Bull. Natl. Mus. Nat. Sci., Ser. B, 41(3), pp. 131–136, August 21, 2015

Phenolic Compounds in the Leaves of Pedicularis chamissonis in Japan
Yoshinori Murai* and Tsukasa Iwashina
Department of Botany, National Museum of Nature and Science,
Amakubo 4–1–1, Tsukuba, Ibaraki 305–0005, Japan* E-mail: murai@kahaku.go.jp(Received 18 May 2015; accepted 24 June 2015)

Abstract We analyzed phenolic compounds in the leaves of Pedicularis chamissonis which wereobtained from a wide range of Japan. Acteoside and luteolin 7-O-glucuronide were found as major
compounds in the all plants from various populations. Luteolin 7-O-glucoside was additionallyfound in the plants sampled from in the southern part of their habitats. Fujii et al. (2013) divided
the plants into two species, i.e., P. chamissonis (Northern type) and P. japonica (Central Honshutype) based on DNA, morphological and ecological data. Our chemical data seem to support thisclassiﬁcation from the view point of chemotaxonomy. In addition, ecological signiﬁcance of theisolated compounds is also described.
Key words : chemical ecology, chemotaxonomy, ﬂavonoids, Pedicularis chamissonis, Pedicularis
japonica, phenylpropanoid.

Introduction
The genus Pedicularis (Orobanchaceae) consists of ca. 600 species that is mainly distributed
in mountain area of Central and East Asia (Mabberley, 2008). Of their species, P. chamissonis
Steven is a perennial herb and widely distributedin subalpine and alpine zone of Japan. The plantshows a variety of morphological features, andthe diversity resulting in several intraspeciﬁctaxa (Yamazaki, 1993). Fujii et al. (1997) havereported that the two major clades of the species,i.e., Central Honshu clade and Northern clade bygene phylogeny using chloroplast DNA. Subsequently, they suggested that the plants should betreated Central Honshu type as P. japonica andNorthern type as P. chamissonis from the evidence obtained of genetic, morphological andecology data (Fujii et al., 2013).
Phenolic compounds such as ﬂavonoids andphenylpropanoids are known as UV protectants,antioxidants and so on. The intensity of UV radiation markedly increases at high altitude (Shibata1992; Murai et al., 2009). Plants that occupy thehighland area are thought to be adaptive to several environmental stresses (e.g. intense UV-Band cold) by accumulating UV-absorbing compounds such as ﬂavonoids and phenylpropanoids(Spitaler et al., 2006; Murai et al., 2009, 2015).
In addition, these compounds are also used as
chemical markers of plant species. Some studies
have been carried out on phenolics of Pedicularis (Yoshitama et al., 1980; Zimin andZhongjian, 1991; Di et al., 2004; Chu et al.,2007), but the number of related study is limited.
In this study, we analyzed P. chamissonis
growing in Central Japan to Hokkaido for theirphenolic compounds, and describe the chemotaxonomical and ecological signiﬁcance of theircompounds.
Materials and Methods
Plant materials
Pedicularis chamissonis was collected from
Nagano to Hokkaido (S1–S8), Japan (Fig. 1),

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Extraction and separation
Fresh leaves of each individual of P. chamissonis (1.5 g) were extracted with MeOH. The concentrated extracts were applied to preparative
paper chromatography using solvent systems:
BAW (n-BuOH/HOAc/H2O＝4 : 1 : 5, upperphase) and 15% HOAc. The compounds werepuriﬁed by Sephadex LH-20 column chromatography (solvent system: 70% MeOH).

Fig. 1. Collection sites of Pedicularis chamissonis. S1: Mt. Hoken-dake. S2: Mt. Norikuradake. S3: Mt. Arakawa-naka-dake. S4: Mt.
Jizo-dake. S5: Mt. Gassan. S6: Mt. Chokai. S7:Mt. Hayachine. S8: Mt. Shari-dake.

i.e., S1: Mt. Hoken-dake, Kiso Mountains,
Nagano Prefecture, S2: Mt. Norikura, Hida
Mountains, Nagano Prefecture, S3: Mt. Arakawa-naka-dake, Akaishi Mountains, Shizuoka
Prefecture, S4: Mt. Jizo-dake, Akaishi Mountains, Yamanashi Prefecture, S5: Mt. Gassan,Dewa Mountains, Yamagata Prefecture, S6: Mt.
Chokai-san, Dewa Mountains, Yamagata Prefecture, S7: Mt. Hayachine, Iwate Prefecture, andS8: Mt. Shari-dake, Hokkaido, in summer of2011-2014. Plant collections were carried out
under the permissions of each prefecture, theMinistry of Environment, the Ministry of Agriculture, Forestry and Fisheries, and the Agency
for Cultural Affairs, Japan, and Tokushu Tokai
Paper Co., Ltd. and Dewa Sanzan Shrine.
Voucher specimens were deposited in the Herbarium of National Museum of Nature and Science, Japan (TNS).

Quantitative HPLC analysis of phenolic compounds
Fresh leaves (0.2 g) from three individuals ofeach population were extracted with 4 ml MeOH.
After ﬁltration with Maisyoridisc H-13-5(Tosoh), the extracts were analyzed using a Shimadzu HPLC system with L-column2 ODS (5μm particle material, I.D. 6.0×150 mm, Chemicals Evaluation and Research Institute, Japan), at
ﬂow-rate: 1.0 mL min−1, detection: 190–400 nm
and eluents: MeCN/H2O/H3PO4 (20 : 80 : 0.2),
injection: 10 μL.
Liquid chromatograph-mass spectra (LC-MS)
LC-MS were measured with Shimadzu
LC-MS system using L-column2 ODS (3 μm particle material, I.D. 2.1×100 mm, Chemicals
Evaluation and Research Institute), at a ﬂow-rate
of 0.2 mL min−1, eluting with HCOOH/MeCN/
H2O (1 : 20 : 79), injection: 3 μL, ESI＋ 4.5 kV,
ESI− −3.5 kV, 250°C.
Identiﬁcation of compounds
In this research, one phenylpropanoid, acteoside (1) (Fig. 3), and two ﬂavonoids, 7-O-glucoside (2) and 7-O-glucuronide (3) of luteolin (Fig.
4), were identiﬁed by UV spectroscopy (Mabryet al., 1970), acid hydrolysis, LC-MS and HPLC
comparisons with authentic standards from theleaves of Plantago spp. (Murai et al., 2008,2009). UV, acid hydrolysis, HPLC and LC-MS
data of the isolated compounds are as follows.
Acteoside (verbascoside) (1). White powder.
UV: λmax (nm) MeOH 292, 330; ＋NaOMe 380;
＋AlCl3 300, 361; ＋AlCl3/HCl 292, 330;
＋NaOAc 290, 340; ＋NaOAc/H3BO3 295, 355.

Phenolic Compounds from Pedicularis chamissonis

133Fig. 2. Pedicularis chamissonis in Mt. Norikura, Nagano Prefecutre (left) and Mt. Chokai, Yamagata Prefecture(right).

Fig. 3. Chemical structure of acteoside from
Pedicularis chamissonis.

Fig. 4. Chemical structures of ﬂavones from
Pedicularis chamissonis. 2: luteolin 7-O-glucoside (R＝glucosyl), 3: luteolin 7-O-glucuronide(R＝glucuronyl).

HPLC: Rt 7.4 min. LC-MS: m/z 647 [M＋Na]＋,623 [M−H]−.
Luteolin 7-O-glucoside (2). Pale yellow powder. UV: λmax (nm) MeOH 255, 267, 343;
＋NaOMe 267, 385 (inc.); ＋AlCl3 273, 426;
＋AlCl3/HCl 262sh, 276, 297 362, 392sh;＋NaOAc 260, 400; ＋NaOAc/H3BO3 260, 373.
HPLC: Rt 8.0 min. Acid hydrolysis: luteolin andglucose. LC-MS: m/z 449 [M＋H]＋, 447[M−H]− (luteolin＋1 mol glucose), and 287[M−162＋H]＋, 285 [M−162−H]− (luteolin).
Luteolin 7-O-glucuronide (3). Pale yellow
powder. UV: λmax (nm) MeOH 256, 268, 348;
＋NaOMe 266, 389 (inc.); ＋AlCl3 274, 296sh,427; ＋AlCl3/HCl 263sh, 272, 295, 364, 386sh;＋NaOAc 260, 401; ＋NaOAc/H3BO3 260, 373.
HPLC: Rt 8.6 min. Acid hydrolysis: luteolin andglucuronic acid. LC-MS: m/z 463 [M＋H]＋, 461[M−H]＋ (luteolin＋1 mol glucuronic acid), and287 [M−176＋H]＋, 285 [M−176−H]− (luteolin).

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Yoshinori Murai and Tsukasa Iwashina

Fig. 5. HPLC patterns of MeOH extracts from Pedicularis chamissonis. (a) Central Honshu type. (b) Northerntype. 1: acteoside, 2: luteolin 7-O-glucoside, 3: luteolin 7-O-glucuronide.

Results and Discussion
The composition of phenolic compounds
One phenylpropanoid, acteoside (1) and twoﬂavones, luteolin 7-O-glucoside (2) and7-O-glucuronide (3) were isolated from theleaves of P. chamissonis. However, the presenceor absence of 2 is different between the northernand southern populations (Fig. 5; Table 1). Compound 2 was exclusively produced by the plants
sampled from S1 (Mt. Hoken-dake) to S5 (Mt.

Gassan), while the other populations locating in
the more northern part of Japan lacked this compound. The other peaks on HPLC chromatograms (Fig. 5) were presumed as other phenylpropanoids and ﬂavonoids from their UV spectraobtained by photo diode array HPLC detector,
and they were not identiﬁed in this study. The
concentration of these minor compounds wasvaried in low level among populations.

Phenolic Compounds from Pedicularis chamissonis
Table 1.

135

Phenolic compounds and DNA type of Pedicularis chamissonis

Population
Mt. Hoken-dake (S1)
Mt. Norikura-dake (S2)
Mt. Arakawa-naka-dake (S3)
Mt. Jizo-dake (S4)
Mt. Gassan (S5)
Mt. Chokai (S6)Mt. Hayachine (S7)Mt. Shari-dake (S8)

Phenolic compounds
12

3＋＋＋＋＋＋＋＋＋＋＋＋＋−−−＋＋＋＋＋＋＋＋

DNA type*CHTCHTCHTCHTCHT/NTCHT/NTNTNT

1: acteoside, 2: luteolin 7-O-glucoside, 3: luteolin 7-O-glucuronide. ＋: presence. −: absence.
*According to Fujii et al. (2013). CHT: Central Honshu type, NT: Northern type.

Flavonoids and phylogeny of Pedicularis chamissonis
Flavonoids are more stable chemotaxonomic
marker than phenylpropanoids among phenolic
compounds (Sahin et al., 2007). Two chemotypes(presense or absence of 2) were found in P.
chamissonis of Japan. These chemotypes seem tosupport the classiﬁcation by Fujii et al. (2013)that divided P. chamissonis in Japan into P.
japonica (Central Honshu type) and P. chamissonis (Northern type). According to Fujii et al.
(2013), two species, P. japonica and P. chamissonis are both distributed in Mt. Gassan and Mt.
Chokai. In the present study, we collected theplants from their large population in each mountain and another chemotype (species) was notdetected in HPLC analysis, but some plants atthe other populations in those mountains might
possess the opposite chemotype.
Ecological signiﬁcanceIn this study, acteoside and luteolin glycosides
were isolated from Pedicularis chamissonis.
Both phenolics are ortho-dihydroxylated phenolics that are strong antioxidants among related
compounds (Rice-Evans et al., 1996; Pietta,2000; Nishibe, 2002). Interestingly, several
plants growing in higher altitudes synthesize
such ortho-dihydroxylated phenolics (Spitaler etal., 2006; Murai and Iwashina, 2010; Murai etal., 2009, 2015). These compounds may play animportant role in protection against several environmental stresses (e.g. intense UV light andcold) in high altitudes. Further studies, e.g. physiological experiment, using alpine plants arerequired for the understanding of their functions.
Acknowledgments
This work was partially supported by JSPS
KAKENHI Grant No. 24570035 (Representative,T. Iwashina).
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