Preparation of Knockout Extract by Immunoaffinity Column and Its Application

Importance of herbal medicines have recently increased owing to rising interest in their health benefits. However, medicinal plant extracts are complex mixtures of phytochemicals that act synergistically or additively on specific and/or multiple molecular and cellular targets. Thus, it is difficult to examine the actual pharmacological roles of active compounds in plant extracts. This review describes a new strategy for isolating target compounds from plant extracts using immunoaffinity columns coupled with monoclonal antibodies (mAbs) against natural compounds. Through one-step purification using mAb-coupled immunoaffinity columns, we succeeded in preparing a knockout (KO) extract, which contains all components except the target compound. Furthermore, we investigated the pharmacological effects of the KO extract to reveal the actual effects of a bioactive compound in the crude extract. This approach may help determine the potential function of target compounds in herbal medicines.


Introduction
Traditional herbal medicines have been used for thousands of years in Asian countries such as China, Japan, and Korea.Recently, global demand for herbal medicines has increased owing to rising interest in the associated health benefits.Studies of bioactive compounds derived from medicinal plant extracts have revealed their molecular actions in vivo and in vitro.However, plant extracts are complex mixtures of effective phytochemicals that act synergistically or additively on specific and/or multiple molecular and cellular targets.Hence, pharmacological evidence of interactions between these natural compounds is required to identify their actual pharmacological activity.
Immunoassay using monoclonal antibodies (mAbs) against small molecular-weight-compounds such as synthetic drugs and natural compounds is a very important tool for investigating receptor binding, enzyme reactions, and quantitative and/or qualitative analysis in both animals and plants.Immunoaffinity purification using mAbs is also commonly used to purify larger molecules, such as peptides and proteins, for research and commercial purposes.However, few studies have targeted small-molecular-weight compounds such as natural bioactive compounds.In previous studies, we established various types of mAbs against natural compounds, including terpenoids [13], saponins [48], alkaloids [9,10], and phenolics [11][12][13][14], and developed several applications using these mAbs.One of the applications is an immunoaffinity column coupled with anti-natural compound-specific mAbs, which can capture target compounds and specifically remove them from crude mixtures.We previously prepared immunoaffinity columns against the terpenoids forskolin [15], solasodine glycosides [16], ginsenoside Rb 1 [17], and glycyrrhizin [18].
In this review, we introduce and describe the preparation of a knockout (KO) extract, which is prepared by eliminating one target compound from the crude extract using immunoaffinity columns.Application of the KO extract is critical to such pharmacological investigations for clarifying the actual effects of the bioactive compound and elucidating interactions between the target compound and the other compounds in the component mixture, such as plant extracts and traditional Chinese medicines (TCMs).

Preparation of the Immunoaffinity Column against Ginsenoside Rb 1 (G-Rb 1 ), and the G-Rb 1 -KO Extract
Ginseng, the root of Panax ginseng C.A. Meyer (Araliaceae), has been one of the most important medicinal plants in Asian countries for >1,000 years [19].The pharmacological and therapeutic effects of ginseng are mainly attributed to ginsenosides, which are triterpernoid saponin glycosides (dammarane-type saponins) [19,20].Recently, 60 additional ginsenosides have been isolated from different ginseng species [21,22].Ginsenoside Rb 1 (G-Rb 1 ) is one of the main ginsenosides obtained from ginseng [23] and reportedly exerts various pharmaceutical actions, such as facilitating acquisition and retrieval of memory [24], scavenging free radicals [25], inhibiting calcium over-influx into neurons [26], and preserving the structural integrity of neurons [27].In our previous work, we established anti-G-Rb 1 mAb and applied it to enzyme-linked immunosorbent assay (ELISA) and a new immunostaining method, named Eastern blotting [4,28].Furthermore, we prepared an immunoaffinity column coupled with anti-G-Rb 1 mAb and performed one-step purification using ginseng extract [17,28].We here report the preparation of the anti-G-Rb 1 mAb-coupled immunoaffinity column and its applications.
Ginseng, the root of Panax ginseng C.A. Meyer (Araliaceae), has been used as one of the most important medicinal plants in Asian countries for more than thousand years [19].The pharmacological and therapeutic effects of ginseng are mainly attributed to ginsenosides, which are triterprnoid saponin glycosides (dammarane-type saponins) [19,20].Recently, more than 60 ginsenosides have been isolated from different species of ginseng [21,22].Ginsenoside Rb 1 (G-Rb 1 ) is one of the main ginsenosides of ginseng [23], and has been reported to exert many pharmaceutical actions such as facilitating acquisition and retrieval of memory [24], scavenging free radicals [25], inhibition of calcium over-influx into neurons [26], and preserving the structural integrity of the neurons [27].In our previous work, we have established anti-G-Rb 1 MAb and its application including enzyme-linked immunosorbent assay (ELISA) and a new immunostaining method named Eastern blotting [4,28].Furthermore, we prepared an immunoaffinity column combined with anti-G-Rb 1 MAb, and then performed the one-step isolation from ginseng extract by this column [17,28].Herein we report the preparation of anti-G-Rb 1 immunoaffinity column and it applications.

Preparation of Anti-G-Rb 1 mAb
Small compounds such as synthetic drugs and natural compounds are insufficiently complex to induce an immune response and antibody production.For antibody production against small compounds, a hapten must be chemically conjugated to carrier proteins such as bovine serum albumin (BSA).The first step for production of such mAbs is the preparation of a hapten-carrier protein conjugate, which directly combines an immune antigen with the carrier protein.Thus, G-Rb 1 was oxidized with NaIO 4 solution to generate aldehyde groups on the sugar moiety, allowing the aldehyde group to react with BSA (Figure 1a) [4].Subsequently, it was necessary to characterize the synthesized antigen G-Rb 1 -BSA conjugate.However, direct and appropriate methods to determine the hapten-conjugated carrier proteins without differential UV analysis, radiochemical, or chemical methods were not reported.To determine the hapten density of immunoconjugates, Wengatez et al. used matrix-assisted UV laser desorption/ionization (MALDI) mass spectrometry [29].We also performed direct analysis of hapten-carrier protein conjugates by MALDI-TOF mass spectrometry [1][2][3][4][5][6][7][8][9][10][11][12][13][14][30][31][32]. Figure 1b indicates the MALDI-TOF mass spectra of the G-Rb 1 -BSA conjugate.A broad peak coinciding with the G-Rb 1 -BSA conjugate appeared between 70,000 and 90,000 m/z and centered at approximately 79,469 m/z.The molecular weight of BSA is 66,433, and the calculated molecular mass of the G-Rb 1 moiety is 1,109.These data suggest that most BSA was effectively conjugated to G-Rb 1 .The experimental results and molecular weight of BSA revealed that the calculated molecular mass of the conjugated G-Rb 1 is between 3,327 and 23,289, suggesting that 3-21 (average of 12) molecules of G-Rb 1 are conjugated to BSA [4].This method is therefore suitable for the characterization of hapten-carrier protein conjugates.The synthesized G-Rb 1 -BSA conjugate was injected into mice [4].Briefly, BALB/c female mice were injected intraperitoneally with G-Rb 1 -BSA dissolved in phosphate buffer saline (PBS) four times.The first immunization (50 g protein) was injected as a 1:1 emulsion in Freund's complete adjuvant.The second and third immunization (50 g protein in each injection) were injected as a 1:1 emulsion in Freund's incomplete adjuvant.On the third day after the final immunization (100 g protein), splenocytes were isolated and fused with a HAT-sensitive mouse myeloma cell line, P3-X63-Ag8-653, by the polyethylene glycol (PEG) method.After cell fusion, we assessed the binding activity against carrier protein BSA, and the hybridomas secreting antibody against BSA were removed.Hybridomas producing mAb reactive to G-Rb 1 were cloned by the limited dilution method [33].Established hybridomas were cultured in eRDF medium supplemented with 10 g/mL insulin, 35 g/mL of transferrin, 20 M ethanolamine and 25 nM selenium (ITES) [34].The mAb was purified using a Protein G FF column [4].The cultured medium containing the IgG was adjusted to pH 7 with 1 M Tris solution and subjected to the column, and washed the column with 10 mM phosphate buffer (pH 7).Absorbed IgG was eluted with 100 mM citrate buffer (pH 3).The eluted IgG was neutralized with 1 M Tris solution, then dialyzed against PBS (pH 7.4) three times, and finally lyophilized.To further examine the binding specificity of this mAb against G-Rb 1 , but not the carrier protein BSA, we immobilized G-Rb 1 to human serum albumin (HSA) and adsorbed the G-Rb 1 -HSA conjugate onto a polystyrene microtiter plate.Subsequently, the specificity of IgG-type mAb, 9G7, for G-Rb 1 was examined by competitive ELISA using a microtiter plate coated with the G-Rb 1 -HSA conjugate.Under these conditions, the free mAb bound to free G-Rb 1 or the G-Rb 1 -HSA conjugate and the full measurement range of the assay using this anti-G-Rb 1 mAb extended from 20 to 400 ng/mL.Since cross-reactivity is the most important factor in determining the utility of antibodies, assay specificity was tested by determining cross-reactivity of mAb with various related compounds.Cross-reactivity against G-Rc and G-Rd, which possess diglucose moieties attached to C-3 hydroxy groups, was weak compared with that against G-Rb 1 (0.024% and 0.020%, respectively), and G-Re and G-Rg 1 showed negligible cross-reactivity (<0.005%).Therefore, it is clear that this mAb reacts predominantly with G-Rb 1 and not with its structurally related compounds or the carrier protein BSA.

Preparation of the Anti-G-Rb 1 mAb-Coupled Immunoaffinity Column
To couple mAb to the Affi-Gel Hz gel, the sugar moiety of the purified anti-G-Rb 1 mAb was oxidized by NaIO 4 to give a dialdehyde group on the sugar moiety.The oxidized anti-G-Rb 1 mAb was then coupled to the Affi-Gel Hz hydrazide gel to form a hydrozone-type immunoaffinity gel [28].The immunoaffinity gel was poured into a plastic mini-column (Figure 2).

One-Step Purification of G-Rb 1 from Ginseng Extract Using the Anti-G-Rb 1 mAb-Coupled Immunoaffinity Column
To examine the anti-G-Rb 1 mAb-coupled immunoaffinity column, a crude extract of P. ginseng roots was loaded into the column.After washing the column with washing buffer to remove the unbound compounds, the bound compounds were eluted with elution buffer.Figure 3 shows that fractions 1-8 contained the overloaded G-Rb 1 , as determined by ELISA.The Eastern blotting procedure, a highly sensitive on-membrane quantitative analysis of anti-G-Rb 1 mAb, indicated that these fractions contained other ginsenosides such as G-Rg 1 , Rc, Re, and Rd.After washing, a single sharp peak was detected in the eluted fractions 21-24, which contained G-Rb 1 .However, these eluted fractions were contaminated with a small amount of malonyl-G-Rb 1 , as detected by Eastern blotting, which indicated that anti-G-Rb 1 mAb has almost the same reactivity with malonyl-G-Rb 1 [28].Therefore, to convert malonyl-G-Rb 1 to pure G-Rb 1 , these fractions were treated with a mild alkaline solution (0.1% KOH in MeOH) at room temperature [17].Overloaded G-Rb 1 in washing buffer was repeatedly loaded, and purified G-Rb 1 was finally obtained.The activity of the anti-G-Rb 1 mAb-coupled immunoaffinity column was stable during all procedures.The capacity of the immunoaffinity column was approximately 20 g of G-Rb 1 /mL of gel, which did not decrease after 10 repeated purification cycles under the same conditions, as reported in one-step purification of forskolin from a crude extract of Coleus forslohlii root [15].This methodology made it possible to purify G-Rb 1 from the crude extract without using complicated procedures and may be applicable to related studies of other families of saponins for which an acceptable one-step purification method has not yet been developed.In addition, it may be possible to separate the total ginseng saponins using a widely cross-reactive mAb against ginsenosides, such as anti-G-Re mAb [35].A combination of immunoaffinity column chromatography, ELISA, and Eastern blotting could be used for high-sensitivity detection of G-Rb 1 from plants and/or experimental animals and humans.Using this combination of methods, we detected G-Rb 1 from Kalopanax pictus Nakai, which was not previously reported to contain ginsenosides [36].

Preparation of the G-Rb 1 -KO Extract Using the Anti-G-Rb 1 mAb-Coupled Immunoaffinity Column
We separated G-Rb 1 from the crude ginseng extract using the anti-G-Rb 1 mAb-coupled immunoaffinity column.The capacity of the immunoaffinity column is 20 g of G-Rb 1 /mL of gel [28].Upon loading the crude extract into the immunoaffinity column without exceeding the G-Rb1 binding capacity, the column specifically eliminated G-Rb 1 from the crude ginseng extract.After loading the ginseng extract into the immunoaffinity column, the washing and eluted fractions were collected.Figure 4 indicates the TLC profile of each fraction stained by H 2 SO 4 .A standard of ginsenosides was spotted on lanes 1 (G-Rd, G-Rc, and G-Rb 1 ) and 2 (G-Rg 1 and G-Re).The crude extract, washing fraction, and eluted fraction were spotted on lanes 3, 4, and 5, respectively.In the crude extract (lane 3), all spots of ginsenosides were clearly detected.In contrast, the washing fraction (lane 4) contained all ginsenosides, except G-Rb 1 , and G-Rb 1 was detected in the eluted fraction (lane 5).These data clearly demonstrate that the G-Rb 1 molecule from the ginseng extract was captured by the anti-G-Rb 1 mAb-coupled immunoaffinity column.Thus, the washing fraction was referred to as the G-Rb 1 -KO extract because it lacked G-Rb 1 [37,38].This G-Rb 1 -KO extract may be useful for pharmacological characterization of G-Rb 1 in ginseng extracts and traditional medicines such as Kampo medicines and TCMs that prescribe ginseng.

Glycyrrhizin (GC)-KO Extract and its Application in Cell-Based Analysis
Licorice (Glycyrrhiza spp.) is an important medicinal plant used in >70% of Kampo medicines and TCMs.Licorice is prescribed with other herbal medicines as a demulcent in the treatment of sore throats, an anti-tussive, an expectorant for coughs and bronchial catarrh, an anti-inflammatory agent for anti-allergic reactions, rheumatism, and arthritis, a prophylactic for liver disease, tuberculosis, and adrenocorticoid insufficiency [39][40][41].A number of phytochemical investigations showed that licorice contains at least 470 constituents, including triterpenes, saponins, flavonoids, isoflavonoids, chalcones, polysaccharides, simple sugars, amino acids, and other substances [42].Accumulated evidence indicates that GC is a major bioactive triterpene glycoside in licorice and has numerous pharmacological uses.It exhibits anti-inflammatory, anti-ulcer, anti-tumor, anti-allergic, and hepato-protective activities [41,43].In addition, it was reported that various licorice constituents, such as flavonoid glycosides and their aglycones, exhibit anti-inflammatory, anti-oxidative, anti-microbial, superoxide scavenging, and anti-carcinogenic activities [41,44].Although the functional effects of individual compounds in licorice were analyzed in vitro and in vivo, the potential function of GC in licorice extract (LE) and interactions between GC and other components in LE were unclear because it was technically difficult to prepare GC-free LE.Previously, we purified GC from LE using an anti-GC mAb-coupled immunoaffinity column and then prepared a GC-KO extract [18].In this section, we review the preparation of this GC-KO extract and its application in in vitro assays.

Preparation of the GC-KO Extract and its Characterization
The anti-GC mAb was prepared using the same procedure as for G-Rb 1 [7].Cross-reactivity of the anti-GC mAb against glycyrrhetic acid-3-O-glucuronide and glycyrrhetic acid was 0.585% and 1.865%, respectively.Cross-reactivity with other related compounds, including deoxycholic acid, ursolic acid, and oleanolic acid, was <0.005%.Thus, we established competitive ELISA and Eastern blotting using anti-GC mAb to specifically detect GC [7,45].In addition, we used the anti-GC mAb-coupled immunoaffinity column to prepare the GC-KO extract.After purifying 60 mg of anti-GC mAb and coupling it to 25 mL of an Affi-Gel Hz gel, the immunoaffinity gel was packed into a plastic column [18].To eliminate GC from LE, 12 mg of LE, which contained 1275.0 g of GC in loading buffer (5% MeOH) was applied to the immunoaffinity column.After circulation of the loading buffer to enhance the binding efficiency of GC, the unbound fraction was collected.The column was washed with washing buffer (5% MeOH) and then eluted with elution buffer (20 mM PB containing 30% MeOH).After elution of bound compounds, each fraction was deionized, the solvent was lyophilized, and the GC concentration was determined by ELISA.The unbound fraction contained 3.50 g of GC (0.27% of the loaded GC), while the bound fraction contained 1269.26g of GC (99.55% of the loaded GC), suggesting that the anti-GC mAb-coupled column eliminated 99.55% of the loaded GC.In the unbound fraction, 3.50 g of GC (0.27% of the applied GC) was detected.In contrast, 1269.26g of GC (99.55% of the applied GC) was found in the bound fraction.These data suggest that the anti-GC mAb-coupled column eliminated 99.55% of the loaded GC and produced a GC-KO extract [46].
To characterize this GC-KO extract, we performed Eastern blotting and TLC analysis [46].As shown in Figure 5a, Eastern blotting using anti-GC mAb demonstrated that GC was detected in LE (lane 2), but the spot corresponding to GC was not present in the GC-KO extract (lane 3).Furthermore, TLC analysis (Figure 5b) showed that many compounds, including GC, were present in LE (lane 2), whereas GC was completely absent in the GC-KO extract (lane 3).Taken together, these data clearly demonstrate that GC molecules in LE can be eliminated using the anti-GC mAb-coupled immunoaffinity column and that the unbound fraction was indeed a GC-KO extract.

Cell-Based Assay Using the GC-KO Extract
The GC-KO extract is a useful tool for clarifying the interaction between GC and other compounds contained in LE.To investigate the potential function of GC in LE, we used macrophages to compare the effects of LE and the GC-KO extract on the production of nitric oxide (NO).The free radical NO has multiple effects on various systems, including host defense against pathogens, inhibition of tumor cells, and neurotransmission [47][48][49].However, excess production of NO, which is synthesized by the inducible NOS (iNOS), can be harmful and can trigger rheumatoid arthritis, gastritis, bowel inflammation, neuronal cell death, and bronchitis [50,51].Macrophages produce large quantities of NO after stimulation with bacterial lipopolysaccharide (LPS), and concomitantly produced inflammatory cytokines participate in the pathogenesis of inflammatory diseases [52].Overproduction of NO by iNOS appears to be involved in the pathogenesis of various inflammatory diseases [53].Therefore, inhibition of NO production by blocking iNOS expression may be useful in treating NO overexpression-mediated diseases.
To examine the inhibitory effect of LE on NO production, we used murine RAW264 macrophage cells, which produce NO upon stimulation with LPS.NO induction by LPS was blocked by treatment with LE in a dose-dependent manner [46].Furthermore, Western blotting and RT-PCR showed that iNOS protein and mRNA expression was downregulated in a dose-dependent manner by LE and was completely suppressed in the presence of 100 g/mL of LE.ELISA using anti-GC mAb demonstrated that 100 g of LE contains 10.6 ± 0.618 g of GC.However, significant inhibition of NO production and iNOS protein expression was not observed with GC at approximately 10.6 g/mL.
Figure 6a shows that LE treatment (100 g/mL) markedly inhibits NO production (inhibition ratio (IR) = 57.7%)compared with LPS treatment alone.In contrast, treatment with GC (10.6 g/mL) did not suppress NO production.Subsequently, we treated cells with the GC-KO extract (89.4 g/mL) or the combination of GC-KO extract (89.4 g/mL) and GC (10.6 g/mL).Interestingly, although GC alone did not block NO production, the inhibitory effect of the GC-KO extract (IR = 17.8%) was weaker than that of LE.Moreover, co-treatment with the GC-KO extract and GC significantly improved inhibition (IR = 33.5%).To determine whether these effects depended on iNOS expression, we performed Western blotting.As shown in Figure 6b, inhibition by the GC-KO extract was weaker than that by LE and addition of GC to the GC-KO extract rescued the inhibition.These data suggest that GC alone cannot suppress iNOS expression; however, in combination with the other constituents of LE, GC suppresses iNOS expression.Thus, in vitro and in vivo analyses using KO extracts can elucidate the functions of target natural compounds in crude extracts.Moreover, we have compared activity using GC contained in elution fraction and commercially available GC when the cells were treated with GC-KO extract.Since the activity was almost same level, the function of purified GC was not lost during the purification step.In future studies, we will investigate the detailed mechanisms including the differences between the original LE and GC-KO extract, the interactions between GC and GC-KO extract, and the action of GC in LE.

Conclusions
This review describes a unique strategy involving one-step purification of target compounds from crude extracts using anti-natural compound-specific mAb-coupled immunoaffinity columns.A G-Rb 1 -KO extract, which contains all compounds except G-Rb 1 , was prepared from the P. ginseng crude extract using the anti-G-Rb 1 mAb-coupled immunoaffinity column.Furthermore, the GC-KO extract was prepared from LE using an anti-GC mAb-coupled immunoaffinity column.Our laboratory has previously prepared mAbs against natural bioactive compounds such as ginsenosides [5,54,55], berberine [10], crocin [2], sennosides [12], and forskolin [1].Moreover, the immunoaffinity columns coupled with anti-G-Rb 1 [17], anti-forskolin [15], anti-solasodine glycoside [16], and anti-GC mAbs [18] achieved one-step purification of target compounds from crude extracts.Therefore, these mAbs and techniques are easily applicable to the preparation of KO extracts that lack only the target compound from crude plant extracts, Kampo medicines, and TCMs.
We also developed an in vitro assay using the GC-KO extract.This assay demonstrated that the GC-KO extract alone did not suppress iNOS expression, but inhibited iNOS when present in combination with the other constituents of LE.Thus, KO extracts may be useful for determining the potential functions of target compounds in crude extracts and Kampo medicines in vitro and in vivo.

Figure 2 .
Figure 2. Scheme of preparation of the anti-G-Rb 1 mAb-coupled immunoaffinity column

Figure 3 .
Figure 3. Elution profile of the P. ginseng crude extract separated using the anti-G-Rb 1 mAb-coupled immunoaffinity column.The G-Rb 1 concentration in each fraction was monitored by ELISA using anti-G-Rb 1 mAb.Inhibition = (A 0 − A)/A 0 ; A 0 is the absorbance in the absence of the test compounds, and A is the absorbance in the presence of the test compounds.

Figure 6 .
Figure 6.Comparison of the effects of the GC-KO extract and the combination of the GC-KO extract and GC on LPS-induced (a) NO production and (b) iNOS expression.Cells were treated with LE (100 g/mL), GC-KO extract (89.4 g/mL), GC (10.6 g/mL), or the combination of GC-KO extract (89.4 g/mL) and GC (10.6 g/mL).* p < 0.05, ** p < 0.01, and *** p < 0.001 indicate significant differences from the effects of LPS alone.