1. Discovery and quantification of leptosperin as a unique component of manuka honey Yoji Kato 1 School of Human Science and Environment, University of Hyogo 2 Research Institute for Nutritional Sciences, University of Hyogo JAPAN 2016 Symposium This is Manuka Honey Auckland

2. 1. A History of Discovery of Leptosperin

3. My Relationship with NZ I had been in New Zealand to study science of free radicals at Free Radical Research Group, University of Otago Christchurch in 2006 to 2007. My project was that "oxidation of serotonin by an inflammatory enzyme, myeloperoxidase". Director of the laboratory was Professor Christine Winterbourn. I worked under the direction of Professor Tony Kettle. Professor Christine Winterbourn, who is the first woman awarded Rutherford Medal (2011) Professor Tony Kettle, who is current director of Centre for Free Radical Research

4. Photos in 2006 – 2007 As a research fellowThe campus, University of Otago CHCH Hagley Park

5. The reason of why I am here During my residence, I often had manuka honey on my toast. Then I have taken an interest in "mysterious" manuka honey, in particular, from the point of view of possible biological functions. I like this Larnach Castle Pancake RocksQueenstown Lake Tekapo

6. During Survey of Inhibitor of Myeloperoxidase … When I came back to Japan, I started on a study about manuka honey. We examined inhibitory effect of some honeys on myeloperoxidase in 2009. Among them, manuka honey showed the most strong inhibitory effect on the enzyme. We then identified a novel glycoside, leptosperin (leptosin), methyl syringe-  - D -gentiobioside, in early 2010 and reported the identification in J. Agric. Food Chem., in 2012. Himeji Castle Myeloperoxidase Leptosperin Methyl Syringate

7. Relationship with UMFHA We published a paper on leptosperin identification in 2012. I met with Mr. John Rawcliffe in June, 2012 in Tokyo for the first time. In 2013, I met with Mr. Rawcliffe again in NZ. I also visited a bee company at that time. In 2014, we have published two papers concerning leptosperin and I visited University of Auckland, Analytica, and Comvita.

8. Leptosperin in Leptospermum Honeys At first, we named "leptosin" for the compound but the name was already used. The fact was pointed-out by Dr. Ralf Schlothauer. Then we have called “leptosperin”, instead. We have found that the leptosperin is the speciality of honey originated from Leptospermum species. It could mean that leptosperin is a possible authentication marker for manuka honey.

9. 2. How to Measure?

10. As a Part of Authentication There are many ways to validate a honey; pollen analyses, chemical profiling of multiple components, and so on. Leptosperin is one of unique compounds in Leptospermum honeys. These honeys also contain methylglyoxal and dihydroxyacetone. The content of leptosperin (with other markers) should be an indicator of “purity” of the honey. The measurement of leptosperin is simple, and consumers are easy to understand the results.

11. An Example of Leptosperin Measurement for Authentication of Manuka Honey sample no. Authentication (UMF®, MGO™, etc.) Purchased inProduced in Leptosperin nmol/g honey B'Not certifiedJPNZ22 BNot certifiedNZ 164 The content is x 7.5 times difference Taste is quite different between the two. Manuka BB' Jars are completely the same. (but each has a different lot no.) Kato et al., JAFC 2014

12. Leptosperin is Stable Marker 37°C50°C Leptosperin is relatively stable during incubation even at 50°C. Because honey is sometimes warmed, it is an important character as a marker for manuka honey. Kato et al., JAFC 2014

13. HPLC-PDA Develosil ODS-HG-5, 4.6 x 150 mm (or Chromolith, 4.6 x 100 mm) Injection volume: 5 µl Detection: 262 nm or 278 nm (photo-diode array, PDA) Sol. A: 0.1% formic acid Sol. B: CH 3 CN Gradient Elution Honey 0.1g/ml in water → centrifuge or filtrate → supernatant (injection) Kato et al., JAFC 2014

14. Quadruple LC-MS/MS For detection of "trace" leptosperin in secondary bee/honey products such as a candy and a soap Kato et al., JAFC 2014

15. ELISA ✦ We have prepared a monoclonal antibody specific to leptosperin. ✦ The data obtained by ELISA shows good correlation with that by HPLC. around 5 hr / 24 samples = 12.5 min/sample Kato et al., JAFC 2014

16. STD curve Kato et al., Food Chem., 2015 Immunochromatography Leptosperin Antibody- colloidal gold Anti-mouse IgG Ab Leptosperin-BSA Time of development is 20 min

17. Fluorescence Detection ✓ Leptosperin has unique fluorescence (ex. 267 nm, em. 362 nm). (Bong et al., Food Chem. 2017) ✓ It is easy and low cost. ✓ We have found that some non-manuka honeys have non-specific fluorescence. ✓ It may be applicable to the 1st screening of leptosperin.

18. Summary of Methods You can choose appropriate method(s) depending on the situation. LC-MS/MS is the best way for analyses of trace amount of leptosperin. Immunochromatography and fluorescence methods are useful for on-site analyses, i.e., at beehives.SpeedEasyNumberLowCostAccuracySensitivityLC-MS/MS+++++ +++ HPLC++ + ++++ Immunochromatography +++++ ELISA +++++ + Fluorescence+++ +++++++

19. 3. Metabolism of Leptosperin and Methyl Syringate Collaboration with Dr. Ishisaka and Dr. Ikushiro Manuka

20. Possible Functions Manuka honey has been expected for beneficial functions. However, the potentials are not well examined except for anti-bacterial activity. Leptosperin is a unique compound in the honey. Therefore, it may explain why the honey has functions. In Japan, we have started a new system for functional food with health claim, which bases on contents of chemicals in food along with human study. To get a hit for these, we have to learn metabolism of leptosperin.

21. Possible Metabolic Pathway of Leptosperin Glycosidase Sulfotransferase UDP- Glucuronosyl transferase Leptosperin Methyl Syringate (MSYR) MSYR-GA MSYR-S Syringate (SYR) Functional metabolites??? Manuka

22. Time-dependent Changes in Profile of Metabolites When we eat manuka honey, sulfate and glucuronate conjugates of methyl syringate appeared in blood in 1 hr. These are excreted into urine in 4 hr. “Trace” leptosperin is found in urine. Urine Plasma confidential

23. Summary Leptosperin is a promising marker for honey from Leptospermum. The study on metabolism of leptosperin may give a hit for beneficial biological functions.

24. Acknowledgements Mr. John Rawcliffe (UMFHA) Dr. Jonathan Stephens Dr. Ralf Schlothauer Dr. Yasue Kikuzaki (identification of leptosperin) Dr. Akari Ishisaka (in vivo study) Dr. Shinichi Ikushiro (synthesis of metabolites) Dr. Hirohito Ishikawa & Mr. Yosuke Takimoto (advices) Students (laboratory): Asuna Maeda, Natsuki Umeda, Daiki Matsumoto, Yukako Araki, Rie Fujinaka, Maki Juri, Mie Takeuchi, Yui Yoshiki This is Photo with my laboratory members This is Photo with my laboratory members