1. Antioxidant and Antihypertensive properties of Reverse-Phase HPLC Partially Purified <1kDa Permeate Hemp seed (Canabis sativa L.) Protein
Derived Peptides
Abraham T. Girgih, Nadith Gunasekara, Moses T. Ukeyima and Rotimi E. Aluko
1Department of Food Science and Technology, Federal University of Agriculture, Makurdi, Benue State
2Department of Human Nutritional Sciences and The Richardson Centre for Functional Foods and Nutraceuticals,
University of Manitoba, Winnipeg, MB R3T2N2 Canada.

2. Outline Background Objectives
Mechanism of Action of Antihypertensive Peptides
Experimental Design & Methods
Results & Discussion
Conclusions
Significance of the work
Future Direction
Acknowledgements

3. Background
Excessive production of reactive oxygen species (ROS) and free radicals beyond the body’s capacity to inactivate and remove them could be responsible for most human degenerative chronic diseases (Sharma, 2012).
Oxidative stress is essentially an imbalance between the production of free radicals and the ability of the body to counteract or detoxify their harmful effects through neutralization by antioxidants
Oxidative stress could promote abdominal inflammation, atherosclerosis, diabetes, obesity, hypertension and cancer etc. resulting from damage impacted on proteins, DNA, enzymes and cell membranes by these radical species.
Progressive oxidative damage could ultimately cause cell, tissue and organ death leading mortality.

4. Background
Hypertension is a condition of sustained increase in systolic blood
pressure (SBP) of 140 mmHg or greater and/or diastolic blood
pressure (DBP) of 90 mmHg or greater (Popescu et al., 2013).
WHO estimates that by 2020, Heart disease and Stroke will become
the leading cause of death and disability globally (Himaya et al. 2012)
Over the past three decades, Hypertension has been controlled via
the Renin-Angiotensin System using enzymatic food derived
peptides from variety of plant and animal food sources (Aluko, 2008)

5. Background
Pharmaceutical drugs such as enalapril, alacepril, lisinopril (ACE
Inhibitors) and Aliskiren (the only approved commercial renin
inhibitor) have been used to manage hypertension, however, they
are associated with undesirable side-effects
Current research is focusing on the development of food derived
peptides for use as therapeutic agents for the prevention and
management of oxidative stress, hypertension and related
morbidities.
Food based protein derived agents are abundant , cheap and with
no known side effects after prolonged usage and could be used for
the prevention and management of oxidative stress & hypertension.

6. Objectives: The objectives of this study therefore were :
i) To determine the in vitro antioxidant properties of < 1 kDa
RP-HPLC partially purified hempseed derived peptides.
ii) To determine the in vitro antihypertensive properties of
< 1 kDa RP-HPLC partially purified hempseed derived peptides
in the prevention and management hypertension.

7. Fig. 1: Experimental Design and Methods
Hempseed protein isolate (HPI)
Enzymatic hydrolysis of HPI
Hempseed Protein Hydrolysate
(HPH)
Membrane Ultrafiltration
1kDa
3kDa
5kDa
10kDa
RP-HPLC Separation
F1; F2; F3 and F4
In vitro antioxidant properties
In vitro Antihypertensive properties
DPPH radical scavenging assay
Superoxide scavenging activity
Metal chelation assay
ACE inhibition assay
Renin inhibition assay
Fig. 1: Experimental Design and Methods

8. Fig. 2: Mechanism of Action of Antioxidant Peptides
Adapted from Young and Woodside, 2001
Possible Mechanisms of Action of Bioactive Peptides as Antioxidants
Intervention
Bioactive peptides
Fe+2
Fe+3
Oxidative
Damage
SOD
o2.
H2o2
OH .
Dismutation
DNA
Cell membranes
Enzymes
Proteins
Tissue damage
2GSH
GPX
GRX
Catalase
GSSG
Development of
chronic diseases:
Diabetes
Cancer
CVD
2H20 H
Conversion to harmless products
Fig. 2: Mechanism of Action of Antioxidant Peptides

9. Figure 3: Mechanism of Action of Antihypertensive Peptides
Angiotensinogen
LIVER
Angiotensin I
Angiotensin II
Vasoconstriction
Angiotensin
Converting
Enzyme (ACE)
Bradykinin
Chymase
Renin
KIDNEY
Inactive Fragments
Increasing blood pressure
Kinin-Nitric Oxide System
(KNOS)
Plant
Bioactive Peptides
Intervention
NO production
Vasodilation
Renin-Angiotensin System
(RAS)
AT1
AT2
Mediates
BLOOD PRESSURE
HYPERTENSION
Figure 3: Mechanism of Action of Antihypertensive Peptides

10. Results and Discussion.

11. Table 1: Amino acid profile and percent yield of RP-HPLC partially purified <1 kDa permeate fractions AA
<1kDa F1 F2 F3 F4
Yield (%)
ASX
9.49 ± 0.06
10.08 ± 0.02
10.94 ± 0.03
11.36 ± 0.04
11.58 ± 0.22
21.2
THR
3.60 ± 0.04
4.02 ± 0.21
4.11 ± 0.31
4.46 ± 0.02
4.94 ± 0.03
37.2
SER
4.73 ± 0.21
6.03 ± 0.23
6.12 ± 0.25
4.90 ± 0.03
4.81± 0.01
29.4
GLX
15.18 ± 0.96
16.26 ± 0.11
16.34 ± 0.24
17.62 ± 0.04
18.75 ± 0.02
23.5
PRO
3.19 ± 0.33
4.03 ± 0.03
4.68 ± 0.04
5.83 ± 0.13
5.96 ± 0.22
86.6
GLY
3.23 ± 0.06
4.13 ± 0.14
4.83 ± 0.05
5.04 ± 0.11
5.48 ± 0.04
69.7
ALA
4.91 ± 0.06
5.82 ± 0.03
3.99 ± 0.14
3.43 ± 0.12
2.10 ± 0.02
18.5
CYS
0.29 ± 0.13
0.31± 0.24
0.45 ± 0.03
0.48 ± 0.01
0.55 ± 0.02
89.7
VAL
5.67 ± 0.14
5.35 ± 0.11
6.62 ± 0.21
6.82 ± 0.31
6.73 ± 0.22
15.8
MET
1.94 ± 0.04
2.87 ± 0.02
3.14 ± 0.04
3.76 ± 0.05
4.00 ± 0.04
100.0
ILE
4.15 ± 0.13
5.79 ± 0.22
5.85 ± 0.12
5.09 ± 0.13
5.07± 0.33
22.2
LEU
9.91 ± 0.06
10.01± 0.04
10.48 ± 0.01
11.33 ± 0.04
11.83 ± 0.12
19.2
TYR
10.11± 0.11
10.46 ± 0.21
11.50 ± 0.02
10.80 ± 0.03
13.8
PHE
4.00 ± 0.15
4.11± 0.01
4.26 ± 0.22
5.02 ± 0.01
39.4
HIS
5.00 ± 0.25
5.08 ± 0.16
5.77± 0.11
6.10 ± 0.32
29.0
LYS
15.18 ± 0.26
15.92 ± 0.31
16.08± 0.22
16.53± 0.06
16.90 ± 0.23
7.0
ARG
3.61± 0.41
3.92 ± 0.33
4.44 ± 0.05
4.72 ± 0.14
48.0
TRP
4.01± 0.30
4.08 ± 0.20
4.31± 0.03
5.87± 0.04
81.7
HAA
46.28
49.70
51.20
53.61
55.77
>100
AAA
16.32
18.12
19.65
21.53
21.69
PCAA
19.82
24.53
25.12
26.74
28.87
84.0
NCAA
34.20
24.67
27.28
28.98
30.33
44.7 .

12. A B C
Fig. 4: (A) DPPH, (B) Superoxide radicals scavenging activities and the (C) Metal chelating properties of RP-HPLC partially purified hempseed 1 kDa peptide fractions Bars with different letters are significantly different at p<0.05.

13. Results and Discussion Cont’dB
Fig. 5: (A) ACE and (B) renin inhibitory activities hempseed RP-HPLC partially purified <1 kDa permeate fractions compared to the crude unpurified sample (<1 kDa) Bars with different letters are significantly different at p<0.05.

14. Conclusions
This study has shown that when peptides are purified, they exert enhanced bioactivity than the parent samples from which they have been produced.
Hempseed RP-HPLC partially purified peptides exhibited improved radical scavenging and metal chelation activities in comparable to that GSH and better than that of <1 kDa permeate.
The partially purified hempseed fractions also showed good blood pressure lowering abilities hence they could be used as antihypertensive agents in hypertension management.
The results of this study suggest that hempseed peptide fractions could be used as therapeutic agents to reduce oxidative stress and be utilized for the formulation of functional foods and nutraceuticals.

15. Significance of the research work
The results from this study will promote the utilization of plant based protein sources for the development of novel functional foods and nutraceutical products.
Studies could encourage the production of industrial hempseed for use in health wellness
Plant peptides could complement pharmaceutical drugs for prevention and management cardiovascular and related morbidities.

16. Acknowledgements:
Professor Rotimi E. Aluko Department of Human Nutritional Sciences, University of Manitoba, Winnipeg, MB
Dr Aluko’s Lab Group and Other Co-authors
Natural Sciences and Engineering Research Council of Canada (NSERC)
Manitoba Agri-Food Research and Development Initiative
Federal University of Agriculture, Makurdi, Benue State Nigeria
Centre of Food Technology and Research, Benue State University, Makurdi,
Benue State, Nigeria