preparartion and in-vitro evaluations on...

澳門大學碩士論文

I

PREPARARTION AND IN-VITRO EVALUATIONS ON

GASTRO-FLORATING TABLETS AND PELLETS OF

GENTIOPICROSIDE

by

Yan WANG

Master of Science

State Key Laboratory of Quality Research in Chinese Medicine

(University of Macau)

Institute of Chinese Medical Sciences

University of Macau

2013


II

PREPARARTION AND IN-VITRO EVALUTATIONS ON GASTRO-FLORATING TABLETS

AND PELLETS OF GENTIOPICROSIDE

by

Yan WANG

A thesis submitted in partial fulfillment of the

requirements for the degree of

Master of Science

State Key Laboratory of Quality Research in Chinese Medicine

(University of Macau)

Institute of Chinese Medical Sciences

University of Macau

2013

Approved by __________________________________________________________

Supervisor

_________________________________________________________

_________________________________________________________

_________________________________________________________

Date


III

In presenting this thesis in partial fulfillment of the requirements for a

Master's degree at the University of Macau, I agree that the Library and the

Institute of Chinese Medical Sciences make its copies freely available for

inspection. However, reproduction of this thesis for any purposes or by

any means shall not be allowed without my written permission.

Authorization is sought by contacting the author at

Address:

Telephone:

Fax:

E-mail:

Signature ______________________

Date __________________________


IV

龍膽苦苷胃漂浮片和胃漂浮微丸的製

備及體外釋藥研究

碩士學位論文

研究生姓名：王焱

導師：鄭穎

專業：中藥學

日期： 2013 年 1 月

中藥品質研究國家重點實驗室(澳門大學)

澳門大學中華醫藥研究院

澳門大學


V

中文摘要

龍膽總苷是龍膽科植物秦艽、龍膽和獐牙菜等藥材的藥用活性成分。龍膽苦

苷（Gentiopicroside）為裂環烯醚萜類化合物，是龍膽總苷中的主要成分之一，

具有抗炎、鎮痛、保肝、健胃、利膽等作用，在臨床上主要以龍膽科植物藥材原

粉的形式入藥，在複方製劑中作為君藥使用，具有清熱燥濕、清瀉肝火和改善肝

功能等功效。製劑形式目前主要為普通固體口服製劑。因龍膽苦苷油水分配係數

小且半衰期短，所以口服後效果較差，生物利用度低。故為提高其生物利用度，

改善其臨床療效，本論文對將龍膽總苷製成胃內滯留漂浮型緩釋製劑，對龍膽總

苷的製備和胃漂浮微丸和胃漂浮片製劑成型進行了探索性研究。

本論文研究內容為：（1）龍膽總苷原料藥的製備，論文對原料藥製備藥材的

選擇、藥用活性成分龍膽總苷的提取、精製和中試工藝制進行了研究；（2）龍膽

總苷胃內滯留漂浮緩釋片的製備及其體外製劑學評價，論文對胃內滯留漂浮型緩

釋片的處方篩選、製劑工藝與關鍵技術參數的確定、小試樣品的體外釋藥曲線進

行了研究；（3）龍膽總苷胃漂浮緩釋微丸的研究及其體外製劑學評價，論文對胃

內滯留漂浮型緩釋微丸輔料處方，成型工藝及體外釋放特性等方面均進行了研

究。

研究方法和結果：本論文根據龍膽與秦艽藥材中龍膽總（苦）苷的提取率，

以及藥材價格等因素進行性價比綜合考慮，確定了以秦艽為龍膽總苷的提取用藥

材；經小試及中試實驗驗證，確定了以水-乙醇熱提工藝並經 D-101 大孔樹脂精

製的龍膽總苷製備工藝，同時制訂了龍膽總苷品質標準草案，為製劑的研究提供

了純度滿足要求且組成相對恒定的原料。


VI

本論文通過對凝膠骨架材料、膨脹劑和產氣物質的篩選等方式確定了龍膽

總苷胃漂浮片所用的輔料種類，採用混料設計方法對各輔料配比進行了優化，並

對 12 個處方的片劑進行了硬度、密度、脆碎度、溶脹溶蝕、起漂時間、釋放度

等體外製劑學性能考察，從而確定了成型工藝研究。

研究結果表明：以秦艽為提取用藥材，以水-醇熱提並經大孔樹脂精製的龍

膽總苷製備工藝產率可達 50%以上，且品質符合擬定標準要求。用優化確定的處

方工藝製備的胃內滯留漂浮型龍膽總苷緩釋片可以滿足其製劑學要求。

本論文的研究結果初步證明：將龍膽總苷製成胃內滯留漂浮型緩釋片，可以

大大延遲其在胃內的滯留時間，從而提高其在吸收部位的濃度，有望達到提高其

生物利用度，改善其臨床療效的目地。同時，本論文還將龍膽苦苷載於胃漂浮微

丸劑型，同樣可以達到定位緩釋的目的。另外，本論文對於同類中藥藥物的製

劑改良和中藥製劑技術的發展都是一種有益的探索嘗試。


VII

Abstract

Total glycosides of Radix Gentianae are the active ingredients of some Chinese

medicinal plants, such as Radix GentianaeMacrophyllae, Radix Gentianae and

Swertiapatens. Gentiopicroside belongs to secoiridoid and is one of the main

components in Gentianae total glycosides. It has some medical effects, for example,

anti-inflammatory, analgesia, and benefit effects for liver, stomach and gallbladder. In

clinical practice, Gentianaceae powder is used as a medicine with heat-clearing and

damp-drying, clearing liver fire and improving liver functions. At present, its main

dosage form is oral solid dosage forms. Because the partition coefficient of

Gentiopicroside is relative low, it could not be well absorbed in-vivo with short

half-life. The low oral bioavailability leads to low efficacy in clinical use. Therefore,

the aim of this study is to develop gastric floating sustained-release solid dosage

forms of Gentiopicroside.

Study in this paper consist of the following parts: 1) Preparing of raw materials

Gentian total glycosides, which includes the choice of crude herbs for extraction and

purification; 2) Preparation and in-vitro evaluation of gastric floating

sustained-release tablets and pellets of Gentian total glycosides. Specifically, it

includes the formulation screening, establishing of the preparing technology and key

technical parameters, as well as the study of in-vitro drug release.

Results showed that based on the extraction yield and the medicinal materials price,

GentianaMacrophylla is selected as the medicinal materials for extraction of

Gentianae Total Glycosides. Both of bench-scale experiment and pilot-scale test have

confirmed that the technology of water-alcohol hot extract and D-101 macroporous

resin could be used to purify Gentianae Total Glycosides. Meanwhile, quality control

standard of Gentianae Total Glycosides has been set up to control raw materials,

which has relative constant composition and meet the purity requirements.

app:ds:extraction

app:ds:purification

app:ds:Preparaing%20Technology


VIII

In this paper, types of excipients were confirmed through screening on hydrophilic

matrix material, swelling agent and aero-genesis materials. Then mixture design was

utilized to optimize the formulation. Meanwhile, pharmaceutical performance of 12

types tablets including the tablet hardness, density, friability, swelling and erosion

capacity, beginning floating time and drug release text, as well as the tablet residence

time in volunteer stomach and animal blood concentration were investigated. The

results also indicated that the extraction yield was satisfied. The optimized

formulation of gastric floating sustained-release tablet could be retained in volunteers’

stomach for at least 6 hours.

As a summary, this study has successfully set up the extraction and purification

technology of Gentianae Total Glycosides from GentianaMacrophylla. The gastric

residence time of GTG in absorption site were extended by preparation as gastric

floating sustained-release tablets or pellets, which may enhance their oral

bioavailability and clinical efficacy. This study has set a good example to the dosage

form modernization of Chinese Medicine.


IX

List of Figures

Figure 1.1 Photographs of Radix Gentianae (material,decoction pieces) .......................................... 1

Figure 1.2 Chemical structure of GTD .............................................................................................. 2

Figure 1.3 GTD metabolic pathway by intestinal flora in human (surmise) ...................................... 6

Figure 2.1 HPLC chromatogram of GTD standard compound(MeOH:H2O=25:75) ...................... 17

Figure 2.2 HPLC chromatogram of GTD in RG (MeOH：H2O=25:75) ........................................ 17

Figure 2.3 GTD standard compound determined by HPLC (ACN:0.1%Acetic acid=13:87) .......... 17

Figure 2.4 GTD HPLC chromatogram of GTD in RGM (ACN:0.1%Acetic acid=13:87) .............. 17

Figure 2.5 HPD300 organic residues chromatogram determined by GC ......................................... 26

Figure 2.6 XDA-1 organic residues chromatogram determined by GC .......................................... 26

Figure 2.7 D-101 organic residues chromatogram determined by GC ............................................ 27

Figure 2.8 Mixed standard compound chromatogram determined by GC ....................................... 27

Figure 2.9 HPD300 organic residues GC chromatogram after conditioning treatment ................... 27

Figure 2.10 XDA-1 organic residues GC chromatogram after conditioning treatment ................... 27

Figure 2.11 D-101organic residues GC chromatogram after conditioning treatment ...................... 28

Figure 2.12 Three kinds of resin ultraviolet absorption curve of preliminary GTG extract purified

with 3 kinds of resin ......................................................................................................................... 30

Figure 3.1 Apparatu for measurement of water uptake ................................................................... 41

Figure 3.2 Hygroscopic balance curve of GTG powders ................................................................. 44

Figure 3.3 Water absorption ratio of GTG gastric floating tablet .................................................... 47

Figure 3.4 Corrosion ratio of GTG gastric floating tablets .............................................................. 48

Figure 3.5 Accumulative release rate of GTG gastric floating tablet ............................................... 49

Figure 3.6 Water absorption of GTG gastric floating tablet ............................................................. 52

Figure 3.7 Corrosion ratio of GTG gastric floating tablet ................................................................ 53

Figure 3.8 Accumulative release curve of GTG gastric floating tablet ............................................ 54

Figure 3.9 Water absorption of GTG gastric floating tablet ............................................................. 58

Figure 3.10 Corrosion ratio of GTG gastric floating tablet .............................................................. 59

Figure 3.11 Accumulative release curve of GTG gastric floating tablet .......................................... 60

Figure 3.12 Four factors profile analysis (HPMC-K15M, HPMC-K4M, PVPP+MCC, CaCO3) ... 67


X

Figure 3.13 Four factors contour profile analysis ............................................................................ 68

Figure 3.14 After 0、1、2、3、4、6h observe the floating tablets in volunteer's stomach by X-ray

technique. ......................................................................................................................................... 71

Figure 4.1 Construction of GTG gastric-flating delayed release pellets........................................ 75

Figure 4.2 Micrograph of sphericity or roundness with Sodium Alginate as framework ................ 79

Figure 4.3 Micrograph of sphericity or roundness with HPMC as pellet framework ...................... 80

Figure 4.4 Particle size distribution with HPMC-K15M as pellet framework ................................. 80


XI

List of Tables

Table 2.1 GTD content in RG and RGM by HPLC ......................................................................... 16

Table 2.2 GTG content in RG and RGM determined by UV ........................................................... 19

Table 2.3 GTD extraction ratio in concentrated solution ................................................................. 21

Table 2.4 GTG extraction ratio in concentrated solution ................................................................. 22

Table 2.5 GTD extraction ration in pilot-scale extract ..................................................................... 23

Table 2.6 GTG extraction ration in pilot-scale extract ..................................................................... 23

Table 2.7 Organic residues in macroporous resin determined by GC .............................................. 26

Table 2.8 Preliminary GTG extract purified with 3 kinds of resin determined by UV .................... 29

Table 2.9 GTG purified by 3 kinds of resin by static saturated adsorption ...................................... 31

Table 2.10 Eluation capacity and eluation ration of 3 kinds of resin after static saturation

adsorption ......................................................................................................................................... 31

Table 2.11a Adsorption ratio of GTD extraction with D-101 resin in bench-scale experiment ....... 33

Table 2.11b Comparison of the eluting effects of four different concentration of EtOH ................. 33

Table 2.12a Adsorption ratio of GTG extraction with D-101 resin in bench-scale experiment ....... 33

Table 2.12b Comparison of the eluting effects of four different concentration of EtOH ................. 34

Table 2.13 Purification result of GTD extraction with D-101 resin in pilot-scale experiment ........ 35

Table 2.14 Purification result of GTG extraction with D-101 resin in pilot-scale experiment ........ 36

Table 2.15 GTD content in GTG with D-101 resin in pilot-scale experiment ................................. 37

Table 3.1 Measurement of bulk density and angle of repose ........................................................... 43

Table 3.2 Hygroscopic balance table of GTG powders .................................................................... 44

Table 3.3 Formulation of GTG gastric floating tablet ...................................................................... 45

Table 3.4 Measurement indicators of GTG gastric floating tablet ................................................... 46

Table 3.5 Expansibility of GTG gastric floating tablets ................................................................... 46

Table 3.6 Corrosion ratio of GTG gastric floating tablets ................................................................ 47

Table 3.7 Accumulative release rate of GTG gastric floating tablets ............................................... 48

Table 3.8 Formulation of GTG gastric floating tablet ...................................................................... 51

Table 3.9 Measurement indicators of GTG gastric floating tablet ................................................... 51


XII

Table 3.10 Water absorption of GTG gastric floating tablet ............................................................ 52

Table 3.11 Corrosion ratio of GTG gastric floating tablet ............................................................... 53

Table 3.12 Accumulative release rate of GTG gastric floating tablet .............................................. 54

Table 3.13 Formulation of GTG gastric floating tablet .................................................................... 57

Table 3.14 Measurement indicators of GTG gastric floating tablet ................................................. 57

Table 3.15 Water absorption of GTG gastric floating tablet ............................................................ 57

Table 3.16 Corrosion ratio of GTG gastric floating tablet ............................................................... 58


Table 3.18 Drug release kinetic model fitting of GTG gastric floating tablet .................................. 62

Table 3.19 Ratio of mixed materials (HPMC- K15M, HPMC-K4M, PVPP, CaCO3 ) .................... 63

Table 3.20 Orthogonal experiment table of mixed materials ........................................................... 64

Table 3.21 Measurement indicators of GTG gastric floating tablet ................................................. 65


Table 3.23 Formulations obtained by SAS analysis software .......................................................... 69

Table 3.24 Result of release testing for formulations obtained by SAS software ............................ 69

Table 3.25 Result of verifying test for the optimal formulation ....................................................... 70

Table 4.1 Investigation on framework materials .............................................................................. 79

Table 4.2 Investigation on ratio of HPMC K15M and GTG ............................................................ 81

Table 4.3 Election of adhesives ........................................................................................................ 82

Table 4.4 Calculation of drug loading for the first batch ................................................................. 83

Table 4.5 Drug loading calculation of the second batch .................................................................. 83

Table 4.6 Drug loading calculation of the third batch ...................................................................... 83

Table 4.7 Average drug loading........................................................................................................ 84

Table 4.8 Effects of rotational speed on pellet performance ............................................................ 84

Table 4.9 Effects of hot-blast temperature on pellet performance ................................................... 85

Table 4.10 Ratio of feeding rate of powder and spraying rate of hinder solution effects on pellet

performance ..................................................................................................................................... 86

Table 4.11 Effects of air flow on pellet performance ....................................................................... 86

Table 4.12 Effects of time on pellet performance ............................................................................ 87

Table 4.13 Effects of spray air pressure on pellet performance ....................................................... 90


XIII

Table 4.14 Effects of materials temperature on pellet performance ................................................. 91

Table 4.15 Effects of spraying rate of hinder solution on pellet performance ................................. 91

Table 4.16 Cumulative percentage dissolution of pellets with different coating weight .................. 92

Table 4.17 Cumulative percentage dissolution of pellets with optimal formula .............................. 95

Table 4.18 Cumulative percentage dissolution of drug-loaded pellets............................................. 98

Table 4.19 In vitro release curve model fitting of GTD gastric-floating pellet .............................. 100


XIV

縮略詞

縮寫英文全稱中文全稱

RG Radix Gentianae 龍膽

GTD Gentiopicroside 龍膽苦苷

GTG Gentianae Total glycosides 龍膽總苷

RGM Radix gentianae macrophyllae 秦艽

HPLC High Performance Liquid

Chromatography 高效液相色譜

L-HPC Low substituted hydroxypropyl

cellulose 低取代羥丙基纖維素

HPMC Hydroxypropyl methylcellulose 羥丙基甲基纖維素

PVP Polyvinyl pyrrolidone 聚乙烯吡咯烷酮

CMC-Na Sodium carboxyl methyl cellulose 羧甲基纖維素鈉

ACN Acetonitrile 乙腈

GC Gas chromatography 氣相色譜


XV

目錄

中文摘要.......................................................................................................................................... V

Abstract ......................................................................................................................................... VII

List of Figures ................................................................................................................................. IX

List of Tables ................................................................................................................................... XI

縮略詞 ......................................................................................................................................... XIV

第一章概述 ........................................................................................................................... 1

1.1 龍膽苦苷的研究概述 ......................................................................................................... 1

1.1.1 龍膽藥材及龍膽苦苷簡介 ...................................................................................... 1

1.1.2 龍膽苦苷的提取分離 .............................................................................................. 2

1.1.3 龍膽苦苷的藥理活性 .............................................................................................. 3

1.1.4 龍膽苦苷的藥代動力學研究 .................................................................................. 6

1.1.5 龍膽苦苷的臨床應用 .............................................................................................. 9

1.2 胃漂浮製劑的研究綜述 ................................................................................................... 11

1.2.1 胃漂浮製劑的原理簡介 ....................................................................................... 11

1.2.2 適合採用胃內漂浮劑型的藥物簡介 ................................................................... 12

1.2.3 不宜設計成胃內漂浮劑型的藥物簡介 ............................................................... 12

1.3 本論文的立題依據與目的 ............................................................................................... 12

第二章龍膽總苷原料的製備 ............................................................................................. 14

2.1 儀器與試藥 ....................................................................................................................... 14

2.2 龍膽總苷提取用藥材的選擇 ........................................................................................... 14

2.2.1 龍膽藥材中龍膽苦苷的含量測定方法 ............................................................... 14

2.2.2 秦艽藥材中龍膽苦苷含量測定方法 ................................................................... 15

2.2.3 上述藥材中龍膽苦苷的含量測定結果 ............................................................... 16

2.2.4 龍膽藥材和秦艽藥材中龍膽總苷含量的測定方法 ........................................... 18

2.2.5 藥材中龍膽總苷的含量測定結果 ....................................................................... 19

2.2.6 藥材選擇的討論與結論 ........................................................................................ 19

2.3 龍膽總苷粗品的提取製備 ............................................................................................... 20

2.3.1 藥材前處理 ............................................................................................................ 20

2.3.2 乙醇提取 ................................................................................................................ 20

2.3.3 測定濃縮液中龍膽苦苷及龍膽總苷的提取率 .................................................... 20

2.3.4 中試規模驗證試驗 ................................................................................................ 22

2.4 龍膽總苷精製工藝研究 .................................................................................................. 23

2.4.1 大孔樹脂的預處理 ................................................................................................ 24

2.4.2 龍膽總苷精製過程大孔吸附樹脂的選擇 ........................................................... 28

2.4.3 D-101 樹脂精製龍膽總苷粗提物小試工藝研究 ................................................. 32

2.4.4 D-101 樹脂精製龍膽總苷粗提物中試工藝驗證研究 ......................................... 34

2.4.5 龍膽總苷乾燥工藝研究 ....................................................................................... 36

2.5 本章小結 ........................................................................................................................... 37

2.5.1 確定提取藥材 ....................................................................................................... 37

2.5.2 確定提取用大孔樹脂種類 ................................................................................... 37

2.5.3 本研究確定的龍膽總苷製備工藝如下： ............................................................ 37


XVI

第三章龍膽總苷胃漂浮片的製備 ..................................................................................... 39

3.1 儀器和試藥 ....................................................................................................................... 39

3.1.1 儀器 ........................................................................................................................ 39

3.1.2 試藥 ....................................................................................................................... 40

3.2 龍膽總苷服用劑量的確定 .............................................................................................. 40

3.3 胃漂浮製劑學評價方法的建立 ....................................................................................... 40

3.3.1 漂浮性能測定方法 ............................................................................................... 40

3.3.2 片重差異和硬度均勻性試驗方法 ....................................................................... 41

3.3.3 脆碎度測定方法 ................................................................................................... 41

3.3.4 溶脹性測定方法 ................................................................................................... 41

3.3.5 溶蝕性測定方法 ................................................................................................... 42

3.3.6 含量測定 ............................................................................................................... 42

3.3.7 體外溶出度測定方法 ........................................................................................... 42

3.4 龍膽總苷製劑處方前研究 ............................................................................................... 43

3.4.1 粉體學基本性質的測定 ........................................................................................ 43

3.5 龍膽總苷胃漂浮片成型工藝研究 ................................................................................... 44

3.5.1 凝膠骨架材料的選擇 ............................................................................................ 44

3.5.2 膨脹劑的選擇 ........................................................................................................ 50

3.5.3 龍膽總苷胃漂浮片產氣物質的選擇 .................................................................... 56

3.5.4 以上 11 種處方配比對藥物釋放動力學影響分析 ............................................. 61

3.5.5 混料設計優化處方用量 ........................................................................................ 63

3.5.6 胃漂浮片胃內狀態評價 ........................................................................................ 70

3.5.7 龍膽總苷胃漂浮片確定的製備工藝 ................................................................... 72

第四章龍膽總苷胃漂浮微丸製劑研究及其體外評價 ..................................................... 73

4.1 材料與儀器 ...................................................................................................................... 74

4.1.1 材料與試藥 ............................................................................................................ 74

4.1.2 儀器 ....................................................................................................................... 74

4.2 試驗方法與結果 .............................................................................................................. 75

4.2.1 龍膽總苷胃漂浮微丸的處方工藝設計 ................................................................ 75

4.2.2 龍膽總苷胃漂浮微丸製劑學評價指標的建立 .................................................... 76

4.2.3 微丸的製備工藝研究 ........................................................................................... 78

4.2.4 龍膽總苷微丸包衣工藝研究 ............................................................................... 87

4.2.4 龍膽總苷胃漂浮微丸製備工藝確定 ................................................................... 92

4.2.5 驗證試驗 ............................................................................................................... 94

4.2.6 討論 ....................................................................................................................... 95

4.3 微丸的體外釋放研究 ...................................................................................................... 97

4.3.1 體外釋放度實驗方法 ................................................................................................... 97

4.3.2 龍膽總苷微丸體外釋藥特性研究 ........................................................................ 98

4.3.3 釋藥機制的研究 ................................................................................................... 98

參考文獻....................................................................................................................................... 101

附錄 .............................................................................................................................................. 105

個人簡介....................................................................................................................................... 109

致謝 .............................................................................................................................................. 110

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