Malays. J. Anal. Sci. Volume 30 Number 1 (2026): 1704

 

Research Article

 

Preparation and characterisation of an anti-benzylacetone monoclonal antibody and its use for histochemical analysis of trans-cinnamic acid in Cinnamomum cassia branches using the dot blot method

 

Kakuyou Ogawa and Osamu Morinaga*

 

Faculty of Pharmacy, Daiichi University of Pharmacy; 22-1 Tamagawa-cho, Minami-ku, Fukuoka 815-8511, Japan

 

*Corresponding author: o-morinaga@daiichi-cps.ac.jp

 

Received: 22 September 2025; Revised: 25 December 2025; Accepted: 5 January 2026; Published: 28 February 2026

 

Abstract

The pharmacological effects of various inhaled aroma compounds have been investigated, such as benzylacetone used as a positive control in behavioural tests. To develop a monoclonal antibody (MAb) for detecting and quantifying the aroma compound benzylacetone in sera obtained from experimental animals, immunogenic conjugates were synthesised using 3-phenylpropanoic acid (3PPa), a benzylacetone-like compound coupled to carrier proteins. Mice were immunised with 3PPa- keyhole limpet haemocyanin, and a MAb-secreting hybridoma was obtained. Cross-reactivities and detection limits were determined for 36 compounds by enzyme-linked immunosorbent assay (ELISA). Although the MAb cross-reacted with several phenylpropanoids, alkaloids, and flavonoids, its detection limits (> 0.1 μg/mL) were unsuitable for ELISA. However, the MAb successfully visualised trans-cinnamic acid and 3PP using the dot blot method. The MAb strongly immunostained trans-cinnamic acid localisation in cinnamon bark. Therefore, this anti-benzylacetone MAb may be a powerful tool for visualising the biosynthesis and distribution of these phenylpropanoids in biological samples.

 

Keywords: trans-cinnamic acid, monoclonal antibody, immunostaining

 

References

1.      Castro, K. P., & Ito, M. (2021). Individual and combined inhalational sedative effects in mice of low molecular weight aromatic compounds found in agarwood aroma. Molecules, 26, 1320.

2.      Tankam, J. M., & Ito, M. (2014). Sedative, anxiolytic and antidepressant-like effects of inhalation of the essential oil of Ocimum gratissimum L. from Cameroon in mice. Journal of Pharmacognosy and Phytochemistry, 2, 1–9.

3.      Ogawa, K., & Ito, M. (2016). Appetite-enhancing effects of trans-cinnamaldehyde, benzylacetone and 1-phenyl-2-butanone by inhalation. Planta Medica, 82, 84–88.

4.      Takamatsu, S., & Ito, M. (2018). Agarotetrol: A source compound for low molecular weight aromatic compounds from agarwood heating. Journal of Natural Medicines, 72, 537–541.

5.      Morita, I., Oyama, H., Yasuo, M., Matsuda, K., Katagi, K., Ito, A., Tatsuda, H., Tanaka, H., Morimoto, S., & Kobayashi, N. (2017). Antibody fragments for on-site testing of cannabinoids generated via in vitro affinity maturation. Biological and Pharmaceutical Bulletin, 40, 174–181.

6.      Ohashi, S., Nagatomo, K., Miyamoto, K., Toko, K., Miura, N., & Matsumoto, K. (2010). Preparation of anti-fragrant monoclonal antibodies by the rat lymph node method and their characterization using enzyme-linked immunosorbent assay. Journal of the Faculty of Agriculture, Kyushu University, 55, 91–96.

7.      National Institute of Standards and Technology. (2018). 2-Butanone, 4-phenyl-. https://webbook.nist.gov/cgi/cbook.cgi?ID=C2550267

8.      Morinaga, O., Tanaka, H., & Shoyama, Y. (2000). Production of monoclonal antibody against a major purgative component, sennoside A, its characterization and ELISA. Analyst, 125, 1109–1113.

9.      Galfrθ, G., & Milstein, C. (1981). Preparation of monoclonal antibodies: Strategies and procedures. Methods in Enzymology, 73, 3–46.

10.   Weiler, E. W., & Zenk, M. H. (1976). Radioimmunoassay for determination of digoxin and related compounds in Digitalis lanata. Phytochemistry, 15, 1537–1545.

11.   Morinaga, O., Uto, T., Sakamoto, S., Putalun, W., Lhieochaiphant, S., Tanaka, H., & Shoyama, Y. (2009). Development of eastern blotting technique for sennoside A and sennoside B using anti-sennoside A and anti-sennoside B monoclonal antibodies. Phytochemical Analysis, 20, 154–158.

12.   Parada, F., & Duque, C. (1998). Studies on the aroma of piρuela fruit pulp (Bromelia plumieri): Free and bound volatile composition and characterization of some glucoconjugates as aroma precursors. Journal of High Resolution Chromatography, 21, 577–581.

13.      Xuan, L. J., Tanaka, H., Xu, Y. M., & Shoyama, Y. (1999). Preparation of monoclonal antibody against crocin and its characterization. Cytotechnology, 29, 65–70.

14.      Lu, Z. H., Morinaga, O., Tanaka, H., & Shoyama, Y. (2003). A quantitative ELISA using monoclonal antibody to survey paeoniflorin and albiflorin in crude drugs and traditional Chinese herbal medicines. Biological and Pharmaceutical Bulletin, 26, 862–866.

15.      Ogawa, K., Honda, M., Tanigawa, A., Hatase, A., Ito, A., Higa, Y., & Morinaga, O. (2020). Appetite-enhancing effects of inhaling cinnamon, clove, and fennel essential oils containing phenylpropanoid analogues. Journal of Natural Medicines, 74, 710–721.

16.      Morinaga, O., Nakajima, S., Tanaka, H., & Shoyama, Y. (2001). Production of monoclonal antibodies against a major purgative component, sennoside B, their characterization and use in ELISA. Analyst, 126, 1372–1376.

17.      Morinaga, O., Uto, T., Yuan, C. S., Tanaka, H., & Shoyama, Y. (2010). Evaluation of a new eastern blotting technique for the analysis of ginsenoside Re in American ginseng berry pulp extracts. Fitoterapia, 81, 284–288.

18.      Morinaga, O., Tanaka, H., & Shoyama, Y. (2006). Enzyme-linked immunosorbent assay for the determination of total ginsenosides in ginseng. Analytical Letters, 39, 287–296.

19.      Morikawa, A., Hamase, K., Ohgusu, T., Etoh, S., Tanaka, H., Koshiishi, I., Shoyama, Y., & Zaitsu, K. (2007). Immunohistochemical localization of D-alanine to β-cells in rat pancreas. Biochemical and Biophysical Research Communications, 355, 872–876.

20.      Li, X. W., Morinaga, O., Tian, M., Uto, T., Yu, J., Shang, M. Y., Wang, X., Cai, S. Q., & Shoyama, Y. (2013). Development of an eastern blotting technique for the visual detection of aristolochic acids in Aristolochia and Asarum species by using a monoclonal antibody against aristolochic acids I and II. Phytochemical Analysis, 24, 645–653.

21.      Osorio, C., Duque, C., Suαrez, M., Salamanca, L. E., & Urueρa, F. (2002). Free, glycosidically bound, and phosphate-bound flavor constituents of badea (Passiflora quadrangularis) fruit pulp. Journal of Separation Science, 25, 147–154.

22.      Sebai, H., Rtibi, K., Selmi, S., Jridi, M., Balti, R., & Marzouki, L. (2019). Modulating and opposite actions of two aqueous extracts prepared from Cinnamomum cassia L. bark and Quercus ilex L. on the gastrointestinal tract in rats. RSC Advances, 9, 21695–21706.

23.      Fan, W. L., & Qian, M. C. (2006). Characterization of aroma compounds of Chinese “Wuliangye” and “Jiannanchun” liquors by aroma extract dilution analysis. Journal of Agricultural and Food Chemistry, 54, 2695–2704.

24.      Prasad, K. N., Yang, B., Dong, X. H., Jiang, G. X., Zhang, H. Y., Xie, H. H., & Jiang, Y. M. (2009). Flavonoid contents and antioxidant activities from Cinnamomum species. Innovative Food Science and Emerging Technologies, 10, 627–632.

25.      He, Z. D., Qiao, C. F., Han, Q. B., Cheng, C. L., Xu, H. X., Jiang, R. W., But, P. P. H., & Shaw, P. C. (2005). Authentication and quantitative analysis on the chemical profile of cassia bark (Cortex Cinnamomi) by high-pressure liquid chromatography. Journal of Agricultural and Food Chemistry, 53, 2424–2428.

26.      Lee, J. M., Lee, D. G., Park, J. Y., Chae, S. W., & Lee, S. H. (2015). Analysis of the trans-cinnamic acid content in Cinnamomum spp. and commercial cinnamon powder using HPLC. Journal of Agricultural Chemistry and Environment, 4, 102–108.

27.      Hsu, K. H., Huang, W. K., Lin, Y. L., Chang, S. T., & Chu, F. H. (2012). A genetic marker of 4-coumarate: Coenzyme A ligase gene in the cinnamaldehyde-chemotype Cinnamomum osmophloeum. Holzforschung, 66, 897–904.

28.      Gao, H. Y., Xu, D. Y., Zhang, H. J., Qian, J. P., and Yang, Q. (2020). Transcriptomics and metabolomics analyses reveal the differential accumulation of phenylpropanoids between Cinnamomum cassia Presl and Cinnamomum cassia Presl var. macrophyllum Chu. Industrial Crops and Products, 148, 112282.

29.      Eilerman, R. G. (2020). Cinnamic acid, cinnamaldehyde, and cinnamyl alcohol. In Kirk-Othmer encyclopedia of chemical technology (p. 5). John Wiley & Sons.