US20170224623A1

US20170224623A1 - Application of andrographolide in the preparation of a pharmaceutical for treatment of inflammatory bowel disease, andrographolide enteric targeting micropellet, and method for preparation thereof

Info

Publication number: US20170224623A1
Application number: US15/469,711
Authority: US
Inventors: Henry Sun; Xiaohui Ma; Zhixin Guo; Sen Lin; Genbei Wang; Lulu Yan; Lihua Zhang; Shuiping Zhou; Shunnan Zhang
Original assignee: Tasly Pharmaceutical Group Co Ltd
Current assignee: Tasly Pharmaceutical Group Co Ltd
Priority date: 2013-08-06
Filing date: 2017-03-27
Publication date: 2017-08-10
Also published as: US9889093B2; US20170224622A1; KR20160040697A; CN104337773B; HK1220909A1; CN104337773A; PH12016500258A1; CN105451734A; CA2920533A1; RU2016107440A; SG11201600933QA; US20160175253A1; JP2016527281A; WO2015018344A1; RU2681930C2; RU2016107440A3; JP6389889B2; AU2014305430A1; AU2014305430B2; EP3031456A1

Abstract

The present invention relates to an andrographolide enteric targeting micropellet and method for preparation thereof; furthermore, the present invention also relates to uses of andrographolide and andrographolide enteric targeting micropellets in the preparation of a pharmaceutical for treatment of inflammatory bowel disease.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No. 14/910,173, filed on Feb. 4, 2016, which is U.S. national phase filing of PCT/CN2014/083810, filed on Aug. 6, 2014, all of which claim priority to Chinese Patent Application No. 20130338444.1, filed on Aug. 6, 2013, the entire contents of each of which are incorporated by reference herein in their entireties.

FIELD OF THE INVENTION

Present invention relates to the field of medicine. More specifically, the invention relates to an andrographolide enteric targeting micropellet and method for preparation thereof. Also, present invention relates to an application of andrographolide and andrographolide enteric targeting micropellet in the preparation of a medicine for treatment of inflammatory bowel disease.

BACKGROUND OF THE INVENTION

Andrographolide (C₂₀H₃₀O₅) is the diterpene lactone compound extracted from the plant Andrographis Paniculata. It is one of the main components in the Andrographis Paniculata Nees (APN) which is crowned as the natural antibiotic because of its effects of anti-pathogenic microorganism, antipyresis, anti-inflammation, improving body immune, protecting liver by normalizing functioning of the gallbladder and anti-tumor etc. The andrographolide belongs to the diterpene lactone compound. Being an herbal extract, it has advantages of less side effects, better anti-inflammation and extensive source with a competitive price.
Inflammatory bowel disease (IBD) is a recurrent chronic inflammatory disease on intestinal, mainly including the ulcerative colitis (UC) and the Crohn's disease (CD). Their definite cause and pathogenesis have not yet been eradicated and therefore there are insufficient effective treatment methods in clinic. The clinical manifestations of UC included: the intestinal injury, most of which firstly appeared in the distal colon and sigmoid colon; mainly the left abdomen dull pain or sustained secret pain, which may be relieved after diarrhea: the myxoid-like and pus-blood-like stool accompanied with the tenesmus. The clinical manifestations of CD included: mostly the abdomen pain; the left abdomen colic pain or spastic sharp pain characterized by paroxysmal occurrence and colic pain occurs post meal; the myxoid-like and watery stool accompanied with constipation alternative with diarrhea. A series of disease may be more likely to be found in CD than in UC, such as the intestinal stenosis, the intestinal obstruction, the intestinal fistula, the intestinal polyp and even the carcinogenesis.
Crohn's disease (CD) is identified as one of the IBD. Usually, the symptoms of inflammation, congestion or swollen lymph may occur in the colon, small intestine or stomach. The main difference from UC lies in inflammation position and the inflammation itself. The Crohn's disease may affect any segment of digestive system, e.g. the small intestine, colon, stomach and esophagus, which is common in terminal ileum and adjacent colon segment and right-half colon. UC, however, just occurs in colon and rectum, which is common in rectum and sigmoid. Microscopically, the Crohn's disease may affect whole inner wall of bowel, while UC is restricted to mucosa.
Crohn's disease is a chronic and recurrent disease. Effectively therapeutical drugs have not yet been developed for its unknown cause. By now, drugs used for treating Crohn's disease mainly include glucocorticoid, salicylic acid, immunosuppressive agents, antibiotics, methotrexate and biological agents (e.g. infliximab). Although these drugs are proven to be able to change the natural process of disease, they can not completely alleviate the conditions of disease and decrease the incidence of complications. Moreover, the chemicals of glucocorticoid and immunosuppressive agents often cause obvious adverse reaction and longtime administration will likely result in damage to the body. Hence, we need to develop a new medicine and its formulation thereof for treatment of the Crohn's disease.
On the other hand, the colon drug delivery has been regarded as a difficult issue in R&D for a long time, which is determined by colon's own physiological characteristics. It is well-known that the colon is located in the bottom half of the digestive tract, that drugs are very difficult to reach the colon when administrated orally and that enema administration is both inconvenient and painful. As a result of this, the enteric targeting preparation technique emerged. Oral colon-specific drug delivery system (OCSDDS) refers to a drug delivery technique, which makes the drug pass through top half of the digestive tract of the stomach and duodenum unchangedly. The medicine is not released until transferred to the ileum to demonstrate local or systematic therapeutic effects. The common-used OCSDDS techniques are divided into the pH-dependent type and the enzyme degraded type.
The pH-dependent OCSDDS is to achieve the colon specific delivery by utilizing the different pH value of each part in human gastrointestinal tract. Usually, the gastric pH value of healthy people is lowest at 1˜3, the duodenum at 4˜6, the jejunum at 6˜7, ileum at 7˜7.5 and colon at 7˜8.
Now, common-used enteric-coating materials have different pH points at which to dissolve. The first type began to dissolve at pH value ≧5.5, the second at ≧6.0 and the third at ≧7.0. Up to now, the drug is wrapped by using the third enteric polymer to coat in the pH-dependent enteric-targeting preparation. It may be achieved that the drug does not release through the top half of the gastrointestinal tract until transferred to the ileum. All Chinese patents (CN1981743, CN101209246, CN103315959) are involved in this technique. As shown in clinical studies, however, gastrointestinal pH values among different individuals are far away from each other. There is a gap between IBD patients and healthy people, and the colon pH value in colitis patients is lower than healthy people. As a result, when using this kind of polymer alone, the drug will not be released in vitro and expelled with stool.
As for the andrographolide enzyme-degraded oral colon-specific delivery, the prior arts include steps: coating blank pellet with the andrographolide to have a drug-loading micropellet and wrapping said micropellet with monosaccharide pore-forming agents-inclusive insoluble polymer. Said polymer film does not release in the stomach and small intestine until reaching the colon. The monosaccharide in the film is degraded by colon enzymes. After the pore is formed, the drug is gradually dissolved and released. Although this technique has overcome defects of difference among individuals in the pH-dependent OCSDDS, there are problems. Because the monosaccharide, e.g. the Guar gum is dissolved in water and the drug will be soon released from pores formed by dissolution of monosaccharide after entering the body, it is difficult to ensure that effective amount of drug reaches the colon. In addition, the molecular of monosaccharide is structurally rigid. Once being embedded into the polymer chain, it will not only affect the extensibility of polymer chain, but also destroy the integrity of polymer membrane, which will make the coating membrane crisped and easily broken. Hence, the risk is increased that the membrane may be early broken during transportation or by gastrointestinal peristalsis. As a result of this, developing new preparations of andrographolide is still needed for treating the IBD.

CONTENT OF THE INVENTION

In the first place, the objective of present invention is to provide a new use of andrographolide, particular to a use of andrographolide in preparation of medicine for treating IBD. Wherein, said IBD includes UC and Crohn's disease.
On the other hand, the objective of present invention is to provide a new pH-dependent enteric targeting preparation. That is to say, two types of pH-dependent polymers are jointly used to achieve the purpose of targeting release in vitro with different pH values. In particular, the present invention relates to an andrographolide enteric targeting micropellet. Said micropellet is used for better treating IBD, for example UC and Crohn's disease. Also, the present invention relates to a method for preparing said pH-dependent enteric targeting preparation.
Preferably, present invention relates to the technical scheme, as follows:
1. An andrographolide enteric targeting micropellet is composed of a blank pellet, a drug layer and an enteric coating layer, wherein said drug layer contains the andrographolide, the polymer A dissolved under condition of pH≧7.0 and the excipient; said ratio of the andrographolide and polymer A is 1:2˜1:0.2 by weight; weight gain of the drug layer is 20 wt %˜100 wt %, preferably 30 wt %˜80 wt %; said enteric coating layer contains the polymer B dissolved under condition of pH≧5.5 and the excipient; weight gain of the enteric coating layer is 5 wt %˜30 wt %, preferably 8 wt %˜20 wt %, most preferably 10 wt %˜18 wt %.
2. The micropellet according to 1^stparagraph, wherein said drug layer contains excipients of the plasticizer, anti-sticking agent, pigment, hydrophilic polymer and surfactant; said enteric coating layer contains excipients of the plasticizer and anti-sticking agent; preferably said excipients optionally include the hydrophilic polymer and pigment.
3. The micropellet according to 1^stparagraph, wherein said polymer A is the copolymer of methacrylic acid and methyl methacrylate and polymer B is the copolymer of methacrylic acid and ethyl acrylate.
4. The micropellet according to 1^stparagraph, wherein said polymer A is the copolymer of methacrylic acid and methyl methacrylate in a ratio of 1:2 and/or the polymer B the copolymer of methacrylic acid and ethyl acrylate in a ratio of 1:1.
5. The micropellet according to 1^stparagraph, wherein said plasticizer is selected from one or more kinds of the triethyl citrate, the dibutyl sebacate, the propanediol and PEG, accounting for 10˜70 wt % of the polymer A, preferably 10˜20 wt %; said anti-sticking agent is the talc, accounting for 25˜100 wt % of the polymer A, preferably 30˜50 wt %; or said anti-sticking agent is the glyceryl monostearate, accounting for 2˜20 wt % of the polymer A, preferably 5˜10 wt %.
6. The micropellet according to 1^stparagraph, wherein the diameter of said blank pellet is 200˜600 μm, preferably 300˜500 μm, accounting for 10˜70 wt % of the formula, preferably 20˜60 wt %.
7. The micropellet according to 2^ndparagraph, wherein said ingredients are present in proportion by weight parts: blank micropellet:andrographolide:polymer A:plasticizer:anti-sticking agent:surfactant=200:(10-100):(10-100):(1-15):(1-30):(0-3), preferably 200:(15-66):(13-74):(2-13.5):(3-27):(0-1.32), most preferably 200:(20-50):(30-60):(5-10):(5-20):(0.5-1.2).
8. The preparation method according to any one micropellet of 1^st˜7^thparagraphs comprising following steps:
(1) applying drug to the blank pellet
a). dispersing the polymer A into a pharmaceutical solvent to let them dissolve fully by mechanical stirring; adding the excipient into the polymer A solution and then adding the andrographolide to have the polymer A coating solution by well stirring;
b). weighing the blank pellet and charging into a fluidized bed; adjusting air flow to such a degree that the micropellet is well fluidized; opening the heating device and until temperature of the material reaches preset value, the peristaltic pump is started to make the polymer A coating solution atomized through a spray gun to obtain a drug-loading micropellet by dispersing on the surface of said blank pellet;
(2) preparation of the enteric coating layer
a). dispersing the polymer B into a pharmaceutical solvent to let them dissolve fully by mechanical stirring; adding the excipient into the polymer B solution to have the polymer B coating solution by well stirring;
b). charging aforesaid drug-loading micropellets into a bottom-spray device of the fluidized bed, and the polymer B coating solution is uniformly spread to form the enteric coating layer; the weight gain is 5 wt %˜30 wt %.
9. The preparation method according to 8^thparagraph comprising following steps:
(1) applying drug to the blank pellet
a). dispersing the polymer A into a pharmaceutical ethanol to make the content of polymer A at 5 wt %; fully dissolving by mechanical stirring and continuing to stir uniformly; adding the excipient of the plasticizer, anti-sticking agent and the surfactant of sodium dodecyl sulfate into the polymer A solution and then adding the andrographolide to have the polymer A coating solution by well stirring;
b). weighing the blank sucrose pellet in a diameter of 200˜600 μm and charging into a fluidized bed; adjusting air flow to such a degree that the micropellet is well fluidized; opening the heating device to keep the temperature of the material at 25˜35° C. and until the temperature reaches preset value, the peristaltic pump is started to make the polymer A coating solution atomized through a spray gun to obtain a drug-loading micropellet by dispersing on the surface of said blank pellet;
(2) preparation of the enteric coating layer
a). dispersing the polymer B into the pharmaceutical ethanol to let them dissolve fully by high-speed shearing mechanical stirring; adding the excipient of the plasticizer and anti-sticking agent into the polymer B solution to have the polymer B coating solution by well stirring;
b). charging aforesaid drug-loading micropellets into a bottom-spray device of the fluidized bed, and the polymer B coating solution is uniformly spread to form the enteric coating layer; the weight gain is 8 wt %˜20 wt %.
10. An andrographolide enteric targeting preparation, characterized in that any one micropellet of 1^st˜7^thparagraphs is prepared into granule or capsule.
11. Use of andrographolide, any one micropellet of 1^st˜7^thparagraphs or the targeting preparation of 10^thparagraph in preparation of medicine for treating the IBD.
12. The use according to 11^thparagraph, wherein said IBD is UC or Crohn's disease.
13. The use according to any one of 11^thor 12^thparagraph including improving colon adhersion, intestinal wall red swelling and thickening and decreased elasticity.
14. The use according to any one of 11^thor 12^thparagraph including reducing colon ulcer surface, hemorrhagic spot and poferation.
15. The use of any one of 11^th˜14^thparagraphs, characterized in that said medicine is prepared into an enteric-coated preparation.
16. The use of any one of 11^th˜14^thparagraphs, characterized in that said medicine is prepared into an enteric targeting micropellet.
17. The use of 16^thparagraph, characterized in that said enteric targeting micropellet is prepared into a granule or capsule.

DESCRIPTION OF THE DRAWINGS

FIG. 1 indicated the effect of the andrographolide on the body weight of TNBS-caused cotilis mouse.

FIG. 2 was the general observation pictures of each group in TNBS-caused UC rats (A-F).

FIG. 3 showed the zebra fish intestine in tested drug group, Crohn's disease model group and blank control group (The region shown by green dotted line was the zebra fish intestine).

FIG. 4 indicated the improvement degree of bowel dilatation in Crohn's disease model group after being administrated with the andrographolide at different doses (compared with the blank control group).

FIG. 5 indicated the therapeutical ratio of andrographolide at different doses in Crohn's disease model group calculated in accordance with the bowel area.

FIG. 6 showed the distribution of neutrophil in zebra fish intestinal tissue after being treated with drugs. (The region shown by yellow dotted green line was the zebra fish intestine and the one by green bright spot was the neutrophil.)

FIG. 7 was the cumulative release amount of drug in the phosphate buffer of simulated intestinal fluid (pH 6.5).

FIG. 8 was the cumulative release amount of drug in the phosphate buffer of simulated intestinal fluid (pH 7.2).

DETAILED DESCRIPTION OF THE INVENTION

In first embodiment of this invention, said IBD chiefly relates to UC and Crohn's disease. By studying, the inventor of present invention concluded: the andrographolide had an improving effect on the colon adhersion, intestinal red swelling and thickening and decreased elasticity. In addition, the andrographolide was able to reduce colon ulcer surface, hemorrhagic spot and poferation.
In one embodiment of present invention, said medicine of present invention includes any one qualified drug prepared by using the andrographolide as an active pharmaceutical ingredient (API). Preferably, said medicine of present invention refers to a pharmaceutical composition comprising the andrographolide alone or in combination with other ingredients.
In one embodiment of present invention, the andrographolide belongs to prior arts, which is either commercially available or prepared by a conventional method. For example, the andrographolide is prepared by the following method: the leaves of Andrographis paniculata is soaked in 95% (v/v) ethanol and the resulting ethanol liquid is decolored with the activated carbon and the ethanol is recovered by distillation to give a concentrated liquid. The liquid is allowed to stand still to have coarse crystal. Said coarse crystal is added with 15 times (15×) 95% (v/v) ethanol, dissolved by heating, decolored with activated carbon and filtered immediately. The liquid is allowed to stand still to give a light-yellow crystal by recrystallization. The obtained crystal is refined by washing with distilled water, chloroform and menthol to have the final product of andrographolide.
In one embodiment of present invention, the andrographolide is desirably administrated in a form of pharmaceutical composition. Said composition may be conventionally used in combination with one or more kinds of physiologically acceptable excipients or carriers. If possible, the andrographolide acts as APT and administrated directly to patients, preferably the API is administrated directly as a preparation. In terms of integration with other ingredients and safety on the subject, said excipient must be accepted pharmaceutically.
In one embodiment of present invention, said pharmaceutical composition can be prepared into any one of pharmaceutically acceptable dosage forms when applied clinically, including but not limited to: the tablet, e.g. the sugar-coated tablet, film-coated tablet and enteric-coated tablets; capsule, e,g, the hard capsule, soft capsule or enteric-coated capsule; injection; suppository, e.g. the intestinal suppository; drop etc, preferably the enteric-coated suppository, e.g. the enteric-coated tablet and enteric-coated capsule.
In one embodiment of present invention, the pharmaceutically acceptable excipients may be added when preparing said pharmaceutical composition.
In one embodiment of present invention, oral preparations may include conventional excipients, e.g. the adhesive, filling agent, diluent, tableting agent, lubricant, disintegrating agent, colorant agent, flavoring agent, wetting agent. If necessary, the tablet may be coated. Said filling agents include cellulose, mannitol, lactose and other analogous filling agent. Competent disintegrating agents include starch, polyvinylpyrrolidone (PVP) and starch derivative (e.g. sodium hydroxyethyl starch). Competent lubricants include magnesium stearate. Competent pharmaceutically acceptable wetting agents include sodium dodecyl sulfate. The oral solid preparations can be prepared by a conventional method of blending, filling, tabletting or granulating etc. Repeated blending is performed to make the API distributed uniformly in compositions in which lots of filling agents are used.
In one embodiment of present invention, as for injections, said preparation unit contains the andrographolide and aseptic excipients. Whether said API is dissolved or suspended in the liquid depends on the type and concentration of excipients. Generally, solution is prepared by dissolving the TCM formulation in the excipients as the API, sterilizing, loading into an appropriate vial or ampoule and sealing. Some pharmaceutically acceptable adjuvant, e.g. local anaesthetic, preservative and buffering agent can be added as required. In order to improving its stability, before loaded into the vial, this TCM formulation of present invention can be frozen and treated in vacuum to remove water.
In one embodiment of present invention, the effective daily dose of the medicine for adult treatment is always in the range of 0.02˜5000 mg when used for prevention and treatment of UC and Crohn's disease, preferably 1˜1500 mg. Said dose needed for treatment is either a single dose or a multidose, at which the medicine is administrated at proper intervals, such as twice, triple, four times or more per day. The preparation of present invention may include 0.1 wt %˜99.9 wt % of the API.
In second embodiment of this invention, a new pH-dependent enteric targeting preparation is provided, which is characterized that said andrographolide enteric targeting micropellet is composed of a blank pellet, a drug layer and an enteric coating layer, wherein said drug layer contains the andrographolide, the polymer A dissolved under condition of pH8≧7.0 and the excipient; said ratio of the andrographolide and polymer A is 1:2˜1:0.2 by weight; weight gain of the drug layer is 20 wt %/˜100 wt %, preferably 30 wt %˜80 wt %; said enteric coating layer contains the polymer B dissolved under condition of pH≧5.5 and the excipient; weight gain of the enteric coating layer is 5 wt %˜30 wt %, preferably 8 wt %˜20 wt %, most preferably 10 wt %˜18 wt %.
Among these, said polymer A is the copolymer of methacrylic acid and methyl methacrylate, preferably the copolymer of methacrylic acid and methyl methacrylate in a ratio of 1:2 and polymer B is the copolymer of methacrylic acid and ethyl acrylate, preferably the copolymer of methacrylic acid and ethyl acrylate in a ratio of 1:1.
In a preferred embodiment of present invention, the polymer A is selected from the Eudragit S100 purchased from Rohm Inc, and the polymer B is the Eudragit L series polymers, most preferably the Eudragit L100-55.
In one embodiment of present invention, said drug layer contains excipients of the plasticizer, anti-sticking agent, pigment, hydrophilic polymer and surfactant. Preferably, the surfactant is selected from the sodium dodecyl sulfate (SDS) or Tween-80 with adding amount of 0˜5 wt % of the andrographolide, preferably 1 wt %˜3 wt %.
In one embodiment of present invention, said plasticizer is selected from one or more kinds of the triethyl citrate, the dibutyl sebacate, the propanediol and PEG, accounting for 10˜70 wt % of the polymer A, preferably 10˜20 wt %; said anti-sticking agent is the talc, accounting for 25˜100 wt % of the polymer A, preferably 30˜50 wt %; or said anti-sticking agent is the glyceryl monostearate, accounting for 2˜20 wt % of the polymer A, preferably 5˜10 wt %.
In one embodiment of present invention, the diameter of said blank pellet is 200˜600 μm, preferably 300˜500 μm, accounting for 10˜70 wt % of the recipe quantity, preferably 20˜60 wt %. Said blank pellet is the conventional pharmaceutical pellet, preferably the blank sucrose pellet or microcrystalline cellulose pellet.
In one embodiment of present invention, said ingredients are present in proportion by weight parts: blank pellet:andrographolide:polymer A:plasticizer:anti-sticking agent:surfactant=200:(10-100):(10-100):(1-15):(1-30):(0-3).
Preferably, said ingredients are present in proportion by weight parts: blank micorpellet:andrographolide:polymer A:plasticizer:anti-sticking agent:surfactant=200:(15-66):(13-74):(2-13.5):(3-27):(0-1.32).
Most preferably, said ingredients are present in proportion by weight parts:blank micorpellet:andrographolide:polymer A:plasticizer anti-sticking agent:surfactant=200:(20-50):(30-60):(5-10):(5-20):(0.5-1.2).
Wherein, excipients in the enteric coating layer include the plasticizer and anti-sticking agent and are selected as depicted before. The plasticizer accounts for 15 wt % of the polymer B and the anti-sticking agent 30 wt %.
Optimized 7 formulas of the blank micropellet and drug layer are present as follows:


	Blank
	micropellet	andrographolide	Eurdragit S		anti-sticking	Surfactant
No.	(g)	(g)	(g)	Plasticizer (g)	agent (g)	(g)

1	200	50	15	2.1	4.5	1.00
2	200	66	20	3	6	1.32
3	200	66	22	3.3	6.6	1.32
4	200	44	13	2	3	0
5	200	58	18	3.6	5.4	1.16
6	200	15.2	40	6	12	0.4
7	200	52	74	13.5	27	1.26

The preparation method for preparing the andrographolide enteric targeting micropellet is present as follows:
(1) applying drug to the blank pellet
a). dispersing the polymer A into a pharmaceutical solvent to let them dissolve fully by mechanical stirring; adding the excipient into the polymer A solution and then adding the andrographolide to have the polymer A coating solution by well stirring;
b). weighing the blank pellet and charging into a fluidized bed; adjusting air flow to such a degree that the micropellet is well fluidized; opening the heating device and until temperature of the material reaches preset value, the peristaltic pump is started to make the polymer A coating solution atomized through a spray gun to obtain a drug-loading micropellet by dispersing on the surface of said blank pellet;
(2) preparation of the enteric coating layer
a). dispersing the polymer B into a pharmaceutical solvent to let them dissolve fully by high-speed shearing mechanical stirring; adding the excipient into the polymer B solution to have the polymer B coating solution by well stirring;
b). charging aforesaid drug-loading micropellets into a bottom-spray device of the fluidized bed, and the polymer B coating solution is uniformly spread to form the enteric coating layer; the weight gain is 5 wt %˜30 wt %.
Preferably, the preparation method for preparing the andrographolide enteric targeting micropellet is present:
(1) applying drug to the blank pellet
a). dispersing the polymer A into a pharmaceutical ethanol to make the content of polymer A at 5 wt %; fully dissolving by high-speed shearing mechanical stirring and continuing to stir uniformly; adding the excipient of the plasticizer, anti-sticking agent and the surfactant of sodium dodecyl sulfate into the polymer A solution and then adding the andrographolide to have the polymer A coating solution by well stirring;
b). weighing the blank sucrose pellet in a diameter of 200˜600 μm and charging into a fluidized bed; adjusting air flow to such a degree that the micropellet is well fluidized; opening the heating device to keep the temperature of the material at 25˜35° C. and until the temperature reaches preset value, the peristaltic pump is started to make the polymer A coating solution atomized through a spray gun to obtain a drug-loading micropellet by dispersing on the surface of said blank pellet;
(2) preparation of the enteric coating layer
a). dispersing the polymer B into the pharmaceutical ethanol to let them dissolve fully by high-speed shearing mechanical stirring; adding the excipient of the plasticizer and anti-sticking agent into the polymer B solution to have the polymer B coating solution by well stirring;
b). charging aforesaid drug-loading micropellets into a bottom-spray device of the fluidized bed, and the polymer B coating solution is uniformly spread to form the enteric coating layer; the weight gain is 8 wt %˜20 wt %.
Besides, present invention is involved in an andrographolide enteric targeting preparation and said preparations include the capsule or granule prepared from aforesaid micropellets by a conventional method.

Advantages

A specific pH-dependent technique has been used, namely the joint use of two pH-dependent polymers, to make it enteric targeting release in bodies of different colon pH values.
{circle around (1)} The first type of enteric coating material, e.g. Eudragit L 100-55, has been used in the enteric coating layer, ensuring that the medicine does not release in the stomach until exposing the drug layer by quick dissolution after reaching the duodenum. {circle around (2)} The second type of coating material (e.g. the Eudragit S 100), as the middle layer, is used as a skeleton in the drug layer, among which the drug is uniformly distributed. Gradually, the drug is released by dissolution of enteric coating layer when the micropellet reaches the duodenum. Under low pH condition, however, the drug is released a little; only when approaching the end of small intestine at pH close to 7, the drug is released quickly, because the Eudragit S 100 in the drug layer has a retarding effect in the low pH condition.
As depicted before, the andrographolide enteric targeting micropellet of present invention has a three-layer structure: the blank pellet, the drug layer and the enteric coating layer. The enteric coating layer is kept intact at pH below 5.5 when the preparation goes into the stomach. After reaching the duodenum, however, the enteric coating layer bursts into dissolving to expose the drug layer. The Eudragit S 100 in the drug layer plays a dual role of sustained release and enteric dissolution. The andrographolide is uniformly distributed in the Eudragit S 100. After reaching the duodenum, the outside layer is dissolved. As soon as exposing to the body fluid, the andrographolide starts to release. Because the pH value on this position is low, the limited amount of Eudragit S 100 is dissolved and very few amount drugs released at very slow rate. With transfer of drug to low half of gastrointestinal tract, the pH value goes up gradually, the dissolution rate of Eudragit S 100 is accelerated and the release rate of drug increased. As a result, most of the drug is not released until approaching ileum and colon, capable of treating intestinal inflammation.
The diameter of said blank pellet of present invention is 200˜600 μm, much less than the clinical commonly-used blank micropellet (500˜1000 μm). This will not only help improving the specific surface area, but also the contact area between the drug and the inflammatory site, ensuring that the andrographolide plays the therapeutical effect on the IBD. The blank pellet accounts for 10 wt %˜70 wt % of the formula quantity.
The Eudragit series polymers are used for coating as a film material in combination with the plasticizer and anti-sticking agent added in the formula. Wherein, the purpose of using plasticizer is for not only reducing glass transition temperature and minimum film forming temperature (MFFT), but also increasing flexibility of the polymer film. The purpose of using anti-sticking agent is for preventing the film from being sticky, causing bonding mutually among the blank pellets. The surfactant may be used for increasing the wetting effect on the drug.
The pharmacological effects of andrographolide and its new preparation are proven by following experiments.

Pharmacological Research 1 Pharmacological Study of Andrographolide on TNBS-Induced UC

1. Aim

The mice IBD model was used to perform a preliminary evaluation of the andrographolide for treating UC and Crohn's disease

2 Materials

2.1 Animals

50 SPF Balbc/c male mice, weighing 18˜24 g, were provided from Beijing Weitonglihua Experimental Animals Inc and Certificate No. 2011-0012
2.2 Raising conditions
Animals were raised in a barrier animal room, 10 mice in each cage, with temperature at 20˜25° C. and relative humidity at 40˜60% free access to water and padding materials replaced daily.
2.3 Tested medicine and reagents
Tested medicine: andrographolide, white dry powder, was provided by Tasly Modern TCM Resource Inc with yield rate of 98% and purity of 98%.
Positive drug: Sulfasalazine was purchased from Shanghai Sanwei Pharmaceutical Inc with the batch No 200206C11 and specification: 250 mg/tablet, 12 tablet×5.
Reagents: TNBS was purchased from Sigma Inc with the batch No 033K5020 and specification: 5% (w/v) and 10 ml/bottle.

3 Method

3.1 Preparation of model
5% (w/v) TNBS solution was diluted with double distilled water and mixed with 50% (v/v) ethanol in equivalent volume to have 1.5% (w/v) TNBS solution. In the model group, the mice were anesthetized with 1 wt % pentobarbital sodium at dose of 0.05 ml/10 g body weight. After being anesthetized, the mice were administrated with 1.5% (w/v) TNBS solution at dose of 0.05 ml/mouse by gently inserting stomach perfusion device to about 3 cm depth of colon via anus and the IBD was induced. In the saline group, the solution was injected into the colon at dose of 0.1 ml mouse. In the normal group, 50% (v/v) ethanol was injected into the colon at 0.1 ml mouse.
3.2 Grouping and administration
After one week adaptive feed, all animals were randomly divided into 5 groups according to body weight, 10 mice in one group: the normal control group, the model group, the andrographolide low-dose group (20 mg/kg/d), the andrographolide high-dose group (40 mg/kg/d) and the positive control group (Sulfasalazine, 300 mg/kg/d). 2 hours after making model, the andrographolide was administrated orally twice daily in each treating groups and once daily in the positive control group. After successive administration for 7 days, the abdomen was opened 24 hours after last administration to observe adhesion between colon and other organs. The colon was separated and weighed.
3.4 Evaluation indices
1. Body weight: after making model, body weight was measured every day to observe the change.
2. Evaluation of inflammation: the abdomen was opened 24 hours after last administration to observe adhesion between colon and other organs. Every segment of colon was taken out and weighed to calculate the ratio of colon weight to body weight as the colon index. The formula was present as follows:
Decrease rate of colon specific weight=(colon index of model group-colon index of treatment group)/colon index of model group×100%
3. Histopathological examination:
0 score, no inflammatory symptoms
1 score, low-grade inflammation with no structure change
2 score, low-grade leukocyte infiltration
3 score, high-grade leukocyte infiltration, high vascular density, crypt extension, thickened colon wall and superficial ulcer
4 score, high-grade leukocyte infiltrating to mucus layer, crypt extension, decrease of goblet cell, high vascular density, thickened colon wall and extensive ulcer

3.5 Statistics

SPSS11.5 software was used for analysis and data were expressed as x±S. Variance analysis was used for comparison of significant difference among groups. P<0.05 showed a statistically significant difference.

4. Results

4.1 Effect of the andrographolide on the BW of TNBS-induced IUC mice
The body weight index was able to generally reflect the overall health status of mice. Compared with the normal control group, the mouse growth was affected after forming UC by rectal administration of TNBS: slow growth of body weight. Being treated with the andrographolide or the sulfasalazine, the body weight grew faster than the model control group. Data were seen in FIG. 1.
4.2 Effect of the colon weight, colon indices and rate of colon specific weight
After UC was indiced by TNBS, the colon weight and colon indices were obviously higher than the normal control group, illustrating the successful modeling. After 7 days of treatment with drugs, compared with the model group, the colon weight in the andrographolide high-dose group and the positive control group was reduced significantly (P<0.01); the colon indices in the andrographolide high-dose group, low-dose group and the positive control group was reduced significantly (P<0.05, P<0.01). The decrease rates of colon specific weight were 33.20%, 58.96% and 47.87. Data were seen in Table 1.

TABLE 1

effect of the andrographolide on colon weight, colon indices and the decrease rate of
colon specific weight in the UC mice

Colon weight	Body		Decrease rate of colon
(g)	weight (g)	Colon indices	specific weight (%)

Normal control	0.17 ± 0.03	22.92 ± 1.01	0.77 ± 0.10	—
group
Model group	0.55 ± 0.15	16.32 ± 1.06	3.35 ± 0.88	—
Andrographolide	0.43 ± 0.12	19.95 ± 2.05	2.24 ± 0.90*	33.20
low-dose group
Andrographolide	0.29 ± 0.09**	20.95 ± 1.09	1.38 ± 0.46**	58.96
high-dose group
Positive control	0.33 ± 0.06**	19.13 ± 0.99	1.75 ± 0.43**	47.87
group

Compared with the model group:
*P < 0.05:
**P < 0.01

4.3 Effect of the andrographolide on histopathological changes of colon in TNBS-induced UC mice

After UC was induced by TNBS, a series of symptoms occurred: colon adhesion, intestinal wall red swelling and thickening, decreased elasticity, UC surface, colon hemorrhagic spot and poferation, indicating extensive inflammatory injuries in colon. The intestinal elasticity in andrographolide and positive control groups was higher than that in the model group and the colon weight much less than the model group, indicating that the inflammatory reaction of colon adhesion and exudation was weaker than the model group. Histopathological injuries of intestinal mucosa and wall caused by Ulcer and hemorrhage were examined with naked eyes; the colon histopathological injuries in the andrographolide high-dose group and the positive control group were much lower than that in the model group. Data were seen in Table 2.

TABLE 2

effect of andrographolide on the colon score in TNBS-induced UC mice

	Colon score

	Normal control group	1
	Model group	3.7
	Andrographolide low-dose	3.2
	group
	Andrographolide	1.8**
	high-dose group
	Positive control group	2.8*

	Compared with the model group:
	*P < 0.05:
	**P < 0.01

5 Conclusions

There were a lot of methods to build animal colitis model, TNBS/ethanol induced model was the most similar with the UC pathological changes in clinic. Ethanol destroyed intestinal mucosal barriers and TNBS, as a hapten, would make T lymphocytes sensitized by combining with tissue proteins to cause the intestinal inflammation after inducing autoimmune reaction. The model rats caused by this method have lots of similarities with clinical symptoms of US patients: the stool change and intestinal general morphological and histological change.
In this study, the UC model mice were used to preliminarily evaluate the treating effect of andrographolide. As shown in the results, the high-dose andrographolide (40 mg/kg/d) was able to delay the descending trend of body weight in mice of model group, which, compared with the model group, could significantly reduce the colon indices (P<0.01), ameliorate colon pathological changes and the decrease rate of colon specific weight was 58.96%. Considering aforesaid indices, the andrographolide had a significantly improving effect on the UC in mice, having a certain therapeutical effect on UC and also capable of treating Crohn's disease.
Pharmacological Research 2 Study on Therapeutical Effect of Andrographolide on DSS-Induced UC

1. Aim

Efficacy of andrographolide on DSS-induced UC was evaluated.

2. Animals

84 SPF Balbc/c male mice, weighing 18˜22 g, were provided from Guangdong Medical Experimental Animal Center and Certificate number SYXK 2008-0002. Raising conditions: 7 mice per cage raised in a group; temperature and humidity: 20-26° C. and 40-70%. The animals were lighted 10 h/14 h days and nights intermittently. The condition of raising room was always remained unchanged, ensuring reliability of experiment results. Animals were fed with the complete pellet feed (provided by Guangdong Medical Experimental Animal Center), free access to water via drinking bottle.
3. Apparatuses and reagents

3.1 Apparatuses

3.1.1 Electronic balance, accuracy: 0.001 g, Zhongshan Hengxin Electronics Inc.
3.1.2 Auto dehydrating machine for organic tissue (TS-12N, Xiaogan Hongye Medical Device Inc.)
3.1.3 Embedding machine for organic tissue (BM-VII, Xiaogan Hongye Medical Device Inc.)
3.1.4 Machine for paraffin section (RM2135, Leica Inc. German)
3.1.5 Machine for spreading and roasting section (CS-V, Xiaogan Hongye Medical Device Inc.)
3.1.6 Auto staining machine for organic tissue (RS-18II, Xiaogan Hongye Medical Device Inc.)

3.2 Reagents

3.2.1 Andrographolide was provided by Tasly Modern TCM Resource Inc of batch number: 20140508 with purity>95%;
3.2.2 Positive drug: mesalazine enteric-coated tablet was purchased from Jiamusi Luling Pharmaceutical Inc, Kuihua Pharmaceutical Group with batch number: 140225.
3.2.3 DDS (dextran sulfate sodium) was purchased from MPBIO Inc.
4 Dose planning and grouping
4.1 Grouping: quarantine-qualified 84 mice were randomly divided into 6 groups: the model group, the positive drug group, the tested drug low-dose intragastric group, the tested drug high-dose intragastric group, the tested drug low-dose intracolonical group and the tested drug high-dose intracolonical group, 14 mice per group. All animals drank 5% DDS solution daily for consecutive 14 days to establish UC model. Animals were administrated with the drug for treatment 2 days after given 5% DDS drinking water.
4.2 Dose: in this study dose of administration of mice in all groups was designed on the basis of client's requirement and the same doses were adopted in both intragastric and intracolonical groups. Animals were not treated in the model group, mesalazine enteric-coated tablet (227.5 mg·kg⁻¹·d⁻¹) in the positive drug group, andrographolide (60 mg·kg⁻·d⁻¹) in the low-dose intragastric group, andrographolide (180 mg·kg⁻¹·d⁻¹) in the high-dose intragastric group, andrographolide (60 mg·kg⁻¹·d⁻¹) in the low-dose intracolonical group and andrographolide (180 mg·kg⁻¹·d⁻¹) in the high-dose intracolonical group.

5 Method

5.1 Method of making UC model: all animals drank 5% DSS solution daily for consecutive 14 days.
5.2 Method of administration: 2 days after modeling, mice were treated intragastrically or intracolonically with drug at 1 mL/100 g once a day for consecutive 14 days.
5.3 Method for sampling in experiment: 2 hours after last administration, mice were killed by cervical vertebra dislocation and abdomen was opened to separate the colon. Along mesenteric side, the cavity scissored off washed with 4° C. normal saline and spread on a plastic plate.
5.4 Method for evaluating disease active index (DAI): the disease active index was assessed at 7^thand 14^thday after administration. The method was present together with following three parameters: the BW loss percentages (unchanged BW: 0 score, 1˜5: 2 score, 5˜10: 2 score, 10˜15: 3 score, more than 15: 4 score), stool viscosity (normal stool: 0 score, loosen stool: 2 score, diarrhea: 4 score) and bloody stool (normal stool: 0 score, occult blood stool: 2 score and positive blood stool: 4 score). Total score of three parameters was divided by 3 to give the DAI, namely DAI=(BW index+stool character score+stool blood score)/3.
6 Observation indices
6.1 Observation: the daily general clinical symptoms in mice were observed from beginning to end of the experiment in mice and the stool, mental status and death status were recorded.
6.2 Body weight: BW was recorded weekly from beginning to end of the experiment
6.3 Calculation of DAI: DAI was calculated at 7^thand 14^thday after administration so as to assess disease activity.
6.4 The colon mucosal tissue 8˜10 cm away from the anus was sampled, embedded with paraffin and stained with routine HE. Colon musco injury was observed by microscope and histological injury was scored: ulcer inflammatory granuloma, fibrosis and pathological degree.

7. Statistics

Data were expressed as x±S. All numerical variables were analyzed with SPSSI1.5 software by one-way anava. T-test analysis was used for inter-group comparison. P<0.05 showed a statistically significant difference.

8 Results

7 days after modeling, loosen stool, diarrhea, bloody stool and BW loss were observed in mice of model group, while the stool was more formed in the treatment groups and occurrence of blood stool was slightly less than the model group. None of mice was found death during the experiment.

8.1 Effect on the DAI

By comparing disease activity in all groups, diarrhea occurred in the DSS group 3^rd˜5^thdays after drinking DSS and the occult blood stool was positive. On 5^th˜7th days, different levels of gross blood stool occurred. As such, diarrhea and occult blood stool occurred on 3^rd˜5^thdays in the positive control group and the andrographolide high-dose intracolonical group, but no obvious gross blood stool was found in mice after 5^thday. 14 days after administration, the number of bloody stool and diarrhea in the positive control group and andrographolide high-dose intracolonical group was less than the model group and DAI was significantly reduced (P<0.01) in comparison with the model group and there was a certain decrease tendency in the number of bloody stool and diarrhea in the positive control group and andrographolide high-dose intragastric group, having no statistically significant difference in comparison with the model group. Data were seen in Table 3.

TABLE 3

DAI score in all groups (x ± SD)

Dose

DAI

Groups	(mg/kg)	7^thday	14^thday

Model group		1.63 ± 0.69	2.27 ± 0.91
Positive control group	227.5	1.31 ± 0.14	1.10 ± 0.55**
Andrographolide low-dose	60	1.63 ± 0.99	2.01 ± 0.80
intragastric group
Andrographolide high-dose	180	1.43 ± 0.38	1.64 ± 0.83
intragastric group
Andrographolide low-dose	60	1.67 ± 0.65	1.77 ± 0.95
intracolonic group
Andrographolide high-dose	180	1.36 ± 0.64	1.35 ± 0.18**
intracolonic group

Compared with the model group:
*P < 0.05,
**P < 0.01

9. Conclusions

Up to now, mechanism of using DSS-induced model has not yet been entirely clear. Perhaps, it is associated with many aspects: macrophage dysfunction; intestinal flora imbalance; influence of DNA synthesis of colonic epithelium cell by DSS negative charge; inhabitation to epithelial cell proliferation and destroying mucosal barrier, which indicated a more ideal model for studying human IBD. As shown in results, incidence of gross blood stool was reduced just 7 days after administration in the andrographolide high-dose intracolonic group (180 mg/kg) and bloody stool and diarrhea ameliorated 14 days after administration. DAI score was significantly lower than the model group, which would be proven to have a certain protective effect on UC.

Pharmacological Research 3 Pharmacological Study of Andrographolide on TNBS-Induced UC

1. Aim

Efficacy of andrographolide on DSS-induced UC was evaluated.

2 Animals

84 SD rats of both sexes, weighing 180˜220 g, were provided from Guangdong Medical Experimental Animal Center and Certificate number SYXK 2008-0002. Raising conditions: 5 rats per cage raised in a group; temperature and humidity: 20-26° C. and 40-70%. The animals were lighted 10 h/14 h days and nights intermittently. The condition of raising room was always remained unchanged, ensuring reliability of experiment results. Animals were fed with the complete pellet feed (provided by Guangdong Medical Experimental Animal Center), free access to water via drinking bottle.
3. Apparatuses and reagents

3.1 Apparatuses

3.2 Reagents

3.2.1 Andrographolide was provided by Tasly Modern TCM Resource Inc of batch number: 20140508 with purity>95%;
3.2.2 Positive drug: mesalazine enteric-coated tablet was purchased from Jiamusi Luling Pharmaceutical Inc, Sunflower Pharmaceutical Group with batch number: 140225.
3.2.3 TNBS was purchased from SIGMA Inc with batch number: SLBG2566V.
4 Dose planning and grouping
4.1 Grouping: quarantine-qualified 84 rats were all used for making UC model. The successful rats were randomly divided into 6 groups: the model group, the positive drug group, the tested drug low-dose intragastric group, the tested drug high-dose intragastric group, the tested drug low-dose intracolonical group and the tested drug high-dose intracolonical group, 14 rats per group.
4.2 Dose: the same doses were adopted in both intragastric and intracolonical groups. Animals were not treated in the model group, mesalazine enteric-coated tablet (420 mg·kg⁻¹·d⁻¹) in the positive drug group, andrographolide (30 mg·kg⁻¹·d⁻¹) in the low-dose intragastric group, andrographolide (90 mg·kg⁻¹·d⁻¹) in the high-dose intragastric group, andrographolide (30 mg·kg⁻¹·d⁻¹) in the low-dose intracolonical group and andrographolide (90 mg·kg⁻¹·d⁻¹) in the high-dose intracolonical group.

5. Method

5.1 Method of making UC model: the rats were anesthetized. 2 mm-diameter latex tube was gently inserted into the position about 8 cm inside rats through anus and 50% ethanol TNBS solution (TNBS 125 mg/kg) was injected into intestinal cavity with an injector one time, 0.5 ml/rat. The tail of rats was lifted up for 30 s upended to make the model making agent fully infiltrate into the intestinal cavity of rats.
5.2 Method of administration: the same dose (1 mL/100 g) was adopted in both intragastric and intracolonical groups, once daily for consecutive 5 days.
5.3 Method for sampling in experiment: 2 hours after last administration, mice were killed by cervical vertebra dislocation and abdomen was opened to separate the colon. Along mesenteric side, the cavity scissored off washed with 4° C. normal saline and spread on a plastic plate.
5.4 Scoring method for colon gross morphological injury was referred to Scoring method for Mucosal Injury (Bjelkengren G, Aronsen K F, Augustsson N E, etc. Radioprotective effect of local administration of lysine vasopressin and triglycyl lysine vasopressin on the rectal mucosa in rats [J]. Acta Oncol, 1995, 34(4):487-92) to record the mucosal injury score: dot splinter blooding and small erythema (<1 mm): 1 score, piece blooding and big erythema (=1 mm): 3 score and erosion and ulcer: 5 score.
6 Observation indices
6.1 Observation: the daily general clinical symptoms in mice were observed from beginning to end of the experiment in mice and the stool, mental status and death status were recorded.
6.2 BW was recorded weekly from beginning to end of the experiment.
6.3 Colon injury was observed with naked eyes to score the colon gross morphological injury, including blood spot, piece blooding and ulcer.

7. Statistics

Data were expressed as x±S. All numerical variables were analyzed with SPSS13.0 software by one-way anava. T-test analysis was used for inter-group comparison. P<0.05 showed a statistically significant difference.

8. Results

8.1 effect on general conditions in rats
In the model group, on 1^stday, formless watery stool occurred in rats, increased stool frequency and accompanied with mucus on 2^nd˜3^rddays, lasting to the end of administration. In the positive control group (420 mg/kg), on 1^stday, formless watery stool occurred in rats and most of watery stool symptoms disappeared in rats on 5^th˜6^thdays. In the andrographolide high-dose intragastric and intracolonical groups (90 mg/kg), on 1^stday, formless watery stool occurred in rats, symptoms disappeared gradually on 2^nd˜3^rddays and totally disappeared on 4^th˜5^thdays.

8.2 Effect on UC

In the model group, the intestinal wall got thickened, ruga disappeared and small area of necrosis appeared, wide mucosal congestion, edma and ulcer seen in many sites. In the positive control group, the intestinal wall was thickened mildly, a part of ruga disappeared and small area of necrosis appeared, mucosal congestion, edma seen in many sites and the ulcer area was a little more reduced than the model group (P<0.05). In the andrographolide high-dose intracolonic group (90 mg/kg), symptoms of colon disease in rats was relieved obviously, no thickened intestinal wall found, the ruga normal, no significant mucusal congestion observed and edma and very small area of necrosis was visible topically only. The ulcer area was significantly more reduced than the model group and positive control group (P<0.01). Compared with the andrographolide high-dose intragastric group, the area was reduced (P<0.05). Data were seen in Table 4 and FIG. 2.

TABLE 4

effect of andrographolide on UC in rats by different administration routes

		Gross
		morphological	Ulcer ratio
Groups	Dose (mg/kg)	score	(%)

Model group		8.87 ± 1.71	61.87
Positive control group	420	3.16 ± 1.97*	35.45*
Andrographolide low-dose	30	6.71 ± 2.57	45.69*
intragastric group
Andrographolide high-dose	90	3.25 ± 1.98*	29.21**
intragastric group
Andrographolide low-dose	30	6.53 ± 2.12	38.54*
intracolonic group
Andrographolide high-dose	90	1.96 ± 1.09**	17.21**
intracolonic group

Compared with the model group:
*P < 0.05:
**P < 0.01

9. Conclusions

As a hapten, TNBS was mostly used in combination with ethanol to build model. The mechanism of this model was set forth as follows: mucosal injury was caused by using ethanol, TNBS, acting as hapten, infiltrated into colon tissue to form complete antigen by combining with high polymeric substance of tissue proteins to cause Th1 immune response and then lead to human CD alike inflammation.
As shown in the results, the andrographolide would ameliorate the diarrhea in TNBS-induced UC model of rats and have a certain effect on improving colon injury and ulcer ratio. Compared with the andrographolide high-dose intragastric group, the andrographolide high-dose intracolonic group had a stronger effect on improving colon injury and ulcer ratio (P<0.05). It was confirmed that rectally topical administration would have a better effect on improving UC than the introgastric administration, having a certain advantage of application.

Pharmacological Research 4 Therapeutical Effect on Zebra Fish Crohn's Disease Model

1. Exploration of concentration for efficacy evaluation in zebra fish Crohn's disease model
Fish farm water of wild-type AB strain zebra fish Crohn's disease model (1 L reverse osmosis water was added with 200 mg instant sea salt with the conductivity of 480˜510 μS/cm, pH value of 6.9˜7.2 and hardness of 53.7˜71.6 mg/L CaCO₃) was added with andrographolide in the concentration of 0 μg/mL, 0.1 μg/mL, 1 μg/mL, 10 μg/mL, 100 μg/mL and 500 μg/mL by volume. At each concentration, 30 zebra fish were treated, during which zebra fish death number was counted daily and the dead fish was removed in time. After treatment, the dearth number of zebra fish in each group was analyzed statistically to draw the optimum concentration-effect curve by using JMP software and calculate MNLC. By exploring concentration, solubility of the drug in DMSO was about 250 mg/ml and the administration method was to dissolve the drug into the fish farm water. It was found that when concentration ≧500 ug/ml, the drug began to precipitate and toxicity and dearth were not found in zebra fish. As a result of this, following four concentrations were selected for efficacy evaluation in Crohn's disease in this study: 50 μg/ml, 100 μg/ml, 250 μg/ml and 500 μg/ml.
2. Efficacy of andrographolide in zebra fish Crohn's disease model
According to the results of aforesaid concentration exploration, there were 4 levels set in treatment group to evaluate the efficacy in zebra fish Crohn's disease model, respectively 50 μg/ml, 100 μg/ml, 250 μg/ml and 500 μg/ml. Meanwhile, the positive control group (prednisolone in fish farm water at 10 μM), blank control group and Crohn's disease model group.
Crohn's disease model (mainly inflammation) was built by using TNBS and 30 zebra fish were randomly divided into each treating group. 48 hours after treating zebra fish with the andrographolide (at aforesaid 4 different concentrations), prednisolone (positive control group) and excipient (the excipient group), 10 zebra fish were randomly taken out in each group to observe and photographed. Image analysis software was used to analyze the pictures. Moreover, the intestine mocuscal thickness, intestinal diameter and intestinal area were observed carefully under the microscope and the intestinal diameter and intestinal area analyzed quantitatively. Therapeutical ratio of tested drug in zebra fish Crohn's disease was calculated in accordance with the intestinal area in each group. The calculation formula was present as follows:
Therapeutical ratio (%)=[1−(treatment group−blank control group)/(model group−blank control group)]×100%
Statistically analyzed results were expressed as mean±SD. Variance analysis was used for comparison among many groups and Dunnett's T test for comparison between two groups. P<0.05 showed a statistically significant difference.
As shown in FIG. 3, in the blank control group and excipient control group, the intestinal mocusa was smooth, morphologically integrated, intestinal tension clear and ruga regular. In the model group (Crohn's disease), zebra fish bowel was dilated, the bowel area increased, the intestinal mocusa thinned and the ruga unregular, flattened or vanished. In the positive control group, Crohn's disease zebra fish bowel dilation was reduced, the bowel area decreased, the intestinal tension and ruga recovered significantly. After being treated with low-dose andrographolide (50 ug/ml), Crohn's disease zebra fish bowel area was reduced to a certain degree, no obvious recovery of the intestinal tension and ruga found. High-dose of andrographolide (100, 250, 500 ug/ml), however, could effectively ameliorate mucosal morphology in colotis, bowel dilation reduced significantly, the bowel area close to normal, the intestinal tension and ruga nearly recovered.
As shown in FIG. 4 and FIG. 5, after being treated with the prednisolone in the positive control group, the bowel area was reduced by (76±9.3) %, namely the therapeutical ratio of 76±9.3% in zebra fish Crohn's disease model. The prednisolone (50 ug/ml) had a certain therapeutic effect on zebra fish Crohn's disease model and bowel area was reduced by (38±12.9)%. Compared with the model group, there was no statistically significant difference (P>0.05). In the andrographolide high-dose group (100, 250 and 500 ug/ml), zebra fish bowel areas were reduced respectively by (49±8.9)%, (65±14.7)% and (65±10.1)%, having statistically or extremely significant difference (P<0.05 and P<0.01) compared with the model group.
As shown in FIG. 6 and Table 5, a small amount of neutrophil was found in zebra fish intestinal tissue of blank control group and excipient control group. In the model group (Crohn's disease), bowel was dilated significantly, a large amount of neutrophil infiltrated in bowel tissue and inflammation obvious. The prednisolone of positive drug could significantly reduce neutrophil in Crohn's disease tissue. The inflammation regression ratio was (72±4.14)%, having extremely statistically significant difference (p<0.001) compared with the model group (Crohn's disease). In the andrographolide high-dose group (100, 250 and 500 ug/ml), the neutrophil infiltration in bowel tissue of Crohn's disease zebra fish was reduced, facilitating inflammation regression. The inflammation regression ratio in bowel tissue of Crohn's disease were (45±3.74)%, (46±3.74)%, (63±4.42)% and (79±8.98)%, having an extremely significant difference (p<0.001) compared with the model group (Crohn's disease).

TABLE 5

fluorescence evaluation on neutrophil in Crohn's disease zebra fish model
(x ± SE, n = 10)

			Inflammation
	Neutrophil	Ratio of blank	regression
Group	count	control group	ratio (%)

Blank control group	9 ± 0.53	1.00 ± 0.06	—
Excipient control group	10 ± 0.27	1.07 ± 0.03	—
Model group	24 ± 1.17	2.70 ± 0.13	—
Positive control group	14 ± 0.60*	1.53 ± 0.07*	72 ± 4.1*
Andrographolide	18 ± 0.54*	1.97 ± 0.06*	45 ± 3.7*
(50 μg/ml)
Andrographolide	17 ± 0.54*	1.96 ± 0.06*	46 ± 3.7*
(100 μg/ml)
Andrographolide	15 ± 0.64*	1.67 ± 0.07*	63 ± 4.4*
(250 μg/ml)
Andrographolide	13 ± 1.30*	1.40 ± 0.15*	79 ± 9.0*
(500 μg/ml)

Compared with the model group,
*P < 0.001

Pharmacological Research 5 Pharmacological Study of pH-Dependent Enteric Targeting Preparation

In Vitro Release Experiment

In vitro release of micropellets was determined, which were prepared by two formulas selected from aforesaid optimized ones. That is to say, 150 mg of andrographolide enteric targeting micropellet was loaded into a capsule to measure in vitro release. Dissolution 1 method of Chinese Pharmacopeia was used at rotating speed of 100 rpm with different pH salt solution (1000 ml) as release medium. According to requirement of Chinese Pharmacopeia, after sampling, HPLC was used to measure release amount of drug during different period of time. The results were seen in FIG. 7 and FIG. 8. Wherein, #1 formula had the fastest release rate and #7 formula slowest. The other formulas stayed somewhere in between.

EXAMPLE

1. Preparative Example for Andrographolide Enteric-Coated Tablet and Capsule

Example 1-1 Preparation of Enteric-Coated Tablet

Extraction of andrographolide: the leaves of Andrographis paniculata was soaked in 95% (v/v) ethanol and the resulting ethanol liquid was decolored with activated carbon and the ethanol is recovered by distillation to give a concentrated liquid. The liquid was allowed to stand still to have coarse crystal. Said coarse crystal was added with 15 times (15×) 95% (v/v) ethanol, dissolved by heating, decolored with activated carbon and filtered immediately. The resultant liquid was allowed to stand still to give a light-yellow crystal by recrystallization. The obtained crystal is refined by washing with distilled water, chloroform and methol to have the final product of andrographolide.
Appropriate amount of excipient was added into afore-obtained andrographolide to prepare the enteric-coated tablet by a conventional method.
The extracting method was the same as the EXAMPLE 1-1.
Appropriate amount of excipient was added into afore-obtained andrographolide to prepare the enteric-coated capsule by a conventional method.

Example 1-3 Preparation of Granule


	andrographolide	100 g
	microcrystalline cellulose	50 g
	lactose	50 g
	starch	50 g
	surcose	250 g

	to prepare 500 g granule

Method:

The extracting method was the same as the EXAMPLE 1-1. In addition, andrographolide and other excipients were screened with 100-mesh sifter, mixed well to prepare into the soft material by using appropriate amount of water, granulated with 14-mesh and sorted out.

Example 1-4 Preparation of Intestinal Suppository

The materials were mixed well according to the formula of EXAMPLE 1-3, into which the matrix was added to prepare the intestinal suppository by a conventional method.

2. Examples and Preparative Examples of Andrographolide pH-Dependent Enteric Targeting Micropellet

It should be noted that the percentage of examples preparative examples referred to percentage by weight.

Example 2-1

Andrographolide enteric targeting micropellet was composed of a blank pellet, a drug layer and an enteric coating layer, wherein said drug layer was composed of following formula (g):


				anti-
Blank		Eurdragit		sticking
pellet	Andrographolide	S100	plasticizer	agent	Surfactant

200	50	15	2.1	4.5	1.00

Wherein, said blank pellet was a blank sucrose pellet with a diameter of 600 μm; said plasticizer was the triethyl citrate; said anti-sticking agent was the talc and said surfactant was the SDS (sodium dodecyl sulfate);
The enteric coating layer included the Eudragit L100-55, plasticizer and anti-sticking agent and they were selected as depicted in the drug layer. The amount of plasticizer was 15 wt % of the Eudragit L100-55, the anti-sticking agent 30 wt % and weight gain of coating 5%.

Example 2-2


				anti-
Blank		Eurdragit		sticking
pellet	Andrographolide	S100	plasticizer	agent	Surfactant

200	66	20	3	6	1.32

Wherein, said blank pellet was a blank sucrose pellet with a diameter of 200 μm; said plasticizer was the dibutyl sebacate; said anti-sticking agent was the glyceryl monostearate and said surfactant was the Tween-80;
The enteric coating layer included the Eudragit L100-55, plasticizer and anti-sticking agent and they were selected as depicted in the drug layer. The amount of plasticizer was 15 wt % of the Eudragit L100-55, the anti-sticking agent 30 wt % and weight gain of coating 30%.

Example 2-3


				anti-
Blank		Eurdragit		sticking
pellet	Andrographolide	S100	plasticizer	agent	Surfactant

200	66	22	3.3	6.6	1.32

Wherein, said blank pellet was a blank sucrose pellet with a diameter of 400 μm; said plasticizer was the propanediol; said anti-sticking agent was the talc and said surfactant was the SDS;
The enteric coating layer included the Eudragit L100-55, plasticizer and anti-sticking agent and they were selected as depicted in the drug layer. The amount of plasticizer was 15 wt % of the Eudragit L100-55, the anti-sticking agent 30 wt % and weight gain of coating 8%.

Example 2-4


				anti-
blank		Eurdragit		sticking
pellet	andrographolide	L30D-55	plasticizer	agent	surfactant

200	44	13	2	3	0

Wherein, said blank pellet was a blank sucrose pellet with a diameter of 500 μm; said plasticizer was the propanediol and said anti-sticking agent was the talc;
The enteric coating layer included the Eudragit L30D-55, plasticizer and anti-sticking agent and they were selected as depicted in the drug layer. The amount of plasticizer was 15 wt % of the Eudragit L30D-55, the anti-sticking agent 30 wt % and weight gain of coating 20%.

Example 2-5


				anti-
blank		Eurdragit		sticking
pellet	andrographolide	S100	plasticizer	agent	surfactant

200	58	18	3.6	5.4	1.16

Wherein, said blank pellet was a blank sucrose pellet with a diameter of 500 μm; said plasticizer was the PEG and said anti-sticking agent was the talc;
The enteric coating layer included the Eudragit L100-55, plasticizer and anti-sticking agent and they were selected as depicted in the drug layer. The amount of plasticizer was 15 wt % of the Eudragit L100-55, the anti-sticking agent 30 wt % and weight gain of coating 28%.

Example 2-6


				anti-
blank		Eurdragit		sticking
pellet	andrographolide	S100	plasticizer	agent	surfactant

200	15.2	40	6	12	0.4

Wherein, said blank pellet was a blank sucrose pellet with a diameter of 500 μm; said plasticizer was the PEG and said anti-sticking agent was the talc;
The enteric coating layer included the Eudragit L100-55, plasticizer and anti-sticking agent and they were selected as depicted in the drug layer. The amount of plasticizer was 15 wt % of the Eudragit L100-55, the anti-sticking agent 30 wt % and weight gain of coating 15%.

Example 2-7


				anti-
blank		Eurdragit		sticking
pellet	andrographolide	S100	plasticizer	agent	surfactant

200	52	74	13.5	27	1.26

Wherein, said blank pellet was a blank sucrose pellet with a diameter of 500 m; said plasticizer was the triethyl citrate and said anti-sticking agent was the talc;
The enteric coating layer included the Eudragit L100-55, plasticizer and anti-sticking agent and they were selected as depicted in the drug layer. The amount of plasticizer was 15 wt % of the Eudragit L100-55, the anti-sticking agent 30 wt % and weight gain of coating 15%.

Example 2-8

Wherein, said blank pellet was a blank sucrose pellet with a diameter of 600 μm; said plasticizer was the triethyl citrate; said anti-sticking agent was the talc and said surfactant was the SDS;
The enteric coating layer included the Eudragit L100-55, plasticizer and anti-sticking agent and they were selected as depicted in the drug layer. The amount of plasticizer was 15 wt % of the Eudragit L100-55, the anti-sticking agent 30 wt % and weight gain of coating 8%.

Example 2-9

Andrographolide enteric targeting micropellet was composed of a blank pellet, a drug layer and an enteric coating layer, wherein said drug layer contained the andrographolide, the polymer A dissolved under condition of pH≧7.0: said ratio of the andrographolide and polymer A is 1:2 by weight; weight gain of the drug layer was 20 wt %;
Said enteric coating layer contained the polymer B dissolved under condition of pH≧5.5 and weight gain of the enteric coating layer was 8 wt %. The proportion of plasticizer and anti-sticking agent was the same with the one of polymer A and plasticizer and anti-sticking agent in the drug layer.
Wherein, said polymer A was the copolymer of methacrylic acid and methyl methacrylate and polymer B is the copolymer of methacrylic acid and ethyl acrylate.
Aforesaid plasticizer was the triethyl citrate and anti-sticking agent the talc.

Example 2-10

Andrographolide enteric targeting micropellet was composed of a blank pellet, a drug layer and an enteric coating layer, wherein said drug layer contained the andrographolide, the polymer A dissolved under condition of pH≧7.0; said ratio of the andrographolide and polymer A is 1:0.2 by weight; weight gain of the drug layer was 100 wt %; said enteric coating layer contained the polymer B dissolved under condition of pH≧5.5 and weight gain of the enteric coating layer was 20 wt %.
Said polymer A was the copolymer of methacrylic acid and methyl methacrylate in a ratio of 1:2 and polymer B was the copolymer of methacrylic acid and ethyl acrylate in a ratio of 1:1.
Aforesaid plasticizer was the dibutyl sebacate and anti-sticking agent the glyceryl monostcarate.

Example 2-11

Andrographolide enteric targeting micropellet was composed of a blank pellet, a drug layer and an enteric coating layer, wherein said drug layer contained the andrographolide, the polymer A dissolved under condition of pH≧7.0; said ratio of the andrographolide and polymer A is 1:1.5 by weight; weight gain of the drug layer was 30 wt %; said enteric coating layer contained the polymer B dissolved under condition of pH≧5.5 and weight gain of the enteric coating layer was 10 wt %. The proportion of plasticizer and anti-sticking agent was the same with the one of polymer A and plasticizer and anti-sticking agent in the drug layer.
Said polymer A was the copolymer of methacrylic acid and methyl methacrylate in a ratio of 1:2 and polymer B was the copolymer of methacrylic acid and ethyl acrylate in a ratio of 1:1.

Example 2-12

Andrographolide enteric targeting micropellet was composed of a blank pellet, a drug layer and an enteric coating layer, wherein said drug layer contained the andrographolide, the polymer A dissolved under condition of pH≧7.0, the plasticizer, anti-sticking agent, pigment, hydrophilic polymer and surfactant; said ratio of the andrographolide and polymer A is 1:2 by weight; weight gain of the drug layer was 100 wt %; said plasticizer was selected from the triethyl citrate, accounting for 10 wt % of the polymer A; said anti-sticking agent was selected from the talc, accounting for 25 wt % of the polymer A.
Said enteric coating layer contained the polymer B dissolved under condition of pH≧5.5 and weight gain of the enteric coating layer was 20 wt %. The proportion of plasticizer and anti-sticking agent was the same with the one of polymer A and plasticizer and anti-sticking agent in the drug layer.
Said polymer A was the copolymer of methacrylic acid and methyl methacrylate in a ratio of 1:2 and polymer B was the copolymer of methacrylic acid and ethyl acrylate in a ratio of 1:1.
Aforesaid plasticizer was the triethyl citrate and anti-sticking agent the talc.

Example 2-13

Andrographolide enteric targeting micropellet was composed of a blank pellet, a drug layer and an enteric coating layer, wherein said drug layer contained the andrographolide, the polymer A dissolved under condition of pH≧7.0, the plasticizer, anti-sticking agent, pigment, hydrophilic polymer and surfactant; said ratio of the andrographolide and polymer A is 1:0.5 by weight; weight gain of the drug layer was 80 wt %; said plasticizer was selected from the dibutyl sebacate, accounting for 70 wt % of the polymer A; said anti-sticking agent was selected from the talc, accounting for 100 wt % of the polymer A.
Said enteric coating layer contained the polymer B dissolved under condition of pH≧5.5 and weight gain of the enteric coating layer was 18 wt %. The proportion of plasticizer and anti-sticking agent was the same with the one of polymer A and plasticizer and anti-sticking agent in the drug layer.
Said polymer A was the copolymer of methacrylic acid and methyl methacrylate in a ratio of 1:2 and polymer B was the copolymer of methacrylic acid and ethyl acrylate in a ratio of 1:1.
Aforesaid plasticizer was the triethyl citrate and anti-sticking agent the talc.

Example 2-14

Andrographolide enteric targeting micropellet was composed of a blank pellet, a drug layer and an enteric coating layer, wherein said drug layer contained the andrographolide, the polymer A dissolved under condition of pH≧7.0, the plasticizer, anti-sticking agent, pigment, hydrophilic polymer and surfactant; said ratio of the andrographolide and polymer A is 1:1 by weight: weight gain of the drug layer was 50 wt %; said plasticizer was selected from the propanediol, accounting for 20 wt % of the polymer A; said anti-sticking agent was selected from the talc, accounting for 30 wt % of the polymer A.
Said enteric coating layer contained the polymer B dissolved under condition of pH≧5.5 and weight gain of the enteric coating layer was 15 wt %. The proportion of plasticizer and anti-sticking agent was the same with the one of polymer A and plasticizer and anti-sticking agent in the drug layer.
Said polymer A was the copolymer of methacrylic acid and methyl methacrylate in a ratio of 1:2 and polymer B was the copolymer of methacrylic acid and ethyl acrylate in a ratio of 1:1.
Aforesaid plasticizer was the triethyl citrate and anti-sticking agent the talc.

Example 2-15

Andrographolide enteric targeting micropellet was composed of a blank pellet, a drug layer and an enteric coating layer, wherein said drug layer contained the andrographolide, the polymer A dissolved under condition of pH≧7.0, the plasticizer, anti-sticking agent, pigment, hydrophilic polymer and surfactant; said ratio of the andrographolide and polymer A is 1:1.5 by weight; weight gain of the drug layer was 60 wt %; said plasticizer was selected from the PEG, accounting for 50 wt % of the polymer A; said anti-sticking agent was selected from the talc, accounting for 80 wt % of the polymer A.
Said enteric coating layer contained the polymer B dissolved under condition of pH≧5.5 and weight gain of the enteric coating layer was 16 wt %. The proportion of plasticizer and anti-sticking agent was the same with the one of polymer A and plasticizer and anti-sticking agent in the drug layer.
Said polymer A was the copolymer of methacrylic acid and methyl methacrylate in a ratio of 1:2 and polymer B was the copolymer of methacrylic acid and ethyl acrylate in a ratio of 1:1.
Aforesaid plasticizer was the triethyl citrate and anti-sticking agent the talc.

Example 2-16

Andrographolide enteric targeting micropellet was composed of a blank pellet, a drug layer and an enteric coating layer, wherein said drug layer contained the andrographolide, the polymer A dissolved under condition of pH≧7.0, the plasticizer, anti-sticking agent, pigment, hydrophilic polymer and surfactant; said ratio of the andrographolide and polymer A is 1:1.5 by weight; weight gain of the drug layer was 60 wt %; said plasticizer was selected from the PEG, accounting for 50 wt % of the polymer A; said anti-sticking agent was selected from the glyceryl monostearate, accounting for 20 wt % of the polymer A.
Said enteric coating layer contained the polymer B dissolved under condition of pH≧5.5 and weight gain of the enteric coating layer was 16 wt %. The proportion of plasticizer and anti-sticking agent was the same with the one of polymer A and plasticizer and anti-sticking agent in the drug layer.
Said polymer A was the copolymer of methacrylic acid and methyl methacrylate in a ratio of 1:2 and polymer B was the copolymer of methacrylic acid and ethyl acrylate in a ratio of 1:1.
Aforesaid plasticizer was the triethyl citrate and anti-sticking agent the talc.

Example 2-17

Andrographolide enteric targeting micropellet was composed of a blank pellet, a drug layer and an enteric coating layer, wherein said drug layer contained the andrographolide, the polymer A dissolved under condition of pH≧7.0, the plasticizer, anti-sticking agent, pigment, hydrophilic polymer and surfactant; said ratio of the andrographolide and polymer A is 1:1.5 by weight; weight gain of the drug layer was 60 wt %; said plasticizer was selected from the PEG, accounting for 50 wt % of the polymer A; said anti-sticking agent was selected from the glyceryl monostearate, accounting for 20 wt % of the polymer A.
Said enteric coating layer contained the polymer B dissolved under condition of pH≧5.5 and weight gain of the enteric coating layer was 16 wt %. The proportion of plasticizer and anti-sticking agent was the same with the one of polymer A and plasticizer and anti-sticking agent in the drug layer.
Said polymer A was the copolymer of methacrylic acid and methyl methacrylate in a ratio of 1:2 and polymer B was the copolymer of methacrylic acid and ethyl acrylate in a ratio of 1:1.
Aforesaid plasticizer was the triethyl citrate and anti-sticking agent the talc.
Micropellets in aforesaid Examples were prepared by following method.

Preparative Example 2-1

(1) applying drug to the blank pellet
a). dispersing the polymer A into a pharmaceutical solvent to let them dissolve fully by mechanical stirring: adding the excipient into the polymer A solution and then adding the andrographolide to have the polymer A coating solution by well stirring;
b). weighing the blank pellet and charging into a fluidized bed; adjusting air flow to such a degree that the micropellet was well fluidized; opening the heating device and until temperature of the material reaches preset value, the peristaltic pump was started to make the polymer A coating solution atomized through a spray gun to obtain a drug-loading micropellet by dispersing on the surface of said blank pellet;
(2) preparation of the enteric coating layer
a). dispersing the polymer B into a pharmaceutical solvent to let them dissolve fully by mechanical stirring; adding the excipient into the polymer B solution to have the polymer B coating solution by well stirring;
b). charging aforesaid drug-loading micropellets into a bottom-spray device of the fluidized bed, and the polymer B coating solution is uniformly spread to form the enteric coating layer; the weight gain is 5 wt %.

Preparative Example 2-2

(1) applying drug to the blank pellet
a). dispersing the polymer A into a pharmaceutical solvent to let them dissolve fully by mechanical stirring; adding the excipient into the polymer A solution and then adding the andrographolide to have the polymer A coating solution by well stirring;
b). weighing the blank pellet and charging into a fluidized bed; adjusting air flow to such a degree that the micropellet was well fluidized; opening the heating device and until temperature of the material reaches preset value, the peristaltic pump was started to make the polymer A coating solution atomized through a spray gun to obtain a drug-loading micropellet by dispersing on the surface of said blank pellet; in
(2) preparation of the enteric coating layer
a). dispersing the polymer B into a pharmaceutical solvent to let them dissolve fully by mechanical stirring; adding the excipient into the polymer B solution to have the polymer B coating solution by well stirring;
b). charging aforesaid drug-loading micropellets into a bottom-spray device of the fluidized bed, and the polymer B coating solution is uniformly spread to form the enteric coating layer, the weight gain is 30 wt %.

Preparative Example 2-3

a). dispersing the polymer A into a pharmaceutical ethanol to make the content of polymer A at 5 wt %; fully dissolving by high-speed shearing mechanical stirring and continuing to stir uniformly; adding the excipient of the plasticizer, anti-sticking agent and the surfactant of sodium dodecyl sulfate into the polymer A solution and then adding the andrographolide to have the polymer A coating solution by well stirring;
b). weighing the blank sucrose pellet in a diameter of 200 μm and charging into a fluidized bed; adjusting air flow to such a degree that the micropellet is well fluidized; opening the heating device to keep the temperature of the material at 25° C. and until the temperature reaches preset value, the peristaltic pump is started to make the polymer A coating solution atomized through a spray gun to obtain a drug-loading micropellet by dispersing on the surface of said blank pellet;
(2) preparation of the enteric coating layer
a). dispersing the polymer B into the pharmaceutical ethanol to let them dissolve fully by high-speed shearing mechanical stirring; adding the excipient of the plasticizer and anti-sticking agent into the polymer B solution to have the polymer B coating solution by well stirring;
b). charging aforesaid drug-loading micropellets into a bottom-spray device of the fluidized bed, and the polymer B coating solution is uniformly spread to form the enteric coating layer, the weight gain is 8 wt %.

Preparative Example 2-4

(1) applying drug to the blank pellet
a). dispersing the polymer A into a pharmaceutical ethanol to make the content of polymer A at 5 wt %; fully dissolving by high-speed shearing mechanical stirring and continuing to stir uniformly; adding the excipient of the plasticizer, anti-sticking agent and the surfactant of sodium dodecyl sulfate into the polymer A solution and then adding the andrographolide to have the polymer A coating solution by well stirring;
b). weighing the blank sucrose pellet in a diameter of 600 μm and charging into a fluidized bed; adjusting air flow to such a degree that the micropellet is well fluidized; opening the heating device to keep the temperature of the material at 35° C. and until the temperature reaches preset value, the peristaltic pump is started to make the polymer A coating solution atomized through a spray gun to obtain a drug-loading micropellet by dispersing on the surface of said blank pellet;
(2) preparation of the enteric coating layer
a). dispersing the polymer B into the pharmaceutical ethanol to let them dissolve fully by high-speed shearing mechanical stirring; adding the excipient of the plasticizer and anti-sticking agent into the polymer B solution to have the polymer B coating solution by well stirring;
b). charging aforesaid drug-loading micropellets into a bottom-spray device of the fluidized bed, and the polymer B coating solution is uniformly spread to form the enteric coating layer the weight gain is 20 wt %.

Preparative Example 2-5

(1) applying drug to the blank pellet
a). dispersing the polymer A into a pharmaceutical ethanol to make the content of polymer A at 5 wt %; fully dissolving by high-speed shearing mechanical stirring and continuing to stir uniformly; adding the excipient of the plasticizer, anti-sticking agent and the surfactant of sodium dodecyl sulfate into the polymer A solution and then adding the andrographolide to have the polymer A coating solution by well stirring;
b). weighing the blank sucrose pellet in a diameter of 400 μm and charging into a fluidized bed; adjusting air flow to such a degree that the micropellet is well fluidized; opening the heating device to keep the temperature of the material at 32° C. and until the temperature reaches preset value, the peristaltic pump is started to make the polymer A coating solution atomized through a spray gun to obtain a drug-loading micropellet by dispersing on the surface of said blank pellet;
(2) preparation of the enteric coating layer
a). dispersing the polymer B into the pharmaceutical ethanol to let them dissolve fully by high-speed shearing mechanical stirring; adding the excipient of the plasticizer and anti-sticking agent into the polymer B solution to have the polymer B coating solution by well stirring;
b). charging aforesaid drug-loading micropellets into a bottom-spray device of the fluidized bed, and the polymer B coating solution is uniformly spread to form the enteric coating layer; the weight gain is 15 wt %.

Preparative Example 2-6

Micropellets obtained from Example 2-1˜2-17 were prepared to have the conventional granule and capsule.

Claims

What is claimed is:

1. A method of preparing an andrographolide enteric micropellet of composed of a blank pellet, a drug layer and an enteric coating layer, wherein said drug layer contains andrographolide, a polymer A that dissolves under a condition of pH≧7.0 and an excipient; where said ratio of the andrographolide and polymer A is 1:2˜1:0.2 by weight; and wherein the weight gain of the drug layer is 20 wt %˜100 wt %; said enteric coating layer containing a polymer B that dissolves under a condition of pH≧5.5 and an excipient; and wherein the weight gain of the enteric coating layer is 5 wt %˜30 wt %,

comprising the following steps:

(1) applying andrographolide to a blank micropellet by

a). dispersing the polymer A into a pharmaceutical solvent to allow the polymer A to dissolve in the solvent fully by mechanical stirring to obtain a polymer A solution: adding an excipient into the polymer A solution and then adding andrographolide to the polymer A solution and then stirring well to provide a polymer A coating solution;

b). weighing the blank micropellet and charging the blank micropellet into a fluidized bed; adjusting the air flow in the fluidized bed to such a degree that the micropellet is well fluidized; starting a heating device and heating the well fluidized blank micropellet until temperature of the well fluidized blank micropellet material reaches a preset value; starting a peristaltic pump which atomizes the polymer A coating solution through a spray gun to uniformly disperse the polymer A coating solution on the surface of the blank micropellet to obtain a drug-loaded micropellet;

(2) preparing the enteric coating layer by

a). dispersing the polymer B into a pharmaceutical solvent to allow the polymer B to dissolve in the solvent fully by mechanical stirring to obtain a polymer B solution; adding an excipient into the polymer B solution and then stirring well to provide a polymer B coating solution;

b). charging aforesaid andrographolide-loaded micropellet into a bottom-spray device of a fluidized bed, and uniformly spreading the polymer B coating solution to form the enteric coating layer; wherein the weight gain of the enteric coating layer is 5 wt %˜30 wt %.

2. The preparation method according to claim 8 comprising following steps:

(1) applying drug to the blank micropellet by

a). dispersing the polymer A into the pharmaceutical solvent which is ethanol such that the content of polymer A is 5 wt %; fully dissolving the polymer A in the ethanol by mechanical stirring to form the polymer A solution and continuing to uniformly stir the polymer A solution; adding excipient which is plasticizer, anti-sticking agent and the surfactant sodium dodecyl sulfate into the polymer A solution, and then adding the andrographolide to the polymer A solution and then stirring well to provide the polymer A coating solution;

b). weighing the blank micropellet which is a sucrose micropellet having a diameter of 200˜600 μm and charging the sucrose micropellet into a fluidized bed; adjusting air flow to such a degree that the sucrose micropellet is well fluidized; starting the heating device and heating the well fluidized sucrose micropellet until the temperature reaches the preset value of 25˜35° C. starting the peristaltic pump which atomizes the polymer A coating solution through a spray gun to uniformly disperse the polymer A coating solution on the surface of the sucrose micropellet to obtain a drug-loaded sucrose micropellet;

(2) preparing the enteric coating layer by

a). dispersing the polymer B into the pharmaceutical solvent ethanol to allow the polymer B to dissolve in the ethanol fully by high-speed shearing mechanical stirring to obtain a polymer B solution; adding the excipient of the plasticizer and anti-sticking agent into the polymer B solution and stirring well to provide the polymer B coating solution;

b). charging aforesaid drug-loaded sucrose micropellet into a bottom-spray device of the fluidized bed, and uniformly spreading the polymer B coating solution to form the enteric coating layer; wherein the weight gain of the enteric coating layer is 8 wt %˜20 wt %.

3. The method of claim 1 wherein the ratio of the andrographolide and polymer A is 1:2.5˜1:0.5 by weight.

4. The method of claim 1 wherein the excipient of the drug layer is selected from the group consisting of plasticizer, antisticking agent, pigment, hydrophilic polymer and surfactant and wherein the excipient of the enteric coating layer is selected from the group consisting of plasticizer and antisticking agent.

5. The method of claim 4 wherein the ingredients are present in proportion by weight parts: blank micropellet:andrographolide:polymer A:plasticizer:antisticking agent:surfactant=200: (10-100):(10-100):(1-15):(1-30):(0-3).

6. The method of claim 4 wherein the ingredients are present in proportion by weight parts: blank micropellet:andrographolide:polymer A:plasticizer:antisticking agent:surfactant=200:(15-66):(13-74):(2-13.5):(3-27):(0-1.32).

7. The method of claim 4 wherein the ingredients are present in proportion by weight parts: blank micropellet:andrographolide:polymer A:plasticizer:antisticking agent:surfactant=200: (20-50):(30-60):(5-10):(5-20):(0.5-1.2).