Effect of caffeic acid and chlorogenic acid in hypertension

1. Presented by:
Moh Aijaz
M.Pharm* (Pharmacology)
Presented to:
Dr. Navneet Khurana
Head of the Department &
Mr Biplab Pal
Cardio-protective and antioxidant properties of caffeic acid and
chlorogenic acid: Mechanistic role of angiotensin converting enzyme,
cholinesterase and arginase activities in cyclosporine induced
hypertensive rats

2. Table Of Content
Introduction
Material and methods
Results
Discussion
Conclusion

3. Hypertension commonly refers to as a persistent
increase in the systolic and diastolic blood pressure
Hypertension is refers to as ‘A Silent Killer”
responsible for around 9.4 million deaths worldwide
The pathogenesis of hypertension involves some
enzymes system such as-
Renin-angiotensin Converting Enzymes
System (RAS), Arginase, Cholinesterase As Well As
Oxidative Stress
Introduction

4. Types Of Hypertension
Primary (Essential) Hypertension
Elevated BP with unknown cause
90% to 95% of all cases
Secondary Hypertension
Elevated BP with a specific cause
5% to 10% in adults

5. Predisposing Factors for HTN
Genetic (Family History)
Sodium intake
Obesity (BMI > 30)
Diabetes Mellitus
Alcohol Consumption
Smoking
Serum Lipid Levels (Cholesterol and LDL)
Sedentary Lifestyle
Age
Socioeconomic Status
Stress

6. Blood Pressure Classification
BP Classification SBP mmHg DBP mmHg
Normal < 120 < 80
Pre-hypertension* 120-139 80-89
Stage 1 Hypertension 140-159 90-99
Stage 2 Hypertension > 160 > 100

9. Cyclosporine
It is a common immunosuppressive agent used in solid organ and bone marrow
transplants and the treatment of some immunological diseases
Treatment with cyclosporine can cause a patient to develop hypertension within a few
weeks of treatment
 The exact mechanism of cyclosporine-induced hypertension is unknown but several
hypotheses have been proposed, including increased prostaglandin synthesis and
decreased water, sodium, and potassium excretion

10. Role of Enzymes in Cardiac activity
 Angiotensin-converting enzyme or ACE, is a central component of the Renin-
angiotensin system (RAS), which controls blood pressure by regulating the volume
of fluids in the body
 It converts the hormone angiotensin I to the active vasoconstrictor angiotensin
II. Therefore, ACE indirectly increases blood pressure by causing blood vessels to
constrict.
Angiotensin-converting enzyme

11. Cholinesterase
 It is an esterase which is responsible for the hydrolysis of Acetylcholine
( ACh) into choline and acetic acid
 It is of two types
(i) Acetylcholinesterase (ii) Butyrylcholinesterase
ACh
Muscarinic receptors
NO from endothelial cells
Relaxation of vascular
smooth muscle
Vasodilation Blood flow rate
Activate
Release

12. Arginase
 It involves in the metabolism of arginine to urea and ornithine
 Competes directly with endothelial nitric oxide synthase
(NOS) for same substrate
NOS
Urea and Ornithine
Arginase
Arginine
Nitric oxide
(NO)
Vascular thickening
Stiffness
Vasodilation
Relaxation of SM
Increase Blood flow rate

13. Catalase
 Catalase is a common enzyme found in nearly all living organisms
 It catalyzes the decomposition of hydrogen peroxide to water and oxygen
 It protect the cell from oxidative damage by reactive oxygen species (ROS)
 It is a substance produced naturally by the liver
 It is also found in fruits, vegetables, and meats
 It is an antioxidant capable of preventing damage to important cellular
components caused by reactive oxygen species such as free radicals,
peroxides, lipid peroxides, and heavy metals
Glutathione

14. Cont….
 It is use for treating cataracts, glaucoma, preventing aging, asthma, cancer,
heart disease, liver disease, memory loss, Alzheimer’s disease, osteoarthritis,
and Parkinson’s disease.
 It is also used for maintaining the body’s defense system (immune system)
and fighting metal and drug poisoning.
 It is the organic colorless liquid compound, It occurs naturally and is a marker
for oxidative stress.
 Malondialdehyde results from lipid peroxidation of polyunsaturated fatty acids
Malondialdehyde (MDA)

15. Chemicals
Material And Methods
 Caffeic Acid
 Chlorogenic Acid
 HHL (Hippuryl-Histidylleucine)
 L-arginine
 Caffeic acid (CAA) and chlorogenic acid (CHA) are important members of
hydroxycinnamic acid with natural antioxidant and cardio-protective properties
 Useful as- Antihypertensive, as well as also useful as inhibits DNA damage,
anti-inflammatory, anti-Alzheimer’s disease and Anti-diabetic,
 Non-toxic dose range CAA:- 10 - 2437 mg/kg
CHA:- 10 - 1250 mg/kg

16. Experimental rats
 The rats were maintained at room temperature (25 °C) with
free access to food and water
 They were allowed to adapt to their new environment for 2
weeks prior to the commencement of induction and treatment.

17. Grouping
S.No Group Treatment Dose Route
1 Control Distilled water 0.2 ml Daily Oral
2 negative control Cyclosporine 25 mg/kg/day ip
3 Positive control Captopril 10 mg/kg/day Oral
4 Treatment Group Caffeic acid 10 mg/kg/day Oral
5 Treatment Group Caffeic acid 15 mg/kg/day Oral
6 Treatment Group Chlorogenic acid (10 mg/kg/day Oral
7 Treatment Group Chlorogenic acid (15 mg/kg/day Oral
n = 6
Treatment continued for 7 days

18. Hemodynamic Parameter Determination
Systolic blood pressure (SBP) and heart rates (HR) were measured by non-
invasive tail-cuff plethysmography
The animals were dissected, and blood from the inferior venacava of the heart
was collected
centrifuged at 3000g for 15 min in an MSC bench centrifuge
The clear supernatant obtained (plasma) was used in estimation of enzyme
activities and other biochemical indices

19. The tissues (lung, heart and kidney) were removed
Cont……
Rinsed in ice-cold normal saline
Blotted and weighed
Minced with scissors
Three volumes of ice-cold 50mM Tris−HCl buffer (pH 7.4)
Homogenates
Homogenized in a Teflon-glass homogenizer
The clear supernatants obtained were
used for various biochemical assays
Centrifuged for 10 min at 5000×g

20. Determination of ACE activity
The amount of cleaved hippuric acid from hippuryl-histidyl-leucine (Substrate)
was measured by the enzymatic method
50 μL of sample + 150 μL of 8.33mM of hippurylhistidylleucine
125mM Tris−HCl buffer (pH 8.3)
Incubated at 37 °C for 30 min.
250 μL of 1M HCl (Gly–His bond was then Cleaved)
Hippuric acid produced
1.5 mL ethyl acetate (Centrifuged)
Ethyl acetate layer

21. Cont….
1 mL of the ethyl acetate layer was transferred to a clean test tube
Residue
Evaporated
Distilled water
absorbance was measured at 228 nm
The plasma ACE activity was expressed as μmol HHL cleaved/min.

22. Determination of arginase activity
Arginase activity in the plasma and heart tissue was determined by
measuring the rate of urea production using α isonitrosopropriophenone (9% in
absolute ethanol)
50 μl of samples + 75 μl of Tris−HCl (50 mmol/l, pH 7.5) containing 10 mmol/ l MnCl2
Hydrolysis reaction of L-arginine by arginase
Incubate the mixture containing activated arginase with 50 μl of L-arginine
(0.5 mol/l, pH 9.7) at 37 °C for 1 h
25 μl, 9% in absolute ethanol
Heated at 100 °C for 45 min
Placed in the dark for 10 min at room temperature

23. Cont….
the urea concentration was determined spectrophotometrically by the absorbance at 550
nm
The amount of urea produced was used as an index for arginase activity.
The arginase activity was expressed as μmol urea produced/min/mg protein.

24. Determination of cholinesterase (acetylcholinesterase
and butrylcholinetrase) activity
Reaction mixture (2 ml final volume)
0.8mM (AcSCh) for acetylcholineterase assay
OR
0.8mM BcSCh was used for butrylcholineterase assay
Measured at absorbance at 412 nm
Incubated for 2 min at 25 °C.
Reaction mixture: 100mM K+-phosphate buffer, pH 7.5 and 1mM 5,5′ dithiobisnitrobenzoic
acid (DTNB)
The method is based on the formation of the yellow anion, 5, 5′- dithio-bis-acid-
nitrobenzoic,
The enzyme activities were expressed in Units/mg of protein.

25. Nitric oxide level (NOx) determination
70 μl sample, 70 μl 2% vanadium chloride (VCl3) In 5% HCl
+
70 μl of 0.1% N-(l-naphthyl) ethylenediamine dihydrochloride and 2%
sulphanilamide (in 5% HCl) in 1:1 ratio
Incubating at 37 °C for 60 min
Absorance at 540 nm
Nitrite levels determined based on the reduction of nitrate to nitrite by VCl3
Nitrite levels is correspond to an estimative level of NOx,
The nitrite and nitrate levels were expressed as nanomole of
NOx/mg protein.

26. Determination of tissue lipid peroxidation
300 μl of tissue homogenate
+
300 μl of 8.1% SDS
500 μl of Acetic acid/HCl (PH=3.4)
++
TBA (Thiobarbituric acid)
Incubated at 100 °C for 1 h.
Mixed
Thiobarbituric acid reactive species was (TBARS) produced and was
measured at 532 nm
It was calculated as Malondialdehyde (MDA) equivalent

27. Catalase (CAT) activity
Tissue + 0.1M potassium phosphate buffer (1:5 w/v)
Homogenized
Homogenate
Supernatant
centrifuged at 2000×g for 10 min
20 μl Supernatant+ 0.1M potassium phosphate buffer (pH 7.4), 10mM H2O2
Absorbance at 240 nm

28. Change in absorbance was observed due to CAT-dependent
decomposition of hydrogen peroxide.
Cont…..
The rate of H2O2 reaction Was monitored at 240 nm for 2 min at room
temperature
The enzymatic activity was expressed in units/mg protein
One unit of the enzyme is considered as the amount of CAT that decomposes 1
mmol of H2O2 per min at pH 7 at 25 °C

29. Reduced glutathione
1 ml of supernatant
3.0 ml of 0.2M phosphate buffer (pH 8.0)
500 μl of Ellman’sreagent
++
Absorbance was read at 412 nm in spectrophotometer
Ellman’s reagent :-19.8 mg of 5,5′dithiobisnitrobenzoic acid in 100 ml
of 0.1% sodium citrate

30. Results
Evaluation of the effect of CAA and CHA on SBP
and heart rate in CSA-induced hypertensive rats

31. Effect of CAA and CHA on ACE activity in
cyclosporine induced hypertensive rats

32. Effect of CAA and CHA on acetylcholinesterase (AChE)
and butrylcholinesterase (BChE) activity in CSA- induced
hypertensive rats

33. Effect of CAA and CHA on arginase activity in CSA- induced
hypertensive rats

34. Effect of CAA and CHA on nitric oxide (NOx) level in
CSA-induced hypertensive rats

35. Effect of CAA and CHA on malondialdehyde (MDA)
level in CSA induced hypertensive rats

36. Effect of on catalase activity in CAA and CHA CSA-
induced hypertensive rats

37. Effect of CAA and CHA on glutathione level in
CSA- induced hypertensive rats

38. Discussion
The CSA involvement in the cardiovascular disorders has been linked to alteration of
renin-angiotensin aldosterone system, inhibition of nitric oxide synthase , impaired renal
function as well as increased the generation of ROS
The intraperitoneal administration of cyclosporine 25 mg/kg
body weight for 7 days caused hypertension in a rat model
The observed increase in the SBP and HR in the induced-hypertensive rats
accomplished with significant increase in the activity of ACE in the plasma
coupled with significant decreased in NO level in plasma and heart these events also
affirm the hypertensive effect of cyclosporine
This study revealed that CAA and CHA exhibited blood
pressure lowering properties justified by SBP and HR lowering ability in
the hypertensive rats

39. Conclusion
This study revealed that caffeic acid and chlorogenic acid possess
anti-hypertensive properties by their blood pressure lowering Ability by reduction
in the activity of some key enzymes such as ACE, cholinesterase, arginase,
involved in the pathogenesis of hypertension as well as offer cellular prevention
against oxidative damage.