Two new diacetylene glycosides: Bhutkesoside A and B from the roots of Ligusticopsis wallichiana

Article abstract of DOI:10.1080/14786419.2015.1118635

Two new diacetylene glycosides: bhutkesoside A (1) and B (2), along with 10 known compounds, i.e. falcarindiol (3), chlorogenic acid (4), 5-O-p-coumaroyl-quinic acid (5), 3,5-di-O-caffeoyl-quinic acid (6), 4-hydroxy-7-methoxy-phenylethanol (7), ferulic acid (8), dehydrodiconiferyl alcohol-4-O-β-d-glucopyranoside (9), 5,7-dihydroxy-2-methylchromone-7-O-rutinoside (10), schumanniofioside B (11) and marmesinin (12) were isolated from the roots of Ligusticopsis wallichiana (DC) Pimenov & Kljuykov (Apiaceae), commonly known as ‘Bhutkesh’ in Nepal. The structures were determined on the basis of spectroscopic data. Compounds 4 and 6 showed potent antioxidant activity on DPPH free radical scavenging assay.

Full text of DOI:10.1080/14786419.2015.1118635

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
Two new diacetylene glycosides: bhutkesoside A
and B from the roots of Ligusticopsis wallichiana
Bibek Adhikari, Hari Prasad Devkota, Khem Raj Joshi, Takashi Watanabe &
Shoji Yahara
To cite this article: Bibek Adhikari, Hari Prasad Devkota, Khem Raj Joshi, Takashi Watanabe &
Shoji Yahara (2015): Two new diacetylene glycosides: bhutkesoside A and B from the roots of
Ligusticopsis wallichiana, Natural Product Research, DOI: 10.1080/14786419.2015.1118635
To link to this article: http://dx.doi.org/10.1080/14786419.2015.1118635
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Date: 21 March 2016, At: 10:56

Natural Product research, 2015
http://dx.doi.org/10.1080/14786419.2015.1118635
Two new diacetylene glycosides: bhutkesoside A and B from
the roots of Ligusticopsis wallichiana
Bibek Adhikaria,b, Hari Prasad Devkotaa,b, Khem Raj Joshia, Takashi Watanabea and
Shoji Yaharaa
aGraduate school of Pharmaceutical sciences, Kumamoto university, Kumamoto, Japan; bProgram for leading
Graduate schools, hIGo Program, Kumamoto university, Kumamoto, Japan
ARTICLE HISTORY
ABSTRACT
received 3 september 2015
accepted 10 october 2015
Two new diacetylene glycosides: bhutkesoside A (1) and B (2), along
with 10 known compounds, i.e. falcarindiol (3), chlorogenic acid
(4), 5-O-p-coumaroyl-quinic acid (5), 3,5-di-O-caffeoyl-quinic acid
(6), 4-hydroxy-7-methoxy-phenylethanol (7), ferulic acid (8), dehy-
drodiconiferyl alcohol-4-O-β-d-glucopyranoside (9), 5,7-dihydroxy-
2-methylchromone-7-O-rutinoside (10), schumanniofioside B (11)
and marmesinin (12) were isolated from the roots of Ligusticopsis
wallichiana (DC) Pimenov & Kljuykov (Apiaceae), commonly known
as‘Bhutkesh’in Nepal. The structures were determined on the basis of
spectroscopic data. Compounds 4 and 6 showed potent antioxidant
activity on DPPH free radical scavenging assay.
KEYWORDS
Ligusticopsis wallichiana;
apiaceae; Bhutkesh;
bhutkesoside a;
bhutkesoside B
1. Introduction
Ligusticopsis wallichiana (DC) Pimenov & Kljuykov (Syns. Selinum tenuifolium Wall. ex C. B.
Clarke, Selinum wallichianum (DC) Raizada & H.O. Saxena) (Family: Apiaceae), commonly
known as‘Bhutkesh’in Nepal, is a perennial aromatic herb, widely distributed in the Himalayan
region of Nepal, India, Pakistan, Bhutan and China between 2700 and 4800 m. The roots are
CONTACT hari Prasad devkota
devkotah@kumamoto-u.ac.jp; shoji Yahara
yaharas1@gpo.kumamoto-u.ac.jp
supplemental data for this article can be accessed at http://dx.doi.org/10.1080/14786419.2015.1118635.
© 2015 taylor & Francis

2
B. ADHIKARI eT Al.
used to treat body pain, fever, cough and cold (Watanabe et al. 2013) and the flowers and
leaves are used in stomach ache, cuts and wounds (Gewali 2008). The root decoction is used
for the treatment of diarrhoea, stomach ache and vomiting. The flowers and stem are used
for stimulant and carminative properties (Padalia et al. 2012). Previous studies on this plant
mainly focused on the volatile constituents of the different plant parts of L. wallichiana (Sood
et al. 1978; Dev et al. 1984; Chauhan et al. 2012; Padalia et al. 2012) but detailed characteri-
sation of non-volatile constituents is not available. Thus, in the present study, we report the
isolation and structure elucidation of two new (1, 2) and 10 known compounds (3–12) from
the roots of L. wallichiana and the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging
activity of the isolated compounds.
2. Results and discussion
The shade dried roots (370 g) were extracted with 70% MeOH at 60 °C and evaporated
under reduced pressure to give 99.2 g extract. The extract was then separated into water-
soluble and insoluble fractions. The water-soluble fraction was applied on repeated col-
umn chromatography (CC) on MCI gel CHP20P, Sephadex lH-20, ODS and silica gel to iso-
late two new compounds, bhutkesoside A (1) and B (2), along with 10 known compounds
(Figure 1). From the detailed spectral analysis and comparison with literature data, the
known compounds were identified as falcarindiol (3) (Tamura et al. 2010), chlorogenic
acid (4), 5-O-p-coumarylquinic acid (5), 3,5-di-O-Caffeoyl-quinic acid (6) (Kwon et al. 2000),
4-hydroxy-7-methoxy-phenylethanol (7) (Kim et al. 2006), ferulic acid (8) (Rollinger et al.
2003), dehydrodiconiferyl alcohol 4-O-β-glucopyranoside (9) (lundgern et al. 1981), 5,7-
dihydroxy-2-methylchromone-7-O-rutinoside(10)(Yoshimatsuetal.2007),schumanniofioside
Figure 1. structures of compounds 1–12.

NATuRAl PRODuCT ReSeARCH
3
B (11) (Tane et al. 1990) and marmesinin (12) (Kitajima et al. 1998). All of these compounds
were isolated for the first time from L. wallichiana.
Bhutkesoside A (1) was obtained as a pale yellow oil, [ꢀ]2D0 −127.8° (c = 0.28, MeOH). Its
molecular formula was determined to be C15H22O6 on the basis of a HR-FAB-MS. The proton
nuclear magnetic resonance (1H NMR) spectrum of compound 1 showed signals attached
to oxygen bearing carbon from δH 3.17 to 4.80 ppm, two methylene proton signals at δH
2.27 (2H, t, J = 7.3 Hz), 1.55 (2H, dq, J = 7.0, 7.3 Hz) and two methyl signals at δH 1.42 (3H,
d, J = 6.7 Hz) and 1.00 (3H, t, J = 7.3 Hz). The carbon nuclear magnetic resonance spectra
(13C NMR) showed total 15 carbons signals. Among these carbon signals, four quaternary
carbon signals at δC 82.0 (C), 75.7 (C), 71.3 (C) and 65.4 (C) were assigned to a disubstituted
diacetylene moiety (Chauhan et al. 2012). Six carbon signals at δC 101.3 (CH), 78.1 (CH), 78.0
(CH), 74.9 (CH), 71.7 (CH) and 62.8 (CH2) were assigned to a β-glucopyranosyl moiety. The
coupling constant of anomeric proton (J = 7.6 Hz at δH 4.55) also suggested β-configuration
of the glucopyranosyl moiety (Roslund et al. 2008). The 1H1H correlation spectroscopy (COSY)
showed correlation between proton at δH 4.80 (1H, overlapped, D2O) and methyl protons at
δH 1.42 (3H, d, J = 6.7). Similarly, the protons at δH 1.55 (2H, dq, J = 7.3) had correlations with
methylene protons at δH 2.27 (2H, t, J = 7.3) and methyl protons at δH 1.00 (3H, t, J = 7.3). This
suggests a substituted ethyl and a propyl moiety present is in the compound. Heteronuclear
multiple bond correlation (HMBC) confirmed the correlation of anomeric proton at δH 4.55
(1H, d, J = 7.6) with oxygenated carbon at δC 64.2 (C-2). Methyl proton at δH 1.42 showed
correlation with acetylenic carbon at δC 75.7 (C-3) and oxygenated carbon at δC 64.2 (C-2).
Similarly, methylene protons δH 2.27 of the propyl moiety showed correlation with acetylenic
carbon at δC 82.0 (C-6) and 65.4 (C-5) suggesting the propyl side chain carbon attached to
next side of the acetylenic chain. Acid hydrolysis of compound 1 gave d-glucose [ꢀ]2D0 +66
1
(c = 0.35 H2O) and aglycone 3,5-nonadiyn-2-ol (1a), confirmed on the basis of H and 13C
NMR data (Chauhan et al. 2012). The relative configuration of compound 1 was confirmed
using β-D-glycosylation shift (Seo et al. 1978). In 13C NMR, the carbon attached with the
β-d-glucopyranosyl moiety (C-2, α-carbon) was shifted downfield 5.4 ppm and the adja-
cent carbons C-1 (β-carbon) and C-3 (β-carbon) were shifted upfield by 2.1 and 3.0 ppm,
respectively, which suggested the R configuration at position C-2. Thus, the structure of 1
was elucidated as 2(R)-hydroxy-3,5-nonadiyn-2-Ο-β-d-glucopyranoside.
Bhutkesoside B (2) was obtained as pale yellow oil, [ꢀ]2D0 −14.5° (c = 0.81, MeOH). The
molecular formula was determined as C20H30O10 on the basis of a HR-FAB-MS. The 1H NMR
spectrum of 2 showed protons attached to oxygen-bearing carbon from δH 3.18 to 4.99 ppm,
two methylene signals δH 2.32 (2H, q, J = 7.3 Hz), 1.79 (2H, dq, J = 7.0, 7.3 Hz) and two methyl
signals δH 1.15 (3H, t, J = 7.3 Hz), 1.01 (3H, t, J = 7.3 Hz. The 13C NMR spectra of 2 showed total
20 carbon signals. Among them, four quaternary carbon signals at δC 83.2 (C), 75.6 (C), 71.4
(C) and 64.9 (C) were assigned to a disubstituted diacetylene moiety as in 1. Six oxygenated
carbon signals at δC 103.2 (CH), 78.1 (CH), 76.8 (CH), 74.9 (CH), 71.5 (CH) and 68.2 (CH2) were
assigned to a C-6-substituted β-glucopyranosyl moiety. In comparison to 1, six extra carbon
signals at δC 110.9 (CH), 80.6 (C), 78.0 (CH), 75.1 (CH2) and 65.8 (CH2) were observed in 2,
which were assigned to a β-apiofuranosyl moiety (Park et al. 2002). The glucose C-6 carbon
was shifted to downfield by 6, which confirmed the apiofuranosyl 1→6 glucopyranoside
linkage of the sugars. In the 1H1H COSY spectrum of 2, protons at δH 1.79 were correlated to
methyl protons δH 1.01 and oxymethine protons at δH 4.43, which suggested the presence
of a propyloxy moiety. A next correlation was seen between methylene protons at δH 2.32

4
B. ADHIKARI eT Al.
and methyl protons at δH 1.15 suggested the presence of ethyl moiety. In the HMBC spec-
trum, the anomeric proton of glucopyranosyl moiety δH 4.39 had correlation with carbon at
δC 72.3 (C-3). The anomeric proton of apiofuranosyl moiety at δH 4.99 was correlated to the
carbon at δC 68.2 (Glc-C6). In addition, this carbon δC 68.2 was shifted downfield by +6 ppm
in comparison to chemical shift in compound 1 (Glc-6, 62.8, CH2). Further, the proton at δH
2.32 was correlated to acetylenic carbon at δC 83.2 (C-3), while the protons at δH 4.43 and
1.79 were correlated to acetylenic carbon at δC 75.6 (C-6) confirming two side chains at
either side of the diacetylenic carbons. Glucose and apiose were confirmed on the basis of
co-TlC of acid hydrolysate of 2 with authentic samples. Thus, the structure of compound 2
was elucidated as 3-hydroxy-4,6-nonadiyn-3-Ο-β-apiofuranosyl-(1→6)-β-glucopyranoside.
All of these isolated compounds were evaluated for their in vitro antioxidant activity by
DPPH free radical scavenging assay. Only compounds 6, 4, 8 and 5 showed potent activity
with eC50 values being 39, 89, 127 and 189 μM, respectively, while Trolox used as a positive
control had eC50 value of 96 μM.
3. Experimental
3.1. Instruments and chemicals
CC was carried out with silica gel 60 (0.040–0.063 mm, Merck (Germany), MCI gel CHP20P
(75–150 μm, Mitsubishi Chemical Industries Co., ltd (Japan), Sephadex lH-20, Amersham
Pharmacia Biotech and Silica NH and Chromatorex ODS (30–50 μm, Fuji Silysia Chemical
Co., ltd, (Japan). TlC was performed on precoated silica gel 60 F254 plates, 0.2 mm, alumin-
ium sheet, Merck (Germany). NMR spectra were measured on a JeOl α-500 spectrometer
(1H: 500 MHz and 13C: 125 MHz) and BRuKeR, AVANCe 600 (1H: 600 MHz and 13C: 150 MHz).
1
Chemical shifts in the H and 13C NMR spectra are given in ppm with reference to TMS at
0 ppm. Mass spectra were recorded on JeOl JMS 700 MStation, FAB mass spectrometer.
Optical rotations were measured with a JASCO DIP-1000KuY polarimeter. Melting points were
measured on Yanaco Micromelting point apparatus (MP-J3, MP-S3) and were uncorrected.
DPPH was obtained from Wako Pure Chemicals (Osaka,Japan). Trolox from Calbiochem
(Denmark) and 2-(N-morpholino)-ethanesulphonic (MeS) acid buffer from Dojindo Chemical
Research (Kumamoto, Japan). Absorbance was recorded on Immuno-MiniNJ-2300 Microtiter
Plate Reader, Biotech Pvt, ltd, (Tokyo, Japan).
3.2. Plant material
Fresh roots of L. wallichiana were collected from forest of Kurikharkha, Dolkha on August
2013 at altitude 2696 m. The roots were shade dried for a month. The plant specimen was
identified by Mr Kuber Jung Malla, Senior Scientific Officer, Department of Plant Resources,
Nepal. The voucher specimen (Voucher No.: KuNP20130809-015) was deposited on Graduate
School of Pharmaceutical Sciences, Kumamoto university, Kumamoto, Japan.
3.3. Extraction and isolation
The shade dried roots (370 g) were extracted with 70% MeOH at 60 °C and the extract was
evaporated under reduced pressure to give 99.2 g extract. The extract was then suspended in
water to separate water soluble (94.2 g) and water insoluble fraction (5.0 g). The water-soluble

NATuRAl PRODuCT ReSeARCH
5
fraction was run over MCI gel CHP20P CC and eluted successively with water, 40%, 60%, 80%,
100% MeOH and CHCl3 to give ten fractions. Fraction 2 (497.0 mg) was subjected to Sephadex
lH-20 CC (H2O) and ODS CC (H2O, 10% MeOH) to give compound 4 (35.4 mg). Fraction 3
(4.22 g, 40% MeOH eluate) was applied on Sephadex lH-20 CC (50% MeOH) and ODS CC
(10% MeOH) to give compound 5 (68.0 mg) and subfraction 3-3-9 (34.5 mg) was subjected
to silica gel CC (CHCl3: MeOH: H2O = 9: 1: 0.1) to afford compound 7 (20.7 mg). Fraction 4
(1.15 g 40% MeOH eluate) was applied on Sephadex lH-20 CC (50% MeOH), ODS CC (25%
MeOH), Sephadex lH-20 CC (100% MeOH) and silica gel CC (CHCl3: MeOH: H2O = 8: 2: 0.1)
to afford compound 10 (20.0 mg) and compound 11 (9.1 mg). Fraction 6 (207.7 mg, 70%
MeOH eluate) was applied over Sephadex lH-20 to give compound 6 (3.3 mg). Subfraction
6-2 (196.0 mg) was applied over ODS column (25–45% MeOH), silica gel column to com-
pound 2 (2.9 mg). Fraction 7 (2.86 g, 70% MeOH eluate) was subjected to Sephadex lH-20
CC (MeOH) and ODS (40% MeOH) to give fraction 7-3-4 which was recrystallized in 20%
MeOH to give compound 8 (84.2 mg). Similarly, subfraction 7-2 (1563.0 mg) was subjected
to ODS (40–70% MeOH) and silica gel column to afford compound 9 (18.4 mg). Subfraction
7-2-4 (93.0 mg) was applied over silica gel column and recrystallized in 50% MeOH to get
compound 12 (3.0 mg). Subfraction 7-2-9 (214.0 mg) was applied over silica gel column
(CHCl3: MeOH: H2O = 9: 2: 0.1) and (CHCl3: MeOH = 10:1) to give of compound 1 (23.0 mg).
Subfraction 7-2-6, (323.0 mg) was applied over silica gel CC and ODS CC (40% MeOH) to
give compound 2. (9.1 mg). similarly, subfraction 7-4 (93.4 mg) was applied over ODS CC
20% MeOH to give compound 6 (14.4 mg). Fraction 8 (1.63 g, 100% MeOH eluate) was sub-
jected to silica gel CC (CHCl3: MeOH = 20:1) and (Hexane:etOAc = 9:1) to give compound 3
(41.6 mg). The water-insoluble fraction was partitioned into hexane and methanol fractions.
The methanol fraction was applied over Sephadex lH20 and silica gel (Hexane: etOAc = 10:1)
to give compound 1 (14.5 mg).
3.4. Bhutkesoside A (1)
A pale yellow oil, [ꢀ]2D0 −127.8° (c = 0.28, MeOH). HR-FAB-MS m/z 321.1314 [M + Na]+ (Calcd
1
for C15H22O6Na, 321.1314). H NMR (500 MHz, CD3OD): δ 4.80 (1H, overlapped, D2O, C2-H),
4.55 (1H, d, J = 7.6, Glc-C1-H), 3.80 (1H, dd, J = 11.5, 1.5, Glc-C6a-H), 3.64 (1H, dd, J = 11.5, 5.4,
Glc-C6b-H), 3.38 (1H, t, J = 9.2, Glc-C3-H), 3.27 (1H, m, Glc-C4-H), 3.27 (1H, m, Glc-C5-H), 3.17
(1H, dd, J = 7.6, 9.2 Glc-C2-H), 2.27 (2H, t, J = 7.3, C7-H), 1.55 (2H, dq, J = 7.3, C8-H), 1.42 (3H,
d, J = 6.7, C1-H), 1.00 (3H, t, J = 7.3, C9-H). 13C NMR (125 MHz, DMSO-d6): 101.3 (C-Glc-1), 82.0
(C-6), 78.1 (C-Glc-5), 78.0 (C-Glc-3), 75.7 (C-3), 74.9 (C-Glc-2), 71.7 (C-Glc-4), 71.3 (C-4), 65.4
(C-5), 64.2 (C-2), 62.8 (C-Glc-6), 22.8 (C-8), 22.3 (C-1), 21.7 (C-7), 13.7 (C-9).
3.5. Acid hydrolysis of bhutkesoside A (1)
Compound 1 (20.0 mg) was hydrolysed with 2.0 ml of 2 M HCl in a water bath at 60 °C for
3 h in a sealed tube. The reaction mixture was extracted with diethyl ether and separated in
to diethyl ether and aqueous layers. The diethyl ether layer was purified in silica gel chroma-
tography with (CHCl3: MeOH: H2O = 9:1:0.1 v/v/v) to isolate the aglycone (1a, 4.0 mg). The
aqueous layer was passed over Chromatorex Silica NH to ensure neutralisation and evap-
orated to give d-glucose (3.5 mg), which confirmed on the basis of co-TlC with authentic
sample and optical rotation, [ꢀ]2D0 +66.0° (c, 0.35, H2O).

6
B. ADHIKARI eT Al.
3.6. 2(R)-hydroxy-3,5-nonadiyne (1a)
A colourless oil,[ꢀ]2D0 −6.5° (c 0.40, MeOH), 1H NMR, (600 MHz, CD3OD): δH 4.50 (1H, q, J = 6.7 Hz,
C2-H), 2.25 (2H, t, J = 7.3 Hz, C7-H), 1.53 (2H, dq, J = 7.3 Hz, C8-H), 1.39 (3H, d, J = 6.7 Hz, C1-H),
0.90 (3H, t, J = 7.3 Hz, C9-H). 13C NMR (CD3OD, 150 MHz): δC 81.6 (C-6), 78.7 (C-3), 69.1 (C-4),
65.4 (C-5), 58.8 (CH-2), 24.4 (C-1), 22.8 (C-7), 21.7 (C-8) and 13.7 (C-9).
3.7. Bhutkesoside B (2)
A yellowish oil, [ꢀ]2D0 −14.5° (c 0.81, MeOH). HR-FAB-MS m/z 453.1737 [M + Na]+ (Calcd for
C20H30O10Na, 453.1752). 1H NMR (500 MHz, CD3OD): δH 4.99 (1H, d, J = 2.1 Hz, Api-C1-H), 4.43
(1H, t like, J = 7.3 Hz, C3-H), 4.39 (1H, d, J = 7.6 Hz, Glc-C1-H), 3.98 (1H,d, J = 9.5 Hz, Api-C4a-H),
3.97 (1H,dd, J = 11.5, 2.1 Hz, Glc-C6a-H), 3.91 (1H, d, J = 2.1 Hz, Api-C2-H), 3.77 (1H, d, J = 9.5 Hz,
Api-C4b-H), 3.61 (1H, dd, J = 11.5, 5.4, Glc-C6b-H), 3.60 (2H, s, Api-C5-H), 3.37 (1H, m, Glc-C5-H),
3.33 (1H, m, Glc-C4-H), 3.31 (1H, m, Glc-C3-H), 3.18 (1H, dd, J = 7.9, 9.5 Hz, Glc-C2-H), 2.32 (2H,
quartet, J = 7.3 Hz, C8-H), 1.79 (2H, dq, J = 7.0,7.3 Hz, C2-H), 1.15 (3H, t, J = 7.3 Hz, C9-H), 1.01
(3H, t, J = 7.3 Hz, C1-H). 13C NMR (CD3OD): δC 110.9 (CH, C-Api-1), 103.2 (CH, C-Glc-1), 83.2
(C-7), 80.6 (C-Api-3), 78.1 (C-Glc-3), 78.0 (C-Api-2), 76.8 (C-Glc-5), 75.6 (C-4), 75.1 (C-Api-4),
74.9 (C-Glc-2), 72.3 (C-3), 71.5 (C-Glc-4), 71.4 (C-5), 68.2 (C-Glc-6), 65.8 (C-Api-5), 64.9 (C-6),
29.7 (C-2), 13.7 (C-9), 13.5 (C-8), 9.7 (C-1).
3.8. Acid hydrolysis of bhutkesoside B (2)
Compound 2 (1.0 mg) was hydrolysed with 2.0 ml of 2 M HCl in a water bath at 60 °C for
3 h in a sealed tube. Co-TlC of the reaction mixture with authentic samples confirmed the
presence of glucose and apiose.
3.9. Known compounds
Falcarindiol (3), a yellowish orange oil,[ꢀ]D25 +99.9° (c 0.88, MeOH); chlorogenic acid (4), a pale
yellow powder,[ꢀ]2D5 −26.3° (c 0.99, MeOH + H2O, 1:1); 5-O-p-coumarylquinic acid (5), a white
amorphous powder,[ꢀ]2D5 −27.6° (c 1.0, MeOH + H2O, 1:1); 3,5-di-O-Caffeoyl quinic acid (6), a
pale yellow powder,[ꢀ]2D5−138.8° (c 0.95, MeOH + H O, 1:1); 4-hydroxy-7-methoxy-phenyleth-
2
anol (7), a crystalline powder (MeOH), mp 92 °C,[ꢀ]2D5 0° (c 0.98, MeOH); ferulic acid (8), nee-
dle-shaped crystals (MeOH), mp 174 °C; dehydrodiconiferyl alcohol 4-O-β-glucopyranoside
(9), a pale yellow mass, [ꢀ]2D5 −31.4° (c 0.98, MeOH); 5,7-dihydroxy-2-methylchromone-7-O-
rutinoside (10), a pale yellow mass [ꢀ]2D5 −91.2° (c 0.99, MeOH); schumanniofioside B (11), a
white crystalline powder (MeOH), mp 250 °C,[ꢀ]2D7 −58.8° (c 0.90, MeOH + H2O); marmesinin
(12), a white crystalline powder (MeOH), mp 258 °C, [ꢀ]2D0 −10° (c 0.16, MeOH).
3.10. Antioxidant activity
The DPPH radical scavenging activity of the isolated compounds was measured by the
method as described previously (Joshi et al. 2014).

NATuRAl PRODuCT ReSeARCH
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4. Conclusion
Two new diacetylenic glycosides, bhutkesoside A (1) and bhutkesoside B (2) and 10 known
compounds (3–12) were isolated from the dried roots of L. wallichiana. All these compounds
were isolated first time from the title plant. Among the phenolic compounds, 3, 5-di-O-
Caffeoyl-quinic acid (6) and chlorogenic acid (4) had potent antioxidant activity against
DPPH free radical.
Acknowledgements
The authors are grateful to the Program for leading Graduate Schools ‘HIGO Program’ (Health life
science: Interdisciplinary and Glocal Oriented), MeXT, Japan for scholarship to Mr Bibek Adhikari. We
are grateful to Ms Teruo Tanaka and Mr Toshiyuki Iriguchi of Institute of Resource Development and
Analysis, Kumamoto university for measurement of NMR and mass spectra, respectively.
Disclosure statement
No potential conflict of interest was reported by the authors.
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