*Tel.:#359-2-988-31-42.
E-mail address:gkitanov@mbox.pharmfac.acad.bg (G.M. Kitanov).
Hypericin and pseudohypericin in some
Hypericum
species
Gerassim M. Kitanov
*
Department of Pharmacognosy, Faculty of Pharmacy, Medical University of Soxa, DunavStr. 2, 1000 Soxa, Bulgaria
Received 24 November 1999; accepted 3 March 2000
Abstract
Hypericin and pseudohypericin were found in 27 of the 36 evaluated species fromHypericum
L., belonging to 17 sections of the genus. Pseudohypericin is reported by us in 15 taxa for the
"rst time. Most of the species contained both components and the amount of pseudohypericin
usually exceeded that of hypericin. InH. hirsutumandH. empetrifoliumonly hypericin was
found, whereasH. formosissimumyielded pseudohypericin only. The total content of hypericins
varied widely from 0.009% in H. empetrifolium to 0.512% in H. boissieri and the largest
amounts were established in taxa of sections Drosocarpium, Hypericum and Thasia. The
distribution of hypericin and pseudohypericin inHypericum species has an important
taxo-nomic value for infrageneric classi"cation of the genus. These components were not found in the
primitive sectionsAscyreia, Androsaemum, Inodora, Roscyna, BupleuroidesandSpachiumbut
occur widely inHypericum, Adenosepalumand the sections fromOlympiagroup. Although the
genera of subfamily Hypericoideae are characterized by the presence of anthrone derivatives, condensed anthrones such as hypericin and pseudohypericin have not been found in these
genera and the remaining subfamilies of the Guttiferae. ( 2001 Elsevier Science Ltd. All rights
reserved.
Keywords: Hypericum; Hypericoideae; Guttiferae; Hypericin; Pseudohypericin; Hypericins content; Chemosystematics
1. Introduction
Hypericin and pseudohypericin, naturally occurring red pigments, have been very intensively studied. They possess photodynamic, antidepressive and antiviral activ-ities (Roth, 1990; Weiss, 1991; Bombardelli and Morazzoni, 1995) and are chemotaxonomically important for the infrageneric classi"cation ofHypericum (sub-family Hypericoideae) (Robson, 1977, 1981). According to Robson the genus includes over 400 species and is divided into 30 sections. Many individual or groups of species have been investigated for the presence of hypericin. Mathis and Ourisson (1963) summarized the results of previous investigations and studied about 150 new taxa. These authors have listed the taxa according to the old infrageneric classi"cation by Keller (1925). From then on few new species have been studied. Twenty-two taxa have been found by other authors to contain pseudohypericin (Brockmann and Sanne, 1957; Netien and Lebreton, 1964; Sakar and Engelshowe, 1990; Zevakova et al., 1991; Makovetskaya and Maksiutina, 1991). Kartnig et al. (1996) reported the presence of both compounds in cell cultures from 6Hypericumspecies.
The aim of this investigation was (i) to examine the distribution of hypericin and pseudohypericin and total hypericin contents in some representatives ofHypericum and (ii) to examine their potential chemotaxonomic signi"cance for the Robson's sectional classi"cation of the genus.
2. Material and methods
2.1. Plant materials
Samples were taken from herbarium material (7 species) or collected from wild Bulgarian habitats (20 species). Nine taxa were gathered at di!erent geographical regions and some of them were donated by Prof. I. Assenov (So"a), Dr. L. Teslov (Sankt Petherburg) and Dr. A. Makovetskaya (Kiev). The origin of the species, botanical identi"cation and voucher specimens at SOM were given in the"rst part of this series (Kitanov and Nedialkov, 1998).
The samples of fresh plants were air dried at room temperature and the relative moisture was 8}10%.
2.2. TLC separation of hypericin and pseudohypericin
The powdered samples (1 mm) were defatted with CHCl
Hypericin was isolated from #owers of H. hirsutum. A mixture of hypericin and pseudohypericin was obtained fromH. perforatumand both compounds were separ-ated by TLC. These have then been used as standards for TLC comparisons with plant extracts.
The relative concentration of the compounds on the chromatograms was evaluated visually by their#uorescence.
2.3. Quantitative determination of hypericins content
The determination of the total hypericin content in plant materials was carried out by classic spectrophotometric method at 590 nm (DAC, 1986). A Shimadzu UV-1203 apparatus (Japan) was used for the measurements. Three determinations were made from each sample and the mean value was calculated.
3. Results and discussion
3.1. Distribution and content of hypericin and pseudohypericin in investigated species
The two compounds have a red #uorescence in UV light and they are easily distinguished from the other phenolic constituents on the chromatograms. Chloro-phylls possess a similar #uorescence and for that reason they were removed with CHCl
3.
The 36 examined species represent about 9% of all taxa in the genus and fall into 17 sections. Both herbarium and fresh samples were used. The results from the chromato-graphic investigation and quanti"cation of the hypericins are presented in Table 1. The taxa are listed according to the infrageneric classi"cation by Robson (1977).
Hypericin and/or pseudohypericin were observed in 27 species and were not detectable in 9 taxa ofHypericum. Most of the taxa contain both components. In two species* H. hirsutum and H. empetrifoliumonly hypericin was found, whereas H. formosissimumcontains pseudohypericin only. In contrast to current assumptions in most cases the pseudohypericin content was signi"cantly higher than that of hy-pericin. Summarizing the results of our earlier investigation (Kitanov, 1988) and of the present study, in 15 taxa pseudohypericin is reported by us for the"rst time (Table 1).
Table 1
Distribution of hypericin (Hp) and pseudohypericin (Pshp) and their total content in someHypericumspp
Section and species Hp Pshp Content (%)!
IIIAscyreiaChoisy (36)"
4.H. xylosteifolium(Spach) N. Robson (H. inodorumWilld.) ! ! ! VIIRoscyna(Spach) R. Keller (3}4)
5.H. ascyronL. (144288) ! ! !
VIIIBupleuroidesStef. (1)
6.H. bupleuroidesGriseb. ! ! !
IXHypericum(48)
7.H. maculatumCrantz (144298) # # 0.058
8.H. tetrapterumFries (144307) # # 0.052
9.H. perforatumL. (144303) # # 0.125
10.H. triquetrifoliumTurra # # 0.090
11.H. attenuatumChoisy$(144922) # # 0.072
12.H. elegansStephan ex Willd. (153305) # # 0.104
XOlympia(Spach) Nyman (2)
13.H. olympicumL. (144301) # # 0.015
14.H. polyphyllumBoiss. et Balansa$ # # 0.012
XICampylopusBoiss. (1)
15.H. cerastoides(Spach) N. Robson$(144295) # # 0.029 XIIOriganifoliaStef. (4)
16.H. origanifoliumWilld. # # 0.064
XIIIDrosocarpiumSpach (12)
17.H. montbretiiSpach (144300) # # 0.174
18.H. umbellatumA. Kerner$(144309) # # 0.063
19.H. richeriVill.$(144304) # # 0.134
20.H. rocheliiGriseb. et Schenk$(144305) # # 0.232
21.H. boissieriPetr.$(144293) # # 0.512
22.H. barbatumJacq. (144292) # # 0.306
23.H. rumeliacumBoiss.$(144306) # # 0.263
24.H. bithynicumBoiss.$ # # 0.056
XVIIITaeniocarpiumJaub. et Spach (22}23)
29.H. hirsutumL. (144299) # ! 0.043
30.H. linarioidesBosse$(144920) # # 0.019
XIX Coridium Spach (5)
31.H. asperuloidesCzernj. Ex Turcz. ! ! !
Table 1
Continued
Section and species Hp Pshp Content (%)!
XXVIIAdenosepalumSpach (33)
33.H. annulatumMoris (H. degeniiBornm.)$(144296) # # 0.066
34.H. montanumL. # # 0.040
35.H. formosissimumTakht.$ ! # 0.054
XXXSpachium(R. Keller) N. Robson (54)
36.H. japonicumThunb. Ex Murray (144918) ! ! !
!Means of three determinations.
"Number of species in the section (Robson, 1977).
#Voucher number at SOM.
$Pseudohypericin reported by us for"rst time.!not detectable;#present.
Table 2
Comparison of the presence of hypericin (Hp) and pseudohypericin (Pshp) in some reinvestigated Hy-pericumspecies reported by us and other authors
Taxa Personal results Results of other authors References
Hp Pshp Hp Pshp
VI sect.Inodora
H. inodorumWilld. ! ! ! # (Brockmann and Sanne,
1957) VII sect.Roscyna
H. ascyronL. ! ! # ! (Zevakova et al., 1991)
X sect.Olympia
H. olympicumL. # # ? # (Brockmann and Sanne,
1957)
H. polyphyllumBois et Balansa# # ! ! (Brockmann and Sanne, 1957)
XIII sect.Drosocarpium
H. barbatumJacq. # # ! # (Brockmann and Sanne,
1957) XVII sect.Hirtella
H. scabrumL. ! ! # ! (Zevakova et al., 1991)
XVIII sect.Taeniocarpium
H. hirsutumL. # ! # # (Makovetskaya and
Maksiutina, 1991)
hypericin and pseudohypericin inH. hirsutun, we have not found the last compound in this species. This is in accordance with the results earlier reported by some other authors (Brockmann and Sanne, 1957; Zevakova et al., 1991).
The total content of hypericins in the investigated taxa varies widely from 0.009% inH. empetrifoliumto 0.512% inH. boissieri. The largest amounts were found in the species ofDrosocarpium, ThasiaandHypericumsections. Moderate quantities of these components were established inH. origanifolium(sect.Origanifolia) and plants from sectionAdenosepalum. The lowest quantities were observed in the taxa of sections Olympia, CoridiumandTaeniocarpium.
Di!erences in the content of hypericins in some reinvestigated species were found when the results were compared with those of other authors. These distinctions are due to the di!erent methods used, and perhaps to geographical and ecological factors, population variability, using herbarium or fresh plant materials and the phases of plant collection. For example, Sakar and Engelshowe (1990) have found 0.157% hypericins in#owering plants ofH. triquetrifolium, whereas we observed only 0.09% of these components in the herb of the same plant, collected during the budding period.
3.2. Chemotaxonomic signixcance at the generic and higher levels
The red and the black glands on the vegetative organs of manyHypericumspecies have been used by botanists as an important distinguishing mark for the classi"cation of the genus (Keller, 1925; Stefano!, 1932}1934; Robson, 1977). Based on this and other characteristics Stefano! has divided the Mediterranean-Oriental species of Hypericuminto 40 sections, whereas Robson has combined the world taxa of this genus into 30 sections. The last author used a modi"cation of Mathis's results on the distribution of hypericin glands in his own classi"cation and found the occurrence of black glands in organs to be an accurate indication of the presence of hypericin in these organs (Robson, 1981).
The results given in Table 1 showed that hypericin and pseudohypericin were not found in the taxa of the primitive sectionsAscyreia (III),Androsaemum(V),Inodora (VI),Roscyna(VII) andBupleuroides(VIII). These components were observed in large quantities in the species of the most phylogenetically advanced sectionsHypericum (IX) and Drosocarpium (XIII) and were presented in all other sections from the
`Olympiaagroup (X-XVI).
We did not observe these metabolites inH. scabrumof sectionHirtella(XVII), but hypericins have been found in some other examined species from this section (Robson, 1981). According to Stefano!(1932,1933) the taxa of sectionHirtellado not contain black glands on the vegetative organs.H. scabrumhas been regarded by this author as an isolated taxon and has been separated in a monotype sectionScabra (Stefano!, 1933).
hypericin glands on the sepals only. Sections XVII}XIX descend from the section Olympia(Robson, 1977) or the sectionAscyreia(Robson, 1981) and form a separate branch in the evolutionary development of the genus. It is evident that taxa of these sections are de"cient in, but not free from, hypericins.
The three analysed species from sectionAdenosepalum(XXVII) were characterized by the presence of moderate amounts of pseudohypericin and/or hypericin. They were absent inH. japonicum, the only evaluated species from the large sectionSpachium (XXX) and this fact was in accordance with the report by Mathis and Ourisson (1963). Our results were in good correlation with the data listed by Robson (1981) from the modi"ed results obtained from these authors.
Although a large number of species have been phytochemically investigated from the other subfamilies of Guttiferae, hypericins have not been encountered in any of them. At the same time, several simple and isoprenylated anthraquinones and dia-nthrones have been found in"ve out of nine genera from subfamily Hypericoideae, but condensed anthrones like hypericin and pseudohypericin have been established only in Hypericumspecies. The genera, in which anthranoid derivatives have been reported belong to the tribes Hypericeae (Hypericum L.) (Brockmann and Sanne, 1953),Vismieae(HarunganaLam., VismiaVand.,PsorospermumSpach) (Ritchie and Taylor, 1964; Marini-Bettolo et al., 1978; Delle Monache et al., 1979; Amonkar et al., 1981; Miraglia et al., 1981; Botta et al., 1986; Marston et al., 1986) andCratoxyleae (CratoxylumBlume) (Dam The Van et al., 1987; Kitanov et al., 1987).
Thus, it may be concluded that: (i) the occurrence of hypericin and pseudohypericin has an important taxonomic value for infrageneric classi"cation ofHypericum*these components are speci"c only for the taxa of more phylogenetically advanced sections (IX}XV, XXVII) and are completely absent in species from the primitive sections (III, V}VIII) of the genus and (ii) the genera of subfamily Hypericoideae are characterized by the presence of anthrone derivatives and they are not known to date in the rest of the family Guttiferae.
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