Application of rhizoma coptidis aqueous extract in preparation of biopesticide for inhibiting melanospora

Abstract

The invention discloses application of a rhizoma coptidis aqueous extract in preparation of biopesticide for inhibiting melanospora, and belongs to the field of biopesticide application of rhizoma coptidis aqueous extracts and monomers thereof. The in-vitro inhibition effect of the rhizoma coptidis aqueous extract and the monomer thereof on the melanospora nigra is researched by adopting an in-vitro model of the melanospora nigra. An in-vitro growth rate method finds that the rhizoma coptidis aqueous extract and a monomer thereof have a remarkable inhibition effect on melanospora sp. Experimental results show that the rhizoma coptidis aqueous extract or the rhizoma coptidis monomeric compound has an exact effect of inhibiting the melanospora nigra and can be applied to preparation of biopesticides for inhibiting the melanospora nigra.

Description

Technical Field

[0001] The invention belongs to the field of biology and new medicine technology, and specifically relates to use of a coptis chinensis water extract and monomers thereof in preparing a biological pesticide for inhibiting black spore mold. Background Art

[0002] Black spore fungus is a fungus that affects crop production worldwide, for example, it causes leaf spot disease in rice, leaf spot disease in watermelon, leaf spot disease and blight in blueberries, etc. This can lead to reduced yields and reduced food quality, resulting in significant economic losses.

[0003] Coptis chinensis is a plant of the genus Coptis chinensis in the family Ranunculaceae. From a pharmacological point of view, it is mainly used to treat diabetes, gastrointestinal diseases, skin damage and other diseases due to its hypoglycemic, antioxidant, anti-inflammatory and anti-tumor effects. There are also reports on its antibacterial effect. Among the many chemical components of Coptis chinensis, the components that have antibacterial effects are mainly alkaloid compounds, while the antibacterial properties of non-alkaloid and polysaccharide components of Coptis chinensis are relatively weak. At present, the research on the antibacterial effect of Coptis chinensis is mainly focused on bacteria, and the mechanism is relatively clear; there is less research on fungi. So far, there is no report on the inhibition of Nigrosporium by Coptis chinensis water extract and its monomers.

[0004] Patent CN201010550374 discloses a preservative composition containing Chinese herbal medicine extracts and its use, wherein the preservative composition contains 0.2-6.0% of Chinese herbal medicine active components, 92-99.6% of solvents and 0-2.1% of synergists, wherein the Chinese herbal medicine active components are specifically berberine hydrochloride, epiberberine, jatrorrhizine, palmatine hydrochloride, levosyringinol glucoside, stigmasterol, 7-methoxy-3-methyl-2,5-dihydroxy-9,10-dihydrophenanthrene, methylcyclodecane, pursenoside, glycyrrhizic acid, pachymic acid, scutellariae acid, palmitic acid, and a single compound or a mixture of multiple compounds; and points out the use of the preservative composition in the preparation of preparations for inhibiting Staphylococcus aureus, molds, yeasts, saprophytes, and Escherichia coli. However, the goal of the patent is preservation, which belongs to the field of preservation technology. Although it also mentions that it has an inhibitory effect on mold, it does not clearly indicate which type of mold is used. Mold is a general term for some filamentous fungi. It is not a taxonomic term. Fungi with mold in their names belong to different categories, and the differences between each category are very large. From the perspective of its use, the mold used in this patent is a type of mold that can cause food to spoil and rot, and the molds that can cause food corruption are generally Aspergillus flavus, Penicillium, etc. This type of mold and the plant pathogenic fungus Black Sporangium, whose main pathogen is crops, obviously do not belong to the same category. In the embodiments of this patent, a large number of compounds are used as a mixture to be added to the product. It does not clearly indicate which specific ingredients are effective, and in the preservative composition mentioned in the patent, the proportion of Chinese herbal extract ingredients is not high. Summary of the invention

[0005] The purpose of the present invention is to provide a new use of a water extract of Coptis chinensis and its monomer compounds in the preparation of a biological pesticide for inhibiting black spore mold on the basis of the prior art. The water extract of Coptis chinensis and its monomer compounds can effectively inhibit the growth of black spore mold.

[0006] The purpose of the present invention can be achieved by the following measures:

[0007] Use of water extract of Coptis chinensis in preparing biological pesticide for inhibiting black spore mold.

[0008] The coptis chinensis water extract of the present invention can be a water extract obtained by heating and extracting the medicinal material coptis chinensis in water, or can be a water extract obtained by further freeze-drying the water extract into a powder, or can be an extract obtained by further concentrating the water extract into an extract.

[0009] In the heating extraction process of the water extract of Coptis chinensis, it is preferably heated to reflux. Coptis chinensis can be extracted with water once or multiple times, and each extraction can be 0.2-5h.

[0010] A specific method for preparing a water extract of Coptis chinensis includes: taking Coptis chinensis slices, extracting once or multiple times by reflux extraction, adding distilled water, each time for 0.2-5 hours, combining the filtrates after extraction, concentrating the combined filtrates, or freeze-drying the combined filtrates to obtain powder.

[0011] The coptis chinensis water extract of the present invention includes at least three of jatrorrhizine, epiberberine, coptis chinensis, palmatine and berberine; preferably, the coptis chinensis water extract includes at least four of jatrorrhizine, epiberberine, coptis chinensis, palmatine and berberine; more preferably, the coptis chinensis water extract includes jatrorrhizine, epiberberine, coptis chinensis, palmatine and berberine. We found that when any three of the above five substances are combined together, they can have a synergistic inhibitory effect on black spore mold, when any four are combined, the synergistic inhibitory effect is stronger, and when the five substances are combined, the best synergistic inhibitory effect can be achieved.

[0012] The content of jatrorrhizine, epiberberine, coptisine, bamipine and berberine in the water extract of Coptis chinensis may vary slightly according to the actual extraction conditions or different medicinal materials. In a preferred embodiment, the content of jatrorrhizine, epiberberine, coptisine, bamipine or berberine in the water extract of Coptis chinensis is greater than 0.03 mg / mg; further, the content of jatrorrhizine is greater than 0.03 mg / mg, the content of epiberberine is greater than 0.1 mg / mg, the content of coptisine is greater than 0.13 mg / mg, the content of bamipine is greater than 0.11 mg / mg, and the content of berberine is greater than 0.4 mg / mg.

[0013] The coptis chinensis water extract of the present invention can be prepared into a water extract when used. Preferably, the concentration of the coptis chinensis water extract in the water extract is greater than 0.5 mg / mL, preferably greater than 0.9 mg / mL.

[0014] The coptis chinensis water extract of the present invention can be made into biological pesticides in various dosage forms, including but not limited to powders, wettable powders, soluble powders, emulsifiable concentrates, aqueous solutions, suspensions, microemulsions, granules or water-dispersible granules.

[0015] The invention also includes the use of the water extract of Coptis chinensis in preparing biological pesticides for resisting plant diseases such as rice leaf spot, watermelon leaf spot, blueberry leaf spot or branch blight.

[0016] Through the study of the aqueous extract of Coptis chinensis, we found the use of a pharmaceutical composition in the preparation of a biological pesticide for inhibiting black spore mold. The pharmaceutical composition includes at least three of jatrorrhizine or jatrorrhizine hydrochloride, epiberberine, coptis chinensis or coptis chinensis hydrochloride, palmatine or palmatine hydrochloride, berberine or berberine hydrochloride; or the pharmaceutical composition includes at least four of jatrorrhizine or jatrorrhizine hydrochloride, epiberberine, coptis chinensis or coptis chinensis hydrochloride, palmatine or palmatine hydrochloride, berberine or berberine hydrochloride; or the pharmaceutical composition includes jatrorrhizine or jatrorrhizine hydrochloride, epiberberine, coptis chinensis or coptis chinensis hydrochloride, palmatine or palmatine hydrochloride and berberine or berberine hydrochloride.

[0017] In the above pharmaceutical composition, the mass proportions of each component can be 0.1-0.5 parts of jatrorrhizine or jatrorrhizine hydrochloride, 0.9-1.5 parts of epiberberine, 1.2-2.0 parts of coptisine or coptisine hydrochloride, 1.0-1.6 parts of palmatine or palmatine hydrochloride, and 4.0-5.5 parts of berberine or berberine hydrochloride.

[0018] In a preferred embodiment, the mass proportions of each component can be 0.2-0.5 parts of jatrorrhizine or jatrorrhizine hydrochloride, 1.0-1.4 parts of epiberberine, 1.3-1.9 parts of coptisine or coptisine hydrochloride, 1.2-1.5 parts of palmatine or palmatine hydrochloride, and 4.5-5.0 parts of berberine or berberine hydrochloride.

[0019] In a more preferred embodiment, the mass proportions of each component can be 0.3-0.4 parts of jatrorrhizine or jatrorrhizine hydrochloride, 1.1-1.3 parts of epiberberine, 1.4-1.7 parts of coptisine or coptisine hydrochloride, 1.2-1.4 parts of palmatine or palmatine hydrochloride, and 4.5-4.9 parts of berberine or berberine hydrochloride.

[0020] In a most preferred embodiment, the composition of the aqueous extract of Coptis chinensis can be simulated, wherein the mass proportions of each component can be 0.38 parts of jatrorrhizine or jatrorrhizine hydrochloride, 1.19 parts of epiberberine, 1.6 parts of coptisine or coptisine hydrochloride, 1.29 parts of palmatine or palmatine hydrochloride, and 4.73 parts of berberine or berberine hydrochloride.

[0021] The present invention also provides a method for analyzing monomer compounds in the water extract of Coptis chinensis, which comprises: detecting the water extract of Coptis chinensis by HPLC (Waters e2695), C 18 The chromatographic column was (DEVOTE 4.6mm*250mm, 5μm), the mobile phase was 0.03mol / L ammonium bicarbonate solution (containing 0.7% ammonia water, 0.1% triethylamine) (A)-acetonitrile (B), and elution was performed according to the gradient (0-10min, 10%-25% B; 10-25min, 25%-30% B; 25-35min, 30%-40% B; 35-70min, 40%-40% B), the flow rate was set to 1.0mL / min, the column temperature was 30°C, the detection was performed at a wavelength of 345nm, and the injection volume was 10μL.

[0022] The invention establishes an in vitro growth model of black spore mold to study the in vitro inhibitory effect of coptis chinensis water extract and its monomer compounds on black spore mold.

[0023] The present invention focuses on the active ingredients in the water extract of Coptis chinensis, analyzes the components of the water extract, clarifies the influence of a single compound on the growth of black spore mold, and investigates the synergistic inhibitory effect of different combinations of monomer compounds in the water extract of Coptis chinensis on black spore mold, thereby clarifying the material basis of the water extract of Coptis chinensis to inhibit black spore mold and its optimal combination of monomer compounds.

[0024] The present invention studies the in vitro inhibitory effect of coptis chinensis water extract, berberine, epiberberine, palmatine, jatrorrhizine and coptis chinensis on black spore mold by growth rate method. The results show that coptis chinensis water extract has a good inhibitory effect on black spore mold at different concentrations. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 HPLC chromatogram of Coptis chinensis extract; A in the figure is a mixed reference substance, B is Coptis chinensis extract, 1. Jatrorrhizine hydrochloride, 2. Epiberberine, 3. Coptis chinensis hydrochloride, 4. Palmatine hydrochloride, 5. Berberine hydrochloride;

[0026] Figure 2 The inhibitory effect of the aqueous extract of Coptis chinensis on the growth of Nigrosporium (n=3, ), P<0.05(*);

[0027] Figure 3The inhibitory effect of the monomer compound on black spore mold (n = 3, ), P<0.05(*);

[0028] Figure 4 The inhibitory effect of each component on black spore mold (n = 3, ), P<0.05(*); In the figure, 1 jatrorrhizine, concentration 0.1 mg / mL; 2 epiberberine, concentration 0.3 mg / mL; 3 coptisine, concentration 0.4 mg / mL; 4 palmatine, concentration 0.3 mg / mL; 5 berberine, concentration 1.2 mg / mL (this concentration is calculated based on the water extract concentration of 2.5 mg / mL). DETAILED DESCRIPTION

[0029] The present invention is further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, it should be understood that the embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements fall within the scope of protection of the present invention.

[0030] Experimental preparation

[0031] 1. Materials

[0032] 1.1 Experimental strains

[0033] Black spore mold was provided by the Collaborative Innovation Center for Industrialization of Traditional Chinese Medicine Resources of Nanjing University of Chinese Medicine.

[0034] 1.2 Drugs and materials

[0035] 1.2.1 Drugs

[0036] Jatrorrhizine hydrochloride standard (ChemFaces, CAS No. 6681-15-8);

[0037] Coptisine hydrochloride standard (ChemFaces, CAS No. 6020-18-4);

[0038] Berberine hydrochloride standard (ChemFaces, CAS No. 633-65-8);

[0039] Bamipine hydrochloride standard (ChemFaces, CAS No. 10605-02-4);

[0040] Epiberberine standard (ChemFaces, CAS No. 6873-09-2);

[0041] Berberine standard (ChemFaces, CAS No. 2086-83-1);

[0042] Coptisine standard (ChemFaces, CAS No. 3486-66-6);

[0043] Jatrorrhizine standard (ChemFaces, CAS No. 3621-38-3);

[0044] Bamipine standard (ChemFaces, CAS No. 3486-67-7);

[0045] Coptis chinensis slices were purchased from Bozhou Zirui Pharmaceutical Co., Ltd.

[0046] 1.2.2 Culture medium and reagents

[0047] Glucose, agar powder, ammonium bicarbonate, ammonia water, triethylamine, and acetonitrile were purchased from Macklin; potato extract powder was purchased from Yuanye.

[0048] Example 1. In vitro inhibitory effect of the aqueous extract of Coptis chinensis and its monomeric compounds on Nigrosporium

[0049] 1. Experimental methods

[0050] 1.1 Preparation of Coptis chinensis aqueous extract

[0051] Weigh 20g of Coptis chinensis slices, use reflux extraction, add 160mL of distilled water, extract twice, each time for 2h, combine the filtrate, and place in a freeze dryer. After freeze-drying into powder, prepare water extracts of different concentrations in turn, place in a 4℃ refrigerator for later use.

[0052] 1.2 Analysis of monomer compounds in the aqueous extract of Coptis chinensis

[0053] The samples of Rhizoma Coptidis were tested by HPLC (Waters e2695). 18 The chromatographic column was (DEVOTE 4.6mm*250mm, 5μm), the mobile phase was 0.03mol / L ammonium bicarbonate solution (containing 0.7% ammonia water, 0.1% triethylamine) (A)-acetonitrile (B), and elution was performed according to the gradient (0-10min, 10%-25% B; 10-25min, 25%-30% B; 25-35min, 30%-40% B; 35-70min, 40%-40% B), the flow rate was set to 1.0mL / min, the column temperature was 30°C, the detection was performed at a wavelength of 345nm, and the injection volume was 10μL.

[0054] Preparation of reference solution: Accurately weigh appropriate amounts of berberine hydrochloride, coptisine hydrochloride, epiberberine, palmatine hydrochloride and jatrorrhizine hydrochloride in a centrifuge tube, dissolve in methanol to prepare a 0.11 mg / mL solution.

[0055] For the preparation of the test solution, accurately weigh 0.1 mg of lyophilized powder of Coptis chinensis extract, put it into a 2 mL centrifuge tube, add 1.5 mL of methanol, dissolve it, and ultrasonicate it for 10 min to obtain the solution.

[0056] 1.3 Establishment of an in vitro model of Black Sporangium

[0057] Black spore mold was cultured and maintained on potato dextrose agar (PDA) in the dark at 30°C.

[0058] 1.4 In vitro inhibitory effects of Coptis chinensis aqueous extract and its monomer compounds on Nigrosporium

[0059] The water extract of Coptis chinensis and its monomer compounds were prepared into solutions of different concentrations and filtered for sterilization. 0.1 mL of the solution was added to the surface of potato dextrose agar (PDA) to prepare a drug-added culture medium. A sterile punch with a diameter of 5 mm was used to evenly cut a 5 mm diameter bacterial block from the cultured test strain, and the blank control group (CK) was directly placed with the bacterial block for culture.

[0060] 1.5 Data processing

[0061] All data are presented with mean and standard deviation Inhibition rates were calculated using one-way ANOVA. Significance was set at P < 0.05. All analyses were performed using Excel 2021.

[0062] 2. Experimental results

[0063] 2.1 Analysis of monomer compounds in the aqueous extract of Coptis chinensis

[0064] The liquid phase analysis revealed that there were five alkaloid components in the water extract of Coptis chinensis. Figure 1 The contents of five components in the water extract were determined, and the results are shown in Table 1.

[0065] Table 1 Contents of five alkaloids in Coptis chinensis extract (mg / mg)

[0066]

[0067] 2.2 In vitro inhibitory effect of Coptis chinensis aqueous extract on Nigrosporium

[0068] The results of the inhibitory effect of the water extract of Coptis chinensis on black spore mold are shown in Figure 2 .Depend on Figure 2It can be seen that the concentrations are set at 125.00, 62.50, 31.25, 15.63, 7.81, 3.91, and 0.98 mg / mL. The coptis extract has a good inhibitory effect at high concentrations or even higher concentrations (7.81 mg / mL to 125 mg / mL) (inhibition rate is 24.73% to 92.07%), and also has an inhibitory effect at lower concentrations (concentration is 0.98 mg / mL, inhibition rate is 13.20%). The higher the concentration of the coptis extract, the more significant the inhibitory effect.

[0069] 2.3 In vitro inhibitory effect of Coptidis chinensis monomer compounds on Nigrosporium

[0070] The inhibitory effect of the monomeric compounds of Coptis chinensis on black spore mold is shown in Figure 3 .Depend on Figure 3 It can be seen that the concentration is set at 0.1mg / mL, 0.05mg / mL, and 0.01mg / mL. At the same concentration, the best effect is jatrorrhizine (inhibition rates are 13.51%, 13.09%, and 9.55%, respectively), followed by coptisine (inhibition rates are 10.84%, 10.60%, and 7.95%, respectively), and the worst effect is bamipine (inhibition rates are 5.83%, 6.07%, and 2.79%, respectively). Berberine and epiberberine have similar effects. At the set low concentration, a single compound has a certain inhibitory effect on black spore mold.

[0071] 2.4 Experiments on the synergistic effects between monomeric compounds of Coptis chinensis

[0072] The best-effect jatrorrhizine was used at a concentration of 0.1 mg / mL. This concentration was returned to the water extract of Coptis chinensis. The concentrations of the five components at this time were calculated and the next step of the experiment was carried out. The results are as follows Figure 4. For individual compounds: component 5 has the best effect (inhibition rate of 37.20%), followed by component 3 (inhibition rate of 18.38%), and finally component 1 (inhibition rate of 12.33%). The inhibition rates of the remaining two components are all below 10%, which may be due to the different contents of each component in the water extract. For the two combinations of compounds, some of the inhibitory effects are not much different from those of the single compounds. The best effect is component 4+5 (inhibition rate of 35.93%), followed by component 3+5 (inhibition rate of 34.41%). In the two-by-two combinations, it can be preliminarily seen that there is a synergistic effect between the compounds, for example, 1+3>1 and 3, 2+3>2 and 3, etc. For the three combinations of compounds, some effects have been significantly improved, 3+4+5 (inhibition rate of 53.15%), 2+3+5 (inhibition rate of 42.11%), and the inhibitory effects of the remaining components have also been improved to a certain extent, further indicating that there is a certain synergistic effect between the compounds. For the four combinations of compounds, the inhibition rate is significantly higher than that of other combinations, 2+3+4+5 (inhibition rate is 83.69%), 1+2+3+4 (inhibition rate is 81.56%), 1+3+4+5 (inhibition rate is 97.44%), and the lowest effect 1+2+3+4 (inhibition rate is 30.71%) is also better than the previous combination. Combining the entire experiment, component 5 has the best effect, followed by component 3 and component 4, and component 1 has a poor effect, which may be related to its lowest content. Through the different combinations of compounds, it can be seen that there is a relatively obvious synergistic effect between the five compounds.

[0073] In summary, the aqueous extract of Coptis chinensis or its monomer compounds have a good inhibitory effect on Black Sporophyte, and are expected to be used in the preparation of biological pesticides that inhibit Black Sporophyte.

[0074] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the experimental conditions without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. Use of water extract of Coptis chinensis in the preparation of biological pesticides for inhibiting black spore mold.

2. The use according to claim 1, characterized in that The coptis chinensis water extract is a water extract obtained by extracting the medicinal material coptis chinensis by heating in water, or a water extract further freeze-dried into a powder, or an extract further concentrated into an extract; Preferably, heating to reflux is performed during the extraction process.

3. The use according to claim 2, characterized in that The preparation method of the water extract of Coptis chinensis comprises: taking Coptis chinensis slices, extracting once or multiple times by reflux extraction, adding distilled water, each time for 0.2-5 hours, combining the filtrates after extraction, concentrating the combined filtrates, or freeze-drying the combined filtrates to obtain powder.

4. The use according to claim 1, characterized in that The coptis chinensis water extract comprises at least three of jatrorrhizine, epiberberine, coptis chinensis, palmatine and berberine; Preferably, the aqueous extract of Coptis chinensis includes at least four of jatrorrhizine, epiberberine, coptisine, palmatine and berberine; More preferably, the aqueous extract of Coptis chinensis includes jatrorrhizine, epiberberine, coptisine, palmatine and berberine.

5. The use according to claim 3, characterized in that The content of jatrorrhizine, epiberberine, coptisine, palmatine or berberine in the water extract of Coptis chinensis is greater than 0.03 mg / mg; Furthermore, the content of jatrorrhizine is greater than 0.03 mg / mg, the content of epiberberine is greater than 0.1 mg / mg, the content of coptisine is greater than 0.13 mg / mg, the content of palmatine is greater than 0.11 mg / mg, and the content of berberine is greater than 0.4 mg / mg.

6. The use according to claim 1, characterized in that The Rhizoma Coptidis water extract is prepared into a water extract, and the concentration in water is greater than 0.5 mg / mL, preferably greater than 0.9 mg / mL.

7. The use according to claim 1, characterized in that The biological pesticide is prepared into powder, wettable powder, soluble powder, emulsifiable concentrate, aqueous solution, suspension, microemulsion, granule or water-dispersible granule.

8. Use of the aqueous extract of Coptis chinensis in the preparation of biological pesticides for resisting plant diseases such as rice leaf spot, watermelon leaf spot, blueberry leaf spot or branch blight.

9. Use of a pharmaceutical composition in the preparation of a biopesticide for inhibiting black spore mold, the pharmaceutical composition comprising at least three of jatrorrhizine or jatrorrhizine hydrochloride, epiberberine, coptisine or coptisine hydrochloride, palmatine or palmatine hydrochloride, berberine or berberine hydrochloride; Or the pharmaceutical composition comprises at least four of jatrorrhizine or jatrorrhizine hydrochloride, epiberberine, coptisine or coptisine hydrochloride, palmatine or palmatine hydrochloride, berberine or berberine hydrochloride; Alternatively, the pharmaceutical composition comprises jatrorrhizine or jatrorrhizine hydrochloride, epiberberine, coptisine or coptisine hydrochloride, palmatine or palmatine hydrochloride and berberine or berberine hydrochloride.

10. The use according to claim 9, characterized in that In the pharmaceutical composition, the mass proportions of each component are 0.1-0.5 parts of jatrorrhizine or jatrorrhizine hydrochloride, 0.9-1.5 parts of epiberberine, 1.2-2.0 parts of coptisine or coptisine hydrochloride, 1.0-1.6 parts of palmatine or palmatine hydrochloride, and 4.0-5.5 parts of berberine or berberine hydrochloride; Preferably, 0.2-0.5 parts of jatrorrhizine or jatrorrhizine hydrochloride, 1.0-1.4 parts of epiberberine, 1.3-1.9 parts of coptisine or coptisine hydrochloride, 1.2-1.5 parts of palmatine or palmatine hydrochloride, 4.5-5.0 parts of berberine or berberine hydrochloride; Further preferably, 0.38 parts of jatrorrhizine or jatrorrhizine hydrochloride, 1.19 parts of epiberberine, 1.6 parts of coptisine or coptisine hydrochloride, 1.29 parts of palmatine or palmatine hydrochloride, and 4.73 parts of berberine or berberine hydrochloride.

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