<?xml version="1.0" encoding="UTF-8"?><!DOCTYPE article  PUBLIC "-//NLM//DTD Journal Publishing DTD v3.0 20080202//EN" "http://dtd.nlm.nih.gov/publishing/3.0/journalpublishing3.dtd"><article xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink" dtd-version="3.0" xml:lang="en" article-type="research article"><front><journal-meta><journal-id journal-id-type="publisher-id">JACEN</journal-id><journal-title-group><journal-title>Journal of Agricultural Chemistry and Environment</journal-title></journal-title-group><issn pub-type="epub">2325-7458</issn><publisher><publisher-name>Scientific Research Publishing</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="doi">10.4236/jacen.2013.21004</article-id><article-id pub-id-type="publisher-id">JACEN-28211</article-id><article-categories><subj-group subj-group-type="heading"><subject>Articles</subject></subj-group><subj-group subj-group-type="Discipline-v2"><subject>Chemistry&amp;Materials Science</subject><subject> Earth&amp;Environmental Sciences</subject></subj-group></article-categories><title-group><article-title>
 
 
  Isolation and identification of alkaloids and anthocyanins from flower and bulb of Lycoris radiata using HPLC and LC-ESI-MS
 
</article-title></title-group><contrib-group><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>in-Hyuk</surname><given-names>Chun</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="corresp" rid="cor1"><sup>*</sup></xref></contrib><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>In</surname><given-names>Hyuk Jang</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Mariadhas</surname><given-names>Valan Arasu</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Naif</surname><given-names>Abdullah Al-Dhabi</given-names></name><xref ref-type="aff" rid="aff3"><sup>3</sup></xref></contrib><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Veeramuthu</surname><given-names>Duraipandiyan</given-names></name><xref ref-type="aff" rid="aff3"><sup>3</sup></xref></contrib><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Dong-Hoon</surname><given-names>Lee</given-names></name><xref ref-type="aff" rid="aff4"><sup>4</sup></xref></contrib><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Sanghyun</surname><given-names>Lee</given-names></name><xref ref-type="aff" rid="aff5"><sup>5</sup></xref></contrib><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Sun-Ju</surname><given-names>Kim</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib></contrib-group><aff id="aff4"><addr-line>Yeonggwang Agricultural Technology Center, Yeonggwang-Gun, Republic of Korea</addr-line></aff><aff id="aff5"><addr-line>Department of Integrative Plant Science, Chung-Ang University, Anseong, Republic of Korea</addr-line></aff><aff id="aff3"><addr-line>Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, Saudi Arabia</addr-line></aff><aff id="aff1"><addr-line>Department of Bio-Environmental Chemistry, Chungnam National University, Daejeon, Republic of Korea</addr-line></aff><aff id="aff2"><addr-line>Department of Formulation, Sungbo Chemicals Co., Ltd., Goyang-si, Republic of Korea</addr-line></aff><author-notes><corresp id="cor1">* E-mail:<email>kimsunju@cnu.ac.kr(IC)</email>;</corresp></author-notes><pub-date pub-type="epub"><day>26</day><month>02</month><year>2013</year></pub-date><volume>02</volume><issue>01</issue><fpage>22</fpage><lpage>26</lpage><history><date date-type="received"><day>20</day>	<month>November</month>	<year>2012</year></date><date date-type="rev-recd"><day>25</day>	<month>December</month>	<year>2012</year>	</date><date date-type="accepted"><day>8</day>	<month>January</month>	<year>2013</year></date></history><permissions><copyright-statement>&#169; Copyright  2014 by authors and Scientific Research Publishing Inc. </copyright-statement><copyright-year>2014</copyright-year><license><license-p>This work is licensed under the Creative Commons Attribution International License (CC BY). http://creativecommons.org/licenses/by/4.0/</license-p></license></permissions><abstract><p>
 
 
  Three anthocyanins (cyanidin 3-diglucoside, cyanidin 3-sambubioside and cyanidin 3-glucoside) together with eleven known alkaloids (lycoricidine, hipppeastrine,O-Demethyllycoramin, lycoricidinol, galanthine, lycorine, lycorenine, lycoramine, galanthamine, homolycorine and pretazettine) were identified in the flower and bulb of 
  <em>Lycoris radiata </em>using high-performance liquid chromatography coupled with UV detection and electrospray ionization mass spectrometry. Anthocyanins play a major role in protecting plant’s DNA from the UV spectrum of sunlight and also in attracting insects for the purpose of pollination. Thus, knowledge on the contents and types of anthocyanins of 
  <em>L. radiata </em>will help to evaluate the adaptive evolution of flowers and provide useful information for the ornamental breeding.
 
</p></abstract><kwd-group><kwd>Lycoris radiata; Anthocyanins;Alkaloids; Flower; Bulb</kwd></kwd-group></article-meta></front><body><sec id="s1"><title>1. INTRODUCTION</title><p>Plants of the family Amaryllidaceae are well known for their ornamental values and also for their alkaloids they produce. Some of these alkaloids exhibit interesting pharmacological and biological activities. Lycoris radiata belongs to Amaryllidaceae family of perennial plants, and 20 different species are distributed in East Asia including China, Japan, Korea and Nepal. The Korean peninsula has seven Lycoris species such as L. flavescens, L. chinensis var. sinuolata, L. uydoensis, L. sanguinea var. koreana, L. chejuensi, L. radiata and L. squamigera respectively [<xref ref-type="bibr" rid="scirp.28211-ref1">1</xref>]. Since 2000, several Korean researches have reported identification of metabolites from Lycoris species [2,3]. L. radiata has ornamental and medical values and therefore the interest of plant breeding is high. Floral colors of L. radiata are due to biological pigments of different kinds of molecules, such as porphyrins, carotenoids, anthocyanins and betalains. Anthocyanins occur only in terrestrial plants and are thought to be evolutionarily adaptive for several different plants biological processes. Four anthocyanin components, pelargonidin- 3-glucosides, pelargonidin-3-xylosylglucosides, cyanidin- 3-glucosides, and cyanidin-3-xylosylglucosides were identified in L. radiata [<xref ref-type="bibr" rid="scirp.28211-ref4">4</xref>]. To our best knowledge, cyanidin 3- diglucoside and cyanidin 3-sambubioside have not been reported in the flower of L. radiata, but they were found to be an important determinant of floral colors in L. radiata.</p></sec><sec id="s2"><title>2. MATERIALS AND METHODS</title><sec id="s2_1"><title>2.1. Plant Materials</title><p>Flowers and bulbs of L. radiata harvested in 2011 were provided by the Yeonggwang Agricultural Technology Center (Yeonggwang-gun, Korea).</p></sec><sec id="s2_2"><title>2.2. Extraction of Alkaloids and Anthocyanins</title><p>For alkaloids, a 100 mg of lyophilized powder was transferred to 2 mL eppendorf tube and mixed with 1 mL of water/sulfuric acid solution (99:1, v/v). The solution was mixed thoroughly by vortexing for 2 min and then allowed to stand at room temperature for 24 h without disturbance. In the case of anthocynins, a 100 mg portion of lyophilized powder was transferred to a 2 mL eppendorf tube, and 2 mL of water/formic acid solution (95:5, v/v) was added. The solution was vigorously mixed for 5 min, sonicated for 20 min, and centrifuged at 8000 rpm for 30 min. The extract was filtered through a 0.45 μm PTFE syringe filter (Advantec DISMIC-13HP, Toyo Roshi Kaisha, Ltd., Tokyo, Japan). The filtrate was analyzed by HPLC and LC-ESI-MS.</p></sec><sec id="s2_3"><title>2.3. LC-ESI-MS Analysis of Alkaloids and Anthocyanins</title><p>HPLC analyses were carried out with a flow rate of 1.0 mL/min at column oven temperature of 40˚C and detector wavelength of 292 nm. The solvent system employed was (A) 10 mM ammonium carbonate and (B) 10% 10 mM ammonium carbonate in acetonitrile. The solvent program used is as follows: 0 min solvent B 0%, 13 min solvent B 7%, then kept constant at solvent B 7% till 18 min, 23 min solvent B 10%, then kept constant at solvent B 10% till 28 min, 35 min solvent B 20%, 50 min solvent B 70%, then kept constant at solvent B 70% till 55 min, rapidly reduce solvent B to 0% at 55.1 min, and then kept constant at solvent B 0% till total 60 min. For the identification of individual alkaloids, the MS analysis was carried out with an ESI interface operating in the positive ion mode [M+H]<sup>+</sup>. The MS operating conditions were as follows: ion spray voltage, 5.5 kV; curtain gas (N<sub>2</sub>), 20 psi; nebulizing gas and heating gas (N<sub>2</sub>), 50 psi; heating gas temperature, 550˚C; spectra range, m/z 100 - 500 (scan time, 4.8 sec).</p><p>For anthocynins, an API 4000 Q TRAP tandem mass spectrometer (Applied Biosystems, Foster City, CA), equipped with an Agilent 1200 series HPLC system and an electrospray ionization-tandem mass spectrometry (ESI-MS) source in positive ion mode was used. The HPLC conditions were the same as those described above, but the mobile phase was changed to (A) water/ formic acid (99:1, v/v) and (B) acetonitrile/formic acid (99:9, v/v) because of low pH of less than 2.0 required.</p></sec></sec><sec id="s3"><title>3. RESULTS AND DISCUSSION</title><p>The MS spectral data by comparing with data of a previous report [5,6] led to identification of eleven known alkaloids (lycoricidine (peak no. 4), hipppeastrine (6), O-demethyllycoramine (7), lycoricidinol (8), galanthine (9), lycorine (10), lycorenine (12), lycoramine (13), gaanthamine (14), homolycorine (16), and pretazettine (18). Seven unknown compounds were also observed. The peak numbers are the elution order in the HPLC chromatogram in <xref ref-type="table" rid="table1">Table 1</xref>. LC-ESI-MS spectra of lycorine and cyanidin 3-sambubioside (anthocyanin) were showed in <xref ref-type="fig" rid="fig1">Figure 1</xref> and <xref ref-type="fig" rid="fig2">Figure 2</xref>(a). Identification of anthocyanins was primarily based on the comparison of their retention time and elution order together with mass spectrometric data with standards and isolation methods described previously [7,8]. Separation of identified anthocyanins on HPLC with their retention time and elution order together with mass spectrometric data were shown in <xref ref-type="table" rid="table1">Table 1</xref>, <xref ref-type="fig" rid="fig2">Figure 2</xref>(b) and <xref ref-type="fig" rid="fig3">Figure 3</xref>. LC-ESI-MS spectra of three anthocyanins were compared with the published reports of other anthocyanins derivatives, which coincide in fragmentation patterns and further confirmed that the identified anthocyanins were cyanidin 3-diglucoside, cyanidin 3-sambubioside and cyanidin 3-glu-coside respectively. HPLC chromatogram of peaks 1 and 3 had exhibited similar MS profiles but showed different retention times (23.8 and 26.0 min). The LC-ESI-MS analysis indicated that peaks 1, 2 and 3 contained aglycone cyanidin (MW, molecular weight, 287) as a common backbone with varied monosaccharide glucose (MW, 180) and peaks 2 had monosaccharide with the combination of D-xylose and D-glucose (MW, 312). Cyanidin 3-diglucoside and cyanidin 3-glucoside exhibited molecular ions at m/z 287, respectively, whereas cyanidin 3-sambubioside showed at m/z 294 because this compound produced only one major fragment [9,10]. Peaks 1 and 3 had the same aglycone (cyanidin) as peak 3 but</p><p><xref ref-type="table" rid="table1">Table 1</xref>. Alkaloids identified in the bulb of Lycoris radiate.</p><disp-formula id="scirp.28211-formula95239"><graphic  xlink:href="4-2750014\9f3aadf8-9be3-411f-85a2-e2943f54dbad.jpg"  xlink:type="simple"/></disp-formula><p><sup>a</sup>The elution order of alkaloids in HPLC chromatogram (<xref ref-type="fig" rid="fig1">Figure 1</xref>); <sup>b</sup> Retention time (min).</p><p><xref ref-type="table" rid="table2">Table 2</xref>. Anthocyanins identified in the flower of Lycoris radiata.</p><disp-formula id="scirp.28211-formula95240"><graphic  xlink:href="4-2750014\3f9276a6-d83f-4ada-bcd9-9ce3775b9f41.jpg"  xlink:type="simple"/></disp-formula><p><sup>a</sup>The elution order of anthocyanins in HPLC chromatogram (<xref ref-type="fig" rid="fig3">Figure 3</xref>). <sup>b</sup>Retention time (min).</p><p>showed different retention times (23.8 vs 26.0 min). Similar types of anthocyanins were found in red leaf lettuce, pistachio, small red bean, red cabbage and red onion respectively [<xref ref-type="bibr" rid="scirp.28211-ref8">8</xref>].</p></sec><sec id="s4"><title>4. ACKNOWLEDGEMENTS</title><p>This research was supported by the Bio-industry Technology Development Program (Grant No. 110139-03-2-HD110), Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea. 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