Effects of Ginkgo biloba extract EGb and its terpene Ginkgo Extract by benbenzhou

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									                       Effects of Ginkgo biloba extract EGb 761 and its terpene-lactones on angiotensin converting enzyme activity / Asian
                                                         Journal of Traditional Medicines, 2008, 3 ( 2 )




Regular Articles

              Effects of Ginkgo biloba extract EGb 761 and its terpene-
          lactones on angiotensin converting enzyme activity and nitric
                             oxide production in human endothelial cells
                           Ingrid A-L Persson *, Erica Lindén, Malin Andersson, Karin Persson
             Department of Medicine and Health Sciences, Division of Pharmacology, Faculty of Health Sciences,
                                              Linköping University, Sweden




Abstract

The effects of Ginkgo biloba (Ginkgoaceae), its terpene-lactones (ginkgolide A, B, C and bilobalide), biflavonols (quercetin),
biflavones (sciadopitysin) and proanthocyanidins (procyanidin) on angiotensin-converting enzyme (ACE) activity and nitric oxide
(NO) production in cultured human endothelial cells from umbilical veins (HUVEC) were investigated. A dose-dependent significant
inhibition of the ACE activity was observed after 10 min incubation with Ginkgo biloba extract EGb 761 and quercetin. No
significant effects due to terpene-lactones or sciadopitysin were seen. Incubation with Ginkgo biloba extract, quercetin, sciadoitysin
and procyanidin for 24 hr significantly increased NO production. No significant effects were seen with ginkgolide A, B and C, while
bilobalide induced a dose-dependent decrease in NO production. In conclusion, this study shows that Ginkgo biloba extract inhibits
ACE activity and increases NO production from HUVEC. A flavonol (quercetin and/or homologs) is the main component responsible
for the inhibitory effect of ACE activity. Quercetin and a proanthocyandin (procyanidin) are responsible for the increases seen in NO
production. These results may explain the positive effects of Ginkgo biloba on the cardiovascular system and on cognitive function.


Key words: angiotensin-converting enzyme; flavonoids; Ginkgo biloba; nitric oxide; terpene-lactones




Introduction                                                                        Ginkgo biloba to document its beneficial effects on
     Ginkgo biloba L. (Ginkgoaceae) is commonly                                     the vascular and the nervous systems. Effects such
used as a phytomedicine and nutritional supplement                                  as cardioprotective properties [3, 4], increased nitric
all over the world. In China, the leaves and the fruit                              oxide (NO) production [5-7], increased peripheral and
have been used in the treatment of asthma, high                                     cerebral blood flow [8] have been described. Reduced
blood pressure, tinnitus and angina pectoris [1]. In                                dementia [9] and reduction of age-related loss of
1578 Ginkgo biloba was mentioned as treatment for                                   cognitive function [10, 11] also have been reported. The
senility and aging [2]. During the past few decades, a                              effects of Ginkgo biloba extract have been ascribed to
number of investigations have been performed with                                   its content of terpene-lactones and to the antioxidative
                                                                                    properties of the extract [3, 4, 12].
                                                                                         The standardized Ginkgo biloba extract EGb 761
* Author to whom correspondence should be addressed. Address:                       is prepared from the dried leaves of the Ginkgo biloba
Department of Medicine and Health Sciences, Division of
Pharmacology, Faculty of Health Sciences, Linköping University,                     tree. This extract, EGb 761 is reliable concerning
SE-58185 Linköping, Sweden; Tel: +46-13221052; Fax:                                 the content of the presumed or proposed most active
+46-13149106; E-mail: ingpe@imv.liu.se
                                                                                    substances; 6 % terpene-lactones (ginkgolides A,
Received: 2007-11-12     Accepted: 2008-02-10




                                                                              42
                   Effects of Ginkgo biloba extract EGb 761 and its terpene-lactones on angiotensin converting enzyme activity / Asian
                                                     Journal of Traditional Medicines, 2008, 3 ( 2 )




B, C, J and bilobalide), 24 % flavonol glycosides                               Sciences, Linköping, Sweden (Dnr 03-602).
(principally derivatives of quercetin, kaempferol
                                                                                Ginkgo biloba extraction
and isorhamnetin), 7 % proanthocyanidins (e. g.,
procyanidin), 13 % carboxylic acids, 2 % catechins                                   Ginkgo biloba, EGb 761, was used for preparation
and < 0.005 % ginkgotoxin [13-15]. Ginkgo leaves, but                           of a solution. One g EGb 761 was dissolved in 10 ml
not the standardized extract EGb 761, also contain                              sterile phosphate-buffered saline (PBS) and placed
small amounts of biflavones (e. g., sciadopitysin) [13].                        in 60°C waterbath for 1 hr. The Ginkgo biloba
       The renin-angiotensin system (RAS) is one of                             suspension was then filtered twice through a standard
the most important systems in the body for regulating                           filter 0.45 µm (Munktell, Grycksbo, Sweden).
blood pressure, fluid and electrolyte balance by                                The obtained filtrate was considered to have a
maintaining the extracellular fluid volume and                                  concentration of 100 mg/ml and was frozen in aliquots
arterial pressure despite wide variations in electrolyte                        at -20 ºC.
intake. The angiotensin-converting enzyme (ACE),                                     Gingkolides A, B, C, bilobalide, quercetin,
a carboxypeptidase, is an important component of                                procyanidin and sciadopitysin were dissolved in
the RAS where it rapidly converts physiologically                               dimethylsulfoxide (DMSO).
inactive angiotensin I to the physiologically active
                                                                                Cultured endothelial cells from human umbilical
octapeptide angiotensin II. Angiotensin II increases                            veins (HUVEC)
systemic blood pressure and renal perfusion pressure.
                                                                                     Human umbilical cords were obtained after
The primary physiological mechanism of RAS is
                                                                                normal vaginal delivery (after informed consent from
the influence on the blood pressure, but angiotensin
                                                                                the mothers) and kept in sterile bottles containing
II is also known to influence neuronal and cognitive
                                                                                PBS, penicillin, streptomycin and gentamicin.
function [16, 17]. RAS has a major influence on the
                                                                                Endothelial cells were then isolated by treatment
circulation and there are several interactions known
                                                                                with 0.5 mg/ml collagenase for 20 min at 37 °C as
between the RAS and nitric oxide (NO). There are
                                                                                described previously [19]. Briefly, the collagenase +
reports that Ginkgo biloba increases NO production
                                                                                cell perfusate was washed twice, then resuspended
[18]
     . However, the NO-mediated antioxidative effects
                                                                                in culture medium (Dulbecco´s modified Eagle´s
of Ginkgo biloba might not be the only explanation of
                                                                                medium, DMEM with normal glucose) supplemented
the pharmacological effects of Ginkgo biloba.
                                                                                with nonessential amino acids (NEAA 1:100),
       The aim of this study was to investigate the
                                                                                oxalacetic acid (1.2 mM), insulin (0.24 IE/ml),
effects of Ginkgo biloba extract on ACE activity
                                                                                penicillin (5 U/ml), streptomycin (0.5 µg/ml), Hepes
and NO production. The aim of this study was also
                                                                                (10 mM), endothelial cell growth factor (ECGF,
to investigate the effect of the unique substances of
                                                                                30 µg/ml) and 17 % inactivated fetal bovine serum
Ginkgo biloba, ginkgolides A, B, C, bilobalide, the
                                                                                (FBS). Resuspended HUVEC were seeded in 25
flavonol, quercetin, the biflavon, sciadopitysin, and the
                                                                                cm2 tissue culture flasks coated with 0.2 % gelatine
proanthocyanidin, procyanidin on ACE activity and
                                                                                (Sigma-Aldrich Chemical Co, MO, USA), and kept
NO production in HUVEC.
                                                                                in an incubation chamber. Medium was replaced
                                                                                every 48-72 hr. At confluence, cells were harvested
Materials and methods
                                                                                with trypsin-EDTA for 5-10 min, and then reseeded
     This study on cultured endothelial cells from                              1:2. The second passage was seeded in a 96-well
human umbilical veins (HUVEC) was approved by                                   microtiter plate and allowed to reach confluence.
the regional ethics committee at the Faculty of Health                          Immediately prior to treating the cells with extract,




                                                                           43
                  Effects of Ginkgo biloba extract EGb 761 and its terpene-lactones on angiotensin converting enzyme activity / Asian
                                                    Journal of Traditional Medicines, 2008, 3 ( 2 )




the medium was removed and replaced with medium                                until nitrite analysis. Nitrite content in the medium was
without FBS to avoid discrepancies in results due to                           analysed as a marker of NO production as proposed by
ACE or NO present in the serum. Cells were treated                             Lauer et al. [20]. Nitrite and nitrate concentration in the
with Ginkgo biloba extract EGb 761 (0.1, 0.5, 1, 5 and                         Ginkgo biloba extract was also analysed. Nitrite and
10 mg/ml); ginkgolides A, B, C, bilobalide, quercetin,                         nitrate concentrations were analysed with a commercially
procyanidin and sciadopitysin (0.1, 0.5, and 1 mg/ml).                         available nitrite/nitrate assay: Nitric oxide (NO2-/NO3-)
NO production was also analyzed in cells treated with                          assay kit obtained from R&D Systems, Abingdon, UK.
Ginkgo biloba extract EGb 761 in the presence of the                           The principle for the colorimetric detection of nitrite is
endothelial nitric oxide synthase (eNOS) inhibitor,                            the Griess reaction. Diazonium ions are produced when
NG-monomethyl-L-arginine (L-NMMA) and induced                                  acidified nitrite reacts with sulfanilic acid. The diazonium
nitric oxide synthase (iNOS), N-3-(aminomethyl)                                ions form chromophore agents after reacting with N-(1-
benzylacetamidine (1400 W) inhibitor. Ginkgo biloba                            naphthyl) ethylenediamine. Then the optical density
extract EGb 761 was dissolved in PBS and all other                             was determined with a spectramax reader at 540 nm
drugs in dimethylsulfoxide (DMSO). Corresponding                               (Molecular Devices, Wokingham, UK).
volumes of PBS or DMSO were used as controls.
                                                                               Chemicals
After incubation with drugs for 10 min, ACE activity
was analysed (see below) and after 24 hr incubation                                All chemicals for culturing cells were obtained from
with drugs, NO production was analysed (see below).                            Life Technologies, Scotland, UK, except endothelial cell
                                                                               growth factor was from Boehringer-Mannheim, Germany,
ACE activity                                                                   heparin (Heparin LEO) from LEO Pharma AB, Malmö,
     ACE activity was analysed with a commercial                               Sweden, NG-monomethyl-L-arginine (L-NMMA) and
radioenzymatic assay (ACE-direct REA, Bühlmann                                 N-3-(aminomethyl) benzylacetamidine (1400W) from
Laboratories, Allschwil, Switzerland) with the                                 Alexis Biochemicals, San Diego, USA, ginkgolides A,
following modifications. Blank and standard serum                              B, C, bilobalide, procyanidin and sciadopitysin from
were added to wells with corresponding volumes                                 Extrasynthese Genay, France, and quercetin from
of medium without FBS. The synthetic substrate                                 Sigma-Aldrich Chemical Co, MO, USA. The Ginkgo
3
  H-hippuryl-glycyl-glycine was then added directly                            biloba extract EGb 761 was a kind gift from Dr
to all the wells of the microtiter plate. Cells were                           Willmar Schwabe, Karlsruhe, Germany mediated via
incubated with substrate for 2 hr at 37 °C to allow                            Cederroth International AB, Upplands Väsby, Sweden.
for the ACE present to cleave the substrate to
                                                                               Viability
3
  H-hippuric acid. Then, 150 µl medium + substrate
was transferred from each well into scintillation vials                            A viability test was performed by incubating
containing 50 µl 1 M HCl (to stop the enzymatic                                HUVEC with Ginkgo biloba extract 10 mg/ml,
reaction). Scintillation liquid from the commercial kit                        ginkgolides A, B, C 1 mg/ml, respectively, and
was added and the radiation levels were determined                             bilobalide 0.1 mg/ml for 24 hr. Then, trypan blue
in a scintillation counter (1217 RackBeta Liquid                               0.05 % was added for 5 min at room temperature
Scintillation Counter from LKB Wallac, Turku,                                  before counting the cells.
Finland).
                                                                               Calculations
NO determination
                                                                                   Results are presented as mean ± s.e.m. (standard
    After incubating HUVEC with the different                                  error of the mean).
substances, medium was removed and stored at –20 °C




                                                                         44
                   Effects of Ginkgo biloba extract EGb 761 and its terpene-lactones on angiotensin converting enzyme activity / Asian
                                                              Journal of Traditional Medicines, 2008, 3 ( 2 )




     1 unit (U) of ACE activity is defined as the                                           Results
amount of the enzyme required to release 1 µmol of
hippuric acid per min and liter. Statistical calculations                                   Effects on ACE activity
were done with Graph Pad PrismTM 5.0 (San Diego,                                                After incubation with Ginkgo biloba extract EGb
CA, USA). One way analysis of variance (ANOVA)                                              761 for 10 min, a dose-dependent inhibition of ACE
for repeated measures was performed followed by                                             activity in HUVEC was seen. The Ginkgo biloba
Dunnett´s post test. Statistical significance is denoted                                    extract at the concentrations of 1 mg/ml, 5 mg/ml and
as * P<0.05, **P<0.01 and ***P<0.001.                                                       10 mg/ml showed significant (***P<0.001) inhibition
                          Figure 1
                                                                                            compared to the PBS control (Fig. 1).

                                                40
                             ACE activity (U)




                                                30

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                                                20


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                                                 G


                                                              G




                                                              Ginkgo biloba (mg/ml)
                                                  Ginkgo biloba for 10 min.
Fig. 1. ACE activity in HUVEC after incubation with Ginkgo biloba extract(mg/ml) Statistics were calculated with one way
ANOVA repeated measures and denoted as ***P<0.001, n=6 from different individuals.


     After 10 min incubation with ginkgolides A, B                                          However, there was a significant difference (**P<0.01)
and C and bilobalide, respectively, no significant                                          between 0.1 and 1 mg/ml procyanidin (Fig. 3).
inhibition of ACE activity in HUVEC was noted due
                                                                                            Effects on NO production
to any of the drugs compared to the DMSO control
(Fig. 2).                                                                                       After 24 hr incubation with 5 and 10 mg/ml
     Ten minutes incubation with quercetin dose-                                            Ginkgo biloba extract EGb 761, a significant increase
dependently and significantly inhibited ACE activity                                        in NO production from HUVEC was seen (***P<0.001)
( *P<0.05 for 0.1 mg/ml quercetin and ***P<0.001                                            compared to the PBS control (Fig. 4).
for 0.5 and 1 mg/ml respectively; Fig. 3). Neither                                              After 24 hr incubation with ginkgolides A, B or C,
sciadopitysin nor procyanidin significantly affected                                        no effect on NO production was seen compared to the
ACE activity compared to the DMSO control.                                                  DMSO control (Fig. 5). However, a dose-dependent




                                                                                    45
                  Figure 2
                                                        Effects of Ginkgo biloba extract EGb 761 and its terpene-lactones on angiotensin converting enzyme activity / Asian
                                                                                          Journal of Traditional Medicines, 2008, 3 ( 2 )




                                        40
                                                                                                                                                             Ginkgolide A
                                                                                                                                                             Ginkgolide B
                     ACE activity (U)




                                        30
Figure 3                                                                                                                                                     Ginkgolide C
                                                                                                                                                             Bilobalide
                                        20


                                        10


                                         0
                                                            DMSO             0.1 mg/ml 0.5 mg/ml                            1 mg/ml
           Fig. 2. ACE activity in HUVEC after incubation with ginkgolides A, B, C and bilobalide for 10 min. Statistics were calculated with
           one way ANOVA repeated measures, n=6 from different individuals.



                                                            10
                                                                                                                                **
                                                                                                                                                        Quercetin
                                                            8                                                                                           Sciadopitysin
                                         ACE activity (U)




                                                                                                                                                        Procyanidin
                                                            6
                                                                             * *
                                                                        *
                                                            4                * *
                                                                             * *
                                                            2

                                                            0
                                                               1 g/ml
                                                                m l

                                                                D l


                                                               1 g/ml
                                                                m l

                                                                D l


                                                               1 g/ml
                                                                m l
                                                                       l
                                                            0. m g O




                                                            0. m g O




                                                            0. mg O
                                                               5 /m


                                                                     m


                                                               5 /m


                                                                     m


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                                                               1 S




                                                               1 S




                                                               1 S
                                                                  g/




                                                                  g/




                                                                  g/
                                                            0 . DM




                                                            0. M




                                                            0. M

                                                                m
                                                                m




                                                                m




           Fig. 3. ACE activity in HUVEC after incubation with quercetin, sciadopitysin or procyanidin for 10 min. Statistics were calculated
           with one way ANOVA repeated measures and denoted as *P<0.05, **P<0.01 and ***P<0.001, n=5 from different individuals.


           significant decrease in NO production was seen with                                                            No significant effect on NO production was seen
           bilobalide at the concentrations 0.5 mg/ml (*p<0.05)                                                      when HUVEC were incubated with Ginkgo biloba
           and 1 mg/ml (***P<0.001) (Fig. 5).                                                                        extract and the eNOS inhibitor, L-NMMA, or the
                  Twenty-four hr incubation with quercetin,                                                          iNOS inhibitor, 1400 W, for 24 hr compared to Ginkgo
           s c i a d o p i t y s i n a n d p r o c y a n i d i n r e s p e c t i v e l y,                            biloba extract alone (Fig. 7).
           significantly and dose-dependently increased NO                                                               Table 1 shows the amount of nitrite and nitrate
           production (Fig. 6).                                                                                      found in the Ginkgo biloba extract.


                                                                                                                       20
                                                                                                               46
                   Figure 4
                     Effects of Ginkgo biloba extract EGb 761 and its terpene-lactones on angiotensin converting enzyme activity / Asian
                                                        Journal of Traditional Medicines, 2008, 3 ( 2 )




                                     25
                                                                                                                          ***
                                     20
                      Nitrite ( M)



                                     15
                                                                                                           ***
                                     10

                                      5

                                      0
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                                                                                                                 10
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                                                             Ginkgo biloba (mg/ml)

                                                            Ginkgo biloba (mg/ml)
Fig. 4. NO production (measured as nitrite) in HUVEC after incubation with Ginkgo biloba extract for 24 hr. Statistics were
calculated with one way ANOVA repeated measures and denoted as ***P<0.001, n=6 from different individuals.



Table 1. The quantity of nitrite, nitrate and nitrite/                             98.5 % ± 1.5, to 1 mg/ml ginkgolide C, 99.5 % ± 0.5
nitrate in the Ginkgo biloba extract compared to PBS.                              and to 0.1 mg/ml bilobalide, 98.5 % ± 1.5.
                      Nitrite             Nitrate      Nitrite/Nitrate
                      (µM)                (µM)              (µM)                   Discussion
       PBS                 0.32                 0.53          0.85
                                                                                        Since Ginkgo biloba is used for its beneficial
  Ginkgo biloba
    10 mg/ml               5.57             12.43            18.00                 effects on the circulation [3, 4, 8, 18] and since the RAS
  Ginkgo biloba                                                                    system is one of the most important systems in the
     5 mg/ml               3.21             13.36            16.57
  Ginkgo biloba                                                                    body that regulates blood pressure and blood flow, we
     1 mg/ml               0.87                 8.20           9.07                examined whether Ginkgo biloba affects ACE activity
  Ginkgo biloba
   0.5 mg/ml               0.32                 4.93           5.25
                                                                                   and NO production. Previous studies have shown that
                                                                                   Ginkgo biloba increases NO production and that this
                                                                                   is one mechanism mediating the vasorelaxation due to
Viability
                                                                                   Ginkgo biloba extract [6, 12]. However, this is the first
    The cell viability was after exposure to 10 mg/ml                              study on Ginkgo biloba determining the effects on
Ginkgo biloba extract, 99.5 % ± 0.5, to 1 mg/ml                                    ACE activity. Here, Ginkgo biloba extract induced
ginkgolide A, 99 % ± 1.0, to 1 mg/ml ginkgolide B,                                             22
                                                                                   dose-dependent inhibition of ACE activity in HUVEC




                                                                              47
                                Effects of Ginkgo biloba extract EGb 761 and its terpene-lactones on angiotensin converting enzyme activity / Asian
                                                                  Journal of Traditional Medicines, 2008, 3 ( 2 )

  Figure 5


                    4                                                                                               *
                                                                                                                                       Ginkgolide A
                                                                                                                    *
                                                                                          *                                            Ginkgolide B
                    3                                                                                               *
                                                                                                                                       Ginkgolide C
     Nitrite ( M)




                                                                                                                                       Bilobalide
                    2


                    1


                    0
                                DMSO             0.1 mg/ml 0.5 mg/ml 1 mg/ml
Fig. 5. NO production (measured as nitrite) in HUVEC after incubation with ginkgolides A, B, C and bilobalide for 24 hr. Statistics
were calculated with one way ANOVA repeated measures and denoted as *P<0.05 and ***P<0.001, n=6 from different individuals.

      Figure 6


                                                                                   *
                           60                                                      *
                                                                                                                                       Quercetin
                                                                                   *
                                                                                                                                       Sciadopitysin
                                                                                                                                       Procyanidin
            Nitrite ( M)




                           40                                               *
                                                                            *
                                           * *                              *                               *
                           20              * *                                                              *
                                             *                                                            * *
                                         * *                                                              *
                           0
                           0. mg O




                           0. mg O




                           0. mg O
                             1 g/ml
                              m l

                              D l


                             1 g/ml
                              m l
                                    l



                             1 g/ml
                              m l
                                    l
                             5 /m


                                   m


                             5 /m


                                   m


                             5 /m


                                   m
                             1 S




                             1 S




                             1 S
                                g/




                                g/




                                g/
                           0. D M




                           0. M




                           0. DM
                              m




                              m




                              m




Fig. 6. NO production (measured as nitrite) in HUVEC after incubation with quercetin, sciadopitysin or procyanidin for 24 hr.
Statistics were calculated with one way ANOVA repeated measures and denoted as *P<0.05, **P<0.01 and ***P<0.001, n=5 from
different individuals.




                                                                                       48
                                                                                        23
                   Effects of Ginkgo biloba extract EGb 761 and its terpene-lactones on angiotensin converting enzyme activity / Asian
                                                     Journal of Traditional Medicines, 2008, 3 ( 2 )

                       Figure 7


                                         50


                          Nitrite ( M)   40

                                         30

                                         20

                                         10

                                          0
                                                                       A
                                                                         l




                                                                       A
                                                                        l
                                                                       W




                                                                       W
                                                                       m




                                                                       m
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                                                                     M


                                                                   00
                                                                   00
                                                                    g/




                                                                    g/
                                                                   M




                                                                  M
                                            m




                                                               5m




                                                                14
                                                                14
                                                                 N




                                                                N
                                         10


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                                                              L-


                                                             l+
                                                             l+


                                                             B
                                                           l+




                                                           l+


                                                           m
                                                           m
                                    B




                                                          G


                                                         m
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                                G




                                                        g/
                                                        g/




                                                      g/
                                                      g/




                                                    5m
                                                      m
                                            m




                                                  5m
                                                   10
                                         10




                                                  B
                                                B




                                               G
                                                B
                                              G
                                         B




                                              G
                                   G




Fig. 7. NO production (measured as nitrite) in HUVEC after incubation with Ginkgo biloba extract (GB) 5 and 10 mg/ml, Ginkgo
biloba extract and L-NMMA (10-4 M), Ginkgo biloba extract and 1400 W (10-4 M) for 24 hr, n=5 from different individuals.




(Fig. 1). These results could, at least partially, explain                      bilobalide present in Ginkgo biloba extract can not
the beneficial effects seen on the cardiovascular                               be excluded [4, 22]. Such a synergistic effect might
system due to Ginkgo biloba treatment.                                          be very important and necessary for the beneficial
    Since there is a known connection between                                   effects of Ginkgo biloba. The fact that Ginkgo biloba
ACE activity and cognitive functions [16, 17, 21]; e.g.,                        extract was dissolved in PBS and the terpene-lactones
concentration and memory, the effects of Ginkgo                                 were dissolved in DMSO might explain some of the
biloba on ACE activity was studied. The inhibitory                              differences in the results due to the Ginkgo biloba
effect of Ginkgo biloba on ACE activity documented                              extract and the terpene-lactones.
here may explain, at least partially, the positive                                     Since this study did not discover any significant
pharmacological effects of Ginkgo biloba on                                     inhibition of ACE activity due to terpene-lactones,
cognition.                                                                      there is presumably some other Ginkgo biloba-
    Effects of Ginkgo biloba extract on the vascular                            substance(s) that inhibits ACE activity. Ginkgo biloba
system has been generally ascribed to terpene-                                  extract contains 6 % terpene-lactones (ginkgolides
lactones, e.g., the ginkgolides, and bilobalide, and                            A, B, C, J and bilobalide), 24 % flavonol glycosides
the flavonoids [6]. No effects on ACE activity was                              (principally derivatives of quercetin, kaempferol
seen by any of the terpene-lactones used (Fig. 2).                              and isorhamnetin), 7 % proanthocyanidins (e.g.,
Therefore, the ginkolides A, B, C and bilobalide are                            procyanidin), 13 % carboxylic acids and 2 % catechins
not responsible for the beneficial effects on ACE                               [13-15]
                                                                                        . This study shows that quercetin significantly
activity due to treatment with the whole extract.                               and dose-dependently inhibits ACE activity (Fig. 3).
However, synergistic effects of the ginkgolides and                             Others have also shown that quercetin and derivatives


                                                                                                         25
                                                                           49
                   Effects of Ginkgo biloba extract EGb 761 and its terpene-lactones on angiotensin converting enzyme activity / Asian
                                                     Journal of Traditional Medicines, 2008, 3 ( 2 )




of kaempferol inhibit ACE activity [23, 24]. Previous                           also explain why NO production from HUVEC was
studies by our group have shown inhibition of ACE                               only slightly, and not significantly, inhibited by NOS
activity by several types of flavonoids: tea catechins                          inhibitors (Fig. 7).
[25]
     , Vaccinium myrtillus (Ericaceae), (anthocyanins;                               In conclusion, Ginkgo biloba extract inhibits ACE
Persson et al. in manus) and procyanins in chocolate                            activity and increases NO production in HUVEC. The
(Persson et al. unpublished data). This might be of                             effect on ACE activity was not seen after treatment
interest as Ginkgo biloba extract contains 2 % catechins                        with terpene-lactones or the biflavonoid, sciadopitysin,
and 7 % proanthocyanidins which are flavonoids also                             but was clearly seen due to treatment with the
found in tea and Vaccinium myrtillus. Our results on                            flavonol, quercetin. The inhibition of the ACE activity
the effect of procyanidin on ACE activity are difficult                         may be one explanation of the pharmacological
to interpret: no signficant effect was seen compared to                         effects of Ginkgo biloba on the cardiovascular system.
control but there was a significant inhibition when 0.1                         As ACE activity, i.e., angiotensin II, is involved in
and 1 mg/ml was compared (Fig. 3). Thus, the most                               cognitive functions, the inhibition of ACE activity
abundant components of Ginkgo biloba, flavonols,                                also might at least partly explain the effects of Ginkgo
proanthocyanidins and catechins are responsible for                             biloba on cognitive functions. Ginkgo biloba extract,
the effects on ACE activity seen in this study.                                 quercetin, sciadopitysin and procyanidin increased
       The pharmacological mechanism of the allopathic                          NO production from HUVEC. This effect on NO
ACE inhibitors is their ability to bind Zn 2+ at the                            production was not seen after treatment with terpene-
active site of ACE. Flavonoids are known to be able                             lactones.
to chelate metal ions such as Zn2+ and Fe2+.
       In confirmation of earlier studies, Ginkgo biloba                        Acknowledgements
extract increased NO production in HUVEC (Fig. 4).                                  This study was supported by grants from
The results of this study did not show any effect of                            Östergötlands Läns Landsting (LIO-8355) and the
the ginkgolides A, B or C on NO production. In fact,                            Cardiovascular Inflammation Research Centre (CIRC)
there was a significant decrease in NO production                               at Linköping University.
when the cells were treated with bilobalide (Fig. 5).                               We thank Cederroth International AB, Upplands
Quercetin, sciadopitysin and procyanidin significantly                          Väsby, Sweden, and Dr Willmar Schwabe, Karlsruhe,
increased NO production (Fig. 6). Sciadopitysin                                 Germany for the kind gift of the Ginkgo biloba extract
is present in ginkgo leaves, but is removed when                                EGb761.
preparing the standardized EGb 761 extract due to
lack of effect according to Van Beck [13]. However,                             References
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                       Effects of Ginkgo biloba extract EGb 761 and its terpene-lactones on angiotensin converting enzyme activity / Asian
                                                         Journal of Traditional Medicines, 2008, 3 ( 2 )




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