screening of spices for antioxidant activity -...

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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

34

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)

36

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


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


Hydroxyl radical scavenging assay



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


Curcumin Water extract Boiling waterextract

Ethanol extract Methanolextract

Hexane extract Petroleumether extract

Different extracts of Cinnamon

% in

hibi

tion


Hydroxyl radical scavenging assay



43

Fig. 2.4 Inhibition of Lipid peroxidation and scavenging of hydroxyl radicals, DPPH radical and superoxide radicals by Cumin (Cuminum Cyminum) extracts


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



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


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


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


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


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


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


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


Boilingwater

extract

Ethanolextract

Methanolextract

Hexaneextract

Petroleumether

extract

Different extracts of Wild Turmeric (Curcuma aromatica)

% in

hibi

tion


Hydroxyl radical scavenging activity




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)


Cinnamon (Cinnamomum verum)


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)


Black Pepper (Piper nigrum)


Turmeric (Curcuma longa)


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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