全 文 ：第 12期 董 岩，等:山东迷迭香挥发油化学成分及抑菌活性研究
第 27卷第 12期
2015年 12月
化 学 研 究 与 应 用
Chemical Research and Application
Vol．27，No．12
Dec．，2015
文章编号:1004-1656(2015)12-1811-06
Studies on Isolation，characterization，and antibacterial
activity of bellidifolin from gentianella acuta
LV Li-juan1，ZHU Hua-ling1，WANG Yan-fang2，JIANG Shuai2，ZHAO Wei1，ZHAO Ya-chan1，LI Min-hui2*
(1．Department of Basic Science，Tianjin Agricultural University，Tianjin 300384，China;
2．Baotou Medical College，Baotou 014060，China)
Abstract:Bellidifolin was isolated from Gentianella acuta，and characterized by IR，UV-vis spectra and X-ray diffraction analysis．It
crystallizes in the triclinic lattice，space group P-1 with a= 7．0602(14)，b= 7．5236(15)，c= 10．662(2)，α= 86．65(3)°，β=
79．84(3)°，γ= 84．96(3)°，V= 554．77(19)3，Z= 2，Dc = 1．642 g /cm
3 and F(000)= 284．Geometrical structure of bellidifolin was
optimized by density functional theory(DFT)using B3LYP method with 6-31g(d，p)as the basis set． The vibrational frequencies
were calculated by the DFT /6-31g(d，p)method and the results are nearly consistent with the observed frequencies．And the elec-
tronic absorption spectra were studied with the time-dependent density functional theory(TD-DFT)and the calculated results are in
good agreement with the corresponding experimental data． In addition，the preliminary antibacterial activities of bellidifolin against
Staphylococcus aureus，Escherichia coli，Salmonella enterica and Pseudomonas aeruginosa were investigated．
Key words:bellidifolin;Gentianella acuta;characterization;DFT calculation;antibacterial activity
尖叶假龙胆中雏菊叶龙胆酮的分离、
表征及抗菌活性研究
吕丽娟1，朱华玲1，王艳芳2，姜 帅2，赵 维1，赵雅婵1，李旻辉2*
(1．天津农学院基础科学学院，天津;2．包头医学院，内蒙古 包头)
摘要:本文对尖叶假龙胆中雏菊叶龙胆酮进行提取分离，采用红外光谱、紫外可见光谱和单晶 X 射线衍射分
析技术对该化合物结构进行了表征;然后，采用密度泛函理论(DFT) ，在 B3LYP /6-31G基组水平上，对化合物
结构进行了量子化学计算，结果表明其理论表征数据与实验表征数据吻合良好。另外，研究了雏菊叶龙胆酮
对金黄色葡萄球菌、大肠杆菌、沙门氏菌和绿脓杆菌的初步抗菌活性。
关键词:雏菊叶龙胆酮;尖叶假龙胆;表征;DFT计算;抗菌活性
中图分类号:O629．1 文献标志码:A
Gentianella acuta(Michx．)Hulten，mainly dis-
tributed in East Asia，Siberia and North America，is
well-known as Guixincao in the Chinese folk and has
been used as a kind of herb tea in the eastern region
of Inner Mongolia due to its health properties of heat-
clearing and detoxifying effect．In addition，the Ewenki
收稿日期:2015-05-04;修回日期:2015-07-23
基金项目:国家自然科学基金(81303306，81160504)资助;天津市应用基础与前沿技术研究计划(15JCQNJC13400)资助;天津市大学生创
新创业训练计划项目(201410061052，201410061159)资助
联系人简介:李旻辉(1978-) ，男，教授，主要从事药用植物资源利用与保护研究。E-mail:li_minhui@ aliyun．com
化 学 研 究 与 应 用 第 27卷
use the aerial parts of G． acuta for the treatment of
heart diseases［1-2］．And bellidifolin(1，5，8-Trihydrox-
y-3-methoxy-9H-xanthen-9-one)is one of the main
active components in G．acuta［2］． The compound also
showed significantly biological activities，such as an-
tidiabetics，antifungal，enhancing rehabilitation of in-
juried sciatic nerve，protective against cerebral dama-
ges induced by ischemia-repefusion［2］，and it posses-
ses the prospect of utilization． However，there? is?
less?research?on?the crystal structure and quan-
tum chemistry calculation of bellidifolin．In this study，
bellidifolin was isolated from G．acuta and character-
ized by IR，UV-vis spectra and X-ray diffraction anal-
ysis．Then，the DFT calculated results of the structure
geometry about bellidifolin at the B3LYP /6-31g(d，
p)level was provided，the vibrational frequencies and
electronic absorption spectra have also been predic-
ted，assigned and compared with the experimental da-
ta． In addition，its preliminary antibacterial activities
were also measured in vitro． It is noteworthy that the
reporting theoretical and experimental results may be
of significant importance for the insight of structures
and macro-scopic properties of xanthones．
1 Experimental Section
1．1 Instruments and reagents
The determination of crystal structure was car-
ried out on a Rigaku Saturn724 CCD diffractometer．
IR spectra(400 ～ 4000 cm－1)were measured on a
Bruker Tensor27 FT-IR spectroscopy．UV-vis absorp-
tion spectra were performed on a Shimadzu UV-2450
spectrophotometer in MeOH．All reagents and chemi-
cals were purchased from commercial sources and
used as received．
1．2 Plant materials
The aerial parts of G．acuta were collected from
Inner Mongolia Virgin Forest near the boundary of
China and Russia in August 2013 and identified by
Professor Li Minhui． A voucher specimen
(2013083006)has been deposited in the Herbarium
of Baotou Medical College．
1．3 Extraction，isolation and crystallization of
bellidifolin
The aerial parts of G．acuta(500 g)were extrac-
ted with 95% EtOH for 3 times． After removing the
solvent，EtOH concrete(99 g)was obtained，and frac-
tionated with petroleum ether and ethyl acetate suc-
cessively．Then the ethyl acetate extract was concen-
trated to 300 mL，after adding anhydrous ethanol，a
yellow precipitate was allowed to filter and dry to get
bellidifolin extract．Bellidifolin extract(21 g)was sub-
jected to silica gel column chromatography，repeatedly
eluted with petroleum ether-ethyl acetate(7 ∶ 3)． The
combined fractions were evaporated and yield yellow
powder were identified by spectral data(1H-NMR，13
C-NMR and ESI-MS)as bellidifolin(1．1 g) ，and the
purity of the compound was above 98%as determined
by HPLC．
The single crystal of bellidifolin for X-ray dif-
fraction analysis was obtained by slow solvent(ace-
tone)evaporation at 4℃ for 16 days．
1．4 Crystallographic data collection and struc-
ture determination
A yellow transparent columnar crystal with di-
mensions of 0．20mm×0．18mm×0．12mm was selected
for data collection on a Rigaku Saturn724 CCD dif-
fractometer equipped with a multilayer-monochromat-
ic Mo-Kα radiation(λ = 0．71073)． A total of 5703
reflections were collected in the range of 1．94≤θ≤
27．92° by using an ω-scan mode at 113(2)K，of
which 2592 were independent with Rint = 0．0376 and
1672 were observed with I ＞ 2σ(I)． Semi-empirical
absorption corrections were applied．The structure was
solved by direct methods with SHELXS-97，and re-
fined by full-matrix least-squares procedure on F2
with SHELXL-97［3-4］． All the non-hydrogen atoms
were refined anisotropically．The hydrogen atoms were
located by geometrical calculations，and their posi-
tions and thermal parameters were fixed during the
structure refinement． During refinement，all H atoms
were geometrically positioned and treated as riding on
their parent atoms，with C-H = 0．95 for the aromat-
ic，0．98 for the methyl with Uiso(H)1．5Ueq(Cm-
ethyl)． The final full-matrix least-squares refinement
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gave S= 1．023，R= 0．0403 and wR= 0．1034．
1．5 Computational methods
The molecular structure of the compound in the
ground state was optimized using DFT(B3LYP)with
the 6– 31G(d，p)basis set．The initial guess of the
model of the compound was first obtained from the X-
ray coordinates． The harmonic vibrational frequencies
and intensities were calculated at the same level of
theory with the analytical evaluation of second deriva-
tives of the energy as a function of atomic coordi-
nates，and the calculated intensities were used to gen-
erate the theoretical spectra．All the quantum chemi-
cal calculations were performed with the Gamess soft-
ware package［5］．
Based on the optimized geometry，electronic
spectra of the title compound was predicted via the
TD-DFT with PCM model in CH3OH．Fig．1 shows the
atomic number for quantitative calculation of bellidi-
folin．
Fig．1 The atomic number for
DFT /B3LYP /6-31g(d，P)of bellidifolin
1．6 Antibacterial activity tests
Preliminary in vitro tests for antibacterial activity
of bellidifolin were carried out by agar diffusion meth-
od［6］against Gram positive Staphylococcus aureus and
Gram negative Escherichia coli，Salmonella enterica，
and Pseudomonas aeruginosa at the concentration of
0． 25，0． 5，1 and 2 mg /mL with N，N-dimethyl-
formamide(DMF)as solvent，respectively． All the
samples were tested for five times with the average
value as the final result．
1．7 Data analysis
Analysis of variance(ANOVA)was carried out
by using the SPSS 17．0 software．The results were sta-
tistically compared using ANOVA．The variables were
presented significant differences among treatments by
F test(P ＜0．05)． The concentration was selected as
factor and diameter of inhibition zone was selected as
dependent factors．
2 Results and discussion
2．1 Crystal structure description(CCDC 1035961)
The single crystal X-ray analysis reveals that the
molecular formula of the crystal is C14 H10 O6(Mr =
274．22)． It crystallizes in the triclinic lattice，space
group P-1 with a = 7．0602(14)，b = 7．5236(15)，
c = 10．662(2)，α= 86．65(3)°，β = 79．84(3)°，γ =
84．96(3)°，V= 554．77(19)3，Z = 2，Dc = 1．642 g /
cm3，F(000)= 284 and μ(MoKa)= 0．131mm－1．The
ORTEP plot of the compound is shown in Fig．2．The
packing diagram of the crystal structure is shown in
Fig．3．In addition，there are four intermolecular hydro-
gen bonds and no intramolecular hydrogen bond ob-
served in the crystal of bellidifolin，and the further
details of these hydrogen bonds are given as below:
O2．．．O3(x，y+1，z) :2．9967(18)，O3．．．O4:2．6529
(15)，O3．．． O4(-x + 1，-y，-z) :2． 9876(18)，and
O5．．．O4:2． 6620(16)． The molecule maintains its
stability in the spatial arrangement by hydrogen bond
force and Van der Waals force．
Fig．2 The ORTEP plot of bellidifolin
Fig．3 The packing diagram of the crystal structure
2．2 The frontier molecular orbitals
According to molecular orbital theory，the fron-
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tier molecular orbitals(highest occupied molecular or-
bital，HOMO and the lowest unoccupied molecular or-
bital，LUMO)and the near molecular orbitals influ-
ence the biological activity of molecule in maximum．
The energy of molecular frontier orbital［eg． E(HO-
MO)or E(LUMO) ］is related to molecular electron i-
onization potential or affinity of a biological active re-
action，the interaction between the active molecules
and receptor of biological macromolecules mainly oc-
curs in the vicinity of frontier molecular orbitals of
them ［7-8］．
The energy difference of frontier molecular orbit-
als［△E = E(HOMO)-E(LUMO) ］is an important
index for molecular stability，the smaller the differ-
ence is，the more stable the compound is．As shown in
the frontier orbitals of bellidifolin molecule(Fig．4) ，
the energy values of all HOMO and two LUMO are
negative，and △E = E(HOMO)-E(LUMO)= -0．
19973AU=-3．6545eV，indicate that bellidifolin mole-
cule is stable under the state．
Fig．4 Frontier orbitals of bellidifolin
molecule in DFT /B3LYP /6-31g(d，p)
2．3 The charge distribution
As shown in Fig．5，the net charges of O(10)-C
(11)-C(13)-C(14)-C(16)-O(1)-C(17)appear
positive-negative alternately，indicating that there are
certain conjugate effects among the 7 atoms，and the
formation of large conjugated system can reduce the
energy of bellidifolin and makes the molecule more
stable．
Fig．5 The charge distribution of bellidifolin
2．4 Vibrational frequencies
The experimental and simulated infrared spectra
are shown in Fig．6．Vibrational frequencies calculated
at the B3LYP /6-31g(d，p)level are scaled by 0．
9555．To facilitate the assignment of observed peaks，
some primary calculated harmonic frequencies are as-
signed by Macmolplt 7．4．2［9］ and compared with the
experimental data．According to the data in Tab．1，the
predicted vibrational wavenumbers are nearly in a-
greement with the experimental results except for the
peak at 3438，3104 and 3017 cm-1 attributed to the
νOH of 1，5，8-trihydroxyxanthone in the experimental
IR spectra，in which the same vibrations are at 3345，
3161 and 3086 cm－1 simulated by the B3LYP meth-
od．The discrepancy between the observed and calcu-
lated wavenumbers is that the calculations have been
actually done on a single molecule contrary to the ex-
perimental values recorded in the presence of intra-or
inter-molecular O– H…O interaction．On the whole，
the theoretical vibrational frequencies are consistent
with the experimental results．
2．5 Electronic spectra
The electronic absorption spectra of bellidifolin
were measured in MeOH at room temperature． In or-
der to compare the experimental spectra with the the-
oretical ones，TD-DFT method was applied to obtain a
predicted electronic spectra based on the B3LYP /6-
31g(d，p)level．The UV-vis spectra are simulated by
fitting to the Lorentzian line with a Half-Width at
half-Maximum of 10．2 nm．Both the experimental and
predicted electronic absorption spectra are shown in
Fig．7．
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第 12期 吕丽娟，等:尖叶假龙胆中雏菊叶龙胆酮的分离、表征及抗菌活性研究
Fig．6 Experimental IR spectrum(a)and Simulated
IR spectrum(b)of bellidifolin
Table 1 Observed and calculated
vibrational frequencies of bellidifolin
Exp．(cm－1) Calcd．(cm－1) Assignment
3438 3345 O2-H3 strength
3104 3161 O4-H5，O7-H8 strength，sym
3017 3086 O4-H5，O7-H8 strength，unsym
2986 2995 CH3 strength，unsym
2950 2930 CH3 strength，sym
1657 1648 C26 =O6 strength
1632 1624 O4-H5，O7-H8 bend，sym
1613 1612 O4-H5，O7-H8 bend，unsym
1594 1599 Phenyl-ring strength
1563 1571 Phenyl-ring strength
1497 1497 O4-H5，O2-H3 bend，sym
1433 1442 Phenyl-H bend
1356 1383 O2-H3 bend
1094 1088 C11-H12，C14-H15 bend，sym
scaled factor = 0．9555
As shown in Fig． 7，the experimental electronic
spectra was simulated approximately by the B3LYP /
6-31g(d，p)method．The observed wavelength，calcu-
lated wavelength and the assignment for the relevant
transitions of electronic absorption bands were listed
in Tab．2．
TD-DFT calculations using B3LYP /6-31g(d，p)
predict five intense electronic transitions at 400，344，
278，252 and 203nm which are in agreement with the
experimental electronic transitions at 391，332，278，
254 and 204 nm respectively． According to the TD-
DFT calculations，the five peaks from high wavelength
to low wavelength are due to the electronic transition
from HOMO to LUMO，HOMO-1 to LUMO，HOMO to
LUMO+1，HOMO-3 to LUMO，and HOMO-1 to LU-
MO+3，respectively．
Fig．7 Experimental(a)and theoretical(b)
electronic spectra for bellidifolin in MeOH
Table 2 Experimental and Theoretical Values
and assignment of Electronic Absorption Spectra
Exp．
wavelenth
(nm) logε
Calcd．
wavelenth
(nm)
Assignment
390．60 3．66 400．34 HOMO→LUMO
332．20 4．16 343．93 HOMO-1→LUMO
278．40 4．34 277．93 HOMO→ LUMO+1
253．80 4．47 251．85 HOMO-3 →LUMO
204．08 4．46 203．20 HOMO-1→LUMO+3
2．6 Antibacterial study
The diameter data of inhibition zone are listed in
Tab．3．It can be observed that bellidifolin exhibits an-
tibacterial activity against Staphylococcus aureus，
Escherichia coli，Salmonella enterica and Pseudomonas
aeruginosa． The data showed the inhibition effect is
strengthened with the increasing concentration(0．25-
2．0 mg /mL) ，wherein the compound exhibited supe-
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化 学 研 究 与 应 用 第 27卷
rior biological activity against Staphylococcus aureus，
followed by Escherichia coli，Salmonella enterica and
Pseudomonas?aeruginosa．
Table 3 The antibacterial activities of bellidifolin(x±s，n= 3)
Concentration(mg /mL) DMF(Blank) 0．25 0．5 1 2
Diameter of
inhibition
zone
(mm)
Staphylococcus aureus
Escherichia coli
Salmonella enterica
Pseudomonas aeruginosa
17．02±0．40
19．51±0．33
17．09±0．13
24．04±0．43
17．49±0．44*
19．84±0．29*
17．45±0．23*
24．40±0．26
18．06±0．16＊＊
20．13±0．17＊＊
17．60±0．26*
24．58±0．31*
18．20±0．26＊＊
20．26±0．15＊＊
17．91±0．23＊＊
24．88±0．39*
18．97±0．38＊＊
20．90±0．28＊＊
18．62±0．25＊＊
25．39±0．56＊＊
Note:Compared with Blank control:* P＜0．05，＊＊P＜0．001．
3 Conclusions
In the study，bellidifolin has been isolated and
characterized by IR，UV-vis spectra and X-ray crys-
tallography． The determination of crystal structure
showed that the compound crystallizes in triclinic with
P-1 space group．Theoretical frontier molecular orbit-
als and atomic charge distribution indicated that bel-
lidifolin molecule is stable． The vibrational frequen-
cies calculated by the DFT /6-31g(d，p)method as-
certained that the structure was stable too(no imagi-
nary frequencies) ，and the results were nearly con-
sistent with the observed frequencies．Meanwhile，the
electronic absorption spectra were studied with the
time-dependent density functional theory(TD-DFT)
and the calculated results were in good agreement
with the corresponding experimental data，and com-
pound molecule have a strong UV absorption at differ-
ent wavelength．It was noted that the preliminary anti-
bacterial activities indicated that bellidifolin possesses
obvious antibacterial activity， especially against
Staphylococcus aureus，which can be used as a poten-
tial antibacterial agent．
Acknowledgements
This work was financially supported by the Na-
tional Natural Science Foundation of China (No．
81303306;81160504) ，the Training Programs of Inno-
vation and Entrepreneurship for Undergraduates(No．
201410061052;201410061159)and Natural Science
Foundation of Tianjin(No．15JCQNJC13400)．
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