto a PDA plate. Invert the plate and incubate it in a 28°C incubator for 72 hours.

[0051] (2) Seed culture preparation: Vigorous mycelia were picked from the activated plate using an inoculation loop and inoculated into a 500 mL Erlenmeyer flask containing 100 mL of PDB liquid medium. The flask was placed in a shaker and cultured for 4 days at 30℃ and 180 rpm in the dark to obtain the *Trichoderma longicornis* seed culture (OD). 600 (between 0.5).

[0052] (3) Weigh 20 mg of the primary indigo naturalis prepared in Example 1 and place it in a 500 mL Erlenmeyer flask. Add 100 mL of purified water to prepare a suspension. Wrap the Erlenmeyer flask and sterilize it in a high-pressure steam sterilizer at 121 °C for 20 minutes. Remove and cool to room temperature. In a clean bench, inoculate 15 mL of the Trichoderma longifolia seed culture prepared in step (2) at an inoculation rate of 15% (v / v). Place the Erlenmeyer flask in a shaker and ferment it in the dark at 30 °C and 180 rpm for 5 days. After fermentation, centrifuge the fermentation broth (8000 rpm, 10 min), collect the precipitate, vacuum dry it at 55 °C, grind it, and pass it through a 100-mesh sieve to obtain the secondary processed indigo naturalis described in this invention.

[0053] Experimental Example 1: Determination of Indigo Content (HPLC Method)

[0054] Instrument: Analytical high performance liquid chromatography (1260 Infinity II)

[0055] Chromatographic conditions: Column: Agilent ZORBAX SB-C18 (4.6) 150 mm), mobile phase: methanol: water (7:3), isocratic elution, flow rate 1 mL / min, detection wavelength: 292 nm.

[0056] Preparation of the reference solution: accurately weigh an appropriate amount of indigo carmine reference substance (purchased from China Institute for Food and Drug Control, purity ≥98%) into a brown volumetric flask, dissolve and dilute quantitatively with N,N-dimethylformamide (DMF) to prepare a solution containing 100 μg of indigo carmine per 1 mL, shake well, and the indigo carmine reference substance mother liquor is obtained. The working solution is obtained by diluting the mother liquor by 10 times.

[0057] Preparation of the test solution: accurately weigh about 10 mg of the secondary processed indigo white prepared in Example 2-4 and the primary indigo white sample of Example 1 into a Soxhlet extractor, add 100 mL of chloroform, and reflux extract until the extract is colorless. Dry the extract, dissolve the residue with DMF and dilute to a 10 mL volumetric flask, pass through a 0.45 μm microporous filter membrane, and take the filtrate as the test solution.

[0058] Determination method: accurately pipette 10 μL of the reference solution and the test solution respectively, inject into the liquid chromatograph, and determine. The content of indigo carmine in the sample is calculated by the peak area according to the external standard method.

[0059] Results: see Figure 1 and Table 1, the peak area of the secondary processed indigo white at the peak position of indigo carmine (about 7.73 min) is significantly larger than that of the primary indigo white of Example 1. By calculation, the content of indigo carmine in the secondary processed indigo white is 0.33%, while the content in the primary indigo white of Example 1 is 0.21%, which is increased by about 157%.

[0060] Table 1 Content of indigo carmine in the samples prepared in Examples 1-4

[0061]

[0062] Test Example 2: In vitro anti-tumor activity test

[0063] 1. Experimental principle

[0064] MTS is a new MTT analog, the full name is 3-(4, 5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfopheny)-2H-tetrazolium, which is a yellow dye. The succinate dehydrogenase in the mitochondria of living cells can metabolize and reduce MTS to generate soluble formazan compound, and the content of formazan can be determined by an enzyme marker at 490 nm. Under normal circumstances, the amount of formazan is proportional to the number of living cells, so the number of living cells can be inferred according to the optical density OD value.

[0065] 2. Experimental materials and reagents

[0066] Cell lines: human chronic myelogenous leukemia cell K562 (suspension cells), human colon adenocarcinoma cell SW480 (adherent cells).

[0067] Samples: secondary processed indigo samples prepared in Example 2-3, primary indigo samples prepared in Example 1 were extracted with chloroform / ethyl acetate and then evaporated to dryness.

[0068] Positive control: doxorubicin (Doxorubicin, DOX), paclitaxel (Paclitaxel, Taxol).

[0069] Main reagents: MTS reagent, RPMI-1640 medium, DMEM medium, fetal bovine serum (FBS), dimethyl sulfoxide (DMSO).

[0070] Main equipment: CO2 cell incubator, multifunctional enzyme label instrument (such as MULTISKAN FC), super clean bench, 96-well cell culture plate.

[0071] 3. Experimental steps

[0072] 3.1 Preparation of sample and positive control solution

[0073] Each indigo sample was accurately weighed, dissolved in DMSO to prepare a stock solution of 50 mg / mL, and diluted to the required working concentration (100 μg / mL) using the corresponding complete medium, ensuring that the final concentration of DMSO in each well did not exceed 0.5% (v / v) to exclude solvent toxicity. The positive controls (doxorubicin, paclitaxel) were prepared and diluted to a series of concentration gradients in the same way.

[0074] 3.2 Cell plating and inoculation

[0075] Take K562 and SW480 cells in the logarithmic growth phase, resuspend them in complete medium containing 10% FBS (K562 uses RPMI-1640, SW480 uses DMEM), adjust the cell density to 5×10 4 cells / mL.

[0076] Add 100 μL of cell suspension to each well of the 96-well plate (i.e. inoculate 5×10 3 cells per well). The following groups are set up:

[0077] Experimental group: add cells and different concentrations of samples to be tested (secondary processed indigo / primary indigo).

[0078] Positive control group: add cells and different concentrations of positive control drugs.

[0079] Solvent control group: add cells and equal volume of 0.5% DMSO medium as experimental groups.

[0080] Blank control group: only add 100 μL complete medium (without cells) for zero setting.

[0081] Each group has 3 replicate wells. Put the 96-well plate in a 37°C, 5% CO2 incubator for 24 hours of pre-culture, so that the cells are fully adherent (SW480) or enter the logarithmic growth phase (K562).

[0082] 3.3 Drug treatment

[0083] After the end of pre-culture, add 100 μL of sample diluted with complete medium (100 μg / mL) or positive drug working solution (10 μM) to each well of the experimental and positive control groups, with a final volume of 200 μL per well. The solvent control group uses equal volume of complete medium containing 0.5% DMSO. Put the cells back in the incubator for 48 hours of continuous culture.

[0084] 3.4 MTS color development and detection

[0085] After 48 hours of culture, perform color development reaction:

[0086] For adherent cells SW480: carefully aspirate the old culture medium in each well to avoid damaging the cell layer. Add 120 μL of fresh complete medium and 20 μL of MTS reagent to each well, and mix gently.

[0087] For suspended cells K562: without discarding the liquid, directly aspirate 100 μL of old culture medium from each well (avoid aspirating the cells), then add 20 μL of MTS reagent to each well.

[0088] Blank zero setting well: add 120 μL of complete medium and 20 μL of MTS reagent.

[0089] Put the 96-well plate back in the incubator for 2-4 hours of continuous incubation in the dark until obvious brown-red formazan crystals appear in the solvent control wells under a microscope.

[0090] 3.5 Absorbance determination and data analysis

[0091] After incubation, use a multifunctional enzyme marker to measure the absorbance value (OD 490nm ) of each well at 490 nm wavelength.

[0092] Data processing:

[0093] Calculate the average OD value of each group of replicate wells.

[0094] The cell proliferation inhibition rate (IR) was calculated according to the following formula:

[0095] Inhibition rate (%) = [1 - (OD 实验组 - OD 空白组 ) / (OD 溶剂对照组 - OD 空白组 )] x 100%,

[0096] IC 50 value calculation: set a series of concentration gradients for the twice processed indigo samples, with the logarithm of the drug concentration as the abscissa (X) and the cell inhibition rate (%) as the ordinate (Y) to draw the dose-effect curve. The half-inhibitory concentration (IC 50 value) was calculated by the Logistic fitting or Reed & Muench method.

[0097] 4 Experimental results

[0098] As shown in Table 1, at a concentration of 50 μg / mL, after treating K562 and SW480 cells for 48 hours, the twice processed indigo showed a significantly stronger cell proliferation inhibition ability than the ordinary indigo. Figure 2 For K562 cells: the inhibition rate of the primary indigo was 48%, while the inhibition rate of the twice processed indigo was as high as 78% (p<0.01), with an inhibition effect increased by about 1.6 times, and the IC 50 was 30.70±0.71 μg / ml.

[0099] For SW480 cells: the inhibition rate of the primary indigo was 43%, while the inhibition rate of the twice processed indigo reached 96% (p<0.01), with an inhibition effect increased by about 2.2 times, and the IC 50 was 43.06±3.04 μg / ml.

[0100] The results showed that after being processed by Trichoderma fermentation, the anti-tumor activity of indigo was fundamentally improved.

[0101] In summary, the twice processing process of Trichoderma fermentation can significantly change the chemical composition of indigo (such as significantly increasing the indigo content, see Test Example 1), which in turn leads to the synergistic enhancement of its anti-tumor biological activity. This provides a solid pharmacological basis for developing the twice processed indigo as a highly effective traditional Chinese medicine preparation for treating leukemia and colon adenocarcinoma.

[0102]

[0103] ​It should be pointed out finally that the above embodiments are only used to illustrate the technical solutions of the present application, but not to limit the same; and although the present application has been described in detail with reference to the foregoing embodiments, it should be appreciated by those skilled in the art that the technical solutions recorded in the foregoing embodiments can be modified, or some technical features thereof can be replaced equivalently; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for processing indigo naturalis using Trichoderma fermentation, characterized in that, The method includes the following steps: (1) Preparation of Trichoderma seed culture; (2) The primary indigo is prepared into a suspension and sterilized; the primary indigo is commercially available indigo or indigo obtained from raw materials such as Isatis indigotica or Isatis tinctoria through soaking, indigo extraction and filtration. (3) The seed liquid prepared in step (1) is inoculated into the sterilized indigo suspension at an inoculation amount of 5% to 15% by volume. The mixture is then fermented in the dark at 25 to 30°C and 120 to 180 rpm for 5 to 9 days. After fermentation, the mixture is centrifuged, the precipitate is collected, and the mixture is dried to obtain the secondary processed indigo.

2. The method according to claim 1, characterized in that, In step (1), the Trichoderma fungus is selected from Trichoderma longifolia, Trichoderma viride or Trichoderma harzianum; the preparation method of the fungal seed liquid includes: activating the Trichoderma strain, inoculating it into a liquid culture medium, and shaking it at 25-30℃ and 120-180 rpm for 2-4 days.

3. The method according to claim 1, characterized in that, The concentration of the suspension in step (2) is 0.2 to 3.33 mg / ml; the sterilization process is high-pressure steam sterilization at 121°C for 15 to 20 min.

4. The method according to claim 1, characterized in that, The fermentation culture time in step (3) is 7 days.

5. A secondary processed indigo naturalis prepared by the method described in any one of claims 1-4.

6. The secondary processing of indigo according to claim 5, characterized in that, Its active ingredient, indirubin, contains ≥0.33%.

7. The use of the secondary processed indigo naturalis as described in claim 5 or 6 in the preparation of antitumor drugs.

8. The application according to claim 7, characterized in that, The tumor is either leukemia or colon adenocarcinoma.

9. A pharmaceutical composition comprising the secondary processed indigo naturalis as described in claim 5 or 6, and one or more pharmaceutically acceptable carriers or excipients.

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