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Can the aggregation be a new approach for understanding the mechanism of Traditional Chinese Medicine?
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Journal of Ethnopharmacology 117 (2008) 378–384
Can the aggregation be a new approach for understanding the mechanism of Traditional Chinese Medicine? Yan Zhuang a , Jingjing Yan a , Wei Zhu b , Lirong Chen a , Dehai Liang a,∗ , Xiaojie Xu a,∗ a
College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China b Guangdong Hospital of Traditional Chinese Medicine, Guangzhou, China
Received 5 October 2007; received in revised form 14 November 2007; accepted 2 February 2008 Available online 10 March 2008
Abstract “Frequent hitter” phenomenon emerged in the high-throughput screening; one of the most common mechanisms behind artifactual inhibition is that some organic molecules formed large colloid-like aggregates which are able to sequester and thereby inhibit enzymes. To investigate the situation in Traditional Chinese Medicine (TCM), 60 medicinal herbs and 24 Chinese herbal formulae were detected by dynamic light scattering (DLS), and aggregates were observed in all the 84 solution mixtures. The aggregates of two Chinese herbal formulae, ‘Xue-Fu-Zhu-Yu Tang’ (XF) and ‘Jing-Guan Tang’ (JG), were not only able to survive in the gastro-intestinal environment, but also had the ability to pass through the monolayer of the Caco-2 cell. The activities of XF and JG against three cardiovascular targets were also aggregates-related. Based on these findings, a new possible mechanism of the action of Chinese medicine was proposed. © 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: Aggregate; Traditional Chinese Medicine; Dynamic light scattering
1. Introduction Biochemical screens are designed to get the competitive, reversible bindings and specific ligands with high-affinity, but many false positives are emerged in the high-throughput screening (Rishton, 1997). One of the most common mechanisms behind artifactual inhibition is that some organic molecules formed large colloid-like aggregates which are able to sequester and thereby inhibit enzymes (McGovern et al., 2002; Feng et al., 2005; Feng and Shoichet, 2006a; Shoichet, 2006). The aggregates formed by single-compound are observed in the libraries of chemical compounds, in leads for drug discovery process (McGovern and Shoichet, 2003), and even in a part of drugs (Seidler et al., 2003). Furthermore, it is reported that multicompound mixtures are able to influence the aggregate-based inhibition with predominated synergistic effect or antagonism (Feng and Shoichet, 2006b). These findings inspired us to investigate the aggregation behaviors in Traditional Chinese Medicine (TCM). Generally,
TCM consists of multiple herbs, so it contains hundreds of constituent diversely in chemical structure and in bioactivity. Since aggregate occurs in the mixtures of compounds or even by single compound under certain conditions, it is expected that aggregation is a common process in TCM. 60 medicinal herbs and 24 Chinese herbal formulae were detected by dynamic light scattering (DLS), and aggregates were observed in all the 84 solution mixtures. In order to study the properties of the aggregates and the effects of aggregate on biological activity, we chose two TCM formulae, ‘Xue-Fu-Zhu-Yu Tang’ (XF) and ‘Jing-Guan Tang’ (JG), both of which are widely used in clinical practice for treating many kinds of cardiovascular diseases in China, to further study. The aggregates of the two formulae were not only able to survive in the gastro-intestinal environment, but also had the ability to pass through the monolayer of the Caco-2 cells. The activities of XF and JG against three cardiovascular targets were also aggregates-related. 2. Materials and methods 2.1. Plant material
∗
Corresponding authors. Tel.: +86 10 6275 7456; fax: +86 10 6275 1708. E-mail addresses: [email protected] (D. Liang), [email protected] (X. Xu). 0378-8741/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2008.02.017
All the medicinal herbs were purchased from the Tong Ren Tang Pharmaceutical Shop, Beijing, China, and authenticated
Y. Zhuang et al. / Journal of Ethnopharmacology 117 (2008) 378–384
by Dr. Wei Zhu, Guangdong Hospital of Traditional Chinese Medicine, China. The name of 60 medicinal herbs and 24 Chinese herbal formulae were listed in the Supplementary Tables S1 and S2. Herbal formula XF is composed of 11 crude herbs: Flos Carthami (9 g), Fructus Aurantii (6 g), Radix Achyranthis Bidentatae (9 g), Radix Angelicae Sinensis (9 g), Radix Bupleuri (3 g), Radix Glycyrrhizae (6 g), Radix Paeoniae Rubra (6 g), Radix Platycodonis (4.5 g), Radix Rehmanniae (9 g), Rhizoma Chuanxiong (4.5 g), and Semen Persicae (12 g). JG contains only four of the 11 herbs: Fructus Aurantii (10 g), Radix Bupleuri (5 g), Radix Paeoniae Rubra (15 g), and Rhizoma Chuanxiong (15 g). The herb resource could refer the Supplementary Table S1. 2.2. TCM processing procedure 10 g of each of the 60 single herbs was added with 50 mL water, and boiled for 30 min. Each of the 24 herbal formulae (amount of ingredients shown in the Supplementary Table S2), was added with 200 mL water, and boiled for 30 min. The upper solution of the decoctions was collected and diluted ten times with double-boiled water and studied by DLS and Transmission Electron Microscope (TEM) (Supplementary Fig. S2). The oral decoctions of XF and JG at the concentration of administration (practical dosage), 23 mg/mL for XF and 10 mg/mL for JG, were prepared and used as the stock solutions. The stock solution was diluted three times, each by a factor of 10 (i.e. 23 mg/mL, 2.3 mg/mL, 0.23 mg/mL and 0.023 mg/mL for XF and 10 mg/mL, 1.0 mg/mL, 0.1 mg/mL and 0.01 mg/mL for JG). We use XF-n or JG-n, where n is the order number of 1–4, to denote the solution at different concentrations. Gastric condition in vitro was simulated by using HCl (pH 1). And intestinal condition was mimicked by Hank’s balanced salt solution (HBSS, pH 7.4). XF and JG were dried and resolved in HBSS at concentration of 2.0 mg/mL for XF and 1.0 mg/mL for JG.
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centrifuge. The supernatant was carefully transferred to a dustfree light scattering cell and studied by DLS. The elemental analysis were performed on Elementar Vario EL (Germany). 2.5. Caco-2 experiment Caco-2 cells were similar to the intestinal epidermic cells in morphological and biochemical characteristics, and here were used to study the absorption of aggregates. The Caco2 experiment was performed in the State Key Laboratory of Natural and Biomimetic Drugs, China, as previously described (Tian et al., 2006). Caco-2 cell monolayers were prepared according to a standard 21 day cultural procedure to reach confluence and differentiation. The integrity of the monolayers was checked by measuring the transepithelial electrical resistance (TEER) with an epithelial voltohmmeter (EVOM, World Precision Instrument, Sarasota, FL) equipped with a chopstick. The measurements were performed before and after transport experiments. Then, 20 mg/mL XF and 10 mg/mL JG in HBSS were added on the apical side and were incubated for 1.5 h in a shaker (37 ◦ C, 50 rpm). The basolateral solutions were collected, diluted 10 times, and then detected by DLS. Detailed Caco-2 experiment was shown in Supplementary. 2.6. PAI-1 assays The PAI-1 Activity Assay Kit came from Department of Molecular Genetics, Medical School, Fudan University, Shanghai, China. The experiment followed the specification except that the decoctions were incubated with the blood for ten minutes before testing. TB (Tris-borate) buffer solution was taken as negative control and Corilagin was taken as positive control (Shen et al., 2003).
2.3. Dynamic light scattering (DLS)
2.7. ACE assays
DLS was performed either on a commercial Zeta Potential Analyzer with particle size analysis (ZetaPALS, Brookhaven Inc.) or on a commercial laser light scattering spectrometer equipped with a BI-200SM goniometer and a BI-TurboCorr digital correlator (Brookhaven Inc.). A 100-mW, vertically polarized solid state laser (GNI, Changchun, China) operating at 532 nm was used as the light source. The detector angle was 90◦ . Data analysis was performed using CONTIN program (Provencher, 1982). To minimize dust interference, the solutions were filtered with Millipore filters. Detailed description about DLS was shown in Supplementary.
The assay method is that N-(3[2-furyl]acryloyl)-Phe-GlyGly (FAPGG) are degraded to FAP and GG by ACE and the absorption decrease at 340 nm was detected. The activities of XF and JG against ACE were examined with Hitachi 7600 automatic analyzer using ‘ACE Activity Detection Kit’ purchased from Anj Biotechnology Corporation, Suzhou, China. The control was distilled water and Captopril was taken as positive control.
2.4. Centrifugation and elemental analysis The four concentrations of XF1-4 and JG1-4 were centrifugated at 3,00,000 × g for 2 h with Beckman L-80XP super-speed
2.8. iNOS assays The activities of specimens against iNOS were tested by ‘Typed Nitric Oxide Synthetase (NOS) Detection Kit’ which was purchased from Jiancheng Bioengineering Institute, Nanjing, China. The control was distilled water and positive inhibitor control was supplied in Kit.
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3. Results 3.1. Aggregates in TCM Sixty medicinal herbs and 24 Chinese herbal formulae (Supplementary Tables S1 and S2) were selected and the corresponding decoctions were prepared by the well-established decocting procedure. As demonstrated by DLS (Fig. 1) and TEM results (Supplementary Fig. S1), aggregates were observed in all the 84 solution mixtures at the concentration 10 times lower than that of the conventional oral dosing. Although all aqueous solutions showed aggregation, the aggregate sizes varied for different samples (Fig. 1). Basically, the formulae of several herbs showed higher scattered intensity than single herbs, indicating that herb mixtures were easier to form aggregates. 3.2. Aggregation behavior of different concentrations of XF and JG The four concentrations solutions of XF and JG were filtered through Millipore filters with pore size of 0.45 m and detected by DLS. As shown in Fig. 2A, multimodal distribution was observed in the solutions at all the concentrations. Obviously, the broad distribution with hydrodynamic radius (Rh) above 100 nm was attributed to the aggregate, and it was the predominant component in the system. The components peaked at below 1 nm and around 30 nm possibly resulted from some single molecules and minor aggregates, respectively. Since their contribution to the scattered intensity was negligible, only the size of the major component as well as the scattered intensity was summarized in Table 1 for further comparison. Fig. 2B shows the DLS results from XF-2 and JG-1 after passing through the filters with different pore size. The size distributions of both XF-2 and JG-1 were shifted downward with decreasing the filter pore size, so did the scattered intensity
(Table 1). As for XF-2, the diameter was 249.6 nm (intensity, 596 kcps) after the 0.45 m filter; it was decreased to 158.8 nm (intensity, 121.2 kcps) after the 0.22 m filter, and further decreased to 108.0 nm (intensity, 54.5 kcps) after the 0.10 m filter (Fig. 2B). Similar results were observed for JG-1, but the diameter was 138.0 nm after the 0.10 m filter, larger than that of XF-2. 3.3. Super-speed centrifugation of XF and JG Besides filtration, centrifugation was also an effective approach to separate the aggregates by means of density difference. After the super-speed centrifugation, the supernatant was studied by DLS and the results were summarized in Table 1. Aggregates were observed in all the supernatants in XF and JG. Unlike the aggregates after filtration with 0.10 m filter, the aggregates after centrifugation showed no significant decrease in size, except those at extremely low concentrations (XF-4 and JG-4). However, the scattered intensity after centrifugation was dramatically decreased, indicating that the aggregates in the supernatants had a much lower density and its content was also low. Elemental analysis was also performed. The C, H, N contents of XF-1 were 38.55%, 6.12%, and 1.85%, respectively, in the original solution. As for the supernatant, they were 37.82%, 6.25%, and 1.74%, respectively, indicating that the composition was changed. 3.4. Aggregation behavior of XF and JG in solutions mimicking the gastrointestinal environment The aggregates of XF at pH 1 showed similar size and size distribution as they did under neutral conditions, with the diameter of 276.4 nm (XF-2) versus 249.6 nm. However, the aggregates of JG at pH 1 were much smaller, with the diam-
Fig. 1. Size distribution (Rh) of examples of Chinese herbal medicines measured by DLS. (a) Three herbs: Flos Caryophylli, Rhizoma Atractylodis, and Herba Taraxaci. (b) Three herbal formulae: ‘Da-Cheng-Qi Tang’, ‘Shen-Fu Tang’, and ‘Sheng-Hua Tang’. The herb source and formulae ingredients could be found in the Supplementary Tables S1 and S2.
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Fig. 2. Size distribution (Rh) of decoctions of XF and JG measured by DLS: (a) after filtration with 0.45 m filter, (b) comparison of XF-2 and JG-1 after filtration with 0.45 m, 0.22 m, and 0.10 m filters.
eter of 145.8 nm (JG-1) and 160.0 nm (JG-2) versus 237.8 nm and 255.0 nm, respectively. Under mimicking the intestinal conditions, 2.0 mg/mL of XF and 1.0 mg/mL of JG in HBSS, the aggregates with diameter of 306.4 nm and 245.8 nm were observed for XF and JG, respectively. 3.5. Caco-2 cells experiment The tightness and integrity of the Caco-2 monolayers as well as the effective differentiation were validated prior to the transport experiments. All the TEER values of the Caco-2 cell monolayers used in the studies were higher than 550 cm2 , indicating sufficient tightness and integrity of the cell monolayers.XF and JG in HBSS were added on the apical side of the Caco-2 cell monolayers and the basolateral solutions were
studied by DLS. As shown in Fig. 3, a bimodal distribution was observed in the basolateral solutions. The smaller component had the same size (0.7 nm) in XF and JG. The average diameter of the aggregate in XF was decreased from 306.4 nm of the apical side to 111.2 nm of the basolateral side, by a factor of almost 3; while it decreased from 245.8 nm to 167.2 nm in JG, the degree of shrinkage was much smaller than that in XF. 3.6. The activities against three cardiovascular targets We compared the activities of XF and JG before and after centrifugation against three different cardiovascular targets—plasminogen activator inhibitor one (PAI-1), angiotensin-converting enzyme (ACE) and inducible nitric oxide synthase (iNOS). All values are mean ± S.D. (n = 3).
Table 1 The DLS data of XF and JG series after filtration and centrifugation 0.45 m filter Intensity (kcps)
0.10 m filter
Centrifugation
Diameter (nm)
Intensity (kcps)
Diameter (nm)
Intensity (kcps)
Diameter (nm)
XF-1 XF-2 XF-3 XF-4
489.0a 596.0 93.4 16.9
239.4 249.6 212.6 212.6
223.9a 54.5 10.5 –b
124.2 108.0 75.0 No particles
189.0 50.7 31.5 11.1
250.6 227.0 208.2 114.0
JG-1 JG-2 JG-3 JG-4
2005.5 538.0 84.5 27.8
237.8 255.0 263.2 173.2
269.7 71.0 9.9 –b
138.4 139.2 97.0 No particles
32.0 43.7 15.4 9.0
181.0 181.0 170.4 92.4
XF-1: 23 mg/mL; XF-2: 2.3 mg/mL; XF-3: 0.23 mg/mL; XF-4: 0.023 mg/mL. JG-1: 10 mg/mL; JG-2: 1 mg/mL; JG-3: 0.1 mg/mL; JG-4: 0.01 mg/mL. a The filters were plugged up when filtered. b The intensity was too weak to detect.
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Fig. 3. Size distribution of XF and JG in HBSS. In Caco-2 cell monolayers experiment the concentration of XF and JG in HBSS were 20 mg/mL and 10 mg/mL, respectively, and they were diluted 10 times in DLS experiment. The postfix “–bl” denoting the solution collected in the basolateral side after XF-HBSS and JG-HBSS incubating with the Caco-2 cell monolayers for 1.5 h.
For PAI-1, the IC50 value of XF was 0.11 ± 0.02 mg/mL without centrifugation. However, the value increased to 1.39 ± 0.36 mg/mL when supernatant was tested, indicating that the activity reduced by a factor of 12.6. The same thing happened to JG, whose IC50 value increased from 9.8 ± 2.9 mg/mL to 199.2 ± 19.6 mg/mL after centrifugation. The activity of the original JG decoction was 20 times higher than that of the supernatant. As for ACE, the IC50 value of positive control Captopril was 0.107 ± 0.011 M. The IC50 value of XF increased from 6.7 ± 0.7 mg/mL to 14.4 ± 1.2 mg/mL, which was corresponding to a decrease in the activity by a factor of 2.2. No obvious difference of activity was observed for JG between the original decoction and the supernatant (10 mg/mL, inhibition 45.5 ± 3.7% versus 41.6 ± 2.9%; 1 mg/mL, inhibition 31.1 ± 3.1% versus 28.6 ± 3.4%). As for iNOS, the inhibition of supernatant of XF-1 against the target was 11% higher than XF-1 in all three times experiments
though at the different times the inhibition was changed about 5% (supernatant of XF-1 was about 25% inhibition and XF-1 was 14% inhibition). 4. Discussion From 60 medicinal herbs and 24 Chinese herbal formulae, aggregation was a common process in TCM. However, TCM contains hundreds of unidentified constituents, at current stage it is impossible to determine how the aggregates were formed and what their nature were. Further studies should be performed to determine whether the aggregates are composed by the primary metabolites (carbohydrate, proteins, lipids, etc.) or by secondary metabolites which are herb-related, or by both of them. However, DLS results showed that the aggregates had a broad size distributions ranging from nanometer to micrometers (Figs. 1 and 2). And the aggregates also possessed different morphology and density. Neither filter nor centrifugation could
Fig. 4. ( ) The colorful dots presenting compounds existed as single molecules. ( ) These massed dots presenting aggregates. A drawing schematically showing the composition of TCM and centrifugation effect. Left panel: aggregates with different size and density as well as the single molecules in the original decoction; right panel: solution after centrifugation, the upper solution denoting supernatant. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
Y. Zhuang et al. / Journal of Ethnopharmacology 117 (2008) 378–384
totally remove them. XF and JG exhibited different behaviors after filtration and centrifugation, indicating that the aggregates in XF and JG were different in type. It was reasonable to believe that each TCM contained many types of aggregate, some of them were stable in shape and size, and some of them were in an equilibrium state with single molecules. The mixture of aggregates as well as the coexisting molecules was schematically shown in Fig. 4. After super-speed centrifugation, the aggregates with low density were remained in the supernatant. Elemental analysis indicated that they were chemically different from those with high density. Arnold and co-workers studied the aggregation behavior of non-nucleoside reverse transcriptase inhibitors (NNRTIs), the hydrophobic drug, under the conditions similar to the gastrointestinal (GI) environment (Frenkel et al., 2005). The change in pH or ion strength could shift the equilibrium between aggregates and single molecules. In extreme conditions, old equilibriums were destroyed and new ones were established. The shift or destroy of equilibrium was related with the chemical nature of the aggregates and molecules. Since XF have seven more herbs than JG, their constituents were different in number and in chemical structure. Therefore, XF and JG exhibited different changes in size and size distribution of aggregates under GI environments. Whether the aggregates can pass through the intestinal tract or not was related with their biological properties in vivo. XF and JG showed different behavior in passing through the Caco2 cell monolayers. After incubation, aggregates were observed in the basolateral side. We attributed the smaller component of the size of 0.7 nm to the proteins secreted by cells. The larger component was the aggregates. The change in size and in size distribution suggested that the aggregates, especially larger aggregates, did not pass through the monolayers intact. The size distribution of the aggregates in XF and JG was much broader than those measured under intestinal conditions without passing the Caco-2 cell monolayers. The size also became smaller. Do the aggregates affect the activity of the TCM? Each TCM contains hundreds of constituents, and only a few of them had the therapeutic activities. Depending on the different TCM formulae and aimed target, such active components exist in aggregation form or non-aggregation form. Our study showed that the active compounds for PAI-1 may be in aggregation form with higher density in XF and JG. Super-speed centrifugation could remove the majority of it from the supernatant, making the supernatant less effective against the PAI-1. On the contrary, the active compounds for iNOS may form aggregates with lower density in XF. Centrifugation relatively increased its purity and quality in the supernatant by means of removing other aggregates. Therefore, supernatant showed slightly higher activity on iNOS than the original solution. As for ACE, the active reagent was in different conformation in XF and JG. In XF, it was possibly formed aggregates with higher density. Therefore the IC50 value of supernatant was increased. While in JG it could be in single molecules format, no obvious difference on activity was observed, since centrifugation had almost no effect on the sedimentation of single molecules.
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How the aggregates interact with the biological target? Shoichet and co-workers have reported that aggregates interacted with protein directly (McGovern et al., 2003). The aggregates in TCM may interact with biological targets by two approaches: no-specific interaction and specific interaction. In non-specific interaction, one aggregate interacted with different targets or different aggregates interacted with the same target; while in specific interaction, one aggregateinteracted with only one specific target. Another specific interaction may come from some compounds diffusing from the aggregates and interacting with the target by single molecules state, especially when the aggregates were close enough to the target. In addition, the presence of aggregates may be one of the false-positive reasons in screening such mixtures as the extracts of Chinese medicinal herbs, other natural products and combinatorial libraries. Unlike precipitates, aggregates could not be visualized by eyes. By forming aggregates, the therapeutic activity of the compound could be changed. Under certain circumstances, the therapeutic activity was generated solely by the formation of aggregation, i.e., the compounds forming the aggregates had no effect by themselves in the conformation of single molecules. Even though the active compounds exist in herb, their activity may also be changed between the single compound form and the aggregate form. Therefore, the aggregation should be taken into account in screening the multi-compound mixtures and in bioassay guiding the isolation of natural products. 5. Conclusions Our results have demonstrated that aggregates commonly existed in TCM and they were able to sustain the GI environments. More importantly, they showed possible profound effect on the biological activity. Overall, based on these results, we proposed a possible Chinese medicine mechanism as follows. Each TCM contained a lot of single molecular compounds and many types of aggregates, and the chemical and biological properties of the compounds and aggregates were TCM specific. Some effective component could be single specific compound, such as ephedrine, indirubin, qinghaosu, and tetramethylpyrazine. Others may act by aggregates through nonspecific adsorption or specific compounds diffusing from aggregates with multiple targets. These results should be helpful for understanding the mechanism of TCM and drug discovery based on TCM. Acknowledgment This work was financially supported by the National Natural Science Foundation of China (20504001). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jep.2008.02.017
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References Feng, B.Y., Shelat, A., Doman, T.N., Guy, R.K., Shoichet, B.K., 2005. Highthroughput assays for promiscuous inhibitors. Nature Chemical Biology 1, 146–148. Feng, B.Y., Shoichet, B.K., 2006a. A detergent-based assay for the detection of promiscuous inhibitors. Nature Protocols 1, 550–553. Feng, B.Y., Shoichet, B.K., 2006b. Synergy and antagonism of promiscuous inhibition in multiple-compound mixtures. Journal of Medicinal Chemistry 49, 2151–2154. Frenkel, Y.V., Clark, A.D., Das, K., Wang, Y.H., Lewi, P.J., Janssen, P.A.J., Arnold, E., 2005. Concentration and pH dependent aggregation of hydrophobic drug molecules and relevance to oral bioavailability. Journal of Medicinal Chemistry 48, 1974–1983. McGovern, S.L., Caselli, E., Grigorieff, N., Shoichet, B.K., 2002. A common mechanism underlying promiscuous inhibitors from virtual and highthroughput screening. Journal of Medicinal Chemistry 45, 1712–1722. McGovern, S.L., Helfand, B.T., Feng, B.Y., Shoichet, B.K., 2003. A specific mechanism of nonspecific inhibition. Journal of Medicinal Chemistry 46, 4265–4272.
McGovern, S.L., Shoichet, B.K., 2003. Kinase inhibitors: not just for kinases anymore. Journal of Medicinal Chemistry 46, 1478–1483. Provencher, S.W., 1982. CONTIN: a general purpose constrained regularization program for inverting noisy linear algebraic and integral equations. Computer Physics Communications 27, 229–242. Rishton, G.M., 1997. Reactive compounds and in vitro false positives in HTS. Drug Discovery Today 2, 382–384. Seidler, J., McGovern, S.L., Doman, T., Shoichet, B.K., 2003. Identification and prediction of promiscuous aggregating inhibitors among known drugs. Journal of Medicinal Chemistry 46, 4477–4486. Shen, Z., Chen, P., Shen, J., Dong, Z., Liu, J., 2003. Effects of the fraction from Phyllanthus Urinaria on thrombolysis and the activity of PAI-1 and tPA. Natural product research and development. 15, 441–445. Shoichet, B.K., 2006. Screening in a spirit haunted world. Drug Discovery Today 11, 607–615. Tian, X.J., Yang, X.D., Wang, K., Yang, X.W., 2006. The efflux of flavonoids morin, isorhamnetin-3-O-rutinoside and diosmetin-7-O-beta-Dxylopyranosyl-(1-6)-beta-D-glucopyranoside in the human intestinal cell line Caco-2. Pharmaceutical Research 23, 1721–1728.
Journal of Ethnopharmacology 117 (2008) 378–384
Can the aggregation be a new approach for understanding the mechanism of Traditional Chinese Medicine? Yan Zhuang a , Jingjing Yan a , Wei Zhu b , Lirong Chen a , Dehai Liang a,∗ , Xiaojie Xu a,∗ a
College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China b Guangdong Hospital of Traditional Chinese Medicine, Guangzhou, China
Received 5 October 2007; received in revised form 14 November 2007; accepted 2 February 2008 Available online 10 March 2008
Abstract “Frequent hitter” phenomenon emerged in the high-throughput screening; one of the most common mechanisms behind artifactual inhibition is that some organic molecules formed large colloid-like aggregates which are able to sequester and thereby inhibit enzymes. To investigate the situation in Traditional Chinese Medicine (TCM), 60 medicinal herbs and 24 Chinese herbal formulae were detected by dynamic light scattering (DLS), and aggregates were observed in all the 84 solution mixtures. The aggregates of two Chinese herbal formulae, ‘Xue-Fu-Zhu-Yu Tang’ (XF) and ‘Jing-Guan Tang’ (JG), were not only able to survive in the gastro-intestinal environment, but also had the ability to pass through the monolayer of the Caco-2 cell. The activities of XF and JG against three cardiovascular targets were also aggregates-related. Based on these findings, a new possible mechanism of the action of Chinese medicine was proposed. © 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: Aggregate; Traditional Chinese Medicine; Dynamic light scattering
1. Introduction Biochemical screens are designed to get the competitive, reversible bindings and specific ligands with high-affinity, but many false positives are emerged in the high-throughput screening (Rishton, 1997). One of the most common mechanisms behind artifactual inhibition is that some organic molecules formed large colloid-like aggregates which are able to sequester and thereby inhibit enzymes (McGovern et al., 2002; Feng et al., 2005; Feng and Shoichet, 2006a; Shoichet, 2006). The aggregates formed by single-compound are observed in the libraries of chemical compounds, in leads for drug discovery process (McGovern and Shoichet, 2003), and even in a part of drugs (Seidler et al., 2003). Furthermore, it is reported that multicompound mixtures are able to influence the aggregate-based inhibition with predominated synergistic effect or antagonism (Feng and Shoichet, 2006b). These findings inspired us to investigate the aggregation behaviors in Traditional Chinese Medicine (TCM). Generally,
TCM consists of multiple herbs, so it contains hundreds of constituent diversely in chemical structure and in bioactivity. Since aggregate occurs in the mixtures of compounds or even by single compound under certain conditions, it is expected that aggregation is a common process in TCM. 60 medicinal herbs and 24 Chinese herbal formulae were detected by dynamic light scattering (DLS), and aggregates were observed in all the 84 solution mixtures. In order to study the properties of the aggregates and the effects of aggregate on biological activity, we chose two TCM formulae, ‘Xue-Fu-Zhu-Yu Tang’ (XF) and ‘Jing-Guan Tang’ (JG), both of which are widely used in clinical practice for treating many kinds of cardiovascular diseases in China, to further study. The aggregates of the two formulae were not only able to survive in the gastro-intestinal environment, but also had the ability to pass through the monolayer of the Caco-2 cells. The activities of XF and JG against three cardiovascular targets were also aggregates-related. 2. Materials and methods 2.1. Plant material
∗
Corresponding authors. Tel.: +86 10 6275 7456; fax: +86 10 6275 1708. E-mail addresses: [email protected] (D. Liang), [email protected] (X. Xu). 0378-8741/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2008.02.017
All the medicinal herbs were purchased from the Tong Ren Tang Pharmaceutical Shop, Beijing, China, and authenticated
Y. Zhuang et al. / Journal of Ethnopharmacology 117 (2008) 378–384
by Dr. Wei Zhu, Guangdong Hospital of Traditional Chinese Medicine, China. The name of 60 medicinal herbs and 24 Chinese herbal formulae were listed in the Supplementary Tables S1 and S2. Herbal formula XF is composed of 11 crude herbs: Flos Carthami (9 g), Fructus Aurantii (6 g), Radix Achyranthis Bidentatae (9 g), Radix Angelicae Sinensis (9 g), Radix Bupleuri (3 g), Radix Glycyrrhizae (6 g), Radix Paeoniae Rubra (6 g), Radix Platycodonis (4.5 g), Radix Rehmanniae (9 g), Rhizoma Chuanxiong (4.5 g), and Semen Persicae (12 g). JG contains only four of the 11 herbs: Fructus Aurantii (10 g), Radix Bupleuri (5 g), Radix Paeoniae Rubra (15 g), and Rhizoma Chuanxiong (15 g). The herb resource could refer the Supplementary Table S1. 2.2. TCM processing procedure 10 g of each of the 60 single herbs was added with 50 mL water, and boiled for 30 min. Each of the 24 herbal formulae (amount of ingredients shown in the Supplementary Table S2), was added with 200 mL water, and boiled for 30 min. The upper solution of the decoctions was collected and diluted ten times with double-boiled water and studied by DLS and Transmission Electron Microscope (TEM) (Supplementary Fig. S2). The oral decoctions of XF and JG at the concentration of administration (practical dosage), 23 mg/mL for XF and 10 mg/mL for JG, were prepared and used as the stock solutions. The stock solution was diluted three times, each by a factor of 10 (i.e. 23 mg/mL, 2.3 mg/mL, 0.23 mg/mL and 0.023 mg/mL for XF and 10 mg/mL, 1.0 mg/mL, 0.1 mg/mL and 0.01 mg/mL for JG). We use XF-n or JG-n, where n is the order number of 1–4, to denote the solution at different concentrations. Gastric condition in vitro was simulated by using HCl (pH 1). And intestinal condition was mimicked by Hank’s balanced salt solution (HBSS, pH 7.4). XF and JG were dried and resolved in HBSS at concentration of 2.0 mg/mL for XF and 1.0 mg/mL for JG.
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centrifuge. The supernatant was carefully transferred to a dustfree light scattering cell and studied by DLS. The elemental analysis were performed on Elementar Vario EL (Germany). 2.5. Caco-2 experiment Caco-2 cells were similar to the intestinal epidermic cells in morphological and biochemical characteristics, and here were used to study the absorption of aggregates. The Caco2 experiment was performed in the State Key Laboratory of Natural and Biomimetic Drugs, China, as previously described (Tian et al., 2006). Caco-2 cell monolayers were prepared according to a standard 21 day cultural procedure to reach confluence and differentiation. The integrity of the monolayers was checked by measuring the transepithelial electrical resistance (TEER) with an epithelial voltohmmeter (EVOM, World Precision Instrument, Sarasota, FL) equipped with a chopstick. The measurements were performed before and after transport experiments. Then, 20 mg/mL XF and 10 mg/mL JG in HBSS were added on the apical side and were incubated for 1.5 h in a shaker (37 ◦ C, 50 rpm). The basolateral solutions were collected, diluted 10 times, and then detected by DLS. Detailed Caco-2 experiment was shown in Supplementary. 2.6. PAI-1 assays The PAI-1 Activity Assay Kit came from Department of Molecular Genetics, Medical School, Fudan University, Shanghai, China. The experiment followed the specification except that the decoctions were incubated with the blood for ten minutes before testing. TB (Tris-borate) buffer solution was taken as negative control and Corilagin was taken as positive control (Shen et al., 2003).
2.3. Dynamic light scattering (DLS)
2.7. ACE assays
DLS was performed either on a commercial Zeta Potential Analyzer with particle size analysis (ZetaPALS, Brookhaven Inc.) or on a commercial laser light scattering spectrometer equipped with a BI-200SM goniometer and a BI-TurboCorr digital correlator (Brookhaven Inc.). A 100-mW, vertically polarized solid state laser (GNI, Changchun, China) operating at 532 nm was used as the light source. The detector angle was 90◦ . Data analysis was performed using CONTIN program (Provencher, 1982). To minimize dust interference, the solutions were filtered with Millipore filters. Detailed description about DLS was shown in Supplementary.
The assay method is that N-(3[2-furyl]acryloyl)-Phe-GlyGly (FAPGG) are degraded to FAP and GG by ACE and the absorption decrease at 340 nm was detected. The activities of XF and JG against ACE were examined with Hitachi 7600 automatic analyzer using ‘ACE Activity Detection Kit’ purchased from Anj Biotechnology Corporation, Suzhou, China. The control was distilled water and Captopril was taken as positive control.
2.4. Centrifugation and elemental analysis The four concentrations of XF1-4 and JG1-4 were centrifugated at 3,00,000 × g for 2 h with Beckman L-80XP super-speed
2.8. iNOS assays The activities of specimens against iNOS were tested by ‘Typed Nitric Oxide Synthetase (NOS) Detection Kit’ which was purchased from Jiancheng Bioengineering Institute, Nanjing, China. The control was distilled water and positive inhibitor control was supplied in Kit.
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3. Results 3.1. Aggregates in TCM Sixty medicinal herbs and 24 Chinese herbal formulae (Supplementary Tables S1 and S2) were selected and the corresponding decoctions were prepared by the well-established decocting procedure. As demonstrated by DLS (Fig. 1) and TEM results (Supplementary Fig. S1), aggregates were observed in all the 84 solution mixtures at the concentration 10 times lower than that of the conventional oral dosing. Although all aqueous solutions showed aggregation, the aggregate sizes varied for different samples (Fig. 1). Basically, the formulae of several herbs showed higher scattered intensity than single herbs, indicating that herb mixtures were easier to form aggregates. 3.2. Aggregation behavior of different concentrations of XF and JG The four concentrations solutions of XF and JG were filtered through Millipore filters with pore size of 0.45 m and detected by DLS. As shown in Fig. 2A, multimodal distribution was observed in the solutions at all the concentrations. Obviously, the broad distribution with hydrodynamic radius (Rh) above 100 nm was attributed to the aggregate, and it was the predominant component in the system. The components peaked at below 1 nm and around 30 nm possibly resulted from some single molecules and minor aggregates, respectively. Since their contribution to the scattered intensity was negligible, only the size of the major component as well as the scattered intensity was summarized in Table 1 for further comparison. Fig. 2B shows the DLS results from XF-2 and JG-1 after passing through the filters with different pore size. The size distributions of both XF-2 and JG-1 were shifted downward with decreasing the filter pore size, so did the scattered intensity
(Table 1). As for XF-2, the diameter was 249.6 nm (intensity, 596 kcps) after the 0.45 m filter; it was decreased to 158.8 nm (intensity, 121.2 kcps) after the 0.22 m filter, and further decreased to 108.0 nm (intensity, 54.5 kcps) after the 0.10 m filter (Fig. 2B). Similar results were observed for JG-1, but the diameter was 138.0 nm after the 0.10 m filter, larger than that of XF-2. 3.3. Super-speed centrifugation of XF and JG Besides filtration, centrifugation was also an effective approach to separate the aggregates by means of density difference. After the super-speed centrifugation, the supernatant was studied by DLS and the results were summarized in Table 1. Aggregates were observed in all the supernatants in XF and JG. Unlike the aggregates after filtration with 0.10 m filter, the aggregates after centrifugation showed no significant decrease in size, except those at extremely low concentrations (XF-4 and JG-4). However, the scattered intensity after centrifugation was dramatically decreased, indicating that the aggregates in the supernatants had a much lower density and its content was also low. Elemental analysis was also performed. The C, H, N contents of XF-1 were 38.55%, 6.12%, and 1.85%, respectively, in the original solution. As for the supernatant, they were 37.82%, 6.25%, and 1.74%, respectively, indicating that the composition was changed. 3.4. Aggregation behavior of XF and JG in solutions mimicking the gastrointestinal environment The aggregates of XF at pH 1 showed similar size and size distribution as they did under neutral conditions, with the diameter of 276.4 nm (XF-2) versus 249.6 nm. However, the aggregates of JG at pH 1 were much smaller, with the diam-
Fig. 1. Size distribution (Rh) of examples of Chinese herbal medicines measured by DLS. (a) Three herbs: Flos Caryophylli, Rhizoma Atractylodis, and Herba Taraxaci. (b) Three herbal formulae: ‘Da-Cheng-Qi Tang’, ‘Shen-Fu Tang’, and ‘Sheng-Hua Tang’. The herb source and formulae ingredients could be found in the Supplementary Tables S1 and S2.
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Fig. 2. Size distribution (Rh) of decoctions of XF and JG measured by DLS: (a) after filtration with 0.45 m filter, (b) comparison of XF-2 and JG-1 after filtration with 0.45 m, 0.22 m, and 0.10 m filters.
eter of 145.8 nm (JG-1) and 160.0 nm (JG-2) versus 237.8 nm and 255.0 nm, respectively. Under mimicking the intestinal conditions, 2.0 mg/mL of XF and 1.0 mg/mL of JG in HBSS, the aggregates with diameter of 306.4 nm and 245.8 nm were observed for XF and JG, respectively. 3.5. Caco-2 cells experiment The tightness and integrity of the Caco-2 monolayers as well as the effective differentiation were validated prior to the transport experiments. All the TEER values of the Caco-2 cell monolayers used in the studies were higher than 550 cm2 , indicating sufficient tightness and integrity of the cell monolayers.XF and JG in HBSS were added on the apical side of the Caco-2 cell monolayers and the basolateral solutions were
studied by DLS. As shown in Fig. 3, a bimodal distribution was observed in the basolateral solutions. The smaller component had the same size (0.7 nm) in XF and JG. The average diameter of the aggregate in XF was decreased from 306.4 nm of the apical side to 111.2 nm of the basolateral side, by a factor of almost 3; while it decreased from 245.8 nm to 167.2 nm in JG, the degree of shrinkage was much smaller than that in XF. 3.6. The activities against three cardiovascular targets We compared the activities of XF and JG before and after centrifugation against three different cardiovascular targets—plasminogen activator inhibitor one (PAI-1), angiotensin-converting enzyme (ACE) and inducible nitric oxide synthase (iNOS). All values are mean ± S.D. (n = 3).
Table 1 The DLS data of XF and JG series after filtration and centrifugation 0.45 m filter Intensity (kcps)
0.10 m filter
Centrifugation
Diameter (nm)
Intensity (kcps)
Diameter (nm)
Intensity (kcps)
Diameter (nm)
XF-1 XF-2 XF-3 XF-4
489.0a 596.0 93.4 16.9
239.4 249.6 212.6 212.6
223.9a 54.5 10.5 –b
124.2 108.0 75.0 No particles
189.0 50.7 31.5 11.1
250.6 227.0 208.2 114.0
JG-1 JG-2 JG-3 JG-4
2005.5 538.0 84.5 27.8
237.8 255.0 263.2 173.2
269.7 71.0 9.9 –b
138.4 139.2 97.0 No particles
32.0 43.7 15.4 9.0
181.0 181.0 170.4 92.4
XF-1: 23 mg/mL; XF-2: 2.3 mg/mL; XF-3: 0.23 mg/mL; XF-4: 0.023 mg/mL. JG-1: 10 mg/mL; JG-2: 1 mg/mL; JG-3: 0.1 mg/mL; JG-4: 0.01 mg/mL. a The filters were plugged up when filtered. b The intensity was too weak to detect.
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Fig. 3. Size distribution of XF and JG in HBSS. In Caco-2 cell monolayers experiment the concentration of XF and JG in HBSS were 20 mg/mL and 10 mg/mL, respectively, and they were diluted 10 times in DLS experiment. The postfix “–bl” denoting the solution collected in the basolateral side after XF-HBSS and JG-HBSS incubating with the Caco-2 cell monolayers for 1.5 h.
For PAI-1, the IC50 value of XF was 0.11 ± 0.02 mg/mL without centrifugation. However, the value increased to 1.39 ± 0.36 mg/mL when supernatant was tested, indicating that the activity reduced by a factor of 12.6. The same thing happened to JG, whose IC50 value increased from 9.8 ± 2.9 mg/mL to 199.2 ± 19.6 mg/mL after centrifugation. The activity of the original JG decoction was 20 times higher than that of the supernatant. As for ACE, the IC50 value of positive control Captopril was 0.107 ± 0.011 M. The IC50 value of XF increased from 6.7 ± 0.7 mg/mL to 14.4 ± 1.2 mg/mL, which was corresponding to a decrease in the activity by a factor of 2.2. No obvious difference of activity was observed for JG between the original decoction and the supernatant (10 mg/mL, inhibition 45.5 ± 3.7% versus 41.6 ± 2.9%; 1 mg/mL, inhibition 31.1 ± 3.1% versus 28.6 ± 3.4%). As for iNOS, the inhibition of supernatant of XF-1 against the target was 11% higher than XF-1 in all three times experiments
though at the different times the inhibition was changed about 5% (supernatant of XF-1 was about 25% inhibition and XF-1 was 14% inhibition). 4. Discussion From 60 medicinal herbs and 24 Chinese herbal formulae, aggregation was a common process in TCM. However, TCM contains hundreds of unidentified constituents, at current stage it is impossible to determine how the aggregates were formed and what their nature were. Further studies should be performed to determine whether the aggregates are composed by the primary metabolites (carbohydrate, proteins, lipids, etc.) or by secondary metabolites which are herb-related, or by both of them. However, DLS results showed that the aggregates had a broad size distributions ranging from nanometer to micrometers (Figs. 1 and 2). And the aggregates also possessed different morphology and density. Neither filter nor centrifugation could
Fig. 4. ( ) The colorful dots presenting compounds existed as single molecules. ( ) These massed dots presenting aggregates. A drawing schematically showing the composition of TCM and centrifugation effect. Left panel: aggregates with different size and density as well as the single molecules in the original decoction; right panel: solution after centrifugation, the upper solution denoting supernatant. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
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totally remove them. XF and JG exhibited different behaviors after filtration and centrifugation, indicating that the aggregates in XF and JG were different in type. It was reasonable to believe that each TCM contained many types of aggregate, some of them were stable in shape and size, and some of them were in an equilibrium state with single molecules. The mixture of aggregates as well as the coexisting molecules was schematically shown in Fig. 4. After super-speed centrifugation, the aggregates with low density were remained in the supernatant. Elemental analysis indicated that they were chemically different from those with high density. Arnold and co-workers studied the aggregation behavior of non-nucleoside reverse transcriptase inhibitors (NNRTIs), the hydrophobic drug, under the conditions similar to the gastrointestinal (GI) environment (Frenkel et al., 2005). The change in pH or ion strength could shift the equilibrium between aggregates and single molecules. In extreme conditions, old equilibriums were destroyed and new ones were established. The shift or destroy of equilibrium was related with the chemical nature of the aggregates and molecules. Since XF have seven more herbs than JG, their constituents were different in number and in chemical structure. Therefore, XF and JG exhibited different changes in size and size distribution of aggregates under GI environments. Whether the aggregates can pass through the intestinal tract or not was related with their biological properties in vivo. XF and JG showed different behavior in passing through the Caco2 cell monolayers. After incubation, aggregates were observed in the basolateral side. We attributed the smaller component of the size of 0.7 nm to the proteins secreted by cells. The larger component was the aggregates. The change in size and in size distribution suggested that the aggregates, especially larger aggregates, did not pass through the monolayers intact. The size distribution of the aggregates in XF and JG was much broader than those measured under intestinal conditions without passing the Caco-2 cell monolayers. The size also became smaller. Do the aggregates affect the activity of the TCM? Each TCM contains hundreds of constituents, and only a few of them had the therapeutic activities. Depending on the different TCM formulae and aimed target, such active components exist in aggregation form or non-aggregation form. Our study showed that the active compounds for PAI-1 may be in aggregation form with higher density in XF and JG. Super-speed centrifugation could remove the majority of it from the supernatant, making the supernatant less effective against the PAI-1. On the contrary, the active compounds for iNOS may form aggregates with lower density in XF. Centrifugation relatively increased its purity and quality in the supernatant by means of removing other aggregates. Therefore, supernatant showed slightly higher activity on iNOS than the original solution. As for ACE, the active reagent was in different conformation in XF and JG. In XF, it was possibly formed aggregates with higher density. Therefore the IC50 value of supernatant was increased. While in JG it could be in single molecules format, no obvious difference on activity was observed, since centrifugation had almost no effect on the sedimentation of single molecules.
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How the aggregates interact with the biological target? Shoichet and co-workers have reported that aggregates interacted with protein directly (McGovern et al., 2003). The aggregates in TCM may interact with biological targets by two approaches: no-specific interaction and specific interaction. In non-specific interaction, one aggregate interacted with different targets or different aggregates interacted with the same target; while in specific interaction, one aggregateinteracted with only one specific target. Another specific interaction may come from some compounds diffusing from the aggregates and interacting with the target by single molecules state, especially when the aggregates were close enough to the target. In addition, the presence of aggregates may be one of the false-positive reasons in screening such mixtures as the extracts of Chinese medicinal herbs, other natural products and combinatorial libraries. Unlike precipitates, aggregates could not be visualized by eyes. By forming aggregates, the therapeutic activity of the compound could be changed. Under certain circumstances, the therapeutic activity was generated solely by the formation of aggregation, i.e., the compounds forming the aggregates had no effect by themselves in the conformation of single molecules. Even though the active compounds exist in herb, their activity may also be changed between the single compound form and the aggregate form. Therefore, the aggregation should be taken into account in screening the multi-compound mixtures and in bioassay guiding the isolation of natural products. 5. Conclusions Our results have demonstrated that aggregates commonly existed in TCM and they were able to sustain the GI environments. More importantly, they showed possible profound effect on the biological activity. Overall, based on these results, we proposed a possible Chinese medicine mechanism as follows. Each TCM contained a lot of single molecular compounds and many types of aggregates, and the chemical and biological properties of the compounds and aggregates were TCM specific. Some effective component could be single specific compound, such as ephedrine, indirubin, qinghaosu, and tetramethylpyrazine. Others may act by aggregates through nonspecific adsorption or specific compounds diffusing from aggregates with multiple targets. These results should be helpful for understanding the mechanism of TCM and drug discovery based on TCM. Acknowledgment This work was financially supported by the National Natural Science Foundation of China (20504001). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jep.2008.02.017
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