Flavan-3-Ols Content in Red Raspberry Leaves Increases Under Blue Led-Light Irradiation
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H OH metabolites OH Article Flavan-3-ols Content in Red Raspberry Leaves Increases under Blue Led-Light Irradiation Ryo Kobori 1, Seiya Hashimoto 2, Hayato Koshimizu 2, Shuich Yakami 1, Mizuki Hirai 1, Kenta Noro 1, Takashi Kawasaki 3 and Akiko Saito 1,2,* 1 Graduate School of Engineering, Osaka Electro-Communication University (OECU), 18-8 Hatsu-cho, Neyagawa-shi, Osaka 572-8530, Japan; [email protected] (R.K.); [email protected] (S.Y.); [email protected] (M.H.); [email protected] (K.N.) 2 Faculty of Engineering, Osaka Electro-Communication University (OECU), 18-8 Hatsu-cho, Neyagawa-shi, Osaka 572-8530, Japan; [email protected] (S.H.); [email protected] (H.K.) 3 Research Institute for Sustainable Humanosphere, Kyoto University, Uji, Kyoto 611-0011, Japan; [email protected] * Correspondence: [email protected]; Tel.: +81-72-824-1131 Received: 22 February 2019; Accepted: 20 March 2019; Published: 21 March 2019 Abstract: Berry fruits are well known to contain large amounts of polyphenol compounds. Among them, flavan-3-ol derivatives are a group of secondary metabolism compounds currently attracting a great deal of attention owing to their health benefits. Not only the fruits, but also the leaves of raspberry plants, are highly esteemed for tea making around the world and are largely used for food. In this report, we discuss the results of our study on the effect of light and temperature on polyphenol accumulation in raspberry leaves. When raspberry was cultivated in a plant factory unit and light intensity, wavelength, and temperature were varied, the amount of total polyphenol increased under blue light. Quantitative determination of (+)-catechin, (–)-epicatechin, procyanidin B4, flavan-3-ol trimer, which are flavan-3-ol derivatives, was carried out using HPLC, whereby we confirmed their increase under blue light. Semi-quantitative RT-PCR showed correlation between chalcone synthase (CHS) gene expression and the amounts of the compounds measured in the leaves. Keywords: polyphenols; light; temperature; controlled environment; biosynthesis; leaf extracts; HPLC; structure-function relations 1. Introduction There is currently great interest in the investigation of compounds from food sources with biological activities, as they are generally considered highly safe because they are consumed as part of the general daily diet. In particular, polyphenols contained in many health foods, as well as in vegetables and fruits, are thought to have various health benefits [1,2]. Among them, flavan-3-ol derivatives are general plant-derived physiologically active compounds known to be highly functional. In particular, the main ingredient of green tea-polyphenols shows various moderate bioactivities without severe toxicity and its health promoting effects have been extensively studied. Although various biologically active flavan-3-ol derivatives are present as minor constituents in plants as well as in green tea, their biological activities have yet to be revealed, mainly owing to their relative unavailability. Therefore, the development of new methods to elucidate the biosynthesis pathway, polymerization mechanisms, and transport of flavan-3-ol derivatives has become an important and active field of research [3,4]. Especially, developing a method to control the biosynthesis of flavan-3-ol derivatives with strong biological activities in plants would be a tool of great value to produce highly functional plants and foods. Metabolites 2019, 9, 56; doi:10.3390/metabo9030056 www.mdpi.com/journal/metabolites Metabolites 2019, 9, x 2 of 14 Metabolites 2019, 9, 56 2 of 14 The production of polyphenolic compounds is affected by light irradiation, and it is thought that this can be used to increase the amount of polyphenol secondary metabolites, including flavanThe-3-ol production derivatives of. polyphenolicFor example, compounds short-term isultraviolet affected by C light irradiation irradiation, was andshown it is thoughtto stimulate that biosyntheticthis can be used pathways, to increase leading the amountto enhancement of polyphenol of the secondaryaccumulation metabolites, of polyphenol including compounds flavan-3-ol [5]. Similarly,derivatives. long For-term example, irradiation short-term of visible ultraviolet blue Clight irradiation showed wasa similar shown effect to stimulate on the biosyntheticamounts of flavpathways,onoids produced leading to in enhancement Chinese cabbage of the [6] accumulation. These reports ofdemonstrate polyphenol the compounds possibility [of5]. controlling Similarly, thelong-term amounts irradiation of specific of visiblepolyphenol blue lightcompounds showed ain similar plants effect, including on the flavan amounts-3-ol of derivatives flavonoids; furthermore,produced in Chinesethey indicate cabbage that [ 6environmental]. These reports conditions demonstrate has a the large possibility influence of on controlling polyphenol the production,amounts of specificwhich causes polyphenol difficulty compounds in the functional in plants, evaluation including of flavan-3-ol plant extracts. derivatives; furthermore, theyFlavan indicate-3-ol that derivative environmentals are thought conditions to promote has ahealth large because influence of their on polyphenol various functionalities. production, whichHowever, causes their difficulty functional in the evaluation functional and evaluation the elucidation of plant extracts. of the mechanism of action involved requireFlavan-3-ol the availability derivatives of these are compounds thought to promotein quantities health large because enough of theirto enable various the functionalities.conduction of theHowever, structure their-activity functional relationship evaluation and(SAR) the elucidationstudies ofneeded the mechanism. Therefore, of actionwe involveddeveloped require the stertheeo availabilityselective synthesis of these method compounds for producing in quantities flavan large-3-ol enough derivatives to enable, oligomeric the conduction compound ofs the[7– 12]structure-activity, galloyled derivatives relationship [13– (SAR)17], and studies acylated needed. derivatives Therefore, [18 we–20 developed] for SARthe studies stereoselective, such as antisynthesis-oxidative method activity forproducing [9,13], radical flavan-3-ol scavenging derivatives, activit oligomericy [14,15], compoundsMaillard reaction [7–12],-inhibitory galloyled activity,derivatives [9] [DNA13–17 ],polymerase and acylated inhibitory derivatives activity [18 [10,13–20] for–15,18] SAR, HeLa studies, S3 suchproliferation as anti-oxidative-inhibitory activity [[91,113,1],6,17 radical,20], and scavenging anti-viral activity activity [14 ,15[21],]. MaillardThese studies reaction-inhibitory showed that activity, fine structur [9] DNAal differences,polymerase such inhibitory as differences activity [ 10in, 13stereochemistry–15,18], HeLa S3and proliferation-inhibitory presence or absence of activity modifications [11,16,17, ,20are], importantand anti-viral to demonstrat activity [21e]. biological These studies activit showedy. In addition that fine, structuralthese organic differences, synthesis such studies as differences allowed us in tostereochemistry store various andstandard presence compounds or absence which of modifications, are not commercially are important avail toable demonstrate and to construct biological a flavanactivity.-3- Inol addition,derivative these library organic for chemical synthesis analysis studies allowed studies usand to storeSAR variousstudies standardwhich, overall, compounds have whichrevealed are that not commerciallyamong polyphenols, available flavan and to-3- constructol derivatives a flavan-3-ol possess derivative high functionality library for and chemical that oligomersanalysis studies exhibit and various SAR studiesfunctionalities which,. overall, have revealed that among polyphenols, flavan-3-ol derivativesOur next possess challenge high functionality was to achieve andthat the oligomerseffective, exhibitselective various production functionalities. of high-functional flavanOur-3-ol next derivatives challenge wasin toplant achieves. Thus, the effective,we selected selective red production raspberry of ( high-functionalRubus idaeus L.) flavan-3-ol as our experimentalderivatives in plants.model Thus,plant we because selected it red is raspberrya species ( Rubuswidely idaeus knownL.) as for our its experimental edible fruits model and plant as polyphenolbecause it is arich species and widelyhighlyknown beneficial for itsfor edible human fruits health and as [2 polyphenol2,23]. Furthermore, rich andhighly they beneficialare most commofor humannly known health [as22 ,food23]. Furthermore,products and theytheir are extracts most are commonly also known known as anti as food-inflammatory products and [24] their and antimicrobialextracts are also properties known as[25 anti-inflammatory]. As mentioned above [24], and numerous antimicrobial studies properties report increasing [25]. As polyphenol mentioned contentabove, numerousin various studies plants report by light increasing irradiation polyphenol management content. inTherefore, various plants we planned by light irradiationstudies to irradiatemanagement. raspberries Therefore, with LED we plannedlight to increase studies polyphenols, to irradiateraspberries especially flavan with- LED3-ol compounds. light to increase polyphenols,In this report especially, we flavan-3-oldiscuss the compounds. method for raspberry cultivation in a plant factory unit kept underIn constant this report, environment we discussal theconditions, method