screening of spices for antioxidant activity -...
TRANSCRIPT
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CHAPTER -2
Screening of spices for antioxidant activity
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Introduction
Reactive oxygen species (ROS) are highly reactive and potentially damaging
chemical species (Frankel and Meyer, 2000, Carpenter et al., 2007, Suk Kim et al.,
2011). The oxidative damages caused by ROS Oxidative stress is one of the major
etiological factors for diseases like Cataract, Cancer, Heart ailments, Arthritis,
Alzheimer’s disease, nutritional deficiencies, bacterial, viral infections (Halliwell B.,
1996). Antioxidants can prevent the oxidation of lipids or other molecules by
inhibiting the initiation or propagation of oxidative chain reactions (Tachakittirungrod
et al., 2007). Spices are strong source of natural antioxidants which known to protect
tissues / cells from oxidative stress, which is generally considered to be a cause of
mutation and leads to cancer (Ringman et al., 2005).
Generally, Spices, like turmeric, fenugreek, mustard, ginger, etc. may offer
many health benefits and have been proven to counteract oxidative stress in vitro and
in vivo (Tachakittirungrod et al., 2007). Most of these spices have been intensely
studied only for their components like phenolic compounds, beta carotene,
curcuminoids and flavonoids (Manda and Adams, 2010, Suk Kim et al., 2011), but
when these so called active components are subjected to thermal stability tests, it is
observed that, their antioxidant ability is considerably reduced. Only few spices have
shown their stability against temperature and retain most of antioxidant activity.
Herein efforts were directed to screen different spices for their best possible
antioxidant activity and also to unravel the underlying mechanism of this protection,
various approaches have been taken because there is a need to identify non toxic,
inexpensive, easily available powerful antioxidant from dietary sources. The above
was the aim to screen different spices in search of source of antioxidants.
Star Anise (Illicium verum), commonly used as spice, obtained from the star-
shaped pericarp of Illicium verum. The star shaped fruits are harvested just before
ripening. It is a major source of the chemical compound shikimic acid. Shikimic acid
is a primary precursor in the pharmaceutical synthesis of anti-influenza drug (Wang et
al., 2011). Star anise has been used in a tea as a traditional remedy for rheumatism,
and the seeds are sometimes chewed after meals for better digestion. Star Anise is
used to relieve cold-stagnation in traditional Chinese medicine (Divya Chouksey et
al., 2010, Cheng HongYang et al., 2012).
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Cloves (Syzygium aromaticum) are the aromatic dried flower buds of a tree in
the family Myrtaceae. Cloves are used in Ayurvedic medicine, Chinese medicine,
and western herbalism and dentistry. Clove oil, applied to a cavity in a decayed tooth,
also relieves toothache (Alqareer et al., 2006). The clove oil is used in aroma therapy
and as painkiller in dental emergencies. Cloves are used to increase hydrochloric acid
in the stomach and to improve absorption. Cloves are also said to be a natural
anthelmintic. Topical application over the stomach or abdomen are said to warm the
digestive tract (Balch et al., 2000). Research results show that, extracts of clove buds
inhibits carbohydrate hydrolyzing enzymes in case of type 2 diabetes (Stephen and Ganiyu
Oboh, 2012, Pradeep Kumar et al. 2011).
Cinnamon (Cinnamomum verum) is obtained from the inner bark of trees of
Cinnamomum that is used in both sweet and savoury foods. Cinnamon is one the
most popular spices used in food preparation (Wu et al., 2013). Cinnamon bark is
used widely as a spice for its attractive flavour. The bark oil used in the manufacture
of perfumes, soaps, toothpastes and flavouring agent for liquors and in dentifrices.
Besides these, Cinnamon and Cassia have a wide range of medicinal and
pharmacological application like appetite stimulation, treatment for arthritis (Denys J.
Charles, 2013).
Cumin (Cuminum cyminum) is of the family Apiaceae, has been used as a
spice and is native to the eastern Mediterranean, extending to East India (Sowbhagya
et al., 2008). Cumin seeds are used for their unique aroma and its medicinal
properties. Cumin is used in foods, beverages, liquors and in medicines (Sowbhagya
et al., 2007). Literature shows that, Cumin rich in vitamin A, beta carotene, calcium
and magnesium. In traditional medicine, cumin is used to treat jaundice, dyspepsia
and diarrhea (Muhammad Namdeem and Riaz, 2012, Thippeswamy and Naidu, 2005,
Ani et al., 2006).
Fenugreek or methi seeds (Trigonella foenum) belong to the subfamily
Papilionacae of the family Leguminosae. Fenugreek is used both as an herb (the
leaves) and a spice (the seed). It is reported that, Fenugreek seeds rich with protein,
crude fibre and crude fat (Nazar and Tinay 2007, Shakuntala et al, 2011). As
mentioned in Ayurveda and Unani, the seeds are bitter, mucilaginous, aromatic,
carminative, tonic, thermogenic, galactogogue, astringent, emollient and an
aphrodisiac. It is used to treat for fever, vomiting, anorexia, cough, bronchitis and
callosities (Anon., 1976, Kirtikar and Basu, 1984, Xue et al. 2007). In Ayurveda, the
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seeds are used to treat smallpox, enlargement of liver and spleen and rickets. The
Fenugreek seeds contain steroidal substance, which is used as a starting material in
the production of sex hormones and oral contraceptives (Sharma, 1986, Narender et
al. 2006, Acharya et al., 2011).
Black pepper (Piper nigrum) belongs to the family of Piperaceae. It is
cultivated for its fruit, dried and used as a spice. It is widely consumed spice through
out the world. It is rich with carbohydrates, protein, vitamin A, phosphorus and
Potassium. It increases villi surface and so it used to adjunct to increase absorption of
other compounds (Ernest Hogson, 2004, Zachariah et al. 2005, Meghwal and
Goswami, 2012). It is used as medicines for analgesic, antiseptic, antispasmodic,
antitoxic, aphrodisiac, diaphoretic, digestive, diuretic, febrifuge, laxative and tonic for
spleen (Gulcin, I., 2005, Nooman et al., 2008, Meghwal and Goswami, 2012).
Turmeric (Curcuma longa) belongs to Zingiberaceae family has been
attributed a number of medicinal properties in the traditional system of medicine for
treating several common ailments (Sivananda, 1958; Nadkarni and Nadkarni, 1976;
Parastoo et al., 2012). It belongs to the genus Curcuma, which consists of several
plant species with underground rhizomes and roots. About 40 species of the genus are
indigenous to India, indicating the Indian origin (Velayudhan et al., 1999, El-Masry,
A.A., 2012). Originally, it had been used as a food additive to improve the
palatability, storage and preservation of food. Literature shows that, it is rich with
Carbohydrates, proteins, Curcumin I, II, III, curcuminoids etc. It has a wide range of
medicinal property like antimutagenic, anticarcinogen, antioxidant, antiviral,
antibacterial, antiparasitic, antiinflammant etc. (Mithra et al., 2012, Ammon and
Wahl, 1991, Araujo and Leon, 2001, Meera and Divya, 2012, Leela Srinivas et al.,
1992, Trinidad, 2012).
Wild Turmeric (Curcuma aromatica) is belongs to the Zingiberaceae family.
In Ayurveda and Unani, it is mentioned that, it has strong antibiotic properties. It is
believed to play a role in preventing and curing cancer in Chinese medicine, in an
effort to remove cell accumulations such as tumors (Lee et al., 2007, Angel et al.,
2012). Curcuma aromatica possesses curcuminoids, which give it natural antibiotic
powers. It is used as anti-inflammant (Amit Kumar et al, 2009, Ahmed et al., 2005),
anti-allergic (Ram et al., 2003) and anti-dementia (Lim et al., 2001, Shahwar et al.,
2012, Srividya et al., 2012).
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Turmeric (Curcuma longa) Wild Turmeric (Curcuma aromatica)
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Materials
Selected spices were obtained from authentic sources, cleaned thoroughly,
dried in shade, powdered and sieved through B.P.100 mesh. Butylated hydroxyl
anisole, α-tocopherol, thiobarbituric acid, ferrous sulphate, ascorbic acid, 2-deoxy
ribose, ferric chloride, sodium carbonate, sodium tartarate, linoleic acid were
purchased from Sigma, US. Sephadex G 10 and G 25, DEAE were from Pharmacia,
Sweden. All other chemical unless otherwise mentioned were purchased from E.
Merck, Germany. The 0.45μm and 0.22μm filter were purchased from Sartorius,
India. The spectro-photometric analysis was done using Shimadzu UV-1601
Spectrophotometer. All organic solvents were of analytical grade and were distilled
prior to use if needed.
Methods
2.1.1 Aqueous extract of spices: Flow Chart – 1
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2.1.2 Solvent extract of spices: Flow Chart - 2
2.3. Thermal stability of spice extracts The stability of the spice extracts were tested against heat treatment at 1000C
up to 60 minutes. The solubility was tested in water, tris buffer (10mM, pH 7.4),
phosphate buffer (10mM, pH 7.4) and saline.
2.4 Biological characterization
2.4.1 DPPH radical scavenging activity of the spice extracts
DPPH radical scavenging activity was assessed according to the method of
Shimada et al. (1992) with minor modifications. The spice extracts at a concentration
of 25 µg was mixed in 1 ml of freshly prepared 0.5 mM DPPH ethanolic solution and
2 ml of 0.1 M acetate buffer pH 5.5. The resulting solutions were then incubated at
37°C for 30 min and measured colorimetrically at 517 nm. Ascorbic acid, BHA and
Curcumin were used as positive control under the same assay conditions. Negative
control was without any inhibitor or spice extracts. Lower absorbance at 517 nm
represents higher DPPH scavenging activity. The % DPPH radical scavenging activity
of spice extracts was calculated from the decrease in absorbance at 517 nm in
comparison with negative control.
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2.4.2 Superoxide radical scavenging activity
The Superoxide radical (O2•-) scavenging activity of spice extracts were
measured according to the method of Lee et al. (Lee et al., 2002) with minor
modifications. The reaction mixture containing 100μl of 30mM EDTA (pH 7.4), 10μl
of 30mM hypoxanthine in 50mM NaOH, and 200μl of 1.42mM nitro blue tetrazolium
with or without spice extracts and SOD serving as positive control at various
concentrations ranging from 50-300μg. After the solution was pre-incubated at
ambient temperature for 3min, 100μl of xanthine oxidase solution (0.5U/ml) was
added to the mixture and incubated for one hour at 37C, and the volume was made
up to 3ml with 20mM phosphate buffer (pH 7.4). The solution was incubated at room
temperature for 20 min; absorbance was measured at 560 nm. Appropriate controls
were included to rule out the artifacts induced reaction. The control was without any
inhibitor. Inhibitory effect of spice extracts on superoxide radicals were calculated as
2.4.3 Lipid peroxidation inhibition activity
The evaluation of antioxidant activity of spice extracts based on the inhibition
of peroxidation according to Shimazaki et al., 1984 with minor modifications. The
evaluation of oxidation was done by measuring the TBA reactive substances (Dahle et
al, 1962). In another model like linolenic acid micelles, 100 μl of linoleic acid was
subjected to peroxidation by ferrous sulphate and ascorbic acid (10:100 μmol)
(Gutteridge, 1984) in final volume of 1 ml of Tris buffered saline (20 mM, pH 7.4,150
mM NaCl). The reaction mixture was treated with or without spice extracts (25µg),
BHA, α-tocopherol and Curcumin (400μM) were used as positive control. The
contents were incubated for 1 h at 37°C. The reaction was terminated by the addition
of 10 μl of 5% phenol and 1 ml of 1% TCA. To each system 1 ml of 1% TBA was
added, the contents were kept in a boiling water bath for 15 min, cooled and
centrifuged at 6000 rpm for 10 min. The absorbance of supernatants was measured
colorimetrically at 535 nm. Appropriate blanks were included for each measurement.
The negative control without any test sample was considered as 100% peroxidation.
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The % inhibition of lipid peroxidation was determined accordingly by comparing the
absorbance of the test samples with negative control.
2.4.5. Hydroxyl radical scavenging activity
The hydroxyl radical scavenging activity of spice extracts were done
according to the method of Halliwell et al., 1981 with minor modifications. The
reaction mixture containing FeCl3
(100 μM), EDTA (104 μM), H2O
2 (1 mM) and 2-
deoxy- D-ribose (2.8 mM) were mixed with or without spice extracts (25µg) in 1 ml
final reaction volume made with potassium phosphate buffer (20 mM pH 7.4) and
incubated for one hour at 37°C. BHA and Curcumin (400μM) were used as positive
control. The mixture was heated at 95°C in water bath for 15 min followed by the
addition of 1 ml each of TCA (2.8%) and TBA (0.5% TBA in 0.025 M NaOH
containing 0.02% BHA). Finally the reaction mixture was cooled on ice and
centrifuged at 5000 rpm for 15 min. Absorbance of supernatant was measured at 532
nm using the negative control without any antioxidant was considered 100%
oxidation. The percentage hydroxyl radical scavenging activity of spice extracts was
determined.
2.5. Statistical Analysis Results are expressed as Means ± S.D of five experiments. Student’s t-test was
used for statistical significance using SPSS (statistical presentation system software)
for windows version 10.0.1 software, Inc. New York. A probability level of P < 0.05
was considered as statistical significance in comparing with relevant controls.
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Fig. 2.1 Inhibition of Lipid peroxidation and scavenging of hydroxyl radicals, DPPH radical and superoxide radicals by Star anise (Illicium verum) extracts
% inhibition of Lipid peroxidation and scavenging of hydroxyl radicals, DPPH radical and superoxide radicals was calculated as described in methods.
The control was without any antioxidant and the results are mean ± SD (n = 5). Standard antioxidants BHA, α-tocopherol and Curcumin used at a dosage of 400µM and Star anise (Illicium verum) extracts used at a
dosage of 25 µg.
0
10
20
30
40
50
60
70
80
90
100
Notreatment
BHA Alphatocopherol
Curcumin Waterextract
Boilingwater
extract
Ethanolextract
Methanolextract
Hexaneextract
Petroleumether
extract
Different extracts of Star Anise
% in
hibi
tion
Lipid peroxidation inhibition activity
Hydroxyl radical scavenging activity
DPPH radical scavening activity
Superoxide radical scavening activity
41
Fig. 2.2 Inhibition of Lipid peroxidation and scavenging of hydroxyl radicals, DPPH radical and superoxide radicals by Clove (Syzygium aromaticum) extracts
% inhibition of Lipid peroxidation and scavenging of hydroxyl radicals, DPPH radical and superoxide radicals was calculated as described in methods.
The control was without any antioxidant and the results are mean ± SD (n = 5). Standard antioxidants BHA, α-tocopherol and Curcumin used at a dosage of 400µM and Clove (Syzygium aromaticum) extracts used at a
dosage of 25 µg.
0
10
20
30
40
50
60
70
80
90
100
No treatment BHA Alphatocopherol
Curcumin Water extract Boiling waterextract
Ethanol extract Methanolextract
Hexane extract Petroleumether extract
Different extracts of Clove
% in
hibi
tion
Lipid peroxidation inhibition activity
Hydroxyl radical scavenging assay
DPPH radical scavening activity
Superoxide radical scavening activity
42
Fig. 2.3 Inhibition of Lipid peroxidation and scavenging of hydroxyl radicals, DPPH radical and superoxide radicals by Cinnamon (Cinnamomum verum) extracts
% inhibition of Lipid peroxidation and scavenging of hydroxyl radicals, DPPH radical and superoxide radicals was calculated as
described in methods. The control was without any antioxidant and the results are mean ± SD (n = 5). Standard antioxidants BHA, α-tocopherol and Curcumin used at a dosage of 400µM and Cinnamon (Cinnamomum verum) extracts used
at a dosage of 25 µg.
0
10
20
30
40
50
60
70
80
90
100
No treatment BHA Alphatocopherol
Curcumin Water extract Boiling waterextract
Ethanol extract Methanolextract
Hexane extract Petroleumether extract
Different extracts of Cinnamon
% in
hibi
tion
Lipid peroxidation inhibition activity
Hydroxyl radical scavenging assay
DPPH radical scavening activity
Superoxide radical scavening activity
43
Fig. 2.4 Inhibition of Lipid peroxidation and scavenging of hydroxyl radicals, DPPH radical and superoxide radicals by Cumin (Cuminum Cyminum) extracts
% inhibition of Lipid peroxidation and scavenging of hydroxyl radicals, DPPH radical and superoxide radicals was calculated as described in methods.
The control was without any antioxidant and the results are mean ± SD (n = 5). Standard antioxidants BHA, α-tocopherol and Curcumin used at a dosage of 400µM and Cumin (Cuminum Cyminum) extracts used at a
dosage of 25 µg.
0
10
20
30
40
50
60
70
80
90
100
No treatment BHA Alphatocopherol
Curcumin Waterextract
Boiling waterextract
Ethanolextract
Methanolextract
Hexaneextract
Petroleumether extract
Different extracts of Cumin
% in
hibi
tion
Lipid peroxidation inhibition activityHydroxyl radical scavenging assayDPPH radical scavening activitySuperoxide radical scavening activity
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Fig. 2.5 Inhibition of Lipid peroxidation and scavenging of hydroxyl radicals, DPPH radical and superoxide radicals by Methi (Trigonella foenum graecum) seeds extracts
0
10
20
30
40
50
60
70
80
90
100
Notreatment
BHA Alphatocopherol
Curcumin Waterextract
Boilingwater
extract
Ethanolextract
Methanolextract
Hexaneextract
Petroleumether
extract
Different extracts of Methi seeds
% in
hibi
tion
Lipid peroxidation inhibition activityHydroxyl radical scavenging assayDPPH radical scavening activitySuperoxide radical scavening activity
% inhibition of Lipid peroxidation and scavenging of hydroxyl radicals, DPPH radical and superoxide radicals was calculated as
described in methods. The control was without any antioxidant and the results are mean ± SD (n = 5). Standard antioxidants BHA, α-tocopherol and Curcumin used at a dosage of 400µM and Fenugreek or methi (Trigonella foenum) seeds
extracts used at a dosage of 25 µg.
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Fig. 2.6 Inhibition of Lipid peroxidation and scavenging of hydroxyl radicals, DPPH radical and superoxide radicals by Black pepper (Piper nigrum) extracts
0
10
20
30
40
50
60
70
80
90
100
Notreatment
BHA Alphatocopherol
Curcumin Waterextract
Boilingwater
extract
Ethanolextract
Methanolextract
Hexaneextract
Petroleumether
extract
Different extract of Black pepper
% in
hibi
tion
Lipid peroxidation inhibition activityHydroxyl radical scavenging activityDPPH radical scavening activitySuperoxide radical scavening activity
% inhibition of Lipid peroxidation and scavenging of hydroxyl radicals, DPPH radical and superoxide radicals was calculated as
described in methods. The control was without any antioxidant and the results are mean ± SD (n = 5). Standard antioxidants BHA, α-tocopherol and Curcumin used at a dosage of 400µM and Black pepper (Piper nigrum) extracts used at a
dosage of 25 µg.
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Fig. 2.7 Inhibition of Lipid peroxidation and scavenging of hydroxyl radicals, DPPH radical and superoxide radicals by Turmeric (Curcuma longa L) extracts
0
10
20
30
40
50
60
70
80
90
100
Notreatment
BHA Alphatocopherol
Curcumin Waterextract
Boilingwater
extract
Ethanolextract
Methanolextract
Hexaneextract
Petroleumether
extract
Different extracts of Turmeric (Curcuma longa)
% in
hibi
tion
Lipid peroxidation inhibition activityHydroxyl radical scavenging activityDPPH radical scavening activitySuperoxide radical scavening activity
% inhibition of Lipid peroxidation and scavenging of hydroxyl radicals, DPPH radical and superoxide radicals was calculated as
described in methods. The control was without any antioxidant and the results are mean ± SD (n = 5). Standard antioxidants BHA, α-tocopherol and Curcumin used at a dosage of 400µM and Turmeric (Curcuma longa L) extracts used at a
dosage of 25 µg.
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Fig. 2.8 Inhibition of Lipid peroxidation and scavenging of hydroxyl radicals, DPPH radical and superoxide radicals by Wild Turmeric (Curcuma aromatica) extracts
0
10
20
30
40
50
60
70
80
90
100
Notreatment
BHA Alphatocopherol
Curcumin Waterextract
Boilingwater
extract
Ethanolextract
Methanolextract
Hexaneextract
Petroleumether
extract
Different extracts of Wild Turmeric (Curcuma aromatica)
% in
hibi
tion
Lipid peroxidation inhibition activity
Hydroxyl radical scavenging activity
DPPH radical scavening activity
Superoxide radical scavening activity
% inhibition of Lipid peroxidation and scavenging of hydroxyl radicals, DPPH radical and superoxide radicals was calculated as
described in methods. The control was without any antioxidant and the results are mean ± SD (n = 5). Standard antioxidants BHA, α-tocopherol and Curcumin used at a dosage of 400µM and Wild Turmeric (Curcuma aromatica) extracts
used at a dosage of 25 µg.
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Table-2.01 Heat stability nature of Star Anise, Clove, Cinnamon and Cumin extracts : A comparison
% inhibition of lipid peroxidation
Minutes
Spices Extracts
0 60
Water
55±2
20±1
Boiling water 52±1 22±3 Ethanol 45±2 19±4 Methanol 49±2 14±3 Hexane 36±3 16±4
Star Anise (Illicium verum)
Petroleum ether 25±1 04±3 Water 67±1 26±1 Boiling water 69±1 37±1 Ethanol 64±2 11±3 Methanol 64±2 16±2 Hexane 55±2 09±3
Clove (Syzygium aromaticum)
Petroleum ether 46±3 11±3 Water 58±1 15±1 Boiling water 52±2 13±3 Ethanol 61±1 11±3 Methanol 59±2 16±3 Hexane 36±3 09±1
Cinnamon (Cinnamomum verum)
Petroleum ether 29±2 05±4 Water 54±1 10±5 Boiling water 59±1 05±2 Ethanol 58±2 13±2 Methanol 53±1 15±4 Hexane 48±2 14±5
Cumin (Cuminum Cyminum)
Petroleum ether 34±2 08±1
Spice extracts were heated in boiling water bath, 60 minutes, the antioxidant
activity was estimated by TBARS method. The antioxidant activity is expressed as %
inhibition.
The results are mean ± SD (n = 5).
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Table-2.02 Heat stability nature of Fenugreek, Black Pepper, turmeric and wild turmeric extracts : A comparison
% inhibition of lipid
peroxidation
Minutes
Spices Extracts
0 60
Water
63±1
23±2
Boiling water 69±2 16±3 Ethanol 63±2 12±1 Methanol 60±1 19±1 Hexane 55±2 13±2
Fenugreek (Trigonella foenum graecum)
Petroleum ether 44±3 07±1 Water 52±1 21±1 Boiling water 56±2 25±1 Ethanol 62±2 12±3 Methanol 49±2 10±3 Hexane 40±1 11±1
Black Pepper (Piper nigrum)
Petroleum ether 28±2 08±3 Water 72±1 68±3 Boiling water 75±1 71±1 Ethanol 79±1 29±2 Methanol 74±3 24±1 Hexane 60±2 20±2
Turmeric (Curcuma longa)
Petroleum ether 55±2 05±3 Water 51±2 36±2 Boiling water 55±1 35±1 Ethanol 53±2 21±4 Methanol 53±1 23±2 Hexane 38±2 21±2
Wild Turmeric (Curcuma aromatica)
Petroleum ether 31±2 13±3
Spice extracts were heated in boiling water bath, at the end of 60 minutes, the
antioxidant activity was estimated by TBARS method. The antioxidant activity is
expressed as % inhibition.
The results are mean ± SD (n = 5).
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Discussion
In the present chapter, ambient temperature water, boiling water, ethanol,
methanol, hexane and petroleum ether extracts of eight spices was done to evaluate
their antioxidant activity against lipid peroxidation inhibition and scavenging of
hydroxyl, DPPH and superoxide radicals.
Linoleic acid micelles were subjected to peroxidation with ferrous sulphate -
ascorbic acid and the extent of lipid peroxidation was estimated by measurement of
TBARS. In the lipid peroxidation inhibition studies, antioxidants like BHA,
Curcumin and alpha tocopherol used as standard antioxidants.
Hydroxyl radicals are known to be the most reactive of all reduced forms of
dioxygen and are thought to initiate cell damage in vivo (Rollet-Labelle et al., 1998).
The hydroxyl radical scavenging method is an assay to determine the rate constants
for reactions of hydroxyl radical. The extracts on hydroxyl radicals generated by Fe3+
ions were measured by determining the degree of deoxyribose degradation, an
indicator of (TBA-MDA) adducts formation. The appropriate controls like BHA,
Curcumin and α-tocopherol at 400µM dose were used.
1,1-Diphenyl-2-picrylhydrazyl, (DPPH) is a cell-permeable, stable free radical
that acts as a hydrogen radical scavenger. It is useful in inducing free-radical injury to
tissues and as a screening tool for detecting free radical scavenging capacity of other
antioxidants.
The superoxide radical is a free radical form of superoxide dismutaste (SOD).
The two oxygen molecules are connected together and one oxygen has an extra
electron. This free radical targets cell membranes, the barrier protecting the inside of
cells. The presence of copper and zinc are six histidine and one aspartate side-chains;
one histidine is shared between the two metals can help SOD from forming into a
superoxide free radical and the manganese SOD present in mitochondria.
Herein we have used different extracts of spices Star anise (Illicium verum),
Clove (Syzygium aromaticum), Cinnamon (Cinnamomum verum), Cumin
(Cuminum cyminum), Methi (Trigonell foenum graecum), Black pepper (Piper
nigrum), Turmeric (Curcuma longa) and Wild turmeric (Curcuma aromatica) to
check its inhibitory activity.
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The water and boiling water extracts of selected eight spices was done as
shown in Flow chart -1. Different solvent extracts of spices was done as shown in
Flow chart-2. As shown in Figure 2.1, the lipid peroxidation inhibition capacity and
hydroxyl radical, DPPH radical and superoxide radical scavenging activity of
different extracts of Star Anise (Illicium verum) was done using standard antioxidants
BHA, α-tocopherol and Curcumin. The ambient temperature water extract, the boiling
water extract, ethanol and methanol extract of Star Anise showed more inhibition of
lipid peroxidation when compared to other extracts. The hydroxyl radical scavenging
activity of methanol extract of Star Anise was more than other extracts. The boiling
water and ambient temperature water extract showed more DPPH radical scavenging
activity. In superoxide radical scavenging activity, the boiling water, ethanol and
methanol extracts are more active. The above results indicate that, the ambient
temperature water extract, boiling water extract, ethanol extract and methanol extracts
of Star Anise are effective towards inhibiting lipid peroxidation and scavenging
hydroxyl, DPPH and Superoxide radical effectively. Figure No. 2.2 shows that,
inhibition of lipid peroxidation, scavenging of hydroxyl radicals, DPPH radicals and
superoxide radicals by aqueous and solvent extracts of Clove (Syzygium aromaticum).
The ability of inhibition of lipid peroxidation by all the extracts of clove is promising
when compared to standard antioxidants. In hydroxyl radical scavenging activity, the
ambient temperature water, boiling water and ethanol extracts are more when
compared to hexane and petroleum ether extracts. In DPPH radical scavenging
activity, the methanol extract, boiling water extract and ambient temperature water
extract showed more activity. In the superoxide radical scavenging studies, all the
extracts showed the inhibitory activity. From the above results, it is clear that, except
petroleum ether extract, all other extracts acts as effective antioxidants.
The lipid peroxidation inhibitory activity and hydroxyl radicals, DPPH
radicals and superoxide radicals scavenging activity results of Cinnamon
(Cinnamomum verum) are as shown in Figure No. 2.3. The ethanol and methanol
extracts of Cinnamon showed inhibition of lipid peroxidation where as the inhibition
shown by Hexane and petroleum ether was negligible. The hydroxyl radical
scavenging activities of ethanol and methanol extracts are more. The DPPH radical
scavenging activity of methanol extract was more. The superoxide radical
scavenging activity of methanol, ethanol and boiling water extract are more. The
52
above results show that, only the ethanol and methanol extract of Cinnamon are acts
as effective antioxidants.
Figure 2.4 shows that, the lipid peroxidation inhibitory activity and hydroxyl
radicals, DPPH radicals and superoxide radicals scavenging activity results of Cumin
(Cuminum Cyminum). Except Petroleum ether extract, all other extracts are showing
good peroxidation inhibitory activity. In hydroxyl radical scavenging activity, both
ambient temperature water extract and boiling water extract are showing more
scavenging activity. But in the DPPH radical scavenging activity, all the extracts
shows comparatively equal activity. The ethanol, boiling water extract, ambient
temperature water extract of Cumin shows good amount of superoxide radical
scavenging activity whereas, petroleum ether extract shows very less. The above
results confirm that, all the extracts of Cumin effective against DPPH radicals than
standard antioxidants like BHA and Curcumin.
As shown in Figure 2.5, the ambient temperature water extract, boiling water
extract, ethanol, methanol, hexane and petroleum extracts of Fenugreek or Methi
(Trigonella foenum graecum) showed very good antioxidant activity by inhibiting
lipid peroxidation and by scavenging hydroxyl radicals. But the DPPH and
Superoxide radical scavenging activity of all the extracts are very less and negligible.
The above results confirm that, all the extracts of Fenugreek are inhibitors of lipid
peroxidation and scavenge hydroxyl radicals.
The figure 2.6 showed that, the ethanol and boiling water extract of Black
pepper (Piper nigrum) showed lipid peroxide inhibitory activity and very less activity
reported by Hexane and petroleum ether extracts. The ambient water extract, boiling
water extract, ethanol extract are showing highest hydroxyl radical scavenging
activity and the extract of petroleum ether was very less. The DPPH radical
scavenging activity of all the extracts are almost equal and having antioxidant
activity. In superoxide radical scavenging activity of black pepper, very less was
reported by petroleum ether and methanol extract and others are negligible. It is
confirmed that, all extracts of Pepper effective in scavenging hydroxyl radicals,
DPPH radicals and inhibits lipid peroxidation.
The Figure 2.7 shows that, among Turmeric (Curcuma longa L) extracts, the
boiling water extract, ethanol extract and methanol extracts showed lipid peroxidation
53
inhibitory activity when compared to standards whereas, the ambient temperature
water extract showed comparatively less activity. Except petroleum ether extract, all
the other extracts are showed hydroxyl radical scavenging activity. In DPPH radical
scavenging activity, only ambient temperature water extract and boiling water extract
are showed radical scavenging activity. The boiling water extract and ambient
temperature water extract are showing superoxide radical scavenging activity. Above
results indicates that, only ambient temperature water extract and boiling water
extracts are effective antioxidants in all model systems.
In Curcuma aromatica extracts, the ambient temperature water extract, the
boiling water extract, ethanol extract and methanol extracts showed good percentage
of lipid peroxidation inhibitory activity when compared to standards as shown in
Figure 2.8. The ambient temperature water extract, boiling water extract, ethanol
extracts are showed hydroxyl radical scavenging activity when compared to other
extracts. The boiling water, ethanol and methanol extracts are showed DPPH radical
scavenging activity and petroleum ether extract showed very less. The ethanol and
methanol extracts are showed superoxide radical scavenging activity when compared
to hexane and petroleum ether extracts. The above result shows that, the ethanol and
methanol extracts of Curcuma aromatica are effective when compared to other
extracts.
Finally all eight spice extracts were subjected to thermal stability test by
keeping all extracts in boiling water bath for 60 minutes; their ability towards
inhibiting lipid peroxidation was examined at the end of 60 minutes as in shown in
Table 2.01 and Table 2.02. At “zero” minute, all the extracts showed their highest
lipid peroxidation inhibitory activity. At the end of 60 minutes of heat treatment, the
boiling water extract of Clove (37 ± 1%), boiling water extract of black pepper (25
1%), ambient temperature water extract of Turmeric (68 ± 3%), boiling water extract
of Turmeric (71 ± 2%), ambient temperature extract of wild turmeric (35±2%) and
boiling water extract of wild turmeric (36±1%) retained their antioxidant potential
against lipid peroxidation inhibition. Among the above extract only boiling water
extract of Turmeric (Curcuma longa L) was retained most of its antioxidant activity.
Hence, Turmeric (Curcuma longa L) was selected for further studies to understand its
antioxidant ability and other biological activities.
54
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