The exposure of human skin to 4-(4-hydroxyphenyl)-2-butanone (raspberry ketone, RK) is known to cause chemical/occupational leukoderma. RK is a carbonyl derivative of 4-(4-hydroxyphenyl)-2-butanol (rhododendron), a skin-whitening agent that was found to cause leukoderma in the skin of many consumers. These two phenolic compounds are oxidized by tyrosinase and the resultant products seem to cause cytotoxicity to melanocytes by producing reactive oxygen species and depleting cellular thiols through o-quinone oxidation products. Therefore, it is important to understand the biochemical mechanism of the oxidative transformation of these compounds. Earlier studies indicate that RK is initially oxidized to RK quinone by tyrosinase and subsequently converted to a side chain desaturated catechol called 3,4- dihydroxybenzalacetone (DBL catechol). In the present study, we report the oxidation chemistry of DBL catechol. Using UV-visible spectroscopic studies and liquid chromatography-mass spectrometry, we have examined the reaction of DBL catechol with tyrosinase and sodium periodate. Our results indicate that DBL quinone formed in the reaction is extremely reactive and undergoes facile dimerization and trimerization reactions to produce multiple isomeric products by novel ionic Diels-Alder type condensation reactions. The production of a wide variety of complex quinonoid products from such reactions would be potentially more toxic to cells by causing not only oxidative stress but also myelotoxicity through exhibiting reactions with cellular macromolecules and thiols. 

According to relevant studies,cistanche is a common herb that is known as "the miracle herb that prolongs life". Its main component is cistanoside, which has various effects such as antioxidant, anti-inflammatory, and immune function promotion. The mechanism between cistanche and skin whitening lies in the antioxidant effect of cistanche glycosides. Melanin in human skin is produced by the oxidation of tyrosine catalyzed by tyrosinase, and the oxidation reaction requires the participation of oxygen, so the oxygen-free radicals in the body become an important factor affecting melanin production. Cistanche contains cistanoside, which is an antioxidant and can reduce the generation of free radicals in the body, thus inhibiting melanin production.

david.deng@wecistanche.com  WhatApp:86 13632399501

rhododendron toxicity; raspberry ketone; 3,4-dihydroxybenzalacetone; DBL catechol; ionic Diels-Alder addition; myelotoxicity; leukoderma; skin lightening compounds 

1. Introduction

Raspberry ketone (4-(4-hydroxyphenyl)-2-butanone, RK), is a phenolic compound widely used as a cosmetic, perfume, and food flavoring agent [1]. Some of the workers engaged in the manufacturing process of this compound in Japan developed occupational leukoderma [2]. In this context, it is important to draw particular attention to rhododendron, 4-(4-hydroxyphenyl)-2-butanol, the derivative of RK with its carbonyl group reduced to a secondary alcohol. Rhododendron was also used widely in the cosmetics industry as a skin-whitening product. Repeated application of rhododendron as a skin color-lightening agent resulted in the development of leukoderma on the face, neck, and hands [3]. These two compounds may be interconvertible in the cell by oxidation-reduction reactions. Therefore, their toxicity may be caused by the same kind of reactions.

Tyrosinase-catalyzed oxidation of RK produced its corresponding quinone which exhibited rapid isomerization via its quinone methide to (E)-4-(3,4-dihydroxyphenyl)-3-buten-2-one, commonly known as 3,4-dihydroxybenzalacetone (DBL catechol) [11]. Such nonenzymatic introduction of the double bond in the side chain by the intermediary formation of quinone and quinone methide is a well-documented reaction for a few catecholamine derivatives[12–15]. Preliminary studies indicated that the resultant DBL catechol is very reactive and the quinonoid products formed from this compound could rapidly react with cellular thiol compounds [11]. In addition, possible oligomerization of the quinonoid product was inferred but was not examined due to the extreme reactivity of the intermediates. In recent years, one of our laboratories has successfully used mass spectral studies to investigate the intricate details of oxidative transformations of several dehydrodopa and dehydrodopamine derivatives [12–20]. Our explorations yielded valuable information on the course of oxidative transformation of several catecholamine derivatives. Therefore, we decided to investigate the reaction course used by DBL catechol also using this technique, and report in this paper the novel oxidative transformations exhibited by DBL quinone.

2. Results and Discussion

Figure 2A shows the UV-visible spectral changes accompanying the oxidation of DBL catechol by mushroom tyrosinase. As soon as tyrosinase is added, rapid changes occur in the absorbance spectra. The UV peak due to DBL catechol at around 320 nm progressively reduced and absorbance in the visible region at about 420 nm increased steadily. The spectral changes accompanying this transformation exhibited two isosbestic points at about 285 nm and 385 nm, indicating the direct transformation of DBL catechol to the compound exhibiting absorbance at about 420 nm. This compound, which has a yellow color, must be the corresponding quinone as tyrosinase is well known to exhibit wide substrate specificity and oxidize many o-diphenolic compounds to their corresponding o-quinones [15]. When the reaction was performed at slightly alkaline conditions (at pH 8), the color of the reaction mixture became lighter as the reaction progressed (Figure 2B). In the end, a compound exhibiting broad absorbance at around 330 nm was formed. One could also witness a slight shift in the spectral scans between the substrate and the end product, suggesting that part of the conjugation in the substrate is lost in the product. These results indicated that the tyrosinase-generated DBL quinone is undergoing further transformation.

To visualize the fate of DBL quinone, we generated this quinone by quantitatively oxidizing DBL catechol with sodium periodate and monitoring the UV and visible absorbance spectral changes. Initial studies conducted at pH 6 or 7 revealed that the oxidation is extremely fast and resulted in the formation of a final product that exhibited an absorbance maximum at around 265 nm with a broad absorbance at the visible region between 380 and 440 nm (Figure 3A)

Since the reaction occurred too fast, monitoring the spectral changes became very difficult. Hence, we decided to slow down the reaction by conducting it at acidic pH values. To slow the reaction and monitor the quinone formation, we conducted the studies in 0.2 M acetic acid. Figure 3B shows the rapid generation of DBL quinone from DBL catechol by the oxidation of sodium periodate in acetic acid. The oxidation was completed in less than a min and the quinone formed could be visualized by its absorbance at the visible region. However, the quinone turned out to be very unstable and exhibited rapid conversion to product(s) that exhibit ultraviolet absorbance at the 280–480 nm range as shown in Figure 4.

Two products—one eluting at 18 min and another eluting at 21 min—showed a mass of m/z 355.1171 (Figure 5B), which is within 3 ppm of the theoretical mass for the addition product of DBL quinone to DBL catechol (C20H18O6). The CID spectrum of the dimer eluting at 18 min (Figure 6) differed significantly from that eluting at 21 min (Figure 7). The dimer eluting at 18 min exhibited major product ions at m/z values of 337 (loss of water), 313 (loss of acetyl group), and 295 (loss of water and acetyl group). The loss of both water and acetyl groups is possible only for the benzodioxan-type dimer shown in the inset. Note this m/z 295 ion peak is not the prominent peak in the CID spectrum of the 21 min peak. This observation coupled with the production observed at 189 (loss of a catholic group and CH2=CO-CH3 group) suggests that the 21 min peak is due to a different dimer whose proposed structure is shown in the inset of Figure 7.

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