Hydroxy-alpha-sanshool

David Schlander, Chiara Schleif, Maxim Schnell, Paul Roumeliotis

1. General information

Hydroxy-alpha-sanshool is a light brown solid that belongs to the class of organic compounds called n- 1 acyl amines. Its IUPAC Name is (2E, 6Z, 8E, 10E)-N-(2-hydroxy-methylpropyle)dodeca-2,6,8,10- tetraenamide but it is mostly called its synonym.2

Hydroxy Alpha-Sanshool is a long, mostly unbranched molecule with several conjugated double bounds. It has an amide group and a terminal tertiary hydroxy group.

• Molar mass: 263,381 g/mol

• Molecular formula: C16H25NO2 • Melting point: not known

Figure 1: Structure of hydroxy-alpha-sanshool. 1.1. The occurrence

It is found in Lychees and plants from the genus Zanthoxylum which are mainly called Sichuan pepper and that is where it got its name from: the Japanese name of the pepper is „Sansho“ which developed to „Sanshool“ in the synonym because of the hydroxy group the molecule has.3 Sichuan Pepper grows in the Asian part of the world. Its fruit is used as a spice because of the tingly and numbing feeling it causes in the mouth. The reason for this phenomenon is hydroxy-alpha-sanshool, that is what makes this molecule interesting.2,3asd4

Figure 2: Dried Sichuan pepper.4 1.2. Extraction methods

To isolate the molecule from the pepper in form of an extract, steam distillation can be used: Dried peels of the fruit are immersed in a mixture of lower alcohols (for example ethanol) and water with a mass percentage between 35-65% of the alcohol. The solution gets heated up in the process of steam distillation where the aqueous part evaporates and takes parts of the hydroxy-alpha- sanshool up, too. The distillate separates in two phases: the aqueous ethanol phase and the oil phase which contains the 5,6 desired molecule.

Problem of this extraction method is the low yield which is most times below 60%.5,7

Figure 3: Schematic diagram of steam distillation.7

2. Synthesis of Hydroxy-alpha-Sanshool

Because of the low yield in extraction methods, different ways to synthesize Hydroxy-alpha-Sanshool got developed to reach a bigger amount of the molecule. The total synthesis shown below is just one of the different possibilities to synthesize Hydroxy-alpha-Sanshool. The wanted product 1 can be synthesized by SUZUKI-MIYUANA-coupling between N-methyliminodiacetic acid (MIDA) boronate 3 and bromoalkyne 4 followed by (Z)-selective reduction of the triple bond of the coupling product 10. The MIDA-boronate 3 can be prepared out of trans-2-bromovinylboronic acid MIDA ester and trans-1-Propen-1-ylboronic acid by SUZUKI-MIYUANA-coupling, bromoalkyne 4 is synthesized from 2-Hydroxy-2-methylpropylamine 6 and Hept-2-en-6-ynoic acid 9 by condensation and following bromination. 9 can be prepared out of 4-pentyn- 1-ol by SWERN-oxidation followed by WITTIG-reaction and hydrolysis of the resulting ester. 6 is the product of addition between 1,2-epoxy-2- methylpropane 5 and dibenzyl amine followed by debenzylation.8

Figure 4: Retroynthesis of Hydroxy-alpha-Sanshool.8

Figure 5: Preparation of fragment 3.8

Figure 6: Synthesis of hydroxy-alpha-sanshool.8

4

Figure 7: Preparation of fragment 4.8

3. Application

Hydroxy-alpha-Sanshool is used as a component in pain management and as a spice ingredient in specific Asian foods.1,9 It is also used as an addition in oral compounds for producing saliva in the mouth. As an advance, already about 0.1% concentration is sufficient for a strong salivating effect.10

Another possible use of Hydroxy-alpha-sanshool is cancer treatment. Several studies suggest, zanthoxylum fruits, leaves, bark, and roots can exert notable anticancer activity. Most of the studies are limited on in vitro studies against various neoplastic cell lines. For example, several alkamides were tested against tumour cell lines in mice. Four of the ten alkamides tested resulted in >50% growth inhibition. However only hydroxy-a-sanshool did so without negatively impacting cell viability relative to untreated cells, as shown in Table 1.11

Table 1: Growth inhibition and cell viability of cells treated with different alkylamides.11

Alkylamide Growth inhibition cell viability

Hydroxy-α-sanshool 53 % 106

Hydroxy-β-sanshool 65 % 56

ZP-amide A 52 % 75

ZP-amide C 31 % 79

ZP-amide D 56 % 66

ZP-amide E 41 % 67

Timuramide A 38 % 97

Timuramide B 13 % 65

Timuramide C 47 % 66

Timuramide D 42 % 70

The only other alkamide showing similar results in cell viability is Timuramide A, but with less growth inhibition compared to Hydroxy-a-Sanshool. Extracts from Zanthoxylum piperitum also demonstrated anticancer activities in three different human cancer cell lines. This is in line with previous studies which showed that Hydroxy-alpha-Sanshool induces apoptosis in human liver cancer cell lines. It is possible that several different components of the extract were responsible, in addition to Hydroxy-a-Sanshool, for the observed pro apoptotic activity. However, it should be noted that, although these in vitro studies clearly demonstrate that certain Zanthoxylum alkamides provide anticancer activities against certain cancer cell lines, it is still too early to attribute any of the substances to anti-cancer activities. It needs more in vitro studies to verify and fully understand the observed in vitro results.11

4. Cause of the numbing and tingling effect

Consumption of hydroxy-alpha-sanshool causes a numbing and tingling sense on the tongue or the whole oral cavity. This sense is mainly caused by affecting somatosensory neurons which normally detect changes in pressure or temperature and therefore are part of the sense of touch.

For further understanding of the molecular mechanism of action it is necessary to take a closer but still strong simplified look on neurons and how resting and action potential are arising. First, neurons, which are cells of the nerve system, contain of a membrane which is basically like the skin of the cell. To understand how hydroxy-alpha-sanshool acts, we assume that the membrane is impermeable for anions. Furthermore, the membrane contains of sodium-potassium-pumps and potassium channels like shown in Figure 8. The pumps exchange three sodium-ions from intracellular space for two potassium ions from extracellular space. Therefore, the intracellular space will electrostatic charge negative compared to the extracellular space. Moreover, a diffusion potential establishes due to the different concentrations of ions in intracellular and extracellular space, so potassium ions diffuse through the potassium channels resulting in the so-called background potassium leak current. In equilibrium the diffusion potential and the electrostatic potential compensate each other resulting in a constant electrostatic potential difference between intracellular and extracellular space, called resting potential.12,13

Figure 8: Membrane of a neuron with potassium channels and sodium-potassium-pumps.13

If a stimulus reaches a neuron in rest, it increases the potential of the neuron. If therefore the potential exceeds a threshold a so-called depolarization will take place and result in a forwarding of the stimulus. The development of the electrostatic potential of the neurone changes during this process like shown in Figure 9.14

Figure 9: Development of cell potential during stimulation.14

Hydroxy-alpha-sanshool is inhibiting pH- and anaesthetic-sensitive two pore potassium channels of the KCNK-family. As shown in Figure 10Fehler! Verweisquelle konnte nicht gefunden werden. the mainly affected channels are KCNK3, KCNK9 and KCNK18. Most of the background potassium leak current is caused by the KCNK18 channel. The inhibition of this channel therefore results in a strongly reduced background potassium leak current.15

Figure 10: Inhibition of potassium channels by hydroxy-alpha- sanshool.15

The reduced background potassium leak current results in a depolarization which means that the neurons are forwarding a stimulus.16 Because the forwarded stimulus is caused by neurons which normally detect pH-decrease or touch, consuming hydroxy-alpha-sanshool (e.g. via Szechuanpepper) results in a sour taste and prickling and numbing sense.

5. References (1) http://www.hmdb.ca/metabolites/HMDB0029567 (accessed June 20, 2019). (2) https://en.wikipedia.org/wiki/Hydroxy_alpha_sanshool (accessed May 25, 2019). (3) https://de.wikipedia.org/wiki/Szechuanpfeffer (accessed May 25, 2019). (4) https://www.backstars.de/szechuan-pfeffer-sichuanpfeffer/a-471/ (accessed June 23, 2019). (5) https://patents.google.com/patent/CN103099163A/en (accessed May 25, 2019). (6) https://de.wikipedia.org/wiki/Wasserdampfdestillation (accessed June 23, 2019). (7) http://collagenrestores.com/apparatus-diagram-photo/10_3/ (accessed June 23, 2019). (8) Igarashi, Y.; Aoki, K.; Nishimura, H.; Mroishita, I.; Usuii, K. Total Synthesis of Hydroxy-α- and Hydroxy-β-sanshool Using Suzuki–Miyaura Couplin. Chem. Pharm. Bul 2012, 60, 1088–1091. (9) https://scienceandfooducla.wordpress.com/2016/01/13/sanshool-seduction-the-science-of- spiciness/ (accessed June 24, 2019). (10) https://worldwide.espacenet.com/publicationDetails/originalDocument?CC=EP&NR=2842428A1& KC=A1&FT=D&ND=4&date=20150304&DB=EPODOC&locale=en_EP (accessed June 24, 2019). (11) Chruma, J. J.; Cullen, D. J.; Bowman, L.; Toy, P. H. Polyunsaturated fatty acid amides from the Zanthoxylum genus - from culinary curiosities to probes for chemical biology. Natural product reports 2018, 35, 54–74, DOI: 10.1039/c7np00044h. (12) Biologie - simpleclub. Ruhepotential - Aktionspotential - einfach erklärt! https://www.youtube.com/ watch?v=lqq6lu3WouY (accessed June 15, 2019). (13) https://upload.wikimedia.org/wikipedia/commons/thumb/4/4f/Sodium-potassium_pump_and_ diffusion.png/800px-Sodium-potassium_pump_and_diffusion.png (accessed May 28, 2019). (14) https://upload.wikimedia.org/wikipedia/commons/thumb/4/4a/Action_potential.svg/1024px- Action_potential.svg.png (accessed June 15, 2019). (15) Bautista, D. M.; Sigal, Y. M.; Milstein, A. D.; Garrison, J. L.; Zorn, J. A.; Tsuruda, P. R.; Nicoll, R. A.; Julius, D. Pungent agents from Szechuan peppers excite sensory neurons by inhibiting two-pore potassium channels. Nature neuroscience 2008, 11, 772–779, DOI: 10.1038/nn.2143. (16) Gierten, J.; Ficker, E.; Bloehs, R.; Schlömer, K.; Kathöfer, S.; Scholz, E.; Zitron, E.; Kiesecker, C.; Bauer, A.; Becker, R. et al. Regulation of two-pore-domain (K2P) potassium leak channels by the tyrosine kinase inhibitor genistein. British journal of pharmacology 2008, 154, 1680–1690, DOI: 10.1038/bjp.2008.213.