Bull. Natl. Mus. Nat. Sci., Ser. B, 47(2), pp. 109–114, May 21, 2021

Phenolic Compounds from Three Alpine Anaphalis Species inJapan and Bhutan
Yoshinori Murai1, *, Rinchen Yangzom2, Choki Wangmo2 and Rinchen Dorji2
1

Department of Botany, National Museum of Nature and Science,
4–1–1 Amakubo, Tsukuba, Ibaraki, 305–0005 Japan
2
National Biodiversity Centre, Ministry of Agriculture and Forests,
Post Box 875 Serbithang, Thimphu, Bhutan*E-mail: murai@kahaku.go.jp(Received 9 February 2021; accepted 24 March 2021)

Abstract Phenolic compounds of Anaphalis alpicola in Japan, and A. nepalensis and A. cooperi
in Bhutan, were surveyed. Three phenylpropanoids (chlorogenic acid, isochlorogenic acid A, andisochlorogenic acid C) were isolated together with several ﬂavonoids. A novel bioactive compound
chlorogenic acid, and its derivative isochlorogenic acid A, were the main components in Anaphalis
leaves. The ﬂavonoid composition of A. alpicola was similar to that of A. cooperi. In addition, theoverall content of phenolic compounds in the leaves of A. alpicola decreased when plants were
cultivated in a glasshouse, compared to plants grown in their natural habitat. Here, we describe thechemotaxonomic and ecological signiﬁcance of the isolated compounds.
Keywords : alpine plants, Asteraceae, chemical ecology, chemical proﬁling.

Introduction
The genus Anaphalis (Asteraceae), comprisingca. 110 species (Mabberley, 2017), is mainly distributed in Asia. Three species are found inJapan, including A. alpicola Makino (Fig. 1),
which is endemic to Japan and distributed inalpine zones from Hokkaido to Central Honshu(Koyama, 1995). In Bhutan, two Himalayan species, A. nepalensis (Spreng.) Hand.-Mazz. and A.
cooperi Grierson & Spring. (Fig. 1), are widelydistributed. Anaphalis cooperi is endemic to
Bhutan (Grierson and Long, 2001).
Anaphalis species are often used as traditional
medicinal plants. Previous phytochemical investigations of the genus have led to the identiﬁcation of a variety of compounds such as ﬂavonoids and triterpenes; several new and rare
ﬂavonoids have been reported (Ikramov et al.,
© 2021 National Museum of Nature and Science

1986; Wollenweber et al., 1993; Wu et al., 2003;Ren et al., 2008, 2009; Joshi et al., 2009; Abdullaeva et al., 2016). However, a limited number ofAnaphalis species were used for the chemical
analyses, and little is known about the composition of a major class of the phenolic compound,phenylpropanoid.
On the other hand, plants that occupy alpine
regions are thought to adapt to several environmental stresses (e.g., intense UV-B and cold) byaccumulating UV-absorbing and antioxidative
phenolics such as phenylpropanoids and ﬂavonoids (Spitaler et al., 2008; Murai et al., 2009;
Murai et al., 2015).
In the present study, we surveyed the phenolic
compounds of alpine Anaphalis species thatgrow in harsh environments, and compared thecompositions in the plants distributed in two geographically distant regions (Japan and Bhutan).

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Fig. 1.

Anaphalis alpicola in Japan (left), A. nepalensis (center) and A. cooperi (right) in Bhutan.

Materials and Methods
Plant materials
Anaphalis alpicola was collected from Mt.
Koakaishi-dake, 3,025 m elev., Shizuoka Prefecture, Japan in July 2014 and Mt. Kita-dake,
2,850 m elev., Yamanashi Prefecture, Japan in
September 2017. Plant collections of A. alpicola
were carried out under the permissions of Shizuoka and Yamanashi Prefectures, the Ministryof the Environment, Japan, and Tokushu Tokai
Paper Co., Ltd. Voucher specimens were deposited in the Herbarium of National Museum ofNature and Science, Japan (TNS). The Himalayan species, A. nepalensis and A. cooperi, werecollected from Dagala trek, 3,900–4,020 m elev.,
Thimphu, Bhutan in August 2019. Voucher specimen was deposited in the National Herbarium(THIM) of National Biodiversity Centre, Bhutan.
Furthermore, a Japanese endemic species, A.
alpicola was cultivated in a glasshouse at Tsukuba Botanical Garden, Tsukuba, Ibaraki Prefecture, Japan under the same growth conditions
according to the methods of Murai (2020).
Extraction and separation
The dried aerial parts of A. alpicola (0.8 g), A.
nepalensis (1.1 g) and A. cooperi (1.9 g) wereextracted repeatedly with MeOH. The concentrated extracts were cleaned up by InertSep C18(5 g/20 mL, GL Sciences Inc., Japan), and thenapplied to preparative high-performance liquid
chromatography (HPLC) with InertSustain
AQ-C18 column [5 µm particle material, I.D.
10×250 mm (GL Sciences Inc.)] using solvent
systems: HCOOH/MeCN/H2O＝1 : 20 : 79. The
separated compounds were puriﬁed by Sephadex
LH-20 column chromatography (solvent system:80% MeOH).
HPLC analysis of phenolic compounds
To compare the composition of phenolic compounds, the dried leaves (0.1 g) of each Anaphalis species were extracted with 2 mL MeOH. Inaddition, to survey and compare the variation ofphenolic compounds in the leaves of A. alpicola
cultivated in the natural habitat as well as in aglasshouse at lower altitude, fresh leaves (0.1 g)
were extracted with 2 mL MeOH. HPLC separation of the extracts ﬁltered with GL Chromatodisk 13N (0.45 µm pore size, GL Sciences, Inc.)
was performed with a Shimadzu Prominence
HPLC system using a SunShell PFP&C18 column [2.6 µm particle material, I.D. 4.6 mm ×100 mm (ChromaNik Technologies Inc., Japan)]at a ﬂow rate of 0.8 mL min−1, detection wavelength at 190–700 nm and H3PO4/MeCN/H2O(0.2 : 15 : 85) as the eluent. Injection volume was2 µL. HPLC chromatograms of phenolic compounds (at 340 nm) in the dried leaves of eachspecies are shown in Fig. 2. The amount of eachphenolic compound was estimated by the absor-Phenolic compounds from three Anaphalis species

111Fig. 2. HPLC chromatograms of MeOH extracts from the leaves of three Anaphalis species. (a) A. alpicola(Japan). (b) A. nepalensis (Bhutan). (c) A. cooperi (Bhutan). P1: chlorogenic acid (2.3 min), P2: isochlorogenic acid A (12.6 min), P3: isochlorogenic acid C (19.4 min), F1: quercetagetin 7-O-glucoside (4.6 min), F2:6-hydroxyluteolin 7-O-glucoside (5.1 min), and F3: scutellarein 7-O-glucoside (8.5 min).

bance at 340 nm (mAU) of HPLC.
Liquid chromatograph-mass spectra (LC-MS)
LC-MS measurement of the isolated compounds was performed with a Shimadzu LC-MS
system using a SunShell C18 column [2.6 µm
particle material, I.D. 2.1 mm × 100 mm (ChromaNik Technologies Inc., Japan)] at a ﬂow-rate
of 0.2 mL min−1, eluting with HCOOH/MeCN/
H2O (1 : 15 : 84), injection: 2 µL, ESI＋ 4.5 kV,ESI− 3.5 kV, 250°C.

Identiﬁcation of compounds
In this survey, three phenylpropanoids (P1–P3) and four ﬂavonoids (F1–F4) were isolated
from the aerial parts of three alpine Anaphalis
species. Phenylpropanoids were characterized by
UV spectroscopy, LC-MS, and HPLC comparisons with authentic samples of chlorogenic acid
(MP Biomedicals, USA), isochlorogenic acid A
and isochlorogenic acid C (ChemFaces, China).
In the case of isolated ﬂavonoids, their chemical
structures were elucidated by UV spectroscopy
according to Mabry et al. (1970), acid hydrolysates (in 12% HCl, 100°C, 30 min), LC-MS, and112

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HPLC comparisons with authentic samples ofquercetagetin 7-O-glucoside (MedChemExpress,USA), 6-hydroxyluteolin 7-O-glucoside, andscutellarein 7-O-glucoside from the leaves ofPlantago asiatica L. (Plantaginaceae) (Murai etal., 2009), and quercetin 3-O-glucoside from theaerial parts of Osyris alba L. (Santalaceae)(Iwashina et al., 2008) (TNS collection number77-5). Chemical data of the isolated compounds
are as follows.
Chlorogenic acid (3-Caﬀeolylquinic acid)(P1). White powder. UV: λmax (nm) MeOH300sh, 330. HPLC: Retention time (Rt) (min)2.3. LC-MS: m/z 355 [M＋H]＋, 353 [M – H]−.
Isochlorogenic acid A (3,5-Dicaﬀeolylquinicacid) (P2). Clear solution. UV: λmax (nm)
MeOH 301sh, 331. HPLC: Rt (min) 12.6.
LC-MS: m/z 515 [M – H]−.
Isochlorogenic acid C (4,5-Dicaﬀeolylquinicacid) (P3). Clear solution. UV: λmax (nm)
MeOH 302sh, 331. HPLC: Rt (min) 19.4.
LC-MS: m/z 515 [M – H]−.
Quercetagetin 7-O-glucoside (F1). Pale yellow
solution. UV: λmax (nm) MeOH 260, 277sh,361; ＋NaOMe 298, 375 (dec.); ＋AlCl3 282,450; ＋AlCl3/HCl 271, 393, 425sh; ＋NaOAc
259, 290sh, 370, 420sh; ＋NaOAc/H3BO3 271,280sh, 376. HPLC: Rt (min) 4.6. Acid hydrolysis: quercetagetin and glucose. LC-MS: m/z 481[M＋H]＋, 479 [M – H]− (quercetagetin ＋ 1 molglucose), 319 [M – 162 ＋ H]＋ (quercetagetin).
6-Hydroxyluteolin 7-O-glucoside (F2). Paleyellow solution. UV: λmax (nm) MeOH 255,284, 344; ＋NaOMe 263, 304, 392 (inc.);＋AlCl3 274, 301, 421; ＋AlCl3/HCl 261, 296,370; ＋NaOAc 260, 302, 392; ＋NaOAc/H3BO3262, 287, 357. HPLC: Rt (min) 5.1. Acid hydrolysis: 6-hydroxyluteolin. LC-MS: m/z 465[M＋H]＋, 463 [M – H]− (6-hydroxyluteolin ＋1 mol glucose), 303 [M – 162 ＋ H]＋ (6-hydroxyluteolin).
Scutellarein 7-O-glucoside (F3). Clear solution. UV: λmax (nm) MeOH 285, 334; ＋NaOMe311, 372 (inc.); ＋AlCl3 302, 365; ＋AlCl3/HCl301, 359; ＋NaOAc 312, 381; ＋NaOAc/H3BO3292, 331. HPLC: Rt (min) 8.5. Acid hydrolysis:

scutellarein. LC-MS: m/z 465 [M＋H]＋, 463[M – H]−.
Quercetin 3-O-glucoside (F4). Pale yellowsolution. UV: λmax (nm) MeOH 256, 267sh,356; ＋NaOMe 273, 331, 409 (inc.); ＋AlCl3274, 425; ＋AlCl3/HCl 269, 299sh, 362sh, 400;
＋NaOAc 273, 327, 394; ＋NaOAc/H3BO3 261,297sh, 377. HPLC: Rt (min) 7.9. Acid hydrolysis: quercetin and glucose. LC-MS: m/z 465[M＋H]＋, 463 [M – H]− (quercetin ＋1 mol glucose), 303 [M – 162 ＋ H]＋ (quercetin).
Results and Discussion
Three phenylpropanoids, chlorogenic acid (1),isochlorogenic acid A (2) (Fig. 3) and isochlorogenic acid C (3), and four ﬂavonoids, quercetagetin 7-O-glucoside (F1), 6-hydroxyluteolin
7-O-glucoside (F2), scutellarein 7-O-glucoside(F3) and quercetin 3-O-glucoside (F4) (Fig. 4)
were isolated from the aerial parts of three
Anaphalis species.
Compared to ﬂavonoids, Asteraceae plants
often contain phenylpropanoids predominantly intheir leaves. The same trend was observed in
Anaphalis species in this study. HPLC analysis
revealed that the main components in leaves ofAnaphalis species were chlorogenic acid (1) andisochlorogenic acid A (2). Chlorogenic acid andderivatives are well known as bioactive compounds isolated from coﬀee. In particular, chlorogenic acid is eﬀective for several chronic andFig. 3. Chemical structures of main phenylpropanoids in the leaves of three Anaphalis species. 1: chlorogenic acid (R＝H) and 2: isochlorogenic acid A (R＝caﬀeoyl).

Phenolic compounds from three Anaphalis species

Fig. 4. Chemical structures of ﬂavonoids identiﬁed
from three Anaphalis species. F1: quercetagetin
7-O-glucoside (R1＝R2＝OH, R3＝glucosyl,R4＝OH), F2: 6-hydroxyluteolin 7-O-glucoside(R1＝H, R2＝OH, R3＝glucosyl, R4＝OH), F3:scutellarein 7-O-glucoside (R1＝H, R2＝OH,R3＝glucosyl, R4＝H), and F4: quercetin 3-Oglucoside (R1＝glucosyl, R2＝ R3＝H, R4＝OH).

degenerative diseases, such as cancer, cardiovascular disorders, diabetes, and Parkinsonʼs disease(Cliﬀord et al., 2017). Moreover, chlorogenic
acid possesses strong antioxidant activity, andmay play important roles in plant cells. Recently,
chlorogenic acid has been isolated from theleaves of several alpine plants, e.g., Campanulachamissonis Fed. (Campanulaceae) (Murai et al.,2014) and Artemisia kitadakensis H. Hara etKitam. (Asteraceae) (Murai, 2020), and from theﬂowers of Himalayan black pea Thermopsis barbata Royle (Fabaceae) (Murai et al., 2017).
The presence of glycosides of quercetagetin,6-hydroxyluteolin and scutellarein were reported
from the Anaphalis genus for the ﬁrst time.
HPLC analysis revealed that F1–F3 were leaf
components accumulated in A. alpicola (Japan)and A. cooperi (Bhutan). The ﬂavonoid composition of the two species distributed in two diﬀerent regions was similar. These ﬂavonoids werenot found from A. nepalensis in this study. A.
alpicola was formerly identiﬁed as A. lactea
Maxim. (Shimizu, 1982). However, the ﬂavonoid
composition of A. alpicola was diﬀerent fromthat of A. lactea as reported by Ren et al. (2008).
Most of the studies on chemical components ofthe Anaphalis genus were carried out using
whole plants or aerial parts, and they did not distinguish between leaves and ﬂowers. F4 was not113found in the leaf extracts of the three Anaphalisspecies, but an additional HPLC survey (data notshown) detected it in ﬂower extracts.
Furthermore, the total phenolic content in theleaves of A. alpicola in the alpine habitat was 2.4
times higher than that of plants cultivated in theglasshouse according to the HPLC analysis (P ＜0.05, t-test). This quantitative variation of phenolic compounds between a natural habitat (alpinezone) and a cultivation site (lowland) was previously observed in some Japanese alpine species(Murai and Iwashina, 2010; Murai et al., 2014;
Murai, 2020). In particular, three ﬂavonoids (F1–F3) in the leaves of three Anaphalis species weredetected at trace levels from the plants grown in
the glasshouse. Since major environmental
stresses such as low temperature and UV radiation were almost excluded under glasshouse conditions, such ﬂavonoids were barely produced inAnaphalis leaves. Some of isolated phenolic
compounds may act as anti-stress compounds
against cold, UV light and oxidative stresses inharsh alpine environments. Further studies (e.g.,gene phylogeny and physiological experiments)
are required for a comprehensive understandingof the distribution and functions of phenolics inAnaphalis species.
Acknowledgments
This work was supported by the integrated
research entitled “Biological Properties of Biodiversity Hotspots in Japan” and “Discretionary
expenses of Director General” of the National
Museum of Nature and Science.
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