# Application of Monoclonal Antibodies against Bioactive Natural

Authors: Hiroyuki Tanaka; Osamu Morinaga; Takuhiro Uto; Shunsuke Fuji; Frederick Asare Aboagye; Nguyen Huu Tung; Xiao Wei Li; Waraporn Putalun; Yukihiro Shoyama
Journal: International Journal of Analytical Chemistry
 (2012)

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## Abstract

Matrix-assisted laser desorption/ionization (MALDI) tof mass spectrometry was
used for the confirmation of hapten number in synthesized antigen. As
application of MAb, the MAbs against ginsenosides and glycyrrhizin have been
prepared resulting in the development of two new techniques that we named the
eastern blotting method and the knockout extract preparation. In eastern
blotting technique, glycosides like ginsenosides and glycyrrhizin separated by
silica gel TLC were blotted to PVDF membrane that was treated with a
NaIO4 solution followed by BSA resulted in glycoside-BSA
conjugate on a PVDF membrane. The blotted spots were stained by MAb. Double
staining of eastern blotting for ginsenosides using antiginsenoside
Rb1 and Rg1 MAbs promoted complete identification of
ginsenosides in Panax species. The immunoaffinity concentration
of glycyrrhizin was determined by immunoaffinity column conjugated with
antiglycyrrhizin MAb resulting in the glycyrrhizin-knockout extract, which was
determined by the synergic effect with glycyrrhizin on NO production using the
cell line.


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## Body

## 1. Introduction

Immunoassay systems using monoclonal antibody (MAb) against drugs and small molecular
weight bioactive compounds have become an important tool for studies on receptor
binding analysis, enzyme assay, and quantitative and/or qualitative analytical
techniques in animals or plants. The immunoblotting method is based on the western
blotting technique that utilizes the antigen-antibody binding properties and
provides a specific and sensitive detection of higher molecule analytes like
peptides and proteins. In our ongoing study on MAb, previously we prepared various
kinds of MAb against natural products like forskolin [1], solamargine [2], crocin [3], marihuana compounds [4], opium alkaloids [5],
ginsenosides [6, 7], berberine [8],
sennosides [9], paeoniflorin [10], glycyrrhizin [11, 12], ginkgolic acid
[13], aconitine alkaloid [14], baicalin [15], and so on and developed individual competitive enzyme-linked
immunosorbent assay (ELISA) as a highly sensitive, specific, and simple
methodology.The confirmation of hapten number in synthesized antigens is most important in the
first stage of MAb preparation. Therefore, its determination method will be
discussed first of all. As an application of MAb, the MAb against ginsenosides and
glycyrrhizin has been prepared resulting in the development of two new techniques
that we have named the eastern blotting method [12] and the knockout extract preparation [16]. They will be introduced in this paper.


## 2. Preparation of MAb against Natural Products

Various methods have been employed for the determination of natural products. They
include spectral methods such as infrared (IR), nuclear magnetic resonance (NMR),
and circular dichroism (CD) and other chromatographic methods such as ion
chromatography (IC), capillary electrophoresis (CE), and high-speed counter current
chromatography (HSCCC), and so on. Compared to TLC, GLC and HPLC methods, the ELISA
method was more sensitive and selective. Moreover, it is possible to study a large
number of natural products. Since natural product extracts consist of various
chemical constituents (e.g., licorice contains 470 components or more), in general,
some pretreatment is necessary for HPLC and other chromatographic analysis methods.
ELISA, however, can determine the concentration of components directly without any
pretreatment. Therefore, ELISA was used to measure the concentration of ginsenoside
Rb1in ginseng and traditional Chinese medicines (TCMs).


### 2.1. Analytical Method for Determination of Hapten Number in Antigen,
Hapten-Carrier Protein Conjugate

For production of MAb, synthesis of hapten, which is derived from an immune
antigen and linker bridge, and the carrier protein conjugate is necessary. There
had been no direct and appropriate methods for the determination of haptens
conjugated with carrier proteins without differential UV analysis,
radiochemical, or chemical methods. Therefore, immunization by the injection of
hapten-carrier protein conjugate was unreliable. Wengatz et al. [17] determined the hapten density of
immunoconjugates by matrix-assisted UV laser desorption/ionization mass
spectrometry. We also reported the direct analytical method of hapten and
carrier protein conjugates by a matrix-assisted laser desorption/ionization mass
spectrometry (MALDI) tof mass spectrometry using an internal standard [18–20].Figure1 shows the MALDI tof mass spectrum
of the most pharmacologically active marihuana compound, tetrahydrocannabinolic
acid (THCA)-bovine serum albumin (BSA) conjugate, and BSA used as an internal
standard [20]. This shows only the
singly, doubly, and triply ionized molecule ions of the intact conjugate. The
sharp peak at m/z 66,465 is the [M + H]+ peak of BSA. A small [M +
H]+ peak of the THCA-BSA conjugate is at m/z 70,792, indicating
that the calculated molecular mass of the THCA-BSA conjugate is 70,581 using a
calculated molecular mass of 66,267 for BSA. The calculated molecule mass of the
THCA moiety is 4,314. From this result, 12.7 molecules of THCA are combined with
BSA [20]. Since this method is suitable
for small molecule natural products, we had been analyzing the hapten number of
all natural products for MAbs including glycosides like ginsenosides and
glycyrrhizin.Figure 1
Matrix-assisted laser desorption/ionization tof mass spectrometry of
tetrahydrocannabinolic-acid-BSA conjugate. [M + H] indicates the
molecular weight of the conjugate, from which the hapten number can be
calculated.


### 2.2. Preparation of MAb against Ginsenosides and ELISA as an Assay
System

Ginseng, the crude drug ofPanax ginseng, is one of the most
important natural medicines in many countries. It has been used to enhance
stamina and capacity to cope with fatigue and physical stress and as a tonic
against cancers, disturbances of the central nervous system (memory, learning,
and behavior), hypothermia, carbohydrate and lipid metabolism, immune function,
the cardiovascular system, and radioprotection [21]. It contains more than 30 kinds of dammarane and oleanane
saponins considered to be pharmacologically active components. Ginsenoside
Rb1 is the main saponin in ginseng. However, since the
concentration in the ginseng root or the root extract varies depending on the
method of extraction, subsequent treatment, or even the season of its collection
[22], standardization of quality is
required. For this purpose, we have prepared anti-ginsenoside Rb1
[6] and Rg1 MAbs [7]. The immunoassay system using MAb is not
frequently used for naturally occurring smaller molecular weight bioactive
compounds. Preparation of MAbs is difficult, but is one of the most important
steps for the analysis of natural products. As a typical natural product, the
preparation of MAb against the ginseng saponin ginsenoside Rb1 will
be discussed.A hybridoma-producing MAb reactive to ginsenoside Rb1 was obtained by
the general procedure and classified into IgG2b which had
κ light chains. The reactivity of IgG type MAb, 9G7
was tested by varying antibody concentration and by performing a dilution curve.
The antibody concentration was selected for competitive ELISA. The free MAb
following competition is bound to polystyrene microtiter plates precoated with
ginsenoside Rb1-HSA. Under these conditions, the full measurement
range of the assay extends from 20 to 400 ng/mL [6].Cross-reactivity is the most important factor in determining the value of an
antibody. Since the ELISA for ginsenoside Rb1 was established for
phytochemical investigations involving crude plant extracts, the assay
specificity was checked by determining the cross-reactivity of the MAb with
various related compounds. The cross-reactivity data of MAb that was obtained
were examined by competitive ELISA and calculated using picomole amounts of
ginsenoside Rb1. The cross-reactivity of ginsenoside Rc and Rd, which
possess a diglucose moiety attached to the C-3 hydroxy group, was weak compared
to ginsenoside Rb1 (0.024 and 0.020%, resp.). Ginsenoside Re and
Rg1 showed no cross-reactivity (less than 0.005%). It is
evident that the MAb reacted only with a small number of structurally related
ginsenoside Rb1 molecules, and very weakly, and did not react with
other steroidal compounds like glycyrrhizin, digitoxin, tigogenin, tigonin, and
solamargine.In our ongoing studies on MAbs against ginseng saponins, anti-ginsenoside
Rg1 MAb [7] and Re [23] have been prepared and their ELISA was
set up. Anti-ginsenoside Rg1 MAb was also highly specific like
anti-ginsenoside Rb1. On the other hand, anti-ginsenoside Re MAb
showed wide cross-reactivity. Therefore, the MAb can be used for the analysis
for the total ginsenoside concentration.


### 2.3. Application of MAb in the Natural Products Field

Although western blotting is a common assay methodology for
high-molecular-weight-substances, this method has not been employed for small
molecules, as direct immunostaining of such compounds on a TLC plate is as yet
unknown. Therefore, a new method for such small-molecular compounds is required.
Moreover, if small molecules can be blotted to a membrane, fixing them also
requires a new methodology. Previously, we succeeded in separating
small-molecule compounds such as solasodine glycosides into a part of an epitope
and fixing on the membrane [24], as
follows.


#### 2.3.1. New Staining Method for Ginsenosides, “Eastern
Blotting”

Figure2 shows the
H2SO4 staining and eastern blotting of ginsenoside
standards and TCM using anti-ginsenoside Rb1 MAb. It is
impossible to determine the ginsenosides by TLC staining by
H2SO4 as indicated in Figure 2(a). On the other hand, clear staining of
ginsenoside Rb1 occurred by eastern blotting, as indicated in
Figure 2(b). Furthermore, it became
evident that Jigengtang and Dahuanggancaotang prescriptions that did not
contain ginseng, as indicated by the absence of a ginsenoside Rb1band. The eastern blotting method was considerably more sensitive than
that of H2SO4 staining. The
H2SO4 staining detected all standard compounds.
The eastern blotting indicated only limited staining of ginsenoside
Rb1, Rc, and Rd, whose cross-reactivities were under
0.02% as shown in Figure 2(b).
We suggest that an aglycone, protopanaxadiol, and a part of the sugars may
be of importance to the immunization and may function as an epitope for the
structure of ginsenosides. In addition, it is suggested that the specific
reactivity of sugar moieties in the ginsenoside molecule against
anti-ginsenoside Rb1 MAb may be modified by the NaIO4
treatment of ginsenosides on the PVDF membrane, causing ginsenoside Rc and
Rd to become detectable by eastern blotting.Eastern blotting of ginsenosides in traditional Chinese medicine
formulas using anti-ginsenoside Rb1 MAb. Samples (1) Jigengtang, (2)
Dahuanggancaotang, (3) Nenshenyangyongtang, (4) Sijunzitang, (5)
Nenshentang, (6) Buanxiaxiexintang, (7) Xiaocaihutang, and (8) Crude
ginseng extract. Lanes 1 and 2 do not contain ginseng. Standard of
ginsenosides indicated, ginsenosidr-Rg1, -Re, -Rd, -Rc,
and -Rb1, respectively, from the upper spot.
(a)
TLC
(b)
Eastern blottingApplication of the eastern blotting method for the detection of glycyrrhizin
in the serum samples was investigated. In general, it is difficult to detect
glycyrrhizin in serum due to the large amount of impurities. The developed
bands of impurities lapped over the band of glycyrrhizin, and we failed to
find a proper developing solvent system that could separate glycyrrhizin and
the impurities apart from each other clearly on the TLC plate. Figure3 shows the detection of glycyrrhizin
by the eastern blotting technique in the rat serum samples. Glycyrrhizin
could not be identified on the TLC plate stained by H2SO4through many bands that were detected (Figure 3(a)). On the other hand, eastern blotting clearly
shows the band of glycyrrhizin even after 1 h (Figure 3(b)). Although the sensitivity of the
eastern blotting method was greatly affected by the impurities, the
detection limit was still at the nanogram level. The results proved that the
eastern blotting technique could be a unique method for identifying
glycyrrhizin against a background of a large amount of impurities.Eastern blotting profile of glycyrrhizin in rat serum after injection
of glycyrrhizin (a) TLC of rat-serum-injected glycyrrhizin TLC was
developed withn-BuOH-H2O-AcOH
(7 : 2 : 1). Spots were detected by
10% H2SO4. (b) Eastern blotting of
rat-serum-injected glycyrrhizin; the band of glycyrrhizin was
detected until 1 h after injection. GA: glycyhrrhetinic acid,
GC: glycyrrhizin, and GG: glycyrrhetinic acid monoglucuronide.
(a)
(b)When the mixture of anti-ginsenoside Rb1 and Rg1 MAbs
and the pair of substrates were tested for staining for ginsenosides, all
ginsenosides, ginsenoside Rb1, -Rc, -Rd, -Re, and -Rg1
were stained blue although the purple color staining for ginsenoside
Rg1 was expected because 3-amino-9-ethylcarbazole and
4-chloro-1-naphotol might be different. Therefore, we performed successive
staining of the membrane using anti-ginsenoside Rg1 and then
anti-ginsenoside Rb1. Finally, we performed the double staining
of ginsenosides indicating that ginsenoside Rg1 and ginsenoside
Re were stained purple and the other blue, as indicated in Figure 4. From this result, both antibodies
can distinguish the individual aglycones, protopanaxatriol, and
protopanaxadiol. For this application, the crude extract of various
Panax species was analyzed by the newly developed
double staining system. Major ginsenosides can be determined clearly by the
double staining method, as indicated in Figure 4.Double Eastern blotting staining of ginsenosides contained in various
ginseng samples using anti-ginseenoside-Rb1 and
anti-ginsenoside-Rg1 MAbs. (a) TLC profile stained by
sulfuric acid. (b) Eastern blotting by
anti-ginsenoside-Rb1 and
anti-ginseside-Rg1 monoclonal antibodies I, II, III,
IV, V, and VI indicated white ginseng, red ginseng, fibrous ginseng
(P. ginseng), P.
notoginseng, P.
quinquefolius and P.
japonicus, respectively. Upper purple color
spots and lower blue color spots were stained by
anti-ginsenoside-Rg1, and
anti-ginsenoside-Rb1 monoclonal antibodies,
respectively.
(a)
(b)Therefore, it is suggested that the staining color shows the pharmacological
activity. As shown in Figure4(b),
the purple bands indicate ginsenosides which have protopanaxatriol as an
aglycone and stimulation activity for the central nervous system (CNS). On
the other hand, the blue color indicates ginsenosides containing
protopanaxadiol as an aglycone that possess a depression effect on the CNS.
Moreover, the Rf value of ginsenosides roughly suggests the number of sugars
attached to the aglycone. Both analyses make it possible to jointly identify
which aglycone attaches and how many sugars it possesses, leading to the
structure of the ginsenosides. In fact, three kinds of ginsenosides
possessing protopanaxadiol-ginsenoside Rh1, -Rf, and
20-O-glucoginsenoside Rf in P. ginseng
root were determined by coloring and Rf value by comparing them with the
structures reported in the previous paper [25].Figure5(a) indicates
immunolocalization of glycyrrhizin in a licorice root slice using
anti-glycyrrhizin MAb as another application of the eastern blotting method.
The phloem (⟷) contained a higher concentration of
glycyrrhizin than the xylem and cork part [26]. On the other hand, no staining occurred on ginseng (Figure
5(c)).Immunocytolocalization of glycyrrhizin in fresh licorice root using
anti-glycyrrhizin MAb. (a) Eastern blotting of fresh licorice slice,
(b) fresh licorice slice, (c) Eastern blotting of fresh ginseng
slice, and (d) fresh ginseng slice.
(a)
(b)
(c)
(d)In the earlier experiments, we carried out the blotted staining on PVDF
membrane using MAb on solasodine glycosides and called it western blotting
[24]. Now we have applied this
new methodology to licorice glycoside, glycyrrhizin and named it eastern
blotting [12] for studying
ginsenosides [23], saikosaponin
[27], and so on.


#### 2.3.2. Immunoaffinity Concentration and One-Step Purification of
Ginsenoside Rb1 by Immunoaffinity Column

A crude extract ofP. ginseng roots was loaded onto the
immunoaffinity column and washed with the washing solution of phosphate
buffer. Figure 6 shows the fraction
1–8 containing overloaded ginsenoside Rb1. The other
ginsenosides Rg1, Rc, Re. and Rd were also detected in these
fractions by eastern blotting (data not shown). A sharp peak appeared around
fraction 20–24 eluted by acetate buffer containing KSCN and methanol
to give pure ginsenoside Rb1.Figure 6
Elution profile ofPanax ginseng crude extract on
immunoaffinity column monitoring by ELISA using
anti-ginsenoside-Rb1 MAb. The column was washed by
phosphate buffer and then eluted by HOAc buffer containing KSCN and
MeOH. Individual fractions (2 mL) were assayed by ELISA using
anti-ginsenoside-Rb1 MAb.Overloaded ginsenoside Rb1 was repeatedly immunoaffinity column
choromatographed to separate ginsenoside Rb1 completely [28]. The antibody was stable when
exposed to the eluent, and the immunoaffinity column showed almost no
decrease in capacity after repeated use more than 10 times under the same
conditions, as was reported for a one-step separation of forskolin from a
crude extract of Coleus forskohlii root [29]. This methodology is effective for
the rapid and simple purification of ginsenoside Rb1 and may open
up a wide field of comparable studies with other families of saponins for
which an acceptable method for one-step separation has not yet been
developed. Furthermore, to separate the total ginseng saponins, a wide
cross-reactive MAb against ginsenoside like anti-ginsenoside Re MAb could be
designed, as was done for the total solasodine glycosides by an
immunoaffinity column using an anti-solamargine MAb [30]. A combination of immunoaffinity column
chromatography, Eastern blotting and ELISA could be used to survey low
concentrations of ginsenoside Rb1 of plant origin and/or in
experimental animals and humans. In fact, we have succeeded in the isolation
of ginsenoside Rb1 from a different plant, Kalopanax
pictus Nakai, which was not known previously to contain
ginsenosides, using this combination of methods [31].


#### 2.3.3. Newly Established Knockout Extract for Glycyrrhizin

Glycyrrhizin has been reported to possess numerous pharmacological effects
like anti-inflammation [32],
antiulcer [33], antitumor [34], antiallergy [35], and hepatoprotective activities [36]. Although glycyrrhizin is supposed
to be a major active principle in licorice crude extract, a large number of
studies have demonstrated that the licorice extract is rich in bioactive
compounds other than glycyrrhizin such as triterpenes, flavonoids, and their
aglycones [37]. In order to confirm
the effect of glycyrrhizin in TCM, previously we purified glycyrrhizin from
TCM using an immunoaffinity column conjugated with anti-glycyrrhizin MAb
[38]. In this section, one-step
purification of glycyrrhizin and its function in the licorice crude extract
have been indicated as previously discussed about ginsenoside
Rb1.To eliminate glycyrrhizin from licorice extract, 12 mg of licorice
extract (glycyrrhizin content: 1275.0μg) in
loading buffer was applied on the anti-glycyrrhizin MAb immunoaffinity
column, and then the loading buffer was continuously circulated through the
column to enhance the binding efficiency. After overnight circulation at
4°C, the unbound column fraction was collected. The column was washed,
and then bound column fraction was eluted by elution solvent. Glycyrrhizin
concentration of bound column fraction was
1269.26 μg of glycyrrhizin (99.5% of
the loading glycyrrhizin). On the other hand, glycyrrhizin content of
unbound fraction was 3.50 μg (0.27% of
the loading glycyrrhizin). These data indicated that anti-glycyrrhizin MAb
immunoaffinity column could eliminate glycyrrhizin from licorice crude
extract with high efficiency. From this evidence, we named this fraction
knockout extract [16, 39].To further characterize glycyrrhizin knockout extract, the TLC analysis and
eastern blotting were performed. As shown in Figure7(a), several spots including glycyrrhizin were
detected in licorice extract. However, the spots of glycyrrhizin were
undetected in glycyrrhizin-knockout extract, although all other spots were
clearly detected (Figure 7(a), lane
2). eastern blotting by anti-glycyrrhizin MAb demonstrated that glycyrrhizin
was detected in licorice extract, but the spot of glycyrrhizin was invisible
in unbound fraction (Figure 7(b),
lane 2). Therefore, these data suggest that glycyrrhizin was specifically
eliminated from licorice extract by anti-glycyrrhizin MAb binding
immunoaffinity column.Preparation of knock-out extract eliminated glycyrrhizin from
licorice crude extract using immunoaffinity column conjugated with
anti-glycyrrhizin MAb. Lines 1, 2, and 3 indicate glycyrrhizin,
crude extract, and knock-out extract, respectively.
(a)
(b)This technique is useful approach to clarify the multicomponent interaction
with the specific protein such as enzymes, but it is unsuitable to
investigate the interaction between one principal component and other
components in herbal medicines. To investigate the role of one principal
compound in the crude extract, we prepared knockout extract by removing
target compound from the crude extract using high-specific MAb
immunoaffinity column. By using this approach, it may become possible to
determine the potential function of one natural compound on the crude
extract or TCM byin vitro and in vivoassays.Nitric oxide (NO) is free radical with multiple physiological functions, such
as vasodilatation, neurotransmission, and inflammation [40]. During inflammatory process, large
amount of NO is produced by inducible nitric oxide synthase (iNOS) by
inflammatory cytokines and/or bacterial lipopolysaccharide (LPS) in various
cell types including macrophages [41]. Overproduction of NO by iNOS triggers the pathogenesis of
septic shock and organ destruction in certain inflammatory and autoimmune
diseases [42–44]. Therefore, the inhibition of NO
production by blocking iNOS expression may be useful strategy for the
treatment of various inflammatory diseases.To determine whether licorice crude extract inhibits NO production in
LPS-stimulated mouse RAW264 macrophages, cells were treated with various
concentrations of licorice extract for 30 min and then stimulated
with LPS. LPS caused a dramatic increase in NO production, and this
induction was inhibited in a dose-dependent manner by treatment with
licorice extract.We next investigated the inhibitory effect of glycyrrhizin-knockout extract
and the combined treatment with glycyrrhizin-knockout extract and
glycyrrhizin on NO production. Since 100μg/mL
of licorice extract contains 10.6 μg of
glycyrrhizin, the cells were pretreated with licorice extract
(100 μg/mL), glycyrrhizin-knockout extract
(89.4 μg/mL), or combination of
glycyrrhizin-knockout extract (89.4 μg/mL) and
glycyrrhizin (10.6 μg/mL). Figure 8(a) indicated that treatment of
licorice extract leads to a marked suppression of NO production as compared
to LPS treatment [inhibition ratio (IR) = 57.7%]. Interestingly,
although glycyrrhizin alone could not block NO production, the inhibitory
effect of glycyrrhizin-knockout extract was decreased compared with licorice
extract (IR = 17.8%). Moreover, the combined treatment with
glycyrrhizin-knockout extract and glycyrrhizin significantly improved the
inhibition of NO production (IR = 33.5%). To determine whether the
combinational effect of glycyrrhizin-knockout extract and glycyrrhizin was
related to iNOS expression, we performed western blotting (Figure 8(b)). Although the licorice extract
strongly suppressed LPS-induced iNOS expression, treatment of
glycyrrhizin-knockout extract reduced this effect [45].Effects of glycyrrhizin-knockout extract and the combination of
glycyrrhizin-knockout extract and glycyrrhizin on LPS-induced NO
production (a) and iNOS protein expression (b).
*P
<
0.05,
**P
<
0.01,
***P
<
0.001.
(a)
(b)


## 2.1. Analytical Method for Determination of Hapten Number in Antigen,
Hapten-Carrier Protein Conjugate

For production of MAb, synthesis of hapten, which is derived from an immune
antigen and linker bridge, and the carrier protein conjugate is necessary. There
had been no direct and appropriate methods for the determination of haptens
conjugated with carrier proteins without differential UV analysis,
radiochemical, or chemical methods. Therefore, immunization by the injection of
hapten-carrier protein conjugate was unreliable. Wengatz et al. [17] determined the hapten density of
immunoconjugates by matrix-assisted UV laser desorption/ionization mass
spectrometry. We also reported the direct analytical method of hapten and
carrier protein conjugates by a matrix-assisted laser desorption/ionization mass
spectrometry (MALDI) tof mass spectrometry using an internal standard [18–20].Figure1 shows the MALDI tof mass spectrum
of the most pharmacologically active marihuana compound, tetrahydrocannabinolic
acid (THCA)-bovine serum albumin (BSA) conjugate, and BSA used as an internal
standard [20]. This shows only the
singly, doubly, and triply ionized molecule ions of the intact conjugate. The
sharp peak at m/z 66,465 is the [M + H]+ peak of BSA. A small [M +
H]+ peak of the THCA-BSA conjugate is at m/z 70,792, indicating
that the calculated molecular mass of the THCA-BSA conjugate is 70,581 using a
calculated molecular mass of 66,267 for BSA. The calculated molecule mass of the
THCA moiety is 4,314. From this result, 12.7 molecules of THCA are combined with
BSA [20]. Since this method is suitable
for small molecule natural products, we had been analyzing the hapten number of
all natural products for MAbs including glycosides like ginsenosides and
glycyrrhizin.Figure 1
Matrix-assisted laser desorption/ionization tof mass spectrometry of
tetrahydrocannabinolic-acid-BSA conjugate. [M + H] indicates the
molecular weight of the conjugate, from which the hapten number can be
calculated.


## 2.2. Preparation of MAb against Ginsenosides and ELISA as an Assay
System

Ginseng, the crude drug ofPanax ginseng, is one of the most
important natural medicines in many countries. It has been used to enhance
stamina and capacity to cope with fatigue and physical stress and as a tonic
against cancers, disturbances of the central nervous system (memory, learning,
and behavior), hypothermia, carbohydrate and lipid metabolism, immune function,
the cardiovascular system, and radioprotection [21]. It contains more than 30 kinds of dammarane and oleanane
saponins considered to be pharmacologically active components. Ginsenoside
Rb1 is the main saponin in ginseng. However, since the
concentration in the ginseng root or the root extract varies depending on the
method of extraction, subsequent treatment, or even the season of its collection
[22], standardization of quality is
required. For this purpose, we have prepared anti-ginsenoside Rb1
[6] and Rg1 MAbs [7]. The immunoassay system using MAb is not
frequently used for naturally occurring smaller molecular weight bioactive
compounds. Preparation of MAbs is difficult, but is one of the most important
steps for the analysis of natural products. As a typical natural product, the
preparation of MAb against the ginseng saponin ginsenoside Rb1 will
be discussed.A hybridoma-producing MAb reactive to ginsenoside Rb1 was obtained by
the general procedure and classified into IgG2b which had
κ light chains. The reactivity of IgG type MAb, 9G7
was tested by varying antibody concentration and by performing a dilution curve.
The antibody concentration was selected for competitive ELISA. The free MAb
following competition is bound to polystyrene microtiter plates precoated with
ginsenoside Rb1-HSA. Under these conditions, the full measurement
range of the assay extends from 20 to 400 ng/mL [6].Cross-reactivity is the most important factor in determining the value of an
antibody. Since the ELISA for ginsenoside Rb1 was established for
phytochemical investigations involving crude plant extracts, the assay
specificity was checked by determining the cross-reactivity of the MAb with
various related compounds. The cross-reactivity data of MAb that was obtained
were examined by competitive ELISA and calculated using picomole amounts of
ginsenoside Rb1. The cross-reactivity of ginsenoside Rc and Rd, which
possess a diglucose moiety attached to the C-3 hydroxy group, was weak compared
to ginsenoside Rb1 (0.024 and 0.020%, resp.). Ginsenoside Re and
Rg1 showed no cross-reactivity (less than 0.005%). It is
evident that the MAb reacted only with a small number of structurally related
ginsenoside Rb1 molecules, and very weakly, and did not react with
other steroidal compounds like glycyrrhizin, digitoxin, tigogenin, tigonin, and
solamargine.In our ongoing studies on MAbs against ginseng saponins, anti-ginsenoside
Rg1 MAb [7] and Re [23] have been prepared and their ELISA was
set up. Anti-ginsenoside Rg1 MAb was also highly specific like
anti-ginsenoside Rb1. On the other hand, anti-ginsenoside Re MAb
showed wide cross-reactivity. Therefore, the MAb can be used for the analysis
for the total ginsenoside concentration.


## 2.3. Application of MAb in the Natural Products Field

Although western blotting is a common assay methodology for
high-molecular-weight-substances, this method has not been employed for small
molecules, as direct immunostaining of such compounds on a TLC plate is as yet
unknown. Therefore, a new method for such small-molecular compounds is required.
Moreover, if small molecules can be blotted to a membrane, fixing them also
requires a new methodology. Previously, we succeeded in separating
small-molecule compounds such as solasodine glycosides into a part of an epitope
and fixing on the membrane [24], as
follows.


### 2.3.1. New Staining Method for Ginsenosides, “Eastern
Blotting”

Figure2 shows the
H2SO4 staining and eastern blotting of ginsenoside
standards and TCM using anti-ginsenoside Rb1 MAb. It is
impossible to determine the ginsenosides by TLC staining by
H2SO4 as indicated in Figure 2(a). On the other hand, clear staining of
ginsenoside Rb1 occurred by eastern blotting, as indicated in
Figure 2(b). Furthermore, it became
evident that Jigengtang and Dahuanggancaotang prescriptions that did not
contain ginseng, as indicated by the absence of a ginsenoside Rb1band. The eastern blotting method was considerably more sensitive than
that of H2SO4 staining. The
H2SO4 staining detected all standard compounds.
The eastern blotting indicated only limited staining of ginsenoside
Rb1, Rc, and Rd, whose cross-reactivities were under
0.02% as shown in Figure 2(b).
We suggest that an aglycone, protopanaxadiol, and a part of the sugars may
be of importance to the immunization and may function as an epitope for the
structure of ginsenosides. In addition, it is suggested that the specific
reactivity of sugar moieties in the ginsenoside molecule against
anti-ginsenoside Rb1 MAb may be modified by the NaIO4
treatment of ginsenosides on the PVDF membrane, causing ginsenoside Rc and
Rd to become detectable by eastern blotting.Eastern blotting of ginsenosides in traditional Chinese medicine
formulas using anti-ginsenoside Rb1 MAb. Samples (1) Jigengtang, (2)
Dahuanggancaotang, (3) Nenshenyangyongtang, (4) Sijunzitang, (5)
Nenshentang, (6) Buanxiaxiexintang, (7) Xiaocaihutang, and (8) Crude
ginseng extract. Lanes 1 and 2 do not contain ginseng. Standard of
ginsenosides indicated, ginsenosidr-Rg1, -Re, -Rd, -Rc,
and -Rb1, respectively, from the upper spot.
(a)
TLC
(b)
Eastern blottingApplication of the eastern blotting method for the detection of glycyrrhizin
in the serum samples was investigated. In general, it is difficult to detect
glycyrrhizin in serum due to the large amount of impurities. The developed
bands of impurities lapped over the band of glycyrrhizin, and we failed to
find a proper developing solvent system that could separate glycyrrhizin and
the impurities apart from each other clearly on the TLC plate. Figure3 shows the detection of glycyrrhizin
by the eastern blotting technique in the rat serum samples. Glycyrrhizin
could not be identified on the TLC plate stained by H2SO4through many bands that were detected (Figure 3(a)). On the other hand, eastern blotting clearly
shows the band of glycyrrhizin even after 1 h (Figure 3(b)). Although the sensitivity of the
eastern blotting method was greatly affected by the impurities, the
detection limit was still at the nanogram level. The results proved that the
eastern blotting technique could be a unique method for identifying
glycyrrhizin against a background of a large amount of impurities.Eastern blotting profile of glycyrrhizin in rat serum after injection
of glycyrrhizin (a) TLC of rat-serum-injected glycyrrhizin TLC was
developed withn-BuOH-H2O-AcOH
(7 : 2 : 1). Spots were detected by
10% H2SO4. (b) Eastern blotting of
rat-serum-injected glycyrrhizin; the band of glycyrrhizin was
detected until 1 h after injection. GA: glycyhrrhetinic acid,
GC: glycyrrhizin, and GG: glycyrrhetinic acid monoglucuronide.
(a)
(b)When the mixture of anti-ginsenoside Rb1 and Rg1 MAbs
and the pair of substrates were tested for staining for ginsenosides, all
ginsenosides, ginsenoside Rb1, -Rc, -Rd, -Re, and -Rg1
were stained blue although the purple color staining for ginsenoside
Rg1 was expected because 3-amino-9-ethylcarbazole and
4-chloro-1-naphotol might be different. Therefore, we performed successive
staining of the membrane using anti-ginsenoside Rg1 and then
anti-ginsenoside Rb1. Finally, we performed the double staining
of ginsenosides indicating that ginsenoside Rg1 and ginsenoside
Re were stained purple and the other blue, as indicated in Figure 4. From this result, both antibodies
can distinguish the individual aglycones, protopanaxatriol, and
protopanaxadiol. For this application, the crude extract of various
Panax species was analyzed by the newly developed
double staining system. Major ginsenosides can be determined clearly by the
double staining method, as indicated in Figure 4.Double Eastern blotting staining of ginsenosides contained in various
ginseng samples using anti-ginseenoside-Rb1 and
anti-ginsenoside-Rg1 MAbs. (a) TLC profile stained by
sulfuric acid. (b) Eastern blotting by
anti-ginsenoside-Rb1 and
anti-ginseside-Rg1 monoclonal antibodies I, II, III,
IV, V, and VI indicated white ginseng, red ginseng, fibrous ginseng
(P. ginseng), P.
notoginseng, P.
quinquefolius and P.
japonicus, respectively. Upper purple color
spots and lower blue color spots were stained by
anti-ginsenoside-Rg1, and
anti-ginsenoside-Rb1 monoclonal antibodies,
respectively.
(a)
(b)Therefore, it is suggested that the staining color shows the pharmacological
activity. As shown in Figure4(b),
the purple bands indicate ginsenosides which have protopanaxatriol as an
aglycone and stimulation activity for the central nervous system (CNS). On
the other hand, the blue color indicates ginsenosides containing
protopanaxadiol as an aglycone that possess a depression effect on the CNS.
Moreover, the Rf value of ginsenosides roughly suggests the number of sugars
attached to the aglycone. Both analyses make it possible to jointly identify
which aglycone attaches and how many sugars it possesses, leading to the
structure of the ginsenosides. In fact, three kinds of ginsenosides
possessing protopanaxadiol-ginsenoside Rh1, -Rf, and
20-O-glucoginsenoside Rf in P. ginseng
root were determined by coloring and Rf value by comparing them with the
structures reported in the previous paper [25].Figure5(a) indicates
immunolocalization of glycyrrhizin in a licorice root slice using
anti-glycyrrhizin MAb as another application of the eastern blotting method.
The phloem (⟷) contained a higher concentration of
glycyrrhizin than the xylem and cork part [26]. On the other hand, no staining occurred on ginseng (Figure
5(c)).Immunocytolocalization of glycyrrhizin in fresh licorice root using
anti-glycyrrhizin MAb. (a) Eastern blotting of fresh licorice slice,
(b) fresh licorice slice, (c) Eastern blotting of fresh ginseng
slice, and (d) fresh ginseng slice.
(a)
(b)
(c)
(d)In the earlier experiments, we carried out the blotted staining on PVDF
membrane using MAb on solasodine glycosides and called it western blotting
[24]. Now we have applied this
new methodology to licorice glycoside, glycyrrhizin and named it eastern
blotting [12] for studying
ginsenosides [23], saikosaponin
[27], and so on.


### 2.3.2. Immunoaffinity Concentration and One-Step Purification of
Ginsenoside Rb1 by Immunoaffinity Column

A crude extract ofP. ginseng roots was loaded onto the
immunoaffinity column and washed with the washing solution of phosphate
buffer. Figure 6 shows the fraction
1–8 containing overloaded ginsenoside Rb1. The other
ginsenosides Rg1, Rc, Re. and Rd were also detected in these
fractions by eastern blotting (data not shown). A sharp peak appeared around
fraction 20–24 eluted by acetate buffer containing KSCN and methanol
to give pure ginsenoside Rb1.Figure 6
Elution profile ofPanax ginseng crude extract on
immunoaffinity column monitoring by ELISA using
anti-ginsenoside-Rb1 MAb. The column was washed by
phosphate buffer and then eluted by HOAc buffer containing KSCN and
MeOH. Individual fractions (2 mL) were assayed by ELISA using
anti-ginsenoside-Rb1 MAb.Overloaded ginsenoside Rb1 was repeatedly immunoaffinity column
choromatographed to separate ginsenoside Rb1 completely [28]. The antibody was stable when
exposed to the eluent, and the immunoaffinity column showed almost no
decrease in capacity after repeated use more than 10 times under the same
conditions, as was reported for a one-step separation of forskolin from a
crude extract of Coleus forskohlii root [29]. This methodology is effective for
the rapid and simple purification of ginsenoside Rb1 and may open
up a wide field of comparable studies with other families of saponins for
which an acceptable method for one-step separation has not yet been
developed. Furthermore, to separate the total ginseng saponins, a wide
cross-reactive MAb against ginsenoside like anti-ginsenoside Re MAb could be
designed, as was done for the total solasodine glycosides by an
immunoaffinity column using an anti-solamargine MAb [30]. A combination of immunoaffinity column
chromatography, Eastern blotting and ELISA could be used to survey low
concentrations of ginsenoside Rb1 of plant origin and/or in
experimental animals and humans. In fact, we have succeeded in the isolation
of ginsenoside Rb1 from a different plant, Kalopanax
pictus Nakai, which was not known previously to contain
ginsenosides, using this combination of methods [31].


### 2.3.3. Newly Established Knockout Extract for Glycyrrhizin

Glycyrrhizin has been reported to possess numerous pharmacological effects
like anti-inflammation [32],
antiulcer [33], antitumor [34], antiallergy [35], and hepatoprotective activities [36]. Although glycyrrhizin is supposed
to be a major active principle in licorice crude extract, a large number of
studies have demonstrated that the licorice extract is rich in bioactive
compounds other than glycyrrhizin such as triterpenes, flavonoids, and their
aglycones [37]. In order to confirm
the effect of glycyrrhizin in TCM, previously we purified glycyrrhizin from
TCM using an immunoaffinity column conjugated with anti-glycyrrhizin MAb
[38]. In this section, one-step
purification of glycyrrhizin and its function in the licorice crude extract
have been indicated as previously discussed about ginsenoside
Rb1.To eliminate glycyrrhizin from licorice extract, 12 mg of licorice
extract (glycyrrhizin content: 1275.0μg) in
loading buffer was applied on the anti-glycyrrhizin MAb immunoaffinity
column, and then the loading buffer was continuously circulated through the
column to enhance the binding efficiency. After overnight circulation at
4°C, the unbound column fraction was collected. The column was washed,
and then bound column fraction was eluted by elution solvent. Glycyrrhizin
concentration of bound column fraction was
1269.26 μg of glycyrrhizin (99.5% of
the loading glycyrrhizin). On the other hand, glycyrrhizin content of
unbound fraction was 3.50 μg (0.27% of
the loading glycyrrhizin). These data indicated that anti-glycyrrhizin MAb
immunoaffinity column could eliminate glycyrrhizin from licorice crude
extract with high efficiency. From this evidence, we named this fraction
knockout extract [16, 39].To further characterize glycyrrhizin knockout extract, the TLC analysis and
eastern blotting were performed. As shown in Figure7(a), several spots including glycyrrhizin were
detected in licorice extract. However, the spots of glycyrrhizin were
undetected in glycyrrhizin-knockout extract, although all other spots were
clearly detected (Figure 7(a), lane
2). eastern blotting by anti-glycyrrhizin MAb demonstrated that glycyrrhizin
was detected in licorice extract, but the spot of glycyrrhizin was invisible
in unbound fraction (Figure 7(b),
lane 2). Therefore, these data suggest that glycyrrhizin was specifically
eliminated from licorice extract by anti-glycyrrhizin MAb binding
immunoaffinity column.Preparation of knock-out extract eliminated glycyrrhizin from
licorice crude extract using immunoaffinity column conjugated with
anti-glycyrrhizin MAb. Lines 1, 2, and 3 indicate glycyrrhizin,
crude extract, and knock-out extract, respectively.
(a)
(b)This technique is useful approach to clarify the multicomponent interaction
with the specific protein such as enzymes, but it is unsuitable to
investigate the interaction between one principal component and other
components in herbal medicines. To investigate the role of one principal
compound in the crude extract, we prepared knockout extract by removing
target compound from the crude extract using high-specific MAb
immunoaffinity column. By using this approach, it may become possible to
determine the potential function of one natural compound on the crude
extract or TCM byin vitro and in vivoassays.Nitric oxide (NO) is free radical with multiple physiological functions, such
as vasodilatation, neurotransmission, and inflammation [40]. During inflammatory process, large
amount of NO is produced by inducible nitric oxide synthase (iNOS) by
inflammatory cytokines and/or bacterial lipopolysaccharide (LPS) in various
cell types including macrophages [41]. Overproduction of NO by iNOS triggers the pathogenesis of
septic shock and organ destruction in certain inflammatory and autoimmune
diseases [42–44]. Therefore, the inhibition of NO
production by blocking iNOS expression may be useful strategy for the
treatment of various inflammatory diseases.To determine whether licorice crude extract inhibits NO production in
LPS-stimulated mouse RAW264 macrophages, cells were treated with various
concentrations of licorice extract for 30 min and then stimulated
with LPS. LPS caused a dramatic increase in NO production, and this
induction was inhibited in a dose-dependent manner by treatment with
licorice extract.We next investigated the inhibitory effect of glycyrrhizin-knockout extract
and the combined treatment with glycyrrhizin-knockout extract and
glycyrrhizin on NO production. Since 100μg/mL
of licorice extract contains 10.6 μg of
glycyrrhizin, the cells were pretreated with licorice extract
(100 μg/mL), glycyrrhizin-knockout extract
(89.4 μg/mL), or combination of
glycyrrhizin-knockout extract (89.4 μg/mL) and
glycyrrhizin (10.6 μg/mL). Figure 8(a) indicated that treatment of
licorice extract leads to a marked suppression of NO production as compared
to LPS treatment [inhibition ratio (IR) = 57.7%]. Interestingly,
although glycyrrhizin alone could not block NO production, the inhibitory
effect of glycyrrhizin-knockout extract was decreased compared with licorice
extract (IR = 17.8%). Moreover, the combined treatment with
glycyrrhizin-knockout extract and glycyrrhizin significantly improved the
inhibition of NO production (IR = 33.5%). To determine whether the
combinational effect of glycyrrhizin-knockout extract and glycyrrhizin was
related to iNOS expression, we performed western blotting (Figure 8(b)). Although the licorice extract
strongly suppressed LPS-induced iNOS expression, treatment of
glycyrrhizin-knockout extract reduced this effect [45].Effects of glycyrrhizin-knockout extract and the combination of
glycyrrhizin-knockout extract and glycyrrhizin on LPS-induced NO
production (a) and iNOS protein expression (b).
*P
<
0.05,
**P
<
0.01,
***P
<
0.001.
(a)
(b)


## 2.3.1. New Staining Method for Ginsenosides, “Eastern
Blotting”

Figure2 shows the
H2SO4 staining and eastern blotting of ginsenoside
standards and TCM using anti-ginsenoside Rb1 MAb. It is
impossible to determine the ginsenosides by TLC staining by
H2SO4 as indicated in Figure 2(a). On the other hand, clear staining of
ginsenoside Rb1 occurred by eastern blotting, as indicated in
Figure 2(b). Furthermore, it became
evident that Jigengtang and Dahuanggancaotang prescriptions that did not
contain ginseng, as indicated by the absence of a ginsenoside Rb1band. The eastern blotting method was considerably more sensitive than
that of H2SO4 staining. The
H2SO4 staining detected all standard compounds.
The eastern blotting indicated only limited staining of ginsenoside
Rb1, Rc, and Rd, whose cross-reactivities were under
0.02% as shown in Figure 2(b).
We suggest that an aglycone, protopanaxadiol, and a part of the sugars may
be of importance to the immunization and may function as an epitope for the
structure of ginsenosides. In addition, it is suggested that the specific
reactivity of sugar moieties in the ginsenoside molecule against
anti-ginsenoside Rb1 MAb may be modified by the NaIO4
treatment of ginsenosides on the PVDF membrane, causing ginsenoside Rc and
Rd to become detectable by eastern blotting.Eastern blotting of ginsenosides in traditional Chinese medicine
formulas using anti-ginsenoside Rb1 MAb. Samples (1) Jigengtang, (2)
Dahuanggancaotang, (3) Nenshenyangyongtang, (4) Sijunzitang, (5)
Nenshentang, (6) Buanxiaxiexintang, (7) Xiaocaihutang, and (8) Crude
ginseng extract. Lanes 1 and 2 do not contain ginseng. Standard of
ginsenosides indicated, ginsenosidr-Rg1, -Re, -Rd, -Rc,
and -Rb1, respectively, from the upper spot.
(a)
TLC
(b)
Eastern blottingApplication of the eastern blotting method for the detection of glycyrrhizin
in the serum samples was investigated. In general, it is difficult to detect
glycyrrhizin in serum due to the large amount of impurities. The developed
bands of impurities lapped over the band of glycyrrhizin, and we failed to
find a proper developing solvent system that could separate glycyrrhizin and
the impurities apart from each other clearly on the TLC plate. Figure3 shows the detection of glycyrrhizin
by the eastern blotting technique in the rat serum samples. Glycyrrhizin
could not be identified on the TLC plate stained by H2SO4through many bands that were detected (Figure 3(a)). On the other hand, eastern blotting clearly
shows the band of glycyrrhizin even after 1 h (Figure 3(b)). Although the sensitivity of the
eastern blotting method was greatly affected by the impurities, the
detection limit was still at the nanogram level. The results proved that the
eastern blotting technique could be a unique method for identifying
glycyrrhizin against a background of a large amount of impurities.Eastern blotting profile of glycyrrhizin in rat serum after injection
of glycyrrhizin (a) TLC of rat-serum-injected glycyrrhizin TLC was
developed withn-BuOH-H2O-AcOH
(7 : 2 : 1). Spots were detected by
10% H2SO4. (b) Eastern blotting of
rat-serum-injected glycyrrhizin; the band of glycyrrhizin was
detected until 1 h after injection. GA: glycyhrrhetinic acid,
GC: glycyrrhizin, and GG: glycyrrhetinic acid monoglucuronide.
(a)
(b)When the mixture of anti-ginsenoside Rb1 and Rg1 MAbs
and the pair of substrates were tested for staining for ginsenosides, all
ginsenosides, ginsenoside Rb1, -Rc, -Rd, -Re, and -Rg1
were stained blue although the purple color staining for ginsenoside
Rg1 was expected because 3-amino-9-ethylcarbazole and
4-chloro-1-naphotol might be different. Therefore, we performed successive
staining of the membrane using anti-ginsenoside Rg1 and then
anti-ginsenoside Rb1. Finally, we performed the double staining
of ginsenosides indicating that ginsenoside Rg1 and ginsenoside
Re were stained purple and the other blue, as indicated in Figure 4. From this result, both antibodies
can distinguish the individual aglycones, protopanaxatriol, and
protopanaxadiol. For this application, the crude extract of various
Panax species was analyzed by the newly developed
double staining system. Major ginsenosides can be determined clearly by the
double staining method, as indicated in Figure 4.Double Eastern blotting staining of ginsenosides contained in various
ginseng samples using anti-ginseenoside-Rb1 and
anti-ginsenoside-Rg1 MAbs. (a) TLC profile stained by
sulfuric acid. (b) Eastern blotting by
anti-ginsenoside-Rb1 and
anti-ginseside-Rg1 monoclonal antibodies I, II, III,
IV, V, and VI indicated white ginseng, red ginseng, fibrous ginseng
(P. ginseng), P.
notoginseng, P.
quinquefolius and P.
japonicus, respectively. Upper purple color
spots and lower blue color spots were stained by
anti-ginsenoside-Rg1, and
anti-ginsenoside-Rb1 monoclonal antibodies,
respectively.
(a)
(b)Therefore, it is suggested that the staining color shows the pharmacological
activity. As shown in Figure4(b),
the purple bands indicate ginsenosides which have protopanaxatriol as an
aglycone and stimulation activity for the central nervous system (CNS). On
the other hand, the blue color indicates ginsenosides containing
protopanaxadiol as an aglycone that possess a depression effect on the CNS.
Moreover, the Rf value of ginsenosides roughly suggests the number of sugars
attached to the aglycone. Both analyses make it possible to jointly identify
which aglycone attaches and how many sugars it possesses, leading to the
structure of the ginsenosides. In fact, three kinds of ginsenosides
possessing protopanaxadiol-ginsenoside Rh1, -Rf, and
20-O-glucoginsenoside Rf in P. ginseng
root were determined by coloring and Rf value by comparing them with the
structures reported in the previous paper [25].Figure5(a) indicates
immunolocalization of glycyrrhizin in a licorice root slice using
anti-glycyrrhizin MAb as another application of the eastern blotting method.
The phloem (⟷) contained a higher concentration of
glycyrrhizin than the xylem and cork part [26]. On the other hand, no staining occurred on ginseng (Figure
5(c)).Immunocytolocalization of glycyrrhizin in fresh licorice root using
anti-glycyrrhizin MAb. (a) Eastern blotting of fresh licorice slice,
(b) fresh licorice slice, (c) Eastern blotting of fresh ginseng
slice, and (d) fresh ginseng slice.
(a)
(b)
(c)
(d)In the earlier experiments, we carried out the blotted staining on PVDF
membrane using MAb on solasodine glycosides and called it western blotting
[24]. Now we have applied this
new methodology to licorice glycoside, glycyrrhizin and named it eastern
blotting [12] for studying
ginsenosides [23], saikosaponin
[27], and so on.


## 2.3.2. Immunoaffinity Concentration and One-Step Purification of
Ginsenoside Rb1 by Immunoaffinity Column

A crude extract ofP. ginseng roots was loaded onto the
immunoaffinity column and washed with the washing solution of phosphate
buffer. Figure 6 shows the fraction
1–8 containing overloaded ginsenoside Rb1. The other
ginsenosides Rg1, Rc, Re. and Rd were also detected in these
fractions by eastern blotting (data not shown). A sharp peak appeared around
fraction 20–24 eluted by acetate buffer containing KSCN and methanol
to give pure ginsenoside Rb1.Figure 6
Elution profile ofPanax ginseng crude extract on
immunoaffinity column monitoring by ELISA using
anti-ginsenoside-Rb1 MAb. The column was washed by
phosphate buffer and then eluted by HOAc buffer containing KSCN and
MeOH. Individual fractions (2 mL) were assayed by ELISA using
anti-ginsenoside-Rb1 MAb.Overloaded ginsenoside Rb1 was repeatedly immunoaffinity column
choromatographed to separate ginsenoside Rb1 completely [28]. The antibody was stable when
exposed to the eluent, and the immunoaffinity column showed almost no
decrease in capacity after repeated use more than 10 times under the same
conditions, as was reported for a one-step separation of forskolin from a
crude extract of Coleus forskohlii root [29]. This methodology is effective for
the rapid and simple purification of ginsenoside Rb1 and may open
up a wide field of comparable studies with other families of saponins for
which an acceptable method for one-step separation has not yet been
developed. Furthermore, to separate the total ginseng saponins, a wide
cross-reactive MAb against ginsenoside like anti-ginsenoside Re MAb could be
designed, as was done for the total solasodine glycosides by an
immunoaffinity column using an anti-solamargine MAb [30]. A combination of immunoaffinity column
chromatography, Eastern blotting and ELISA could be used to survey low
concentrations of ginsenoside Rb1 of plant origin and/or in
experimental animals and humans. In fact, we have succeeded in the isolation
of ginsenoside Rb1 from a different plant, Kalopanax
pictus Nakai, which was not known previously to contain
ginsenosides, using this combination of methods [31].


## 2.3.3. Newly Established Knockout Extract for Glycyrrhizin

Glycyrrhizin has been reported to possess numerous pharmacological effects
like anti-inflammation [32],
antiulcer [33], antitumor [34], antiallergy [35], and hepatoprotective activities [36]. Although glycyrrhizin is supposed
to be a major active principle in licorice crude extract, a large number of
studies have demonstrated that the licorice extract is rich in bioactive
compounds other than glycyrrhizin such as triterpenes, flavonoids, and their
aglycones [37]. In order to confirm
the effect of glycyrrhizin in TCM, previously we purified glycyrrhizin from
TCM using an immunoaffinity column conjugated with anti-glycyrrhizin MAb
[38]. In this section, one-step
purification of glycyrrhizin and its function in the licorice crude extract
have been indicated as previously discussed about ginsenoside
Rb1.To eliminate glycyrrhizin from licorice extract, 12 mg of licorice
extract (glycyrrhizin content: 1275.0μg) in
loading buffer was applied on the anti-glycyrrhizin MAb immunoaffinity
column, and then the loading buffer was continuously circulated through the
column to enhance the binding efficiency. After overnight circulation at
4°C, the unbound column fraction was collected. The column was washed,
and then bound column fraction was eluted by elution solvent. Glycyrrhizin
concentration of bound column fraction was
1269.26 μg of glycyrrhizin (99.5% of
the loading glycyrrhizin). On the other hand, glycyrrhizin content of
unbound fraction was 3.50 μg (0.27% of
the loading glycyrrhizin). These data indicated that anti-glycyrrhizin MAb
immunoaffinity column could eliminate glycyrrhizin from licorice crude
extract with high efficiency. From this evidence, we named this fraction
knockout extract [16, 39].To further characterize glycyrrhizin knockout extract, the TLC analysis and
eastern blotting were performed. As shown in Figure7(a), several spots including glycyrrhizin were
detected in licorice extract. However, the spots of glycyrrhizin were
undetected in glycyrrhizin-knockout extract, although all other spots were
clearly detected (Figure 7(a), lane
2). eastern blotting by anti-glycyrrhizin MAb demonstrated that glycyrrhizin
was detected in licorice extract, but the spot of glycyrrhizin was invisible
in unbound fraction (Figure 7(b),
lane 2). Therefore, these data suggest that glycyrrhizin was specifically
eliminated from licorice extract by anti-glycyrrhizin MAb binding
immunoaffinity column.Preparation of knock-out extract eliminated glycyrrhizin from
licorice crude extract using immunoaffinity column conjugated with
anti-glycyrrhizin MAb. Lines 1, 2, and 3 indicate glycyrrhizin,
crude extract, and knock-out extract, respectively.
(a)
(b)This technique is useful approach to clarify the multicomponent interaction
with the specific protein such as enzymes, but it is unsuitable to
investigate the interaction between one principal component and other
components in herbal medicines. To investigate the role of one principal
compound in the crude extract, we prepared knockout extract by removing
target compound from the crude extract using high-specific MAb
immunoaffinity column. By using this approach, it may become possible to
determine the potential function of one natural compound on the crude
extract or TCM byin vitro and in vivoassays.Nitric oxide (NO) is free radical with multiple physiological functions, such
as vasodilatation, neurotransmission, and inflammation [40]. During inflammatory process, large
amount of NO is produced by inducible nitric oxide synthase (iNOS) by
inflammatory cytokines and/or bacterial lipopolysaccharide (LPS) in various
cell types including macrophages [41]. Overproduction of NO by iNOS triggers the pathogenesis of
septic shock and organ destruction in certain inflammatory and autoimmune
diseases [42–44]. Therefore, the inhibition of NO
production by blocking iNOS expression may be useful strategy for the
treatment of various inflammatory diseases.To determine whether licorice crude extract inhibits NO production in
LPS-stimulated mouse RAW264 macrophages, cells were treated with various
concentrations of licorice extract for 30 min and then stimulated
with LPS. LPS caused a dramatic increase in NO production, and this
induction was inhibited in a dose-dependent manner by treatment with
licorice extract.We next investigated the inhibitory effect of glycyrrhizin-knockout extract
and the combined treatment with glycyrrhizin-knockout extract and
glycyrrhizin on NO production. Since 100μg/mL
of licorice extract contains 10.6 μg of
glycyrrhizin, the cells were pretreated with licorice extract
(100 μg/mL), glycyrrhizin-knockout extract
(89.4 μg/mL), or combination of
glycyrrhizin-knockout extract (89.4 μg/mL) and
glycyrrhizin (10.6 μg/mL). Figure 8(a) indicated that treatment of
licorice extract leads to a marked suppression of NO production as compared
to LPS treatment [inhibition ratio (IR) = 57.7%]. Interestingly,
although glycyrrhizin alone could not block NO production, the inhibitory
effect of glycyrrhizin-knockout extract was decreased compared with licorice
extract (IR = 17.8%). Moreover, the combined treatment with
glycyrrhizin-knockout extract and glycyrrhizin significantly improved the
inhibition of NO production (IR = 33.5%). To determine whether the
combinational effect of glycyrrhizin-knockout extract and glycyrrhizin was
related to iNOS expression, we performed western blotting (Figure 8(b)). Although the licorice extract
strongly suppressed LPS-induced iNOS expression, treatment of
glycyrrhizin-knockout extract reduced this effect [45].Effects of glycyrrhizin-knockout extract and the combination of
glycyrrhizin-knockout extract and glycyrrhizin on LPS-induced NO
production (a) and iNOS protein expression (b).
*P
<
0.05,
**P
<
0.01,
***P
<
0.001.
(a)
(b)


## 3. Conclusions

Two unique applications using MAb, eastern blotting and knockout extract have been
introduced in this paper. The eastern blotting method has great potential
applications for the wide range of natural products, especially glycosides like
ginsenosides of ginseng and glycyrrhizin in licorice. When two kinds of MAbs,
against ginsenoside Rb1 and Rg1 can be used, the double
staining that enhanced the separate staining of ginsenosides having protopanaxatriol
or protopanaxadiol in a molecule, occurred. The staining color can be used to
monitor the pharmacological activity suggesting that the purple spots contain
protopanaxatriol as an aglycone indicating central nervous system (CNS) stimulatory
activity [46]. On the other hand, the blue
color indicates ginsenosides having protopanaxadiol which possess a depression
effect on the CNS [37]. Furthermore, the Rf
value of ginsenosides suggests the number of sugars attached to the aglycone. Both
evidences make it possible to confirm which aglycone is attached and how many sugars
are combined with the aglycone.We demonstrated that knockout extract prepared by anti-natural compound-specific MAb
immunoaffinity column is a useful approach for determination of potential function
of natural compound onin vitro and in vivoassays. The pharmacological analysis by knockout extract might be directly
applicable to both other crude extracts and various TCMs to clarify the real
pharmacologically active components.A ginsenoside Rb1 knockout extract can be prepared using an immunoaffinity
column conjugated with anti-ginsenoside Rb1 MAb. In this extract, all
compounds except only ginsenoside Rb1 are contained. Furthermore,
glycyrrhizin-knockout extract prepared from licorice crude extract using an
immunoaffinity column conjugated with anti-glycyrrhizin MAb. These knockout extracts
may be able to support the pharmacological investigation for finding out a really
active component in a crude drug and/or TCM. In fact, addition of glycyrrhizin to
glycyrrhizin-knockout extract could improve the inhibition of iNOS expression
resulting that glycyrrhizin may exert combinational inhibition of iNOS expression
when coexisting with the other constituents contained in licorice extract.



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*Source: 260425-2012-02-14.xml*