Anal. Chem. 2003, 75, 3994-3998
Frontal Immunoaffinity Chromatography with Mass Spectrometric Detection: A Method for Finding Active Compounds from Traditional Chinese Herbs Hongpeng Luo, Lirong Chen, Zhengquan Li, Zhensheng Ding, and Xiaojie Xu*
College of Chemistry and Molecular Engineering, Modern Research Center for Traditional Chinese Medicine, Peking University, Beijing, China 100871
Frontal affinity chromatography (FAC) using immobilized polyclone antibodies of compound A coupled with mass spectrometry was used for the screening of affinity compounds from an extract of Phyllanthus urinaria L. Mass spectrometry was used as an analyzer of FAC. It can analyze the frontal affinity chromatogram of each compound of the extract in one program. The extract was dissolved in 2 mM NH4OAc at a concentration of 10 µg/ mL, then loaded on the immobilized antibody column, and data were collected from mass spectrometry to get a frontal affinity chromatogram. The screening of extract resulted in brevifolin, brevifolin carboxylic acid, corilagin, ellagic acid, and phyllanthusiin U. Activity analyses give high inhibitory activities to these compounds. This research work afforded us a new approach to find new leading compounds from nature or a man-made combinatorial library that have different structure styles or to find substitutes for the synthetic active compound that has high toxicity.
throughput screening methods.4-7 In this method, a column is prepared containing an immobilized biological receptor such as a protein. A sample containing potential ligands is continuously infused through the column. The order of elution of compounds from the column depends on their affinities for the receptor.8 It is reported that FAC/MS can be used to screen man-made mixtures of enzyme inhibitors.7 In this report, polyclonal antibodies are used to mimic hepatitis C virus (HCV) NS3 protease in FAC/MS for screening of the extract of Phyllanthus urinaria L. HCV causes chronic infection, often leading to liver cirrhosis and hepatocellular carcinoma. The number of carriers worldwide has reached into the millions, while current treatments have only limited success and preventative vaccination or antiviral drug is not available. In recent researches, HCV NS3-NS4A protease is often used as a useful enzyme target for anti-HCV compounds.9 RD3-4078 (Figure 1) has high inhibitory activity to HCV NS3NS4A protease (IC50 8.5 µg/mL).10 Compound A was synthesized by add a linker to the benzene ring of RD3-4078. The carboxyl of the linker helps compound A to link with carrier protein BSA.
Traditional Chinese medicine herbs are a rich natural resource and have been used for thousands of years in China. We consider the Chinese medicine herb system as a natural combinatorial chemical library. Compared to a synthetic combinatorial chemical library, it has more abundant diversities in chemical structures with potent pharmacological activities and it is easy and inexpensive to make. But it is very difficult to isolate and purify the active compounds from traditional Chinese medicine herbs. The traditional methods for separation and identification of active compounds from herbs are time-consuming and expensive. Sometimes it is easy to miss the low-content active compounds. This report addresses a novel approach to isolate, screen, and identify the active compounds from traditional Chinese medicine herbs in single-step frontal immunoaffinity chromatography with mass spectrometry detection (FIAC/MS). There are some methods currently in use to solve the problem of screening for potential therapeutics from a combinatorial library.1-3 Frontal affinity chromatography coupled on-line to electrospray mass spectrometry (FAC/MS) is one of the high-
EXPERIMENTAL SECTION Materials. BSA, OVA, and Sepharose CL 4B were purchased from Hualvyuan Co. 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was purchased from Sigma. White female New Zealand rabbits were supplied by the College of Life Science. ID Cartridge and ID Self-Pack Device were purchased from Perseptive Biosystems. P. urinaria L was collected from Fujian province. Marina was purchased from Applied Biosystem Co.
* Corresponding author. Tel: 86-10-62757456. Fax: 86-10-62751708. E-mail
[email protected]. (1) Shen, K.; Keng, Y. F.; Wu, L.; Guo, X. L.; Lawrence, D. S.; Zhang, Z. Y. J. Biol. Chem. 2001, 276 (50), 47311-47319.
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(2) Planells, C. R.; Montoliu, C.; Humet, M.; Fernandez, A. M.; Garcia, M. C.; Valera, E.; C.; Merino, J. M.; Perez, P. E.; Messeguer, A.; Felipo, V.; Ferrer, M. A. J. Pharmacol. Exp. Ther. 2002, 302 (1), 163-173. (3) Lau, D. H.; Guo, L. L.; Liu, R. W.; Song, A. M.; Shao, C. K.; Lam, K. S. Biotechnol. Lett. 2002, 24 (6), 497-500. (4) Schriemer, D. C.; Bundle, D. R.; Li, L.; Hindsgaul, O. Angew. Chem., Int. Ed. Engl. 1998, 37, 3383-3387. (5) Schriemer, D. C.; Hindsgaul, O. Comb. Chem. High Throughput Screening 1998, 1, 155-170. (6) Zhang, B.; Palcic, M. M.; Mo, H.; Goldstein, I. J.; Hindsgaul, O. Glycobiology 2001, 11, 141-147. (7) Zhang, B.; Palcic, M. M.; Schriemer, D. C.; Alvarez-Manilla, G.; Pierce, M.; Hindsgaul, O. Anal. Biochem. 2001, 299 (2), 173-182. (8) Kasai, K. I.; Oda, Y. J. Chromatogr. 1986, 376, 33-47. (9) Takeshita, N.; Kakiuchi, N.; Kanazawa, T.; et al. Anal. Biochem, 1977, 247, 242-246. (10) Sudo, K.; Matsumoto, Y.; Matsushima, M.; et al. Antiviral Chem. Chemather. 1977, 8(6), 541. 10.1021/ac034190i CCC: $25.00
© 2003 American Chemical Society Published on Web 06/28/2003
Figure 1. Structures of RD3-4078 and compound A. The carboxyl of the linker helps compound A to link with carrier protein BSA.
Figure 2. FAC device used in this research. Syringe A contain sample solution, and syringe B contain acetonitrile. These two solutions are mixed in the tee valve and then enter the mass spectrograph.
Preparation of Immunoaffinity Column. Compound A was linked to BSA at a weak base (0.01 M NaHCO3) in an air bath under 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide catalysis. Mass analysis shows an antigen has on average ∼18 residues of compound A. Two white female New Zealand rabbits were immunized every 10 days for a total of 4 times. The blood was collected from an artery in the rabbit’s neck one week after the last immunization shot. A 50-mL serum sample was collected from each rabbit, and the ELISA test showed that the potency of the serum was 16 000. The antibody was purified by using the saturation ammonium sulfate method and then was immobilized to Sepharose by using the chloroepoxypropane method first developed by Porath et al.11 After reaction, the ID self-pack device packing ID cartridge (diameter 2.1 × 30 mm) was used, followed by equilibrium of the cartridge with 2 mM pH 6.7 NH4OAc. Analysis of the Immunoaffinity Column Effect. Compound A was dissolved in 2 mM pH 6.7 NH4OAc at concentrations of 1, 2, 4, and 8 µg/mL. The FAC device is shown in Figure 2.4 Syringes A and B contained compound A solution and acetonitrile. The flow rate was 5 µL/min. A Mariner electrospray ionization/time-of-flight (ESI-TOF) mass spectrometry (PE Biosystems) was used to online analyze the concentration of each compound flowing through the column. For ESI, the mass spectrometer was operated in the negative mode using the following conditions: spray tip potential, 4500 V; nozzle potential, 200 V; detector voltage, 2000 V; nozzle temperature, 140 °C; Quad temperature, 140 °C. All the other parameters were set as default values. Run time of one test was from 100 to 150 min. The column was washed by 500 µL of 2 mM pH 6.7 NH4OAc, 1000 µL of 0.1 M CHCOOH + 0.5 M NaCl and 2500 µL of 2 mM pH 6.7 NH4OAc after each run. Preparation and Analysis of the Extract of P. urinaria L. Acetone-water (1:1, v/v) was used to extract P. urinaria L three times, and then the acetone in the extract was boiled off to get a water solution. After ligroin was used to extract the water solution, the mixture was extracted with ethyl acetate. The extract of P. urinaria L used in this report was obtained when the ethyl acetate was boiled off. Figure 5 shows the mass spectrogram of the extract. The extract was dissolved in 2 mM pH 6.7 NH4OAc at a concentration of 10 µg/mL, and then the solution was loaded (11) Porath, J. C.; Jansson, J. C.; et al. J. Chromatogr. 1971, 60, 167.
Figure 3. Frontal affinity chromatograms of methanol (total ion chromatogram) for 8, 4, 2, and 1 µg/mL compound A (select ion chromatogram, from left to right). The time/volume of the front was gotten by re-forming the area under the curve to a rectangle that has a height equal to equilibrium intensity (Figure 4). V0 can get from the chromatogram of methanol.
in syringe A. The parameters of the FIAC/MS of the extract were same as noted above. RESULTS AND DISCUSSION Characteristic Parameters of the Immunoaffinity Column. A total ion chromatogram (TIC) was constructed when compound A was infused through the column. The concentration of compound A at each time was derived from the selected ion chromatogram of compound A that generated from TIC (Figure 3). The ideal front (long-dash front in Figure 4) of compound A breakthrough on the column was gotten by re-forming the area under the curve to a rectangle that had a height equal to equilibrium intensity12 (Figure 4). The relationship between V (the volume of compound A breakthrough on the column), V0 (the volume of nonretained compound breakthrough on the column), C (the concentration of compound A), Lt (the values of dynamic binding capacity of the column) and Kd (ligand binding affinity) is shown in eq 1:8
V - V0 )
Lt C + Kd
(1)
It also can be shown in another form:
1 1 Kd 1 ) + C(V - V0) Lt Lt C
(2)
Equation 2 is a linear equation, so Kd and Lt can be gotten from the intercept and slope of the linear relation figure of 1/C(V (12) Arata, Y.; Hirabayashi, J.; Kasai, K. I. J. Chromatogr., A 2001, 905, 337343.
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Figure 4. Calculation of the breakthrough time (frontal) of a selected ion frontal chromatogram. t1 is one point on the baseline, and t2 is another point on the equilibrium line. An average intensity (hav) of the selected ion between t1 and t2 can be calculated by the software (Data Explorer 3.5) of Mariner, so the area of the short-dash rectangle (S1, has a value of hav(t2 - t1)) is equal to the area under the solid curve (S0, between t1 and t2). The long-dash line is the wanted ideal front of the chromatogram, which is broken at the equilibrium intensity (heq), and tx is the wanted time of the front. The area of the longdash rectangle (S2, has a value of heq(t2 - tx)) is equal to the area under the solid curve (S0), so it is also equal to the area of the shortdash rectangle (S1). This equation can be derived to tx ) t2 - hav(t2 - t1)/heq.
Figure 6. Mass spectrogram of the extract solution. The 13 labeled peaks are major compounds in the extract.
Table 1. Results of FAC for Compound A C (µg/mL)
C (µmol/L)
1/C (µmol/L)-1
V (µL)
V - V0 (µL)
1/C(V - V0) (µmol-1)
8 4 2 1
17.094 8.547 4.274 2.137
0.0585 0.1166 0.2340 0.4679
283.6 335.7 376.0 400.5
183.6 235.7 276.0 300.5
318.6 496.4 847.8 1557.4 Figure 7. Frontal affinity chromatograms of the compounds in the extract. The numbers refer to the 13 compounds shown in Figure 5. Table 2. Results of FAC and Inhibitory Activity Test of Compounds in the Extract t V V - V0 IC50 (min) (µL) (µL) (mg/mL) 1 2 3 4 5 6 7
20.32 21.97 45.33 46.74 59.42 21.59 21.30
101.6 109.8 226.6 233.7 297.1 108.0 106.5
1.6 9.8 126.6 133.7 197.1 8.0 6.5
>1 0.35 0.10 0.03 0.003 0.74 0.74
t V V - V0 IC50 (min) (µL) (µL) (mg/mL) 8 9 10 11 12 13
20.57 32.89 35.61 57.82 29.29 63.78
102.8 164.4 178.0 289.1 146.4 318.9
2.8 64.4 78.0 189.1 46.4 218.9
>1 0.20 0.18 0.009 0.25 0.001
Figure 5. Column capacity and ligand binding affinity for compound A are determined by FIAC/MS, with a linear regression analysis for the experimental data. Y ) A + BX, A ) 142.1(1.6, B ) 3023.5(5.9. Lt and Kd can be calculated by eq 2, Lt ) 1/A ) 32.95 µg/mL and Kd) B/A ) 21.31 µM.
V0) and 1/C. The results of FIAC/MS of compound A are shown in Table 1. Figure 5 was drawn by using the data from Table 1. The column capacity and ligand binding affinity for compound A were calculated from Figure 5 by using eq 2, Lt ) 1/A ) 32.95 µg/mL and Kd) B/A ) 21.31 µM. It shows that the affinity column is working. Application of the FIAC/MS Method To Find Other AntiHCV NS3 Protease Inhibitors from Herbs. Figure 6 is the mass spectrogram of the extract solution of P. urinaria L. The peaks show the primary compounds in the solution. The frontal immunoaffinity chromatograms (the select ion chromatogram of each compound) of the compounds given in Figure 6 are shown in Figure 7. The frontal time and volume of the compounds’ breakthrough on the column are shown in Table 2. These 3996 Analytical Chemistry, Vol. 75, No. 16, August 15, 2003
Figure 8. Relationship of V - V0 and Log(IC50) of the compounds in Table 2. It is shown that the delayed volume (V - V0) of a compound breakthrough on the column is relative to its inhibitory activity.
compounds have been separated from the extract using traditional methods (use silicon gel and Sephadex LH-20) and have been
Figure 9. Structures of 3, 4, 5, 11, and 13.
measured by the inhibitory activity to HCV NS3 protease9 in order to test the effect of frontal immunoaffinity chromatography. The result is also shown in Table 2. Figure 8 was drawn to analyze the relationship of Log(IC50) and V - V0 of the compounds in Table 2. The relationship of Log(IC50) and V - V0 shown in Figure 8 is obviously inverse. This means that the longer it takes a compound to break through the immunoaffinity column at its infusion concentration, the higher the inhibitory activity it has to HCV NS3 protease. On the basis of this result, the immunoaffinity column can be used with the FIAC/MS method to screen the extract of plants to find compounds with high inhibitory activity to HCV NS3 protease. Figure 9 shows the structures of 3, 4, 5, 11, and 13. These five compounds are all polyphenols, but their structures and sizes are different from that of RD3-4078. It means some new active structure styles can be found by using the FIAC/MS method. This result extends the range for finding new drugs. More structures can be modified to find compounds that have higher activity and lower toxicity, but these compounds cannot be found only by modifying RD3-4078. These results can be explained by the key-lock theory. The acceptor (enzyme) is like a lock and the leading compound is like a key, so the structure characteristics of the contact area of the two compounds relate to each other. Antibody and hapten are also lock and key, but their contact area is not big enough to include the whole hapten,13 so each clone of the antibody can only contain part of the structure characteristic of the hapten. As a statistical result, polyclonal antibodies can contain all of the structure characteristic of the hapten. This means the polyclonal antibodies can simulate the acceptor of the hapten, so the column containing immobilized polyclonal antibodies can be used to (13) Richards, F. F.; Lifter, J.; Hew, C. L.; Yoshioka, M.; Konigsberg, W. H. Biochemistry 1974, 13 (17), 3572-3575.
Figure 10. Relationship of acceptor, hapten (inhibitor), and polyclonal antibody. The hapten is an inhibitor of the acceptor, so it contains the structureal information of the active site of the acceptor. At the same time, each clone of the antibody contains part of the structure information of the hapten, so the polyclonal antibody can mimic the acceptor as a statistical result.
screen compounds by their activity to the acceptor. It also can explain why the affinity column can screen compounds that have different structure types and sizes, because each clone of an antibody can only contain part of the structure characteristic of the hapten. The simulation is only at the level of statistics. Figure 10 shows the relationship of acceptor and polyclone antibody. The influence of the concentration on V - V0 is described in eq 1. For most compounds in this experiment, the concentrations are lower than 2 µM, but the values of Kd are all bigger than that of compound A’s, 21 µM. So for the factor C + Kd, the effect of Kd is bigger than that of C. It can explain why the volumes of active compounds’ breakthrough on the column mainly depend on their activities (binding affinities). CONCLUSIONS The polyclone antibodies can simulate the enzyme, so frontal immunoaffinity chromatography with mass spectrometric detection can be used to rapidly isolate, screen, and identify various active compounds in the extract of a plant in a single step and to sequence them by their activities. This method can be applied to Analytical Chemistry, Vol. 75, No. 16, August 15, 2003
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find new leading compounds from nature or a man-made combinatorial library that have different structure types or to find substitutes for the synthetic active compound that has high toxicity.
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Analytical Chemistry, Vol. 75, No. 16, August 15, 2003
Received for review February 26, 2003. Accepted May 7, 2003. AC034190I
