Hypericin: Source, Determination, Separation, and Properties

Abstract

Hypericin is a naturally occurring compound synthesized by certain species of the genus Hypericum, with various pharmacological effects. It is used as a natural photosensitizing agent with great potential in photodynamic therapy. This review discusses the latest results about the biosynthetic pathways and chemical synthetic routes to obtain hypericin. Although many analysis methods can be used for the determination of hypericin purity, HPLC has become the method of choice due to its fast and sensitive analyses. The extraction and purification of hypericin are also described. Hypericin can be used as a photosensitizer due to a large and active π-electron conjugated system in its structure. Medical applications of hypericin are not easy due to several unsolved practical problems, which include hypericin phototoxicity, poor solubility in water, and extreme sensitivity to light, heat, and pH.

Full text

Hypericin: Source, Determination, Separation, and Properties
Jie Zhang
a
, Ling Gao
a
, Jie Hu
a
, Chongjun Wang
a
, Peter-Leon Hagedoorn
b
, Ning Li
a
, and Xing Zhou
c
a
Chongqing Engineering Research Center for Processing, Storage and Transportation of Characterized Agro-Products, College of Environment and
Resources, Chongqing Technology and Business University, Chongqing, China;
b
Department of Biotechnology, Delft University of Technology, Delft,
The Netherlands;
c
Chongqing Academy of Chinese Materia Medica, Chongqing, China
ABSTRACT
Hypericin is a naturally occurring compound synthesized by certain species of the genus Hypericum, with
various pharmacological eects. It is used as a natural photosensitizing agent with great potential in
photodynamic therapy. This review discusses the latest results about the biosynthetic pathways and
chemical synthetic routes to obtain hypericin. Although many analysis methods can be used for the
determination of hypericin purity, HPLC has become the method of choice due to its fast and sensitive
analyses. The extraction and purication of hypericin are also described. Hypericin can be used as
a photosensitizer due to a large and active π-electron conjugated system in its structure. Medical
applications of hypericin are not easy due to several unsolved practical problems, which include hypericin
phototoxicity, poor solubility in water, and extreme sensitivity to light, heat, and pH.
ARTICLE HISTORY
Received 8 February 2020
Revised 4 July 2020
Accepted 7 July 2020
KEYWORDS
Hypericin; synthesis;
extraction; photosensitivity;
solubility; stability
INTRODUCTION
Hypericum or Saint John’s wort, is one of the nine genera
belonging to the Clusiaceae Lindl family widely spread
throughout the world. A large number of Hypericum species,
including Hypericum perforatum L., Hypericum perfoliatum L.,
Hypericum ascyron L., Hypericum androsaemum L., and
Hypericum chinense L., have been identified in Europe, Asia,
North Africa, and North America.
[1]
In China, there are 55
species of Hypericum, 18 of which have been used as local
resources for medicinal purposes in traditional Chinese
medicine.
[2,3]
The plants of the genus Hypericum contain
numerous bioactive substances, such as naphthodianthrones,
flavonoids, phloroglucinols, and polyphenols.
[1,4]
Naphthodianthrones are considered as characteristic constitu-
ents for the identification of Hypericum species
[5]
and one of
the most important kinds of compounds, which includes
hypericin and its biosynthetic precursors: protohypericin,
pseudohypericin, and protopseudohypericin (Figure 1).
Hypericin (4,5,7,4′,5′,7′-hexahydroxy-2,2′- dimethylnaphto-
dianthrone, C
30
H
16
O
8
, m.w. 504) is a brownish-black powder
with a unique bitter taste that is mainly found in Hypericum
plants.
[6]
Bucher first discovered that hypericin was an active
ingredient of Hypericum perforatum, and it was renamed
hypericin by Cerny in 1911.
[7]
Hypericin is one out of the most biologically active sub-
stances in the genus Hypericum,
[1,4,8]
and has drawn much
interest in recent years. Evidence of antidepressant properties
has been reported.
[9]
Hypericin was active against chronic
unpredictable mild stress-induced depression and metabolic
dysfunction by affecting excitatory amino acids and monoa-
mine neurotransmitters.
[10]
It also exhibits antitumor activity
as an antineoplastic and photocytotoxic agent, a property
attributed to its photosensitivity.
[11–14]
Studies demonstrated
that hypericin possesses immunomodulatory properties and
can induce the production of interferon.
[15,16]
It was found to
be particularly effective as an antiviral agent against the herpes
virus,
[17]
infectious bronchitis virus,
[18]
hepatitis C virus,
[19]
human immunodeficiency virus,
[20]
and novel duck
reovirus.
[21]
Finally, hypericin was considered as an antimicro-
bial agent, antioxidant, and as a promising candidate for
photodynamic diagnosis.
[22,23]
Recently, investigations on the pharmaceutical and clinical
purposes of hypericin surged, and Hypericum perforatum as
a source of hypericin has gradually become one of the three
most popular Chinese herbal medicines.
[24]
The aim of this review
is to describe the recent advances on hypericin research, focusing
on biosynthesis, chemical synthesis, analysis, extraction, purifica-
tion, photosensitivity, solubility in water and stability.
HYPERICIN SOURCES
Natural Sources
Hypericin as a natural bioactive compound can be obtained
from plants, insects, and protozoa.
[3]
It is found in the integu-
ment of Australian Lac insects of the Coccoidea family,
[25,26]
and
the blue-green ciliate, Stentor coerulus, which is a form of
protozoa.
[27]
However, the Hypericum genus has spread
throughout the temperate and tropical regions worldwide, and
is therefore the leading natural source of hypericin. The genus
contains 484 species divided into 36 subgroups.
[28]
The
Hypericum genus has 30 species in Italy
[29]
and 89 in
Turkey.
[5,30]
In China, the 55 various Hypericum species are
widely spread across the country, but the main Hypericum con-
taining areas are concentrated in southwest China. An early
CONTACT Jie Zhang lang19880107@hotmail.com Chongqing Technology and Business University, College of Environment and Resources, Chongqing 400067,
China; Xing Zhou sheya2624@163.com Chongqing Academy of Chinese Materia Medica, Chongqing, China
SEPARATION & PURIFICATION REVIEWS
https://doi.org/10.1080/15422119.2020.1797792
Copyright © Taylor & Francis Group, LLC

survey of circa 200 species of Hypericum indicated that almost all
hypericin-containing species belong to the sections Euhypericum
and Campylosporus of Keller’s classification.
[31]
The most impor-
tant and well-known species is Hypericum perforatum which is
commonly known as St. John’s wort.
[32]
Hypericum perforatum
is a perennial herbaceous plant widely distributed in the world
and it has been included in numerous pharmacopeia. Hypericin
is produced in specialized minute glands on all aerial parts of the
plant, predominantly in flowers and leaves. The hypericin con-
centration varies depending on the species, the geographical
locations of Hypericum,
[1,8]
and the part of the plant,
[30]
as
shown in Table 1. Additionally, the developmental stage of the
plant and seasonal variations also influences the hypericin
concentration.
[30,33]
Although there are numerous other
Hypericum species known to contain approximately similar
amounts of hypericin as Hypericum perforatum,
[34]
information
from the literature on these species is scarce.
Biosynthesis of Hypericin
The biosynthesis of hypericin in Hypericum is more compli-
cated than known chemical synthetic routes and involves the
OOH OH
HO
HO R
OH O OH
OOH OH
HO
HO R
OH O OH
R=CH3 Hypericin
R=CH2OH Pseudohypericin
R=CH3 Protohypericin
R=CH2OH Protopseud ohypericin
Figure 1. Chemical structures of hypericin, protohypericin, pseudohypericin, and protopseudohypericin.
Table 1. The hypericin concentration in some species of the genus Hypericum.
Hypericum species Provenance [ref] Plant part
Hypericin
(mg·g
−1
)
Hypericum perforatum Italy (Sicily)
[1]
Flowering tops 15–20 cm 3.69
Italy (Latium)
[9]
Flowering tops 0.27
Italy (Trentino)
[9]
Flowering tops 0.22
Italy (Tuscany)
[9]
Flowering tops 0.16
Italy (Molise)
[9]
Flowering tops 0.13
Turkey (Samsun)
[5]
Top 1/3 of the crown 2.82
Japan (Tokyo)
[4]
Flowering tops 1.20
China (Hubei)
[89]
Above the ground 1.50
China (Guizhou)
[90]
Above the ground 0.25
Hypericum aviculariifolium Turkey (Gumus)
[5]
Top 1/3 of the crown 2.14
Hypericum aegypticum Italy (Sicily)
[1]
Flowering tops 15–20 cm 0.03
Hypericum enshiense China (Hubei)
[89]
Above the ground 3.00
Hypericum empetrifolium Greece (Crete)
[6]
Above the ground 0.09
Hypericum faberi China (Guizhou)
[90]
Above the ground 0.05
Hypericum hirsutum Italy (Bulgaria)
[6]
Flowers 0.43
Italy (Sicily)
[1]
Flowering tops 15–20 cm 0.15
Italy (Siena)
[9]
Flowering tops 0.02
China (Xinjiang)
[91]
Above the ground 0.06
Serbia (Rudina Planina)
[92]
Above the ground 0.024
Hypericum linarioides Serbia (Rudina Planina)
[92]
Above the ground 0.02
Hypericum lydium Turkey (Havza)
[5]
Top 1/3 of the crown 0.18
Hypericum maculatum Serbia (Rudina Planina)
[92]
Above the ground 0.03
Hypericum montanum Italy (Sicily)
[1]
Flowering tops 15–20 cm 1.42
Hypericum montbretii Turkey (Samsun)
[5]
Top 1/3 of the crown 1.39
Hypericum origanifolium Turkey (Samsun)
[5]
Top 1/3 of the crown 1.43
Hypericum patulum Italy (Sicily)
[1]
Flowering tops 15–20 cm 0.02
Hypericum perfoliatum Turkey (Samsun)
[30]
Top 1/3 of the crown, floral budding 1.06
Turkey (Samsun)
[30]
Top 1/3 of the crown, full flowering 0.96
Turkey (Samsun)
[30]
Top 1/3 of the crown, fresh fruiting 0.41
Italy (Sicily)
[1]
Flowering tops 15–20 cm 0.93
Hypericum pruinatum Turkey (Cumus)
[5]
The top 1/3 of the crown 0.79
Hypericum rumeliacum Serbia (Rudina Planina)
[92]
Above the ground 0.18
Hypericum sampsonii China (Jiangxi)
[90]
Flowering tops 20 cm 0.04
Hypericum scabrum China (Xinjiang)
[91]
Above the ground 0.06
Hypericum tetrapterum Italy (Stia)
[9]
Flowering tops 0.84
Italy (Sicily)
[8]
Flowering tops 15–20 cm 0.40
Serbia (Rudina Planina)
[92]
Above the ground 0.09
Hypericum wightianum China (Guizhou)
[90]
Above the ground 0.023
2J. ZHANG ET AL.

expression of multiple genes. The precise regulation of hyper-
icin biosynthesis remains uncertain until today. The generally
accepted biosynthetic hypericin pathway can be divided into
two main parts: the formation of emodin anthrone and the
conversion of emodin anthrone to hypericin (Figure 2).
[35]
Emodin anthrone is most likely the immediate precursor of
hypericin. Early studies presumed that emodin anthrone
synthesis followed the polyketide pathway.
[36,37]
The cycliza-
tion of a linear polyketide starting with the condensation of
acetyl-CoA and malonyl-CoA results in the formation of emo-
din anthrone catalyzed by polyketide synthase (PKS). Two
cDNAs encoding for PKS designated as HpPKS1 and
HpPKS2 were cloned and identified from Hypericum
perforatum.
[38]
Although the expression of HpPKS2 was corre-
lated with the concentrations of hypericin, the recombinant
HpPKS2 protein failed to convert the acetylated substrates to
emodin or hypericin under in vitro conditions.
[39]
Pillai and
Nair
[40]
provided direct biochemical and molecular evidence in
support of the PKS hypothesis of hypericin biosynthesis in
2014. Auxin inducible culture systems of Hypericum hookeria-
num were applied as a model system to study the metabolic
pathway of hypericin synthesis. The results demonstrated the
presence of additional protein components besides PKS
activity.
In later steps of hypericin biosynthesis, emodin anthrone is
oxidized to emodin by emodin anthrone oxygenase.
[35]
Emodin dianthrone can be produced by a condensation reac-
tion with emodin and emodin anthrone. This subsequently
undergoes oxidation to form protohypericin, then protohyper-
icin produces hypericin on irradiation. Bais, et al.
[41]
discov-
ered that hypericin biosynthesis is related to a gene termed
hyp-1. Based on an in vitro study, the phenolic oxidative
coupling protein (Hyp-1) was shown to catalyze the dimeriza-
tion of emodin and emodin anthrone, the dehydration of the
intermediate to emodin dianthrone, and further phenolic oxi-
dation to protohypericin and hypericin. A high-resolution
crystal structure of the Hyp-1 protein indicated that it has
a pathogenesis-related class 10 protein structure.
[42]
However,
it is unable to dimerize emodin to hypericin using Hyp-1
protein as biocatalyst. Kosuth, et al.
[43]
also found that the
hyp-1 gene is not a limiting factor for hypericin biosynthesis.
The low expression of the genes in the early stages of the
hypericin biosynthetic route may be the potential key factors
in the accumulation and biosynthesis of hypericin.
[44]
The role
of the hyp-1 gene should be further verified by functional
validation experimental approaches. In addition, Kimakova
et al.
[45]
identified new compounds present in the genus
Hypericum and proposed that the anthraquinone skyrin is the
key intermediate in hypericin biosynthesis. Further research on
the role of anthraquinone derivatives in plant metabolism
should be performed.
Chemical Synthesis of Hypericin
In 1957, Brockmann, et al.
[46]
published the first multistep
chemical synthesis method for the production of hypericin.
This synthesis route starts with the reaction of 3,5-dimethox-
ybenzoic acid methyl ester and chloral hydrate. The procedure
with 12 steps is complicated, and the overall yield is 6% - 9%.
Such a low yield and complex synthetic route are no longer
acceptable for the industrial production of hypericin, and new
synthetic pathways have been studied to increase the synthesis
yield and simplify the route. Hypericin can be obtained from
2-methyl anthraquinone in eight steps .
[47]
The key step in this
process is to obtain emodin by steps such as nitration, reduc-
tion, bromination, deamination, and substitution. Emodin is
condensed in the presence of hydroquinone under alkaline
conditions to give protohypericin, which is subsequently
photochemically converted to hypericin by irradiation with
a halogen lamp. Although the synthetic route was optimized
to perform under mild conditions, it involves multiple synth-
esis steps resulting in a low yield.
In 2007, Motoyoshiya, et al.
[48]
proposed a six-step method
for synthesizing hypericin (Figure 3). The regioselective two-
fold Diels-Alder reaction of 1,4-benzoquinone with (1-meth-
oxy-3- methylbuta-1,3-dienyloxy)trimethylsilane results in
7-methyljuglone in the first step. Emodin and its
O-methylated derivative are subsequently produced from
7-methyljuglone and (1,3-dimethoxybuta- 1,3-dienyloxy)tri-
methylsilane. The reduction of both compounds with SnCl
2
in acidic media was accompanied by an acid hydrolysis that
produced emodin anthrone, which after oxidative dimerization
with FeCl
3
hydrate gave bianthrone in high yield. Bianthrone is
oxidized by N-ethyldiisopropylamine (i-Pr
2
EtN) to produce
protohypericin, which is then converted to hypericin by irra-
diation. This shorter route realizes the straightforward synth-
esis of hypericin from a simple compound. However, the yield
in the final step is low and should be improved.
In general, the synthesis of emodin is a necessary step in the
synthesis of hypericin. The synthesis methods for emodin are
Figure 2. The proposed biosynthesis of hypericin. Figure 3. Synthesis of hypericin from 1,4-benzoquinone.
SEPARATION & PURIFICATION REVIEWS 3

numerous and well optimized. Therefore, it has also been
reported that emodin can be used as the starting
material.
[49,50]
After interaction with SnCl
2
, emodin is first con-
verted into emodin anthrone. Then, emodin anthrone is used as
an intermediate reactant. It is reacted with pyridine, piperidine,
pyridine N-oxide and FeSO
4
· 7H
2
O to form hypericin.
[51]
Some
scholars have developed an even more direct method to synthe-
size hypericin. Emodin was converted to hypericin
[52,53]
using
hydroquinone as catalyst under nitrogen and light illumination
after 2 weeks (Figure 4). A large number of scientists have
focused their attention on the synthesis of hypericin, and the
synthesis technology of hypericin has gradually matured.
However, the methods can still be improved further in terms
of atom economy and environmental impact (E-factor).
DETERMINATION AND SEPARATION OF HYPERICIN
Analysis of Hypericin
Various hypericin determination methods have been devel-
oped including: ultraviolet-visible spectroscopy (UV-VIS),
chemiluminescence-flow injection analysis (CL-FIA), thin-
layer chromatography (TLC), and high-performance liquid
chromatography (HPLC).
UV-VIS spectroscopy was applied to determine hypericin
and pseudohypericin in the extracts of Hypericum perforatum,
and the solution of the compounds in methanol was measured
at 588 nm.
[54]
The molar extinction coefficient was difficult to
establish because none of the routine purity criteria can be
applied to hypericin and pseudohypericin. However, specific
reference spectra can successfully be used to analyze hypericin
and pseudohypericin.
Shi
[55]
combined chemiluminescence and flow injection
analysis to establish a CL-FIA method to detect the hypericin
content in Hypericum perforatum. Hypericin has a sensitizing
effect on the chemiluminescence intensity of the Luminol-
KMnO
4
system in an alkaline medium. Under optimized
experimental conditions, the hypericin mass concentration is
linearly related to the luminescence intensity in the range of
1.9 × 10
−5
- 3.8 × 10
−4
g·L
−1
with a limit of detection (LOD) of
3.8 µg·L
−1
. The content of hypericin in Hypericum perforatum
was detected as 0.492 mg·g
−1
. The experimental results also
indicated that CL-FIA and UV methods are equally sensitive.
Moreover, CL-FIA shows some advantages in terms of larger
linear range, higher sensitivity, and higher speed of analysis.
Mulinacci, et al.
[56]
established a TLC-densitometry method
with fluorescence detection to detect the hypericin content in
Hypericum perforatum, and this method was compared with
reversed-phase HPLC-DAD (diode array detection). The
mobile phase was optimized by adjusting the ratio of toluene,
ethyl acetate, and formic acid. The TLC densitometry was
performed without the use of spray or dipping reagents
which improved the speed of the analytical procedure. The
method is cost-effective because of the short analysis time
and the low solvent consumption. The accuracy and reprodu-
cibility of TLC densitometry were comparable with those
obtained by HPLC-DAD. However, HPLC-DAD does provide
much more information than TLC densitometry.
Currently, various HPLC methods for hypericin analysis are
gradually emerging. Wang, et al.
[57]
established an HPLC-
visible spectroscopy method for hypericin determination in
the extracts of Hypericum perforatum. The flow rate of mobile
phase was 1.0 mL·min
−1
(Table 2, A). The linear relationship of
hypericin was good in the range of 6–36 mg·L
−1
(r = 0.9996),
the average recovery rate was 98.86% (n = 6), the relative
standard deviation of the peak area was 3.15%. This HPLC
detection method has a good reproducibility and accuracy and
is suitable to determine hypericin in complex samples. In 2006,
Ruckert, et al.
[58]
presented an HPLC method for the quantita-
tion of hypericin using a new and sensitive amperometric
detection. Using Ag/AgCl as a reference electrode in the detec-
tor, hypericin was eluted isocratically using a mobile phase
consisting of ammonium acetate, methanol, and acetonitrile
(Table 2, B). Hypericin eluted at a retention time of 12 min.
Linearity was obtained over the range 0.035–1.30 mg·L
−1
(r = 0.9994). The LOD of hypericin was 0.010 ng injected on-
column. These parameters show that the method is selective,
simple, rapid, and accurate. Zhang, et al.
[59]
established a high-
resolution HPLC method for determining hypericin by com-
paring different chromatographic conditions. The different
methods showed different detection efficiencies depending on
the mobile phase and detection wavelength. A purchased crude
extract of Hypericum perforatum was applied to optimize the
chromatographic conditions for the determination of hyperi-
cin in a complex sample. The results demonstrated that the best
resolution was obtained at 590 nm and with the mobile phase
composition: methanol/acetonitrile/0.1 mol·L
−1
sodium dihy-
drogen phosphate 200/300/100 v/v/v (Table 2, C). The hyper-
icin calibration curve showed a good linearity in the range of
4–14 mg·L
−1
(r = 0.9986), and the established method is fast
and easy to use.
[59]
In addition, the stationary phase in the column has
a significant effect on the resolution of HPLC. Dolezal, et al.
[60]
selected four stationary phases modified by the pentafluoro-
phenyl group to investigate the contribution of π-π interactions
to the improvement of hypericin separation in comparison to
the separation obtained with a conventional C18 reversed-
phase column. The best analytical method employing the pen-
tafluorophenyl stationary phase (Table 2, E) showed sufficient
linearity, accuracy, and precision and was used for the deter-
mination of hypericin in Hypericum perforatum. Besides the
HPLC methods discussed above, other reported HPLC meth-
ods with different mobile and stationary phases are presented
in Table 2 (F-I). For routine hypericin analyzes, UV detection
at 278 and 284 nm, and VIS detection at 579–590 nm, have
been proposed. However, the VIS detection has a better sensi-
tivity and selectivity than the UV detection.
[58,59,61]
HPLC
Figure 4. The direct synthesis of hypericin with emodin.
4J. ZHANG ET AL.

analyzes with various detection modes will replace the other
method for hypericin analysis because it is fast, accurate, and
sensitive.
[58,60]
Separation of Hypericin
The hypericin content in Hypericum is extremely low, in most
cases below 3 mg·per gram of dry weight of plant material.
Therefore, effective hypericin extraction and purification
methods are needed. The most common method is solvent
extraction using methanol, ethanol, and polar alcohols. In
addition, microwave-assisted extraction has been used to
reduce the extraction time and enzyme-assisted extraction to
increase the extraction yield. The lye (sodium hydroxide)
extraction method has been rarely used because of its higher
energy consumption and longer extraction time. Separation
and purification of hypericin has been performed by using
macroporous resin column chromatography (MRCC) and
molecular imprinting techniques. Additionally, counter-
current chromatography (CCC) has been used widely due to
its high purification rate.
Extraction of Hypericin
Cossuta et al.
[62]
used Soxhlet extraction to extract hypericin
from Hypericum perforatum with four different solvents
(n-hexane, ethyl acetate, 2-propanol, and ethanol), and the
extracts were analyzed by UV-HPLC. Ethanol was the best
solvent to extract hypericin producing a maximum of
0.060 mg·g
−1
. However, the 16 h Soxhlet extraction time was
discouraging needing to be optimized.
Xing
[63]
took a two-step impurity removal method to
extract hypericin. Ether was first used to remove apolar impu-
rities including chlorophyll, and water-soluble impurities
including sugars were removed by suspension in warm water.
After that, ethyl acetate was utilized to extract hypericin. Using
40% and 80% ethanol as eluents, respectively, the extracts were
separated by MRCC. The red MRCC effluent was concentrated
under reduced pressure to obtain a hypericin paste. Finally, the
content of hypericin in the paste, determined by UV-HPLC,
was 1.2%, which far exceeds the 0.3% hypericin content
required for the international market of medicinal products.
This hypericin optimized extraction method has a high
extraction rate and is simple and effective. Moreover, the
MRCC product has the characteristics of low hygroscopicity
and low tackiness.
Punegov, et al.
[64]
studied the extraction of hypericin and
pseudohypericin from raw Hypericum perforatum using
microwave activation. The maximal extraction efficiency was
achieved when 55% ethanol or isopropanol was used as extrac-
tant at 0.0205 W·cm
−3
microwave irradiation power density
and a microwave frequency of 2450 MHz for 60 s. Microwave
activation was found to improve 10-fold the extraction effi-
ciency, reducing the time necessary to fully extract hypericin
and pseudohypericin compared to classical extraction meth-
ods. Zhang, Feng, Xu, Tan, Hagedoorn, and Ding
[59]
used
a xylanase-assisted associated with a microwave-assisted
extraction to improve the hypericin extraction. This method
was found to improve the extraction yield of hypericin signifi-
cantly compared to unassisted extraction. Microwave-assisted
extraction after xylanase-assisted extraction was found to be
the most efficient strategy for extracting hypericin. The yield
was 0.32 ± 0.006 mg·g
−1
, which was a 210% increase over
unassisted extraction. Compared to conventional solvent
extraction, enzyme-assisted extraction can be accomplished
using a low enzyme concentration. It reduces energy consump-
tion due to less wastewater generation and lower temperatures.
In addition, the unique microwave heating method decreases
the extraction time and increases the yield even further. In
conclusion, enzyme and microwave assistance are effective
strategies to improve the mass transfer rate of hypericin during
the extraction process. Scalability of the equipment has to be
addressed in order to use microwave assistance on an industrial
scale.
Purification of Hypericin
The purity of hypericin in the extracts is not sufficient to meet
the requirements for pharmaceutical products. Further
research on how to purify hypericin is necessary. Xue, et al.
[65]
selected six macroporous adsorption resins (D101, AB-8, HZ-
801, HZ-818, HZ-806, X-5) with different specific surface areas
and pore sizes to separate hypericin. The crude extracts of
Hypericum perforatum were used to investigate the hypericin
separation and purification performance of six resins. Optimal
conditions were determined using a medium operating
Table 2. Different chromatographic conditions for determining hypericin.
Methods
[ref]
Mobile phase
v:v:v Stationary phase
Temperature
(ºC)
Detection
wavelength
(nm)
A
[57]
Acetonitrile, 0.02 M sodium dihydrogen phosphate, volume ratio of 85: 15 v:v ODS-A 25 588
B
[58]
Ammonium acetate, methanol and acetonitrile, volume ratio of 10: 40: 50 LiChroCart
Purospher RP18e
22 254
C
[59]
Methanol, acetonitrile, sodium dihydrogen phosphate solution, volume ratio of 200: 300: 100 v:v:v Kromasil C18 40 590
D
[59]
Methanol, acetonitrile, sodium dihydrogen phosphate solution, volume ratio of 200: 300: 100 v:v:v Kromasil C18 40 284
E
[60]
Solvent A: H
2
O, 0.1 M acetic acid, 0.1 M trimethylamine, solvent B: acetonitrile, 0.1 M acetic acid,
0.1 M trimethylamine
0–1 min (A: B = 15: 85), 1–8 min (A: B = 0: 100), 8.0–10.0 min (A: B = 15: 85).
Pentafluorophenyl 25 278
F
[93]
Methanol, ethyl acetate, 0.1 M Na
2
PO
4
, volume ratio of 72: 23: 5 v:v:v Ultracarb 7 ODS 23 579
G
[94]
Ethylacetate, 15.6 g L
−1
sodium dihydrogen phosphate (pH = 2 with phosphoric acid) and
methanol, volume ratio of 39: 41: 160 v:v:v
ACE C18 40 590
H
[95]
Methanol, acetonitrile, water and 3% aqueous phosphoric acid, volume ratio of 45: 50: 4.5: 0.5 with
triethylamine adjusted to pH = 6
Supelcosil ODS 25 590
I
[96]
Methanol, ethyl acetate and phosphate buffer (pH = 2, 0.1 M), volume ratio of 60: 20: 20 v:v:v VP-ODS C18 25 590
SEPARATION & PURIFICATION REVIEWS 5

pressure separation method. The results showed that HZ-801
resin is the preferred separation material with a high adsorp-
tion rate and a high elution rate, and the purity of hypericin
after elution with ethanol solution was 79%. Hypericin separa-
tion using MRCC is feasible on industrial scale due to its easy
operation and recyclability. However, contaminating sub-
stances which are similar in structure to hypericin affect the
results of hypericin separation due to the physical adsorption
principle of the resins.
Molecular imprinting is a newly established method for
chemical separation and purification in recent years. The syn-
thetic molecularly imprinted polymer used for separation
shows high selectivity and specificity for the template (target)
molecule. A core-shell structure molecularly imprinted mag-
netic nanospheres of hypericin (Fe
3
O
4
@MIPs) were prepared
by mercapto-alkyne click polymerization.
[66]
The Fe
3
O
4
@MIPs
showed a good adsorption capacity of 3.43 mg·g
−1
, high fast
mass transfer rates, and good reusability. Hypericin, acryla-
mide, and pentaerythritol triacrylate were used as a template
molecule, functional monomer, and molecular imprint pre-
assembly cross-linker, respectively.
[67]
A cooperative hydro-
gen-bonding complex between hypericin and acrylamide was
formed at the ratio of 1:6 in the prepolymerized system. A high
recovery of 82.3% was achieved by molecular-imprinted poly-
mers to extract hypericin from Hypericum perforatum extracts.
Molecular imprinting is simple, rapid, accurate, and reliable.
The main disadvantage is the amount of pure hypericin which
has to be used to prepare the molecular-imprinted polymers.
Cao, et al.
[68]
found that CCC combined with pre-separation
by ultrasonic solvent extraction was successful for the separation
of series of bioactive compounds from the crude extracts of
Hypericum perforatum. The ethyl acetate extract was separated
by using the solvent system hexane-ethylacetate- methanol-
water (1:1: 1:1 and 1:3: 1:3) in gradient through both reverse
phase and normal phase elution mode. The hypericin purity was
determined to be 95% by HPLC-DAD. CCC does not require
a solid carrier, and the hypericin obtained in less than 5 hours by
this method has high purity. However, CCC is difficult to realize
on an industrial scale and it is not environmentally friendly due
to the amount of organic solvents used.
The extraction and purification technologies that have been
developed provide an indispensable foundation to prepare
hypericin products with different purities. Further studies are
required to achieve a method that combines low cost and
a green process. Furthermore, preparative liquid chromatogra-
phy is a feasible technology to prepare a high purity product,
can be utilized to separate hypericin.
[69–71]
This technology is
also amenable to industrial scale-up.
PROPERTIES OF HYPERICIN
Photosensitivity
Hypericin is one of the most effective natural photosensitizers,
and shows a good photosensitivity due to the extensive elec-
tron-conjugated system in its structure (Figure 1). Hypericin is
extremely sensitive to light and it is photoactivated to produce
peroxides. The photosensitive mechanism of hypericin has
been described as follows: in the photodynamic action, light
quanta are absorbed by the sensitizer, generating the excited
singlet state. This excited singlet state may undergo intersystem
crossing to the triplet excited state. The triplet sensitizer will
excite singlet oxygen that is produced when the energy is
transferred to ground state triplet oxygen, and the singlet oxy-
gen forms a peroxide subsequently.
[72]
Moreover, it has been
confirmed that the photosensitivity of hypericin induces cell
apoptosis and inhibits the growth of cancer cells. This optical
activity of hypericin has been widely used in optical
diagnostics.
[73]
Hypericin can produce superoxide-free radicals under the
irradiation of visible light and in the presence of oxygen.
[74]
Hypericin shows an electron paramagnetic resonance (EPR)
signal caused by a semiquinone-like radical formed by inter-
molecular electron transfer in the absence of light and electron
donors. The amplitude of the radical EPR signal for the water-
dispersed lysine salt of hypericin is significantly increased
under visible light irradiation. This finding indicates that the
free hypericin radicals and the superoxide radicals are formed
during light irradiation, and may also be implicated in the
biological activities.
The photosensitivity of hypericin is commonly used in
photodynamic therapy, providing an effective treatment of can-
cer. Rabbits and mice xenografted with P3 human squamous cell
carcinoma were used to assess the usefulness of hypericin for
laser photoinactivation of solid tumors.
[75]
The tissue uptake and
distribution of hypericin in rabbits and mice were measured.
The degree of absorption of hypericin by intravenous injection at
4 and 24 hours in both animal tissues was determined by ethanol
extraction and quantitative fluorescence spectrophotometry.
Experimental results show that elimination of hypericin was
rapid in most animal organs with residual hypericin under
10% of the maximum after 7 days. The retention rate of squa-
mous cell tumors is only 25% to 30%. It indicated that photo-
dynamic therapy using hypericin can, to a certain extent,
eliminate some cancer cells.
[76]
In addition, hypericin and laser
irradiation induced cell death mediated by the intracellular
reactive oxygen species and mitochondrial damage. These data
demonstrate that hypericin is an effective photosensitizer with
potential for human cancer therapy.
Hypericin can absorb light in the ultraviolet and visible
range. Although hypericin has potential for cancer treatment,
some studies have shown that hypericin is phototoxic to the
skin and human eye. Ingestion of hypericin containing drugs is
potentially phototoxic to the retina, which may lead to retinal
or early macular degeneration.
[77]
In addition, hypericin is not
cytotoxic in the dark.
[78]
Further research into the practical
application of hypericin in photodynamic therapy should be
performed.
Water Solubility of Hypericin
Hypericin is readily dissolved in dimethylsulfoxide, methanol,
ethanol, and alkaline aqueous solution, and it is red-colored at
pH <11.5 and green-colored above pH 11.5.
[79]
However, it is
a serious drawback that hypericin exhibits low level of solubi-
lity in neutral water because of its hydrophobicity.
[80,81]
Hypericin forms nonsoluble aggregates in an aqueous
environment.
6J. ZHANG ET AL.

Fluorescence spectroscopy and diffusion coefficient mea-
surements were used to investigate the self-association of
hypericin molecules in DMSO/water mixtures.
[82]
Fluorescence measurements revealed that hypericin remained
in its monomeric form in DMSO/water mixtures containing up
to 20% - 30% water. As the proportion of water passes 30%,
hypericin gradually formed non-fluorescent aggregates, and
the size of the aggregates increased with increasing water con-
centrations. Hypericin presumably produces large molecular
weight stacked aggregates in a neutral aqueous environment.
In addition, molecules of hypericin remain in the monomeric
state in an aqueous environment at alkaline pH.
The insoluble hypericin aggregates in aqueous solutions do
not possess biological activity. This characteristic restricts
hypericin applications in medicine. Polymeric micelles made
with polyethylene glycol (PEG) have been utilized to improve
the solubility of hypericin.
[83]
PEGs with low molecular weight
(<1000 g·mol
−1
) did not significantly contribute to the hyper-
icin solubilization. However, PEGs with molecular weight
>2000 g·mol
−1
efficiently transformed hypericin aggregates to
the monomeric state. The solubility of hypericin in water
increased significantly by adding cromolyn disodium salt
(DSCG).
[81]
The monomerization of hypericin under these
conditions can be explained as a result of the hydrotropic effect
of DSCG. This hydrotropic effect is most likely a result of
interactions between the two relative rigid aromatic rings of
DSCG and a delocalized charge on the surface of the hypericin
molecule. Kubin, et al.
[84]
prepared a non-covalently bound
hypericin-polyvinylpyrrolidone (PVP) complex to enhance the
water solubility of hypericin. The hypericin-PVP complex
bound more than 1000 mg of hypericin in presence of 100 g
PVP and the resulting complex was soluble in 1 L of pure
water. The proposed methods provide strategies to improve
the solubility of hypericin in water, which will facilitate medical
applications. The effects of these additives on the biological
activities of the resulting hypericin preparations have to be
investigated.
Hypericin Stability
Due to its photosensitivity, light inevitably affects the stability
of hypericin. Therefore, exposure to light has a significant
impact on the biological activities of hypericin. The stability
of hypericin in extract solutions of Hypericum perforatum and
standard solutions has been evaluated under different light
conditions monitored by HPLC-VIS.
[85]
Hypericin was extre-
mely unstable after exposure to light, and light was the main
factor reducing the effective hypericin concentration.
Additionally, temperature was a factor affecting hypericin sta-
bility. Wang, et al.
[86]
investigated the effects of light and
temperature on the long-term stability of hypericin extracts.
After 8 weeks, the content of hypericin in the extracts
decreased by 49% under constant light at room temperature,
and it declined by only 8.5% in the dark. The hypericin content
of the extracts was unchanged under dark and low temperature
(−24°C) conditions. Long-term storage is possible for hyper-
icin dissolved in a polar solvent, under a nitrogen atmosphere
at freezing temperature (<-30°C).
[87]
Wang and Zhang
[88]
stu-
died the effects of visible light, temperature, pH, Na
2
SO
3
and
ascorbic acid on the stability of hypericin by UV-VIS spectro-
scopy. The results indicated that light is the major factor
influencing the stability of hypericin. Light and temperature
were found to have a greater effect on stability under alkaline
conditions than acidic conditions. Therefore, alkaline solvents
should be avoided during the extraction of hypericin. However,
the stability of hypericin improved when ascorbic acid or Na
2
SO
3
was added. The instability of hypericin has always been
a major challenge in the separation and purification process.
CONCLUSIONS
Hypericin is one of the effective bioactive substances primarily
extracted from the Hypericum plants, and has various pharma-
cological activities such as anti-depressive, anti-tumor, and
anti-viral. The biosynthetic pathways in the plants are known,
but the precise regulation of these pathways remains uncertain.
Chemical synthesis routes for hypericin from different starting
compounds have been developed. However, a novel synthesis
route combining a high overall yield, low cost, and less envir-
onmental pollution is still desired. HPLC is widely used for
hypericin analysis, and it may replace all other analytical
methods due to its fast analysis times and high sensitivity.
Extraction of hypericin from Hypericum can provide low pur-
ity products or extracts. Microwave-assisted extraction and
enzyme-assisted extraction contribute to a higher hypericin
yield. Macroporous adsorption resin, molecular imprinting
techniques, CCC, and preparative liquid chromatography sys-
tems were used to prepare high purity hypericin. Especially,
preparative liquid chromatography system is a feasible strategy
to realize the industrial production of high purity hypericin.
Hypericin is photosensitive due to its extensive system of
conjugated C = C double bonds. It can be utilized for photo-
dynamic therapy. However, the phototoxicity of hypericin to
the skin and human lens should be considered. Hydrophobic
groups in hypericin account for its low solubility in water.
PEG, DSCG, and PVP can significantly improve the solubility.
In addition, the storage and operation conditions, such as light,
temperature, and pH will affect the hypericin stability. Without
a doubt, the disadvantages of hypericin can be overcome with
technical solutions, which are worthwhile of investigation
because its great medicinal value.
Funding
The study has been carried out with financial support from the Natural
Science Foundation Project of CSTC [No. cstc2017shms-xdny100003];
Project of China Scholarship Council [No. 201808500035].
ORCID
Jie Zhang http://orcid.org/0000-0002-3938-0878
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