The necessity of providing evidence for the therapeutic effectivity of herbal
preparations A success journey for STW 5 in gastro-
intestinal disorders.
Mohamed T. KhayyalFaculty of Pharmacy, Cairo University
Herbal Remedies used by the
Ancient Egyptians
Acacia (acacia nilotica) : vermifuge , eases diarrhoea and internal bleeding, also used to treat skin diseases .
Aloe vera : worms, relieves headaches, soothes chest pains, burns, ulcers and for skin disease and allergies .
Basil (ocimum basilicum) : excellent for heart .
Balsam Apple (malus sylvestris) : laxative, skin allergies, headaches, gums and teeth, for asthma, liver stimulant, digestion.
Bayberry (Myrica cerifera) : stops diarrhoea, ulcers, haemorrhoids .
Liquorice (Glycyrrhiza glabra): laxative, expels phlegm, liver, pancreas and chest respiratory problems .
Fenugreek (Trigonella foenumgraecum): respiratory disorders, cleanses the stomach, calms the liver, soothes pancreas, reduces swelling .
Caraway (Carum carvi): flatulence, digestive, breath freshener .
Herbal Combinations
• Early in the history of use of medicinal agents, it was realized that the presence of one herb may alter the effect of the other when they are co-administered.
• The combined effect, either complementary or antagonistic, would be manifested in the clinical
outcome.
* Herb-Herb Combination for Therapeutic Enhancement and Advancement: Theory, Practice and Future PerspectivesChun-Tao Che, Zhi Jun Wang, Moses Sing Sum Chow and Christopher Wai Kei Lam Molecules 2013, 18, 5125-5141
Need for evidence based medicine
• Many herbal medicines have been developed empirically and their therapeutic usefulness proven clinically.
• Developing models for human disease conditions helps to establish evidence of mechanisms of action and therapeutic usefulness.
• Evidence increases credibility for both patients and physicians.
• Synergism between academia and industry mandatory
Shown clinically/experimentally to be a multi-target preparation for gastrointestinal disorders, such as:
• Ulcers (anti-inflammatory & ↓ hyperacidity)1
• Functional dyspepsia2
• Irritable bowel syndrome3
• DSS induced colitis4
(1) Khayyal MT et al. (2006), Phytomedicine. 2006;13 Suppl 5:56-66. (2) Schmulson MJ (2008) Nature clinical practice gastroenterology & hepatology, 5, 136-137.(3) Madisch A et al. (2004) Aliment Pharmacol Ther, 19: 271-279. (4) Wadie,W. et al. (2012) Int J Colorectal Dis. 2012 Nov;27(11):1445-53.
STW5 (Iberogast®)
STW 5 (Iberogast®, Steigerwald, Germany)
Anti-ulcerogenic activityin
Pyloric – ligated rats
Anti-ulcerogenic effect of STW5 & components
Effect of STW5 & components on gastric acidity
Effect of STW5 & components on gastric mucin
Effect of STW5 & components on PGE2 in gastric juice
Effect of STW5 & components on LTD4 in gastric juice
Conclusions
• Individual components exert effects in their own right on various parameters relevant to gastric ulcers.
• Combined effect as STW 5 gives evidence to its beneficial activity
Novel mechanisms underlying the effectiveness of STW 5 in functional
dyspepsia.
Introduction• Functional dyspepsia often correlated with subjection to
stress at some stage in life.
• Clinical evidence suggests that stress in early life followed by stress in adulthood could lead to functional dyspepsia (FD)1,2, involving derangement in gastric function.
• Present study assesses the value of a novel developed stress model for determining mechanisms involved in the effectiveness of STW 5 in FD.
• 1Kim HI et al. J Korean Med Sci 2013;28:431-7; 2Monnikes H et al. Dig Dis 2001;19:201-11
Assessment of stress in animal models: Plasma CRF
• Neonatal maternal stress (NMS)Weanling Wistar rats exposed to (NMS) by removing pups from mother cage for 3 h/day from postnatal day 2 till 21 then weaned.
• Restraint stress (RS)Adult Wistar rats subjected (RS) by confining them restrained in tightly fitting ventilated Perspex cylinders 90 min/day for 1 week.
Plasma CRF measured 24h after last subjection to stress
Stress induces ↑ plasma CRF, normalization by STW 5
Sequential model: NMS/RS
• Weanling rats exposed to NMS then weaned. Once adult, subjected to RS for 1 week.
• STW 5 (Iberogast® Steigerwald, Darmstadt, Germany) was administered orally at 2 dose levels (2 and 5 ml/Kg) for 1 week before and during exposure to RS.
• Experimentation started 24 h after last restraining session.
Parameters studied• Gastric emptying using a phenol red meal*.
• Corticosterone and ghrelin in plasma
• Stomach fundus strips tested ex-vivo for sensitivity towards carbachol, KCl, serotonin and adrenaline.
• Duodenal homogenates examined using qPCR for expression of some signalling and tight junction proteins, including CSE, RelA, Nrf-2, ZO-1, occludin.
• * Scarpignato C, et al. Arch Int Pharmacodyn Ther1980; 246: 286-94
Gastric emptying inhibited by model but normalized by STW5
Plasma corticosterone ↑model tended to be normalized by STW5
Normal NMS + RS STW 5 (2ml/kg) STW 5 (5 ml/kg)0
50
100
150
200
250
300
350
400
450
Co
rtic
ost
ero
ne
(n
g/m
l)
#
*
Plasma ghrelin raised by model hardly affected by STW5
NORMAL NMS+RS STW5(2ml/kg) STW5(5ml/kg)0
10
20
30
40
50
60
70
Gh
relin
co
nc.
(fm
ol/m
l)
Stress induced ↓ fundus sensitivity to carbachol/amelioration by STW5
Stress induced ↓ fundus sensitivity to KCl amelioration by STW5
Stress induced ↓ fundus sensitivity to serotonin / improvement by STW5
Stress induced ↓ fundus sensitivity to adrenaline improvement by STW5
Stress ↓duodenal ZO1, RelA, Nrf2 & ↑Occludin, CSE expression/ partial improvement by STW5
Summary & Conclusions
• Stress model led to marked delay in gastric emptying, raised plasma level of active ghrelin and corticosterone, but reduced expression of Nrf-2 and ZO-1 and raised occludin in duodenal homogenates: changes normalized by STW5.
• Stress markedly reduced gastric fundus sensitivity of parasympathetic, sympathetic and serotonergic receptors.
• Since deranged gastric functions (emptying, fundus sensitivity) are among symptoms of FD, present findings help to elucidate mechanisms underlying therapeutic usefulness of STW 5 in that condition.
• Sequential stress offers a new model for studying agents with potential usefulness in FD
Novel Therapeutic Potential for STW 5: Prophylactic Measure against Intestinal
Mucositis induced by Radiation
Background
• Exposure to radiotherapy often leads to mucositis of intestinal epithelium, possibly as a result of release of ROS and induction of apoptosis. This severely limits continuation of treatment.
• Treatment of mucositis usually carried out with agents with anti-inflammatory properties. So far, no standard medication has proved effective in preventing mucositis.
• STW 5 was studied for its potential usefulness.
Induction & Assessment of Mucositis
• Male Wistar rats exposed to whole body gamma radiation levels of 4, 6 and 8 Gy from Cs 137 source.
• Animals sacrificed 3 days after exposure, segments of intestine examined histologically.
• Intestinal homogenates and serum examined for parameters of apoptosis, inflammation and oxidative stress.
Rats were divided into the following groups
Non-irradiated control Acute Irradiated
4 Gy 6 Gy 8 Gy
STW 5 + 8 Gy
STW 5 (2, 5 or 10 ml/kg)
orally for 5 days before and 2 days after radiation exposure. Rats sacrificed one day later.
Experimental Design
At Sacrifice
• Segments of small intestine examined histologically.
• Intestinal homogenates and serum samples
used to assess relevant parameters for:
a) mucosal damage and apoptosis
b) inflammation
c) oxidative stress.
Histological Results
STW 5 inhibits radiation induced histological changes dose-dependently
normal 8 Gy : shortened villi, activated mucous glands, apoptotic
bodies.
STW 5 (2 ml/kg):Short villi, activation of muc.
glands, inflam. cell infiltration
STW 5 (5ml/kg):Epith. denudation, few inflam.cell
infilt. activation of muc. glands.
STW 5 (10 ml/kg): very few histological changes
Semi-quantitative histological assessment of intestinal damage
A total score for each proximal jejunal specimen was derived from the sum of scores for 9 histological criteria.
1) number of apoptotic bodies
2) villus fusion and stunting (atrophy)
3) epithelial denudation and erosion
4 ) activation of glandular epithelium
5) reduction in goblet cell number
6) activation of nuclei of enterocytes
7) inflammatory cell infiltration
8) oedema
9) haemorrhage in lamina propria
Each histological variable was scored from 0 (normal) to 3 (maximal damage) for each rat intestine.
Acute irradiation induced intestinal mucosal damage
Normal 4 Gy 6 Gy 8 Gy
0
5
10
15
20
25
Ove
rall
scor
e of
dam
age
seve
rity
STW 5 dose dependently suppresses radiation-induced mucosal damage
Concluding Remarks
• Acute exposure to radiation dose levels of 4, 6, and 8 Gy produced graded extents of intestinal mucositis as judged by light and elecron microscopy with evidence of apoptotic cells with the highest exposure level.
• STW 5 in a dose of 10 ml/kg was most effective in reducing the severity of degenerative changes observed after acute irradiation.
Markers for Mucosal Damage
• LDH in serum
• Diamine oxidase in serum
• TNF α in intestinal homogenates
Radiation induces release of LDH in serum
0
50
100
150
200
250
300
350
400
LDH
seru
m a
ctivi
ty (U
/l)
*
*
Non-irradiated control
8 Gy acute irradiation
6 Gy acute irradiation
4 Gy acute irradiation
Significantly different at P≤0.05 from control group (*)
STW 5 protects against rise in LDH in Serum
0
50
100
150
200
250
300
350
400
450
500
STW 5 (5 ml/kg) treatment
STW 5 (10 ml/kg) treatment
8 Gy acute irradiation Non-irradiated control
DAO
seru
m le
vel (
pg/m
l) * #
* #
*
Significantly different at P≤0.05 from control group (*) or from irradiated group (#)
STW 5 suppresses radiation induced rise in serum diamine oxidase (DAO)
0
50
100
150
200
250
300
TNF-
α c
onte
nt (p
g/g
wet
tiss
ue)
* #
* #
*
Non-irradiated control 8 Gy acute irradiation
STW 5 (5 ml/kg) treatment
STW 5 (10 ml/kg) treatment
Significantly different at P≤0.05 from control group (*) or from irradiated group (#)
STW 5 suppresses radiation induced rise in intestinal TNF-α
Apoptotic markers
- Cytosolic calcium content
- Mitochondrial cytochrome c (pro-apoptotic protein)
- Mitochondrial B-cell lymphoma-2 (Bcl-2) (anti-apoptotic protein)
- Mitochondrial respiratory chain complex I activity.
Radiation induces release of cytosolic Calcium in Intestinal Tissue
0
50
100
150
200
250
300
Calc
ium
con
tent
in c
ytos
ol (
µg/g
wet
tiss
ue)
Non-irradiated control
8 Gy acute irradiation
4 Gy acute irradiation
6 Gy acute irradiation
*
*
Significantly different at P≤0.05 from control group (*)
STW 5 prevents radiation induced rise of cytosolic Calcium
STW 5 supresses radiation induced depletion in mitochondrial cytochrome c
0
100
200
300
400
500
600
700
800
900
1000
Cyto
chro
me
c co
nten
t (pg
/mg
mito
chon
dria
)
Non-irradiated control
8 Gy acute irradiation
STW 5 (5 ml/kg) treat-ment
*
* #
* # STW 5 (10 ml/kg) treatment
Significantly different at P≤0.05 from control group (*) or from irradiated group (#)
0
5
10
15
20
25
30
35
40
45
50 Non-irradiated control
8 Gy acute irradiation
STW 5 (5 ml/kg) treatment
STW (10 ml/kg) treatment
*
Bcl-2
con
tent
(pg
/mg
mito
chon
dria
)
* #
* #
Significantly different at P≤0.05 from control group (*) or from irradiated group (#)
STW 5 supresses radiation induced depletion in mitochondrial Bcl-2
0
10
20
30
40
50
60
70
80
90
100
Com
plex
I ac
tivity
(nm
ol/m
g m
itoch
ondr
ia)
Non-irradiated control
8 Gy acute irradiation
STW 5 (10 ml/kg) treatment
STW 5 (5 ml/kg) treatment
* #*
#
*
Significantly different at P≤0.05 from control group (*) or from irradiated group (#)
STW 5 supresses radiation induced depletion in mitochondrial complex I
Oxidative stress biomarkers
- Reduced glutathione (GSH) content in intestinal homogenate
- Malondialdehyde (MDA) content in intestinal homogenate
0
10
20
30
40
50
60
GSH
cont
ent (
ug/g
wet
tiss
ue)
* #
* #
*
Non-irradiated control
8 Gy acute irradiation
STW 5 (5 ml/kg) treatment
STW 5 (10 ml/kg) treatment
Significantly different at P≤0.05 from control group (*) or from irradiated group (#)
STW 5 suppresses radiation induced depletion in intestinal GSH
0
5
10
15
20
25
30
MDA
con
tent
(nm
ol/g
wet
tiss
ue)
* # *
#
*
Non-irradiated control
8 Gy acute irradiation
STW 5 (5 ml/kg) treatment
STW 5 (10 ml/kg) treatment
Significantly different at P≤0.05 from control group (*) or from irradiated group (#)
STW 5 suppresses radiation induced rise in intestinal MDA
Summary & Conclusions
STW 5 protected to a large extent in a dose dependent manner against histological changes induced by gamma radiation and counteracted to different extents the derangement in all the relevant parameters measured suggesting that it may be useful as an adjuvant during radiotherapy to reduce the tendency to develop radiation enteritis.
Effects of STW 5 and STW 5-II in dextran sodium sulfate-induced
colitis
How does STW 5-II differ from STW 5
Iberis amara15%
tonicisinganti-inflammatory
Liquorice
10%
spasmolyticanti-inflammatory
Lemon balm
15%
spasmolyticanti-inflammatory
Chamomille30%spasmolyticanti-inflammatory
Caraway20%
spasmolyticbacteriostatic
Peppermint10%
spasmolytic,anti-emetic
Inflammatory Bowel Disease (IBD)
• 1. Crohn‘s Disease: STW 5 found effective in TNBS induced colitis.
Abdel-Aziz H, Wadie W, Abdallah DM, Vinson B, Kelber O, Weiser D, Khayyal MT.
Z.Gastroenterol. 2008, 46, 362
• 2. Ulcerative Colitis: STW 5 found effective in DSS induced colitis.
Wadie W, Abdel-Aziz H, Zaki HF, Kelber O, Weiser D, Khayyal MT (2012)
Int.J.Colorectal Dis. 27, 1445 – 1453
Experimental Design• Male Wistar rats (n=7-9) administered 5% Dextran Sodium
Sufate (DSS) in drinking water. Lesions in the colon develop within 7 days.
• Drugs given orally for 1 week before starting adding DSS in the drinking water and continued for a further week.
• Rats sacrificed. Colon length and weight recorded. The colon was cut longitudinally into 2 segments. One was examined histologically, and the other homogenized and tested for various relevant parameters.
Normal 5% DSS Sulfasalazine STW5 (2ml/kg) STW 5 (5 ml/kg)0
5
10
15
20
25
30
35
Bo
dy
Wei
gh
t in
crea
se (
g)
#
#
@@
STW5 improved abnormal body weight changes.
STW 5 II prevents body weight loss
Normal 5% DSS STW 5 II (2 ml/kg)0
5
10
15
20
25
30
35
40
45
Bo
dy
Wei
gh
t in
crea
se (
g)
#
@
Normal 5% DSS Sulfasalazine STW5 (2ml/kg) STW 5 (5 ml/kg)10
11
12
13
14
15
16
17
18
Co
lon
Len
gth
(cm
)
#
@@ @
STW5 prevents DSS induced colon shortening.
STW 5 II tends to prevent colon shortening
Normal 5% DSS STW 5 II (2 ml/kg)0
5
10
15
20
25
Co
lon
Len
gth
(cm
)
## @
Normal 5% DSS Sulfasalazine STW5 (2ml/kg) STW 5 (5 ml/kg)0
20
40
60
80
100
120
140
Co
lon
Wt
(mg
) /
colo
n l
eng
th (
cm)
#
##
# @
STW5 protects against increase in colon weight (oedema formation).
STW 5 II protects against rise in colon mass index
Normal 5% DSS STW 5 II (2 ml/kg)0
1
2
3
4
5
6
7
8
9
Co
lon
Mas
s In
dex
(m
g/g
)
#
STW5 guards against rise in myeloperoxidase activity induced by DSS (measure of neutrophilic infiltration).
Normal 5% DSS Sulfasalazine STW5 (2ml/kg) STW 5 (5 ml/kg)0
1
2
3
4
5
6
7
Tis
sue
Mye
lop
ero
xid
ase
(U/g
)
#
# @
# @
# @
STW 5 II prevents rise in MPO
Normal 5% DSS STW 5 II (2 ml/kg)0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
MP
O [U
/g]
#
# @
Normal 5% DSS Sulfasalazine STW5 (2ml/kg) STW 5 (5 ml/kg)0
200
400
600
800
1000
1200
Re
du
ce
d G
luta
thio
ne
(n
mo
l/g
)
STW 5 prevents DSS induced decrease in glutathione levels.
STW 5 II prevents DSS induced fall in GSH levels
Normal 5% DSS STW 5 II (2 ml/kg)0
200
400
600
800
1000
1200
GS
H [
nm
ol/g
]
#
#
STW5 largely prevents DSS induced fall in glutathione peroxidase
Normal 5% DSS Sulfasalazine STW5 (2ml/kg) STW 5 (5 ml/kg)0
100
200
300
400
500
600
700
800
900
Glu
tath
ion
e P
ero
xid
as
e (
mU
/g)
#
# @ # @
# @
STW 5 II protects against DSS induced fall in GPx
Normal 5% DSS STW 5 II (2 ml/kg)0
100
200
300
400
500
600
700
800
900
1000
GP
x [m
U/g
]
#
STW5 completely prevents DSS induced reduction of SOD levels.
Normal 5% DSS Sulfasalazine STW5 (2ml/kg) STW 5 (5 ml/kg)0
500
1000
1500
2000
2500
Su
pe
rox
ide
Dis
mu
tas
e (
U/g
)
@
@
@
#
STW 5 II prevents DSS induced fall in colonic SOD levels
Normal 5% DSS STW 5 II (2 ml/kg)0
500
1000
1500
2000
2500
3000
SO
D [
U/g
]
#
Normal 5% DSS Sulfasalazine STW5 (2ml/kg) STW 5 (5 ml/kg)200
250
300
350
400
450
500
550
TN
F-α
(p
g/g
)
#
@@
@
STW5 completely protects against DSS induced rise in TNF-α.
Histological Examination
DSS induced following changes:• marked necrosis of the epithelium• intra-luminal accumulation of mucous exudates• sub-mucosal oedema• massive inflammatory cell infiltration in the lamina
propria and sub-mucosa. The infiltrated inflammatory cells included polymorphonuclear leucocytes, lymphocytes and plasma cells.
• Treatment with either STW 5 or STW 5 II largely prevented the above changes
Histology Scores after treatment with STW 5 or STW 5 II
Conclusions I• DSS induced marked colitis, as evidenced by changes in
both histological and biochemical parameters.
• Earlier studies showed that treatment with either STW 5 or sulfasalazine was effective in preventing such changes (Abdel-Aziz H et al. Gastroenterology 2011; 14: S-608).
• STW 5 II was developed to contain only 6 of the original components of STW 5 but with modified concentrations.
• Present findings show that STW 5 II in a dose of 2 ml/kg was as effective as STW 5 in protecting against DSS-induced changes in both histological and biochemical parameters.
• .•
Conclusions II
The results point to the good anti-oxidant and anti-inflammatory properties of STW 5 II, imaging those of STW 5.
This lends support to its potential therapeutic usefulness in inflammatory conditions of the GIT, such as ulcerative colitis.
Final concluding remarks• Experimental models help to establish credibility
for therapeutic usefulness of herbal combinations for both patients and physicians.
• The models may help to develop novel therapeutic applications for established preparations.
• Close interaction between academia and industry is imperative for better development of herbal medications.
Thank you for your kind attention
STW 5-II
STW5