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

Abstract 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.

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 characterisation 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.
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 eC 50 values being 39, 89, 127 and 189 μM, respectively, while Trolox used as a positive control had eC 50 value of 96 μM.

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.

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 chromatography with (CHCl 3 : MeOH: H 2 O = 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 evaporated to give d-glucose (3.5 mg), which confirmed on the basis of co-TlC with authentic sample and optical rotation, [ ] 20 D +66.0° (c, 0.35, H 2 O).

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.

Antioxidant activity
The DPPH radical scavenging activity of the isolated compounds was measured by the method as described previously (Joshi et al. 2014).