Method for processing indigo naturalis through trichoderma fermentation, obtained product and application

Abstract

The invention discloses a method for processing indigo naturalis through trichoderma fermentation, an obtained product and application. The method comprises the following steps: preparing a trichoderma seed solution; and sterilizing primary indigo naturalis, inoculating the primary indigo naturalis into the seed solution, and fermenting for 5-9 days in a dark place at 25-30 DEG C and 120-180 rpm to obtain secondarily processed indigo naturalis. Trichoderma is used for biotransformation of indigo naturalis for the first time, the content of indirubin serving as an anti-tumor key component of indigo naturalis can be specifically and remarkably increased, and the amplification reaches 150% or above. In-vitro anti-tumor experiments show that the inhibition activity of the secondarily processed indigo naturalis prepared by the invention on human leukemia K562 cells and human colon adenocarcinoma SW480 cells is obviously superior to that of common indigo naturalis. The product disclosed by the invention can be used for preparing medicines for preventing and / or treating leukemia and colon cancer, is controllable in process and suitable for industrial production, and provides a new way for developing a new efficient indigo naturalis anticancer medicine.

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 cancer. Background Technology

[0002] Indigofera tinctoria is a plant of the Acanthaceae family, specifically the stork (Indigofera tinctoria). Baphicacanthus cusia (Nees) Bremek. ), Polygonaceae plant Polygonum indica ( Polygonum tinctorium Ait. ) or Isatis indigotica (a cruciferous plant) 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 the 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 cancer 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 object, the present application adopts the following technical solutions: In the first aspect, the present application provides a method for fermenting and processing indigo with Trichoderma, comprising the following steps: (1) preparing a Trichoderma seed solution; (2) preparing primary indigo into a suspension and sterilizing the suspension; (3) inoculating the seed solution prepared in step (1) into the sterilized indigo suspension at an inoculation amount (v / v) of 5-15%, and carrying out fermentation culture in the dark at 25-30℃ and 120-180 rpm for 5-9 days; after the fermentation, centrifuging, collecting the precipitate and drying to obtain the secondary processed indigo.

[0009] Preferably, the Trichoderma in step (1) is Trichoderma longibrachiatum, Trichoderma longibrachiatum Trichoderma harzianum, Trichodermaharzianum or Trichoderma atroviride. Trichoderma atroviride The method for preparing the seed solution comprises inoculating Trichoderma spores on PDA flat plate culture medium for activation, and culturing at 28-37℃ for 48-72 hours; then picking the activated colonies with an inoculation loop and inoculating into PDB liquid culture medium, and carrying out vibration culture in the dark at 25-30℃ and 120-180 rpm for 2-4 days.

[0010] Preferably, the primary indigo in step (2) is commercially available indigo or indigo prepared from Baphlanum root by the process of soaking, adding lime milk for indigo preparation, and filtering.

[0011] Preferably, the concentration of the suspension is 0.2-3.33 mg / ml.

[0012] Preferably, the sterilization treatment in step (2) is high-pressure steam sterilization at 121℃ for 15-20 min.

[0013] Preferably, the bacterial concentration of the seed solution in step (3) is OD 600 between 0.5 and 0.7.

[0014] Preferably, the fermentation culture time in step (3) is 7 days.

[0015] In the second aspect, the present application provides secondary processed indigo prepared by any of the above methods.

[0016] Preferably, the content of active ingredient indirubin in the secondary processed indigo is ≥0.33%.

[0017] In the third aspect, the present application provides the use of the above secondary processed indigo in the preparation of anti-tumor drugs.

[0018] Preferably, the tumor is leukemia or human colon adenocarcinoma.

[0019] In a fourth aspect, the present application provides a pharmaceutical composition comprising the above-mentioned twice-processed indigo naturalis and one or more pharmaceutically acceptable carriers or excipients.

[0020] Preferably, the pharmaceutically acceptable carriers or excipients include any one of sustained-release agents, excipients, fillers, binders, humectants, disintegrants, absorption promoters, surfactants, lubricants, or a combination of at least two of them, 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 here one by one.

[0021] Preferably, the dosage form of the pharmaceutical composition can be a conventional dosage form such as tablets, capsules, granules, powders, or oral solutions.

[0022] Compared with the prior art, the present application has the following beneficial effects: Specific synergistic effect: the present application first uses Trichoderma to ferment and process indigo naturalis, and the strain and its metabolic enzyme system can specifically and directionally transform some components in indigo naturalis, 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.

[0023] Significant increase in activity: the indigo naturalis 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 activity than ordinary indigo naturalis, with a significantly reduced IC 50 value, indicating a significant improvement in drug efficacy.

[0024] 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 anticancer drugs.

[0025] High safety: the fermentation process is carried out in a closed condition and through pre-sterilization treatment, effectively controlling the contamination of miscellaneous bacteria, and the microbial limit of the final product meets the requirements, ensuring the safety of drug use. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 Comparison of HPLC chromatograms of twice-processed indigo naturalis and once-processed indigo naturalis of the present application; Figure 2 Comparison of the inhibitory effects of twice-processed indigo naturalis and once-processed indigo naturalis on K562 and SW480 cell proliferation of the present application, where twice-processed product 1 is the sample prepared in Example 2, twice-processed product 2 is the sample prepared in Example 3, and once-processed product is the sample prepared in Example 1. DETAILED DESCRIPTION

[0027] The present application will be further described in the following with reference to the accompanying drawings and examples, but in no way limits the present application, any transformation or improvement based on the teaching of the present application falls into the protection scope of the present application.

[0028] The process, conditions, reagents, experimental methods, etc. for implementing the present application, except for the following specifically mentioned contents, are the general knowledge and common sense in the art, and the present application has no special limitation. The experimental methods not specified in the specific conditions in each example are usually carried out according to the conventional conditions or according to the conditions recommended by the manufacturer.

[0029] Unless otherwise specified, the meanings of all professional terms and scientific terms used in the present specification are the same as those generally understood by the person skilled in the art to which the present application belongs. However, if there is a conflict, the present specification containing the definition shall prevail.

[0030] The following are the sources of some main raw materials in the examples: Trichoderma longibrachiatum Trichoderma longibrachiatum Purchased from China General Microbiological Culture Collection Center, with the preservation number of CGMCC No. 3.8877. Trichoderma atroviride Trichoderma atroviride Purchased from China General Microbiological Culture Collection Center, with the preservation number of CGMCC No. 3.12963. Trichoderma harzianum Trichoderma harzianum Purchased from China General Microbiological Culture Collection Center, with the preservation number of CGMCC No. 3.12069.

[0031] The south radix isophorae is the fresh branch of radix isophorae planted in Yuanyang, Yunnan.

[0032] Example 1 Preparation of primary indigo 80g of dry mixture of south radix isophorae branches and leaves was weighed and placed in a 2000mL beaker, 1000mL of tap water was added, and it was soaked at room temperature, and stirred every 5 hours. After 3 days, it was filtered with gauze, the branches were discarded, and the filtrate was collected. 20mL of lime milk solution containing 5g of lime was added to the filtrate, and stirred uniformly. Then it was stirred at high speed with 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℃, to obtain primary indigo.

[0033] Example 2 Secondary processing of indigo (1) Strain activation: In the clean bench, the frozen bacteria powder of Trichoderma longibrachiatum was picked up with an inoculation loop, and streaked on PDA flat plate culture medium. The flat plate was inverted in a 28℃ constant temperature incubator for 72 hours.

[0034] (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 at 28°C, 150 rpm, and in the dark for 3 days to obtain the Trichoderma longibrachiatum seed liquid (OD 600 between 0.6).

[0035] (3) 1 g of the primary indigo blue prepared in Example 1 was placed in a 1000 mL triangular flask, 300 mL of purified water was added to configure a suspension. The triangular flask was wrapped and sterilized in a 121°C high-pressure steam sterilization pot for 15 minutes, and then taken out and cooled to room temperature. In a clean bench, 10 mL of the 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 at 28°C, 150 rpm, and in the dark for 7 days. 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 the indigo was beaten to precipitate. After vacuum drying at 55°C, grinding and passing through a 100-mesh sieve, the secondary processed indigo blue of the application was obtained.

[0036] Example 3 Secondary processing of indigo blue (1) Strain activation: the frozen bacteria powder of Trichoderma atroviride was picked up with an inoculation loop in a clean bench and streak inoculated on PDA plate medium. The plate was inverted and cultured in a 28°C constant temperature incubator for 72 hours.

[0037] (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 at 25°C, 120 rpm, and in the dark for 4 days to obtain the Trichoderma longibrachiatum seed liquid (OD 600 between 0.7).

[0038] (3) 50 mg of the primary indigo blue prepared in Example 1 was placed in a 500 mL triangular flask, 100 mL of purified water was added to configure a suspension. The triangular flask was wrapped and sterilized in a 121°C high-pressure steam sterilization pot for 20 minutes, and then taken out and cooled to room temperature. In a clean bench, 5 mL of the Trichoderma longibrachiatum seed liquid prepared in step (2) was inoculated at a 5% inoculation amount (v / v). The triangular flask was placed in a shaker and cultured at 25°C, 120 rpm, and in the dark for 9 days. After the fermentation was completed, the fermentation liquid was centrifuged (8000 rpm, 10 min), the precipitate was collected, vacuum dried at 55°C, ground, and passed through a 100-mesh sieve, to obtain the secondary processed indigo blue of the application.

[0039] Example 4 Secondary processing of indigo blue (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.

[0040] (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).

[0041] (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.

[0042] Experimental Example 1: Determination of Indigo Content (HPLC Method) Instrument: Analytical high performance liquid chromatography (1260 Infinity II) Chromatographic conditions: Agilent ZORBAX SB-C18 column (4.6*150 mm), mobile phase: methanol:water (7:3), isocratic elution, flow rate 1 mL / min, detection wavelength: 292 nm.

[0043] Preparation of reference solution: Accurately weigh an appropriate amount of indirubin reference standard (purchased from the National Institutes for Food and Drug Control, purity ≥98%), place it in a brown volumetric flask, add N,N-dimethylformamide (DMF) to dissolve and quantitatively dilute to prepare a solution containing 100 μg of indirubin per 1 mL, shake well, and the indirubin reference standard stock solution is obtained. The working solution is obtained by diluting the stock solution 10 times.

[0044] Preparation of the test solution: Accurately weigh approximately 10 mg each of the secondary processed indigo naturalis obtained in Examples 2-4 and the primary indigo naturalis sample from Example 1, place them in a Soxhlet extractor, add 100 mL of chloroform, and reflux until the extract is colorless. Evaporate the extract to dryness, dissolve the residue in DMF and dilute to a 10 mL volumetric flask, filter through a 0.45 μm microporous membrane, and use the filtrate as the test solution.

[0045] Determination method: Accurately pipette 10 μL each of the reference solution and the test solution into the liquid chromatograph and determine the content. Calculate the indirubin content in the sample based on peak area using the external standard method.

[0046] Results: See Figure 1 As shown in Table 1, the peak area of ​​the secondary processed indigo in this invention at the indigo red peak position (approximately 7.73 min) is significantly larger than that of the primary indigo in Example 1. Calculations show that the indigo red content in the secondary processed indigo is 0.33%, while the content in the primary indigo in Example 1 is 0.21%, representing an increase of approximately 157%.

[0047] Table 1. Indigo content in samples prepared in Examples 1-4

[0048] Experimental Example 2: In vitro antitumor activity assay 1. Experimental Principle MTS is a novel MTT analog, its full name being 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfopheny)-2H-tetrazolium, a yellow dye. Succinate dehydrogenase in the mitochondria of living cells can metabolize and reduce MTS, generating a soluble formazan compound. The formazan content can be measured using a microplate reader at 490 nm. Under normal circumstances, the amount of formazan produced is directly proportional to the number of living cells; therefore, the number of living cells can be inferred from the optical density (OD) value.

[0049] 2. Experimental Materials and Reagents Cell lines: human chronic myeloid leukemia cell line K562 (suspension cell) and human colon cancer cell line SW480 (adherent cell).

[0050] Samples: The secondary processed indigo samples prepared in Examples 2-3 and the primary indigo samples prepared in Example 1 were extracted with chloroform / ethyl acetate and then evaporated to dryness before use.

[0051] Positive controls: Doxorubicin (DOX) and Paclitaxel (Taxol).

[0052] Main reagents: MTS reagent, RPMI-1640 medium, DMEM medium, fetal bovine serum (FBS), dimethyl sulfoxide (DMSO).

[0053] Main equipment: CO2 cell culture incubator, multi-functional microplate reader (such as MULTISKAN FC), clean bench, 96-well cell culture plate.

[0054] 3. Experimental Procedure 3.1 Preparation of Sample and Positive Control Solutions Accurately weigh each indigo sample and dissolve it in DMSO to prepare a stock solution of 50 mg / mL. Before use, dilute with the corresponding complete culture medium to the required working concentration (100 μg / mL), ensuring that the final concentration of DMSO in each well does not exceed 0.5% (v / v) to eliminate solvent toxicity. Positive controls (doxorubicin, paclitaxel) are prepared and diluted to a series of concentration gradients using the same method.

[0055] 3.2 Cell Plating and Inoculation K562 and SW480 cells in the logarithmic growth phase were taken and resuspended in complete medium containing 10% FBS (RPMI-1640 for K562 and DMEM for SW480), and the cell density was adjusted to 5 × 10⁻⁶ cells / year. 4 cells / mL.

[0056] Add 100 μL of cell suspension to each well of a 96-well plate (i.e., seed 5 × 10⁶ cells per well). 3 (cells). Groups were set as follows: Experimental group: Added cells and different concentrations of test samples (second-processed indigo / primary indigo).

[0057] Positive control group: cells and different concentrations of positive control drugs were added.

[0058] Solvent control group: Added cells and the same volume of 0.5% DMSO medium as the experimental group.

[0059] Blank control group: only 100 μL of complete culture medium (cell-free) was added for zeroing.

[0060] Each group had 3 replicates. The 96-well plates were pre-cultured in a 37°C, 5% CO2 incubator for 24 hours to allow the cells to adhere completely (SW480) or enter the logarithmic growth phase (K562).

[0061] 3.3 Drug administration After pre-culture, 100 μL of sample diluted with complete culture medium (100 μg / mL) or positive control working solution (10 μM) was added to each well of the experimental group and the positive control group, with a final volume of 200 μL per well. The solvent control group was replaced with an equal volume of complete culture medium containing 0.5% DMSO. The cells were then returned to the incubator and cultured for another 48 hours.

[0062] 3.4 MTS color development and detection After 48 hours of incubation, a colorimetric reaction was performed: For adherent SW480 cells: Carefully aspirate the old culture medium from each well to avoid damaging the cell layer. Add 120 μL of fresh complete culture medium and 20 μL of MTS reagent to each well and mix gently.

[0063] For K562 suspension cells: No need to discard the medium. Directly aspirate 100 μL of old culture medium from each well (avoid aspirating the cells), and then add 20 μL of MTS reagent to each well.

[0064] Blank zeroing well: Add 120 μL of complete culture medium and 20 μL of MTS reagent.

[0065] Return the 96-well plate to the incubator and continue incubation in the dark for 2–4 hours until obvious brownish-red formazan crystals are observed in the wells of the solvent control group under a microscope.

[0066] 3.5 Absorbance Measurement and Data Analysis After incubation, the absorbance (OD) of each well was measured at 490 nm using a multi-functional microplate reader. 490nm ).

[0067] Data processing: Calculate the average OD value of each group of replicates.

[0068] The cell proliferation inhibition rate (IR) is calculated using the following formula: Inhibition rate (%) = [1 - (OD) 实验组 - OD 空白组 ) / (OD 溶剂对照组 - OD 空白组 )] × 100%, IC 50 Value calculation: A series of concentration gradients were set for the second-processed indigo naturalis samples. A dose-response curve was plotted with the logarithm of drug concentration as the abscissa (X) and the cell inhibition rate (%) as the ordinate (Y). The half-maximal inhibitory concentration (IC50) was calculated using logistic regression or the Reed & Muench method. 50 value).

[0069] 4. Experimental Results like Figure 2 As shown, after treating K562 and SW480 cells with a concentration of 50 μg / mL for 48 hours, the second-processed indigo naturalis showed a significantly stronger cell proliferation inhibition ability than the ordinary indigo naturalis.

[0070] In K562 cells: the inhibition rate of primary indigo naturalis was 48%, while that of secondary processed indigo naturalis was as high as 78% (p<0.01), representing an approximately 1.6-fold increase in inhibitory effect. Its IC50 value was [not specified in the original text]. 50It was 30.70±0.71μg / ml.

[0071] In SW480 cells: the inhibition rate of primary indigo naturalis was 43%, while that of secondary processed indigo naturalis reached 96% (p<0.01), representing an approximately 2.2-fold increase in inhibitory effect. Its IC50 value was [not specified in the original text]. 50 The value was 43.06 ± 3.04 μg / ml.

[0072] The results showed that the antitumor activity of indigo was fundamentally enhanced after fermentation with Trichoderma.

[0073] In summary, the secondary processing of Indigofera fermentation significantly alters the chemical composition of Indigofera tinctoria (e.g., substantially increasing the indirubin content, see Experimental Example 1), thereby synergistically enhancing its antitumor bioactivity. This provides a solid pharmacodynamic basis for developing secondary-processed Indigofera tinctoria into a highly effective traditional Chinese medicine preparation for treating leukemia and colon cancer.

[0074] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

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) Prepare the primary indigo naturalis into a suspension and sterilize it; (3) The seed liquid prepared in step (1) is inoculated into the sterilized indigo suspension at an inoculation rate of 5% to 15%, 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.

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 longifolia 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 primary indigo described in step (2) is commercially available indigo or indigo obtained from raw materials such as Isatis indigotica or Isatis tinctoria through soaking, indigo extraction, and filtration.

4. 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.

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

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

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

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

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

10. A pharmaceutical composition comprising the secondary processed indigo naturalis as described in claim 7 or 8, and one or more pharmaceutically acceptable carriers or excipients.

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