Natural Product Research
969
3. Results and discussion
Chloragin (1) showed a molecular ion peak at m/z 1173 [M þ Na]þ corresponding to the
molecular formula C55H90O25 in its FABMS. It showed the losses of one pentostyl, one
pentosyl plus one hexosyl, two pentosyl plus one hexosyl and two pentosyl plus one
hexosyl plus one methyl pentosyl moieties. The fragment ion at m/z 438 [M þ Na]þ
corresponded to the molecular ion of the aglycone (tigogenin). The proton NMR spectrum
of the compound in DMSO-d6 showed signals for four steroidal methyls, two of them as
tertiary methyls showing signals at ꢂ 0.82 and 0.86 (3H each, s), and rest of the secondary
methyls at ꢂ 0.70 (3H, d, J, 6.1 Hz) and 1.14 (3H, d, J, 6.7 Hz) along with methyl pentose
1
secondary methyl at ꢂ 1.70 (3H, d, J, 6.2 Hz). Further, H NMR spectrum displayed five
anomeric protons at ꢂ 5.12, 4.60, 4.48, 4.36 and 4.29, respectively. This corresponded with
13C NMR cross peaks, which were observed at ꢂ 99.9, 104.0, 103.5, 103.6 and 98.4,
respectively, in the HMQC spectrum, confirming that chloragin (1) contains five sugar
units. Acid hydrolysis of 1 with 2N HCl in 80% aqueous ethanol at 100ꢂC for 2 h afforded
D-glucose, L-rhamnose and D-xylose in the ratio of 2 : 1 : 2, and the aglycone was identified
as tigogenin by comparing spectral data with those given in the literature (Agarwal et al.,
1985) and the co-TLC with an authentic sample. The proton and carbon assignment of
each sugar unit was unambiguously assigned, starting by the combined use of COSY,
TOCSY and NOESY, NMR spectra (Table 3), which was further confirmed by HMQC
and HMBC NMR spectra. The sequences of the sugar chains were determined by HMBC
and NOESY spectra. Cross peak correlations observed in the HMBC spectrum between
signals at ꢂH 5.12 and ꢂc 76.6 (aglycone-3) showed that the L-rhamnosyl moiety is linked at
C-3 of the aglycon (tigogenin). This was confirmed by the NOESY correlation observed
between signals at ꢂ 5.12 (1H, d, J 7.1 Hz) and ꢂH 3.89 (aglycon-3). The glucosyl-1 H-1 at ꢂ
4.60 and rhamnosyl C-4 at ꢂ 75.9, xylosyl-1 at ꢂ 4.48 and glucosyl-1 C-3 at ꢂ 85.7; glucosyl-
2 H-1 at ꢂ 4.29 and xylosyl-1 C-4 at ꢂ 75.9 and xylosyl-2, H-1 at ꢂ 4.36 and glucosyl-2,
C-4 at 79.5 correlated in the HMBC spectrum confirmed that the structure of chloragin (1)
is tigogenin-3-O-ꢀ-L-rhamnopyranosyl-(1 ! 4)-ꢁ-D-glucopyranosyl-(1 ! 3)-ꢁ-D-xylopy-
ranosyl-(1 ! 4)-ꢁ-D-glucopyranosyl-(1 ! 4)-ꢁ-D-xylopyranoside.
3.1. Antihyperglycaemic activity
The crude extract showed promising antihyperglycaemic activity in the streptozotocin-
induced diabetic rat model on further fractionation of the crude extract into four fractions;
the activity was found to be localised in only the n-butanol soluble fraction. Therefore,
only the n-butanol soluble fraction was taken up for detail chemical investigations. The
major pure compound chloragin (1) was isolated and characterised, which is a new
structure and also showed promising antihyperglycaemic effect in the streptozotocin-
induced diabetic rat model. The results are summarised in Table 1.
3.2. Antihyperlipidaemic activity
The antihyperlipidaemic activity of chloragin (1) in triton and hamster models was
evaluated. The results are summarised in Tables 4–6. Values expressed as mg dLꢁ1 are
the mean ꢀ SD of six hamsters. Values in parentheses are percentage change as
compared to HFD group. Data in Table 4 showed that administration of triton