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Arch Pharm Res Vol 31, No 3, 265-273, 2008

DOI 10.1007/s12272-001-1151-3

http://apr.psk.or.kr

Biochemical Pharmacology of Biflavonoids: Implications for


Anti-inflammatory Action
Hyun Pyo Kim, Haeil Park, Kun Ho Son1, Hyeun Wook Chang2, and Sam Sik Kang3
College of Pharmacy, Kangwon National University, Chunchon 200-701, Korea, 1Dept. Food Nutr., Andong National
University, Andong 760-749, Korea, and 2College of Pharmacy, Yeungnam University, Gyongsan 712-749, Korea,
and 3Natural Products Res. Inst., Seoul National University, Seoul 110-640, Korea

(Received November 25, 2007)


Biflavonoids belong to a subclass of the plant flavonoid family. Distribution of biflavonoids in
the plant kingdom is limited to several species. Previously, some pharmacological activities of
biflavonoids were described such as inhibition of histamine release from mast cells and inhibition of lymphocyte proliferation, suggesting the anti-inflammatory/antiallergic potential of the
biflavonoids. Furthermore, several natural biflavonoids including ochnaflavone and ginkgetin
inhibit phospholipase A2. Most importantly, certain biflavonoids exhibit anti-inflammatory activity through the regulation of proinflammatory gene expression in vitro and in vivo. Recently,
several synthetic approaches yielded new biflavonoid molecules with anti-inflammatory potential. These molecules also exhibit phospholipase A2 and cyclooxygenase-2 inhibitory activity.
Although the bioavailability needs be improved, certain biflavonoids may have potential as new
anti-inflammatory agents. This is the first review of biflavonoid pharmacology to date.
Key words: Biflavonoid, Flavonoid, Anti-inflammation, Phospholipase, Cyclooxygenase,
Proinflammatory gene

Chemistry and distribution of biflavonoids


Basically, biflavonoids are flavonoid-flavonoid dimers
with varied chemical structures (Fig. 1). Many different
flavonoid dimer combinations are possible. For example,
flavanone-flavone, flavone-flavone, flavone-flavonol are
among the possible structures. In addition, there are two
general types of bond connections between the flavonoids:
C-C bond or C-O-C bond. Moreover, a connecting bond
may have diverse positions: 3-4, 4-4, etc. In natural
biflavonoids, many hydroxyl/methoxyl groups are substituted at different positions. Therefore, theoretically,
numerous biflavonoids may exist. However, plants that
contain biflavonoids as major constituents are not widelydistributed. Indeed, only a few plant species have
biflavonoids. The examples are Ginkgo biloba, Selaginella
species, and Garcinia kola. Amentoflavone and ginkgetin
(Fig. 2) are the most common biflavonoids reported. In
recent years, there have been findings of new biflavonoids
with unique chemical structures from plants. Anti-inflammatory mechanisms of monomeric flavonoids were pre-

Fig. 1. General chemical structures of biflavonoids. Two flavonoid


monomers are connected with a C-C or C-O-C bond

viously summarized (Kim et al., 2004a). The present review


summarizes the anti-inflammatory activity and cellular
mechanisms of biflavonoid action.

Correspondence to: Hyun Pyo Kim, College of Pharmacy, Kangwon National University, Chunchon 200-701, Korea
Tel: 82-33-2506915, Fax: 82-33-255-9271
E-mail: hpkim@kangwon.ac.kr

265

266

General biochemical pharmacology of naturally


occurring biflavonoids
The biological/pharmacological activities of biflavonoids
are diverse. They include antibacterial, antifungal, antiallergic, antiviral, antihepatotoxic, anticancer, and immune
suppressive activities. For example, amentoflavone was
first reported to strongly inhibit cAMP and cGMP phosphodiesterases with IC50s of 0.66 and 0.54 M, respectively
(Ruckstuhl et al., 1979). Ginkgo biflavones including
amentoflavone, bilobetin, sequoiaflavone and ginkgetin
also inhibit cAMP phosphodiesterase from rat adipose
tissue (Saponara and Bosisio, 1998), supporting the initial
finding. Ginkgo biflavones were also recently found to inhibit
cGMP-specific phosphodiesterase-5 (PDE5) (DellAgli et
al., 2006).
Some biflavones have been shown to inhibit other
enzyme systems. Amentoflavone inhibits lens aldose reductase (Shimizu et al., 1984; Iwu et al., 1990; Felicio et
al., 1995). It is noteworthy that amentoflavone and several
of its derivatives are potent inhibitors of human cathepsin

H. P. Kim et al.

B (Pan et al., 2005). Cathepsin B is a protease involved in


several inflammation-related disorders. The spatial arrangement of amentoflavone has also been reported. In
addition, two biflavanones (Masuda et al., 2005) and 2,3dihydro-4,4-di-O-methylamentoflavone (Cheng et al.,
2007) were recently found to inhibit tyrosinase.
Amentoflavone strongly antagonizes nicotine-, acetylcholine- and barium chloride-induced spasmolytic activity
of isolated guinea-pig ileum (Chakravarthy et al., 1981).
Amentoflavone was repeatedly found to produce vasorelaxation (Kang et al., 2004). The biflavones from Ginkgo
biloba leaves, including sequoiaflavone, stimulate lipolysis
in 3T3-L1 adipocytes (DellAgli and Bosisio, 2002). Interestingly, bilobetin, sciadopitysin and 7,4,7,4-O-methylamentoflavone from Cephalotaxus koreana enhance
osteoblast differentiation (Lee et al., 2006), suggesting
their therapeutic potential in osteoporosis.
Some biflavonoids possess antioxidative activity. For
example, amentoflavone was reported to scavenge
superoxide radicals (Huguet et al., 1990) and to inhibit
nonenzymatic lipid peroxidation (Mora et al., 1990). This
compound was also found to inhibit CCl4-induced microsomal lipid peroxidation (Cholbi et al., 1991). Furthermore,
sciadopitysin and ginkgetin/isoginkgetin protect human
skin fibroblasts from UVB-induced cytotoxicity, probably
by antioxidative mechanism (Kim, 2001).

Anticancer action of biflavonoids


Some biflavonoids exhibit cytotoxic/anticancer activity.
Ginkgetin is cytotoxic to human ovarian adenocarcinoma
(OVCAR)-3 cells, but not to other cells like Hep G2 and
HeLa (Sun et al., 1997). Taiwanhomoflavone-A (3-8 CC) (Fig. 3) shows cytotoxicity against several cancer cell
lines (Kuo et al., 2000). Apoptotic cell death by caspase
activation is involved in the cytotoxic effects of ginkgetin
(Su et al., 2000). In our experiment, several hinokiflavonetype biflavonoids such as cryptomerin B and isocryptomerin exhibited potent cytotoxic effects, probably by apoptotic death, at low micromolar concentrations (unpublished
results). In contrast, some biflavones such as ginkgetin
and sciadopitysin enhance proliferation of normal human
skin fibroblasts and increase collagen production (Kim et
al., 1997). All these results indicate that certain biflavonoids
more profoundly affect cancer cells/cancer cell lines with
reduced effects on normal cell proliferation, suggesting
therapeutic potential against cancer.

Fig. 2. Chemical structures of most common biflavonoids, amentoflavone derivatives and ochnaflavone. Amentoflavone derivatives are
commonly found in Ginkgo biloba leaves and ochnaflavone is
distributed in Lonicera species.

Antimicrobial and antiviral activity of biflavonoids


Some naturally-occurring and synthetic biflavonoids show
antibacterial, antifungal and antiviral activities. Several
biflavones have antituberculosis activity (Lin et al., 2001).
The biflavanone, 7,7-di-O-methyltetrahydroamentoflavone,
exhibit weak anti-malarial activity (Ahmed et al., 2001). 7,4-

Anti-inflammatory Biflavonoids

Fig. 3. Several naturally-occurring biflavonoids mentioned in this review

267

268

Dimethylamentoflavone (putraflavone) inhibits Leishmania


mexicana promastigotes (Suarez et al., 2003). In addition,
lanaroflavone has antimalarial and leishmanicidal activities
(Weniger et al., 2004). Several ethylene glycol-ligated
flavonoid dimers based on the apigenin moiety inhibit
Leishmania (Wong et al., 2007). It is interesting to note
that ginkgetin-sialic acid conjugates have significant antiviral activity (Miki et al., 2007). Recently, ochnaflavone 7O-methyl ether and 2,3-dihydroochnaflavone 7-O-methyl
ether were found to inhibit HIV-1 activity as well as HIV-1
reverse transcriptase activity (Reutrakul et al., 2007).

Anti-inflammatory activity of biflavonoids


Cellular mechanisms of anti-inflammatory biflavonoids
The biflavonoids including amentoflavone, bilobetin,
sciadopitysin and ginkgetin inhibit mast cell histamine
release in the micromolar range (Amella et al., 1985),
suggestive of their antiallergic action. Podoverine B, a
flavanone-flavonol dimer isolated from plant callus culture,
inhibits macrophage chemiluminescence with an IC50 of
6.4 M (Arens et al., 1986). Some biflavonoids inhibit
lymphocyte proliferation in vitro at 10-100 M (Lee et al.,
1995). The biflavonoids including ochnaflavone, ginkgetin
and isoginkgetin inhibit both T-cell and B-cell proliferation
induced by mitogenic stimulation without affecting cell
viability. The inhibition is irreversible, in contrast to the
completely reversible inhibition of T-cell proliferation by
flavones/flavonols. These results suggest that certain biflavonoids are general inhibitors of lymphocyte proliferation. Biflavonoids also inhibit mixed lymphocyte reaction.
These previous studies may indicate that certain biflavonoids are possible therapeutic agents against some
allergic and deleterious autoimmune disorders such as
rheumatoid arthritis and lupus erythematosus.
The effects of biflavonoids on arachidonate metabolic
pathways have also been examined since the reaction
products, arachidonic acid (AA) and eicosanoids, are pivotal
mediators of inflammation. For the first time, ochnaflavone
and several other biflavones were found to inhibit secretory
phospholipase A2 (sPLA2-IIA) (Chang et al., 1994), and
some of them actually inhibit arachidonic acid release
from mouse peritoneal macrophages in culture (Lee et al.,
1997). Another biflavonoid, morelloflavone, also inhibits
sPLA2 and has in vivo anti-inflammatory activity in animal
models of 12-O-tetradecanoylphorbol-13-acetate (TPA)induced ear edema and carrageenan (CGN)-induced paw
edema in mice (Gil et al., 1997). Notably, this compound
shows in vivo activity by oral administration. Later, ginkgetin
was found to inhibit cytosolic PLA2 (cPLA2). It inhibits
epidermal cPLA2 of guinea-pig skin (Kim et al., 2001).
Cyclooxygenase (COX) produces prostanoids, and lipoxygenase (LOX) synthesizes HETEs and leukotrienes (LT).
Many nonsteroidal anti-inflammatory drugs (NSAID) and

H. P. Kim et al.

some anti-allergic drugs that are used clinically are


inhibitors of COX/LOX. When the effects of biflavonoids
on COX-1/12,15-LOX were examined using guinea-pig
epidermal homogenate as an enzyme source, amentoflavone was revealed as a potent COX-1 inhibitor (IC50 = 3
M) compared to indomethacin (IC50 = 1 M), while
ginkgetin only weakly affected COX-1 (Kim et al., 1998b).
Another similar finding of COX-1 inhibition by amentoflavone
has also been reported (Bucar et al., 1998). Later, tetrahydroamentoflavone was also demonstrated to be a weak
COX-1/COX-2 inhibitor (Selvam and Jachak, 2004). Recently, we found that some synthetic biflavonoids without
substitution(s) on the molecules are COX-2 inhibitors
without COX-2 down-regulatory effects, and these inhibitors
will be discussed at the end of this review. On the other
hand, there has been only one report of inhibitory activity
by biflavonoids against LOXs. Ginkgetin was revealed as
an effective 5-LOX inhibitor at the cell level (Son et al.,
2005). However, it is not clear at present whether
ginkgetin really inhibits 5-LOX at the enzyme level. Thus,
the detailed LOX inhibitory activity of biflavonoids remains
to be elucidated. All of these previous results demonstrate
that certain natural biflavonoids possess anti-inflammatory
activity via inhibition of eicosanoid metabolizing enzyme
activities, thereby reducing concentrations of proinflammatory mediators.
One important anti-inflammatory mechanism of biflavonoids is transcriptional regulation of proinflammatory
molecules. The biflavonoids, bilobetin and ginkgetin, were
initially found to suppress inducible nitric oxide synthase
(iNOS) and COX-2 expression in LPS-treated RAW 264.7
cells (Baek et al., 1999). For further elucidation, we examined their effects on iNOS expression. Several biflavones such as ginkgetin, isoginkgetin, bilobetin and
ochnaflavone down-regulated iNOS expression in LPSinduced RAW 264.7 cells, whereas amentoflavone did not
(Cheon et al., 2000). Moreover, ginkgetin was shown to
inhibit COX-2 induction in LPS-treated RAW cells without
affecting COX-1 levels (Kwak et al., 2002). After these
reports, continual findings of regulatory properties of biflavonoids on proinflammatory gene expression have been
described as summarized in Table I.
Some of these suppressive properties of biflavonoids
against proinflammatory molecules were confirmed in
vivo. For example, topical treatment with ginkgetin reduced
COX-2 induction in TPA-treated mouse skin (Kwak et al.,
2002). Furthermore, topical application of ginkgetin reduced chronic skin inflammation provoked by multiple TPA
treatments, with concomitant reduction of edematic response (Lim et al., 2006). This reduction was also observed
by histological comparison. Therefore, it is now clear that
certain biflavonoids down-regulate expression of proinflammatory molecules such as COX-2 and iNOS in vitro

Anti-inflammatory Biflavonoids

269

Table I. Transcriptional regulation of proinflammatory molecules by biflavonoids

Biflavonoids

Target cells

Stimulant

Target genes/enzymes affecteda)

References

Amentoflavone
Amentoflavone
Ginkgetin
Ochnaflavone
Ochnaflavone
Isoginkgetin

M1
A549
mouse BMMC
RAW 264.7
HASMC
HT1080

TNF-
cytokines
LPS
TNF-
-

ICAM-1
PPAR- (), COX-2, NF-B
COX-2
iNOS, ERK1/2, NF-B
ERK1/2, MMP-9
TIMP-1 (), MMP-9, PI3K/Akt

Tanaka et al. (2001)


Banerjee et al. (2002)
Son et al. (2005)
Suh et al. (2006a)
Suh et al. (2006b)
Yoon et al. (2006)

These proinflammatory molecules are down-regulated or inhibited, whereas indicates up-regulation.


M1: mouse myeloid leukemia cells, HASMC: human aortic smooth muscle cells, HT1080: human fibrosarcoma cells
a)

as well as in vivo.
In vivo anti-inflammatory activity of biflavonoids
In vivo anti-inflammatory activities of biflavonoids have
been demonstrated. The Garcinia biflavanones, GB1 and
GB2, showed in vivo anti-inflammatory activity at 50 mg/
kg i.p. against CGN-induced edema (Iwu, 1986). When
topically applied, the Ginkgo biflavonoids, amentoflavone,
ginkgetin and sciadopytisin, showed anti-inflammatory
activity against croton-oil-induced ear edema (Della Loggia
et al., 1996). They exhibited higher anti-inflammatory
activity when a liposome formulation was used. Amentoflavone, a biflavone isolated from Ginkgo leaves and
Selaginella species, showed potent anti-inflammatory
activity in vivo (Kim et al., 1998a). By the i.p. route, it
possessed approximately 1/2-1/5 of the anti-inflammatory
activity of indomethacin or prednisolone against several
animal models of acute inflammation. By intraperitoneal
injection, amentoflavone also possessed potent analgesic
activity against acetic acid-induced writhings in mice.
However, amentoflavone did not significantly reduce
adjuvant-induced arthritis (AIA) in rats.
In particular, ginkgetin dose-dependently reduced arthritic
inflammation (secondary inflammation) at 5-20 mg/kg/day
by intraperitoneal injection in AIA rats, an animal model of
chronic inflammation (Kim et al., 1999). This finding is the
first demonstrating the anti-arthritic potential of biflavonoids. The inhibitory activity of ginkgetin against AIA was
also confirmed by histologic comparison of the affected
paws, in which there were fewer infiltrating inflammatory
cells and almost no signs of inflammation at the synovial
membrane. The potency of inhibition by ginkgetin was 1/2
-1/3 that of prednisolone. No severe side effects were
observed during the 25-day experiment. While prednisolone
produced potent thymus and spleen atrophy due to its
suppressive action on the pituitary-adrenal axis, ginkgetin
did not reduce thymus and spleen weights in AIA rats.
This finding that ginkgetin possesses significant antiarthritic
activity without the typical steroidal systemic side-effects
suggests a potential use for biflavonoids as a new class of

safer anti-inflammatory agents. Ginkgetin also possessed


analgesic activity comparable to that of indomethacin (IC50
for ginkgetin = 8.7 mg/kg, IC50 for indomethacin = 3.8 mg/
kg). Many investigators, including Pelzer et al. (1998),
have claimed that flavonoids possess anti-inflammatory
activity against acute inflammation, but they are not suitable
against chronic inflammation. However, the above finding
demonstrates that biflavonoids can be used as antiinflammatory agents against chronic disorders. If the oral
or topical bioavailability of biflavonoids can be improved,
our study may lead to the development of new types of
anti-inflammatory drugs from natural products, especially
drugs against chronic inflammation.
Along with the anti-inflammatory activity, it should be
mentioned that biflavonoids including amentoflavone,
ginkgetin and isoginkgetin were recently shown to possess potent neuroprotective activity in vitro (Kang et al.,
2005). Similarly, the biflavone fraction from Araucaria
bidwillii protected against rat cerebral ischemia-induced
oxidative stress (Mukherjee et al., 2007).

Analgesic activity of biflavonoids


As an anti-inflammatory agent, the compound with
analgesic activity is favored. The initial in vivo study of
amentoflavone did not reveal any neuropharmacological
and neuroanalgesic effects by i.p. injection, suggesting a
lack of penetration through the blood-brain barrier (BBB)
(Chakravarty et al., 1981). Amentoflavone is nontoxic at
doses of up to 1.5 g/kg i.p. However, in vitro experiments
suggested that amentoflavone might be able to penetrate
the BBB by passive diffusion (Gutman et al., 2002).
Biflavonoids such as amentoflavone and isoginkgetin also
showed neuroprotective effects in vitro (Kang et al.,
2005). Although it is still not clear whether amentoflavone
or other biflavonoids penetrate the BBB, their peripheral
analgesic activities have been demonstrated several
times.
Some biflavonoids possess peripheral analgesic activity
by i.p. injection. Our investigations have shown that
amentoflavone and ginkgetin possess potent analgesic

270

H. P. Kim et al.

activity against writhings by i.p. injection, but not by oral


administration (Kim et al., 1998a, 1999). Similarly, 3-8
binaringenin showed antinociceptive activity by i.p. injection on writhing test and formalin test (Bittar et al., 2000).
I3-Naringenin-II8-4-OMe-eriodictyol isolated from Rheedia
gardneriana leaves also showed analgesic activity by i.p.
injection (Filho et al., 2000). Thus it is clear that some
biflavonoids possess analgesic activity, which may lead to
the development of better anti-inflammatory agents.

Bioavailability and metabolism of biflavonoids


There has been no available data for absorption and
distribution of biflavonoids in animals/humans. Morelloflavone and tetrahydroamentoflavone showed in vivo antiinflammatory activity by oral administration (Gil et al.,
1997; Selvam and Jachak, 2004). But in our experiments,
oral treatment produced much reduced or no activity,
indicating that the oral bioavailability of biflavonoids may
be very low. In contrast, intraperitoneal administration
resulted in higher anti-inflammatory activity. Topical treatment also yielded positive results (Della Loggia et al.,
1996; Kim et al., 1998; Kwak et al., 2002; Lim et al.,
2006).
Anti-inflammatory activity of synthetic biflavonoids
There have only been a few trials to synthesize a
biflavonoid library. Several synthetic C-C biflavonoids
were synthesized by Ullmann condensation, and some of
them showed antituberculosis activity (Lin et al., 2001).
Recently, our group prepared a C-C biflavonoid library
(Fig. 4) and evaluated the compounds for anti-inflammatory activity. These compounds inhibited sPLA2-IIA to
varying degrees depending on the position of the C-C
connection (Chen et al., 2006). Continuing research has
shown that they differentially inhibit various isoforms of
PLA2 including sPLA2 and cPLA2 (Moon et al., 2007).
Using the same C-C biflavonoid library, their inhibitory
activity against COX-2-mediated PGE2 production in LPStreated RAW 264.7 cells was examined. And the results
demonstrated that several derivatives such as A-A and AB ring biflavonoids have higher inhibitory activity against
PGE2 production in LPS-treated RAW cells (Park et al.,
2006). In particular, in the micromolar concentration range,
6-6 C-C biflavone potently inhibited COX-2 without
affecting COX-2 expression. This biflavone also exhibited
in vivo anti-inflammatory activity against rat CGN-induced
paw edema. However, it did not significantly inhibit iNOSmediated NO production. These studies suggest that C-C
biflavones without substitution on the molecule do not
have the capacity to regulate the transcription of proinflammatory molecules such as COX-2 and iNOS. They
simply behave as PLA2 and COX-2 inhibitors, in contrast

Fig. 4. Some synthetic C-C biflavonoid library. Note: Two simple


flavones are connected with a C-C bond at different positions.

to certain natural biflavonoids that have hydroxyl/methoxyl


substitutions on the molecule. Several 6-6 biflavone derivatives with hydroxyl/methoxyl substitution(s) were also
successfully synthesized and their anti-inflammatory properties are under investigation. We have also synthesized
some bichalcone derivatives and examined their PGE2
inhibitory activity. However, they did not considerably inhibit
PGE2 production (unpublished results).
Anti-inflammatory biflavonoid research is now in its early
stages. More intensive studies with structurally diverse
biflavonoids will be carried out in the near future in order
to find optimum structures and to elucidate their antiinflammatory activity.

Perspectives
The research on anti-inflammatory biflavonoids is in the
early stages and now expanding. Based on initial studies,
biflavonoids utilize multiple anti-inflammatory mechanisms
(Scheme I). They affect inflammatory cells such as mast
cells and lymphocytes. They inhibit proinflammatory enzymes such as PLA2 and COX. Recent investigations also
demonstrate that they suppress proinflammatory molecule
expression. Due to these unique properties, biflavonoids
have potential as anti-inflammatory drugs especially for
treating chronic inflammatory disorders. Through more
intensive studies with modern pharmacological techniques,
new types of anti-inflammatory agents based on biflavonoid structures may be successfully developed.

Anti-inflammatory Biflavonoids

271

Scheme I. Multiple anti-inflammatory actions of biflavonoids Symbol (T) denotes inhibition or down-regulation of target molecules. NSAID:
nonsteroidal anti-inflammatory drug.

ACKNOWLEDGEMENTS
This study was supported by research grant No. R012004-000-10134-0 from the Basic Research Program of
the Korea Science & Engineering Foundation and postBK21 program.

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