Medical nutrition therapy

1. Hepatoprotective Effect of Ginger and
Cardamom Extracts Against Diet Induced
NAFLD (Non-alcoholic Fatty Liver Disease) in
Rat Models
MUHAMMAD AWAIS
2011-AG-4268
1

2. Outline
■ Introduction
■ Types
■ Causes
■ Pathogenesis
■ Biomarkers
■ Ginger and cardamom therapeutics
■ Objectives
■ Material and methods
■ Results and discussion
■ Conclusion
■ References 2

3. ■ NAFLD is the buildup of extra fats (TG,CHOLESTEROL) in the liver
■ not taking alcohol and other hepatotoxic drugs or toxins
(Zivkovic et al., 2007)
Healthy Liver Cirrhosis (Type-4 NAFLD)
Introduction
3

4. Types
■ On the basis of state and impairment of liver functioning the NAFLD has
variable types
– Type-1 NAFLD : only accumulation of fats (simple steatosis)
– Type-2 NAFLD : steatosis + inflammation of hepatocytes
– Type-3 NAFLD : steatosis + damage of hepatocytes
– Type- 4 NAFLD: steatosis + fibrosis and mallory bodies
4

5. Causes
■ Obesity, diabetes and metabolic syndrome
■ Poor consumption of antioxidants (vitamin C, E, poly unsaturated fatty acids
(PUFAs) and dietary fiber )
■ High energy diets (high carb especially fructose and high fat diets)
– Elevate the triglycerides level in blood and adipose tissues
■ Alteration in intestinal micro biota (IM)
(Pollock et al., 2012;)
5

6. Pathogenesis
■ Theories:
– Insulin resistance
– “Two-Hit” Hypothesis
– Gut-Liver Axis
(vos et al., 2013)
6

7. Two Hit Hypothesis
7

8. Gut-Liver Axis
8

9. Biomarkers
■ Biochemical analysis
– serum liver enzymes (ALT, AST, ALP)
– Insulin resistance
– Tumor necrosis factor alpha (TNF-α)
– Liver index (liver weight/ body weight)
■ Non destructive methods Imaging techniques used for detection of fatty
liver
– Ultrasonography
– Tomography
– Nuclear magnetic resonance (NMR)
■ Destructive method
– Liver histopathology
■ Noble destructive method for the assessment NAFLD
■ Also characterized the phases of the NASH 9

10. Treatment
■ There is no specific cure for NAFLD at present
■ controlled by handling the possible causative agents e.g. insulin
resistance, obesity etc.
■ DYSLIPIDIMIA , INSULINE RESISTANCE , OXIDATIVE STRESS , INFLAMATION
■ NAFLD prevented by suppressing the key biomarker of NAFLD
■ People preferred the traditional medicine over modern medicine
– Due to least side effect , low cost and more efficiency
(Schwenger et al., 2014; Khosravani et al., 2016))
10

11. Ginger and cardamom therapeutic role
■ Ginger (Zingiber officinale) and cardamom (Elettaria cardamomum)
■ Both relates to family Zingiberaceae
■ They are well assessed in many researches:
– To cure the different diseases e.g. cardiovascular disease, liver
disorders, cancer inflammatory disorders and diabetes
Bioactive components
Ginger Cardamom
Gingerols
Paradols
Shogaols
Zingerone
Indol-3-carbinol
Phenolic acids
Tocopherol
Volatile compound
(Aboubakr and Abdelazem; Lai et al., 2016)
11

12. ■ Dyslipidemia : Lowered the serum lipids by escalating the function of liver enzyme
alpha-hydroxylase
■ Insulin resistance Gingerol improved insulin sensitivity in adipocytes and enhanced
insulin secretion
– Amplify protein levels in GLUT-4 and receptors of insulin
– Increase in function of beta cells
– Glycated hemoglobin
■ Oxidative stress : Inhibition of free radicals production by stimulation the
antioxidant enzymes activity
– Inhibit of lipid peroxidation
– reduced cellular damage in both liver and pancreatic cells
■ Inflammation : Reduced the inflammation by targeting inflammatory cytokines and
free radicals
– Down-regulated the gene expression for TNF-α
(Henriksen, 2006; Stoilova Et Al., 2007; Lee Et Al., 2009; Rahimlou Et Al., 2016)
Ginger targets
12

13. Cardamom targets
■ Inflammation : Activation of NF-κB is reversed
■ oxidative stress : Strengthen antioxidant defense mechanism by
enhancing antioxidant enzymes:
– Glutathione-S transferase (GST), superoxide dismutase (SOD),
glutathione peroxidase (GPx) and glutathione reductase
■ Dyslipidemia Reduce the ALT, AST, ALP, TG, LDL-C By Increasing the
lipoprotein lipase activity
(Aboubakr and Abdelazem,
2016)
13

14. Objectives
■ Under the knowledge of above featured benefits of ginger and
cardamom extracts, the research study was planned to target the
following stated objectives:
– To determine the anti-oxidant and hepato-protective features of
ginger and cardamom
– To assess the synergistic effects of cardamom and ginger against
NAFLD
14

15. Material and methods
Procurement of
raw material
Extraction
Characterization
of sample
Preparation of
experimental
diets
Animal model
and Induction of
NAFLD
Treatment plan
Biological
assessment
Statistical
analysis
15

16. Procurement of raw material
■ Research work was done in the National Institute of Food Science and Technology,
University of Agriculture, Faisalabad
■ For the purpose, ginger rhizome and dry cardamom were procured from local
market.
Extraction of raw material
■ 500g ginger rhizome and dry cardamom were cleaned and grounded into powdered
form
■ Mix the ginger and cardamom powder with 70% ethanol by 3:1 ratio
■ Ethanol extraction was done by flask and shake method
■ Extracts were purified by evaporation of ethanol by using rotary evaporator
16

17. Phytochemical characterization of extracts
Phenolics , flavonoids and antioxidant activity of Both extracts were determined
spectrophotometrically by following methods:
■ Total phenolic content by Folin-ciocalteu (FC) reagent assay
■ Total flavonoid contents by Dowd method
■ Free radical scavenging activity by DPPH assay
(Atoui et al., 2005 ;Ghosh et al., 2016)
17

18. Experimental diets
■ Normal diet : 6% fat, 17% protein ,77 %
carbohydrates
■ High fat diet : 55% fat, 8% protein, 37%
carbohydrates
Animal model and Induction of
NAFLD
■ 25 rats were acquired in animal room
of NIFSAT(UAF)
■ acclimatized by providing water ad
libitum and regular diet for a period
of 1 week
■ Induction of NAFLD was done by
high fat diet (>50% fat of total
energy)
(Qamar et al., 2016)
Composition of Diets g/100g
Ingredient Normal diet High fat diet
Wheat flour 65 g 45.5 g
Chickpea flour 20 g 14 g
Milk powder 15 g 10.5 g
Fat ---- 30 g *
Energy 360.5 kcal 522.4 kcal
* Animal fat 20g, vegetable fat 10g
18

19. Groups Description Treatments
G0 Normal Normal Diet
G1 Control High Fat Diet
G2 Ginger Fed High Fat Diet + Ginger Extract
G3 Cardamom Fed High Fat Diet + Cardamom Extract
G4 Ginger +
Cardamom Fed
High Fat Diet + Ginger Extract +
Cardamom Extract
Treatment plan
• Five groups G0, G1, G2, G3, and G4
• Five rats in each group
• Duration : 6 weeks
• Dosage of extracts: 200mg/kg body
weight
Adaptation : 1st week for
Induction (2-3 weeks)
• G0 received normal diet
• G1, G2, G3, and G4 received
High Fat Diet
Treatment (From 3-6 week )
• G2 High Fat Diet + Ginger Extract
• G3: High Fat Diet + Cardamom
Extract
• G4 High Fat Diet + Ginger Extract +
Cardamom Extract
19

20. Biological assessment
■ Body weight and diet consumption was evaluated on weekly basis
■ On completed, gain in body weight and liver index were calculated
■ Liver tissue and blood sample were analyzed
■ Following parameter were determined by colorimeterically
– liver triglycerides, serum lipid profile and liver enzymes
Statistical analysis
■ All parameter of study was measured accurately and statistically considered
■ Done via analytical software (statistix 8.1)
■ Assessment of Level of significance via one-way ANOVA by tukey test
■ Set standard for statistical significance as p<0.05
20

21. Results and discussion
Extraction yield
■ The percentage of yield of ethanol extracts of
■ ginger 4.2% w/w
■ Cardamom 5.6% w/w
Phytochemicals Characterization
Extract TPC mg/100g TFC g/100g DPPH µg/100g
Ginger 567.00±1.18 0.278±0.001 5.76±0.0145
Cardamom 124.96±0.52 279.33±0.33 191.42±0.0569
21

22. Effect on
body weight
gain
19.33
44.00
38.08
40.55
34.33
0.00
10.00
20.00
30.00
40.00
50.00
60.00
G0 G1 G2 G3 G4
Body
weight
gain
(g/month)
Treatments
G0 Normal Diet G1 High Fat Diet (control group) G2 High Fat Diet+Ginger
G3 High Fat Diet +Cardamom G4 High Fat Diet +Ginger+Cardamom
• Ginger is more effective in weight loss
than cardamom
• As maximum weight loss in G4
(blend)
• As in previous study both Ginger and
Cardamom extracts improved the
weight loss in rats against
hyperlipidemic diets
(Ahmida and Abuzogaya, 2009 ;Bhat et
al., 2015)
22

23. Effect on Feed
Intake (g/day) 18.65
11.95
13.68 13.28
14.9
0
5
10
15
20
25
G0 G1 G2 G3 G4
Average
Feed
Intake
(g/day)
Treatments
Both ginger and cardamom
significantly improved the feed intake
But there is no significant difference
Similar outcomes were reported by
Gao et al. (2012) and extensive
research of Bhaswant et al. (2015)
G0 Normal Diet
G1 High Fat Diet
G2 High Fat Diet +Ginger
G3 High Fat Diet +Cardamom
G4 High Fat Diet +Ginger+Cardamom 23

24. Effect on Liver Index
(Liver weight / Body
weight)
0.0348
0.0378
0.0308 0.0315 0.0302
0
0.005
0.01
0.015
0.02
0.025
0.03
0.035
0.04
G0 G1 G2 G3 G4
Liver
Index
Treatments
• Both extract decrease the liver index
in a similar trend
• Present data confirmed the previous
the study of Bhat et al. (2016) and
Gao et al. (2012) G0 Normal Diet
G1 High Fat Diet
G2 High Fat Diet +Ginger
G3 High Fat Diet +Cardamom
G4 High Fat Diet +Ginger+Cardamom
24

25. Liver Triglycerides
(mg/dL)
• Ginger was more potent to control
the Liver triglycerides
• 6-shogaol from ginger acts as a
PPAR λ agonist in fat cells (Isa et
al., 2008)
46
80.5
58
74
61.6
0
10
20
30
40
50
60
70
80
90
G0 G1 G2 G3 G4
Liver
Triglycerides
(mg/dL)
Treatments
G0 Normal Diet
G1 High Fat Diet
G2 High Fat Diet +Ginger
G3 High Fat Diet +Cardamom
G4 High Fat Diet +Ginger+Cardamom
25

26. Effect on lipid profile
82.5
191.5
162.5
145.5
72.5
34
19.5 24.5 22.5 27
46.5
99.5
79.5
91
71
62
135.5
104.5
116
98
0
50
100
150
200
250
G0 G1 G2 G3 G4
mg/dL
Treatments
TC HDL-C LDL-C TG
G0 Normal Diet
G1 High Fat
G2 High Fat Diet + Ginger Extract
G3 High Fat Diet + Cardamom Extract
G4 High Fat Diet + Ginger Extract + Cardamom Extract 26

27. Effect on liver enzymes
0
200
400
600
800
1000
1200
G0 G1 G2 G3 G4
U/L
Treatments
ALP ALT AST
G0 Normal Diet
G1 High Fat
G2 High Fat Diet + Ginger Extract
G3 High Fat Diet + Cardamom Extract
G4 High Fat Diet + Ginger Extract + Cardamom Extract 27

28. Summary
■ Diet and lifestyle habits have the central role in the prevention and
progression of diseases
■ objective of study is to assess the effect of ginger and cardamom against
high-fat diet induced NAFLD
■ Results drawn after conclusion of study showed that
■ liver tissue TG, liver enzymes, serum TG and cholesterol, body weight gain,
and liver index decreased significantly in response to ginger, cardamom and
their combined extracts
■ Ginger group exerted 14% more reduction in liver fats in contrast to
cardamom
■ Reduction in Liver index was 19, 17, and 20% among ginger, cardamom and
their combination, respectively
28

29. Conclusion
■ Ingestion of ginger is more effective remedy than cardamom extract
for the prevention of non-alcoholic fatty liver disease but a further
research should be conducted in order to assess their some other
potential effects at genetic and molecular level
29

30. REFRENCES
■ Aboubakr, M. and A.M. Abdelazem. 2016. Hepatoprotective effect of aqueous
extract cardamom against gentamicin induced hepatic damage in rats. Int.
J. B. Appl. Sci. 5:1-32.
■ Atoui, A.K., A. Mansouri, G. Boskou and P. Kefalas. 2005. Tea and herbal
infusions: Their antioxidant activity and phenolic profile. Food Chem. 89:27-
36
■ Bhaswant, M., H. Poudyal, M.L. Mathai, L.C. Ward, P. Mouatt and L. Brown.
2015. Green and black cardamom in a diet-induced rat model of metabolic
syndrome. Nutrients. 7:7691-7707.
■ Bhatt, B. and J. Chatterjee. 2016. Identification of proapoptopic, anti-
inflammatory, anti-proliferative, anti-invasive and anti-angiogenic targets of
essential oils in cardamom by dual reverse virtual screening and binding
pose analysis. Asian Pac. J. Cancer Prev. 14:3735-3742. 30

31. CONTINUE
■ Festi, D., R. Schiumerini, L. Marzi, A.R. Di Biase, D. Mandolesi, L.
Montrone, E. Scaioli, G. Bonato, G. Marchesini-Reggiani and A.
Colecchia. 2013. Review article: The diagnosis of non-alcoholic fatty
liver disease availability and accuracy of noninvasive methods. Aliment.
Pharmacol. Ther. 37:392-400.
■ Gao, H., T. Guan, C. Li, G. Zuo, J. Yamahara, J. Wang and Y. Li. 2012.
Treatment with ginger ameliorates fructose-induced Fatty liver and
hypertriglyceridemia in rats: modulation of the hepatic carbohydrate
response element-binding protein-mediated pathway. Evid. Based
Complement. Alternat. Med. 2012:1-12.
■ Ghosh, S., D. Chatterjee, S. Mishra and P. Bhattacharjee. 2016. Food
application of an encapsulated phytochemically rich SC-CO2 extract of a
polyherbal mix of tulsi, bay and cardamom: Shelf-life and frying stability
of soybean oil. J. Food Eng. 171:194-199. 31

32. CONTINUE
■ Henriksen, E.J. 2006. Exercise training and the antioxidant α-lipoic acid
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T. Tsuda. 2008. 6-Shogaol and 6-gingerol, the pungent of ginger, inhibit
TNF-α mediated down regulation of adiponectin expression via different
mechanisms in 3T3-L1 adipocytes. Biochem. Biophys. Res. Commun.
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■ Khosravani, M., M.A. Azarbayjani, M. Abolmaesoomi, A. Yusof, N.Z.
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32

33. CONTINUE
■ Lai, Y.S., W.C. Lee, Y.E. Lin, C.T. Ho, K.H. Lu, S.H. Lin and L.Y. Sheen.
2016. Ginger Essential Oil Ameliorates Hepatic Injury and Lipid
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■ Lee, T.Y., K.C. Lee, S.Y. Chen and H.H. Chang. 2009. 6-Gingerol inhibits
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■ Pollock, N.K., V. Bundy, W. Kanto, C.L. Davis, P.J. Bernard, H. Zhu, B.
Gutin and Y. Dong. 2012. Greater fructose consumption is associated with
cardiometabolic risk markers and visceral adiposity in adolescents. J. Nutr.
142:251-257
■ Qamar, A., A. Usmani, H. Waqar, A. Siddiqui and H. Kumar. 2016.
Ameliorating effect of Allium Sativum on high-fat diet induced fatty liver
33

34. CONTINUE
■ Rahimlou, M., Z. Yari, A. Hekmatdoost, S.M. Alavian and S.A.
Keshavarz. 2016. Ginger supplementation in nonalcoholic fatty liver
disease: a randomized, double-blind, placebo-controlled pilot
study. Hepat. Mon. 16:1-5.
■ Stoilova, I., A. Krastanov, A. Stoyanova, P. Denev and S. Gargov. 2007.
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■ Vos, M.B. and J.E. Lavine. 2013. Dietary fructose in nonalcoholic fatty
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fatty liver disease. Am. J. Clin. Nutr. 86:285-300.
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