Bull. Natl. Mus. Nat. Sci., Ser. B, 46(2), pp. 89–93, May 22, 2020

Phenolic Compounds from Artemisia kitadakensis Endemic to Japan
Yoshinori Murai1,*1

Department of Botany, National Museum of Nature and Science, 4–1–1 Amakubo,Tsukuba, Ibaraki, 305–0005 Japan*E-mail: murai@kahaku.go.jp(Received 7 February 2020; accepted 25 March 2020)

Abstract Phenolic compounds in the leaves of Artemisia kitadakensis, a rare and endangered
alpine species endemic to Japan, were surveyed. Two phenylpropanoids, chlorogenic acid and isochlorogenic acid A, and ﬁve ﬂavonoids, luteolin 7-O-glucoside, 7-O-glucuronide and 7-O-pentosylhexoside, chrysoeriol 7-O-glucuronide and quercetin 3-O-glucoside, were isolated from A. kitadakensis leaves. HPLC analysis revealed that the overall content of bioactive phenolic compounds
decreased when plants were cultivated in a glasshouse, compared to plants grown in their natural
habitat. This suggests that some of these phenolics may act as anti-stress compounds in the harshalpine environment. Insights into the glasshouse cultivation of A. kitadakensis are also discussed.
Keywords : Artemisia kitadakensis, Asteraceae, chemical ecology, chemical proﬁling, phenolic
compounds.

Introduction
The genus Artemisia consists of 540 species(Mabberley, 2017), and 30 species are growingin Japan (Koyama, 1995). Among them, A. kitadakensis H.Hara et Kitam. (Fig. 1) is distributed
only in a few sites in the alpine zone (2600–3000 m elev.) of Akaishi Mountains, CentralJapan. Owing to its limited distribution, the plant
has been listed as endangered species (EN) bythe Ministry of the Environment, Japan (2015).
Artemisia species are rich sources of bioactivenatural products such as terpenoids and ﬂavonoids (Tan, et al., 1998). Recently, ﬂavonoid proﬁling has been carried out using Japanese Artemisia species (Uehara et al., 2018). However,chemical constituents of A. kitadakensis have notbeen surveyed to date. On the other hand, thesigniﬁcant decreases of phenolic compounds
have been observed in some common Japanesealpine plants, Geum calthifolium var. nipponicum(F.Bolle) Ohwi (Rosaceae) and Campanula© 2020 National Museum of Nature and ScienceFig. 1. Artemisia kitadakensis in Mt. Kita-dake,
Yamanashi Prefecture.

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Yoshinori Murai

chamissonis Fed. (Campanulaceae) cultivated inlowland (Murai and Iwashina, 2010; Murai et al.,2014). To clarify the biological properties concerning anti-stress components of A. kitadakensis, the variation of phenolic compounds undercultivation was also surveyed.
Materials and Methods
Plant materials
Artemisia kitadakensis was collected from Mt.
Kita-dake, 2960 m elev., Yamanashi Prefecture,
Japan in July 2015 and September 2019, and Mt.
Senmai-dake, 2860 m elev., Shizuoka Prefecture,
Japan in August 2015. Plant collections were carried out under the permissions of Yamanashi Prefecture, the Ministry of the Environment, Japan,and Tokushu Tokai Paper Co., Ltd. Voucher specimens were deposited in the herbarium ofNational Museum of Nature and Science, Japan(TNS).
Plant cultivation
A. kitadakensis sampled from Mt. Kita-dake
was cultivated in a glasshouse of Tsukuba Botanical Garden (ca. 25 m elev.), National Museum
Nature and Science, Tsukuba City, Ibaraki Prefecture, Japan. Plants were grown for twomonths at a photosynthetic photon ﬂux density ofca. 150 µmol photons m−2 s−1 during the daytime, and the temperatures regulated between
18°C (night) and 25°C (daytime). Relative
humidity was kept around 50–60%. Mature
leaves were randomly selected and sampled forchemical analysis. This experiment was repeatedusing three individuals. In addition to chemicalanalysis, propagation of the endangered species,
A. kitadakensis was successful by division under
the same growth conditions.
Extraction and separation of phenolic compounds
Fresh leaves (6.1 g) of A. kitadakensis wereextracted with MeOH. The concentrated extracts
were cleaned up by InertSep C18 (10 g/ 60 mL,GL Sciences Inc., Japan), and then applied toPreparative HPLC with an Inertsil ODS-4 column [5 µm particle material, I.D. 10×250 mm(GL Sciences Inc.)] using solvent system:HCOOH/MeCN/H2O＝1 : 18 : 81. The compounds
were puriﬁed by Sephadex LH-20 column chromatography (solvent system: 80% MeOH).
Quantitative HPLC analysis of phenolic compounds
Fresh leaves (0.2 g) from cultivation and natural habitats were extracted with 4 mL MeOH.
High performance liquid chromatography

Fig. 2. HPLC chromatogram of MeOH extracts from Artemisia kitadakensis. 1: chlorogenic acid, 2: isochlorogenic acid A, F1: luteolin 7-O-pentosylhexoside, F2: quercetin 3-O-glucoside, F3: luteolin 7-O-glucoside, F4:luteolin 7-O-glucuronide and F5: chrysoeriol 7-O-glucuronide. Detection: 340 nm.

Phenolic compounds from Artemisia kitadakensis

91(HPLC) separation of the extracts ﬁltered withGL Chromatodisk 13N (0.45 µm pore size, GLSciences, Inc.) was performed with a Shimadzu
Prominence HPLC system using a SunShell C18column [2.6 µm particle material, I.D.
4.6×100 mm (ChromaNik Technologies Inc.,Japan)] at a ﬂow rate of 0.8 mL min−1, detection
wavelength at 190–700 nm and MeCN/H2O/
H3PO4 (15 : 85 : 0.2) as eluent. Injection volume
was 2 µL. HPLC chromatogram of phenolic compounds is shown in Fig. 2.
Liquid chromatograph-mass spectra (LC-MS)
LC-MS was measured with a Shimadzu
LC-MS system using an Inertsil ODS-4 (3 µm
particle material, I.D. 2.1×100 mm, GL SciencesInc.), at a ﬂow-rate of 0.2 mL min−1, eluting withHCOOH/MeCN/H2O (1 : 20 : 79), injection: 3 µL,ESI＋ 4.5 kV, ESI− 3.5 kV, 250°C.
Identiﬁcation of compounds
In this research, two phenylpropanoids (1 and2) (Fig. 3a) and ﬁve ﬂavonoids (F1–F5) (Fig. 3b)
were isolated from the leaves of A. kitadakensis.
Phenylpropanoids were characterized by UV
spectroscopy, LC-MS and HPLC comparisonswith authentic specimens. In the case of isolated
ﬂavonoids, their chemical structures were further
estimated by UV spectroscopy according toMabry et al. (1970) and characterization of acidhydrolysates (in 12% HCl, 100°C, 30 min).
Chemical data of the isolated compounds are as
follows.
Chlorogenic acid (3-Caffeolylquinic acid) (1).
Clear solution. UV: λmax (nm) MeOH 300sh,329. HPLC: Rt 1.9 min. LC-MS: m/z 355[M＋H]＋, 353 [M−H]−.
Isochlorogenic acid A (3,5-Dicaffeolylquinicacid) (2). Clear solution. UV: λmax (nm) MeOH301sh, 329. HPLC: Rt 10.3 min. LC-MS: m/z 515[M−H]−.
Luteolin 7-O-pentosylhexoside (F1). Pale yellow solution. UV: λmax (nm) MeOH 255, 265,347; ＋NaOMe 267, 388 (inc.); ＋AlCl3 273,421; ＋AlCl3/HCl 273, 295sh, 357sh, 387sh;＋NaOAc 263, 402; ＋NaOAc/H3BO3 261, 372.

Fig. 3. Chemical structures of phenolic compounds from Artemisia kitadakensis.
(a) 1: chlorogenic acid (R＝H), 2: isochlorogenic acid A (R＝caffeoyl). (b) F1: luteolin
7-O-pentosylhexoside (R1＝pentosylhexosyl,R2＝R3＝H), F2: quercetin 3-O-glucoside(R1＝R2＝H, R3＝glucosyl), F3: luteolin 7-Oglucoside (R1＝glucosyl, R2＝R3＝H), F4: luteolin 7-O-glucoronide (R1＝glucuronyl, R2＝R3＝H), F5: chrysoeriol 7-O-glucoside (R1＝glucosyl, R2＝CH3, R3＝H).

HPLC: Rt 5.0 min. Acid hydrolysis: luteolin.
LC-MS: m/z 581 [M＋H]＋, 579 [M−H]− (luteolin＋1 mol hexose＋1 mol pentose), 449[M＋H]＋ (luteolin＋1 mol hexose), 287[M＋H]＋ (luteolin).
Quercetin 3-O-glucoside (F2). Pale yellowsolution. UV: λmax (nm) MeOH 256, 266sh,357; ＋NaOMe 273, 325, 410 (inc.); ＋AlCl3274, 303sh, 415; ＋AlCl3/HCl 270, 298sh,362sh, 399sh; ＋NaOAc 272, 324, 396;
＋NaOAc/H3BO3 262, 302sh, 379. HPLC: Rt
6.1 min. Acid hydrysis: quercetin and glucose.
LC-MS: m/z 465 [M＋H]＋, 463 [M−H]− (quer-92

Yoshinori Murai

cetin＋1 mol glucose), 303 [M＋H]＋ (quercetin).
Luteolin 7-O-glucoside (F3). Pale yellow solution. UV: λmax (nm) MeOH 255, 268sh, 348;
＋NaOMe 265, 301sh, 389 (inc.); ＋AlCl3 273,297sh, 409; ＋AlCl3/HCl 266sh, 274, 295sh,360sh, 388sh; ＋NaOAc 260, 365sh, 401;
＋NaOAc/H3BO3 260, 372. HPLC: Rt 6.5 min.
Acid hydrolysis: luteolin and glucose. LC-MS:m/z 449 [M＋H]＋, 447 [M−H]− (luteolin＋1 mol glucose), 287 [M＋H]＋ (luteolin).
Luteolin 7-O-glucuronide (F4). Pale yellowsolution. UV: λmax (nm) MeOH 255, 266sh,348; ＋NaOMe 266, 302sh, 390 (inc.); ＋AlCl3273, 302sh, 415; ＋AlCl3/HCl 272, 296sh,362sh, 387sh; ＋NaOAc 260, 403; ＋NaOAc/
H3BO3 260, 373. HPLC: Rt 7.1 min. Acid hydrolysis: luteolin and glucuronic acid. LC-MS: m/z
463 [M＋H]＋, 461 [M−H]− (luteolin＋1 molglucuronic acid), 287 [M＋H]＋ (luteolin).
Chrysoeriol 7-O-glucuronide (F5). Pale yellow solution. UV: λmax (nm) MeOH 267, 343;
＋NaOMe 262, 277sh, 393 (inc.); ＋AlCl3263sh, 272, 298sh, 359sh, 385sh; ＋AlCl3/HCl
260sh, 276, 296sh, 351, 382sh; ＋NaOAc 259,406; ＋NaOAc/H3BO3 266, 348. HPLC: Rt
16.8 min. Acid hydrolysis: chrysoeriol and glucuronic acid. LC-MS: m/z 477 [M＋H]＋, 475[M−H]− (chrysoeriol＋1 mol glucuronic acid),
301 [M＋H]＋ (chrysoeriol).
Results and Discussion
Two phenylpropanoids, chlorogenic acid (1)and isochlorogenic acid A (2), and ﬁve ﬂavonoids, luteolin 7-O-pentosylhexoside (F1), quercetin 3-O-glucoside (F2), luteolin 7-O-glucoside(F3) luteolin 7-O-glucuronide (F4) and chrysoeriol 7-O-glucuronide (F5), were isolated fromthe leaves of A. kitadakensis. The amount of luteolin 7-O-pentosylhexoside (F1) was too small toidentify the sugars. Though chlorogenic acid (1),quercetin 3-O-glucoside (F2) and luteolin
7-O-glucoside (F3) were common compounds in
Artemisia species (Tan et al., 1998; Iwashina,
2018; Uehara et al., 2018), other compounds
were isolated from Japanese Artemisia species

for the ﬁrst time. Chrysoeriol 7-O-glucuronide(F5) is a relatively rare ﬂavone glycoside, but it
was reported from A. judaica L. which is distributed from north Africa to Middle East (Saleh etal., 1987). Furthermore, HPLC survey of MeOH
extracts was carried out using a different eluent:
MeCN/H2O/H3PO4 (25 : 75 : 0.2). Flavone aglycones, luteolin and jaceosidin which wereinferred from their UV spectra and retentiontimes, were detected at trace levels. The phenolic
composition in the leaves of A. kitadakensis wasapproximately the same within each population(Mt. Kita-dake and Mt. Senmai-dake). Plant collections in Mt. Kita-dake were carried out in2015 and 2019, but there was no difference inphenolics composition between two years.
Main phenolics in the leaves of A. kitadakensis
were ortho-dihydroxylated compounds. Thisaccumulation pattern was quite similar to otheralpine species such as Campanula chamissonis,C. lasiocarpa Cham. (Campanulaceae) (Murai etal., 2014) and Pedicularis chamissonis Steven(Orobanchaceae) (Murai and Iwashina, 2015) inJapan. Such phenolics may act as anti-stresscompounds against UV radiation and oxidativestresses in harsh alpine environment. Interestingly, total phenolic content of A. kitadakensis at
alpine habitat (Mt. Kita-dake, n＝3) was 1.8
times higher than that of plants cultivated in aglasshouse (Tsukuba Botanical Garden, n＝3) byHPLC analysis. This quantitative variation ofphenolic compounds between natural habitat(alpine zone) and cultivation site (lowland) waspreviously observed in Geum calthifolium var.
nipponicum and C. chamissonis, which arewidely distributed in the Japanese alpine zone
(Murai and Iwashina, 2010; Murai et al., 2014).
It is also to be noted that the glasshouse cultivation of A. kitadakensis led to a 66% decreasein leaf ﬂavonoid content, compared to natural
habitat.
The synthesis of phenolic compounds isaffected by several environmental factors, e.g.
low temperature and UV radiation (Dixon andPaiva, 1995). Phenolics play important roles inchemical defenses of plants (Lattanzio et al.,

Phenolic compounds from Artemisia kitadakensis

2006). In the present study, low temperature andUV radiation were almost excluded under glasshouse conditions. Because of the lower concentrations of phenolics, the plants cultivated inglasshouse may become vulnerable to some environmental stresses such as insect pests andpathogen attacks (Mazza et al., 1999; Lattanzioet al., 2006). Further studies (e.g. physiologicalexperiment) are required for understanding theactual functions of phenolic compounds in A.
kitadakensis.
Acknowledgments
This work was supported by a project of “Integrated Analysis of Natural History Collections
for Conservation of Highly Endangered Species”
conducted by the National Museum of Natureand Science, Tokyo.
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