Bull. Natl. Mus. Nat. Sci., Ser. B, 40(3), pp. 113–118, August 22, 2014

Flavonoids from Two Alpine Campanula Species in Japan
Yoshinori Murai1*, Junichi Kitajima2 and Tsukasa Iwashina1
1

Department of Botany, National Museum of Nature and Science,
Amakubo 4–1–1, Tsukuba, Ibaraki 305–0005, Japan
2
Laboratory of Pharmacognosy, Showa Pharmaceutical University,
Higashi-tamagawagakuen 3, Machida, Tokyo 194–8543, Japan*E-mail: murai@kahaku.go.jp(Received 19 May 2014; accepted 25 June 2014)

Abstract Two alpine Campanula species, C. lasiocarpa and C. chamissonis, were surveyed forﬂavonoids in their leaves. Two major ﬂavones, 7-O-gentiobioside and 7-O-glucoside of luteolin,
and two minor ﬂavones, apigenin 7-O-glucoside and luteolin were isolated from C. lasiocarpa. On
the other hand, 3-O-galactoside, 3-O-glucoside and 3-O-glucuronide of quercetin, and 3-O-galactoside and 3-O-glucoside of kaempferol were isolated from C. chamissonis. These species are distantly-related from the standpoint of chemotaxonomy. In addition, ecological signiﬁcance of them
was also described.
Key words : Campanula chamissonis, Campanula lasiocarpa, chemical ecology, chemotaxonomy, ﬂavonoids.

Introduction
The genus Campanula consists of ca. 300 species that is distributed in temperate to tropicalzone, and is famous as bellﬂowers (Mabberley,2008). Of their species, C. lasiocarpa Cham. andC. chamissonis Al.Fedr. exclusively occur inalpine habitats in Japan (Fig. 1). Since both species possess similar morphology and growthenvironment, they were classiﬁed as the same
section within the genus Campanula (Okazaki,1997). In recent years, gene phylogeny, however,revealed that they were divided into different
well-supported clades each other (Roquet et al.,2008; Wendling et al., 2011).
In the late 20th century, 45 Campanula species
have been investigated for their ﬂavonoids byTeslovʼs and Dzhumyrkoʼs groups (e.g., Dzhumyrko et al., 1969; Teslov and Blinova, 1972,1974; Dzhumyrko, 1973, 1985; Teslov, 2000).
Over 50 ﬂavonoids were isolated from Campan-ula species by the studies mentioned above, andsome of them exhibited antioxidant activity(Touafek et al., 2011).
On the other hand, ﬂavonoids are one of themost effective UV-absorbing compounds that aresynthesized in plants (Caldwell et al., 1983).
These compounds can absorb both UV-B (280–320 nm) and UV-A (320–400 nm) regions.
Hashiba et al. (2006) have been surveyed thefoliar ﬂavonoid composition of Campanulapunctata Lam. in relation to its UV-absorbing
property using coastal (higher intensities ofUV-B) and inland (lower intensities of UV-B)
populations in Suzaki Peninsula, Shizuoka Prefecture, Japan. The work concluded that there
was little relationship between UV-intensities
and ﬂavonoid contents in the leaves of C. punctata sampled from coastal and inland populations. However, the intensity of UV radiation
markedly increases at high altitude (Shibata,1992; Murai et al., 2009). Plants that occupy theYoshinori Murai et al.

114

Fig. 1.

Campanula lasiocarpa (left) and C. chamissonis (right).

highland 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 phenolic acids(Spitaler et al., 2006; Murai et al., 2009).
In this study, we analyzed two alpine Campanula species for their ﬂavonoids, and describe thechemotaxonomical and ecological signiﬁcance of
their compounds.
Materials and Methods
Plant materials
Campanula lasiocarpa was collected from Mt.
Norikura, Hida Mountains, Nagano Prefecture,
Japan in September 2005, August 2006 andAugust 2011, Mt. Chokai, Asahi Mountains,Yamagata Prefecture and Mt. Aka-dake, Daisetsu
Mountains, Hokkaido, Japan in August 2012. On
the other hand, Campanula chamissonis was collected from Mt. Norikura and Mt. Hoken-dake,
Kiso Mountains, Nagano Prefecture, Japan in
July–August 2011, Mt. Gassan, Dewa Mountains, Yamagata Prefecture and Mt. Shari-dake,Shiretoko Peninsula Range, Hokkaido, Japan inAugust 2012. Plant collections were carried out
under the permissions of Nagano, Yamagata andHokkaido Prefectures, and the permission of theMinistry of Environment, Japan (No. 050823005,No. 060719001, No. 110624004, No. 120720004
and No. 120620003). Voucher specimens weredeposited in the Herbarium of National Museumof Nature and Science, Japan (TNS).
Extraction and separation
Fresh leaves of C. lasiocarpa (1.2 g), C.
chamissonis (2.1 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), and further
isolated by preparative HPLC using Inertsil
ODS-4 column (5 μm particle material, I.D.
10×250 mm, GL Sciences Inc., Japan), at ﬂowrate: 3.0 ml min−1, detection: 350 nm, and eluent:
MeCN/H2O/HCOOH (18:81:1).
Quantitative HPLC analysis of ﬂavonoids
Fresh leaves (0.2 g) of each population innature and the plants grown in Tsukuba, Ibaraki
Prefecture, Japan 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(3 μm particle material, I.D. 3.0×150 mm,
Chemicals Evaluation and Research Institute,Japan), at ﬂow-rate: 0.3 ml min−1, detection:
190–400 nm and eluents: MeCN/H2O/H3PO4(16:84:0.2), injection: 2 μl.
Liquid chromatograph-mass spectra (LC-MS)
LC-MS were measured with Shimadzu

Flavonoids from Two Alpine Campanula Species in Japan

LC-MS system using 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 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, nine ﬂavonoids from theleaves of C. lasiocarpa and C. chamissonis wereidentiﬁed. Flavonoids 1, 2, 5–7 were characterized by UV spectroscopy according to Mabry etal. (1970), LC-MS, HPLC comparisons withauthentic standards and characterization of acidhydrolysates (in 12% HCl, 100°C, 30 min). In thecase of ﬂavonoid 1, its chemical structure wasfurther estimated by 1H and 13C NMR. Flavonoids 3, 4, 8 and 9 were minor compounds andidentiﬁed by HPLC comparison with authentic
specimens and LC-MS. UV, acid hydrolysis,LC-MS, 1H and 13C NMR data of the isolated
compounds are as follows.
Luteolin 7-O-gentiobioside (1). Pale yellow
powder. UV: λmax (nm) MeOH 256, 266, 350;
＋NaOMe 266, 389 (inc.); ＋AlCl3 273, 427;
＋AlCl3/HCl 267sh, 270, 295, 362, 382sh;＋NaOAc 260sh, 402; ＋NaOAc/H3BO3 258,373. HPLC: Rt 7.7 min. Acid hydrolysis: luteolinand glucose. LC-MS: m/z 611 [M＋H]＋, 609[M−H]− (luteolin ＋ 2 mol glucose), 449[M−162＋H]＋ (luteolin ＋ 1 mol glucose), and287 [M−324＋H]＋, 285 [M−324−H]− (luteolin). 1H NMR (600 MHz, pyridine-d5): δ 13.64(1H, s, 5-OH), 8.02 (1H, d, J＝2.2 Hz, H-2′),
7.60 (1H, dd, J＝2.2 and 8.4 Hz, H-6′), 7.43 (1H,d, J＝8.3 Hz, H-5′), 7.07 (1H, d, J＝2.1 Hz, H-8),
6.91 (1H, s, H-3), 6.91 (1H, d, J＝2.2 Hz, H-6),
5.76 (1H, d, J＝7.7 Hz, i-glucosyl H-1), 5.09
(1H, d, J＝7.7 Hz, t-glucosyl H-1), 4.83 (1H, d,
J＝10.5 Hz, i-glucosyl H-6a), 4.55 (1H, dd,J＝2.1 and 11.8 Hz, t-glucosyl H-6a) 4.41 (1H,m, t-glucosyl H-3), 4.40 (1H, m, i-glucosylH-6b), 4.39 (1H, m, i-glucosyl H-4), 4.38 (1H, m,i-glucosyl H-3), 4.36 (1H, m, t-glucosyl H-6b),4.29 (1H, m, i-glucosyl H-2), 4.28 (1H, m, t-glucosyl H-4), 4.27 (1H, m, t-glucosyl H-5), 4.17(1H, t, J＝16.5 Hz, t-glucosyl H-2), 3.95 (1H, m,115

i-glucosyl H-5). 13C NMR (150 MHz, pyridined5): δ (luteolin) 162.5 (C-2), 104.1 (C-3), 182.9(C-4), 164.1 (C-5), 100.8 (C-6), 165.5 (C-7),
95.6 (C-8), 157.9 (C-9), 106.6 (C-10), 122.6(C-1′), 117.0 (C-2′), 147.7 (C-3′), 152.0 (C-4′),
114.7 (C-5′), 120.0 (C-6′); δ (i-glucose) 102.1(C-1), 74.7 (C-2), 77.5 (C-3), 70.8 (C-4), 78.6(C-5), 69.9 (C-6); δ (t-glucose) 105.6 (C-1), 75.2(C-2), 78.2 (C-3), 71.7 (C-4), 78.4 (C-5), 62.7(C-6).
Luteolin 7-O-glucoside (2). Pale yellow powder. UV: λmax (nm) MeOH 255, 266, 348;
＋NaOMe 266, 389 (inc.); ＋AlCl3 273, 427;
＋AlCl3/HCl 266sh, 272, 294, 360, 383sh;＋NaOAc 259, 402; ＋NaOAc/H3BO3 259, 372.
HPLC: Rt 13.8 min. Acid hydrolysis: luteolin andglucose. LC-MS: m/z 449 [M＋H]＋, 447[M−H]− (luteolin ＋1 mol glucose) and 287[M−162＋H]＋ (luteolin).
Apigenin 7-O-glucoside (3). HPLC: Rt
24.7 min. LC-MS: m/z 433 [M＋H]＋, 431[M−H]− (apigenin ＋ 1 mol glucose) and 271[M−162＋H]＋, 269 [M−162−H]− (apigenin).
Luteolin (4). HPLC: Rt 43.7 min. LC-MS: m/z287 [M＋H]＋, 285 [M−H]− (luteolin).
Quercetin 3-O-galactoside (5). UV: λmax (nm)
MeOH 257, 266sh, 360; ＋NaOMe 273, 329,412 (inc.); ＋AlCl3 274, 435; ＋AlCl3/HCl 269,299, 365sh, 405sh; ＋NaOAc 274, 325, 391;
＋NaOAc/H3BO3 262, 381. HPLC: Rt 12.1 min.
Acid hydrolysis: quercetin and galactose. LCMS: m/z 465 [M＋H]＋, 463 [M−H]− (quercetin＋1 mol galactose) and 303 [M−162＋H]＋, 301[M−162−H]− (quercetin).
Quercetin 3-O-glucoside (6). UV: λmax (nm)
MeOH 257, 267sh, 357; ＋NaOMe 272, 330,409 (inc.); ＋AlCl3 270, 434; ＋AlCl3/HCl 270,300, 365sh, 400sh; ＋NaOAc 273, 325, 400;
＋NaOAc/H3BO3 262, 297, 378. HPLC: Rt
13.0 min. Acid hydrolysis: quercetin and glucose.
LC-MS: m/z 465 [M＋H]＋, 463 [M−H]− (quercetin＋1 mol glucose) and 303 [M−162＋H]＋(quercetin).
Quercetin 3-O-glucuronide (7). UV: λmax(nm) MeOH 257, 267sh, 359; ＋NaOMe 273,329, 408 (inc.); ＋AlCl3 271, 420; ＋AlCl3/HCl

Yoshinori Murai et al.

116269, 300, 363sh, 396sh; ＋NaOAc 273, 325,391; ＋NaOAc/H3BO3 262, 296, 379. HPLC: Rt
13.2 min. Acid hydrolysis: quercetin and glucuronic acid. LC-MS: m/z 479 [M＋H]＋, 477[M−H]− (quercetin ＋ 1 mol glucuronic acid)and 303 [M−176＋H]＋ (quercetin).
Kaempferol 3-O-galactoside (8). HPLC: Rt
17.6 min. Acid hydrolysis: kaempferol and galactose. LC-MS: m/z 449 [M＋H]＋, 447 [M−H]−(kaempferol＋1 mol galactose) and 287 [M−162＋H]＋, 285 [M−162−H]− (kaempferol).
Kaempferol 3-O-glucoside (9). HPLC: Rt
21.1 min. Acid hydrolysis: kaempferol and glucose. LC-MS: m/z 449 [M＋H]＋, 447 [M−H]−(kaempferol ＋ 1 mol glucose) and 287 [M−162＋H]＋, 285 [M−162−H]− (kaempferol).
Results and Discussion
Flavonoid composition
Two major ﬂavones, luteolin 7-O-gentiobioside (1) and luteolin 7-O-glucoside (2), and twominor ﬂavones, apigenin 7-O-glucoside (3) andluteolin (4) were isolated from the leaves of C.
lasiocarpa (Fig. 2). On the other hand, threemajor ﬂavonols, quercetin 3-O-galactoside (5),quercetin 3-O-glucoside (6) and quercetin
3-O-glucuronide (7), and two minor ﬂavonols,kaempferol 3-O-galactoside (8) and kaempferol

Fig. 2. Chemical structures of ﬂavones from Campanula lasiocarpa. 1: luteolin 7-O-gentiobioside (R1＝gentiobiosyl, R2＝OH), 2: luteolin
7-O-glucoside (R1＝glucosyl, R2＝OH), 3: apigenin 7-O-glucoside (R1＝glucosyl, R2＝H), 4:luteolin (R1＝H, R2＝OH).

Fig. 3. Chemical structures of ﬂavonols fromCampanula chamissonis.
5: quercetin 3-O-galactoside (R1＝galactosyl,R2＝OH), 6: quercetin 3-O-glucoside (R1＝glucosyl, R2＝OH), 7: quercetin 3-O-glucuronide(R1＝glucuronyl, R2＝OH), 8: kaempferol
3-O-galactoside (R1＝galactosyl, R2＝H), 9:
kaempferol 3-O-glucoside (R1＝glucosyl, R2＝H).

Fig. 4. HPLC patterns of MeOH extracts from two
alpine Campanula species in Japan.
(a) C. lasiocarpa. (b) C. chamissonis. 1: luteolin 7-O-gentiobioside, 2: luteolin 7-O-glucoside, 3: apigenin 7-O-glucoside, 5: quercetin
3-O-galactoside, 6: quercetin 3-O-glucoside, 7:quercetin 3-O-glucuronide, 8: kaempferol
3-O-galactoside, 9: kaempferol 7-O-glucoside.
Peak of 4 appears at 43.7 min of C. lasiocarpa(a).

Flavonoids from Two Alpine Campanula Species in Japan

3-O-glucoside (9) were found in C. chamissonis(Fig. 3). MeOH extracts from each species of all
populations were analyzed by HPLC (Fig. 4).
Geographical chemical variation of C. chamissonis was not observed among the plants collectedfrom Mts. Norikura, Hoken-dake, Gassan andShari-dake. C. lasiocarpa collected from Mts.
Norikura, Chokai and Aka-dake also showed thesame ﬂavonoid composition. Furthermore, HPLC
proﬁles of C. lasiocarpa in Mt. Norikura in2005, 2006 and 2011 showed the same pattern.
These results suggested that ﬂavonoids are stablechemotaxonomic markers in both species.
Flavonoids and phylogeny of the genus Campanula
In this study, four ﬂavones and ﬁve ﬂavonols
were isolated from C. lasiocarpa and C. chamissonis, respectively. Though these ﬂavonoids havepreviously been reported from the aerial parts ofCampanula species (Dzhumyrko, 1973, 1974;
Teslov and Blinova, 1974; Teslov, 1976), the ﬂavonoid composition of C. lasiocarpa and C.
chamissonis were elucidated for the ﬁrst time.
Dzhumyrko (1985) suggested that ﬂavones andﬂavonols are alternatively distributed in the subsection level within the genus Campanula. Bothspecies surveyed in this study have been previously classiﬁed into the section Rapunculus(Okazaki, 1997), but the ﬂavonoid compositionof them was clearly different from each other.
The ﬂavonoid composition of C. chamissonis
was similar to that of C. puncutata, which is distributed in Japan and contains quercetin andkaempferol glycosides (Hashiba et al., 2006).
Recently, ITS and trnL-F data revealed that C.
chamissonis belongs to the Campanula s. str.
clade along with C. punctata (Roquet et al.,2008), whereas C. lasiocarpa belongs to theRapunculus clade as before (Wendling et al.,2011). C. lasiocarpa and C. chamissonis aremorphologically-resemble, and are growing insimilar environmental condition in alpine zone.
Our chemical data also suggested that both species are not closely-related within the genus
Campanula in addition to the gene phylogeny

117

mentioned above. Furthermore, four species
belonging to the Campanula s. str. contained ﬂavonol glycosides, whereas nine species belonging to the Rapunculus contained ﬂavone glycosides.
Ecological signiﬁcanceC. lasiocarpa and C. chamissonis mainly contained luteolin and quercetin glycosides, respectively. They are B-ring ortho-dihydroxy lated ﬂavonoids, which have been reported as efﬁcientantioxidants in ﬂavonoid compounds (RiceEvans et al., 1996; Pietta, 2000). Several plants
growing in higher altitude synthesize such B-ringortho-dihydroxy lated ﬂavonoids (Spitaler et al.,2006; Murai et al., 2009; Murai and Iwashina,2010). In the present study, we collected C.
chamissonis at ca. 2,600 m elevation in Mt. Norikura and then cultivated it in lowland, Tsukuba
city (25 m alt.) with the same method asdescribed by Murai and Iwashina (2010). Interestingly, total ﬂavonoid content of C. chamissonis at alpine habitat (n＝3) was 1.57 times higher
than that of the plants cultivated at low altitude(n＝3) by HPLC analysis. These compounds
may play an important role in protection against
several environmental stresses in high altitudes.
Further studies (e.g., physiological experiment)using alpine plants are required for the understanding of their functions.
Acknowledgments
Authors would like to thank Mr. Chikao
Machino for helping the plant collection in Mt.
Norikura. This work was partly supported by
JSPS KAKENHI Grant Number 24570035.
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