the necessity of providing evidence for the therapeutic effectivity of herbal preparations a success...

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