全 文 ：Received 26 Aug. 2003 Accepted 3 Nov. 2003
Supported by the National Natural Science Foundation of China (30370088) and the Natural Science Foundation of Zhejiang Province (302101).
* Author for correspondence. Tel: +86 (0)579 2282968; E-mail: .
http://www.chineseplantscience.com
FTIR Spectrum of Hypericum and Triadenum with Reference to
Their Identification
LÜ Hong-Fei1, 2*, CHENG Cun-Gui2, TANG Xi3, HU Zheng-Hai4
(1. Institute of Botany, The Chinese Academy of Sciences, Beijing 100093, China;
2. College of Life Sciences, Zhejiang Normal University, Jinhua 321004, China;
3. Fujian Institute of Education, Fuzhou 350001, China;
4. Institute of Botany, Northwest University, Xi’ an 710069, China)
Abstract: The infrared spectroscopic study of leaves of nine sections, 43 species, one subspecies and
one variety in Hypericum L. and two species in Triadenum Raf. were conducted directly and rapidly with
FTIR and OMNI-sampler accessory. The results showed that the infrared spectra of Hypericum and Triadenum
were fingerprint-like patterns which were highly typical for different taxa. Significant differences in the
infrared spectra were found between these two genera and among the nine sections of Hyper icum. And
certain differences exist in the spectra among species in the same section of Hypericum or in the Triadenum.
Furthermore, no significant difference was found in the infrared spectral patterns in the leaves at various
developmental stages and in leaves of the same species collected from different geographic regions, although
occasionally geographic difference did exist in the same species. The results indicated that the infrared
spectra of leaves are of taxonomic value at the level of species and sections in these two genera, and this
technique can be widely used for identification and classification of other taxa when standard spectra
are available.
Key words: Hypericum ; Triadenum ; leaf; infrared spectra figures; classification; identification
Acta Botanica Sinica
植 物 学 报 2004, 46 (4): 401-406
Hypericum L. and Triadenum Raf. are subordinate to
Glusiaceae. There are about 400 species in Hypericum world-
wide except in desert and most of the tropic lowlands. Fifty-
five species and eight subspecies have been reported in
China which were concen trated in Southwest China. H.
androseamum was in t roduced from U.K., and H.
densiflorum was int roduced from U.S.A. There are about
six species of Triadenum from India, Japan, Korea, the Far
Area o f Russia, the East of America and Canada in the
world . There are two species in China (Delectis Florae
Reipublicae Popularis Sinicae Agendae Academiae Sinicae
Edita, 1990). Up to now, the composition of most species in
Hypericum was determined, 70 species were stud ied in
detail, and 140 components were identified (Kitanov and
Blinova, 1987; Liang, 1998). The stability of the characteris-
tic chemical composition renders wide application of these
species in the taxonomical and evolutionary studies. And
infrared spectra images represent the information of plant
components as a whole. Sometimes it is difficult to differ-
entiate two species with similar morphological characters
as Robson (1995) pointed out that it was very difficult to
morphologically differentiate H. uralum from its clos ely
related species— H. henryi subsp. henryi and H. patulum.
Fourier transform infrared (FTIR) spectroscopy allows
to detect the whole range of infrared spectrum s imulta-
neously providing speed and accuracy in measurements of
biolog ical specimens (Griffiths and de Has eth , 1986).
Recently, the technique has been successfully explo ited
for clas sification o f normal and aged s oybean s eeds
(Kusama et al., 1997). Alsberg et al. (1998) reported the
classification of Eubacterium species using the diffuse re-
flectance-abs orbance Fou rier t rans form in frared
spectros copy. Wang et a l. （2003）reported the effect
of boron on the change of FTIR spectra of cell wall during
pollen germination and pollen tube growth. Wu et al. (2003)
studied the chemical characterization of cas parian strip in
needles of Pinus bungeana with this technique. However,
FTIR spectroscopy has not been employed in the classifi-
cat ion of plant leaves so far. In this paper we report the
identification and classification of leaves of Hypericum and
Triadenum by using FTIR technique.
1 Materials and Methods
1.1 Materials
Nine sect ions , fo rty-th ree species one subs pecies
and one variety of Hypericum (Lü and Hu, 2001) and
two species of Triadenum were included in this s tudy
(Tab le 1).
Acta Botanica Sinica 植物学报 Vol.46 No.4 2004402
Table 1 Source of material studied and vouchers
Taxa Vouchers
Hypericum
Sect. 1. Ascyreia
1. H. elliptifolium Gongshan, Yunnan, H. F. Lü 00041 (WNU)
2. H. augustinii Jinghong, Yunnan, H. F. Lü 00043 (WNU)
4. H. cohaerens Daguan, Yunnan, H. F. Lü 00042 (WNU)
5. H. monogynum Jinhua, Zhejiang, H. F. Lü 98001 (WNU)
6. H. prattii Kangding, Sichuan, H. F. Lü 00024 (WNU)
7. H. longistylum subsp. giraldii Taibaishan, Shanxi, H. F. Lü 00035 (WNU)
8. H. subsessile Dali, Yunnan, H. F. Lü 00014 (WNU)
9. H. acmosepalum Lijiang, Yunnan, H. F. Lü 00015 (WNU)
10. H. lagarocladum Fengjie, Chongqing, H. F. Lü 00025 (WNU)
11. H. hookerianum Dali, Yunnan, H. F. Lü 00013 (WNU)
12. H. addingtonii Yunlong, Yunnan, H. F. Lü 00020 (WNU)
13. H. wilsonii Wushan, Chongqing, H. F. Lü 00026 (WNU)
14a. H. Henryi subsp. henryi Kunming Botanical Garden, H. F. Lü 00003 (WNU)
14b. H. henryi subsp. uraloides Songpan，Sich uan， H. F. Lü 0 0021 (WNU)
15. H. patulum Hanzhou Botanical Garden， Zhejiang， H. F. Lü 00032 (WNU)
16. H. uralum Fengqin， Yun nan，J. Zheng 68 (KUN)
17. H. maclarenii Kangding， Sich uan，H. F. Lü 0 0023 (WNU)
18. H. choisianum Gon gshan，Yunnan，L. M. Fon g 24 131 (KUN)
19. H. bellum subsp. bellum Kunmin g Botanical Garden, CAS，H. F. Lü 00004 (WNU)
21. H. stellatum Leibo， Sichuan，H. F. Lü 0 0027 (WNU)
23. H. curvisepalum Dali， Yunnan， H. F. Lü 0 0017 (WNU)
24. H. forrestii (Chittenden) Kunmin g Botanical Garden, CAS，H. F. Lü 00006 (WNU)
25. H. pseudohenryi Kunming Botanical Garden, H. F. Lü 00005 (WNU)
26. H. beanii Kunming Botanical Garden, H. F. Lü 00007 (WNU)
Sect. 2. Roscyna
31. H. macrosepalum Kan gdin g，Sichuan，H. F. Lü 000 28 (WNU)
32a. H. ascyron Northwest University Garden， Shaanxi，H. F. Lü 00001 (WNU)
32b. H. ascyron var. longistylum Northwest University Garden， Shaanxi，H. F. Lü 00033 (WNU)
33. H. przewalskii Qinling，Shaanxi， H. F. Lü 0 0034 (WNU)
Sect. 3. Spachium
34. H. japonicum Jinhua， Zhejiang， H. F. Lü 98002 (WNU)
35. H. gramineum Dali， Yunnan， H. F. Lü 0 0016 (WNU)
Sect. 4. Taeniocarpium
36. H. hirsutum Gongliu， Xinjiang， D. Y. Tan 00001 (WNU)
Sect. 5. Hirtella
37. H. scabrum A’er tai， Xinjiang，D. Y. Tan 000 02 (W NU)
Sect. 6. Adenosepalum
38. H. filicaule Gongshan， Yunnan， H. F. Lü 0 0019 (WNU)
39. H. monanthemum Dali，Yunnan，H. F. Lü 000 12 (WNU)
40. H. wightianum subsp. wightianum Kunming Botanical Garden, H. F. Lü 00008 (WNU)
42. H. elodeoides E’shan， Sichuan， H. F. Lü 00018 (WNU)
Sect. 7. Hypericum
44. H. sampsonii Jinhua， Zhejiang， H. F. Lü 98003 (WNU)
45. H. erectum Jinhua， Zhejiang， H. F. Lü 99001 (WNU)
47. H. faberi Qingyuan， Zhejiang， H. F. Lü 00029 (WNU)
48. H. petiolulatum subsp. yunnanense Wush an，Cho ngqing，sine coll . 0 672 852 (KUN)
49. H. seniavinii Jinhua， Zh ejiang，H. F. Lü 9 8004 (WNU)
50. H. perforatum Northwest University Garden， Shaanxi，H. F. Lü 99002 (WNU)
51. H. attenuatum Jinhua， Zh ejiang，H. F. Lü 0 0053 (WNU)
Sect. 8. Androsaemum
56. H. androsaemum Kunming Botanical Garden, H. F. Lü 00002 (WNU), introduced from U. K.
Sect. 9. Mysiandra
57. H. densiflorum Hanzhou Botanical Garden, Zhejiang，H. F. Lü 00013 (WNU), introduced from U. S. A.
Triadenum
60. T. japonicum Jilin，H. F. Lü 000 52 (W NU)
61. T. breviflorum Jinyun，Zhejian g， H. F. Lü 0 0005 (WNU)
*, the species are arranged and numbered according to the classification system in Flora Reipublicae Popularis Sinicae, 50(2); 56, 57 are two
species introduced to China from U.K. and U.S.A. respectively. WNU, Northwest University specimen chamber; KUN, Kunming Botanical
Institute specimen chamber.
LÜ Hong-Fei et al.: FTIR Spectrum of Hypericum and Triadenum with Reference to Their Identification 403
1.2 Methods
The apparatus employed was the Fourier transform in-
frared (FTIR) spectrometer (American NICOLET NEXUS
Model 670) with a resolving power of 0.125 cm-1, spectrum
range of 650-4 000 cm-1 and scanning accumulative limita-
tion of 128 times. All spectra were obtained with the aid of
an OMNI s ampler and data were computerized with an
OMNIE 5.1 Brainpower Operation software.
The central portion and the edge of a leaf, with the abaxial
side facing up, was respectively placed directly on the ger-
manium piece of the infrared spectrometer and data of in-
frared absorbance were computerized for analyses.
The wave-peak pat tern (s ) o f each s pecies (o r
subs pecies) within the indiv idual s ections or among the
eight sections of Hypericum or even between Hypericum
and Triadenum were overall compared by count ing the
number of wave peaks of all individual species in the two
genera that identically matched (at the level of 0.2 wave-
number error) the peak of the species under concern.
The correlation coefficient of that species within that
section was obtained by dividing the total number of the
identically matched wave peaks by the total number of spe-
cies except the species under concern. This was expressed
by the formula as

Spi=Σ(SEn)/(n-1)
Or the correlation coefficient of that species with an-
other section was obtained by dividing the total number of
the identically matched wave peaks by the total number of
species of another section which of that species matched.
This was expressed by the formula as
Spi=Σ(SEm)/m
Whereas Spi denotes the correlation coefficient, SEn is
the number of matched wave peaks of that species within
that section and SEm is the number of matched wave peaks
of that species with another section. n is the number of that
section which o f that species. m is the number of another
section which o f that species matched that have identical
matches among species.
Then the correlat ion coefficient with in sect ion or be-
tween two sections was calculated by dividing the average
number of identically matched absorpt ion peaks in each
section (Spi) of Hypercum or in Triadenum that had been
counted by the total number of species of that s ection.
That was expressed by the formula as
SEi=Σ(Spi)/n
Whereas SEi denotes the correlation coefficient between
sections or within that section.
2 Results
2.1 The infrared spectra of different parts of a leaf
Comparing the infrared spectrum of central portion and
that o f edge of the same leaf, similar o f waves and wave-
number of wave-peaks were found , but the absorption
peaks of the leaf edge were higher than that of central por-
tion of leaf (Fig.1).
2.2 The infrared spectra of leaves at different develop-
mental stages
There were similar infrared spectral patterns and wave-
number o f wave peaks, but the absorp tion peaks of the
mature leaf were h igher than that of the young leaf. The
high peaks of the mature leaf, which emerged at the spec-
trum at 3 300 and 1 050 cm-1 representing the absorbance
of OH and C-O bond respectively, indicating that the ma-
ture leaf contains more cellulose than that of the young leaf
(Fig.2).
n-1
m
Fig.1. The infrared spectral profiles in different parts of leaves
of Hypericum seniavinii. a, edge section of matured leaf; b, central
section of matured leaf; c, the edge section of young leaf.
Fig.2. The infrared spectral profiles at different developmental
stages in Hypericum sampsonii. a, matured leaf; b, maturing leaf;
c, young leaf.
Acta Botanica Sinica 植物学报 Vol.46 No.4 2004404
2.3 The infrared spectra of leaves of the same species but
grown in different geographic regions
Similar infrared spectral patterns and wave-number of
absorbing peaks were demonstrated in leaves of the same
species collected from differen t geographic regions, but
the height of the abs orption peaks varied, the closer the
regions, the more similar the wave-peak height (Fig.3).
2.4 The infrared spectra of leaves among sections and
species in Hypericum
The in frared spectral patterns and the matched wave-
number of peaks among sections of Hypericum were differ-
ent (Fig.4), but were similar among species within individual
sections (Figs.5, 6), showing bigger correlation coefficient
of the latter (Table 2).
2.5 The correlation coefficient between two sections in
Hypericum (or Triadenum or H. elliptifolium)
Comparison of the number of identically matched wave
peaks (correlation coefficient) between two sections in
Fig.3. The infrared spectral profiles of leaves of the same spe-
cies in Hypericum patulum grown in different geographic regions.
a, Qingyuan, Zhejiang; b, Hanzhou Botanical Garden, Zhejiang; c,
Dali, Yunnan; d, Chengdu, Sichuan.
Fig.4. The infrared spectral profiles of leaves of sections. a, Hyperi-
cum elliptifolium; b, H. beanii (Section 1); c, H. przewalskii (Section
2); d, H. japonicum (Section 3); e, H. hirsutum (Section 4).
Fig.5. The infrared spectral profiles of leaves between sections
or species in the same section. a, Hypericum scabrum (Section 5);
b, H. filicaule; c, H. monanthemum; d, H. wightianum subsp.
wightianum; e, H. elodeoides ( b-e, Section 6).
Fig.6. The infrared sp ectral p rofiles of leaves of species in
Section Hypericum. a, Hypericum sampsonii; b, H. faberi; c, H.
petiolulatum subsp. yunnanense; d, H. perforatum (Section 7).
Hypericum or between a section in Hypericum and
Triadenum or H. elliptifolium.
3 Discussion
In general, no significant difference in infrared spectral
pattern was found between the center and the edge of the
same leaf, during different developmental stages, or in leaves
of the same species grown in various geographic regions.
In addition , the absorp tion peaks of the leaf edge were
higher than those of the leaf center and those of the old leaf
were higher than those of the young leaf, suggesting that
the leaf edge o f the mature leaf is preferab le for infrared
spectroscopic studies.
Seven identically matched infrared spectral absorption
peaks were seen between H. henryi subsp. henryi and H.
henryi subsp. uralo ides that were b igger than those be-
tween H. henryi and other species in Sect. 1. Ascyreia. This
implicated that the two subspecies are more in common in
LÜ Hong-Fei et al.: FTIR Spectrum of Hypericum and Triadenum with Reference to Their Identification 405
leaf composition and are most closely related. The result
also supports the conclusion that the two subspecies were
classified into one species (Delectis Flo rae Reipublicae
Popularis Sinicae Agendae Academiae Sinicae Ed ita,
1990)，and is consonant with their anatomical character-
istics as reported (Lü and Hu, 2001).
The number of matched absorption peaks among spe-
cies of the same section, such as Sect. 1. Ascyreia, Sect. 2.
Roscyna, Sect. 3. Spachium, was bigger than that between
two sections (Table 1). This means that these sections have
more common composition within its section. These re-
su lts support the class ificat ion of sections of 《Flora
Reipublicae Popularis Sinicae》，and are consonant with
their anatomical characteristics (Lü and Hu, 2001) .
No prominent d ifference was found in the number of
matched abs orption peaks between Sect. 1. Ascyreia and
Sect . 2. Rosc yna , Sect . 2. Rosc yna an d Sect .4.
Taeniocarpium, Sect. 2. Roscyna and Sect. 6. Adenosepalum,
Sect. 2. Roscyna and Sect. 7. Hypericum, Sect. 3. Spachium
and Sect. 4. Taeniocarpium, Sect . 3. Spach ium and
Triadenum. This means that leaf composition is more in
common between these section pairs. However, these ab-
sorption peaks were obviously different between Sect. 1.
Ascyreia and Sect. 9. Mysiandra, Sect. 1. Ascyreia and Sect.
8. Androsaemum, Sect . 1. Ascyreia and Sect. 5. Hirtella,
Sect. 2. Roscyna and Sect. 9. Mysiandra, and there is no
common wave peak between Sect. 4. Taeniocarpium and
Sect. 9. Mysiandra, Sect. 5. Hirtella and Sect. 9. Mysiandra,
Sect. 8. Androsaemum and Sect. 9. Mysiandra , Sect. 5.
Hirtella and Sect. 8. Androsaemum. The results showed
that these sections have their own different composition
and are distantly related between these section pairs, simi-
lar to the conclusion drawn by Robson’s (Robson, 1977;
1981) on the evolutionary trends about these sections.
It was believed that there is a d istant relationship be-
tween Sect. 6. Adenosepa lum and Sect. 7. Hypericum
(Robson, 1977; 1981). However, it appeared a much closer
relationship exis ting between the two sections, because
the correlation coefficient between Sect. 6. Adenosepalum
and Sect. 7. Hypericum is bigger than that among species
within it s sect ion, indicating that the two sect ions have
more s imilar compos it ion , es pecially hypericin and
pseudohypericin that exist in nodules (Lü et al., 2001).
Besides, the bract, calyx and petal-base in H. seniavin ii
have nodule-tine, s imilar to the nodu le-hair in Sect. 6.
Adenosepalum. Therefore, we consider H. seniavinii has
common characteristics of transitional species of the two
sections.
The existence of so many identically matched wave
peaks of the infrared spectra between these two genera
suggests a close relationship of the two genera, especially
between Triadenum and H. el lipt i fo lium or Sect . 3.
Spachium. This result supported the fact that Triadenum
comes under Hypericum (Robson, 1977). Therefore, we sug-
gest that the changes of the infrared spectra are o f some
taxonomic value at the level of species and section in these
two genera, and this technique can be widely used for iden-
tification and class ificat ion o f other taxa when standard
spectra were established.
Acknowledgements: Thanks are due to Prof. LI Xi-Wen,
LI Jing-Xiu, LI Xue-Dong, XU Hua, YANG Jian-Bin, ZAO
Shi-Zheng, XU Jie-Mai, YU Xiang-Yu, DING Bin-Yang, TAN
Deng-Yan, SHEN Guan-Mian, CHU Qing-Gang, LIU Wen-
Zhe for their kind guidance and help during the materials
collection. Thanks are due to Prof. N. K. B. Robson (The
Natural History Museum, U.K.) for his kind guidance and
his Hypericum monograph.
Table 2 The correlation coefficient (SEi) between sections in Hypericum (or H. elliptifolium and Triadenum)
H. ellip-
Sect. 1 Sect. 2 Sect. 3 Sect. 4 Sect. 5 Sect. 6 Sect. 7 Sect. 8 Sect. 9 Triadenum
tifolium
H. ellip- 1 0783 1.5 0 3 2 2.5 2 1 0 2
tifolium
Sect. 1 0.783 1.2 0.891 0.891 0.609** 0.565** 0.957 0.758** 0.609** 0.13** 1.403
Sect. 2 1.5 0.913 1.583 1.125 1.5 0.5* 1.5 1.25 1 0.5* 1.375
Sect. 3 0** 0.913* 1** 3 3 1.5 1.5** 1.143** 1** 1** 2
Sect. 4 3 0.783 1.25 2.5 10 1 1 1.143 2 0 1
Sect. 5 2 0.609 0.5 1 1 10 1 1 0 0 0.5
Sect. 6 2.5 0.957* 1.5 1.5 1.5 1 1.834 2.357 1 1.25 1.375
Sect. 7 2 0.808* 1.268 1.143* 1.286 0.857** 2.357 2.119 0.571** 1.286* 1.214*
Sect. 8 1 0.217 1 1 2 0 1 0.714 10 0 0.5
Sect. 9 0 0.087 0.5 1 0 0 1.25 1.286 0 10 1
Triadenum 2 1.087* 1.125* 2 1.5 1** 1.25** 1.43 0.5 1** 2
*, P < 0.05; **, P < 0.01.
Acta Botanica Sinica 植物学报 Vol.46 No.4 2004406
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