Process lochemisry 552017} 224-251 Contents ists available at ScienceDirect . . lotik-aal Process Biochemistry P| journal homepage: www.elsevier.com/locate/procbio ELSEVIER Kinetic and thermodynamic characteristics of fractional precipitation of (+)-dihydromyricetin Dons Ji-Na Park, Jin-Hyun Kim? Deparment of Chena Engines. Kngh atonal Unser, Chonan 350717, South Korea Received 31 August 2016 ‘Accepted 25 November 2015 This study investigated the effect ofthe precipitation tine and temperature onthe efficiency affractonal precipitation forthe purification of{+)-dihydromyrictin, and analyzed the kinetic and thermadynamics ofthe fractional precipitation process. The tie required obtain a given yielé was the shortest a 277K in 2 time-temperature-transformation (TTT) diagram. Whea the Johnson-Mehl-Avrami-Xomelgorov LUMAK} equation was applied to experimental data simultaneous mechanism of nucleation and growth Were determined at precipitation temperatures of 277, 280, and 283 K while consecutive mechanisms of ‘ueleaton and growth were determined at 263K and 281 K Athermedynamic analysis showed thatthe cn enthalpy change (AH?) and entropy change (AS°) were both negative, pls the Gibbs fre energy change (Ac) was negative and decreased when decreasing te temperature (283,280, and 277K) Thus, the pe ‘ipitation was more feasible wien using lowe temperate. and the results indicated that the fractional precipitation process or purlving (+)-linydromyricetin was exothermic, reversible. and spontaneous inyeremye Jhon Mec avram Kalmogarv MAK) "© 2016 Elsevier Ld. Alright reserved. 1. Introduction, (¢}-Dihydromyricetin [(+}ampelopsin] (chemical formula: CisHy20s, molecular weight: 320.25) (Fig. 1) is a bioactive com- pound derived from Hovenia dulcis and Ampelopsts grossedentata ‘As a functional substance, it is eflective for the treatment of alcohol-related liver diseases and ts used as a raw substance in functional foods and pharmaceutical products for relieving hang- ‘vers and liver protection [1-5]. Furthermore, in 2002, Duet al. [5] reported aninhibition of hypertension by Ampelopsisgrossedentara- derived (+)-dihydromyricetin, while Yoshikawa et al. (7| reported an inhibition of muscle relaxation, metabolic stimulation, and liver protection. Pius, anti-allergic effects have also been noted (8) However, the isolation and purification of +)-dihydromyricetin for its commercalization as a functional food or pharmaceutical product have not been extensively studied. especially methods of, Separation and purification that can be used for industrial produc- tion, A few limited studies have focused on obtaining low-purity (<10x) (+}-dinydromyricetin by extraction followed of chromato- graphic resolution with organic solvents, or acquiring a crude Covesponding autor, Erma edérerntynekongia ack oH. Kim). ‘extract containing terpenoid. lipid, chlorophyll, and phenol [810 However, such studies have applied an expensive chromatographic ‘method (0 the pre-purification process for the final purification, stage, ora crude extract as been directly used in the final putifi- cation by HPLC, whieh consumes a large amount of organic solvent and reduces the lifetime of the column packing materials and ‘throughput, making this process uneconomical for mass produc- tion (5) In contrast, fractional precipitation that uses the difference in solubility is an easier method for the efficient separation and purification of (+}-dihydromyricetin The first representative pretreatment process to obtain high-purity and high-yield (+) dlihydromyricetin using fractional precipitation was developed in 2008 |!) Thereater, various studies have attempted 0 reduce the ‘operating time (~32h) requited for fractional precipitation [12], "Notwithstanding, the kinetic and thermodynamic characteristics fof the precipitation process have not yer been investigated. The ‘netic characteristics can help with understanding or predicting the reaction path, reaction rate, reaction extent, all of which can. impact the development ofthe precipitation process, optimization, and testing [13-15], Meanwhile, the thermodynamic characteris tics can reveal the spontaneity ofthe reaction, heat of the reaction, and reversibility, representing the pattern of the precipitation pro- ‘cess [15-18] Accordingly, this study investigated the effect of thePark JH Kin roe Bochemity 83 (2017) 224-231 as OHO Fi 1. Cheri sructre of(+}-thyéromyicetin precipitation time and temperature on the efficiency of the frac- tional precipitation of (+}-dihydromyricetin, and then analyzed the kinetics and thermodynamics of the fractional precipitation pro- cess, The Johnson-Mehi-Avrami-Komolgorov (JMAK) equation was applied to the precipitation process for the kinetic analysis. The activation energy, Gibbs free energy change, enthalpy change, and entropy change were investigated as thermodynamic parameters for a detailed analysis of the reaction pattern for the fractional precipitation of (+)-dihydromyricetin. 2, Materials and methods 2.1. (+}Dihydromyricetin materials The crude extracts of Ampelopsis grossedentara for the frac- tional precipitation were purchased from Guilin Natural Ingredient, Inc. (Guilin, China). (#)-Dihydromyricetin was the major com- ponent of the extracts and constituted approximately 500% of the total sample based on weight. Authentic (+)-dihydromyricetin, (purity: 98%) was also purchased from Guilin Natural Ingredient, Inc. (Gull, Chine) and used as the standard for the analysis of (#-dihydromyticetin [11 22. Analysis of(+}-dihydromyricetin The (+)-dihydromyricetin content was analyzed using a HPLC system (SCL-10 AVP, Shimadzu, Japan) equipped with a Cap- cell Pak C18 column (250.4.6mm, Sjm, Shiseido, Japan). The lution was performed based on a gradient using 2 distilled wates- acetonitrile mixture varying from 80:10 to 30:70 within 6O min (low rate= 1.0mimin). The injection volume was 20 ul. and the effluent was monitored at 254m using a UV detector. The reten- tion time of (+)-dihydromyricetin was 15min, Each sample was analyzed in triplicate 23. Fractional precipitation After dissolving the (+}dihydromyricetin sample (purity 800%) in acetone (0.1g/mL), distilled water (distilled ‘water/acetone solution ratio=5/t, vjv) at pH 9.0 was dropped uring stiring (350rpm) to induce the precipitation of (+)- dihydromyricetin using the solubility difference. The reactor volume was 20mL and the working volume was 6mL. The solution for fractional precipitation was then placed in a thermo- hhygrostat (KCL-2000W, EYELA. Japan) at ~10°C (263K) for different times (4, 8, 12, 16, 20, 4, 28, 22h) for the precipitation, of (+)-dihydromyricetin. The same method was used at tem peratures of 4°C (277K), 7°C (280K), 10°C (283K), and 18°C (291K). A schematic diagram of the fractional precipitation of (-ihydromyricetin is shown in Fig 2 Alter the fractional precip- station, the precipitate was filtered (Whatman Grade 4, 20-25 ym patticle retention, 10mm diameter), dried in a vacuum oven (UP-2000, EYELA Japan) at 40°C for 24h, and analyzed by HPLC. 24. Analysis of+)-ditydromyricetin precipitate ‘The shape and size of the (+}-lihydromyricetin precipitate in the fractional precipitation were measured using an SV-35 Video Microscope System (Some Tech, Korea) at a high magnification (100) [19], The measurements were also verified in dynamic images using IT-Pius software (Some Tech). The particle size mea- sured by microscopy was expressed as an average value 25. Kinetic analysis ‘The JMAK equation is generally applied to crystallization or precipitation as it is effective in the case of a random particle distribution or independent particle growth, plus i can copy the phase transition behavior in an isothermal process (20-22). The JMAK equation is expressed as Eq, (1) and is rearranged as a linear ‘equation in Ea (2). X()= 1 exp [-k"] 's(" (==) X(t) the yield of precipitated (+)-dihydromyricetin overtime. In Eq, (2), the JMAK exponent, n, and constant, k, can be calculated Using the slope and y-intercept, respectively logk+m log t @ 26. Validity of kinetic mode! ‘The applicability of a kinetic model can be identified using the coefficient of determination (r2) and root mean square deviation (RMSD). The RMSD can be expressed a5 Fa (2) Msp where nis the number of experimental runs 27. Thermodynamic analysis ‘The activation energy (Fe, kllmol) is the minimum energy. required for a reaction and can be calculated using the Arthenius equation shown in Ea. (4). Ey @ Ink na Fe 4) ‘The thermodynamic parameters were used as follows: the stan- dard enthalpy change (AH, kl/mol) was used to determine the reaction heat that was absorbed or emitted, the standard entropy change (AS. Jmol) was used an indicator of entropy to deter- mine reversibility, and the standard Gibbs free energy change (AG, kimol) was used to determine the spontaneity of the vexe- on. AG" was calculated using the equilibrium constant (Ky) a8 shown in Eq. (5), where K, is the concentration ratio of precipi- tated (#)-dihydromyricetin to (+)-lihydromyricetin remaining in the supernatant. As shown in Eq. (0), AS* and AH’ can be caleu- lated using the Van't Hoff equation that shows the relation between lnk, and, AG’ = -RTInKe 6) an as. ar ‘The equilibrium state between an activated complex and a reac- tantiscalled the transition state, When an activated complex passes Ink. 6)2s 1 Par Ki Proce Bhs (2017) 224-231 Wp 300m Distilled water —-> 0 ° . ) 277K), 260K (2), 2654 mand 290K (aA) usrauon purposes en. ‘able ‘Values of nee parameter or precipitation f+} dromietin at eferent tenpecstucs, a Suge 149 conld 0980 co2070 because the supersaturation was higher due to the lower precip- itation temperature [26], Notwithstanding. one straight line was obtained at 277, 280, and 283K in which case the value of n was 10.814, 0.792, and 0.819, respectively. This confirmed thatthe pre- cipitation reaction at 277, 280, and 283K was not phased over the precipitation time, implying simultaneous mechanisms of nucle- ation and growth. The kinetic analysis results revealed that the [IMAK model was suitable with the high value of ? (0.985) and low value of RMSD (<0.02332), “The precipitate was also observed using an electron microscope todetermine the precipitation pattern ateach temperature over the precipitation time. The shapes ofthe precipitate at various precip-itation temperatures (263, 277, 280, 283, 291K) and precipitation times (4, 12,20, 28h) are shown in Fis, 5. At 263K, the precipitate showed an irregular shape, where the size was 43.51, 43.57, 48.36, and 51.02 um after 4, 12,20, and 28 hof precipitation, respectively ‘As such, the size showed a minimal change up to 12h, and then login (14-X00)) toa tn 11e-x00)) fog In (111-X09)) JN. Pan JH. Ki Poces Bloc 522017) 24-281 lea) “| (8) ; 7 g~ | ae Z loot leg af © oO) i bos log t on : “© | bogt increased after 20h, confirming that the nucleation and growth ‘were phased. In addition, the precipitate was smaller at 263K than, at the other temperatures. This may have been because the mave- ‘ment of the (+}-dihydromyricetin particles was limited due to the lower precipitation temperature, thereby interrupting the growth ig. ohinon-Mehl-AvamKomolorov MAK) pt at dierent temperatures. (A} 262K; (8277; {C) 280K; (0) 283K. (E) 291JN ark JK Paces Biches 53 (2017) 224-281 a2 ah ah Fig 6. Flecon micrograph of(~)-yeromyreetin relates oes by fractional reiptation 4, 12h, 20h, 26h (A) 255K; (8}277 (6) 280K: (0) 265: (291K, The (inyrorynetn concentration in aetone. ited waterlaeone sl of the precipitate [25]. Meanwhile, at 277K, the precipitate was ‘varied in size after 4h of precipitation, and a similar pattern was ‘observed at 12-28 h asthe precipitate became larger and spherical over the precipitation time. Plus, at each precipitation time, new small precipitates were observed among the larger precipitates, confiming simultaneous nucleation and growth. This pattern was also observed 2t 280K and 283 K suggesting a similar precipitation process at 277, 280, and 283K. At 291 K, the shape of the preci {ate was irregular at 4 and 12 and then became arod at 20h. This same pattern was also observed at 28h when a more clear shape appeared. Therefore, this verified that the nucleation and growth occurred stage by stage at 291 K 33. Thermodynamic analysis ‘The thermodynamic analysis was conducted atthe precipitation temperature (277, 280, 283 K) at which the precipitation reached an equilibrium state. Ink vs, 1/T was linearized using k obtained at each precipitation temperature (277, 280, 283K) (Table 1). Fe ‘was ~25.89K}/mol based on the slope in Fig. 7 (t#: 0991) In the case of a negatives, the precipitation increases as the tempera- ‘ure decreases, which indicates an exothermic process |23).In other ‘words, as the precipitation temperature decreases, the solubility is, reduced whereas the supersaturation increases. Asa result of that the precipitation yield of +)-dihydromyricetin increases. AG was, calculated using Eq (5),and AS" and AM were calculated using Ea (6) (Table 2) Ke was 1435, 1.215, and 1.074 at 277, 280, and 283K, respectively. As the temperature decreased, K, tended to rise. This ‘was because the precipitation reaction was more active at a lower temperature due to the negatively. The AGe values were nega- fon fata pH and siting sped were 01g [ts 860, and 350 rpm. respetvey Table? ‘Thermodynamic parameters or atonal preepation of -dbydromyietin. Texpentare CAG & air a: ey (met) (inet) let) eet) a7 vas 0a 2989S os tom Tan _Acivaton parameters factonalpecptation of +}. eromysicetin. Tenpentare ae ae ae « mot (timo ioe os eat tive (~0833 kjfmol at 277K, -0.495 kjmol at 280K, -0.168 kjimol at 283K) and decreased when decreasing the temperature, Thus, the precipitation was more spontaneous and feasible when using, lower temperature, The AH? vale was negative (31.52 Km, and the precipitation process was exothermic, meaning heat is temitted and the temperature decreases. The AS” value was nega- tive (1108 Jmol). and the precipitation process was irreversible ‘To investigate the thermodynamic change during the tran tion state of the precipitation reaction, the Eyring equation using k obtained at each precipitation temperature (277. 280, 283 K) (Table 1} waslinearized Fig 8), AH" and AS* were calculatedusing the slope and an intercept (7: 0.992), respectively, andl AG* was determined using Eq. (8) (Table 3). The AH* value was negative20 JN Par J. Kim Pres 290 Ink 3.00 ‘0.00360 310 ‘otoss2 —_—000aee 0.00386 ‘00388 4 (1K) 0062 040 248 350 255 InkiT ae 975 Fig 8 eto nk/ versus tora (~22.22Kj/mal), and the precipitation was an exothermic process [23], The AS* value was also negative (~383 8 Jmol, and the pre- ipitates were formed by associative mechanisms 2327. The AG* vvalue was 74.11, 75.26, and 76.41 kjmol ata precipitation temper- ature of277, 280, and 283 K, respectively. This result was confirmed by the AG* value, which has a positive value for all precipitation reactions that receive external energy in order to convert the reac- tant to a product [23] 4, Conclusions This study investigated the effect ofthe precipitation time and temperature on the efficiency of the fractional precipitation of (+)- ‘dibydromyricetin, and conducted a kinetic and thermodynamic analysis ofthe precipitation process. At precipitation temperatures (0f277, 280, and 283K, the yield of (+)-dihydromyricetin increased, as the temperature decreased (58.77% (277K). 54.95% (280K) AIT (11K) onal precipi of -thydromyiein. 51.71% (283K), and the reaction was equilibrated after 28h of pre- cipitation, However, at 263 K and 291K, the yield increased rapidly for up to 16h and then became minimal for up to 32h. In partic- ular, the yield was the lowest at 291K for each precipitation time. In a time-temperature-transformation (TTT) diagram, the given yield was obtained fastest at 277K. A longer precipitation time ‘was required at a higher (280, 283, 291 K) or lower temperature (263K), When applying the experimental data to the JMAK equa- tion, nucleation and growth occurred simultaneously at 277, 280 and 283K, and consecutively at 263 and 291 K. The precipitation ‘pattern was also visually identified using an electron microscope. From the thermodynamic analysis at 277,280, and 283K, the stan- dard Gibbs fee energy change had negative values(-0.833,0.455, =0.168Kjjmol) and the absolute value increased as the precipi- lation temperature decreased, Thus, the precipitation was more spontaneous and feasible when using a lower temperature. Plus, both the standard enthalpy change (~31.52)/mol) and the stan-J-N ark J Paces Biochem 53 (2017) 224-281 a dard entropy change (-110 8 /mol) had negative values, indicating. that the precipitation process was exothermic and irreversible. Acknowledgement ‘This work was supported by the research grant of the Kongiu. National University in 2016. References [n) 5. An ¥6. Kum, ¥.G. Km S08 Km BL Lee SH Le, HL won 8 Hang. FY tee, Compaen of hepatic était atiy and reducing seu tlcoel concentration of Hove ust Thunb ad As pone Steed orean} Mee Crops 7 (1999 253-258 [ay Kstavet Barnes Aaeol, | Namba, ect of Hove dn lpopotsaccandeindved Iver inry inet labled ats | Tae Nea 041997) 28-33, 1B] NOC ee ¥ 61, SW An ML Kim, LH Le. HY Le Boloicl aces of Hoven desu, Krean Me Crop Seu 7 (1898) 185-192 [al sata Yamane. Saitoh lean, Nihat, tet of wate exact of ‘rae grin ecressng blood ethane oncenttons nats, chem Pare buinos ios) aso7—aaaa [51 SM Yoo 5 Mn JH Kim Recovery ad re-prifestion of [hatveromricesin rm oven al Proce ober. $1 (2006) [61 0 De W. Cai i, ¥ te, Pariieation of +-lnyromyricein fom eaves Sint ef dnt puobete cing igre! comtceareat fronton slept colar) choatag: AS72002) 171 Ke Ytssawa.T Marans Four metiybigated 16,17-sca-dammarane Sauipe snes om chine al ei, ovens (ooes) 1736-17 [o) Ne Yotlawa 1 sturkag T eda 5. Yosh K Ninomiya. Makar nets Sut Wu Tapa, louie onsets of cee Datura medicine I Absolute stereostucutes of new ihydofsvenol ventas. and I olted om hveniae seinen suture eed and Hitt vende Tun (Rhanaacae labret oo dlcolinduced muscular eataton and hepatoprtecve sti, Yakuza ‘ss 117 (1987) Tost [91 X song. en Preparation an apavon of eyéromyricein CN Patent. 28892. 2001 (00) ¥. Zhang Process fr preparing dydromyricein rm Porcelain ampelopsis, (cueatent 395-43, 2005, 11] Kit Lee Ki Developient and optimization of frat forthe pre-paniston of -chveromicet tech (2oos) 2042278 (12) 6 Li, Km, rpeovemen ofthe ational precipitation proces fete Pusan chant Ron eri etn na] ¥e_ cheng ]¥. Wu, Kine del and proces parameters for Ulsasound assed extraction ef water-soluble component and pivsaccharies rom a edi ung, Bide Eg. 79 (2013) 214-220 [nay ¥c cheung Ke stu j¥ Wa, Kies models a lsaseind-assstes ‘entiacon sf water-soluble components an plysucchaties orm medicinal ft Fooewapcocess Technol 8 (2013) 2658-2505, [n5) Siktanovie 0. enowe Vs Veliov Empirical knee mades forthe Fesna extract rom acl pris oft ohs wort ypecun Perna US btecnem eng 10 2008 211 116) Nt Hoste NA Jokove. 08 Siamenkovg KM Rakove PS Mie V8 ‘Veykovee Tne nei: a thermody of hermpseee ol entrain by rene nd Crops ro $2 (2014) 679-68, [171 DI Saxena 3 Sharma 88. sam ines anéthemadynamis of Eetspa extraction rm efted cottonseed mel by ehana Pl | Chem ‘Teche. (4 2012) 29-38, [10] Db Fasnone 5 hie A Kas MIN Mie AT Stoanavé 1 Fao Kinet and thermosynare fhe rié.sigi extracion proces ta Paphenal from bare, Ad Teel (2014) Sous [a9] [¥ Tee Kin, Desieasein hepatic sizeof pata by inresed tear area acsonal precipitation Koren} Maced Sterna 40 2013) 151-162 fy WYER Cc Sunni, Atty le etitin tee (au FR aD Ways Akkari dlr quan he Seng A464 (2007) 6675, (22) $4 Rang, Pte element investigation of alt phase trasfrmation sii ced carton el) Me ces Fecal 189207) (23) PS. howdy sgh iv adsorption thermodynaris, ‘hetmodvoanies Pot Mita Tadashi Ed) SBN 978-955-507 526-0, {neat Avalable rom: tp. rr ntechapencombooksthermoaynamie Insghtante-aserpuen-benedynanits. 2011 [24] | Ryect yum, Kneis and mechani of preciptation in an ALO2WeX Sealy Mater sels 396 2005) 03-222 [25) ST June Kl Lee KW. Seo, studies on hyazothermal synths condivons or preparation 21 powdes | Karean Cyst Growth Cyst Tech 8 (1895) Beane [261 IY Zhang. 26 shen}. Zhang 1.7 Hu LE. Cen, 2. Ma}. Yun, reparation of ‘uarpots efroune set nanoparticles by controled nanopecpiion inetd witout surfaces In] Pharm 323006) 153-160, [an Alpe. teraca, cam carbonate and cl silt rei ‘raliostion and dieoivon: evidence forthe setvsted ep ane the Imechans fromthe enthalpy sné entropy fata vals Chem. ea Seraova 148-153,