Establishing the Pharmaceutical Quality of ... - ACS Publications

Article pubs.acs.org/molecularpharmaceutics

Establishing the Pharmaceutical Quality of Chinese Herbal Medicine: A Provisional BCS Classification Sophia Y. K. Fong,† Mary Liu,‡ Hai Wei,§ Raimar Löbenberg,∥ Isadore Kanfer,⊥ Vincent H. L. Lee,† Gordon L. Amidon,‡ and Zhong Zuo*,† †

School of Pharmacy, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR College of Pharmacy, The University of Michigan, Ann Arbor, Michigan 48109, United States § Center for Chinese Medical Therapy and System Biology, Shanghai University of Traditional Chinese Medicine, Shanghai, P. R. China ∥ Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB T6G 2R3, Canada ⊥ Faculty of Pharmacy, Rhodes University, Grahamstown 6139, South Africa ‡

S Supporting Information *

ABSTRACT: The Biopharmaceutical Classification System (BCS), which is a scientific approach to categorize active drug ingredient based on its solubility and intestinal permeability into one of the four classes, has been used to set the pharmaceutical quality standards for drug products in western society. However, it has received little attention in the area of Chinese herbal medicine (CHM). This is likely, in part, due to the presence of multiple active components as well as lack of standardization of CHM. In this report, we apply BCS classification to CHMs provisionally as a basis for establishing improved in vitro quality standards. Based on a top-200 drugs selling list in China, a total of 31 CHM products comprising 50 official active marker compounds (AMCs) were provisionally classified according to BCS. Information on AMC content and doses of these CHM products were retrieved from the Chinese Pharmacopoeia. BCS parameters including solubility and permeability of the AMCs were predicted in silico (ACD/Laboratories). A BCS classification of CHMs according to biopharmaceutical properties of their AMCs is demonstrated to be feasible in the current study and can be used to provide a minimum set of quality standards. Our provisional results showed that 44% of the included AMCs were classified as Class III (high solubility, low permeability), followed by Class II (26%), Class I (18%), and Class IV (12%). A similar trend was observed when CHMs were classified in accordance with the BCS class of AMCs. Most (45%) of the included CHMs were classified as Class III, followed by Class II (16%), Class I (10%), and Class IV (6%); whereas 23% of the CHMs were of mixed class due to the presence of multiple individual AMCs with different BCS classifications. Moreover, about 60% of the AMCs were classified as high-solubility compounds (Class I and Class III), suggesting an important role for an in vitro dissolution test in setting quality control standards ensuring consistent biopharmaceutical quality for the commercially available CHM products. That is, provisionally, more than half of the AMCs of the top-selling CHMs included in this study would be candidates for a bioequivalence (BE) biowaiver, based on WHO recommendations and EMEA guidelines. Thus a dissolution requirement on these AMCs would represent a significant advance in the pharmaceutical quality of CHM today. KEYWORDS: Biopharmaceutical Classification System, traditional Chinese medicine, Chinese herbal medicine, active marker compound, solubility, permeability, dose number

■

INTRODUCTION The assurance of the pharmaceutical quality of herbal medicines has long been challenging. Identification of the likely pharmacologically active components and their therapeutic efficacy is highly debated and controversial. The complexity of the human response to “natural” medicines, usually a complex mixture of ingredients, may preclude establishing efficacy in a manner analogous to single active component of Western medicines. Further, public policies regulating medicines inhibit the establishment of efficacy due © 2013 American Chemical Society

the high cost of such development and the limited, or no, commercial exclusivity. While this situation may prevail for some time, in this manuscript we focus on the further development of pharmaceutical quality standards that can be used to provide, to the best of our knowledge today, a Received: Revised: Accepted: Published: 1623

April 4, 2012 January 23, 2013 March 8, 2013 March 8, 2013 dx.doi.org/10.1021/mp300502m | Mol. Pharmaceutics 2013, 10, 1623−1643

Molecular Pharmaceutics

Article

the BCS has been extended from Western drugs to Western herbs, in which the marker components of herbs were classified into one of the BCS classes.14 Despite the validity and applicability of BCS to Western drugs, it has received little attention in the area of CHM. Like many other “alternative therapies”, CHM does not fit easily within the contemporary scientific paradigm to which Western drugs adhere. It is regarded as dietary supplements in the US under the Dietary Supplement Health and Education Act of 1994 and is thus excluded from the rigorous approval process required for standard prescription drug products. In Europe and Canada, a simplified registration procedure is required for traditional herbal medicines.15,16 In China, herbal medicines are considered as medicinal products, but registration of CHM products is restricted to new drugs according to the Drug Administration Law of the People’s Republic of China.17,18 Since the usual approval process is generally not required for CHM in contrast to Western medicines, traditional bioavailability/bioequivalence in vivo studies have not been performed. Furthermore, CHM is not vigorously regulated in most regions of the world, which results in a lack of consistency in the manufacturing process, content, and performance of herbal products in the different countries. This may lead to adverse effects, toxicity, and herb−drug interactions encountered by consumers as well as overall variable efficacy. Furthermore, concerns due to the different practices of pesticides use, undeclared Western drugs, heavy metals, inconsistent excipients, time and location of harvest, contaminants, and incorrectly labeled herbs also suggest important reasons to regulate the pharmaceutical standards of these products.19 The current study proposes a step forward in the regulation, and hence performance, of CHM based on the advances in the biopharmaceutical sciences and recent scientific recommendation and implementation of BCS based BE requirements. By using the BCS to classify CHMs, it is possible to form a more reliable standard, in particular a dissolution standard, with which we can ensure the consistency in oral performance of herbal products over time with minimal cost. From a scientific point of view, BCS and the in vitro dissolution standard offer the potential to set in vitro quality standards that will ensure minimum consistent oral performance among CHM products. While the therapeutic efficacy of the CHM is very difficult to establish, the consistency of individual products distributed and sold worldwide can be ensured. This would be an important step toward the regulation of the quality of CHMs by ensuring a consistent product is delivered to the patient. However, in contrast to the chemically defined Western drug products, employing BCS to classify CHMs faces significant challenges. The principle challenge is that CHMs have multiple components and multiple indications which make it difficult to chemically define the active marker compounds. However, as AMCs are defined, standards can be set, and multiple dissolution criteria can be used for the various AMCs in CHM. While the in vivo biopharmaceutical quality of herbal medicines, in particular, is not well-documented, the setting of BCS based in vitro dissolution standards provides the best approach today for ensuring that patients receive products of quality. In this report, all information regarding the active marker compound (AMC), standard content, and standard dose of the selected CHMs were obtained from the Chinese Pharmacopoeia. This pharmacopoeia is regarded as the official compendium of drugs in China. In addition, the scientific

consistent Chinese herbal medicine (CHM) product over time to the patient. CHM is a major component of traditional Chinese medicine (TCM), with thousands of years of practice and is popular, not only among the Chinese population, but also among US, UK, Australia, and European populations.1−5 The question of therapeutic efficacy begs the question: “efficacy of what?” The Pharmacopoeia of the People’s Republic of China (Chinese Pharmacopoeia) and others have been taking positive steps to define the content of CHMs.6 The determination of active marker compounds (AMCs) as defined components of CHMs is clearly a step forward in increasing the pharmaceutical quality of CHMs. The AMCs may possess pharmacological activity in in vitro or animal tests, though this is rarely known in humans. Further, the presence of multiple components and their potential in vivo interactions, both pharmacological, and through drug metabolism and/or pharmacokinetic (membrane transporter) pathways is unknown and difficult to unravel. Thus we must rely on pharmaceutical quality standards for providing the best available evidence and assurance of product quality today, i.e., that the patient receives a pharmaceutical product that is of consistent quality over time. As scientific and medical research investigates components of CHMs and uncovers additional AMCs, these can be added to the list of components in CHMs for which we set pharmaceutical quality standards. In regulating the therapeutic quality, interchangeability, and substitutability of Western medicine, the role of bioequivalence (BE) is a critical standard. Certainly, products must be manufactured to quality standards established by regulatory bodies; however it is the BE standard that sets the highest “hurdle” for evidencing this therapeutic equivalence. The most common approach to establishing BE is determination of blood or plasma levels of the drug, from the product and the demonstration of equivalence (in a statistically valid manner) of in vivo drug levels. This becomes an increasingly challenging and expensive set of in vivo tests with multiple components being tested. On the other hand, in vitro dissolution testing has been used in oral drug products to establish in vitro/in vivo correlations between the release of drug from the dosage form and drug absorption.7 This test is less expensive and can be conducted more routinely, thus providing the patient with the greatest assurance possible today that they are receiving a quality pharmaceutical product that is consistent over time. The Biopharmaceutical Classification System (BCS), introduced in 1995 by Amidon et al.,8 categorizes drugs into one of four biopharmaceutical classes based on two main properties: aqueous solubility and gastrointestinal membrane permeability, and such classification allows prediction of the rate-limiting step in the intestinal absorption process following oral drug product administration. Over the past decade, drug regulatory agencies including the World Health Organization (WHO), US Food and Administration (FDA), and European Medicines Agency (EMA) have adapted the BCS effectively in setting bioavailability/bioequivalence standards for immediate-release (IR) oral drug products to obtain market approval. Currently, a waiver of in vivo bioequivalence testing of IR oral solid dosage forms can be granted for BCS Class I, high-solubility, highpermeability drugs (by FDA and EMEA) and Class III, highsolubility, low-permeability drugs (by EMA).9,10 Drug products had been provisionally classified into BCS classes, including 123 IR drugs on the WHO Essential Medicines List,11 the top 200 drugs on the US, UK, Spain, and Japan lists12 and 135 national essential medicines in Pakistan.13 Recently, the application of 1624

dx.doi.org/10.1021/mp300502m | Mol. Pharmaceutics 2013, 10, 1623−1643


Article

Figure 1. Selection overview of CHMs included in current study.

These CHMs were divided into three genres according to the Chinese Pharmacopoeia. The first one is (i) prepared slices of Chinese crude drugs, including banlangen, breviscapine, Chinese arborvitae twig and leaf, leonurus, manyprinckle Acanthopanax root, and red sage. The prepared slices of Chinese crude drugs are herbal slices produced after their crude herbs have undergone various processing methods according to the Chinese Pharmacopoeia. These slices are intended for administration in the form of a decoction. Another genre is (ii) traditional Chinese patent medicines, which are proprietary medicines prepared with standard herbal formulas which contain a combination of dried herbs and other ingredients and are then ground into powder and formulated into different dosage forms, including tablets, capsules, pills, and many others. From the top-selling list, 10 CHMs were categorized into this genre, including blister beetle oral pill, gongxuening capsule, fufang danshen oral tablet, Ginkgo biloba leaf extract tablet, notoginseng total saponins, pingxiao pian,

literature was searched to support that the marker compounds listed in the Chinese Pharmacopoeia contribute to the therapeutic activities of the selected CHMs. The aim of the present study was to provisionally classify active marker compounds (AMCs) of the top-selling TCM products in China according to the BCS paradigm to determine the potential impact of implementing a dissolution standard on CHM. The classification is provisional, similar to previous publications11,12 in that it is based on the computed properties of the AMCs.

■

MATERIALS AND METHODS

Chinese Herbal Medicine List. A list of current 200 topselling drug products in China was obtained from Sinopharm Group Co. Ltd., Shanghai, P. R. China. Among the top 200, 42 were identified as CHM products. A total of 24 of these contained complete information on their official active marker compound, standard content, and dose from the Chinese Pharmacopoeia20,21 and were included in the current study. 1625



Article

Table 1. CHM List with Information on the Indications of CHMs, Molecular Formulae, Chemical Structures, and Pharmacological Activities of Their Corresponding AMCs

1626



Article

Table 1. continued

1627



Article

Table 1. continued

1628



Article

Table 1. continued

1629



Article

Table 1. continued

1630



Article

Table 1. continued

1631



Article

Table 1. continued

a

NLT: not less than.

partition of ionizable species between the n-octanol/water system and is considered as a pH-dependent version of log P.24 A physiological relevant pH of 6.0 (mean upper intestinal pH in humans) was used when calculating log D. Dose Number Calculations. Dose number (Do) is defined by

qingkailing pian, shexiang wan, shuanghuanglian tablet, and xueshuan xinmaining capsule. The last genre is (iii) CHM products containing a single compound, including allicin, andrographolide, anisodine, asiaticosid, butylphthalide, esculin, lappaconitine, and silybin meglumine. These CHM products are more “modern” and contain a single active principle extracted from TCM plant material. Unlike the above two genres, these products contain one single AMC and have definitive standard contents. To include more CHMs for our provisional BCS classification, seven commonly used Chinese herbs proposed by Dharmananda22 were also included as an additional to genre (i), namely atractylodes, cinnamon, licorice, ma-huang, milkvetch root, red peony root, and tang-kuei. A final list comprising 31 CHMs and 50 AMCs were included in the current study for BCS classification (Figure 1). Detailed information concerning each studied CHM including the molecular formula, chemical structure, and the pharmacological activities of its corresponding AMCs are listed in Table 1. In Silico Solubility and Permeability Estimation. Since limited experimental data are available for physical and biopharmaceutical properties of CHMs, ACD/Laboratories Software (ACD/Laboratories, version 11.02; Advanced Chemistry Development Inc., Toronto, Canada) was used to calculate the aqueous solubility (Cs), partition coefficient (log P), and partition coefficient at pH 6 (log D6.0) of the AMCs. The calculated Cs covered pH 1, 4, and 7, approximating the physiological solubility at pH 1.2 (stomach), 4.5 (duodenum), and 6.8 (ileum), respectively. The algorithm for log P (noctanol−water partition coefficient) is based on individual fragment contributions to lipophilicity which were obtained from experimental data.23 To determine the number of CHMs for which the permeability classification would change with pH, log D6.0 of AMCs is also included. Log D is a measure of the

Do = (Mo/Vo)/Cs where Mo is the maximum dose strength (mg), Vo is 250 mL, and Cs is the aqueous solubility (mg/mL).8 Unlike Western drugs, Mo of CHMs is less straightforward to obtain. For prepared slices of Chinese crude drugs and traditional Chinese patent medicine, Mo is calculated by the standard content of AMC (calculated on the dried herb basis for prepared slices) multiplied by its stated dose from the Chinese Pharmacopoeia (Table 1). If the recommended dose of CHMs are of wide range (e.g., 1−6 g), both highest and lowest Mo of AMC were included for calculation to determine the number of CHMs for which the classification would change with dose strength. Taking tang-kuei as an example, the Chinese Pharmacopoeia 2010 states that the prepared slices of tang-kuei should “contain not less than 0.05% of ferulic acid calculated on the dried herb basis” and that the standard dose is “6−12 g”. Therefore highest Mo(ferulic acid) = 12 g × 0.05% = 6 mg while the lowest Mo(ferulic acid) = is 6 g × 0.05% = 3 mg. As for CHM products containing a single compound, Mo is simply given as its maximum dose recommended for this single compound from the package insert and literature. BCS Classification. AMCs were categorized into one of the four BCS classes according to two parameters, aqueous solubility at its highest therapeutic dose within the physiological pH ranges of 1 to 7 and gastrointestinal membrane permeability estimated by the partition coefficient, Log P. If Do was equal to or lower than 1, the AMC was classified as 1632



Article

prediction of in vivo human intestinal permeability of orally administered drugs.28

high-solubility. The AMC was classified as low-solubility if Do was greater than 1. If the Do changed over physiological pH, the lowest solubility would be used for BCS classification of AMC. Metoprolol with a log P of 1.632 was used as a reference drug. Log P of each AMC was compared to that of metoprolol. If log P of AMC was greater than 1.632, the AMC was classified as high-permeability, whereas if it was equal to or below 1.632, then that AMC was considered low-permeability. This allows a comparison with previously published provisional BCS classifications of Western pharmaceutical products. Similarly, labetalol with a log D6.0 of −0.15 was used as a reference drug to classify permeability class of AMCs, based on log D6.0. Table 2 summarizes the provisional BCS classification into the four classes.

■

RESULTS Classification of Solubility. Of the 50 included AMCs, 30 (60%) were classified as high solubility, 13 (26%) were low solubility, and the remaining 7 (14%) had variable solubility classification depending on the physiological pH and/or the dose strength. Among the 32 AMCs with both highest and lowest maximum dose strength, the percentage of highsolubility AMCs increased from 59% to 66% when dose number was calculated using the lowest dose strength compared to highest dose strength (Figure 2I). Physiological

Table 2. BCS Classification of AMC According to Solubility and Log P/Log D6.0 class I II III IV

solubility Do Do Do Do

≤1 >1 ≤1 >1

log P

log D6.0

>1.632 ≤1.632 >1.632 ≤1.632

> −0.15 ≤ −0.15 > −0.15 ≤ −0.15

Correlations of Experimentally Determined Solubility and Permeability with in Silico Solubility and Permeability. Since the BCS classification of AMCs was based on in silico descriptors, the reliability of such descriptors was validated. This was done by correlating the experimentally determined solubility and permeability of AMCs with the in silico solubility and log P/log D6.0, respectively. Information on the experimental aqueous solubility at pH 7 and Caco-2 apparent permeability (Papp) of all the AMCs was identified through an extensive literature search of the following databases: SciFinder Scholar, Medline, and Google scholar. We then calculated the Do for those AMCs with available experimental solubility information using their standard dose (including both maximum and minimum dose) and plotted against the corresponding Do of AMCs calculated based on in silico aqueous solubility at pH 7. AMCs with both the experimental Do and predicted Do equal to or lower than 1 were considered correctly classified as high solubility. Conversely, AMCs with both experimental Do and predicted Do higher than 1 were classified as low solubility. AMCs that exhibit experimental Do lower than 1 but with corresponding predicted Do higher than 1 were termed false negatives. On the other hand, AMCs with experimental Do higher than 1 but with corresponding predicted Do lower than 1 were regarded as false positives. Similarly, for the validation of the permeability descriptors, information on the Caco-2 cell monolayer apical to basolateral Papp values of each AMC was gathered from a literature search and plotted against their corresponding log P or log D6.0. Metoprolol with Papp of 18 × 10−6 cm/s served as the reference compound which set the high-low permeability boundary in the correlation plot of Papp with log P,25 whereas labetalol with Papp of 9.31 × 10−6 cm/s was set as a cutoff of classifying AMCs with high/low permeability in the correlation plot of Papp with log D6.0.26 The permeability coefficients in Caco-2 cells were used to validate our in silico permeability descriptors since Papp was shown to demonstrate a good correlation with fraction absorbed of drugs in humans27 and is considered to be the industry reference standard for in vitro

Figure 2. Effect of (I) dose strength and (II) pH on percentage of AMC having a high solubility.

pH also caused a change of solubility class of AMCs. The percentage of AMCs with a high solubility increased from 60% (pH 1) to 66% (pH 4) and increased further to 70% (pH 7) (Figure 2II). Classification of Permeability. Using log P with metoprolol as the reference drug, 22 (44%) AMCs were classified as high permeability with the rest being of lowpermeability AMCs. When using log D6.0 with labetalol as the reference drug for permeability classification, 30 (60%) AMCs were high permeability. Correlations of Experimentally Determined Solubility and Permeability with in Silico Solubility and Permeability. From our extensive literature search, we were able to identify 36 out of 50 of the included AMCs with experimental aqueous solubility at pH 7. The experimental solubility values and the calculated Do of these AMCs using their standard doses 1633



Article

were presented in Table 1 of the Supporting Information (SI) along with a complete reference list. A total of 55 experimental Do values were obtained (as some AMCs had both maximum and minimum doses) and plotted against their corresponding predicted Do (Figure 3). The plot indicated that the

Figure 3. Plot of experimentally determined Do versus predicted Do of the AMCs.

classification of solubility based on in silico predictor was correct for 42 out of 55 AMCs (76%), with 35 of them (64%) being correctly classified as high-solubility (lower left quadrant). Ten of the incorrectly classified AMCs were false positive (upper left quadrant), while three of them were false negative (lower left quadrant). On the other hand, 29 AMCs with available data on experimentally determined Caco-2 apical to basolateral Papp values were identified from literature and were listed in SI Table 2 along with in silico log P and log D6.0. A plot of the Papp against log P (Figure 4I) indicated that the classification of permeability based on metoprolol as the reference compound was correct for 20 out 29 of AMCs (69%). Among the nine incorrectly classified AMCs, eight of them were false positive (lower left quadrant), while one of them was false negative (upper left quadrant). It should be noted that, except for tanshinone IIA and strychnine, all other included AMCs have experimental Papp values lower than that of metoprolol. A similar plot of the Papp against log D6.0 indicated that 13 out of 29 (45%) of AMCs were correctly classified based on labetalol as the reference compound (Figure 4II). Majority of the incorrectly classified AMCs were false positive (11 out of 29), and the remaining five were false negative. BCS Classification of Prepared Slices of Chinese Crude Drugs. The calculated maximum and minimum standard dose, dose number at pH 1, 4, and 7, log P/log DpH 6.0, and the provisional BCS Class of the prepared slices of Chinese crude drugs, traditional Chinese patent medicines, and CHM products containing a single compound are listed in Table 3. Of the 13 selected prepared slices of Chinese crude drugs which constitute 17 AMCs, only cinnamon was classified as a Class I herb (8%). Actractylodes and red sage, which were considered to be high-solubility and low-permeability AMCs, were classified as Class II herbs (15%). The majority of the prepared slices were Class III herbs (54%), including banlangen, breviscapine, Chinese arborvitae twig and leaf, leonurus, ma-huang, manyprinckle acanthopanax root, and tang-kuei, whereas red peony root was classified as a Class IV herb (8%). The remaining two herbs have mixed classification due to different classifications of multiple individual AMCs, or

Figure 4. Plot of experimentally determined Caco-2 Papp versus predicted log P (I) and log D6.0 (II) of the AMCs.

to differing classifications because of maximum and minimum dose. Licorice has two official AMCs from Chinese Pharmacopoeia: liquiritin and glycyrrhizic acid. Its standard dose range is considerably wide (2−10 g) since it has multiple indications, e.g. peptic ulcer, cough, bronchitis, carbuncles, sores, and so forth.29 The official requirement of liquiritin content is “NLT 0.5%”, making the maximum and minimum doses of liquiritin 50 mg and 10 mg, respectively. The calculated Do of the low dose liquiritin is less than one while that of high dose is more than one; therefore, given that liquiritin has low permeability, its BCS class changed from Class III to Class IV when the dose increased from 10 mg to 50 mg. Glycyrrhizic acid, another AMC of licorice, was even more complicated as its solubility differed over both the dose and physiological pH range. It had a low solubility at pH 1 but a high solubility at pH 7. At pH 4, it had high solubility at low dose but low solubility at high dose. According to current FDA guidance, the classification would go for the lowest solubility over the physiological pH; so that glycyrrhizic acid was classified as Class II provided that it has high permeability. Overall, licorice contained AMCs which covered Class II, III and IV. Milkvetch root is another CHM falling into the mixed classified category. Similar to licorice, milkvetch root also has two official AMCs. One of AMCs calycosin-glucoside belonged to Class III but the other one astragaloside IV had varied classes over dose ranges. It was classified as Class III in low dose (3.6 mg) but changed to Class IV in high dose (12 mg). The distribution of AMCs and CHMs of prepared slices of Chinese crude drugs among the four BCS classes is summarized in 1634


quercitrin

cinnamaldehyde

leonurine hydrochloride

liquiritin

Chinese arborvitae twig and leaf

cinnamon

leonurus

licorice

1635

ferulic acid

gongxuening capsule

polyphyllin VI

(2) Traditional Chinese Patent Medicines blister beetle oral pill cantharidin

tang-kuei

tanshinone IIA

red sage

salvianolic acid B

paeoniflorin

calycosin-7-glucoside

astragaloside IV

syringoside

pseudoephedrine hydrochloride

ephedrine hydrochloride

red peony root

milkvetch root

manyprinckle acanthopanax root

ma-huang

scutellarin

breviscapine

glycyrrhizic acid

(R,S)- goitrin

AMC

banlangen

(1) Prepared Slices of Chinese Crude Drugs atractylodes atractylodin

CHM

max: 0.21 min: 0.105 max: 1.04 min: 0.52

0.0290 0.0145 2.0800 1.0400

0.00032 0.000064 0.00032 0.000064 0.0045 0.0015 1.2632 0.3789 0.0414 0.0124 6 3 57.1429 38.0952 2.9508 1.9672 0.0062 0.0031

333.3

min: 200 max: 80 min: 16 max: 80 min: 16 max: 13.5 min: 4.5 max: 12 min: 3.6 max: 6 min: 1.8 max: 180 min: 90 max: 30 min: 20 max: 450 min: 300 max: 6 min: 3

30 10 0.0019 0.0011 0.1856 0.1113 0.0480 0.0240 0.01 0.002 0.00007 0.00002 1.5385 0.3077 666.6

pH 1

max: 18 min: 16 max: 4.5 min: 2.7 max: 45 min: 27 max: 12 min: 6 max: 7.5 min: 1.5 max: 12 min: 3.6 max: 50 min: 10 max: 400

standard dose (mg)

0.0290 0.0145 2.0800 1.0400

0.00032 0.000064 0.00032 0.000064 0.0045 0.0015 1.2632 0.3789 0.0414 0.0124 6 3 57.1429 38.0952 0.0106 0.0071 0.0043 0.0021

1

30 10 0.0031 0.0018 0.0062 0.0037 0.0480 0.0240 0.01 0.002 0.00013 0.00004 1.5385 0.3077 2

pH 4

Do

0.0290 0.0145 2.0800 1.0400

0.00032 0.000064 0.00032 0.000064 0.0045 0.0015 1.2632 0.3789 0.0414 0.0124 6 3 57.1429 38.0952 0.0018 0.0012 0.000024 0.000012

0.0008

30 10 0.0031 0.0018 0.0002 0.0001 0.0034 0.0017 0.01 0.002 0.0571 0.0171 1.3333 0.2667 0.0016

pH 7

3.97

−0.41

0.96

2.14

4.93

III

−0.70

3.97

II

III

II (pH 1) I (pH 4−7)

−5.41

−0.41

II

IV

IV III III

III

III

4.93

0.25

−0.94

−0.94 0.25

0.63

−1.55

−1.55 0.63

−1.86

−1.86

−0.95

(pH (pH (pH (pH

1−4) 7) 1) 4−7)

II

III

III

IV (pH I) III (pH 4−7)

II

II

II I III

III

III

II I IV III IV III III

III

I

I

III

I

II

log D6.0

BCS class of AMC log P

IV III II (pH 1−4 I (pH 7) II (pH 1) I (pH 4−7) III

III

−0.31 0.27

I

1.90

III

III

−3.19 0.26

III

II

1.52

4.58

log D6.0

1.08

1.08

4.64

0.28

0.72

1.90

0.58

0.43

1.52

4.58

log P

Table 3. Standard Dose (mg), Dose Number (Do) at pH 1, 4, and 7, Log P/Log D6.0, and Provisional BCS Classification of the AMCs

II

III

III

II

IV

mixed

III

III

Mixed

III

I

III

III

III

II

log P

II

III

III

Mixed

II

mixed

III

III

Mixed

III

I

I

III

I

II

log D6.0

BCS class of CHM

Molecular Pharmaceutics Article


AMC

notoginseng total saponins

1636

chlorogenic acid baicalin

geniposide

cholic acid

baicalin

strychnine

gensenoside Rd

gensenoside Re

notoginsenoside R1

anisodine tablet

anisodine

forsythin xueshuan xinmaining capsule anhydrous rutin (3) CHM Products Containing a Single Compound allicin tablet allicin andrographolide tablet andrographolide

shuanghuanglian tablet

qingkailing tablet

pingxiao tablet

gensenoside Rg1

moschus pill

gensenoside Rb1

bilobalide ginkgolide A ginkgolide B ginkgolide C muscone

(2) Traditional Chinese Patent Medicines fufang danshen tablet tanshinone IIA salvianolic acid B Ginkgo biloba leaf extract tablet quercetin kaempferol isorhamnetin

CHM

Table 3. continued

0.1333 0.2866 0.0031 0.1765 0.1176 0.00002 0.0000

20 max: 150 min: 100 max: 4 min: 1

0.0310 0.0031 0.0196 0.0175 0.0727 0.0218 1000.0 333.3 6792.45 2264.15 285.714 95.2381 230.769 76.9231 1666.67 555.56 0.00005 0.00001 0.6286 0.2571 4.6222 1.5407 0.00004 0.00002 0.0049 2.8571

1.1429 0.0984 0.3339 0.8930 1.2190

pH 1

6 48

4.8 4.8 4.8 4.8 max: 2 min: 0.6 max: 750 min: 250 max: 900 min: 300 max: 150 min: 50 max: 75 min: 25 max: 150 min: 50 max: 2.8 min: 0.8 max: 44 min: 18 max: 31.2 min: 10.4 max: 2 min: 1 22 200

0.6 15 19.2 19.2 19.2

standard dose (mg)

Do

0.0031 0.1765 0.1176 0.00002 0.0000

0.1333 0.2866

0.0310 0.0031 0.0196 0.0175 0.0727 0.0218 1000.0 333.3 6792.45 2264.15 285.714 95.2381 230.769 76.9231 1666.67 555.56 0.00007 0.00002 0.0207 0.0085 4.0258 1.3419 0.00004 0.00002 0.0009 0.0941

1.1429 0.0004 0.3200 0.8930 1.2190

pH 4

0.0031 0.1765 0.1176 0.0006 0.0001

0.1333 0.0287

0.0310 0.0031 0.0196 0.0175 0.0727 0.0218 1000.0 333.3 6792.45 2264.15 285.714 95.2381 230.769 76.9231 1666.67 555.56 0.00303 0.00086 0.00018 0.00007 0.0277 0.0092 0.00004 0.00002 0.0001 0.0008

1.1429 0.0001 0.0404 0.1219 0.1506

pH 7

0.38

III III III

−1.60

III IV (pH = 1) III (pH 4−7) III III

II (pH 1−4) I (pH = 7) III

1.13 0.42

−0.31 −1.13 −0.31 −0.90 1.13 0.42

−2.25 −2.19

−2.08

−2.08 0.37 1.43

1.61

III

III

−0.74 −2.19

II

II

IV

II

IV

III

I I

III IV (pH = 1) III (pH 4−7) III III

II (pH 1−4) I (pH = 7) III

III

III

II

II

II

II

II

II III I I II (pH 1−4) I (pH 7) I I I I I

log D6.0

BCS class of AMC log P II I I I II (pH 1−4) I (pH 7) I III I I I

3.38

1.89

1.35

1.88

1.50

4.46 −0.04 2.07 1.72 6.10

4.93 −5.41 1.78 2.49 2.58

log D6.0

2.88

1.43

1.59

3.38

1.89

1.35

1.88

1.50

11.7 −0.036 2.07 1.72 6.10

4.93 2.14 2.08 2.69 2.79

log P

III

III III

III

mixed

mixed

III

mixed

III

I I

III

mixed

mixed

III

II

I

mixed

mixed

I

mixed

log D6.0

mixed

log P

BCS class of CHM

Molecular Pharmaceutics Article


II

II

Figure 5I and II, respectively. When using log D6.0 with labetalol as the reference standard, five AMCs changed class

II

I I I

II II III IV II III II II III

log D6.0 log P log D6.0

2.593 4.23

II

1.59 1.79

I

0.09 3.05 −1.35

lappaconitine

silybin meglumine

lappaconitine tablet

silybin meglumine tablet

32 6.1538 0.0414 0.0207 0.0001 0.00007 727.273 363.636 (3) CHM Products Containing a Single Compound asiaticosid tablet asiaticosid butylphthalide capsule butylphthalide extract of horse chestnut seeds tablet esculin

12 200 max: 300 min: 150 max: 10 min: 5 max: 200 min: 100

32 6.1538 0.0414 0.0207 0.0033 0.00167 727.273 363.636

32 6.1538 0.0207 0.0103 0.1739 0.08696 421.053 210.526

0.089 3.05 −1.31

IV II III

BCS class of AMC

log D6.0 log P pH 7 Do

pH 4 pH 1 standard dose (mg) AMC CHM

Table 3. continued

Article

log P

BCS class of CHM


Figure 5. Percentage of (I) AMC and (II) CHM categorized into BCS classes.

from low permeability to high permeability, namely, (R,S)goitrin, quercitrin, liquiritin, astragaloside IV, and paeoniflorin, while one AMC (salvianolic acid) changed to low permeability. The BCS classes of four Chinese crude drugs were changed accordingly with their corresponding AMCs, including banlangen, Chinese arborvitae twig and leaf, red peony root, and red sage. BCS Classification of Traditional Chinese Patent Medicines. Of the 10 traditional Chinese patent medicines considered, half of them have a single official AMC which enables the CHMs to be BCS classified; whereas the other half have multiple AMCs with different BCS classes, resulting in 5 mixed classified CHMs (50%). Moschus pill and gongxuening capsule were classified as Class I (10%) and Class II (10%), respectively. Blister beetle oral pill, pingxiao tablet, and xuenshuan xinmaining capsule were Class III (30%) while no patent CHM was classified as entirely Class IV (0%). Ginkgo biloba leaf extract tablet has 7 AMCs, with quercetin, kaempferol, isorhamnetin (at pH 7), bilobalide, ginkgolide B, and ginkgolide C being Class I, isorhamnetin (pH 1−4) being Class II, and ginkgolide A being Class III. Similarly, fufang 1637



Article

compounds. For CHM products containing a single compound, they are modern CHM products which contain one single active marker compound contributing to efficacy, and thus their marker compounds are treated like an active pharmaceutical ingredient in Western medicines. For prepared slices of Chinese crude drugs and traditional Chinese patent medicines, questions still exist on whether the selected marker compounds in the Chinese Pharmacopoeia for these products contribute to therapeutic activity of the CHMs. Although the traditional indications of CHMs are of great variety, research on investigating the pharmacological activities of the marker compounds of CHMs and establishing a relationship between the activity of the markers and their corresponding CHMs continues. From literature search most of the marker compounds of our included CHMs do contribute to, if not all, the main pharmacological activity of their corresponding CHMs (Table 1), and thus they are excellent compounds upon which to set in vitro standards for ensuring pharmaceutical quality. For example, atractylodin, the marker compound of Atractylodis Rhizome (atractylodes), was found to improve delayed gastric emptying.31 This matches the traditional indication of the herb for treating hypofunction of the spleen with anorexia and abdominal distension. Another example is Isatidis Radix (banlangen), a CHM used traditionally to treat infections. Its marker compound (R,S)-goitrin was found to possess antiviral activity.32 With advanced research, some marker compounds may contribute to new indications instead of the traditional indication, for instance, the antibacterial and antiviral activity of cinnamon.33,34 The marker compounds selected for the current study are therefore considered to be the AMCs. High Percentage of AMCs Were Classified as High Solubility. In silico calculations of aqueous solubility of the AMCs by ACD/Laboratories software was used for Do calculation in the current study. In view of the predictive nature of such in silico data, we identified experimental solubility values of individual AMCs from extensive literature search and correlated them with the in silico solubility values. Of the 36 AMCs with available experimental solubility data, 76% are correctly classified. Furthermore, in a study comparing the accuracy of solubility class prediction of drugs in the Pakistan national medicine list by different commonly available software, ACD/Laboratories was able to correctly predict 86% in the solubility class, which is higher than with the other studied software such as Drugbank (76.3%) and ALOGPS (78%).13 The use of in silico solubility for BCS classification of the AMCs is thus considered reliable. In our provisional classification, the percentage of AMCs categorized as having high solubility, Do ≤ 1, calculated using maximum dose strength was 60%. This number is comparable to the percentage of high-solubility oral IR drugs on the WHO drug list (67%) and top-200 US drug list (68%).11,12 It is noted that from the correlation plot (Figure 3) 64% of the AMCs are correctly classified as high-solubility. Of the 32 AMCs with both highest and lowest Mo, the percentage of AMCs with Do ≤ 1 increased from 59% to 66% calculated using highest and lowest dose strengths, respectively. Thus, roughly 7% of the AMCs change BCS class over the recommended dose strength range. Moreover, physiological pH also contributes to a change in BCS class in some of the AMCs. The percentage of AMCs having Do ≤ 1 increased with pH, from 60% at pH 1 to 66% at pH 4 and to 70% at pH 7. Therefore, about 10% of the AMCs change BCS class over the physiological pH, with generally higher solubility at higher pH.

danshen, notoginseng total saponins tablets, qiankailing tablets, and shuanghuanlian tablets contain several AMCs with different BCS classes, thus making the classification of the patent CHMs more difficult. The distribution of AMCs and CHMs of traditional Chinese patent medicines among the four BCS classes is shown in Figure 5I and II, respectively. Three AMCs changed permeability class when using log D6.0 instead of log P for classification. Salvianolic acid B changed from high to low permeability, while gensenoside Rg1 and notoginsenoside changed from low to high permeability. The BCS class of notoginseng total saponins was changed accordingly with its corresponding AMCs. BCS Classification of CHM Products Containing a Single Compound. A total of eight CHM products containing a single compound were included in the current study of which lappaconitine tablets were classified as Class I (12.5%), butylphthalide capsules and silybin meglumine tablets as Class II herbs (25%), and asiaticosid tablets as Class IV (12.5%). The majority of the TCM products with high solubility and low permeability were thus classified as Class III (50%), namely, allicin tablets, andrographolide tablets, anisodine tablets, and extract of horse chestnut seeds tablets. Figure 5I and II summarizes the distribution of AMCs and CHMs of CHM products containing a single compound into the four BCS classes. The permeability class and thus the BCS class of three AMCs changed when using log D6.0 instead of log P for classification, including allicin tablet, andrographolide tablet, and asiaticosid tablet. Overall BCS Classification of Top-Selling CHM Products in China. A total of 31 CHMs comprising 50 AMCs were included in the current study. Overall, the majority of AMC’s had high solubility and low permeability, i.e., classified as Class III (44%), followed by Class II (26%) and Class I (18%). Only a few AMCs belonged to Class IV (12%) (Figure 5). A similar trend was observed when we categorized the CHMs into their relevant BCS Classes. The majority of the CHMs were Class III (45%), followed by Class II (16%), Class I (10%), and Class IV (6%). It should be noted that a significant number of CHMs were mixed classified (23%) (Figure 5II). When using log D6.0 for permeability classification, the trend is also similar to that using log P: Class III (CHM: 32%, AMC: 37%) > Class I (23%, 28%) > Class II (23%, 27%) ≥ Class IV (0%, 4%).

■

DISCUSSION Identifying the Active Marker Compounds of CHMs. Traditional Chinese medicine has a history of more than 4000 years. Traditionally, TCM was administered in the form of decoctions with a combination of different herbs. Most recently, modernization and internationalization of TCM development has occurred, with different dosage forms and products now available, including capsules, tablets, injections, single active principles extracted from TCM plant materials, combination preparations of TCM and Western drugs, and so forth.30 In the present study, twenty-four top-selling CHMs in China and seven popular CHMs were included which could be categorized into three product genres: prepared slices of Chinese crude drugs, traditional Chinese patent medicines, and CHM products containing a single compound. Unlike Western drugs, CHMs often lack well-defined single active principles which make it difficult to define excellent AMCs. In view of this, we consulted the national compendium of CHMsthe Chinese Pharmacopoeia for selecting appropriate marker 1638



Article

Predictor (Simulations Plus, Inc.) are also popular alternatives for estimating the permeability for BCS classification of drugs.14,36 Majority of the Included AMCs and CHMs Were Provisionally Classified as BCS Class I and III. The 31 topselling CHMs comprising 50 AMCs were classified according to BCS on the basis of Do and log P criteria. The distribution of these AMCs and their respective herbal drug products follows the same trend: Class III (CHM: 45%, AMC: 44%) > Class II (16%, 26%) ≥ Class I (10%, 18%) > Class IV (6%, 12%). When using log D6.0 for permeability classification, the percentage of AMCs and CHMs in Class I and Class III was 65% and 55%, respectively. These results did not differ much from that using log P for classification where 62% and 55% for AMCs and CHMs corresponded to Class I and III in total, respectively. The majority of the studied Chinese herbal products fell into Class III, i.e. having high solubility but low permeability. On the basis of dose numbers alone, about 60% of AMCs and 55% of the CHMs had high solubility (Class I plus Class III) where a suitable in vitro dissolution test procedure may serve as the standard for setting up specifications for quality control purposes. Interestingly, the number of CHMs with high solubility coincides with the percentage of drug products in the US, UK, Spain, and Japan classified as Class I and Class III, which was also around 55%.12 It should be noted that about a quarter of the CHMs were of mixed classification due to various reasons such as differing classifications of multiple individual AMCs or differing classifications because of maximum and minimum doses. The main challenge of categorizing CHM into BCS classes remains the fact that CHMs have multiple active components, while the BCS classification system is designed to classify single active compounds. For quality control purposes, the Chinese Pharmacopoeia provides the official AMC(s) of each herbal product. However, in most cases, CHM have two or more official AMCs. If the AMCs have different biopharmaceutical properties, it is still difficult to conclude into which BCS class a particular CHM should belong to, and in fact we should have multiple classifications and multiple standards. In the current study, half of the included traditional Chinese patent medicines and three of the prepared slices of Chinese crude drugs have AMCs with multiple classifications. Taking Ginkgo biloba leaf extract tablet as an example, it has seven AMCs bilobalide, ginkgolide A, ginkgolide B, ginkgolide C, isorhamnetin, quercertin, and kaempferol which cover BCS Class I to III. On the other hand, all CHM products containing a single compound can be BCS classified. Another noticeable phenomenon is that sometimes two or more different CHMs have the same AMC. For example, baicalin is one of the official AMCs in both of the patent medicines, qingkailing and shuanghuanglian tablets. Due to their different recommended daily dose, the Do of baicalin of these two products differed, resulting in different BCS Classes. The maximum dose of baicalin in qingkailing tablets is 44 mg and is classified as class III AMC, while the maximum dose of baicalin in shuanghuanglian tablets is 200 mg and becomes a Class IV AMC. Proposing the Use of in Vitro Dissolution Test To Set Up an Improved Quality Control Standard for CHM Products. Understanding the biopharmaceutical properties of AMCs is a key to regulation of the quality of CHM products. It is proposed that in vitro dissolution tests can be used to set up improved quality control standards for commercially available CHM products. For CHM products containing a single AMC,

According to the current US FDA regulatory guidelines, the BCS class is defined by the lowest solubility across the physiological pH. A newer classification of drugs into Class IIA, B, and C and IV-A, B, C has recently been proposed for distinguishing drugs with varying solubility over the physiological pH range.35 Less than Half of the AMCs Were Classified as High Permeability According to Log P. Permeability estimations of AMCs were based on correlations with the n-octanol/water partition coefficient. AMCs exhibiting log P values greater than that of metoprolol (log P 1.632) were categorized as high permeability since metoprolol is known to be 95% (or more) absorbed from the GI tract and hence may be used as a reference standard for the low/high class boundary.36 To validate the reliability of permeability classification of AMCs based on in silico log P values, experimental Caco-2 cell monolayer apical to basolateral Papp values of individual AMCs were collected through a literature search and correlated with in silico log P values. Of the 29 AMCs with available data on Papp, 69% are correctly classified based on metoprolol as the reference compound. These results resemble the study conducted by Takagi et al., in which log P was successfully used to classify 70% of the studied drugs into the correct BCS permeability compared to human jejunal permeability measures using metoprolol as a reference.12 The use of log P for permeability classification of the AMCs is thus considered reliable. Log D, a pH-dependent on log P, was recently found to reflect the true behavior of ionizable drugs in solution at a given pH range and thus can represent the lipophilic properties of molecules in physiological systems and gave better permeability predictions than log P.24,37 Log D(5.5−6.5) was also found to be correlated with the human jejunal effective permeability (Peff) and the intestinal absorption rate constant (Ka).38 Therefore, in addition to log P, log D6.0 was used to provisionally classify AMCs into BCS classes in the current study. Labetalol with a log D6.0 value of −0.15 was used as reference standard for estimating permeability of AMCs as it is known to be 90% or more absorbed from the GI tract, and it was recently suggested to be a better internal standard in the permeability comparisons than metoprolol.39 From our correlation plot of Papp against log D6.0, 45% of the AMCs were correctly classified based on labetalol as the reference compound, which is surprisingly lower than that when Papp correlates with log P (69%). It appears that log P has a better predictability on permeability of AMCs than log D6.0 for the current provisional BCS classification. However, two limitations of such correlation plots should be noted. First, the Papp values of individual AMCs were collected from multiple laboratories. Differences in methodologies among laboratories may exist, and the loading concentrations of AMCs onto Caco-2 cells varied from 10 to 150 μM. Therefore, for a more appropriate comparison, experiments should be conducted in future to obtain Papp values of different AMCs from the same experiment. Second, the permeability correlation plot was based on the 29 (out of 50) AMCs which had reported experimental Papp values from literature. Most of these AMCs (27 out of 29) had experimental Papp values lower than that of metoprolol (Figure 4a). It is possible that the numbers of AMCs having Papp values higher than that of metoprolol would increase should there be more available experimental data. From our provisional permeability classification, 44% of the total 50 included AMCs have high permeability using log P classification while 60% have high permeability using log D6.0 classification. Calculated Peff using software such as ADMET 1639



Article

indications and standards. Third, the lack of standardization of CHM products has been problematic and has impeded the development of CHM.41 In the Chinese Pharmacopoeia, the stated content for prepared slices of Chinese crude drugs and patent medicines is in the format of “not less than” (NLT) certain percentage. The AMC dose used for Do calculation in this study is thus the lowest stipulated amount. Due to varied manufacturing methods or the herbs being cultivated in different regions or harvested in different seasons, the AMC content may vary. This would definitely affect the calculated Do and thereby the BCS class. In this sense, the BCS could have regulatory advantages in assisting the standardization of CHMs. The information presented serves to provide an indication and potential utility of the application of the BCS classification system in Chinese herbal products. Performing this study on dissolution standards for CHM hopefully encourages in vitro studies to be done to regulate CHM and formula standards for the contents and safety of these products. It is necessary to emphasize that CHMs are based on a different paradigm than Western medicine, and standard doses may not be feasible or realistic. The concentrations of AMCs tend to vary based on the exact part of the herb that is used, when the herb was harvested, and how the herb was processed. All of these variables make standardization very difficult to enforce with CHMs. Identifying these barriers and the setting of a dissolution standard allows us to ensure the quality of a product, to the best of our ability for a patient. The differences between Western and Eastern medicine will likely always remain, and the setting of scientifically based quality standards for Chinese herbal products is the best path for TCM to safely and successfully thrive in a heavily evidence-based medicine system.

only one specification is needed for the dissolution test, and this case resembles the single active ingredient of Western drugs. For example, allicin (the AMC of allicin tablet) belongs to Class I, and a high-solubility specification would be the standard in testing allicin tablets existing on the market. It is a more complicated matter if we are to use in vitro dissolution test(s) to conduct quality assurance between CHM products with multiple AMCs. For CHM products which have multiple AMCs but with the same BCS solubility class, such as notoginseng total saponins having five AMCs with BCS Class II and IV, one specification could still be used in setting up a dissolution standard. It would be more complex for CHM products having multiple AMCs with different BCS classes. One possible solution may be that for the same CHM product with different AMCs where the BCS Classes are different, a dissolution test could be carried out but different AMCs follow different specifications. For example, six of the AMCs of ginkgo biloba leaf extract tablets are Class I or III (i.e., high solubility) while one of the AMCs is Class II (i.e., low solubility).Thus there will be two specifications for the Ginkgo biloba leaf extract tablet dissolution test. The feasibility of the utilization of such in vitro dissolution tests as a quality control standard for CHM products is proposed, but one that we believe is the best approach to ensure that the patient receives, to our knowledge today, the best and most consistent product. It is a far simpler test to ensure quality than an in vivo test and can be more routinely conducted. Challenges and Promises in Using Dissolution Tests for Improving Quality Control Standard of CHM Products. Several important issues relating to the current study should be highlighted. First, the data presented are in silico aqueous solubility and permeability estimations based on correlations with log P (and log D6.0). As such, the classifications are “provisional” and can be revised as more experimental data become available. More extensive solubility and permeability (ideally human jejunal permeability data or a validated surrogate) determinations are required to officially classify these drugs into relevant BCS classes. In reality, in silico calculations serve as a much simpler alternative method to using experimental human intestinal permeability data for their high reliability and availability.40 Although CHMs generally have more than one active ingredient in each herb, there is a possibility that solubility and (tissue culture) permeability may be a feasible and inexpensive method to ensure the quality of these products. The second issue is that neither safety nor efficacy of a CHM product has been researched in this study. As mentioned, most CHMs have more than one AMC, and these AMCs together with many other co-occurring components present in the matrix of the CHM may work synergistically or independently; therefore, it is indeed a research question that is yet to be studied extensively in these herbal drugs. Nevertheless, the efficacy due to the interacting AMCs is beyond the realm of our study. Getting the AMC to dissolve and permeate into the body, however, is the initial challenge. Therefore, our attention is focused on the solubility and permeability of individual active ingredients in standardized quantities and utilization of this information to classify these herbal products into a BCS classification. We have to assume that the in vivo performance of any additional components in the CHM track the AMCs. Furthermore, they can be classified and added to the standard as these additional components are determined. This is analogous to the “off label” uses of Western medicines which we have to assume follows the label

■

CONCLUSION The current study has demonstrated that it is feasible to provisionally categorize Chinese herbal medicines into the relevant BCS classes according to the biopharmaceutical properties of their active marker compounds. The majority of the CHMs included in this study were classified as Class III, followed by Class II, Class I, and Class IV. A quarter of the CHMs, however, were of mixed classification. The main challenge in applying BCS to classify CHMs is the fact that many CHMs contain multiple active marker ingredients. More than half of the studied AMCs were classified as high-solubility AMCs (Class I and Class III), suggesting that appropriate in vitro dissolution tests may provide a valuable tool and play a significant role in ensuring product quality for the majority of CHM products.

■

ASSOCIATED CONTENT

S Supporting Information *

Predicted dose number based on in silico solubility and dose number based on experimentally determined solubility of AMCs (Table 1) and predicted log P, log D6.0, and Caco-2 cell monolayer apical to basolateral permeability coefficient (Papp) of AMCs (Table 2). This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*Tel.: 852 3943 6832. Fax: 852 2603 5295. E-mail: joanzuo@ cuhk.edu.hk. 1640



Article

Notes

(17) Although, F., Regulatory Situation of Herbal Medicines A Worldwide Review. World Health Organization, 1998. From http:// apps.who.int.medicinedocs/en/d/Jwhozip57e/ (accessed on Oct 10, 2011). (18) Drug Administration Law of the People’s Republic of China. State Food and Drug Administration, P. R. China. From http:// former.sfda.gov.cn/cmsweb/webportal/-W45649037/A48335975. html (accessed on Oct 10, 2011). (19) Li, G. Q.; Duke, C. C.; Roufogalis, B. D. The Quality and Safety of Traditional Chinese Medicines. Aust. Prescr. 2003, 26, 128−130. (20) Pharmacopoeia of the People’s Republic of China, Chinese ed.; The Pharmacopoeia Commission of the People’s Republic of China; China Medical Science and Technology Press: Beijing, China, 2010; Vol. 1. (21) Pharmacopoeia of the People’s Republic of China, Chinese ed.; The Pharmacopoeia Commission of the People’s Republic of China; People’s Medical Publishing House, 2005; Vol. 1. (22) Dharmananda, S. An Introduction to Chinese Herbs. 1996. From http://www.itmonline.org/arts/herbintro.htm (accessed on Sep 27, 2011). (23) Bhal, S. K. LogPMaking Sense of the Value. Advanced Chemistry Development Inc. (ACD/Labs). From http://www.acdlabs. com/resources/knowledgebase/app_notes/-physchem/making_ sense.php (accessed on Sep 27, 2011). (24) Bhal, S. K.; Kassam, K.; Peirson, I. G.; Pearl, G. M. The Rule of Five Revisited: Applying Log D in Place of Log P in Drug-Likeness Filters. Mol. Pharmaceutics 2007, 4, 556−560. (25) Grès, M.-C.; Julian, B.; Bourrié, M.; Meunier, V.; Roques, C.; Berger, M.; Boulenc, X.; Berger, Y.; Fabre, G. Correlation Between Oral Drug Absorption in Humans, and Apparent Drug Permeability in TC-7 Cells, A Human Epithelial Intestinal Cell Line: Comparison with the Parental Caco-2 Cell Line. Pharm. Res. 1998, 15, 726−733. (26) Yazdanian, M.; Glynn, S. L.; Wright, J. L.; Hawi, A. Correlating Partitioning and Caco-2 Cell Permeability of Structurally Diverse Small Molecular Weight Compounds. Pharm. Res. 1998, 15, 1490− 1494. (27) Pade, V.; Stavchansky, S. Link Between Drug Absorption Solubility and Permeability Measurements in Caco-2 Cells. J. Pharm. Sci. 1998, 87, 1604−1607. (28) Hubatsch, I.; Ragnarsson, E. G.; Artursson, P. Determination of Drug Permeability and Prediction of Drug Absorption in Caco-2 Monolayers. Nat. Protoc. 2007, 2, 2111−2119. (29) Olukoga, A.; Donaldson, D. Liquorice and Its Health Implications. J. R. Soc. Promot. Health. 2000, 120, 83−89. (30) Gao, Q.; Li, J.; Cheung, J. K.; Duan, J.; Ding, A.; Cheung, A. W.; Zhao, K.; Li, W. Z.; Dong, T. T.; Tsim, K. W. Verification of the Formulation and Efficacy of Danggui Buxue Tang (A Decoction of Radix Astragali and Radix Angelicae Sinensis): an Exemplifying Systematic Approach to Revealing the Complexity of Chinese Herbal Medicine Formulae. Chin. Med. 2007, 2, 12. (31) Luszczki, J. J.; Zuchora, M.; Sawicka, K. M.; Kozinska, J.; Czuczwar, S. J. Acute Exposure to Caffeine Decreases the Anticonvulsant Action of Ethosuximide, But Not That of Clonazepam, Phenobarbital and Valproate Against Pentetrazole-Induced Seizures in Mice. Pharmacol. Rep. 2006, 58, 652−659. (32) Xu, L. H.; Huang, F.; Chen, T.; Wu, J. Anti-Virus Constituents in Radix Isatidis. Chin. J. Nat. Med. 2005, 3, 359−360. (33) Hayashi, K.; Imanishi, N.; Kashiwayama, Y.; Kawano, A.; Terasawa, K.; Shimada, Y.; Ochiai, H. Inhibitory Effect of Cinnamaldehyde, Derived from Cinnamomi Cortex, on the Growth of Influenza A/PR/8 Virus In Vitro and In Vivo. Antiviral Res. 2007, 74, 1−8. (34) Bae, K. H.; Ji, J. M.; Park, K. L. The Antibacterial Component from Cinnamomi Cortex Against a Cariogenic Bacterium Streptococcus mutans OMZ 176. Arch. Pharmacal Res. 1992, 15, 239−241. (35) Amidon, G. L. In A New Era in BA/BE World, International Symposium on BA/BE of Oral Drug Products, Kobe, Japan, 2011. (36) Kim, J. S.; Mitchell, S.; Kijek, P.; Tsume, Y.; Hilfinger, J.; Amidon, G. L. The Suitability of an In Situ Perfusion Model for

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors gratefully acknowledge Mr. Rongfu Gan and Mr. Xinchang Lin (Sinopharm Group Co. Ltd., P. R. China) for providing the top-selling Chinese Herbal Medicine List in China.

■

REFERENCES

(1) Chen, F. P.; Chen, T. J.; Kung, Y. Y.; Chen, Y. C.; Chou, L. F.; Chen, F. J.; Hwang, S. J. Use Frequency of Traditional Chinese Medicine in Taiwan. BMC Health Serv. Res. 2007, 7, 26. (2) Cassidy, C. M. Chinese Medicine Users in the United States. Part I: Utilization, Satisfaction, Medical Plurality. J. Altern. Complement. Med. 1998, 4, 17−27. (3) Ernst, E.; White, A. The BBC Survey of Complementary Medicine Use in the UK. Complement. Ther. Med. 2000, 8, 32−36. (4) MacLennan, A. H.; Wilson, D. H.; Taylor, A. W. The Escalating Cost and Prevalence of Alternative Medicine. Prev. Med. 2002, 35, 166−173. (5) Molassiotis, A.; Fernadez-Ortega, P.; Pud, D.; Ozden, G.; Scott, J. A.; Panteli, V.; Margulies, A.; Browall, M.; Magri, M.; Selvekerova, S.; Madsen, E.; Milovics, L.; Bruyns, I.; Gudmundsdottir, G.; Hummerston, S.; Ahmad, A. M. A.; Platin, N.; Kearney, N.; Patiraki, E. Use of Complementary and Alternative Medicine in Cancer Patients: a European Survey. Ann. Oncol. 2005, 16, 655−663. (6) Li, S.; Han, Q.; Qiao, C.; Song, J.; Cheng, C. L.; Xu, H. Chemical Markers for the Quality Control of Herbal Medicines: an Overview. Chin. Med. 2008, 3, 7. (7) Dressman, J. B.; Amidon, G. L.; Reppas, C.; Shah, V. P. Dissolution Testing as a Prognostic Tool for Oral Drug Absorption: Immediate Release Dosage Forms. Pharm. Res. 1998, 15, 11−22. (8) Amidon, G. L.; Lennernäs, H.; Shah, V. P.; Crison, J. R. A Theoretical Basis for a Biopharmaceutic Drug Classification: the Correlation of In Vitro Drug Product Dissolution and In Vivo Bioavailability. Pharm. Res. 1995, 12, 413−420. (9) FDA Guidance for Industry. Waiver of In Vivo Bioavailability and Bioequivalence Studies for Immediate-Release Solid Oral Dosage Forms Based on a Biopharmaceutics Classification System; Federal Food and Drug Administration: Washington, DC, 2000. (10) Guideline on the Investigation of Bioequivalence; European Medicines Agency (EMEA), Committee for Medicinal Products for Human Use (CHMP): London, UK, 2008; p 29. (11) Kasim, N. A.; Whitehouse, M.; Ramachandran, C.; Bermejo, M.; Lennernäs, H.; Hussain, A. S.; Junginger, H. E.; Stavchansky, S. A.; Midha, K. K.; Shah, V. P.; Amidon, G. L.Molecular Properties of WHO Essential Drugs and Provisional Biopharmaceutical Classification. Mol. Pharmaceutics 2004, 1, 85−96. (12) Takagi, T.; Ramachandran, C.; Bermejo, M.; Yamashita, S.; Yu, L. X.; Amidon, G. L. A Provisional Biopharmaceutical Classification of the Top 200 Oral Drug Products in the United States, Great Britain, Spain, and Japan. Mol. Pharmaceutics 2006, 3, 631−643. (13) Shawahna, R.; Rahman, N. Evaluation of the Use of Partition Coefficients and Molecular Surface Properties as Predictors of Drug Absorption: a Provisional Biopharmaceutical Classification of the List of National Essential Medicines of Pakistan. DARU J. Pharm. Sci. 2011, 19, 83−99. (14) Waldmann, S.; Almukainzi, M.; Chacra, N.; Amidon, G. L.; Lee, B. J.; Feng, J.; Kanfer, I.; Zuo, Z.; Bolger, M.; Löbenberg, R. Provisional Biopharmaceutical Classification of Some Common Herbs Used in Western Medicine. Mol. Pharmaceutics 2012, 9, 815−822. (15) De Smet, P. A. Herbal Medicine in Europe-Relaxing Regulatory Standards. N. Engl. J. Med. 2005, 352, 1176−1178. (16) Health Canada, Natural Health Products Directorate. From http://www.hc-sc.gc.ca/dhp-mps/prodnatur/index-eng.php (accessed on Sep 27, 2011). 1641



Article

Permeability Determinations: Utility for BCS Class I Biowaiver Requests. Mol. Pharmaceutics 2006, 3, 686−694. (37) Winiwarter, S.; Bonham, N. M.; Ax, F.; Hallberg, A.; Lennernas, H.; Karlen, A. Correlation of Human Jejunal Permeability (In Vivo) of Drugs with Experimentally and Theoretically Derived Parameters. A Multivariate Data Analysis Approach. J. Med. Chem. 1998, 41, 4939− 4949. (38) Linnankoski, J.; Makela, J. M.; Ranta, V.-P.; Urtti, A.; Yliperttula, M. Computational Prediction of Oral Drug Absorption Based on Absorption Rate Constants in Humans. J. Med. Chem. 2006, 49, 3674− 3681. (39) Benet, L. Z.; Larregieu, C. A. The FDA Should Eliminate the Ambiguities in the Current BCS Biowaiver Guidance and Make Public the Drugs for Which BCS Biowaivers Have Been Granted. Clin. Pharmacol. Ther. 2010, 88, 405−407. (40) Pade, D.; Stavchansky, S. Selection of Bioavailability Markers for Herbal Extracts Based on In Silico Descriptors and Their Correlation to In Vitro Permeability. Mol. Pharmaceutics 5, 665−671. (41) Xie, Y.; Ma, Y. M.; Wang, C. H.; Tao, J. S. Overview and Consideration on Improving Oral Bioavailability of Traditional Chinese Medicine Preparation. J. Int. Pharm. Res. 2011, 38, 195−200. (42) Zhu, J. T.; Choi, R. C.; Li, J.; Xie, H. Q.; Bi, C. W.; Cheung, A. W.; Dong, T. T.; Jiang, Z. Y.; Chen, J. J.; Tsim, K. W. Estrogenic and Neuroprotective Properties of Scutellarin from Erigeron breviscapus: a Drug Against Postmenopausal Symptoms and Alzheimer’s Disease. Planta Med. 2009, 75, 1489−1493. (43) Camuesco, D.; Comalada, M.; Rodriguez-Cabezas, M. E.; Nieto, A.; Lorente, M. D.; Concha, A.; Zarzuelo, A.; Galvez, J. The Intestinal Anti-inflammatory Effect of Quercitrin is Associated with an Inhibition in iNOS Expression. Br. J. Pharmacol. 2004, 143, 908−918. (44) Liu, X. H.; Pan, L. L.; Gong, Q. H.; Zhu, Y. Z. Antiapoptotic Effect of Novel Compound from Herba leonuri−Leonurine (SCM198): a Mechanism Through Inhibition of Mitochondria Dysfunction in H9c2 Cells. Curr. Pharm. Biotechnol. 2010, 11, 895−905. (45) Li, X.; Yuan, F. L.; Zhao, Y. Q.; Chen, F. H.; Lu, W. G.; Li, C. W.; Li, J. P. Effects of Leonurine Hydrochloride on Medically Induced Incomplete Abortion in Early Pregnancy Rats. Eur. J. Obstet. Gynecol. Reprod. Biol. 2011, 159, 375−380. (46) Asl, M. N.; Hosseinzadeh, H. Review of Pharmacological Effects of Glycyrrhiza sp. and Its Bioactive Compounds. Phytother. Res. 2008, 22, 709−724. (47) Roth, R. P.; Cantekin, E. I.; Bluestone, C. D.; Welch, R. M.; Cho, Y. W. Nasal Decongestant Activity of Pseudoephedrine. Ann. Otol. Rhinol. Laryngol. 1977, 86, 235−242. (48) Silver, H. M.; Ohsaki, Y. The Effects of Pseudoephedrine in Brochospastic Subjects. Curr. Ther. Res. Clin. Exp. 1963, 5, 1−6. (49) Lin, C. C.; Hsieh, S. J.; Hsu, S. L.; Chang, C. M. Hot Pressurized Water Extraction of Syringin from Acanthopanax senticosus and In Vitro Activation on Rat-Blood Macrophages. Biochem. Eng. J. 2007, 37, 117−124. (50) Navarrete, A.; Arrieta, J.; Terrones, L.; Abou-Gazar, H.; Calis, I. Gastroprotective Effect of Astragaloside IV: Role of Prostaglandins, Sulfhydryls and Nitric Oxide. J. Pharm. Pharmacol. 2005, 57, 1059−64. (51) Wang, S.; Li, J.; Huang, H.; Gao, W.; Zhuang, C.; Li, B.; Zhou, P.; Kong, D. Anti-Hepatitis B Virus Activities of Astragaloside IV Isolated from Radix Astragali. Biol. Pharm. Bull. 2009, 32, 132−135. (52) Tang, D.; He, B.; Zheng, Z. G.; Wang, R. S.; Gu, F.; Duan, T. T.; Cheng, H. Q.; Zhu, Q. Inhibitory Effects of Two Major Isoflavonoids in Radix Astragali on High Glucose-Induced Mesangial Cells Proliferation and AGEs-Induced Endothelial Cells Apoptosis. Planta Med. 2011, 77, 729−732. (53) Ohta, H.; Matsumoto, K.; Shimizu, M.; Watanabe, H. Paeoniflorin Attenuates Learning Impairment of Aged Rats in Operant Brightness Discrimination Task. Pharmacol., Biochem. Behav. 1994, 49, 213−217. (54) Liu, D. Z.; Zhu, J.; Jin, D. Z.; Zhang, L. M.; Ji, X. Q.; Ye, Y.; Tang, C. P.; Zhu, X. Z. Behavioral Recovery Following Sub-Chronic Paeoniflorin Administration in the Striatal 6-OHDA Lesion Rodent Model of Parkinson’s Disease. J. Ethnopharmacol. 2007, 112, 327−332.

(55) Yu, H. Y.; Mu, D. G.; Chen, J.; Yin, W. Suppressive Effects of Intrathecal Paeoniflorin on Bee Venom-Induced Pain-Related Behaviors and Spinal Neuronal Activation. Pharmacology 2011, 88, 159−166. (56) Zhang, X. J.; Li, Z.; Leung, W. M.; Liu, L.; Xu, H. X.; Bian, Z. X. The Analgesic Effect of Paeoniflorin on Neonatal Maternal SeparationInduced Visceral Hyperalgesia in Rats. J. Pain 2008, 9, 497−505. (57) Sun, D.; Shen, M.; Li, J.; Li, W.; Zhang, Y.; Zhao, L.; Zhang, Z.; Yuan, Y.; Wang, H.; Cao, F. Cardioprotective Effects of Tanshinone IIA Pretreatment via Kinin B2 Receptor-Akt-GSK-3beta Dependent Pathway in Experimental Diabetic Cardiomyopathy. Cardiovasc. Diabetol. 2011, 10, 4. (58) Liu, J. Q.; Lee, T. F.; Miedzyblocki, M.; Chan, G. C.; Bigam, D. L.; Cheung, P. Y. Effects of Tanshinone IIA, a Major Component of Salvia miltiorrhiza, on Platelet Aggregation in Healthy Newborn Piglets. J. Ethnopharmacol. 2011, 137, 44−49. (59) He, H. B.; Yang, X. Z.; Shi, M. Q.; Zeng, X. W.; Wu, L. M.; Li, L. D. Comparison of Cardioprotective Effects of Salvianolic acid B and Benazepril on Large Myocardial Infarction in Rats. Pharmacol. Rep. 2008, 60, 369−381. (60) Yin, Z. Z.; Zhang, L. Y.; Xu, L. N. The Effect of Dang-Gui (Angelica sinensis) and its Ingredient Ferulic Acid on Rat Platelet Aggregation and Release of 5-HT. Yaoxue Xuebao 1980, 15, 321−326. (61) Wang, X. H.; Yin, Y. Q.; Sui, C. G.; Meng, F. D.; Ma, P.; Jiang, Y. H. Inhibitory Effect of Cantharidin on Proliferation of A549 Cells. Chin. J. Cancer Res. 2007, 19, 283−286. (62) Li, W.; Xie, L.; Chen, Z.; Zhu, Y.; Sun, Y.; Miao, Y.; Xu, Z.; Han, X. Cantharidin, a Potent and Selective PP2A Inhibitor, Induces an Oxidative Stress-Independent Growth Inhibition of Pancreatic Cancer Cells Through G2/M Cell-Cycle Arrest and Apoptosis. Cancer Sci. 2010, 101, 1226−1233. (63) Tian, Y.; Zheng, L. H.; Xu, Z. Y.; Sun, L. Q.; Gao, C. K.; Zheng, Q. Z.; Zhang, Z. H.; Shu, Y. Clinical and Pharmacological Study of the Hemostatic Action of Rhizoma paridis by Contraction of Uterus. J. Trad. Chin. Med. 1986, 6, 178−182. (64) Hen, J.; Lee, W.; Gu, Y.; Tong, Y.; Fung, P. C.; Tong, L. Ginkgo biloba Extract (EGb761) Inhibits Mitochondria-Dependent Caspase Pathway and Prevents Apoptosis in Hypoxia-Reoxygenated Cardiomyocytes. Chin. Med. 2011, 6, 8. (65) Smith, P. F.; Maclennan, K.; Darlington, C. L. The Neuroprotective Properties of the Ginkgo biloba Leaf: a Review of the Possible Relationship to Platelet-Activating Factor (PAF). J. Ethnopharmacol. 1996, 50, 131−139. (66) Sun, R.; Zhang, Z. P.; Huang, W.; Lv, L.; Ren, H. Y. Protective Effects of Muskone on Rats with Complete Cerebral Ischemia. Trad. Chin. Drug Res. Clin. Pharmacol. 2009, 3, 197−200. (67) Zhang, S.; Song, Y. Q.; Ju, C. X.; Liu, Z. R.; Yue, W. Comparative Study of Survival Effects of Moschus and Muscone on Rat Retinal Neurons In Vitro. Nat. Prod. Res. Dev. 2007, 19, 415−419. (68) Chan, R. Y..; Chen, W. F.; Dong, A.; Guo, D.; Wong, M. S. Estrogen-Like Activity of Ginsenoside Rg1 Derived from Panax notoginseng. J. Clin. Endocrinol. Metab. 2002, 87, 3691−3695. (69) Li, S. H.; Chu, Y. Anti-inflammatory Effects of Total Saponins of Panax notoginseng. Zhongguo Yao Li Xue Bao 1999, 20, 551−554. (70) Shi, Q.; Hao, Q.; Bouissac, J.; Lu, Y.; Tian, S.; Luu, B. Ginsenoside-Rd from Panax notoginseng Ehances Astrocyte Differentiation from Neural Stem Cells. Life Sci. 2005, 76, 983−995. (71) Deng, X. K.; Yin, W.; Li, W. D.; Yin, F. Z.; Lu, X. Y.; Zhang, X. C.; Hua, Z. C.; Cai, B. C. The Anti-tumor Effects of Alkaloids from the Seeds of Strychnos nux-vomica on HepG2 Cells and its Possible Mechanism. J. Ethnopharmacol. 2006, 106, 179−186. (72) Shen, Y. C.; Chiou, W. F.; Chou, Y. C.; Chen, C. F. Mechanisms in Mediating the Anti-inflammatory Effects of Baicalin and Baicalein in Human Leukocytes. Eur. J. Pharmacol. 2003, 465, 171−181. (73) Liu, C. G.; Zhang, T. H.; Ding, R. G.; Zhao, J.; Huang, M. A Study on the Anti-inflammatory Effect of Bilirubin and Cholic Acid Analogs. Pharm. J. Chin. People’s Liberation Army 2008, 24, 479−481. (74) Zheng, X.; Yang, D.; Liu, X.; Wang, N.; Li, B.; Cao, H.; Lu, Y.; Wei, G.; Zhou, H.; Zheng, J. Identification of a New Anti-LPS Agent, 1642



Article

Geniposide, from Gardenia jasminoides Ellis, and its Ability of Direct Binding and Neutralization of Lipopolysaccharide In Vitro and In Vivo. Int. Immunopharmacol. 2010, 10, 1209−1219. (75) Lu, B. Q. Studies on Pharmacological Constituents of Shuanghuanglian Powder Injection. PhD Thesis. First Military Medical University, Guangzhou, China, 2007. (76) Stanely Mainzen Prince, P.; Karthick, M. Preventive Effect of Rutin on Lipids, Lipoproteins, and ATPases in Normal and Isoproterenol-induced Myocardial Infarction in Rats. J. Biochem. Mol. Toxicol. 2007, 21, 1−6. (77) Yang, C. M.; Sui, D. Y.; Yu, X. F.; Qu, S. C. Protective Effect of Xueshuan Xinmaining Capsules on Experimental Myocardial Infraction in Rats. Wujing Yixue 2005, 16, 352−354. (78) Song, H. P.; Guan, R. F. Pharmacology and Clinical Usage of Allicin. Chin. Comm. Doctor 2007, 24, 9−10. (79) Ried, K.; Frank, O. R.; Stocks, N. P.; Fakler, P.; Sullivan, T. Effect of Garlic on Blood Pressure: a Systematic Review and MetaAnalysis. BMC Cardiovasc. Disord. 2008, 8. (80) Lin, F. L.; Wu, S. J.; Lee, S. C.; Ng, L. T. Antioxidant, Antioedema and Analgesic Activities of Andrographis paniculata Extracts and Their Active Constituent Andrographolide. Phytother. Res. 2009, 23, 958−64. (81) Shen, Y. C.; Chen, C. F.; Chiou, W. F. Andrographolide Prevents Oxygen Radical Production by Human Neutrophils: Possible Mechanism(s) Involved in its Anti-inflammatory Effect. Br. J. Pharmacol. 2002, 135, 399−406. (82) Varma, D. R.; Yue, T. L. Adrenoceptor Blocking Properties of Atropine-like Agents Anisodamine and Anisodine on Brain and Cardiovascular Tissues of Rats. Br. J. Pharmacol. 1986, 87, 587−94. (83) Cai, J. X. The Clinical Usage of Asiaticosid. Strait Pharm. J. 2007, 19, 92−93. (84) Kimura, Y.; Sumiyoshi, M.; Samukawa, K. I.; Satake, N.; Sakanaka, M. Facilitating Action of Asiaticoside at Low Doses on Burn Wound Repair and its Mechanism. Eur. J. Pharmacol. 2008, 584, 415− 423. (85) Peng, Y.; Xu, S.; Chen, G.; Wang, L.; Feng, Y.; Wang, X. l-3-nButylphthalide Improves Cognitive Impairment Induced by Chronic Cerebral Hypoperfusion in Rats. J. Pharmacol. Exp. Ther. 2007, 321, 902−910. (86) Liu, X.; Liao, Q. D.; Cao, H. X.; Huang, J. J. Effect of Extract of Horse Chestnut Seeds(aescuven forte) on IL-6, IL-1β and TNF in Patients with Limb Trauma. Zhongnan Yaoxue 2009, 7, 777−780. (87) Ono, M.; Satoh, T. Pharmacological Studies on Lappaconitine: Antinociception and Inhibition of the Spinal Action of Substance P and Somatostatin. Jpn. J. Pharmacol. 1991, 55, 523−530. (88) Pei, Y. P.; Chen, J.; Li, W. L. Progress in Research and Application of Silymarin. Med. Aromatic Plant Sci. Biotechnol. 2009, 3, 1−8.

1643


Establishing the Pharmaceutical Quality of ... - ACS Publications

Recommend Documents