2
860
Z. Gao et al. / Phytochemistry 69 (2008) 2856–2861
ꢀ
Varian AS 400 or Bruker DRX-400 NMR spectrometers in C
ESIMS and HRESIMS data were obtained on an Agilent Series
100 SL mass spectrometer. GCMS analyses were carried out on
5
D
5
N.
analyses, see Table 1; HRESIMS, m/z 1209.7792 [M + Cl] ,
16 + Cl, requires 1209.7795.
67 114
C H O
1
a ThermoQuest Trace 2000 GC, equipped with a single split/split-
less capillary injector, a ThermoQuest AS2000 autosampler, and
4.3.2. (2S)-1-O-palmitoyl-2-O-linolenoyl-3-O-[
a
-
D
-galactopyranosyl-
-galactopyranosyl]glycerol (2)
max 3370,
0
0
0
00
(1 ? 6 )-(3 -O-linoleoyl)-b-
D
2
7
an Agilent DB-5ms column (30 m ꢂ 0.25 mm ꢂ 0.25
column chromatography (CC) was performed using silica gel (J.T.
Baker, 40 for flash chromatography) and reversed-phase
RP-C18 silica (Polarbond, J.T. Baker). Thin layer chromatography
TLC) was carried out on silica gel 60 F254 plates (Merck, Germany).
Waring Heavy Duty Blender was used for extraction. Sugar
samples, Lipase from Pseudomonoas sp. type XIII, and -cysteine
methyl ester hydrochloride were purchased from Sigma–Aldrich
St. Lousis, MO).
lm). Gravity
Colorless gum; ½
a
ꢁ
þ 39:0 (c, 1.0, MeOH); IR (NaCl)
m
D
ꢀ
1
1
13
3010, 2924, 2853, 1738, 722 cm ; for H and C spectroscopic
ꢀ
lm
analyses, see Table 1; HRESIMS, m/z 1211.7951 [M + Cl] ,
C H O16 + Cl, requires 1211.7952.
67 116
(
4.3.3. (2S)-1-O-palmitoyl-2-O-linolenoyl-3-O-[
a
-D
-galactopyranosyl-
-galactopyranosyl]glycerol (3)
max 3375,
0
0
0
00
L
(1 ? 6 )-(3 -O-palmitoyl)-b-D
2
D
7
Colorless gum; ½
aꢁ
þ 41:0 (c, 1.0, MeOH); IR (NaCl)
m
ꢀ1
1
13
(
3011, 2923, 2852, 1737, 721 cm ; for H and C spectroscopic
ꢀ
analyses, see Table 1; HRESIMS, m/z 1187.7988 [M + Cl] ,
4.2. Plant material
65 116
C H O16 + Cl, requires 1187.7952.
4.3.4. (2S)-1-O-palmitoyl-2-O-linoleoyl-3-O-[
a
-
D
-galactopyranosyl-
-galactopyranosyl]glycerol (4)
max 3377,
0
0
0
00
tic Tibetan Himalayan Dried Goji Berries”. The authentication was
done by Dr. V. Joshi, Plant Taxonomist at the National Center for
Natural Products Research, University of Mississippi, where the
specimen (No. 3469) has been deposited.
(1 ? 6 )-(3 -O-palmitoyl)-b-D
2
D
7
Colorless gum; ½
a
ꢁ
þ 40:0 (c, 1.0, MeOH); IR (NaCl)
m
ꢀ
1
1
13
3010, 2923, 2853, 1737, 722 cm ; for H and C spectroscopic
ꢀ
analyses, see Table 1; HRESIMS, m/z 1189.8124 [M + Cl] ,
C H O16 + Cl, requires 1189.8108.
65 118
4.3. Extraction and isolation
4.4. Sugar analysis
Fruit of Lycium barbarum (4.8 kg) was extracted with MeOH
Compound 1 (3.0 mg) was hydrolyzed with 2N HCl (1 mL) for
3 h at 95 °C. The reaction mixture was cooled, neutralized with
(
5.0 L ꢂ 1, 4.0 L ꢂ 4) at room temperature by using a blender. The
combined extracts were evaporated under reduced pressure to af-
ford a dark-brown gummy residue (A, 2.4L, 2.9 kg). The residue A
NH
layer after drying was dissolved in pyridine (0.3 mL) and 0.1 M
-cysteine methyl ester hydrochloride in pyridine (1.0 mL) was
added. The mixture was heated at 60 °C for 1 h. An equal volume
of Ac O was added with heating continued for another 1 h (Hara
4 2
OH and partitioned between EtOAc and H O. The aqueous
(
2.8 kg) was dissolved in MeOH and precipitated with CH
3
CN. Fol-
L
lowing removal of the precipitates, the solution was evaporated
under reduced pressure to give part B (523 g). A portion of part B
2
(
(
(
500 g) was subjected to flash silica gel CC (40
95 mm ꢂ 370 mm), CHCl –MeOH, gradients of 19:1 (5 L), 9:1
5 L), 17:3 (8 L), 4:1 (5 L), 0:1 (6 L)] to afford seven fractions B1–
m,
–MeOH, gra-
dients of 19:1 (2 L), 93:7 (3 L), 23:2 (2 L), 9:1 (1 L), 15:2 (1 L), 0:1
l
m, 1040 g, column
et al., 1987; Ali and Khan, 2008). Acetylated thiazolidine deriva-
tives were subjected to GC analysis [Capillary Column: DB-5 ms
3
(30 m ꢂ 0.25 mm ꢂ 0.25
lm); Carrier gas He; Pressure: 19.98
psi; Injection temperature 250 °C; Detection temperature
B7. Fraction B3 (5.7 g) was appeared to flash silica gel CC (40
03 g, column, 45 mm ꢂ 960 mm), eluted with CHCl
l
6
3
280 °C; Column temperature: 70 °C 1 min, 20 °C/min to 300 °C].
The sugars were identified as
tion time (t -galactose 13.48 min) with acetylated thiazolidine
derivatives prepared in a similar way from the standard -galact-
D-galactose by comparing its reten-
(
(
1 L)] to afford 12 sub-fractions B3a–B3l. Compounds 13
30.6 mg, 0.00064%), 14 (52.4 mg, 0.00109%) and 15 (10.8 mg,
R
D
D
0
.00022%) were obtained from sub-fraction B3h (1.28 g) by re-
peated CC [flash silica gel (40 m, 250 g), column
–MeOH, 24:1 (1 L) and 19:1 (1 L) and
ose (Sigma–Aldrich). Compounds 2–15 were treated in a similar
manner.
l
(
40 mm ꢂ 550 mm), CHCl
3
RP-C18 (155 g), column (25 mm ꢂ 620 mm), MeOH (1.4 L)]. Com-
4.5. Alkaline hydrolysis
pounds 1 (18.7 mg, 0.00039%), 2 (6.5 mg, 0.00013%), 3 (21.0 mg,
0
.00045%), 4 (14.1 mg, 0.00029%) and 5 (14.2 mg, 0.0003%) were
obtained from fraction B3i (0.76 g) by repeated CC [flash silica
gel (40
m, 130 g), column (25 mm ꢂ 650 mm), EtOAc–CHCl
MeOH–H O, 30:16:5:1 (0.6 L) and RP-C18 (155 g), column
25 mm ꢂ 620 mm), MeOH (3.6 L)]. Fraction B5 (8.36 g) was sub-
jected to CC [flash silica gel (40 m, 316 g), column
–MeOH–H O, 60:32:12:1
1.9 L), 15:8:4:1 (0.9 L), 6:4:4:1 (1 L)] to afford 10 sub-fractions
(18.3 mg, 0.00038%), (71.8 mg,
.0015%), 8 (44.3 mg, 0.00092%), 9 (171.0 mg, 0.00356%), 10
168.5 mg, 0.00351%), 11 (17.9 mg, 0.00037%), and 12 (11.6 mg,
.00024%) were obtained from sub-fraction B5g (1.25 g) by re-
peated CC [flash silica gel (40 m, 130 g), column
25 mm ꢂ 650 mm), EtOAc–CHCl –MeOH–H O, 30:16:5:1 (0.6 L)
and RP-C18 (155 g), column (25 mm ꢂ 620 mm), MeOH (3.3 L)].
A solution of compound 1 (2.7 mg) in 5% dry NaOMe–MeOH
(2 mL) was stirred at room temperature for 5 h. The reaction mix-
ture was neutralized with 2N HCl–MeOH and partitioned between
MeOH and n-hexane. The n-hexane layer containing the fatty acid
methyl esters was analyzed by GCMS [Capillary Column: DB-5 ms
l
2
3
–
(
l
(30 m ꢂ 0.25 mm ꢂ 0.25
lm); Carrier gas He; Pressure: 19.98 psi;
(
(
50 mm ꢂ 390 mm),
EtOAc–CHCl
3
2
Injection temperature 250 °C; Detection temperature 280 °C; Col-
umn temperature: 70 °C 1 min, 20 °C/min to 300 °C]. Alkaline
hydrolysis of compounds 2–15 was performed similarly.
B5a–B5j. Compounds
0
(
0
6
7
4.6. Enzymatic hydrolysis
l
A solution of compound 1 (1.0 mg) and Lipase type XIII (from
Pseudomonoas sp., 1.3 mg, 19.5 units) in 0.7 mL dioxane-H O
2
(1:1) was stirred and incubated at 37 °C for 3 h. The reaction was
quenched by 5% HOAc (0.15 mL) and MeOH (1.5 mL) was added
to the reaction mixture. Following removal of the solvent the res-
(
3
2
4.3.1. (2S)-1-O-palmitoyl-2-O-linolenoyl-3-O-[a-D-galactopyranosyl-
0
0
0
00
(
1 ? 6 )-(3 -O-linolenoyl)-b-
D
-galactopyranosyl]glycerol (1)
þ 40:0 (c, 1.0, MeOH); IR (NaCl) max 3380,
012, 2924, 2853, 1737, 721 cm ; for 1H and C spectroscopic
idue was dissolved in dry MeOH (0.5 mL) and 1 drop of BF
was added. The mixture was stirred at room temperature for
1.5 h and analyzed by GCMS (see alkaline hydrolysis). Enzymatic
3
ꢃOEt
2
2
D
7
Colorless gum; ½
aꢁ
m
ꢀ1
13
3