This study investigated the anti-inflammatory effects of extracts and compounds isolated from Hypericum perforatum and Hypericum gentianoides in RAW 264.7 macrophages. The ethanol extracts of both plants significantly reduced LPS-induced production of PGE2 and NO. Fractionation of the extracts identified several active fractions containing compounds like pseudohypericin, quercetin, and acylphloroglucinols. The most potent fractions from H. perforatum and H. gentianoides inhibited PGE2 by over 67% and 93%, and NO by over 85% and 76%, respectively. Uliginosin A accounted for some of the anti-inflammatory activity in H. gentianoides extract and its active fractions.

1. Studies of The
Immunostimulant Effect of
Hypericum Perforatum
Hatice Kübra Tamer

2. Hypericum perforatum (St. John’s wort) as an antidepressant
supplement (serotonin reuptake inhibitor).
H. perforatum possesses anti-inflammatory and anti-viral
activity
H. perforatum ethanol extract inhibited LPS-induced PGE2
and nitric oxide (NO) production in RAW 264.7
macrophages, and attributed part of the activity of a highly
active fraction of the extract to a group of four compounds
including pseudohypericin (1), quercetin (2),
amentoflavone (3),
and chlorogenic acid (4)

3. Anthrone
Naphthodianthrone

4. Flavonoid:
a 15-carbon
skeleton, containing
2 benzene rings
connected by a 3-
carbon linking
chain.

5. Flavonoid:
a 15-carbon skeleton,
containing 2 benzene
rings connected by a
3-carbon linking
chain.

6. phenolic acid

7. None of the extracts, fractions, or pure compounds reduced cell viability with treatments at the
maximum concentrations, so anti-inflammatory activity was not compromised by cytotoxicity

8. Extracts made from different Hypericum species and accessions were applied to RAW 264.7 macrophages at
10 lg/mL. All extracts significantly reduced LPS-induced PGE2 and NO production.
inhibited both
PGE2 and NO by
more than 30%
compared to the
vehicle control.

9. In the current study,
a DMSO vehicle
control was applied,
H. perforatum
extract, the 4
compounds (1–4) or
pseudohypericin (1)
alone
The extract has stronger
decreasing effect on
PGE2 and NO than
compounds (1–4). ).

10. The later inhibited TNF-a in the cell line and the primary cells, while the extract only decreased
tumor necrosis factor (TNF)-a in peritoneal macrophages
Interleukin (IL)-1b was inhibited by the extract and compounds 1–4 to a similar degree.
Pseudohypericin (1) by itself was only able to mildly lower PGE2 and NO in RAW cells, as well
as NO and TNF-a in the peritoneal macrophages.
Quercetin (2) as a positive control at 10 lM, significantly decreased all inflammatory mediators
in both cell types

11. The extract was subsequently fractionated into eleven fractions according to the peaks at different
retention time using semipreparative HPLC, as shown.

12. The eleven fractions made from H. perforatum extract were applied to RAW 264.7 macrophages at
concentrations in proportion to their yield.
Table 3
LPS-stimulated PGE2 and NO production by RAW 264.7 mouse macrophages treated
with fractions of H. perforatum extract.
Treatment Concentration Inflammatory mediator production
PGE2(ng/mL) NO (lM)
Media + DMSO – 4.3 ± 0.2 21.5 ± 1.3
H. perf extract 58.9 lg/mL 1.4 ± 0.3** 8.4 ± 0.4**
Fractions
1 33.2 lg/mL 1.1 ± 0.2** 7.1 ± 0.6**
2 29.7 lg/mL 1.5 ± 0.1** 8.6 ± 0.6**
3 6.7 lg/mL 2.5 ± 0.4** 11.4 ± 1.3**
4 1.5 lg/mL 2.2 ± 0.2** 10.3 ± 1.1**
5 44.7 lg/mL 0.8 ± 0.1** 7.3 ± 0.6**
6 16.5 lg/mL 1.4 ± 0.1** 9.5 ± 0.4**
7 6.7 lg/mL 1.7 ± 0.2** 11.8 ± 0.6**
8 10.5 lg/mL 3.1 ± 0.2* 14.2 ± 0.6**
9 2.1 lg/mL 3.0 ± 0.2* 15.7 ± 0.9*
10 23.5 lg/mL 1.9 ± 0.1** 10.5 ± 1.3**
11 12.6 lg/mL 1.2 ± 0.2** 7.3 ± 1.1**
Quercetin (2) 10 lM 0.8±0.0** 6.4±1.0**
All fractions
significantly inhibited
LPS-induced PGE2and
NO production.
DECREASİNG
DECREASİNG
Had the most potent
inhibitory activity at
relatively lower
concentrations, all
decreased PGE2 and
NO by more than 40%
compared to vehicle
control

13. In order to see whether the fractions were lowering the inflammatory mediators in a dose-
dependent manner, the cells were treated with the selected fractions 6, 7 and 11 at 1, 5, and
15 lg/mL. Figure below indicates that the inhibition of both inflammatory mediators by
these three fractions become stronger with higher dose.
All three
fractions
inhibited NO
at as low as 1
lg/ mL, with
fraction 11
also able to
significantly
decrease
PGE2 at this
concentration
At 15 lg/mL,
fraction 11 was
the most potent
inhibitor of
PGE2 and NO
among the
three, inhibiting
PGE2 by 67%
and NO by 85%.

14. Major known compounds identified in the fractions are listed below the corresponding fractions.
Pseudohypericin (compound 1) was found in all three active fractions, while rutin (5) and
hyperoside (6) were found in two of them.

15. LC–MS chromatogram of a H. gentianoides ethanol extract highlights a distinctive group of acylphloroglucinols
including uliginosin A (9), saroaspidin A (10), and hyperbrasilol A (11). This extract also contains typical
Hypericum constituents such as chlorogenic acid (4) and quercetin (2) and isoquercetin.
Phloroglucinol is a
spasmolytic agent to treat
colic, as well as spastic pain of
the digestive and biliary
tracts.

16. The ethanol extract of H. gentianoides was fractionated into ten fractions based on semi-preparative
HPLC detection peaks at different retention time, as shown in figure.

17. All ten fractions made from H. gentianoides extract were applied to RAW 264.7 macrophages at
concentrations proportional to their yield.
Table4
LPS-stimulated PGE2 and NO production byRAW264.7 mousemacrophages treated
with fractions ofH.gentianoides extract.
Treatment Concentration Inflammatory mediator production
PGE2(ng/mL) NO (lM)
Media+DMSO – 2.7±0.4 15.5 ±1.3
H. gentianoidesextract 48.5 1.0±0.1** 9.9±0.5**
Fractions
1 176lg/mL 2.7±0.4 9.1 ±1.6**
2 75.1 lg/mL 1.7±0.2** 7.3±0.9**
3 25.5lg/mL 1.5±0.2** 5.6±0.3**
4 13.4lg/mL 1.4±0.2** 13.8±0.8
5 6.1 lg/mL 2.0±0.4* 6.8±1.4**
6 8.6lg/mL 1.7±0.3** 6.7±1.4**
7 13.7lg/mL 1.6±0.2** 7.1 ± 1.1**
8 10.8 lg/mL 0.2±0.0** 3.7±0.1**
9 9.3lg/mL 1.5±0.1** 6.8±0.9**
10
Quercetin (2)
6.8lg/mL
10 lM
2.6±0.5
0.6±0.1**
12.2 ± 1.9*
5.7± 0.6**
All but fractions 1
and 10 significantly
inhibited LPS-
induced PGE2 at the
tested
concentrations.
Relatively stronger
PGE2 inhibitory
activity at
concentrations less
than 15 lg/mL
With regards to NO,
fraction 4 was the only one
that did not decrease NO
production.

18. Table4
LPS-stimulated PGE2 and NO production byRAW264.7 mousemacrophages treated
with fractions ofH. gentianoides extract.
Treatment Concentration Inflammatory mediator production
PGE2(ng/mL) NO (lM)
Media+ DMSO – 2.7±0.4 15.5 ± 1.3
H. gentianoidesextract 48.5 1.0±0.1** 9.9±0.5**
Fractions
1 176 lg/mL 2.7±0.4 9.1 ±1.6**
2 75.1 lg/mL 1.7±0.2** 7.3±0.9**
3 25.5lg/mL 1.5±0.2** 5.6±0.3**
4 13.4lg/mL 1.4±0.2** 13.8±0.8
5 6.1 lg/mL 2.0±0.4* 6.8±1.4**
6 8.6lg/mL 1.7±0.3** 6.7±1.4**
7 13.7lg/mL 1.6±0.2** 7.1 ± 1.1**
8 10.8 lg/mL 0.2±0.0** 3.7±0.1**
9 9.3lg/mL 1.5±0.1** 6.8±0.9**
10
Quercetin (2)
6.8lg/mL
10 lM
2.6±0.5
0.6±0.1**
12.2 ± 1.9*
5.7± 0.6**
All ten fractions made from H. gentianoides extract were applied to RAW 264.7 macrophages at
concentrations proportional to their yield.
Inhibited NO by
>50% at below 15
lg/mL concen-
trations.
Fraction 8 was the
apparent most
potent fraction by
inhibiting PGE2 by
93% and NO by 76%.

19. Fractions 5, 8 and 9 at 1, 5, and 15 lg/mL were applied to cells to establish dose–response relationship
The fraction 8 was
the only one
among the three
that could inhibit
PGE2 at 1 lg/mL
and NO at 5
lg/mL.
All three
fractions
inhibited PGE2
and NO in a
dose dependent
fashion.
Fractions 8 and 9
contained
acylphloroglucinols,
while fraction 5
contained
chlorogenic acid (4)
and quercetin

20. The concentrations of uliginosin A (9), the most abundant of the acylphloroglucinols in H. gentianoides, were
quantified in the H. gentianoides extract, and fractions 5, 8 and 9 of the extract. RAW 264.7 macrophages
were treated with the plant materials or pure uliginosin A (9) at the concentration as it was in the
extract and fractions. The resulting inhibition in PGE2 and NO are shown in the Table
Table 5
LPS-stimulated inflammatory mediator and cytokine release by
RAW 264.7 mouse macrophages treated with H. gentianoides
extract, fractions and uliginosin A.
Treatments Concentration Levels
in supernatant (mean ± SEM)
PGE2
ng/mL
NO
lM
TNF-a
ng/mL
IL-1b
pg/mL
Media + DMSO
Media + DMSO + LPS
H. gentianoides extract 48.5 lg/mL
0.1 ± 0.0
4.9 ± 0.1a
1.2 ± 0.3c
0.05 ± 0.1
15.6 ± 0.6a
10.5 ±0.4c
0.8 ± 0.2
12.3 ± 1.7a
8.1 ± 0.5c
69 ± 9
572 ± 23a
305 ± 31c
Fraction
5 6.1 lg/mL 3.5 ± 0.1b 8.7 ± 0.2c 11.4 ± 0.2 393 ± 22c
8 10.8 lg/mL
9 9.3 lg/mL
UliginosinA(9) 0.6 lM
0.04 lM
2.0lM
2.6lM
Quercetin(2) 10 lM
0.4 ± 0.1c
2.7 ± 0.2c
4.5 ± 0.2
4.9 ± 0.2
2.0 ±2.1c
1.8 ±0.3c
0.9 ± 0.1c
6.3 ± 0.3c
8.3 ± 0.3c
9.8 ± 0.6c
14.8 ± 0.6
8.2 ± 0.2c
7.7 ±0.2c
4.0 ±0.3c
9.3 ± 0.4c
10.5 ±0.3b
10.4 ± 0.3b
11.8 ± 0.1
10.1 ± 0.2b
10.8 ± 0.1b
5.9 ± 0.5c
248 ±42c
436 ±29c
438 ±26c
530 ± 19
347 ± 47c
332 ± 37c
399 ± 33c
The abundance of
uliginosin A (9) in
48.5 lg/mL H.
gentianoides
extract was 0.6 lM.
At concentrations
used here,
fractions 5, 8 and
9 contained 0.04,
2.0, and 2.6 lM
uliginosin A (9),
respectively.

21. In general, uliginosin A
(9) accounted for a part
of the inhibitory activity
of the extract and
fraction 8. The trace
amount of uliginosin A
(9) did not reduce the
release of any
inflammatory mediator
or cytokine.
Table 5
LPS-stimulated inflammatory mediator and cytokine release by
RAW 264.7 mouse macrophages treated with H. gentianoides
extract, fractions and uliginosin A.
Treatments Concentration Levels
in supernatant (mean ± SEM)
PGE2
ng/mL
NO
lM
TNF-a
ng/mL
IL-1b
pg/mL
Media + DMSO
Media + DMSO + LPS
H. gentianoides extract 48.5 lg/mL
0.1 ± 0.0
4.9 ± 0.1a
1.2 ± 0.3c
0.05 ± 0.1
15.6 ± 0.6a
10.5 ±0.4c
0.8 ± 0.2
12.3 ± 1.7a
8.1 ± 0.5c
69 ± 9
572 ± 23a
305 ± 31c
Fraction
5 6.1 lg/mL 3.5 ± 0.1b 8.7 ± 0.2c 11.4 ± 0.2 393 ± 22c
8 10.8 lg/mL
9 9.3 lg/mL
UliginosinA(9) 0.6 lM
0.04 lM
2.0lM
2.6lM
Quercetin(2) 10 lM
0.4 ± 0.1c
2.7 ± 0.2c
4.5 ± 0.2
4.9 ± 0.2
2.0 ±2.1c
1.8 ±0.3c
0.9 ± 0.1c
6.3 ± 0.3c
8.3 ± 0.3c
9.8 ± 0.6c
14.8 ± 0.6
8.2 ± 0.2c
7.7 ±0.2c
4.0 ±0.3c
9.3 ± 0.4c
10.5 ±0.3b
10.4 ± 0.3b
11.8 ± 0.1
10.1 ± 0.2b
10.8 ± 0.1b
5.9 ± 0.5c
248 ±42c
436 ±29c
438 ±26c
530 ± 19
347 ± 47c
332 ± 37c
399 ± 33c
PGE2 was inhibited by
uliginosin A (9) only at
2.0 and 2.6 lM, while
NO, TNF-a and IL-1b
were inhibited at 0.6
lM as well.

22. https://doi.org/10.1016/j.phytochem.2011.07.016