Development of eastern blotting technique for sennoside A and sennoside B using anti-sennoside A and anti-Sennoside B monoclonal antibodies

Osamu Morinaga, Takuhiro Uto, Seiichi Sakamoto, Waraporn Putalun, Sorasak Lhieochaiphant, Hiroyuki Tanaka, Yukihiro Shoyama

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

Introduction - Rhubarb, senna and sennoside-containing preparations are currently widely employed as purgatives. The major active components of these medications are sennoside A (SA) and sennoside B (SB). Objective - To develop an eastern blotting technique for the specific visualisation and easy determination of SA and SB in plant extracts for application in the standardisation and authentication of rhubarb and senna. Methodology - SA and SB were separated by TLC, transferred to a PVDF membrane, treated with 1-ethyl-3-(3′-dimethylaminopropyl)-carbodiimide hydrochloride solution and finally treated with bovine serum albumin (BSA). The resulting membrane-bound SA-BSA and SB-BSA conjugates were linked to anti-SA and anti-SB monoclonal antibodies (MAbs) and then to secondary antibodies labelled with peroxidase. SA and SB were detected by visualisation of the peroxidase reaction products. Results - The limit of detection of the eastern blotting was 62.5 ng for both sennosides. The method was applied to the immunohistochemical localisation of SA in fresh rhubarb root. Phloem and radiate wood were found to contain higher concentrations of SA compared with other tissues (pith and bud) in agreement with results obtained by ELISA. The concentrations of SA in the phloem, radiate wood, pith and bud were 64.4, 48.1, 15.0 and 1.8 ng/mg fresh weight, respectively. Conclusion - The technique described permitted the visualisation of small molecular weight compounds that had been bound to a membrane, using immunostaining. Owing to the specificity of the MAbs, the eastern blotting may prove to be a useful method for the identification of SA and SB in a background containing large amount of impurities.

Original languageEnglish
Pages (from-to)154-158
Number of pages5
JournalPhytochemical Analysis
Volume20
Issue number2
DOIs
Publication statusPublished - Jun 1 2009

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rhubarb
monoclonal antibodies
Monoclonal Antibodies
bovine serum albumin
pith
phloem
peroxidase
buds
laxatives
methodology
Rheum
standardization
plant extracts
drug therapy
Bovine Serum Albumin
detection limit
Phloem
sennoside A&B
enzyme-linked immunosorbent assay
molecular weight

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry
  • Food Science
  • Biochemistry
  • Molecular Medicine
  • Plant Science
  • Drug Discovery
  • Complementary and alternative medicine

Cite this

Development of eastern blotting technique for sennoside A and sennoside B using anti-sennoside A and anti-Sennoside B monoclonal antibodies. / Morinaga, Osamu; Uto, Takuhiro; Sakamoto, Seiichi; Putalun, Waraporn; Lhieochaiphant, Sorasak; Tanaka, Hiroyuki; Shoyama, Yukihiro.

In: Phytochemical Analysis, Vol. 20, No. 2, 01.06.2009, p. 154-158.

Research output: Contribution to journalArticle

Morinaga, Osamu ; Uto, Takuhiro ; Sakamoto, Seiichi ; Putalun, Waraporn ; Lhieochaiphant, Sorasak ; Tanaka, Hiroyuki ; Shoyama, Yukihiro. / Development of eastern blotting technique for sennoside A and sennoside B using anti-sennoside A and anti-Sennoside B monoclonal antibodies. In: Phytochemical Analysis. 2009 ; Vol. 20, No. 2. pp. 154-158.
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AU - Sakamoto, Seiichi

AU - Putalun, Waraporn

AU - Lhieochaiphant, Sorasak

AU - Tanaka, Hiroyuki

AU - Shoyama, Yukihiro

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AB - Introduction - Rhubarb, senna and sennoside-containing preparations are currently widely employed as purgatives. The major active components of these medications are sennoside A (SA) and sennoside B (SB). Objective - To develop an eastern blotting technique for the specific visualisation and easy determination of SA and SB in plant extracts for application in the standardisation and authentication of rhubarb and senna. Methodology - SA and SB were separated by TLC, transferred to a PVDF membrane, treated with 1-ethyl-3-(3′-dimethylaminopropyl)-carbodiimide hydrochloride solution and finally treated with bovine serum albumin (BSA). The resulting membrane-bound SA-BSA and SB-BSA conjugates were linked to anti-SA and anti-SB monoclonal antibodies (MAbs) and then to secondary antibodies labelled with peroxidase. SA and SB were detected by visualisation of the peroxidase reaction products. Results - The limit of detection of the eastern blotting was 62.5 ng for both sennosides. The method was applied to the immunohistochemical localisation of SA in fresh rhubarb root. Phloem and radiate wood were found to contain higher concentrations of SA compared with other tissues (pith and bud) in agreement with results obtained by ELISA. The concentrations of SA in the phloem, radiate wood, pith and bud were 64.4, 48.1, 15.0 and 1.8 ng/mg fresh weight, respectively. Conclusion - The technique described permitted the visualisation of small molecular weight compounds that had been bound to a membrane, using immunostaining. Owing to the specificity of the MAbs, the eastern blotting may prove to be a useful method for the identification of SA and SB in a background containing large amount of impurities.

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