全 文 ：·快讯 Short Communication·
金丝桃属植物分泌结构的类型和金丝桃素含量的相关性
吕洪飞1 ,2＊　沈宗根1　李景原1　胡正海1＊＊
(1.西北大学植物研究所 , 西安 710069;2.浙江师范大学生命与环境科学学院 , 金华 321004)
摘要:　利用整体透明 、石蜡制片和半薄切片法 ,对金丝桃属(Hypericum L.)8 组 20 种 1 变种植物的分泌结构进行
了比较解剖研究 ,结果表明:该属植物的分泌结构可分为分泌细胞团和分泌囊两种类型。但在不同植物种和不同
器官内 , 分泌结构的类型和分布密度存在差异。对上述植物的提取物进行薄层层析和高效液相层析检测 , 结果表
明:具有分泌细胞团的植物器官含有金丝桃素;而无分泌细胞团的植物器官 , 则不含金丝桃素 ,从而证明金丝桃素
由分泌细胞团合成和贮藏。在前者中 ,其金丝桃素的含量与其分泌细胞团密度成正相关。
关键词:　金丝桃属;分泌细胞团;分泌囊;金丝桃素;高效液相层析;薄层层析
中图分类号:Q944.5;Q946.8　　　文献标识码:A　　　文章编号:0577-7496(2001)10-1085-04
The Patterns of Secretory Structure and Their Relation
to Hypericin Content in Hypericum
L Hong_Fei1 ,2＊ , SHEN Zong_Gen1 , LI Jing_Yuan1 , HU Zheng_Hai1＊＊
(1.Institute of Botany , Northwest University , Xi an 710069 , China;
2.College of Life and Environment Science , Zhejiang Normal University , Jinhua 321004 , China)
Key words:　Hypericum;nodule;cavity;hypericin;high_performance liquid chromatography;thin layer
chromatography
　　There are about 400 species in Hypericum world-
wide , and 55 species and 8 subspecies have been reported
in China[ 1] .A medicinal constituent , the hypericin , was
found in this genus[ 2 ,3] .Hypericin can be used as an an-
tidepressant drug[ 4] .In the last decade , hypericin was
also used in the treatment of AIDS
[ 6 , 7] , because of its im-
munotropic activity and marked anti_RNA virus and anti_
DNA virus effects[ 5] .Liu and Hu[ 8] , Lǜ et al[ 9-11] have
examined the histochemistry , types and distributional
density of the secretory structures in the genus , but phy-
tochemical evidence is still lacking.Southwell and Camp-
bell[ 12] have already found the correlation between the hy-
pericin content and the number of leaf gland (dark and
translucent dots)in H.perforatum.Cellarova et al[ 13]
reported that hypericin and its derivatives accumulate in
the multicellular nodules , which are mostly arranged
around the leaf margin and the margins of petals.But
they did not point out the statistically significant relation-
ship between the hypericin content and the mean value of
the gland density per area(mm2)of leaf or the total num-
ber of hypericin_contained glands per leaf.Berghofer(cit-
ed by Pan et al)[ 2] reported that both hypericin and pseu-
dohypericin were found in H.perforatum , but H.hirsu-
tum only contained hypericin.In this paper , the relation-
ship between the patterns of secretory structure and hyper-
icin content of the genus is reported by using phytochemi-
cal methods.
1　Materials and Methods
1.1　Materials
Eight sections , 20 species and 1 variety (Table 1)
of Hypericum L.were used for study.
1.2　Methods
1.2.1　Anatomical observation of microstructure　
Leaves were cleared with 5%NaOH solution[ 9] and were
treated with routine graded ethyl series for paraffin sec-
tions[ 10] , thin sections and semithin(1μm in thickness)
sections[ 11] .Semi_thin sections were cut with a glass
knife on a rotary ultramicrotome (Reichert_Jung).The
sections were stained with toluidine blue O or azure B_
methylene blue , and observed and photographed under a
Leica DMLB microscope.
1.2.2 　Extraction and determination of hypericin
and pseudohypericin　One gram of dried and homoge-
nized leaves of the respective 20 species and 1 variety.In
addition , the leaf margins with nodules , the central por-
tion of leaf with secretory cavities , calyxes , petals and
fruits of H.perforatum were respectively prepared in the
same way for extraction of hypericin and pseudohypericin.
The materials were extracted with chloroform (to remove
the chlorophyll)in a sonicator for 30 min and filtered[ 13] .
The dried powder was then extracted with methanol in a
sonicator for 30min , and the volume of a tract was deter-
Received:2001-05-14　Accepted:2001-06-25
Supported by the National Natural Science Foundation of China(30070045).
＊ The present address:College of Life and Environment Science , Zhejiang Normal University , Jinhua 321004 , China;E_mai l:.
＊＊Author for correspondence.E_mail:.
植　物　学　报　2001 , 43(10):1085-1088 　 　 　 　 　 　 　
Acta Botanica Sinica
mined after filtration[ 14] .
The extracts were then subjected to TLC (thin layer
chromatography)and HPLC assay .In the TLC , a mixture
of n_butyl alcohol_acetic acid_H2O (4∶1∶1)was used as
the spreading solution and the chromatographic profiles
were scanned and photographed.
Hypericin and pseudohypericin were also determined
from methanol extract with an HPLC (Hitachi Pump L_
7110 , UV_Vis L_7420)at 590 nm.A 20_μL sample or
standard hypericin was applied to an ODSRP C18 (4.6
mm×250 mm)column with a flow phase of methanol_
acetonitrile_0.1 mol/L NaH2PO4(800∶100∶100)and a
flow rate of 1 mL/min at a temperature of 22 ℃.The
time of appearing hypericin absorption peak (peak Ⅰ)of
a sample was the same as that of standard hypericin.The
time of appearing pseudohypericin absorption peak(peak
Ⅱ)referred to the paper of Zhou et al reported[ 14] .
2　Results and Discussion
2.1　Correlation between types , location , distribu-
tive density of secretory structures and hypericin con-
tent of leaf in Hypericum
When the translucent leaf , paraffin sections and thin
sections of leaf of the 20 species and 1 variety were inves-
tigated respectively , the secretory structures of leaves were
found to be divided into the nodule(Fig.1)and the cavi-
ty (Fig.2)types.The types differed from each other in
location and distributional density among the Hypericum
species under study (Table 1;Figs.3-6).The nodular
type was found in 2 sections(Hypericum and Adenosepa-
lum)of 5 species.There were no nodules in the leaf of
H.hirsutum , however , nodules were found in the calyx
and petal of this specie.In another 5 sections , only cavi-
ties in leaves were observed.
The results of TLC and HPLC demonstrated that 5
species with nodules contained hypericin(Fig.7), and in
the other 15 species and 1 variety without nodule , no hy-
pericin was detected(Fig.9), suggesting that the nodule
is the secretory structure that produces and stores hyper-
icin.
The ratio of nodule density of H.erectum to that of
H.sampsonii was 3.443 , and the ratio of their hypericin
content was 3.878.The ratio of nodule density of H.
erectum to that of H.perforatum was 3.239 , and the ra-
tio of their hypericin content was 2.890.The ratio of nod-
ule density of H.perforatum to that of H.sampsonii was
1.066.However , if the ratio is multiplied by (75/70)3 ,
i.e.the size coefficient of the nodule , a value of 1.230
was obtained which means that it included nodule density
and nodule size factors.The ratio of their hypericin con-
tent was 1.343.And the ratio of nodule density and size
of H.wightianum to that of H.monanthemum was 0.85
and 0.85×(77.5/75.0)3=0.937 8 respectively , their
ratio of hypericin content was 0.973 4.The above results
indicate a positive correlation between nodule density and
hypericin content in leaves of the genus.The correlation
factor was 0.996 2 , and the regression equation was:hy-
pericin content=0.000 19+0.076 31×nodule density.
The P_values are shown in Table 1.The hypericin con-
tent was also affected by the size of nodule , but it had no
relationships with the cavity density.
Table 1　Distribution and density of secretory structures and the hypericin content in the leaves of the Hypericum
No. Species
Nodule Cavity
L
ADN
(μm)
Density
(/mm2)
Diameter
(μm)/MV L
Density
(/mm2)
Hypericin
content
(%)
5 H.monogynum Ab Ab Ab Ab SC:M 47.1 0
8 H.subsessile Ab Ab Ab Ab SC:PMS 9.0 0
9 H.acmosepalum Ab Ab Ab Ab SC:PMS 11.0 0
11 H.hookerianum Ab Ab Ab Ab SC:PMS 33.0 0
14 H.henryi Ab Ab Ab Ab SC:PMS 11.4 0
15 H.patulum Ab Ab Ab Ab SC:MS 2.3 0
19 H.bellum Ab Ab Ab Ab SC:PM 7.5 0
24 H.forrestii Ab Ab Ab Ab SC:M 11.8 0
25 H.pseudohenryi Ab Ab Ab Ab SC:PMS 20.8 0
26 H.beanii Ab Ab Ab Ab SC:PMS 19.8 0
32a H.ascyron var.ascyron Ab Ab Ab Ab SC:S 10.5 0
32b H.ascyron var.longistylum Ab Ab Ab Ab SC:S 6.4 0
34 H.japonicum Ab Ab Ab Ab SC:PS 22.0 0
36 H.hirsutu m Ab Ab Ab Ab SC:PMA 6.8 0
39 H.monanthemum LE:A 0.68 Ab 70-80/ 75 SC:AS 9.4 0.0661
40 H.wightianum LE:S(A) 0.80 Ab 70-90/ 77.5 SC:S 10.5 0.0646
44 H.sampsonii LE:A ,SC:S 0.72 0.61＊ 60-80/ 70 SC:APS 3.5 0.0413＊＊
45 H.erectum LE, SC:S 0.32 2.10＊ 70-80/ 75 Ab Ab 0.1603＊＊
50 H.perforatum LE ,SC:S 2.20 0.65＊ 60-80/ 75 SC:AS 5.2 0.0555＊＊
56 H.androsaemum Ab Ab Ab Ab SC:PMS 17.1 0
57 H.densiflorum Ab Ab Ab Ab SC:P 27.4 0
No., numbers;L , localization;ADN , average distance between two nodules in leaf edge;MV , mean value;Ab , absent;LE , leaf edge;SC , scattered;P , pal-
isade tissue;S , spongy t issue;M , median type between palisade tissue and spongy tissue;A , across mesophyll tissue.
＊ P_value for density:H.sampsonii vs H.perforatum <0.05;H.sampsonii and H.perforatum vs H.erectum <0.001;＊＊ P_value for hypericin con-
tent:H.sampsonii vs H.perforatum <0.01;H.sampsonii and H.perforatum vs H.erectum <0.001.
1086　 植　物　学　报　　Acta Botanica Sinica 43 卷
Figs.1-6.　1 , 2.Thin sections , ×276.1.Nodule(N)of Hypericum erectum .2.Cavity(C)of H.perforatum.3-6.Translucent leaf ,
×38.3.Nodules(right←)of leaf margin and cavities(left←)of H.wightianum.4.Cavities(←)of H.monogynum.5.Nodules(←)
of H.erectum.6.Nodules(left←)and cavities(right←)of H.sampsonii.
Figs.7-9.　Photographs of TLC.Ⅰ , hypericin;Ⅱ , pseudohypericin;the numbers of species are the same as in Table 1;F , flower;L,
leaf;K , calyx;P , petal;S , stem;R, root;LM , leaf margin;LC , leaf central part;Fr , fruit;HS , hypericin standard sample.8 , H.per-
foratum.
Table 2　Nodule density of organs and parts and hypericin content in Hypericum
Species Organs or parts Nodules density Hypericin content(%)　　
H.perforatum Leaf margin having nodules Ab 0.145 6
Central part s of leaf only having cavit ies Ab 0
Stem Ab 0
Root Ab 0
Fruit Ab 0
Calyx 0.833/mm2 0.065 3
Petal 26.45/ piece 1.268 2
H.hirsutum Flowers Calyx 2.004/mm2 , petal 1.125/ piece 0.257 9
H.monanthemum Flowers Calyx 2.503/mm2 , petal 3.025/ piece 0.343 0
H.erectum Flowers Calyx 3.125/mm2 and 5-8 tubular nodules/ piece ,
petal 21.36/ piece 0.706 8
Ab , absent.
2.2　Relationship between the nodule density in or-
gans of the genus and hypericin content
Nodules was found in the calyx , petal and leaf mar-
gin of H.perforatum in which hypericin was all detect-
ed.The hypericin content was positively correlated with
the nodule density (Table 2;Figs.8 , 10 , 13 , 14).In
contract , nodules were not observed in fruits , roots ,
stems and the central part of the leaves;and likewise hy-
pericin was not detected in any of these organs(Table 2;
Figs.8 , 11).Likewise positive correlation between hy-
pericin content and nodule density was also found in flow_
ers of certain species(H.hirsutum , H.monanthemum ,
10 期 吕洪飞等:金丝桃属植物分泌结构的类型和金丝桃素含量的相关性(英) 1087　
Figs.10-15.　HPLC profiles.10.Calyx of H.perforatum.11.Fruit of H.perforatum .12.Flower of H.hirsutum.13.Hypericin stan-
dard.14.Petal of H.perforatum .15.Flower of H.erectum.Ⅰ , hypericin;Ⅱ , pseudohypericin.
H.erectum)(Table 2;Figs.7 , 12 , 13 , 15).In fact ,
there is no nodule in the leaf of H.hirsutum , so hyper-
icin was not detected either.
Both hypericin and pseudohypericin were detected in
H.monanthemum , H.wightianum , H.sampsonii , H.
erectum , H.perforatum (Figs.7 , 8 , 10 , 14 , 15).But
the hypericin was only detected in the nodules of H.hir-
sutum flower;the pseudohypericin was not detected in
this species(Fig.12).Our results are the same as those
of Berghfere s[ 2] .
In the 3 sections that contain hypericin , Sect.Tae-
niocarpium has distant evolutionary relationship with Sect.
Hypericum and Sect.Adenosepalum , but the relationship
between Sect.Hypericum and Sect.Adenosepalum is
closer.Our results suggested that an understanding of the
anatomic structure of Hypericum might be helpful in the
selection of species with high hypericin productivity.Fur-
thermore , our HPLC data may also provide some system-
atic basic information of the Hypericum genus.
Acknowledgements:　The phytochemical experiments
were done in Xida_Yonghe Key Opening Laboratory of Bi-
ology andMedicine of Shaanxi Province.Thanks are due
to Prof.SHEN Wen_Ji for his kind guidance and com-
ments.
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1088　 植　物　学　报　　Acta Botanica Sinica 43 卷