Cross-reactivities (%) of MAb-6G8 against sennosides and other compounds.
1. Introduction
Recently, medical usage of Japanese traditional medicine has been expanded by reaching aging society and increasing various chronic diseases. Therefore, the demand of crude drugs prescribed for Japanese traditional medicine has been increased. However, over 90% of crude drugs are imported in our country, and those over 70% are supplied by the collection of wild species. It is well known that the natural resources bring the difficulty of quality control depending on collection season, cultivation place, a variety of species and so on. The other problem, shortage of crude drug comes up. For these general environment, micropropagation and clonal propagation systems using tissue and cell culture were investigated in this laboratry.
Sennoside A (SA) and B (SB) have the strong catharsis activity and contained in rhubarb and senna (Figure 1) [1]. The concentration of sennosides in rhubarb and senna is variously dependent on the genetic heterogeneity of species, differences in soil condition and climate influence. Sennosides are metabolized by intestinal bacteria to rheinanthrone which acts in the intestines as a direct purgatives [2, 3] and functions as similar to a natural prodrug (Figure 2). Despite the rising availability of a number of synthetic cathartics, sennoside- containing prescriptions are still among the most widely used today, and their importance is increasing.
Rhubarb, the rhizome and root of
Senna, the leaf and pod of
In the breeding research on the plant, a lot of stages are required as follows : dedifferentiation, extension of mutation by the mutagen, redifferentiation, analysis of the redifferentiated plant, mass propagation of the higher yielding plant and transplanting to soil. Therefore, it is very important to study a large number of plant samples in the phytochemical field and a small sample size
Recently, the immunological assay method is widely developed for the purpose of analysis for a small amount of constituent. In general immunological methodologies in particular enzyme-linked immunosorbent assay (ELISA) have promoted the development of higher sensitive assay system.
On the one hand, monoclonal antibodies (MAbs) have many potential uses in addition to immunological methods in plant sciences. MAbs are superior to polyclonal antibodies (PAbs) in the antigenic specificity and stability. Therefore, immunoassay using MAbs against pharmacologically active compound having small molecular weight has become an important tool for the studies on receptor binding analysis, enzyme assay and quantitative and/or qualitative analytical techniques in plants owing to its specific affinity, and possesses an extremely high possibility in the phytochemical analysis. Up to now, immunological approach for assaying quantities of sennosides in
Production of MAb against SA, its characterization and use for ELISA.
Production of MAbs against SB, their characterization and use for ELISA.
Establishments of a new eastern blotting, double staining and immunohistochemical staining using anti-SA and SB MAbs.
2. Production of MAb against SA, its characterization and use for ELISA
2.1. Preface
In the immunologically analytical methodology, there are two measuring methods using the antiserum (polyclonal antibody ; PAb) and MAb in general. PAb is a heterogeneous mixture of antibody molecules arising from a variety of constantly evolving B lymphocytes. Therefore, PAb can often show high affinity because different antibody populations react with the variety of epitopes that characterize the antigen. On the other hand, there are some problems of PAb that the extensive cross-reactivity occurs between the antibody and the multiple antigens which have the same antigenic determinant, and it is impossible to supply for identical antibody permanently. In the meantime, MAb is produced from a single B lymphocyte and can react with one antigenic determinant of the specific antigen. Besides MAb has identical specificity and affinity. There are some advantages that the complete purity of the immunized antigen is not required and the hybridoma cells can be preserved as freeze stock, and it is possible to get MAb depending on necessary respond.
There are several formats for ELISA like direct ELISA, competitive ELISA, sandwich ELISA and competitive ELISA according to the immune complexes formed during manipulation. Analysis of low molecular weight compound by immunoassay is still limited to competitive format.
Quality control of the Japanese herbal medicine is necessary because it is believed that approximately 70% of these crude drugs prescribed are collected from natural resource. Furthermore, since MAbs become necessary for the assay of concentrations of active constituents in our on-going plant biotechnological projects, we have already produced MAbs against natural compounds such as forskolin [6], solamargine [7], opium alkaloids [8], marihuana compounds [9], glycyrrhizin [10], crocin [11], ginsenoside Rb1 [12] and Rg1 [13], and developed individual competitive ELISAs. An immunological approach for assaying quantities of sennosides using a PAbs has been investigated by Atzorn
2.2. Experimental
2.2.1. Chemicals and immunochemicals
SA was purchased from Wako Pure Chemical Ind., Ltd. (Osaka, Japan). 1-Ethyl-3-(3'-dimethylaminopropyl)-carbodiimide HCl (EDC) was purchased from Nacalai Tesque Inc. (Kyoto, Japan). BSA and HSA were provided by Pierce (Rockford, IL, USA). Peroxidase-labeled anti-mouse IgG was provided by Organon Teknika Cappel Products (West Chester, PA, USA). Enriched RPMI1640-Dulbecco’s-Ham’s F12 (eRDF) medium and RD-1 additives (containing 9 μg/mL insulin, 20 μg/mL transferrin, 20 μM ethanolamine, 25 μM sodium selenite) were purchased from Kyokuto Pharmaceutical Industrial Co., Ltd. (Tokyo, Japan). Hypoxanthine-aminopterin-thymidine (HAT) additives were obtained from Sigma Chemical Company (St. Louis, MO, USA). Fetal calf serum (FCS) was purchased from Cambrex Corporation (Walkersville, MA, USA). All other chemicals were standard commercial products of analytical grade. Samples of various rhubarb roots were purchased from the Tochimototenkaido Corporation (Osaka, Japan).
2.2.2. Extraction of various rhubarb samples
Dried samples (30 mg) of various rhubarb roots were powdered, and then extracted five times with MeOH containing 0.1% (w/v) NH4OH (0.5 mL) with sonication, filtered using a Cosmonice Filter W (0.45 μm Filter Unit, Nacalai Tesque Inc., Kyoto, Japan), and the combined extracts were diluted with 10 mM NaHCO3 to prepare a solution suitable for the ELISA.
2.2.3. Synthesis of antigen conjugates
To SA (6 mg) dissolved in 1 mL of tetrahydrofuran-20 mM phosphate buffer of pH 5.5 (7:3), 0.3 mL of 20 mM phosphate buffer (pH 5.5) containing 6 mg of EDC was added. Then, 0.3 mL of 20 mM phosphate buffer (pH 5.5) containing 6 mg of BSA was added, with stirring at room temperature for 14 hr. The reaction mixture was dialyzed five times against H2O, and then lyophilized to give 5.8 mg of SA conjugate (SA-BSA). SA-HSA conjugate was also synthesized in the same manner.
2.2.4. Determination of hapten density in SA-carrier protein conjugate by matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF) mass spectrometry
The hapten number in the SA-carrier protein conjugate was determined by MALDI-TOF mass spectrometry as previously described [15]. A small amount (1-10 pmol) of antigen conjugate was mixed with a 103-fold molar excess of sinapinic acid in an aqueous solution containing 0.15% trifluoroacetic acid (TFA). The mixture was subjected to a JEOL Mass Spectrometers (JMS) time-of-flight (TOF) mass monitor (model Voyager Elite, PerSeptive Biosystems Inc., Framingham, MA, USA) and irradiated with a N2 laser (337 nm, 150 ns pulse). The ions formed by each pulse were accelerated by a 20 kV potential into a 2.0 m evacuated tube and detected using a compatible computer as previously reported [15].
2.2.5. Competitive ELISA for SA
SA-HSA (five molecules of SA per molecule of HSA) (100 μL, 1 μg/mL) dissolved in 50 mM carbonate buffer (pH 9.6) was adsorbed to the wells of a 96-well immunoplate then treated with 300 μL S-PBS for 1 hr to reduce non-specific adsorption. Fifty μL of various concentrations of SA or samples dissolved in 10 mM NaHCO3 solution were incubated with 50 μL of MAb solution (0.218 μg/mL) for 1 hr. The plate was washed three times with T-PBS, and then incubated with 100 μL of a 1:1000 dilution of POD-labeled anti-mouse IgG for 1 hr. After washing the plate three times with T-PBS, 100 μL of substrate solution [0.1 M citrate buffer (pH 4) containing 0.003% H2O2 and 0.3 mg/mL of ABTS] was added to each well and incubated for 15 min. The absorbance was measured by a micro plate reader at 405 nm and 490 nm.
The cross-reactivities (CR) of sennosides and related compounds were determined as following.
where
2.3. Results and discussion
2.3.1. Direct determination of SA-carrier protein conjugate by MALDI-TOF mass spectrometry
In general, the low molecular weight compounds (hapten) like plant secondary metabolite have no immunogenicity. Therefore, it should be conjugated with some high molecular compound like protein resulting in immunogenic. The specificity of immunoassay method is dependent on the site of linkage between hapten and carrier protein moiety, and enumeration of hapten in immunogen conjugate. SA-BSA and SA-HSA conjugates were synthesized as immunogen and the immobilization antigen for ELISA, respectively. Figure 3 shows the typical synthetic pathway of SA-BSA conjugate. The commonly used methods to link carboxyl group and amino group in a hapten or carrier involve activation by carbodiimides, isobutylchloroformate or carbonyldiimidazole. Carbodiimides react with carboxyl groups to form an unstable
Figure 4 shows the MALDI-TOF mass spectrum of the antigen, SA-BSA conjugate. A broad peak coinciding with the conjugate of SA and BSA appeared from
2.3.2. Production and characteristic of MAb against SA
After the cell fusion and HAT selection, hybridoma producing MAb reactive to SA was obtained, and classfied into IgG1 which had
2.3.3. Assay sensitivity and assay specificity
The free MAb 6G8, following incubation with competing antigen, was bound to the polystyrene microtitre plates precoated with SA-HSA. Under these conditions, the full measuring range of the assay extended from 20 to 200 ng/mL as indicated in Figure 5.
SA is a unique anthraquinone having individual double of carboxylic acid-, hydroxyl-, carbonyl- and
Table 1 indicates the cross-reactivities of anti-SA MAb against related anthraquinone, anthrone and phenol carboxylic acid. MAb 6G8 cross-reacted with rhein and SB weakly; 0.28 and 0.35%, respectively. However, the other related anthraquinone and anthrone did not have appreciable cross-reactivities. From these results it is suggested that a basal structure of rhein and sugar moiety caused immunization. In addition the most important property of MAb 6G8 is an ability of stereochemical recognition because the differences of structure between SA and SB are only the stereochemical configuration at the C-10 and C-10' positions. Therefore, it is suggested that
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sennoside A | 100 |
sennoside B | 0.28 |
rhein | 0.35 |
emodin | < 0.04 |
aloe-emodin | < 0.04 |
barbaloin | < 0.04 |
1,4-dihydroxy-anthraquinone | < 0.04 |
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rhaponticin | < 0.04 |
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gallic acid | < 0.04 |
vanillic acid | < 0.04 |
caffeic acid | < 0.04 |
homogentisic acid | < 0.04 |
2.3.4. Correlation of results of SA determination in crude extracts of rhubarb roots between HPLC and ELISA using MAb 6G8
The ELISA was utilized to measure the concentrations of SA in various rhubarb (Table 2). Oshio and Kawamura determined sennoside concentrations in various crude rhubarbs by HPLC [17]. More recently Seto
Table 2 shows the SA concentrations in various rhubarbs. Shinshu Daio bred by crossing
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Shinshu Daio | 13.69±0.69 | 12.28±0.41 |
Ga-wo | 6.62±0.42 | 6.93±0.02 |
Kinmon Daio | 3.34±0.02 | 0.85±0.04 |
Itto-Ga-wo (powder) | 3.27±0.20 | 3.69±0.32 |
Itto-Ga-wo (refuse) | 3.43±0.16 | 3.69±0.28 |
3. Production of MAbs against SB, their characterization and use for ELISA
3.1. Preface
SB is a very important natural bioactive component of rhubarb and senna as well as SA. Total sennoside (SA and SB) concentrations are important, when rhubarb and senna are used as a raw material of medical supply and traditional Japanese herbal medicine for the purgative effect.
A number of methods for the quantification of SB have been published, most of which have been performed by HPLC [17]. Immunological approaches for assaying quantities of sennosides and SA using PAb and MAb have been investigated by Atzorn
3.2. Experimental
3.2.1. Plant materials
Samples of various rhubarb roots were purchased from the Tochimototenkaido Corporation (Osaka, Japan). Samples of leaves of
3.2.2. Sample preparation
Dried samples (30 mg) of various rhubarb roots,
3.2.3. Synthesisi of antigen conjugates
To SB (6 mg) dissolved in 1 mL of tetrahydrofuran-20 mM phosphate buffer of pH 5.5 (7:3), 0.3 mL of 20 mM phosphate buffer (pH 5.5) containing 6 mg of EDC was added. Then, 0.3 mL of 20 mM phosphate buffer (pH 5.5) containing 6 mg of BSA was added, with stirring at room temperature for 14 hr. The reaction mixture was dialyzed five times against H2O, and then lyophilized to give 5.5 mg of SB-BSA conjugate. SB-HSA conjugate was also synthesized in the same manner.
3.2.4. Determination of hapten density in SB-carrier protein conjugate by MALDI-TOF mass spectrometry
The hapten number in the SB-carrier protein conjugate was determined by MALDI-TOF mass spectrometry as previously described [15].
3.2.5. Competitive ELISA for SB
SB-HSA (four molecules of SB per molecule of HSA) (100 μL, 1 μg/mL) dissolved in 50 mM carbonate buffer (pH 9.6) was adsorbed to the wells of a 96-well immunoplate then treated with 300 μL S-PBS for 1 hr to reduce non-specific adsorption. Fifty μL of various concentrations of SB or samples dissolved in 10 mM NaHCO3 solution were incubated with 50 μL of MAb solution (0.121 μg/mL) for 1 hr. The plate was washed three times with T-PBS, and then incubated with 100 μL of a 1:1000 dilution of POD-labeled anti-mouse IgG for 1 hr. After washing the plate three times with T-PBS, 100 μL of substrate solution [0.1 M citrate buffer (pH 4) containing 0.003% H2O2 and 0.3 mg/mL of ABTS] was added to each well and incubated for 15 min. The absorbance was measured by a micro plate reader at 405 nm and 490 nm.
3.3. Results and discussion
3.3.1. Direct determination of SB-carrier protein conjugate by MALDI-TOF mass spectrometry
It is well known that hapten number in an antigen conjugate is important for immunization against low molecular weight compounds. Figure 7 shows the MALDI-TOF mass spectrum of the antigen, SB-BSA conjugate. A broad peak coinciding with the conjugate of SB and BSA appeared from
3.3.2. Production and characteristics of Mabs against SB
The immunized BALB/c mice yielded splenocytes which were fused with P3-X63-Ag8-653 myeloma cells by the routinely established procedure in this laboratory [6]. Hybridoma producing MAbs reactive to SB were obtained, and classified as IgG1 (5G6, 7H12) and IgG2b (5C7) which had
3.3.3. Assay sensitivity and assay specificity
The free MAb 7H12 following competition was bound to the polystyrene microtitre plates precoated with SB-HSA. Under these conditions, the full measuring range of the assay extends from 0.5 ng/mL to 15 ng/mL as indicated in Figure 8 and the ELISA using a MAb 7H12 is more sensitive than those using MAb 5C7 and 5G6.
SB is a unique anthraquinone having individual double-carboxylic acid-, hydroxyl-, carbonyl- and
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sennoside B | 100 | 100 | 100 |
sennoside A | 2.45 | 2.30 | 8.53 |
rhein | 0.012 | 0.030 | 0.007 |
emodin | < 0.004 | < 0.023 | < 0.006 |
aloe-emodin | < 0.040 | < 0.023 | < 0.006 |
barbaloin | < 0.004 | < 0.023 | < 0.006 |
1,4-dihydroxy-anthraquinone | < 0.004 | < 0.023 | < 0.006 |
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rhaponticin | < 0.004 | < 0.023 | < 0.006 |
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gallic acid | < 0.004 | < 0.023 | < 0.006 |
vanillic acid | < 0.004 | < 0.023 | < 0.006 |
caffeic acid | < 0.004 | < 0.023 | < 0.006 |
homogentisic acid | < 0.004 | < 0.023 | < 0.006 |
3.3.4. Correlation of results of SB determination in crude extracts of rhubarb roots between HPLC and ELISA using MAb 7H12
The concentrations of SB in various rhubarb samples were determined by ELISA (Table 4). Shinshu Daio, bred by crossing
Shinshu Daio | 6.01±0.18 | 6.15±0.59 |
Ga-wo | 3.14±0.27 | 3.80±0.16 |
Kinmon Daio | 0.35±0.01 | 0.38±0.02 |
Itto-Ga-wo (powder) | 1.44±0.12 | 1.52±0.18 |
Itto-Ga-wo (refuse) | 1.42±0.07 | 1.40±0.11 |
3.3.5. Determination of concentrations of SA and SB in various Cassia species
The concentrations of SA and SB in leaves of various
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4.56±0.25 | 5.10±0.15 | 9.66±0.40 |
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1.19±0.12 | 1.16±0.15 | 2.35±0.27 |
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0.40±0.03 | 0.44±0.02 | 0.84±0.05 |
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1.14±0.08 | 0.75±0.08 | 1.89±0.16 |
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2.04±0.32 | 1.52±0.12 | 3.56±0.44 |
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1.90±0.16 | 2.05±0.24 | 3.95±0.40 |
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0.10±0.01 | 0.13±0.00 | 0.23±0.01 |
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(1.30±0.24)×10-2 | (1.88±0.29)×10-4 | (1.32±0.24)×10-2 |
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(2.78±0.11)×10-3 | (1.04±0.03)×10-4 | (2.88±0.11)×10-3 |
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(1.15±0.18)×10-2 | (2.44±0.17)×10-4 | (1.17±0.18)×10-2 |
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(2.13±0.21)×10-3 | (3.64±0.21)×10-5 | (2.17±0.23)×10-3 |
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(4.45±0.14)×10-3 | (1.87±0.13)×10-3 | (6.32±0.27)×10-3 |
4. Establishments of a new eastern blotting, double staining and immunohistochemical staining using anti-SA and SB MAbs
4.1. Preface
Thin-layer chromatography (TLC) is most widely used for detection, separation and monitoring of small molecular compounds like sennosides. If the direct TLC immunostaining with MAb can be done, this procedure must be contributive to the development of structural analysis of small molecular compounds. However, this procedure cannot be used for the direct detection of small molecular compounds on a TLC plate because the silica gel is sloughed off from the plate and the compounds on the plate are easily washed out without fixing during treatment. If the compounds are transferred from the TLC plate to a plastic membrane with hydrophobic properties and immobilized on the membrane, these difficulties can be solved. Therefore, I examined the transfer of sennosides from a TLC plate to a plastic membrane. Towbin
4.2. Experimental
4.2.1. Chemicals and immunochemicals
Polyvinylidene difluoride (PVDF) membranes (Immobilon-N) were purchased from Millipore Corporation (Bedford, MA, USA). Glass microfiber filter sheets (GF/A) were purchased from Whatman International Ltd. (Maidstone, England). All other chemicals were standard commercial products of analytical grade.
4.2.2. EB and Double staining
Sennosides were applied to a TLC plate and developed with 1-propanol-ethyl acetate-water-acetic acid (40:40:30:1, by volume). The developed TLC plate was dried and then sprayed with a blotting solution mixture of isopropanol-methanol-water (1:4:16, by volume). It was placed on a stainless steel plate and then covered with a PVDF membrane sheet. After covering with a glass microfiber filter sheet, the whole assembly was pressed evenly for 70 s with a 120 ˚C hot plate as previously described with some modifications [24, 25]. The PVDF membrane was separated from the TLC plate and dried.
The blotted PVDF membrane was dipped in 20 mM carbonate buffer solution (pH 9.6) containing BSA (1%) and EDC (20 mg/mL), and stirred at room temperature for 14 hr. After washing the PVDF membrane twice with T-PBS for 5 min and then treated with S-PBS for 3 hr to reduce non-specific adsorption. The PVDF membrane was washed with T-PBS twice for 5 min, and then immersed in anti-SA MAb (6G8) and stirred at room temperature for 3 hr. After washing the PVDF membrane twice with T-PBS for 5 min, a 1:1000 dilution of POD-labeled goat anti-mouse IgG in PBS cotaining 0.2% of gelatin (G-PBS) was added and stirred at room temperature for 1 hr. The PVDF membrane was washed twice with T-PBS and water, then exposed to 1 mg/mL 4-chloro-1-naphtol-0.03% H2O2 in PBS solution which was freshly prepared before use for 10 min at room temperature. The protocol of the EB technique is shown in Figure 9.
For successive staining by anti-SB MAb (7H12), the PVDF membrane stained by anti-SA MAb was treated in the same way as anti-SA MAb (6G8) except that it was exposed to 2 mg/10 mL 3-amino-9-ethylcarbazole-0.03% H2O2 in acetate buffer (0.05 M, pH 5.0) containing 0.5 mL of
4.2.3. EB for immunohistochemical staining of SA
A piece of PVDF membrane was placed on a glass microfiber filter sheet. A sliced fresh rhubarb root was placed on the PVDF membrane, and they were pressed together evenly for 1 hr. The blotted PVDF membrane was stained using the same procedure described for the EB method.
4.3. Results and discussion
4.3.1. EB of SA using anti-SA MAb
Previously we established a new immunostaining method named as eastern blotting for several glycosides like solasodine glycosides [21], ginsenosides [26, 27] and glycyrrhizin [22, 28] by using individual MAbs. In this methodology we separated the sugar moiety in a molecule into two functions, the epitope part and fixation ability part on a membrane after blotted to a PVDF membrane from a TLC plate, since small molecular compounds can not be fixed on the membrane. Although I followed the previous methodology for SA, unfortunately staining was not succeeded. Therefore, a new blotting method onto a PVDF membrane from the developed TLC plate is required. SA was transferred to the PVDF membrane by the same way as previously described, and treated with EDC solution followed by the addition of BSA as indicated in Figure 9. This reaction enhanced the fixation of SA via SA-BSA conjugate on the PVDF membrane and the pathway was indicated diagrammatically in Figure 11. When the blotted PVDF membrane was incubated in the absence of EDC, it was essentially free of immunostaining (data not shown).
Figure 12 shows the EB of sennosides and other structurally related compounds using anti-SA MAb (A) and the H2SO4 staining (B). The EB indicated only limited staining of SA as shown in Figure 12A, lane 7. Moreover, the EB method was considerably more sensitive than that of H2SO4 staining. Since anti-SA MAb cross-reacts against SB and rhein as 0.28 and 0.35%, respectively, they can be stained very weakly by anti-SA MAb, as described in the previous section. Previously Fukuda
4.3.2. Double staining of sennosides using anti-SA and SB MAbs
Previously, I used 4-chloro-1-naphthol for staining of SB. However, since it could not function well for SB, the combination of 4-chloro-1-naphthol and 3-amino-9-ethylcarbazole was selected to improve double staining of sennosides as indicated in Figure 10. SA and SB were stained clearly by the purple and red color, respectively (Figure 13). From this result both antibodies can distinguish stereochemical configurations,
4.3.3. Detection of SA and SB in various Cassia species using double staining with a new EB technique
The crude extracts of various
4.3.4. Validation of EB for immunohistochemical staining of SA
As an other application of the EB method, the immunohistochemical staining of SA in rhubarb root, was investigated. A sliced fresh rhubarb root was placed on the PVDF membrane, and they were pressed together evenly for 1 hr. The blotted PVDF membrane was stained using the same procedure described for the EB method. Figure 15II illustrates the immunohistochemical staining of SA in fresh Hokkai Daio root. The phloem and cambium contained a higher concentration of SA compared to other tissues, pith and bud. To confirm this result, I analyzed these tissues individually by ELISA and HPLC. The concentrations of SA were determined by ELISA to determine 64.4±4.5, 48.1±8.2, 15.0±1.6 and 1.8±0.3 ng/mg fresh wt. in phloem, cambium, pith and bud, respectively. This result was a good agreement with those of HPLC resulting in 58.4±2.6, 49.0±3.9 and 13.3±0.5 ng/mg fresh wt. in phloem, cambium and pith, respectively.
5. Conclusion
The recent developments of molecular biosciences and their biotechnological applications have opened up many new avenues of pharmaceutical areas. MAbs have many potential uses in addition to immunological methods to plant sciences. Therefore, immunoassay system using MAbs against pharmacologically active natural products having low molecular weight have become an important tool for the studies on receptor binding analysis, enzyme assay, and quantitative and/or qualitative analytical techniques in plants owing to their specific affinity.
In order to analyze the stereochemical isomers, SA and SB in plants, medicaments, prescriptions, health foods and patients’sera, I have produced MAbs against them. These MAbs have the most important ability to distinguish between SA and SB, which differ only in the stereochemical configuration at the C-10 and C-10’ positions, respectively. Moreover, they have no detectable cross-reaction with the other related anthraquinone and anthrone.
Analytical systems of SA and SB by competitive ELISA using anti-SA and SB MAbs were established. These ELISA systems are capable of measuring SA and SB in complex matrics without any pretreatments. Furthermore, these ELISA methods are approximately 2,000 times for SA and 10,000 times for SB more sensitive than that of HPLC method.
The newly developed EB methodology can be theoretically expanded for all compounds having carboxylic acid such as phenol carboxylic acids, glucuronides, furthermore compounds having only a carboxylic group in a molecule. A new double staining with EB method for sennosides using anti-SA and SB MAbs was established. SA and SB were stained purple and red color, respectively. This system visualized sennosides on a PVDF membrane. In fact, SA and SB in the crude extracts of various
Acknowledgments
We thank Dr. Hiroyuki Tanaka (Faculty of Pharmaceutical Sciences, Kyushu University) for useful suggestions in this work. This research was supported in part by Japan Science and Technology Agency, Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan, the research grant from Takeda Science Foundation.
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