Discovery and Current Status of Evaluation System of Bioavailability and Related Pharmaceutical Technologies for Traditional Chinese Medicines—Flos Lonicerae Japonicae—Fructus Forsythiae Herb Couples as an Example

Traditional Chinese medicines (TCMs) have attracted extensive interest throughout the world due to their long history of health protection and disease control, and the internalization of TCM preparations or patented drugs has been considered a wind vane in the process of TCM modernization. However, multi-target effects, caused by multiple components in TCMs, hinder not only the construction of the quality evaluation system (bioavailability), but also the application of pharmaceutical technologies, which results in the poor efficacy in clinical practice. This review describes the methods in the literature as well as in our thoughts about how to identify the marker components, establish the evaluation system of bioavailability, and improve the bioavailability in TCM preparations. We expect that the current study will be positive and informative.


Introduction
Traditional Chinese medicines (TCMs), utilized in the prevention and treatment of various diseases for thousands of years in China, have been gradually accepted and employed in other countries. TCM preparations or patented drugs, defined by the utilization of herbs, animals, and minerals, with their respective dosages in accordance with the guidance of Chinese medicine theory and the rule of "King, Vassal, Assistant and Delivery servant", have different dosage forms, such as capsules, tablets, pills, powders, oral liquids, etc. [1]. It was reported that the Chinese export of herbal medicines and extracts was significantly higher than that of preparations in the recent years. As shown in 2014, the export of herbal medicines and extracts was worth 2.95 billion dollars, but that of preparations was little, only 250 million dollars [2], which was mainly due to the unsound quality evaluation system (bioavailability) and poor efficacy in clinical practice. For example, Shuang-Huang-Lian oral liquid, a well-known TCM preparation composited of Flos Lonicerae Japonicae, Fructus Forsythiae, and Radix Scutellariae, is usually used as the treatment for acute upper respiratory tract infection caused by bacteria and viruses, but its clinical efficacy was unstable and far lower than that of injection [3]. It was found that multi-target effects, caused by multiple components in TCMs, hindered not only the construction of the evaluation system of bioavailability, but also the formulation, designation, and technologies application. How to establish a quantifiable evaluation system of bioavailability and find suitable pharmaceutical technologies to improve the bioavailability was not only a basic scientific problem of bio-pharmaceutics for TCM preparations, but it was also the key factor in modernizing TCMs.
The following essential problems that refer to the evaluation system of bioavailability construction and pharmaceutical technology applications for TCM preparations exist. Firstly, the network pharmacological effects and the complex structure-effect and dose-effect relationships in TCMs contributed to difficulty in identifying the effective components; Secondly, biological active and pharmacokinetic (absorption, distribution, metabolism, and excretion) diversity of effective constituents resulted in obstacles for setting up weight coefficients for integrating bioavailability; Thirdly, pharmaceutical technologies were hardly applied for TCM preparations due to their complicated physico-chemical properties for both active ingredients and associated constituents.
Therefore, the current problems about how to identify the active components promptly; how to establish a reasonable mathematics model to calculate the weight coefficient to integrate bioavailability; and how to improve the integral bioavailability using related pharmaceutical technologies in TCM preparations need to be further investigated.

Classic Separation and Analysis
The classic separation and analysis model was performed to identify the active components according to the procedures of extraction, separation, purification, characterization, pharmacological tests, etc., and it was applied to new Chinese herbal monomer or Chinese herbal extract development. For example, artemisinin isolated from the plant Artemisia annua, sweet wormwood, and its derivatives possess the most rapid actions against Plasmodium falciparum malaria [4]. Digoxin was a purified cardiac glycoside, extracted from Digitalis lanata, and was occasionally used to treat various heart diseases, namely atrial fibrillation and atrial flutter [5]. Morphine, a pain medication of the opiate type extracted from papaver somniferum L., can decrease feelings of pain through acting directly on the central nervous system (CNS) [6]. Paclitaxel, extracted from the Yew tree, is an anti-cancer drug. It was the first-line treatment for cancers of the breast, colon, lung, etc., and the second-line treatment for AIDS-related Kaposi's sarcoma [7]. The chemotherapy agent (vincristine), extracted from Catharanthus roseus, was utilized as the treatment of leukemias, lymphomas, etc. [8]. The total lactones, a Chinese herbal extract from the Ginkgo leaf, contained mainly ginkgo lactone A, ginkgo lactone B, ginkgo lactone C, and ginkgo seed lactone, which were prepared as a medicine for preventing or treating deafness and tinnitus [9]. The tea polyphenols, included catechins, theaflavins, tannins, and flavonoids, can prevent coronary heart disease and cancer [10].

Knock-in and Knock-out
Xiao et al., 2009 [45], put first forward that constituents knock-out/knock-in, inspired by functional genetic methods, are novel patterns of efficient component recognition and quality control for TCMs, which include marker compounds identified by studying the effect of the constituents knocked out on efficacies, and the dosage-effect or dosage-toxicity relationships studied by observing the effect of marker compounds knocked in on efficacies ( Figure 2; Table 2). For example, Yan et al., 2014 [46], and Li, 2013 [47], reported the identification of the major active constituents for bacterial diarrhea treatment evaluated by the growth of Shigella dysenteriae using microcalorimetry in Rhizoma coptidis by the knock-out and knock-in method, and found that coptisine and berberine were the important components with bacteriostatic activities of 54.10% and 39.75%, respectively, by the knock-out method, and their suitable concentration ranged from 8.08% to 31.92% and from 4.05% to 14.45% of the total, respectively, by the knock-in method. Jin et al., 2013 [48], showed the identification of bioactive compounds for osteoporosis treatment evaluated by osteoblasts cell proliferation and differentiation in Herba Epimedii by the knock-out method, and found that epimedin A, epimedin B, epimedin C, and icariin were the main active constituents. Yu et al., 2009 [49], studied the assessment of effective components for anti-tumor activity evaluated by the synergistic effects of cyclophosphamide on chemotherapy for S180 tumor-bearing mice in Shenmai formulae composited of Radix Ginseng and Radix Ophiopogonis by the knock-out method, and found that panoxadiol and a type of ginseoside were the active components.  Xiao et al., 2009 [45], put first forward that constituents knock-out/knock-in, inspired by functional genetic methods, are novel patterns of efficient component recognition and quality control for TCMs, which include marker compounds identified by studying the effect of the constituents knocked out on efficacies, and the dosage-effect or dosage-toxicity relationships studied by observing the effect of marker compounds knocked in on efficacies ( Figure 2; Table 2). For example, Yan et al., 2014 [46], and Li, 2013 [47], reported the identification of the major active constituents for bacterial diarrhea treatment evaluated by the growth of Shigella dysenteriae using microcalorimetry in Rhizoma coptidis by the knock-out and knock-in method, and found that coptisine and berberine were the important components with bacteriostatic activities of 54.10% and 39.75%, respectively, by the knockout method, and their suitable concentration ranged from 8.08% to 31.92% and from 4.05% to 14.45% of the total, respectively, by the knock-in method. Jin et al., 2013 [48], showed the identification of bioactive compounds for osteoporosis treatment evaluated by osteoblasts cell proliferation and differentiation in Herba Epimedii by the knock-out method, and found that epimedin A, epimedin B, epimedin C, and icariin were the main active constituents. Yu et al., 2009 [49], studied the assessment of effective components for anti-tumor activity evaluated by the synergistic effects of cyclophosphamide on chemotherapy for S180 tumor-bearing mice in Shenmai formulae composited of Radix Ginseng and Radix Ophiopogonis by the knock-out method, and found that panoxadiol and a type of ginseoside were the active components.   Shenmai formulae Panoxadiol, panaxotriol, ophiopogonpolysaccharide, ophiopogonin Antitumor effect S180 bearing mice Panoxadiol, panaxotriol, ophiopogonpolysaccharide [49] HUVEC: human umbilical vein endothelial cells; DPPH: 2,2-diphenylpicrylhydrazyl; PC12: pheochromocytoma.

Knock-in and Knock-out
We found above that the knock-in method can be suitable for identifying the effective components in Chinese herbal extracts and Chinese herbal compounds, but the application for the knock-out method is limited due to the fact that the target constituents are difficult to remove from Chinese herbal compounds.

Pharmacokinetics (PK)-Pharmacodynamics (PD)
Pharmacokinetics (PK)-pharmacodynamics (PD), put forward first by Sheiner et al., 2009 [56], are extensively applied for effective constituent identification in the field of TCMs, which mainly includes the correlation analysis between PK (the blood-drug concentration method) and PD (the pharmacology-effect method) ( Figure 3 and Table 3). For example, Liu et al., 2014 [43], reported the PK profiles of multiple components after oral administration of Da-Huang-Fu-Zi-Tang and the PD profiles evaluated by the effect of the serum at different time points on pancreatic acinar cells (AR42J) from injury, and found that rhein isomer methylation, rhein glucoside, hydroxyl-chrysophanol, hypaconine, talatisamine, chysophanol glucuronide conjugation, and chysophanol glucuronide conjugation might be the principle constituents analyzed by CCA. Peng, 2014 [57], studied the PK of baicalin, geniposide, cholalic acid, hyodeoxycholic acid, chlorogenic acid, and neochlorogenic acid in a Qingkailing injection composed of Cholalicacid, Conchamargaritifera, Hyodeoxycholic acid, Gardeniae Fructus, Cornububali, Radix isatidis, Baicalin, and Flos Lonicerae Japonicae using UPLC-ESI-MS/MS and studied the PD by evaluating temperature changes in rats, and found that baicalin and geniposide were the main effective ingredients by using Winnonlin software analysis. Wang et al., 2014 [58], showed that Tanshinone IIA was the main ingredient for anti-oxidant activity in a Yin-Teng-Gu-Bi-Kang prescription composed of Radix Salviae Miltiorrhiae, Angelicae Sinensis Radix, Paeoniae Radix Alb, and Celastrusorbiculatus Thunb. analyzed by the PK (Tanshinone IIA concentration in plasma)-PD (malondialdehyde (MDA) level in serum) model. 9 We found above that the knock-in method can be suitable for identifying the effective components in Chinese herbal extracts and Chinese herbal compounds, but the application for the knock-out method is limited due to the fact that the target constituents are difficult to remove from Chinese herbal compounds.

Pharmacokinetics (PK)-Pharmacodynamics (PD)
Pharmacokinetics (PK)-pharmacodynamics (PD), put forward first by Sheiner et al., 2009 [56], are extensively applied for effective constituent identification in the field of TCMs, which mainly includes the correlation analysis between PK (the blood-drug concentration method) and PD (the pharmacology-effect method) (Figure 3 and Table 3). For example, Liu et al., 2014 [43], reported the PK profiles of multiple components after oral administration of Da-Huang-Fu-Zi-Tang and the PD profiles evaluated by the effect of the serum at different time points on pancreatic acinar cells (AR42J) from injury, and found that rhein isomer methylation, rhein glucoside, hydroxyl-chrysophanol, hypaconine, talatisamine, chysophanol glucuronide conjugation, and chysophanol glucuronide conjugation might be the principle constituents analyzed by CCA. Peng, 2014 [57], studied the PK of baicalin, geniposide, cholalic acid, hyodeoxycholic acid, chlorogenic acid, and neochlorogenic acid in a Qingkailing injection composed of Cholalicacid, Conchamargaritifera, Hyodeoxycholic acid, Gardeniae Fructus, Cornububali, Radix isatidis, Baicalin, and Flos Lonicerae Japonicae using UPLC-ESI-MS/MS and studied the PD by evaluating temperature changes in rats, and found that baicalin and geniposide were the main effective ingredients by using Winnonlin software analysis. Wang et al., 2014 [58], showed that Tanshinone IIA was the main ingredient for anti-oxidant activity in a Yin-Teng-Gu-Bi-Kang prescription composed of Radix Salviae Miltiorrhiae, Angelicae Sinensis Radix, Paeoniae Radix Alb, and Celastrusorbiculatus Thunb. analyzed by the PK (Tanshinone IIA concentration in plasma)-PD (malondialdehyde (MDA) level in serum) model.

Evaluation System of Bioavailability Establishment for TCMs
The construction of the evaluation system of bioavailability is one of the most important scientific issues in the modernization of TCMs. Hao et al., 2009 [64], first reported that an area under curve (AUC)-weighting method could obtain the integral PK properties based on the same type of components in TCMs (Figure 4). The weighting coefficient for each constituent was calculated using Equations (1) and (2). The integral concentrations (C T ) at each time point were then calculated by Equation (3), where w represented the weighting coefficient, AUC 1 -AUC n represented bioavailability in vivo and C 1 -C n represented the plasma concentration of each constituent studied. The evaluation system establishment could comprehensively estimate the correlation between integral PK and PD, especially for the TCMs with a narrow therapeutic window, to ensure safety in practical applications. As seen in Table 4, Dong et al., 2014 [65], showed the integral PK profiles of Rhodojaponin I, II, and III, the active components in Rhododendri Mollis Flos, and found that the correlation with the potential markers of myocardial injury ((creatine kinase-measurement blood) (CK-MB) and lactate dehydrogenase (LDH)) was fairly strong, which can be conductive to fully understanding the relationship between the PK behaviors and the compound's efficacy. Guo [69], showed the integrated PK of baicalin, baicalein, geniposide, palmatine, and berberine, the main effective ingredients in Huang-Lian-Jie-Du-Tang in middle cerebral artery occlusion (MCAO) rats, and found that the correlation with the anti-ischemia index (Interleukin 6 (IL-6), tumor necrosis factor (TNF-α), superoxide dismutase (SOD), glutamic acid (Glu), and MDA) in the serum was good, which would provide comprehension better understanding of cerebrovascular disease as Huang-Lian-Jie-Du-Tang is used in clinical practice. Xie et al., 2010 [70], reported the holistic PK of Schisandrin, schisantherin A, deoxyschisandrin, and γ-schisandrin, the four main lignin components in Schisandra, and found that the integral AUC and CYP3A activities correlated well with hepatic injury biomarkers (ALT and aspartate aminotransferase (AST)) in serum. However, the integral PK calculated by an AUC-weighting method was established on the basis of the fact that the bioavailability of the integral components was positively correlated with their efficacy. For example, the AUC value of compound A was higher than that of compound B, but their efficacy was opposite. It means that the effect of the bioavailability fluctuation of compound B on the integral AUC was far less than that of compound A, but that its effect on the pharmacology was far stronger than that of compound A, which resulted in the integral PK parameters being negatively or not correlated with pharmacology. Therefore, an AUC-weighting method might not be well suited for studying the integral PK of all TCMs. It was presumed that efficacy as a weight coefficient might be more reasonable if the efficacy we chose could represent the pharmacological effects of TCMs. LDH: lactate dehydrogenase; CK-MB: creatine kinase-measurement blood; E-C: effect-concentration.

Study on the Evaluation System of Bioavailability Establishment and Related Pharmaceutical Technologies-Flos Lonicerae Japonicae-Fructus Forsythiae Herb Couples as an Example
We have previously studied the evaluation system of bioavailability establishment and related pharmaceutical technology applications based on the Flos Lonicerae Japonicae-Fructus Forsythiae (FLJ-FF) herb couple as a model drug ( Figure 6).

Study on the Evaluation System of Bioavailability Establishment and Related Pharmaceutical Technologies-Flos Lonicerae Japonicae-Fructus Forsythiae Herb Couples as an Example
We have previously studied the evaluation system of bioavailability establishment and related pharmaceutical technology applications based on the Flos Lonicerae Japonicae-Fructus Forsythiae (FLJ-FF) herb couple as a model drug ( Figure 6).

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Thirdly, the integral PK for caffeic acid derivatives based on an AUC-weighting approach was established, but the bioavailability of chlorogenic acids was negatively correlated with their efficacy. For example, the AUC of chlorogenic acid in the FLJ-FF herb couple was much higher than that of forsythoside A, but the IC50 was lower than that of forsythoside A (data not shown). It meant that the effect of the bioavailability fluctuation of phenylethanoid glycosides on the integral AUC calculated by an AUC-weighting approach was far less than that of the chlorogenic acids, but the effect on the pharmacology was far more than that of the chlorogenic acids, which resulted in the antiviral activity, not the integral AUC, being improved significantly as forsythoside A knocked in the FLJ-FF herb couple (data not shown). However, the integral AUC calculated by IC50 as follows (W represents the weighting coefficient and the C 1 -C n represents the plasma concentration of the components studied) was increased gradually as the antiviral activity was improved by the FLJ-FF herb couple knocked-in forsythoside A, showing a strong positive correlation, and the integral PK parameters using IC50 as the weight coefficient index could fully take eight caffeic acid derivatives' PK parameters into account (data not shown). The results above indicated that IC50 as a weight coefficient was more reasonable than AUC.
Finally, the antiviral activity of commercially available FLJ-FF herb couple preparations (Shuang-Huang-Lian oral liquid, Yin-Qiao-Jie-Du tablet, Fufang-Qin-Lan oral liquid, and Qing-Re-Jie-Du oral liquid) was regulated based on the integrated AUC calculated by IC50. The antiviral effect was decreased significantly as the four preparations knocked out the FLJ-FF herb couple, but increased significantly as the FLJ-FF herb couple was knocked in (data not shown). Besides, the integral absorption of caffeic acid derivatives in the four preparations was improved significantly both in vitro and in vivo by the chito-oligosaccharide (COS) (data not shown), which was consistent with the fact that the absorption of caffeic acid derivatives in monomers, the FLJ-FF herb couple, or its preparations was mainly restricted by tight junctions (TJs) [211][212][213][214], and COS was an absorption enhancer based on tight junctions with high effectiveness and low mucosal toxicity [215]. In addition, the treatment with FLJ-FF herb couple preparations with COS can restrain the MDCK cell damage upon influenza virus propagation better than that of the control [216], but the treatment with the preparations with the COS-knocked-out FLJ-FF herb couple showed non-significance compared to that of control (data not shown). The results above illustrated not only the antiviral activity improvement due to the COS in FLJ-FF herb couple preparations resulting from the improvement of the integrated AUC of caffeic acid derivatives, but also showed the reasonability of the weight coefficient calculated by IC50, not AUC. Absorption-enhancer COS has been successfully applied for the second development of FLJ-FF herb couple preparations.

Conclusions and Future Perspective
TCM preparations, extensively recorded in Chinese Pharmacopoeia, have long history with applications for protecting health and controlling disease [207]. The present quality assessment of TCM preparations mainly focused on single chemical constituents, not biological indicators, as markers, and novel pharmaceutical excipients were hardly applied for TCM preparations due to their complicated physico-chemical properties, which resulted in poor effects in clinical practice. Here, we attempted to propose a plan (Figure 7) to deal with the obstacles in order to carry out the bio-pharmaceutical explorations of TCM preparations better. Firstly, both the spectrum-effect relationship and PK-PD model can be simultaneously performed to identify the chemical markers and to be verified by the "knock-in" method; Secondly, the weight coefficient calculated by AUC or the efficacy should be compared to decide which one is more suitable for the integral PK; Thirdly, an absorption enhancer might be considered the preferred pharmaceutical technology in Chinese herbal compound preparations, such as the preparations recorded in Chinese Pharmacopeia (Volume I) as the active constituents recognized as belonging to those classified III in the BCS [207].
attempted to propose a plan (Figure 7) to deal with the obstacles in order to carry out the biopharmaceutical explorations of TCM preparations better. Firstly, both the spectrum-effect relationship and PK-PD model can be simultaneously performed to identify the chemical markers and to be verified by the "knock-in" method; Secondly, the weight coefficient calculated by AUC or the efficacy should be compared to decide which one is more suitable for the integral PK; Thirdly, an absorption enhancer might be considered the preferred pharmaceutical technology in Chinese herbal compound preparations, such as the preparations recorded in Chinese Pharmacopeia (Volume I) as the active constituents recognized as belonging to those classified III in the BCS [207]. In recent years, LC-MS was rapidly accepted by the analytical community, and it was gradually applied for qualitative and quantitative analysis [217][218][219][220], PK study [221,222], metabolite in vivo identification [223,224], metabolomics [225,226], quality control [227,228], and pharmacological studies [229,230] in TCMs. The novel methods, such as aggregation morphology [231] and magnetic molecularly imprinted polymer [232], were also helpful in understanding the mechanism of TCMs and discovering drugs based on TCMs. Besides, systems biology (genomics, proteomics, metabolomics, and bioinformatics), a new subject in the field of life sciences, provided a comprehensive resource for the modernization and advancement of TCMs as well as general drug discovery efforts, which proposed a system-to-system research methodology to study the interaction between TCMs and the human body and their applications in drug research and development [233]. In addition, some promising excipients can also accelerate the development of TCM preparations. For example, Kollidon CL, manufactured by BASF, the largest chemical producer in the world, can produce the highest disintegration speed (18 min), which is 50% faster than croscarmellose sodium (CMC-Na: 27 min) and almost three times faster than sodium starch glycolate (CMS-Na: 50 min), and which can be applied for surmounting the obstacles of poor solubility and long disintegration times In recent years, LC-MS was rapidly accepted by the analytical community, and it was gradually applied for qualitative and quantitative analysis [217][218][219][220], PK study [221,222], metabolite in vivo identification [223,224], metabolomics [225,226], quality control [227,228], and pharmacological studies [229,230] in TCMs. The novel methods, such as aggregation morphology [231] and magnetic molecularly imprinted polymer [232], were also helpful in understanding the mechanism of TCMs and discovering drugs based on TCMs. Besides, systems biology (genomics, proteomics, metabolomics, and bioinformatics), a new subject in the field of life sciences, provided a comprehensive resource for the modernization and advancement of TCMs as well as general drug discovery efforts, which proposed a system-to-system research methodology to study the interaction between TCMs and the human body and their applications in drug research and development [233]. In addition, some promising excipients can also accelerate the development of TCM preparations. For example, Kollidon CL, manufactured by BASF, the largest chemical producer in the world, can produce the highest disintegration speed (18 min), which is 50% faster than croscarmellose sodium (CMC-Na: 27 min) and almost three times faster than sodium starch glycolate (CMS-Na: 50 min), and which can be applied for surmounting the obstacles of poor solubility and long disintegration times in oral solid dosage forms of TCM preparations such as tablets or capsules when the active constituents recognized belonged to those classified II in the BCS. Chitosan derivatives, such as N-trimethyl chitosan chloride [234] and chito-oligosaccharide [215], synthesized with remarkable solubility at neutral pH in an aqueous environment, were not only non-toxic, biocompatible, and biodegradable, but also performed as intestinal absorption enhancers by reversible opening of the tight junctions, which can be applied for improving the permeability of active constituents as the active constituents recognized belonged to those classified III in the BCS. We expect that the current study will be positive and informative.