Phytochemical, Antioxidant and Antibacterial Activity of Garam Masala, the Quintessential Indian Spice Mix

1. Phytochemical, Antioxidant and Antibacterial Activity
of Garam Masala, the Quintessential Indian Spice Mix
Presented by:
PUJA BISAWS
M.Sc. 4th Semester in Biotechnology
Registration Number: 02174 of the year 2020-2021
Roll: PG/VUOGP57/ BIT- IVS No. 046
Oriental Institute of Science and Technology
Affiliated to
Vidyasagar University

2. OBJECTIVE
 To assess the Phytochemical content, antioxidant activity and antibacterial activity
of Garam Masala, and its three components viz. Clove (Syzygium aromaticum),
Cinnamon (Cinnamomum verum ) , Cardamom (Elettaria cardamomum).
 To explore the Phytochemicals present within the plants which can serve as
potential new source of natural antioxidants.
 To find out a potent antibacterial agent that can be further used as an alternative
medicines to cure diseases.

3. INTRODUCTION
 Spices
 Dried aromatic plant parts.
 Play important role as flavouring agents & used as a medicine.
 Aroma & flavour derived from phytochemical compounds.
 Possess antioxidant, anticancer, anti inflammatory, antimicrobial & antidiabetic properties.
 Garam Masala
 Hot mixture of spices.
 Helps in digestion, provides antioxidants, improves heart health, reduces inflammation &
lowers cancer risk.
Clove (Syzygium aromaticum):
 Great source of beta-carotene with full of antioxidants.
 Phytochemicals (bioactive compound) - Eugenol
 Helps in digestion, heart disease, cancers. Improved gastrointestinal problem & reduced
inflammation.

4. INTRODUCTION
Cinnamon (Cinnamomum verum):
 Used as a spice & flavouring agent.
 Phytochemicals – Cinnamaldehyde , Cinnamic acid & Eugenol.
 Have anti-inflammatory, antibacterial antioxidant & antidiabetic properties.
Cardamom (Elettaria cardamomum):
 known as “Queen of Spices”.,
 Used for flavouring confectionery.
 May help lower blood pressure & cholesterol levels, improve heart health, prevent
inflammation & toothache.

5. METHODS
 Collection of spices:
 Clove (Syzygium aromaticum), Cinnamon (Cinnamomum verum), and Cardamom (Elettaria
cardamomum) were collected from local market.
 Cleaned and washed and air dried.
 Dried spices were powdered separately using mortar and pestle.
 Preparation of spice extracts:
Each powdered spice(2gm) + distilled water(20ml).
 Formulation of spice mix:
The spice mix were formulated as M1, M2 and M3 and A, B, and C represents cloves, cinnamon and
cardamom respectively.

6. METHODS
Qualitative Estimation of Phytochemicals:
 Test for Carbohydrates (Molisch’s Test):
2ml of spice extract + few drops of Alcoholic α Napthol Solution (Molisch’s reagent) + 1ml
concentrated H2SO4 = Reddish violet ring at the junction of the two layers.
 Tests for Alkaloids: (Wagner’s Test):
2ml of extract +1ml of potassium iodide (Wagner’s reagent) = reddish brown precipitate.
 Test for Tannins : (Ferric Chloride Test):
2ml of sample extract +few drops of 10% Ferric chloride solution = greenish yellow or dark
blue colour.
 Test for Flavonoids:
2ml of extract + few drops of 10% lead acetate = yellow precipitate.

7. METHODS
Qualitative Estimation of Phytochemicals:
 Tests for Proteins :
2ml of extract + 1ml of conc.H2SO4. = white precipitate which on boiling turned yellow. On
addition of NH4OH, yellow ppt. turned orange.
 Test for Coumarins:
2ml of sample extract + few drops of alcoholic NaOH+ conc.HCl = yellow colour.
 Test for Steroids :
2ml solution of the extract+2ml of chloroform +2ml of concentrated H2SO4 = chloroform
layer turned red and acid layer showed greenish yellow fluorescence.
 Test for Glycosides (Legal’s Test):
2ml of sample extract + equal volume of Sodium Nitroprusside + few drops of sodium
hydroxide solution = blood red precipitate.

8. METHODS
Quantitative Determination of Phytochemicals:
 Determination of Total Phenolic Content (TPC) and Total Tannin Content (TTC) of
samples:
 Determined by using modified Folin-Ciocalteu’s (FC) method.
 value of absorbance was recorded by using colorimeter at 720 nm wavelength.
 Determination of Total Flavonoid Content (TFC) of samples:
 determined by using the Aluminium Chloride Assay method.
 optical density was recorded using colorimeter at 520 nm wavelength.
 Determination of Total Antioxidant Activity (TAA)-DPPH free radical scavenging assay:
 Determined using 2, 2- diphenyl-1-picrylhydrazyl (DPPH).
 The absorbance of mixture was measured by using colorimeter at 520 nm wavelength.
 Inhibition (%) = Absorbance of control- Absorbance of test sample x100
Absorbance of control
 Determination of Antibacterial Activity:
 Performed by Agar well diffusion method.
 Two bacterial strains Gram-negative bacteria (Escherichia coli) & Gram-positive bacteria
(Bacillus subtilis) are used to perform the method.

9. RESULTS
Qualitative Phytochemical Analysis of Spices:

10. RESULTS
Quantitative Phytochemical Analysis of Spices:
Quantitative Determination of Total Phenol Content (TPC) (mg GAE/g extract):
clove cinnamon cardamom M1 M2 M3
Series1 14.04 13.6 10.93 60.41 56.19 47.75
0
10
20
30
40
50
60
70
mg
TPC
GAE/g
extract
Samples
Total Phenol Content
Series1
Fig : Total Phenol Content (TPC) of spices & spice mix
Table: Concentration of sample extracts of spices & spice mix.

11. RESULTS
Quantitative Determination of Total Tannin Content (TTC) (mg GAE/g extract)
clove cinnamon cardamom M1 M2 M3
Series1 14.04 13.6 10.93 60.41 56.19 47.75
0
10
20
30
40
50
60
70
mg
TTC
GAE/g
extract
Samples
Total Tannin Content
Series1
Fig : Total Tannin Content (TTC) of spices & spice mix
Table: Concentration of sample extracts of spices & spice mix.

12. RESULTS
Quantitative Determination of Total Flavonoid Content (TFC) (mg QE/g extract)
Table : Concentration of sample extracts of spices & spice mix
CLOVE CINNAMON CARDAMOM M1 M2 M3
Series1 2.9 2.17 1.5 17.6 14.67 12
0
2
4
6
8
10
12
14
16
18
20
mg
QE/g
extract
Samples
Total Flavonoid Content
Series1
Fig : Total Flavonoid Content (TFC) of spices & spice mix

13. RESULTS
Assay of antioxidant activity (DPPH Free Radical Scavenging Assay) :
Table : Total Antioxidant Activity percentage of spices & spice mix
clove cinnamon cardamom M1 M2 M3
Series1 67.19 45.83 30.73 76.04 56.77 40.1
0
10
20
30
40
50
60
70
80
90
Antioxidant
(%)
Samples
Total Antioxidant Activity
Series1
Fig : Total Antioxidant Activity (TAA) of Spices and Spice Mix

14. RESULTS
Fig : Total Antioxidant Activity (TAA) for- a. spices extracts and
b. spice mix containing test tubes

15. RESULTS
Antibacterial Activity (Agar Well Diffusion Method):
• According to this method, zones of growth inhibition (including well diameter 10 mm ±SD)
showing the antibacterial activity.
Table : Antibacterial activity of spice extracts, spice mix & antibiotic

16. clove cinnamon cardamom sample mix antibiotic
Series1 17.33 14.33 10.67 20.33 28.67
0
5
10
15
20
25
30
35
zone
of
inhibition
(mm
)
Samples & antibiotic
Antibacterial Activity against Escherichia. coli
Series1
Fig : Antibacterial activity against (Escherichia coli)
clove cinnamon cardamom sample mix
Series1 18.33 16 11.67 27.33
0
5
10
15
20
25
30
zone
of
inhibition
(mm
)
Samples
Antibacterial activity against Bacillus subtilis
Series1
Fig : Antibacterial activity against (Bacillus subtilis)

17. Fig : Effects of different sample extracts against Gram Negative Bacteria: (Escherichia coli):-
a. spices extract (clove, cinnamon, cardamom) and control, b. spice mixture & antibiotic
Fig : Effects of different sample extracts against Gram Positive Bacteria: (Bacillus subtilis):-
a. spices extract (clove, cinnamon, cardamom) and control, b. spice mixture

18. CONCLUSION
• Quantitative estimation of Phytochemicals and antioxidants showed highest Total phenolic
content (TPC), Total Flavonoid Content (TFC), Total Tannin Content (TTC) & Total Antioxidant
Activity (TAA) in Garam Masala-M1 spice mix (having clove 0.6 parts and rest spices 0.1
parts).
• Spice mixture of Garam Masala showed highest antibacterial activity and highest inhibition
zone against two bacteria.
• Individual component Clove (Syzygium aromaticum) showed highest Phytochemicals,
antioxidant activity & strong antibacterial activity.
• Syzygium aromaticum could be utilized for commercial production of natural antioxidants
after further research.
• These results provide a scientific basis for further studies to explore the potential antioxidant
and search of new antimicrobials as an alternative to the antibiotics.
• Garam Masala is rich in anti-oxidative and anti inflammatory properties when it consumed
in a mixture form. The excessive consumption may lead to problems. So, further study is
required to determine the right amount of Garam Masala consumption for human health
benefits.

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