Dhasingreoside: New flavonoid from the stems and leaves of Gaultheria fragrantissima

Article abstract of DOI:10.1080/14786419.2014.1003932

A new flavonoid, dhasingreoside (1) and seven known compounds, quercetin 3-O-β-d-galacturonopyranoside (2), quercetin 3-O-β-d-galactopyranoside (3), quercetin 3-O-β-d-glucuronopyranoside (4), quercetin 3-O-α-l-rhamnopyranoside (5), (-)-epicatechin (6), salicylic acid (7) and gaultherin (8), have been isolated from the shade-dried stems and leaves of Gaultheria fragrantissima, commonly known as Dhasingre in Nepal. The structures were elucidated on the basis of physical, chemical and spectroscopic methods. Among known compounds, five compounds (3-6 and 8) were isolated for the first time from G. fragrantissima. In vitro antioxidant activity of all the isolated compounds was evaluated by 1,1-diphenyl-2-picrylhydrazyl free radical-scavenging assay. Dhasingreoside (1) and other compounds (2-6) showed significant free radical-scavenging activity.

Full text of DOI:10.1080/14786419.2014.1003932

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Dhasingreoside: new flavonoid from
the stems and leaves of Gaultheria
fragrantissima
Fei Conga, Khem Raj Joshiabc, Hari Prasad Devkotaab, Takashi
Watanabecd & Shoji Yaharaac
a Graduate School of Pharmaceutical Sciences, Kumamoto
University, 5-1 Oe-honmachi, Chuo ku, Kumamoto 862-0973,
Japan
b Program for Leading Graduate Schools “HIGO (Health life
science: Interdisciplinary and Glocal Oriented) Program”,
Click for updates
Kumamoto University, Kumamoto, Japan
c Faculty of Science and Technology, School of Health and Allied
Sciences, Pokhara University, P.O. Box 427 Lekhnath, Kaski, Nepal
d Research Organization for Regional Alliances, Kochi University
of Technology, 185 Miyanokuchi, Tosayamada, Kami City, Kochi
782-0003, Japan
Published online: 27 Jan 2015.
To cite this article: Fei Cong, Khem Raj Joshi, Hari Prasad Devkota, Takashi Watanabe
& Shoji Yahara (2015): Dhasingreoside: new flavonoid from the stems and leaves of
Gaultheria fragrantissima, Natural Product Research: Formerly Natural Product Letters, DOI:
10.1080/14786419.2014.1003932
To link to this article: http://dx.doi.org/10.1080/14786419.2014.1003932
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Natural Product Research, 2015
http://dx.doi.org/10.1080/14786419.2014.1003932
Dhasingreoside: new flavonoid from the stems and leaves of Gaultheria
fragrantissima
Fei Conga, Khem Raj Joshiabc, Hari Prasad Devkotaab, Takashi Watanabecd and Shoji Yaharaac*
aGraduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo ku,
Kumamoto 862-0973, Japan; Program for Leading Graduate Schools “HIGO (Health life science:
b
c
Interdisciplinary and Glocal Oriented) Program”, Kumamoto University, Kumamoto, Japan; Faculty of
Science and Technology, School of Health and Allied Sciences, Pokhara University, P.O. Box 427
Lekhnath, Kaski, Nepal; dResearch Organization for Regional Alliances, Kochi University of Technology,
185 Miyanokuchi, Tosayamada, Kami City, Kochi 782-0003, Japan
(Received 9 December 2014; final version received 30 December 2014)
A new flavonoid, dhasingreoside (1) and seven known compounds, quercetin 3-O-b-D-
galacturonopyranoside (2), quercetin 3-O-b-D-galactopyranoside (3), quercetin 3-O-b-
D-glucuronopyranoside (4), quercetin 3-O-a-L-rhamnopyranoside (5), (–)-epicatechin
(6), salicylic acid (7) and gaultherin (8), have been isolated from the shade-dried stems
and leaves of Gaultheria fragrantissima, commonly known as ‘Dhasingre’ in Nepal.
The structures were elucidated on the basis of physical, chemical and spectroscopic
methods. Among known compounds, five compounds (3–6 and 8) were isolated for the
first time from G. fragrantissima. In vitro antioxidant activity of all the isolated
compounds was evaluated by 1,1-diphenyl-2-picrylhydrazyl free radical-scavenging
assay. Dhasingreoside (1) and other compounds (2–6) showed significant free radical-
scavenging activity.
Keywords: Gaultheria fragrantissima; dhasingreoside; Dhasingre; in vitro; DPPH
1. Introduction
Gaultheria fragrantissima Wall. (Family: Ericaceae) is an evergreen aromatic shrub, about 3 m
tall, called Wintergreen in English and locally called ‘Dhasingre’ in Nepal and
‘Fangxiangbaizhu’ in China. The plant is widely distributed in East Asia from India to Nepal
and West China, Sri Lanka and North Vietnam, particularly in shaded woodland between 1200–
2600 m (Watanabe et al. 2005). The plant is aromatic, antiseptic, carminative, stimulant and also
used in rheumatic, scabies, neuralgia and arthritis treatment (Ranyaphi et al. 2012). Fruits are
*Corresponding author. Email: yaharas1@gpo.kumamoto-u.ac.jp
q 2015 Taylor & Francis

2
F. Cong et al.
eaten to cure stomachache and crushed leaves are used to treat cough (Watanabe et al. 2005).
G. fragrantissima is famous for Wintergreen essential oil, obtained by steam distillation of the
leaves (Liu et al. 2013). Wintergreen oil is externally applied in liniments to counteract their
irritating effects. Wintergreen oil is vermicide towards hookworm and also rubbed on body for
rheumatic pain. Very little chemical investigation was performed till the date on title plant. Thus,
the detailed chemical analysis and biological activity evaluation on this plant is important from
the aspects of natural product chemistry. Therefore, this study was aimed for a detailed chemical
analysis of stems and leaves of G. fragrantissima and 1,1-diphenyl-2-picrylhydrazyl (DPPH)
free radical-scavenging activity of the isolated compounds.
2. Results and discussion
Successive extraction of the shade-dried stems and leaves (60 g) of G. fragrantissima with 70%
MeOH (three times) gave total 12 g of extract. The extract was suspended in water and applied to
repeated column chromatography (CC) on MCI gel CHP20P, Sephadex LH-20, ODS and silica
gel to isolate compounds 1–8.
21
Compound 1 was obtained as a pale-yellow amorphous powder, ½aꢀD 2 41.68 (c ¼ 0.50,
CH3OH–H2O ¼ 1:1). Its molecuþlar formula was determined as C27H28O18 on the basis of an
HR-FAB-MS peak of [M þ Na] at 663.1218 (calcd for C27H28O18Na, 663.1173), indicating
the molecular weight to be 640. Compound 1 was positive towards ferric chloride reagent
(greenish colour) and gave pink colour with magnesium–hydrochloric acid (Mg–HCl) reagent,
suggesting a flavonol derivative. Acid hydrolysis of compound 1 afforded glucose and
1
galacturonic acid confirmed on the basis of co-TLC with authentic samples. The H NMR
(DMSO-d6) spectrum of compound 1 showed proton signals due to an ABX system: dH 7.64
(1H, d, J ¼ 1.8 Hz), 7.54 (1H, dd, J ¼ 1.8, 8.2 Hz), 6.85 (1H, d, J ¼ 8.2 Hz) and two meta-
coupled aromatic protons at dH 6.34 (1H, d, J ¼ 1.5 Hz) and dH 6.19 (1H, d, J ¼ 1.5 Hz) which
were assignable to 1,3,4-trisubstituted B ring and 5,7-disubstituted A ring of flavonol backbone
(Park et al. 2012). The proton signals at dH 5.35 (1H, d, J ¼ 7.3 Hz) and dH 4.59 (1H, d,
J ¼ 7.9 Hz) were assignable to two anomaric protons of two monosaccharide moieties and
coupling constants (J ¼ 7.3, 7.9 Hz) suggesting that both are in b-configuration. Moreover, two
proton signals at dH 3.71 (1H, dd, J ¼ 2.1, 12.2 Hz) and dH 3.62 (1H, dd, J ¼ 4.8, 12.2 Hz) (in
CD3OD þ D2O, 1:1) were assignable to the methylene (CH2) protons of glucopyranosyl moiety
´
(Calis et al. 1999; Andersen & Fossen 2006; Dıaz et al. 2008).
The 13C NMR (DMSO-d6) spectra of compound 1 showed signals equivalent to a total of 27
carbons, in which 15 carbon signals at dC (177.3, 164.1, 161.0, 156.1, 155.6, 148.4, 144.7, 133.0,
121.6, 120.9, 116.2, 115.3, 103.7, 98.5, 93.4) were assignable to a 3-O-glycosyl substituted
quercetin moiety (Markham and Chari 1982). Other characteristic signals of the 13C NMR and
distortionless enhancement by polarisation transfer spectra displayed the presence of C-200-
substituted glucopyranosyl [98.3 (CH), 82.3 (CH), 76.6 (CH), 76.4 (CH), 69.4 (CH), 60.5 (CH2)]
(Andersen & Fossen 2006; Wang et al. 2010) and a galacturonopyranosyl unit [104.1 (CH), 71.3
(CH), 74.2 (CH), 69.4 (CH), 76.4 (CH), 171.0 (ZOZCvO)]. Substitution by glucopyranosyl
moiety at C-3 position of quercetin was supported by the downfield shift of C-2 carbon signal
(Markham and Chari 1982). Connectivities of galacturonopyranosyl moiety with C-200 position
of glucopyranose was supported by glycosidation shifts, showing an a-effect on C-200 and b-
effects on C-100 and C-300 (Faizi et al. 2011). Thus, C-200 resonated at a higher frequency (dC 82.3)
and C-100 and C-300 appeared at lower frequencies (dC 98.3 and dC 76.4, respectively). From the
1H–1H correlation spectroscopy (COSY), the anomaric glucopyranosyl proton signal at dH 5.35
(Glc-1H) was correlated with the proton signal at dH 3.58 (Glc-2H). From the heteronuclear
multiple quantum coherence (HMQC) spectrum, these two protons, dH 5.35 and 3.58, were
correlated to the carbons at dC 98.3 (Glc-1C) and 82.3 (Glc-2C), respectively. This was also

Natural Products Research
3
supported by the upfield of anomaric carbon Glc-1C (dC 98.3) and downfield shift of Glc-2C (dC
82.3). The anomaric proton of galacturonopyranosyl moiety at dH 4.59 (1H, d, J ¼ 7.9 Hz) was
correlated to the anomaric carbon at dC 104.1 by the HMQC spectrum. Heteronuclear multiple
bond correlation (HMBC) correlated to the anomaric proton of GalA-1H (dH 4.59) with Glc-2C
(dC 82.3). Thus, the complete assignment of the proton and carbon atoms, their positions, and the
linkage between the glucopyranosyl and galacturonopyranosyl moiety in the compound 1 were
determined on the basis of chemical shifts, 1H–1H COSY, HMQC and HMBC spectra. Thus, from
the physical and spectral data, the structure of compound 1 was elucidated as quercetin 3-O-(200-O-
b-D-galacturonopyranosyl)-b-D-glucopyranoside and named as dhasingreoside (Figure 1).
The structures of known compounds were elucidated on the basis of their physical and
spectral data and comparison with literature values as quercetin 3-O-b-D-galacturonopyranoside
(2) (Hansen et al. 1999), quercetin 3-O-b-D-galactopyranoside (3) (Bouktaib et al. 2002),
quercetin 3-O-b-D-glucuronopyranoside (4) (Markham and Chari 1982), quercetin 3-O-a-L-
rhamnopyranoside (5) (Lu & Foo 1997), (–)-epicatechin (6) (Zhang et al. 2012), salicylic acid
(7) and gaultherin (8) (Moon et al. 1996).
All of the isolated compounds were tested for their in vitro antioxidant activity towards
DPPH free radical-scavenging assay. The 50% inhibition concentration (IC50) for DPPH radical-
scavenging activity was calculated and provided in Table 1. The results were compared with
Trolox as a positive control (IC50, 96.1 mM). Dhasingreoside (1), quercetin 3-O-b-D-
galacturonopyranoside (2), quercetin 3-O-b-D-galactopyranoside (3), quercetin 3-O-b-D-
glucuronopyranoside (4), quercetin 3-O-a-L-rhamnopyranoside (5) and (–)-epicatechin (6)
showed potent free radical-scavenging activity. These findings were similar to the previous
reports regarding the free radical-scavenging activity of flavonoids (Okawa et al. 2001; Cho
et al. 2003). Thus, the presence of such strong antioxidants in the stems and leaves of
G. fragrantissima might be able to protect against oxidative damage.
3. Experimental
3.1. Instruments and chemicals
Optical rotations were measured with a JASCO DIP-1000KUY polarimeter (JASCO
Corporation, Tokyo, Japan). NMR spectra were measured on a JEOL JNM a-500 spectrometer
Figure 1. Structures of compounds 1–8.

4
F. Cong et al.
Table 1. IC50 (mM) value of compounds 1–6 and Trolox.
Compounds
IC50 (mM)
1
2
3
4
41.6
48.6
51.4
54.6
48.6
138.3
96.1
5
6
Trolox
(JEOL Ltd, Tokyo, Japan) (1H: 500 MHz and 13C: 125 MHz). Chemical shifts are given in ppm
with reference to TMS. Mass spectra were recorded on a JEOL JMS 700 MStation mass
spectrometer (JEOL Ltd., Tokyo, Japan). CC was carried out with silica gel 60 (0.040–
0.063 mm, Merck, Tokyo, Japan), MCI gel CHP20P (75–150 mm, Mitsubishi Chemical
Industries Co., Ltd, Tokyo, Japan), Sephadex LH-20 (Amersham Pharmacia Biotech, Tokyo,
Japan) and Chromatorex ODS (30–50 mm, Fuji Silysia Chemical Co., Ltd, Aichi, Japan). TLC
was performed on a precoated silica gel 60 F254 (0.2 mm, aluminium sheet, Merck). Authentic
sample of sugars were obtained from Sigma-Aldrich, Tokyo, Japan. DPPH was used from Wako
Pure Chemicals, Osaka, Japan. Trolox was purchased Calbiochem, Tokyo, Japan, and 2-(N-
morpholino)ethanesulphonic acid (MES) buffer was obtained from Dojindo Chemical Research,
Kumamoto, Japan.
3.2. Plant material
The fresh stems and leaves of G. fragrantissima were collected from Nagarkot, Bhaktapur,
Nepal in July 2014. The voucher specimen (Voucher No.: KUNP20140709-002) was deposited
on Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan.
3.3. Extraction and isolation
The shade-dried stems and leaves (60 g) were extracted successively with 70% MeOH (3 L
three times), at 558C (5 h) and room temperature (208C, for 22 h) each time. The combined
extract was evaporated under reduced pressure to give 12 g extract. The extract (21 g) was
suspended in water and was subjected to MCI gel CHP20P CC and eluted successively with
water, 40%, 60%, 80% and 100% MeOH and CHCl3 to give 13 fractions (1–13). Fraction 3
(3.15 g, 40% MeOH eluate) was subjected to Sephadex LH-20 CC (50% MeOH) to give five
subfractions (3-1–3-5). Fraction 3-2 (399 mg) was subjected to ODS CC (H2O, 5–8% MeOH)
to give 11 subfractions (3-2-1–3-2-11). Fraction 3-2-3 was obtained as compound 7 (12 mg).
Fraction 3-2-10 (33 mg, 8% MeOH eluate) was applied on Sephadex LH-20 CC (H2O)
followed by MCI gel CHP20P CC to afford compound 1 (10 mg). Fraction 4 (3.01 g, 40%
MeOH eluate) was subjected to Sephadex LH-20 CC (50% MeOH) to give six subfractions (4-
1–4-6). Fraction 4-2 (1.31 g) was subjected to ODS CC (10–15% MeOH) to give six
subfractions (4-2-1–4-2-6). Fraction 4-2-6 (1.13 g, 15% MeOH eluate) was applied on silica
gel CC (CHCl3–MeOH–H2O ¼ 8:2:0.1) to afford compound 8 (165 mg). Fraction 4-5
(123 mg) was applied on ODS CC (15% MeOH) to afford compound 4 (17 mg). Fraction 4-6
(208 mg) was applied on ODS CC (25% MeOH) to afford compounds 2 (3 mg) and 6 (36 mg).
Fraction 7 (424 mg, 60% MeOH eluate) was subjected to Sephadex LH-20 CC (MeOH) to
afford compound 3 (57 mg). Similarly, fraction 9 (243 mg, 60% MeOH eluate) was subjected
to Sephadex LH-20 CC (MeOH) to afford compound 5 (29 mg).

Natural Products Research
5
3.4. Dhasingreoside (1)
21
Pale-yellow amorphous powder; ½aꢀD 2 41.68 (c ¼ 0.50, CH3OH–H2O ¼ 1:1); HR-FAB-MS
þ
(positive mode) [M þ Na]
at 663.1218 (calcd for C27H28O18Na, 663.1173); 1H NMR
(500 MHz, DMSO-d6): d 7.64 (1H, d, J ¼ 1.8, H-20), 7.54 (1H, dd, J ¼ 1.8, 8.2, H-60), 6.85
(1H, d, J ¼ 8.2, H-50), 6.34 (1H, d, J ¼ 1.5, H-8), 6.13 (1H, d, J ¼ 1.5, H-6), 5.65 (1H, d,
J ¼ 7.3, Glc H-1), 4.59 (1H, d, J ¼ 7.9, GalA H-1), 3.58 (1H, t, J ¼ 7.3, Glc H-2), 3.46 (1H, dd,
J ¼ 4.5, 11.6, Glc H-6a), 3.12–3.55 (7H, m, Glc H-3, Glc H-4, Glc H-5, Glc H-6b, GalA H-3,
GalA H-4, GalA H-5); 13C NMR (125 MHz, DMSO-d6): 177.3 (C-4), 171.0 (C-GalA-6), 164.1
(C-7), 161.0 (C-5), 156.1 (C-2), 155.6 (C-9), 148.4 (C-40), 144.7 (C-30), 133.0 (C-3), 121.6
(C-60), 120.9 (C-10), 116.2 (C-50), 115.3 (C-20), 104.1 (C-GalA-1), 103.7 (C-10), 98.5 (C-6), 98.3
(C-Glc-1), 93.4 (C-8), 82.3 (C-Glc-2), 76.6 (C-Glc-5a), 76.4 (C-Glc-3a), 76.2 (C-GalA-5), 74.2
a
(C-GalA-3), 71.3 (C-GalA-2), 69.4 (C-Glc-4), 69.4 (C-GalA-4), 60.5 (C-Glc-6); assignments
may be reversed.
3.5. Known compounds
20
Quercetin 3-O-b-D-galacturonopyranoside (2): pale-yellow amorphous powder, ½aꢀD 2 42.68
20
(c ¼ 0.13, CH3OH); quercetin 3-O-b-D-galactopyranoside (3): pale-yellow amorphous powder,
20
½aꢀD 2 43.58 (c ¼ 0.44, pyridine); quercetin 3-O-b-D-glucuronopyranoside (4): pale-yellow
20
amorphous powder, ½aꢀD 2 18.68 (c ¼ 0.44, CH3OH), quercetin 3-O-a-L-rhamnopyranoside
20
(5): pale-yellow amorphous powder, ½aꢀD 2 17.58 (c ¼ 0.98, CH3OH); (–)-epicatechin (6):
20
½aꢀD 2 50.38 (c ¼ 0.97, CH3OH); salicylic acid (7): white needle crystals; and gaultherin (8):
white amorphous powder, ½aꢀD 2 52.38 (c ¼ 1.14, CH3OH).
3.6. Antioxidative activity
The DPPH radical-scavenging activity was measured by the method described by Li and Seeram
(2011), with slight modifications. Briefly, 80 mL of each compound at various concentrations (in
DMSO–EtOH ¼ 1:1) was mixed with 40 mL of MES buffer (200 mM, pH 6.0) and 40 mL of
DPPH solution (800 mM in EtOH) in a 96-well plate. The reaction mixture was shaken
vigorously and left for 30 min at room temperature in the dark. The antioxidative activity
corresponding to the scavenging of DPPH radicals was measured at 510 nm with a UV
spectrophotometer using the following formula:
A 2 B
Radical scavenging activity ð%Þ ¼ 100 £
;
A
where A is the control absorbance of DPPH radicals without samples and B is the absorbance
after reacting with samples. Trolox was used as the positive control. The result is expressed as
the mean of four experiments. From these data, a curve was plotted and the concentration
(mM) of the sample required for 50% reduction of the DPPH radical absorbance (IC50) was
calculated.
3.7. Acid hydrolysis of compounds 1
Compound 1 (1.0 mg) in 2 M HCl (0.5 mL) was heated at 708C for 3 h in a sealed tube. Co-TLC
in silica gel together with the authentic samples was performed using CHCl3–MeOH–H2O
(6:4:1, v/v/v) and n-BuOH–AcOEt–H2O (5:1:4, v/v/v, upper phase) as the developing solvents.
Aqueous H2SO4 (10%) was used as the detection reagent. Glucose, galacturonic acid and
quercetin were detected from compound 1.

6
F. Cong et al.
4. Conclusion
Eight phenolic compounds (1–8) were isolated as major constituents of leaves and stems of G.
fragrantissima: a new compound, dhasingreoside (1) and seven known compounds; quercetin 3-
O-b-D-galacturonopyranoside (2), quercetin 3-O-b-D-galactopyranoside (3), quercetin 3-O-b-
D-glucuronopyranoside (4), quercetin 3-O-a-L-rhamnopyranoside (5), (2)-epicatechin (6),
salicylic acid (7) and gaultherin (8). Among them, five compounds (3–6 and 8) were isolated for
the first time from G. fragrantissima. In vitro antioxidant activity of all the isolated compounds
from G. fragrantissima was evaluated by DPPH free radical-scavenging assay. New compound,
dhasingreoside (1) and other phenolic compounds (3–6) showed significant free radical
scavenging activity. Thus, these phenolic compounds may contribute to the antioxidant activities
of the leaves and stems of G. fragrantissima and might be used as natural antioxidants. This
study provides some evidences for the use of G. fragrantissima plant as traditional medicine.
Supplementary material
Supplementary material relating to this paper is available online, alongside Figures S1–S7 and
Tables S1–S8.
Acknowledgements
The authors thank KOHNAN Asia Scholarship Foundation for the scholarship to Ms Fei Cong and the
Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan for the scholarship to
Mr Khem Raj Joshi. This work was supported in part by the Program for Leading Graduate Schools ‘HIGO’
(Health life science: Interdisciplinary and Glocal Oriented), MEXT, Japan.
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