thesis pdf-s.r. senthilkumar -...

EXPERIMENTAL RESULTS

The results obtained from various assays, characterization

techniques and evaluation methods are furnished. The data and

information’s derived from these results are presented in the form of

figures, plates and tables

4.1. GREEN SYNTHESIS OF SILVER NANOPARTICLES

4.1.1. Preliminary screening tests of greengram sprout

Phytochemicals present in greengram sprout extract, qualitatively

detected using standard screening tests are presented in Table 4.1

and the results of the tests are shown in Plate 4.1.

4.1.2. GC-MS analysis of phytochemicals in greengram sprout

The chromatogram of the greengram sprout is shown in Fig. 4.1.

Using NIST library 24 compounds were detected, out of which

22 were identified. The peak report derived from the chromatogram is

presented in Table 4.2. Among these identified phytochemicals,

11 compounds with peak area (%) greater than 1 are reported in

Table 4.3. The chemical formula, molecular weight, compound type

and bioactivity are also furnished against each compound.

67

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1 Tannin ---

2 Saponin ---

3 Flavonoids ++

4 Steroids +++

5 Terpenoids ++

6 Alkaloids +++

7 Amino acids +++

8 Polyphenol +++

9 Glycoside +++

10 Protein ++ � � � � � � � � � � � ! � " # � � � � � � � ! ! $ ! ! ! � % � & % � ' ( � � % � ( � � � % ( ) * + � * , * - #

68

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69

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3.257 588627 0.69 1-Butanamine, 2-methyl-N-(2-methylbutylidene)

3.781 564406 0.67 Cyclopentane, 1-acetyl-1,2-epoxy-

4.650 5036211 5.94 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl

5.485 2425030 2.86 1-Deoxy-d-arabitol

6.460 388872 0.46 2-ETHYLHEXYL PENTENOATE

6.883 231882 0.27 1-Heptanol, 2-propyl

7.752 295200 0.35 3-(4-AMINOBUTYL)PIPERIDINE

8.249 493866 0.58 Unknown

10.349 144343 0.17 1H-Pyrrole, 2-(2,4,6-cycloheptatrienyl)

11.212 9582380 11.31 Ethyl .alpha.-d-glucopyranoside

12.714 26132753 30.83 MOME INOSITOL

13.413 7436423 8.77 Caffeine

13.708 895705 1.06 1H-PURINE-2,6-DIONE, 3,7-DIHYDRO-3,7-DIMETHYL

13.908 335443 0.40 Hexadecanoic acid, methyl ester

14.350 12098730 14.27 n-Hexadecanoic acid

16.043 9915501 11.70 Unknown

16.198 3239788 3.82 Octadecanoic acid

17.915 308432 0.36 9-OCTADECENOIC ACID (Z)

18.839 197121 0.23 TRIMETHYLSILYL ESTER OF TETRACOSANOIC ACID

18.967 200087 0.24 PREGNANE, SILANE DERIV.

19.150 896508 1.06 Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester

27.997 488264 0.58 Cholest-5-en-3-ol (3.beta.)-, carbonochloridate

28.676 979558 1.16 Stigmasterol

30.027 1886587 2.23 STIGMAST-5-EN-3-OL, (3.BETA.)

84761717 100.00

71

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74

4.1.3. Visual and UV-Vis monitoring of Ag NPs formation

The greengram plant, seeds, germinated seeds with sprout and

the various stages of silver nanoparticle formation are shown in Plate 4.2(a-d).

It can be observed that significant colour change appeared after 30 min

and the final brownish red colour was obtained after 2 h. The final colour

change confirmed the completion of the synthesis process.

The UV-Vis spectrum of the reaction medium recorded in

the range 300 to 600 nm is shown in Figure 4.2. The UV-Vis spectrum

showed an intense absorption band with a maximum at 445 nm is

attributed to the surface plasmon resonance (SPR) which is characteristic

of the silver nanoparticles (Umashankari et al., 2012). The slight broadening

of the peak indicated that the particles are slightly poly-dispersed.

4.1.4. XRD pattern of Ag NPs

The XRD pattern of the synthesized Ag NPs is illustrated in

Fig. 4.3. Since thin film technique had been used to record the

pattern, the amorphous characteristics of the glass were also present

in the XRD pattern overlapping the Ag NPs pattern. The XRD

spectrum confirmed the crystalline nature of the nanoparticles with

peaks appearing at 2 values of 32 , 38 , 46 corresponding to (111),

(200), (220) and (311) Bragg reflections, respectively. The average

crystallite size (D) of silver nanoparticles, calculated using the

Debye-Scherrer equation (Singh et al., 2010) was 5.6 nm. The XRD

parameters are provided in Table 4.4.

75

� � � � � � � 0 � N � R S � � � � � � � � � � � � � T N � R � � � 2 � T N R � � � � � � � � � 2 � � � 2 � U � � � � � � � �� 2 N 2 R 3 � � � � � � � � � � � � � � V � W � � � � � � � � � � �

(a) (b) (c)

(d)

76

X Y Z [ \ [ ] [ ^ _ ` _ Y a b c a d e f g Y d h a f i j g e k l d m n Z o p a a q h g r i a Y s i t k a Y h ZZ e i i h Z e b l a f e d k g i u g e b j g

X Y Z [ \ [ v [ w x y f b g g i e h d m n Z o p a a q h g r i a Y s i t k a Y h Z Z e i i h Z e b l a f e d k g i u g e b j g

77z b c { i \ [ \ [ w x y f b e b l i g i e a d m n Z o p aPeak No.

Position ( 2 ) FWHM, ( ) Particle size, D (nm)

1 32 2 4.1

2 38 2 4.2

3 46 1 8.5

Mean = 5.6

4.1.5. FE-SEM morphology of Ag NPs

The FE-SEM micrograph revealing the morphology of the

green synthesized Ag NPs is shown in Fig. 4.4.

4.1.6. FT-IR spectra of greengram sprout and Ag NPs

In order to identify the biomolecules present in the

greengram sprout which are responsible for the synthesis of Ag NPs,

the FT-IR spectrum was recorded and is shown in Fig. 4.5a. To study

the biomolecules of greengram sprout extract bound to the surface of

Ag NPs, FT-IR spectrum of the synthesized Ag NPs was recorded and

is shown in Fig. 4.5b. The position and intensity of the absorption

bands in the spectrum provide information about the various chemical

groups present and their concentration. The vibrational frequency

assignments for the prominent bands in the FT-IR spectrum of the

sprout sample are provided in Table 4.5. It can be observed that the

amide-I linkages in protein, amino acids, glycoside linkages and

polyphenols are responsible for the reduction and stabilization

processes in the synthesis of Ag NPs.

78

X Y Z [ \ [ \ [ X | ` } | ~ l Y j e d Z e b f r d m Z e i i h a q h g r i a Y s i t n Z o p a

� � � � � � � � � � � � � � � � � ��

79

X Y Z [ \ [ � [ X z ` � x a f i j g e b d m � b � Z e i i h Z e b l a f e d k g i u g e b j g b h t � c � a q h g r i a Y s i t n Z o p a

80

z b c { i \ [ � [ n c a d e f g Y d h c b h t b a a Y Z h l i h g m d e g r i X z ` � x a f i j g e k l d m Z e i i h Z e b la f e d k g a b l f { iAbsorption band

(cm–1)Vibrational group and

mode Chemical compound References

O–H stretch (hydroxyl) Phenols

Vanaja et al. (2013)

Awwad et al. (2013)

Rajathi and Sridhar (2013) 3398*

N–H stretch Amino group Awwad et al. (2013)

Alkanes Vanaja et al. (2013)

Rajathi and Sridhar (2013) C–H stretch

Aldehydes Umashankari et al. (2012) 2929

O–H stretch Carboxylic acid Vanaja et al. (2013)

2349 N–H stretch Amino acids Umashankari et al. (2012)

Polyphenols

1641 C=O (carbonyl)

Amide-I in protein links

Rajathi and Sridhar (2013)

Jayaseelan et al. (2013)

Prakash et al. (2013)

N–H bend Amino acids Theivasanthi and Alagar (2013)

C–O stretch Amino acids Theivasanthi and Alagar (2013)1408

C–C stretch Aromatics Mallikarjuna et al. (2012)

1249 C–O–C stretch Glycoside linkages Raghavandra et al. (2013)

Amide-I in proteins Awwad et al. (2013) 1081 C–N stretch (carbonyl)

Aliphatic amines Vanaja et al. (2013)

C–O stretch Carboxylic acid 669

C–H bend Phenyl ring substituents

Mallikarjuna et al. (2012)

Jayaseelan et al. (2013) � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �

81

4.1.7. Antibacterial assays

The antibacterial activity of Ag NPs against the studied

bacterial strains are shown in Plate 4.3. The values of zone of

inhibition obtained from the assay are presented in Table 4.6. All

Gram-negative bacteria had shown good sensitivity towards the green

synthesized Ag NPs for the concentrations 10 and 15 g mL–1, while

the Gram-positive bacteria showed almost equal sensitivity as that of

the positive control (Imipenem).

4.1.8. Antifungal assays

Regarding the antifungal activity, all four fungal strains

used in this study are found to be sensitive to the green synthesized

Ag NPs as well as to the commercially available antifungal drug Itraconozole.

The antifungal activities of Ag NPs are shown in Plate 4.4 and the zone

of inhibition values are presented in Table 4.7. The encouraging aspect of

this study is that all the fungal species are relatively more sensitive to

the Ag NPs compared to the positive control. This may be due to the

individual organisms response and their genotypic characters which

differs in their sensitivity pattern towards the single testing agent.

82

p { b g i \ [ v [ n h g Y c b j g i e Y b { b j g Y ¡ Y g q d m n Z o p a b Z b Y h a g � b � ¢ £ ¤ ¥ ¦ § ¨ © ¥ ª « ¦ ¬ ® ¯ ° ± ² ³ ´ µ ¶ · ¸ ¹ º ² ¬ » ¯ ¼ ± ½ ¹ ¾ · ¿ À Á Á ¯ Â ± ² µ ´ ³ µ º ¿ Ã Á Ä Å Å Æ Ç Æ À Ã » È À » Æ À Ã Ç ¿ Ã Ä È À É ÊË Ì Ë È É Ä Ã Ä Í Æ » È À Ã Ç È Î

83

Ï ¿ ® Î Æ Ð Ê Ñ Ê Ò À Ã Ä ® ¿ » Ã Æ Ç Ä ¿ Î È Å Ó Ç Æ Æ À É Ô À Ã Õ Æ É Ä Ö Æ Á Ò Ó × Ë É ¿ Ã Á Ä Å Å Æ Ç Æ À Ã » È À » Æ À Ã Ç ¿ Ã Ä È À É¿ Ó ¿ Ä À É Ã Ø ¿ Ã Õ È Ó Æ À Ä » ® ¿ » Ã Æ Ç Ä ¿ Î É Ø Æ » Ä Æ É*Zone of inhibition (mm)

Concentrations ( g mL–1)

Ag NPs P

Label Bacteria

5 10 15 10

Gram-negative

a K. pneumoniae 5.0 0.3 7.0 0.3 9.5 0.6 6.0 0.5

b P. aeruginosa 6.5 0.6 9.5 0.5 14.5 0.6 6.0 0.5

c E. coli 6.0 0.6 6.0 1.0 8.0 1.0 5.0 0.5

Gram-positive

d S. aureus 5.5 0.3 6.0 0.3 6.5 0.5 5.0 0.2 Ù Ú Û Ü Ý Ü Þ ß à Ú á Ý â Ú ã ä Ù å æ Ü ã ã Ü æ ß Ý ß â ä æ æ å æ Ü à â Ú ç â è æ é æ Ü ã ã Ü ã Ü Ý ß â ä ç æ ê ë ì íî ï Ú á ß Ú ð Ü á ñ Ü ò Ü Ý Ü Ú á Þ è ã ó ß Û è â ß ß ô õ â ß Û Û ß ö è Û æ ß è á Ú ð Ý â Ü õ ã Ü à è Ý ß æ ß è Û ó â ß æ ß á Ý Û Û Ý è á ö è â ö ö ß Þ Ü è Ý Ü Ú á í

84

Ë Î ¿ Ã Æ Ð Ê Ð Ê Ò À Ã Ä Å ÷ À Ó ¿ Î ¿ » Ã Ä Í Ä Ã Ô È Å Ò Ó × Ë É ¿ Ó ¿ Ä À É Ã ¿ ¯ ø ± ù ¾ ² ú µ º ¬ ® ¯ ø ± ¸ · ¶ ³ ´ ¬ » ¯ ø ± ù µ û · ¶ ² ü µ º ¿ À Á Á ¯ ý ± ¶ þ ÿ º ³ µ û ¿ Ã Á Ä Å Å Æ Ç Æ À Ã » È À » Æ À Ã Ç ¿ Ã Ä È À É ÊË Ì Ë È É Ä Ã Ä Í Æ » È À Ã Ç È Î

85

Ï ¿ ® Î Æ Ð Ê � Ê Ò À Ã Ä Å ÷ À Ó ¿ Î È Å Ó Ç Æ Æ À É Ô À Ã Õ Æ É Ä Ö Æ Á Ò Ó × Ë É ¿ Ã Á Ä Å Å Æ Ç Æ À Ã » È À » Æ À Ã Ç ¿ Ã Ä È À É¿ Ó ¿ Ä À É Ã Ø ¿ Ã Õ È Ó Æ À Ä » Å ÷ À Ó ¿ Î É Ø Æ » Ä Æ É*Zone of inhibition (mm)

Concentrations ( g mL–1)

Ag NPs P

Label Fungi

5 10 15 10

a A. flavus 10.3 0.6 10.6 0.1 17.0 0.6 5.5 0.6

b A. niger 11.0 0.5 11.0 0.5 14.0 0.6 9.0 0.6

c A. fumigatus 11.3 0.5 13.5 0.6 15.0 0.5 7.3 0.6

d M. gypseum 11.3 0.6 14.3 0.6 15.0 0.5 9.3 0.6 Ù Ú Û Ü Ý Ü Þ ß à Ú á Ý â Ú ã ä Ù å æ Ü ã ã Ü æ ß Ý ß â ä æ æ å æ Ü à â Ú ç â è æ é æ Ü ã ã Ü ã Ü Ý ß â ä ç æ ê ë ì íî ï Ú á ß Ú ð Ü á ñ Ü ò Ü Ý Ü Ú á Þ è ã ó ß Û è â ß ß ô õ â ß Û Û ß ö è Û æ ß è á Ú ð Ý â Ü õ ã Ü à è Ý ß æ ß è Û ó â ß æ ß á Ý Û Û Ý è á ö è â ö ö ß Þ Ü è Ý Ü Ú á í

86

4.2. GREEN SYNTHESIS OF ZINC OXIDE NANOPARTICLES

4.2.1. Photographs of green tea plant, dried leaves and synthesized ZnO NPs

The photograph of the green tea plant, leaves in dried form

and synthesized ZnO NPs is shown in Plate 4.5. The pale-white colour

of the ZnO NPs arise due to capping action of biomolecules on the

surface of the nanoparticles.

4.2.2. Preliminary screening test of green tea

Phytochemicals present in green tea extracts, qualitatively

detected using standard screening tests are presented in Table 4.8.

The results of the tests are shown in Plate 4.6. Intense colour

changes reveal the presence of terpenoids, polyphenols, proteins and

flavonoids as major constituents in the green tea sample.

4.2.3. GC-MS analysis of green tea

One of the six green tea samples (MOON tea) studied for

the antioxidant potentials (Section 4.3.1) was used to green synthesize

ZnO nanoparticles. The chromatogram obtained from GC-MS analysis,

the peak report and the major phytochemicals identified for this green

tea samples are provided in Figs. 4.10 – 4.15 and Tables 4.12 - 4.18

respectively.

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S. No. Phytochemical Result (Qualitative)

1 Tannin ---

2 Saponin ---

3 Steroids +

4 Terpenoids +++

5 Alkaloids ++

6 Amino acids ----

7 Glycoside ++

8 Polyphenols +++

9 Protein +++

10 Flavonoids +++

89

4.2.4. UV-Vis spectrum of ZnO nanoparticle

Confirmation of the synthesized ZnO product in nano-scale

was done by recording UV-Vis spectrum. The highly blue-shifted

absorption maximum occurring around 325 nm is an indication of

nano-sized ZnO particles. For bulk ZnO the absorption maximum is

around 385 nm approximately. The UV-Vis spectrum of ZnO particles

is shown in Fig. 4.6.

4.2.5. XRD analysis of ZnO NPs

The XRD spectra of the ‘as prepared’ and calcined at 100 C

ZnO NPs are shown in Fig. 4.7. Calcination at 100 C is essential for

complete removal of water and to obtain higher crystallinity. The prominent

peaks corresponding to the diffraction planes (100), (002), (101), (102),

(110), (103) and (112) agree well with the JCPDS Card No. 36-1451,

confirming the hexagonal Wurtzite structure of the ZnO NPs. The

average particle size (D) of synthesized nanoparticles was calculated

using the well known Scherrer formula [24] is nearly 16 nm. The XRD

parameters are given in Table 4.9.

4.2.6. FE-SEM morphology of ZnO NPs

The morphology of the green synthesized ZnO NPs is

illustrated in Fig. 4.8. Nanoparticles are spherical in shape and

uniformly distributed.

90

0 � � � � � / � 1 2 3 2 � � � 4 � � � � � � � � � � � � � � 5 � 6 7 � 8 9 �

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91

Peak No. Position ( 2 ) FWHM, ( ) Particle size, D (nm)

1 31.7 0.49 16.8

2 34.4 0.41 20.2

3 36.2 0.51 16.3

4 47.5 0.62 15.98

5 56.5 0.65 13.30

6 62.8 0.71 13.07

Mean 16

92

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93

4.2.7. FT-IR spectra of-green tea extract and ZnO NPs

The FT-IR spectrum of the green tea extract and that of

the synthesized ZnO NPs are shown in Fig. 4.9.

In the IR spectrum of green tea, the band at 3394 cm-1 is

due to stretching vibrations of O–H groups in water, alcohol and

phenols and N–H stretching in amines. The C–H stretch in alkanes

and O–H stretch in carboxylic acid appear at 2926 and 2864 cm-1

respectively. The strong band at 1627 cm-1 is attributed to the C=C

stretch in aromatic ring and C=O stretch in polyphenols. The C–N

stretch of amide-I in protein gives the band at 1396 cm-1. The C–O–C

stretching in polysaccharides gives a band at 1741 cm-1 and C–O

stretching in amino acid causes a band at 1037 cm-1. Finally the

weak band at 819 cm-1 is the result of C–H out of plane bending.

Thus from the IR spectrum it can be observed that green tea sample

is rich in polyphenols, carboxylic acid, polysaccharide, amino acid

and proteins.

94

0 � � � � � H � 0 � 3 I < � 4 � � � � � � � > � � � � � � � � � � � � � � � � � 6 � > � � � � � � � 5 � 6 7 � 8 9 �

95

4.2.8. Antibacterial assays

The antibacterial activity of ZnO NPs against the studied

pathogenic strains are shown in Plate 4.7. The values of zone of

inhibition obtained from the assay are presented in Table 4.10. All

Gram-negative bacteria had shown good sensitivity towards the green

synthesized ZnO NPs for the concentration 20 g mL–1. It is quite

interesting to note that all bacterial species tested in this study

showed resistance to the synthetic antibiotic drug which in turn

indicates the better antibacterial activity of the ZnO NPs than the

commercially available synthetic drug.

4.2.9. Antifungal assay

Regarding the antifungal activity, all four fungal strains

used in this study are found to be sensitive to the green synthesized

ZnO NPs as well as to the commercially available antifungal drug

Itraconozole. The antifungal activities of ZnO NPs are shown in

Plate 4.8 and the zone of inhibition values are presented in Table 4.11.

The fungal species Aspergillus flavus had shown medium sensitivity to

ZnO NPs with a concentration of 20 g mL–1, whereas the remaining

three fungal species showed good sensitivity to the ZnO NPs

concentration of 20 g mL–1.

96

� � � � � � : � J � � � � � � � � � � � � � � � � K � � � � 7 � 8 9 � � � � � � � � � > L M N O P Q R S P T U O VW X Y Z [ \ ] ^ _ ` a b c [ V d X e Z f b g ` h i j j X k Z [ ^ ] \ ^ c h l j m n n o p o i l d q i d o i l p h l m q i r st u t q r m l m v o d q i l p q w x y u y o z h l m v o d q i l p q w

97

{ h W w o | s } ~ s � i l m W h d l o p m h w h d l m v m l � q n z p o o i r � i l � o r m � o j � i � y t r h l j m n n o p o i ld q i d o i l p h l m q i r h z h m i r l � h l � q z o i m d W h d l o p m h w r � o d m o r q n d w m i m d h w r q � p d o rZone of inhibition (mm)

Concentration of ZnO NPs ( g mL–1)Label Bacteria

5 10 20 P

Gram-negative

a K. pneumoniae – – 10 –

b P. aeruginosa – – 3 –

c E. coli – – – –

Gram-positive

d S. aureus – 2 5 – � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �

98

t w h l o | s s � i l m n � i z h w h d l m v m l � q n � i � y t r h z h m i r l h X ¡ Z ¢ ^ £ ` _ [ ¤ ^ c V W X Y \ a ` f ` g g ` ^ £ r � s Vd X ¡ Z ¢ g [ ¥ ^ c h i j j X ¡ Z a ` _ \ ] h l j m n n o p o i l d q i d o i l p h l m q i r s t u t q r m l m v o d q i l p q wy u y o z h l m v o d q i l p q w

99{ h W w o | s } } s � i l m n � i z h w h d l m v m l � q n z p o o i r � i l � o r m � o j � i � y t r h l j m n n o p o i ld q i d o i l p h l m q i r h z h m i r l � h l � q z o i m d W h d l o p m h w r � o d m o r q n d w m i m d h w r q � p d o rZone of inhibition (mm)

Concentration of ZnO NPs ( g mL–1)Label Fungi

5 10 20 P

a A. fumigatus – – 5 3

b Penicillium sp. – – 7 2

c A. flavus – – 3 3

d A. niger – – 3 2 � � � � � � � � � � � � � � � � � � � � � ¦ � � � � § � � � � � � � � � � � � �4.3. PHYTOCHEMICAL ANALYSIS OF INDIAN GREEN TEAS

4.3.1. GC-MS analysis of green teas

The chromatograms of the six Indian green teas are

presented in Figs. 4.10 through 4.15. The corresponding peak reports

representing the detected phytochemicals in the green tea samples are

provided in Tables 4.12 through 4.17. From these Tables the major

phytochemicals identified with peak area percentage >1 are furnished

in Tables 4.18 through 4.23. Finally, the important phytochemicals

with health beneficial potentials are short listed and presented in

Table 4.24.

100

¨ � p q © h l q z p h © q n ª � � y l o h

101

Peak# R.Time Area Area% Name

3.179 429249 0.57 6-Azabicyclo[3.2.1]octane

4.191 213357 0.28 1-(N,N-DIETHYL)AMINOPROPYNE

4.638 308979 0.41 1,5-ANHYDRO-6-DEOXYHEXO-2,3-DIULOSE

5.540 134717 0.18 2-(HYDROXYMETHYL)-2-METHYL-1-PYRROLIDINECA

6.259 56189 0.07 Decane, 3,7-dimethyl

6.878 83478 0.11 Decane, 3,7-dimethyl

7.757 83077 0.11 1-Undecanol

8.136 13940180 18.40 1,2,3-BENZENETRIOL

9.092 101901 0.13 Nonane, 5-(2-methylpropyl)

9.427 67326 0.09 PHENOL, 2,6-BIS(1,1-DIMETHYLETHYL)-4-METHYL-, M

9.594 56928 0.08 Benzoic acid, 4-ethoxy-, ethyl ester



10.260 88087 0.12 1-Tridecene

11.158 10575793 13.96 1,3,4,5-TETRAHYDROXY-CYCLOHEXANECARBOXYLI

11.622 74425 0.10 Dodecane, 2,6,11-trimethyl



12.219 86505 0.11 DECANOIC ACID

12.515 42399 0.06 Cyclotetradecane

12.571 52148 0.07 7-Oxabicyclo[4.1.0]heptane, 1,5-dimethyl

12.942 71115 0.09 9-ANTHRACENOL, 1,4,4A,5,8,8A,9,9A,10,10A-DECAHYD

13.022 52262 0.07 3,7,11,15-Tetramethyl-2-hexadecen-1-ol

13.520 35035587 46.25 Caffeine




15.564 95617 0.13 7,10-Hexadecadienoic acid, methyl ester

15.633 305636 0.40 9,12,15-Octadecatrienoic acid, methyl ester, (Z,Z,Z)

15.742 1015236 1.34 Phytol

15.825 60320 0.08 HEXADECANOIC ACID, METHYL ESTER

16.027 4082911 5.39 9,12,15-Octadecatrienoic acid, (Z,Z,Z)


18.835 298639 0.39 Cyclohexanol, 4-[(trimethylsilyl)oxy]-, cis

18.961 154801 0.20 PREGNANE, SILANE DERIV.


19.441 58928 0.08 1,2-BENZENEDICARBOXYLIC ACID

19.844 94901 0.13 Olean-12-ene-3,28-diol, (3.beta.)

20.208 196573 0.26 Cyclopropane, 1,1-dichloro-2,2,3,3-tetramethyl

20.283 114145 0.15 ETHYL (9Z,12Z)-9,12-OCTADECADIENOATE #

20.393 113266 0.15 1,3,5-Trisilacyclohexane

21.267 94317 0.12 TRICYCLO[20.8.0.0E7,16]TRIACONTAN, 1(22),7(16)-DIE

21.478 42001 0.06

44 21.722 78484 0.10 Squalene

45 25.942 505852 0.67 Vitamin E

46 29.984 661584 0.87 Stigmasterol

75759248 100.00

102

¨ � p q © h l q z p h © q n { � y l o h

103{ h W w o | s } « s t o h ¬ p o � q p l q n { � y l o h d q © � q � i j rPeak# R.Time Area Area% Name

3.190 213737 0.44 5-METHYL-5,6-DIHYDRO-2(1H)-PYRIDINONE

4.192 247017 0.51 1,2,5,6-Tetrahydropyridin-2-one, 5-methyl

4.425 117973 0.24 3-Azetidin-1-yl-propionic acid, methyl ester

4.644 141301 0.29 2,3-DIHYDRO-3,5-DIHYDROXY-6-METHYL-4H-PYRAN


6.265 55549 0.11 Butane, 2,2-dimethyl

6.883 92787 0.19 Decane, 3,7-dimethyl

7.758 104253 0.21 Pentafluoropropionic acid, octyl ester

8.127 7955212 16.28 1,2,3-BENZENETRIOL


9.207 38178 0.08 1-CHLOROHEXADECANE



10.261 88262 0.18 1-Tridecene


11.486 43687 0.09 1,2,4-BUTANTRIOL, 4-O-OCTYL



12.514 44508 0.09 Cyclotetradecane

13.489 27927732 57.17 Caffeine




14.547 35770 0.07 n-Heptadecanol-1

15.560 90492 0.19 9,12-Octadecadienoic acid (Z,Z)


15.739 164577 0.34 2-HEXADECEN-1-OL, 3,7,11,15-TETRAMETHYL-, [R-[R*


16.012 1393467 2.85


18.142 54843 0.11 Nonadecane

18.829 112398 0.23 6-Ethyl-3-trimethylsilyloxydecane

18.946 173062 0.35 Oxalic acid, 3,5-difluorophenyl tetradecyl ester


19.436 55082 0.11 1,2-BENZENEDICARBOXYLIC ACID, DIISOOCTYL EST

19.708 76496 0.16 Heptadecane, 2,6,10,15-tetramethyl


21.714 67917 0.14 Squalene

25.924 120810 0.25 Vitamin E

29.944 230808 0.47 Trihydroxycholanic acid, (3.alpha., 7.beta., 12.alpha.)

48853778 100.00

104

® ¯ ° ± ² ³ ° ´ ¯ ² ± ° µ ¶ · ³ ¸ ²

105· ² ¹ º ¸ » ¼ ½ » ¼ ¾ ¸ ² ¿ ¯ ¸ À ° ¯ ³ ° µ ¶ · ³ ¸ ² Á ° ± À ° Â Ã Ä ÅPeak# R.Time Area Area% Name



5.549 61451 0.07 4(1H)-Pyrimidinone, 6-hydroxy-

6.261 60165 0.07 Butane, 2,2-dimethyl

6.882 94796 0.11 Decane, 3,7-dimethyl

7.759 78978 0.09 1-Tridecene

8.141 25575712 28.36 1,2,3-BENZENETRIOL



10.260 71745 0.08 1-Tridecene



12.096 80875 0.09 Dodecane, 4,6-dimethyl

13.564 50942083 56.48 1,3,7-TRIMETHYL-3,7-DIHYDRO-1H-PURINE-2,6-DIONE




15.737 773773 0.86 Phytol




18.950 75521 0.08 Butanedioic acid, 2-hydroxy-2-methyl-, dimethyl ester



20.567 166821 0.18 (Z)6,(Z)9-Pentadecadien-1-ol

20.754 208632 0.23 Octadecanoic acid, 2,3-dihydroxypropyl ester

90190842 100.00

106

® ¯ ° ± ² ³ ° ´ ¯ ² ± ° µ · Æ · ³ ¸ ²

107· ² ¹ º ¸ » ¼ ½ Ç ¼ ¾ ¸ ² ¿ ¯ ¸ À ° ¯ ³ ° µ · Æ · ³ ¸ ² Á ° ± À ° Â Ã Ä ÅPeak# R.Time Area Area% Name

4.650 169104 0.21 2,3-DIHYDRO-3,5-DIHYDROXY-6-METHYL-4H-PYRAN

6.264 63534 0.08 Butane, 2,2-dimethyl

6.527 104454 0.13 NONANE, 3,7-DIMETHYL

6.883 98655 0.12 Decane, 3,7-dimethyl

7.708 28881 0.04 DL-Proline, 5-oxo-, methyl ester

7.760 61530 0.08

7.859 1297151 1.62 Tetradecane

8.146 17281888 21.55 1,2,3-BENZENETRIOL

9.134 589430 0.74 PENTADECANE



10.263 67130 0.08 1-Undecanol

10.344 143181 0.18 Tetradecane


11.489 45360 0.06 2-Undecene, 5-methyl



12.568 166863 0.21 5-ISOPROPENYL-2-METHYL-7-OXA-BICYCLO[4.1.0]HE







15.740 155362 0.19 2-HEXADECEN-1-OL, 3,7,11,15-TETRAMETHYL-, [R-[R*


16.014 1599955 2.00 cis,cis,cis-7,10,13-Hexadecatrienal


16.398 70989 0.09 17-Octadecynoic acid

18.832 48886 0.06 trans-9-Octadecenoic acid, trimethylsilyl ester

18.957 72319 0.09 Floxuridine



80184244 100.00

108

® ¯ ° ± ² ³ ° ´ ¯ ² ± ° µ È É Ê ³ ¸ ²

109· ² ¹ º ¸ » ¼ ½ Ë ¼ ¾ ¸ ² ¿ ¯ ¸ À ° ¯ ³ ° µ È É Ê ³ ¸ ² Á ° ± À ° Â Ã Ä ÅPeak# R.Time Area Area% Name

3.194 192821 0.39 6-Azabicyclo[3.2.1]octane


4.433 152281 0.31 1-(2-Hydroxyethyl)-1,2,4-triazole

4.644 305056 0.62 1,5-ANHYDRO-6-DEOXYHEXO-2,3-DIULOSE

5.550 78518 0.16 4(1H)-Pyrimidinone, 6-hydroxy-

6.263 47682 0.10 Hexane, 3,3-dimethyl

6.883 98672 0.20 Decane, 3,7-dimethyl

7.760 93674 0.19 1-Tridecene

8.128 8502784 17.41 1,2,3-BENZENETRIOL

8.649 87500 0.18 1,6,10-DODECATRIENE, 7,11-DIMETHYL-3-METHYLEN




9.767 77480 0.16 Sulfurous acid, hexyl octyl ester

10.033 47175 0.10 HEXANE, 3,3,4-TRIMETHYL

10.263 83867 0.17 1-Undecanol


11.489 40592 0.08 Hexadecane


12.098 93455 0.19 Dodecane, 4,6-dimethyl

12.512 60180 0.12 7-Heptadecene, 1-chloro








16.016 1173311 2.40 cis,cis,cis-7,10,13-Hexadecatrienal


18.834 98067 0.20 6-Ethyl-3-trimethylsilyloxydecane

18.957 93835 0.19 Tridecanoic acid, 3-hydroxy-, ethyl ester



48833185 100.00

110

® ¯ ° ± ² ³ ° ´ ¯ ² ± ° µ Ì Í Í ³ ¸ ²

111· ² ¹ º ¸ » ¼ ½ Î ¼ ¾ ¸ ² ¿ ¯ ¸ À ° ¯ ³ ° µ Ì Í Í ³ ¸ ² Á ° ± À ° Â Ã Ä ÅPeak# R. Time Area Area% Name


3.771 794821 0.77 Cyclopentane, 1-acetyl-1,2-epoxy-


4.640 2198879 2.12 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl


5.911 148928 0.14 1,2,3-Propanetriol, monoacetate

6.486 319524 0.31

7.760 109192 0.11 1-Tridecene

8.127 3744334 3.62


9.645 268760 0.26 Nonane, 5-butyl




12.568 141270 0.14 2(4H)-BENZOFURANONE, 5,6,7,7A-TETRAHYDRO-6-HY




15.558 47963 0.05 (Z)6,(Z)9-Pentadecadien-1-ol

15.608 160823 0.16 9-Octadecenoic acid (Z)-, methyl ester

15.737 328082 0.32 Phytol





20.759 191200 0.18 Octadecanoic acid, 2,3-dihydroxypropyl ester

25.925 111883 0.11 dl-.alpha.-Tocopherol

29.936 607158 0.59 Stigmasterol

103489825 100.00

112

· ² ¹ º ¸ » ¼ ½ Ï ¼ Ð ² Ñ ° ¯ À ® Ò ³ ° Á ® ¸ ± Ó Á ² º Å Ô ² ¯ ¸ ² À ¸ ¯ Á ¸ Ã ³ ² ´ ¸ Õ ½ Ö ° µ Ð É É × ³ ¸ ²Peak R.T.

Area(%)

Molecularformula

M. wt. Name of the compound Type Therapeutic use

8 8.135 18.40 C6H6O3 126.11 1,2,3-Benzenetriol Pyrogallol

AntioxidantAntiseptic fungicide,

Antidermatitic insecticide, candidicide

15 11.158 13.96 C7H12O6 192.16 1,3,4,5-Tetrahydroxycyclo-

hexanecarbonyl Quinic acid

Antimicrobial, anti-inflammatory

induces antioxidant

24 13.520 46.25 C8H10N4O2 194.19 Caffeine Alkaloid Both pro and antioxidant

25 13.746 3.00 C7H8N4O2 180.16 1H-Purine-2,6-dione,

3,7-Dihydro-3,7-Dimethyl (Theobromine)

Xanthinealkaloide

Vasodilaton both pro-and antioxidant

natural diuretic diuretic

27 14.299 2.90 C16H32O2 256.42 n-Hexadecanoic acid Palmitic acid

AntioxidantNematicide pesticide

hemolytic anti-inflammatory

hemolytic

30 15.742 1.34 C20H48O 296.53 Phytol Diterpene

AntioxidantAntimicrobial,

anticancer, diuretic

32 16.027 5.39 C18H30O2 278. 9,12,15-Octadecatrienoic acid

(z,z,z) -linolenic acid

Antiarthritic, antihistaminic, anticoroanary, antiandrogenis, antinematicide,

anticancer, antibacterial

113

· ² ¹ º ¸ » ¼ ½ Ø ¼ Ð ² Ñ ° ¯ À ® Ò ³ ° Á ® ¸ ± Ó Á ² º Å Ô ² ¯ ¸ ² À ¸ ¯ Á ¸ Ã ³ ² ´ ¸ Õ ½ Ö ° µ · Ì × ³ ¸ ²Peak R.T.

Area(%)

Molecularformula

M.wt.

Compound name Type/nature Therapeutic use

9 8.127 16.28 * * 1,2,3-Benzenetriol Pyrogallol *

15 11.022 9.64 * * 1,3,4,5-Tetrahydroxycyclo-

hexanecarbonyl Quinic acid *

20 13.480 57.17 * * Caffeine Alkaloid *

21 13.698 3.25 * * 1H-Purine-2,6-dione,

3,7-Dihydro-3,7-Dimethyl (Theobromine)

Xanthine, Alkaloid

*

23 14.289 1.53 * * n-Hexadecanoic acid Palmitic acid *

34 19.141 1.19 C18H36O2 284 9,12,15-Octadecatrienoic acid

(z,z,z) Palmitic acid,

ethyl ester

Antioxidant, nematicide pesticide, hypocholesterolemic,

hemolytic Ù Ú Û Ü Ý Þ ß à á â ã ä ß å æ å

114

· ² ¹ º ¸ » ¼ ç è ¼ Ð ² Ñ ° ¯ À ® Ò ³ ° Á ® ¸ ± Ó Á ² º Å Ô ² ¯ ¸ ² À ¸ ¯ Á ¸ Ã ³ ² ´ ¸ Õ ½ Ö ° µ ¶ · ³ ¸ ²Peak R.T.

Area(%)

Molecular formula

M.wt.

Compound name Type/nature Therapeutic

use


11 11.139 1.67 * * 1,3,4,5-Tetrahydroxy-cyclohexanecarboxyl

Quinic acid *

14 13.564 56.48 * *

Caffine

1,3,7-Trimethyl-3,7-dihydro-1H-Purine-2,6-

dione

Alkaloid *

15 13.745 4.33 * * 1H-Purine-2,6-dione,

3,7-Dihydro-3,7-Dimethyl Xanthine,Alkaloid

*


20 16.024 2.07 * * 9,12,15-Octadecatrienoic

acid (z,z,z) Lainolenic

acid *

23 19.134 1.03 * * Hexadecanoic acid

ethylester

Palmitic acid, ethyl

ester *Ù Ú Û Ü Ý Þ ß à á â ã ä ß å æ å

115

· ² ¹ º ¸ » ¼ ç ½ ¼ Ð ² Ñ ° ¯ À ® Ò ³ ° Á ® ¸ ± Ó Á ² º Å Ô ² ¯ ¸ ² À ¸ ¯ Á ¸ Ã ³ ² ´ ¸ Õ ½ Ö ° µ · Æ · ³ ¸ ²Peak R.T.

Area(%)

Molecular formula

M.wt.


use

7 7.859 1.62 * * Tetradecane Alkane *


14 11.080 8.35 * * 1,3,4,5-Tetrahydroxy-cyclohexanecarboxyl

Quinic acid *

19 13.552 57.52 * *

Caffine

1,3,7-Trimethyl-3,7-dihydro-1H-Purine-2,6-

dione

Alkaloid *

20 13.730 3.57 * * 1H-Purine-2,6-dione,

3,7-Dihydro-3,7-Dimethyl

Xanthine,Alkaloid

*

22 14.293 1.18 * n-Hexadecanoic acid Palmitic acid *

27 16.014 2.00 C6H26O 234 Cis, cis, cis-7,10,13-

Hexadecatriental Alcohol *

116

· ² ¹ º ¸ » ¼ ç ç ¼ Ð ² Ñ ° ¯ À ® Ò ³ ° Á ® ¸ ± Ó Á ² º Å Ô ² ¯ ¸ ² À ¸ ¯ Á ¸ Ã ³ ² ´ ¸ Õ ½ Ö ° µ È É Ê ³ ¸ ²Peak R.T.

Area(%)

Molecular formula

M. wt. Compound name Type/nature Therapeutic

use


17 11.024 10.10 * * 1,3,4,5-

Tetrahydroxy-cyclohexanecarboxyl

Quinic acid *

22 13.490 59.79 * * 1,3,7-Trimethyl-3,7-dihydro-1H-Purine-

2,6-dione Caffeine *

23 13.70 2.23 * * 1H-Purine-2,6-dione,

3,7-Dihydro-3,7-Dimethyl

Xanthine,Alkaloid

*


29 16.016 2.40 C6H26O 234 Cis, cis, cis-7,10,13-

Hexadecatriental Fatty

aldehydes –é ê ë ì í î ï ð ï ñ ò ï ó ò ô

117

· ² ¹ º ¸ » ¼ ç õ ¼ Ð ² Ñ ° ¯ À ® Ò ³ ° Á ® ¸ ± Ó Á ² º Å Ô ² ¯ ¸ ² À ¸ ¯ Á ¸ Ã ³ ² ´ ¸ Õ ½ Ö ° µ Ì Í Í ³ ¸ ²Peak R.T.

Area(%)

Molecular formula

M.wt.


use

1 3.189 2.13 – – 5-Methyl-5,6-dihydro-

2(1H)-pyridinone Unknown –

3 4.185 1.98 – – 1-(N,N-

Dimethyl)aminoporpyne Unknown –

4 4.640 2.12 * * 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydro-6-

methyl

Flavonoid fraction

*

12 11.441 31.46 * * 1,3,4,5,-tetrahydroxy-cyclohexanecarboxyl

Quinic acid *

16 13.547 42.75 * * 1,3,7,-trimethyl-3,7-

dihydro-1H-purine-2,6-dione

Coffeinealkaloid

*

17 13.849 4.16 * * 1H-Purine-26-Dione, 3,7-

Dihydro-3,7-dimethyl Xanthine,Alkaloid

*


23 16.026 1.50 * * 9,12,15-Octadecatrienoic

acid (z,z,z) Linolenic

acid *é ê ë ì í î ï ð ï ñ ò ï ó ò ô

118

· ² ¹ º ¸ » ¼ ç » ¼ Ð ² Ñ ° ¯ ® ¸ ² º ³ ® ¹ ¸ Ã ¸ µ Ó Á Ó ² º À ® Ò ³ ° Á ® ¸ ± Ó Á ² º Å ° µ ´ ¯ ¸ ¸ Ã ³ ¸ ² Å ² ± À º ¸ Å² Ã ² º Ò ö ¸ Ä ¹ Ò ¶ ÷ Ð ÍPeak area (%) of green tea samples

Compounds

MOON TAN GT TET KOL ASS

Pyrogallol 18.40 16.28 28.36 21.55 17.61 -

Quinic acid 13.96 9.64 1.67 8.35 10.10 31.46

Caffeine (alkaloid) 46.25 57.17 56.48 57.52 59.79 42.75

Xanthine (Alkaloid) 3.00 3.25 4.33 3.57 2.23 4.16

Palmitic acid 2.90 1.53 2.11 1.18 1.17 3.58

Palmitic acid - 1.19 1.03 - - -

Phytol ethyl ester 1.34 - - - - -

-linolenic acid 5.39 - 2.07 - - -

Total 91.24 89.06 96.05 92.17 90.9 83.45 ø ù ø Þ ß Ý ú á ß Û Ý ú á Þ ß á ß û á ß Þ

119 ® ¸ ± Ó Á ² º Å ³ ¯ Â Á ³ Â ¯ ¸ ° µ Å ¸ º ¸ Á ³ ¸ Ä Á ° ± À ° Â Ã Ä Å ° µ ´ ¯ ¸ ¸ Ã ³ ¸ ² Å ² ± À º ¸ Å Ô ü ° µ ² ¯ ¸ ² Õ ½ ¼ è è Ö

121

4.3.2. FT-IR analysis of phytochemical green teas

The FT-IR spectra of the six green tea samples are shown

in Fig. 4.16 (a) through (f).

123

ý Ó ´ ¼ » ¼ ½ Ë ¼ ý · ÷ þ ÿ Å À ¸ Á ³ ¯ ² ° µ ´ ¯ ¸ ¸ Ã ³ ¸ ² Å ² ± À º ¸ Å � Ô ² Ö Ð É É × � Ô ¹ Ö · Ì × � Ô Á Ö ¶ · �Ô Ä Ö · Æ · � Ô ¸ Ö È É Ê ² Ã Ä Ô µ Ö Ì Í Í

124

The vibrational band assignment for the prominent peaks

and the chemical compounds identified are provided in Table 4.25. · ² ¹ º ¸ » ¼ ç Ç ¼ þ Ã µ ¯ ² ¯ ¸ Ä � Ó ¹ ¯ ² ³ Ó ° Ã ² º ¹ ² Ã Ä Å ° µ ´ ¯ ¸ ¸ Ã ³ ¸ ² Å ² ± À º ¸ ÅWavenumber

(cm–1)Vibration band/group Chemical compound Reference

3270 ~ 3320 O–H stretch H–Bonded

Phenols, alcohols

Awwad et al. (2013) Umashankari et al. (2012) Vanaja et al. (2013) Theivasanthi et al. (2013)

C–H stretch (asym.) Alkanes Vanaja et al. (2013) 2946

O–H stretch Carboxylic acid Sharmin et al. (2013)

2833 C–H stretch (sym.) Alkanes Umashankari et al. (2012) Arokiyaraj et al. (2013)

Flavonoids Heneczkowski et al. (2001) C=O stretch (carbonyls)

Polyphenols, catechins Rajathi and Sridhar (2013) 1629 ~ 1663

C=C stretch Aromatics Kong and Yu (2007)

1449 C–C stretch (in ring) Aromatics Umashankari et al. (2012) Heneczkowski et al. (2001) Mallikarjuna et al. (2012)

1239 C–N stretch Aliphatic amines Heneczkowski et al. (2001) Nagajyothi et al. (2013)

1113 C–O stretch Alcohols, esters, carboxylic acids

Mallikarjuna et al. (2012)

C–O stretch Alcohols, esters, carboxylic acids

Rajathi and Sridhar (2013) Mallikarjuna et al. (2012)

C–N stretch Aliphatic amines Nagajyothi et al. (2013) 1014 ~ 1019

C–OH stretch Secondary alcohols Jayaseelan et al. (2013)

The FT-IR spectra of the green tea samples clearly reveals

the presence of polyphenols, flavonoids, amines as major phytochemicals.

This fact enables to speculate higher antioxidant potentials of the

green tea samples.

125

4.4. ANTIOXIDANT POTENTIALS OF INDIAN GREEN TEAS

4.4.1. Total phenolic contents-FCR method

The optical density values representing the total phenolic

contents (TPC) of the six green tea samples and the standard galic

acid for various concentrations (15, 30, 60, 125 and 250 mg mL-1)

measured using spectrophotometer are provided in Table 4.26.

The dose response curves representing TPC of the green tea are shown

in Fig. 4.17. · ² ¹ º ¸ » ¼ ç Ë ¼ É À ³ Ó Á ² º Ä ¸ Ã Å Ó ³ Ò � ² º Â ¸ Å ¯ ¸ À ¯ ¸ Å ¸ Ã ³ Ó Ã ´ ³ ° ³ ² º À ® ¸ Ã ° º Ó Á Á ° Ã ³ ¸ Ã ³ Å ° µ ´ ¯ ¸ ¸ Ã³ ¸ ² Å ² Ã Ä ´ ² ¯ º Ó Á ² Á Ó Ä µ ° ¯ � ² ¯ Ó ° Â Å Á ° Ã Á ¸ Ã ³ ¯ ² ³ Ó ° Ã ÅOptical density values

Green tea samples

Concentrationof extracts

g mL-1 Gallic acid (standard)


15 0.062 0.031 0.015 0.020 0.045 0.021 0.018

30 0.289 0.082 0.074 0.066 0.059 0.045 0.042

60 0.667 0.168 0.147 0.141 0.119 0.095 0.081

125 0.982 0.467 0.435 0.376 0.340 0.287 0.243

250 1.780 1.320 1.294 0.146 0.870 0.812 0.612 � � � � � � � � � � � � � � � � � � � �

126

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127

4.4.2. Total flavonoids-aluminium chloride method

The optical density values of the six green tea samples

and the standard quercetin for various concentrations (15, 30, 60,

125 and 250 g mL-1) are provided in Table 4.27. The dose response

curves representing TF of the green tea are shown in Fig. 4.18. ^ Z _ [ P J I ` L I a S Y G U Z [ b P T O G Y c N X W Z [ V P O R P S R P O P T Y G T H Y N Y Z [ X [ Z W N T N G b O N X Y \ PH R P P T Y P Z O X N R W Z R G N V O U N T U P T Y R Z Y G N T OOptical density values

Green tea samples

Concentration of extracts

g mL-1


15 0.042 0.035 0.026 0.021 0.018 0.017

30 0.084 0.072 0.054 0.042 0.03 0.025

60 0.145 0.131 0.112 0.088 0.071 0.062

125 0.264 0.245 0.215 0.171 0.155 0.132

250 0.561 0.543 0.473 0.399 0.384 0.324 � � � � � � � � � � � � � � � � � � � �

128

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129

4.4.3. DPPH-RSA assay

The optical absorbance percentage values of the six green

tea samples and the standard quercetin for various concentrations

(15, 30, 60, 125 and 250 g mL–1) are provided in Table 4.28. The

dose-response curves representing the DPPH-radical scavenging

activity (%) for the six green tea sample and the standard ascorbic acid

are shown in Fig. 4.19. The DPPH scavenging activity of all samples

appeared to depend on the extract concentration up to 60 g mL–1,

above which the activity approaches saturation level. This is due to

the quantity of DPPH used in the test reaction. The DPPH-radical

scavenging activity values expressed in ascorbic acid equivalents are

furnished in Table 4.30. ^ Z _ [ P J I ` e I M f f g R Z b G U Z [ O U Z W P T H G T H Z U Y G W G Y G P O N X Y \ P h T b G Z T H R P P T Y P Z OAbsorbance (%)

Green tea samples


g mL-1Ascorbic

acid(standard) MOON TAN GT TET KOL ASS

15 8 2 4 4 5 4 2

30 42 23 18 18 19 16 14

60 83 55 49 49 46 43 38

125 104 88 87 83 80 78 61

250 108 96 94 91 87 85 73 � � � � � � � � � � � � � � � � � � � �

130

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131

4.4.4. FRAP – assay

The optical density values of the six green tea samples and

the standard ascorbic acid for various concentrations (15, 30, 60, 125

and 250 g mL–1) are provided in Table 4.29. The dose-response

curves representing the ferric reducing power of the six green teas and

that of the standard ascorbic acid are illustrated in Fig. 4.20.

The FRAP activity values expressed in ascorbic acid equivalents are

furnished in Table 4.30. ^ Z _ [ P J I ` p I F t u f Z O O Z c W Z [ V P O N X Y \ P h T b G Z T H R P P T Y P Z OAbsorbance (%)

Green tea samples


g mL-1 Ascorbicacid

(standard)MOON TAN GT TET KOL ASS

15 0.08 0.08 0.06 0.07 0.04 0.04 0.05

30 0.16 0.13 0.12 0.13 0.09 0.08 0.06

60 0.26 0.21 0.20 0.18 0.14 0.12 0.08

90 0.32 0.28 0.27 0.21 0.22 0.18 0.15 � � � � � � � � � � � � � � � � � � � �

132

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133

^ Z _ [ P J I x w I M f f g R Z b G U Z [ O U Z W P T H G T H Z U Y G W G Y c q t y u s z X P R R G U R P b V U G T H Z T Y G N { G b Z T YS N | P R q F t u f s z Y N Y Z [ S \ P T N [ G U U N T Y P T Y q ^ f } s Z T b Y N Y Z [ X [ Z W N T N G b O q ^ F sN X Y \ P H R P P T Y P Z O Z ] S [ P OS.

No. Green tea sample

DPPH-RSA (mg AE g-1)

FRAP (mg AE g-1)

TPC (mg QAE g-1)

TF (mg QE g-1)

1 MOON 651 12.7* 731 20.2 267 22.6 27.4 1.2

2 TAN 610 14.1 683 15.5 238 25.0 24.2 1.0

3 GT 556 8.5 652 16.4 219 12 19.4 1.1

4 TET 521 9.9 424 10.0 191 18.3 15.4 1.2

5 KOL 492 8.5 381 11.5 159 9.9 11.9 1.7

6 ASS 385 7.8 319 12.0 135 1.4 9.8 1.6 � ~ � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � ��

134

4.5. CORRELATION STUDIES

4.5.1. Correlation analysis on the antioxidant potentials of Indian green teas with their phenolic and flavonoid contents

The relationship existing between the content of phenolic

compounds and antioxidant activities can be well understood by

carrying out correlation analysis. The linear and positive correlation

existing between the DPPH-RSA values and the TPC and TF values are

shown in Fig. 4.21. Similarly Fig. 4.22 illustrates the relationship

between the antioxidant activity assayed by FRAP method and the

TPC and TF values. Further the significant correlation observed

between the antioxidant activities assayed by the DPPH and FRAP

methods is illustrated in Fig. 4.23.

The results of the correlation analysis are presented in the

form of Pearson’s correlation coefficient (R) matrix in Table 4.31 � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � ¡ � � � � � � ¢ � � �£ � � � � ¤ � ¥ � � � � � � � � � � � ¦ � � � � � � � � � � ¥ � �Variables DPPH-RSA FRAP TPC TF

DPPH-RSA 1

FRAP 0.925 1

TPC 0.974 0.966 1

TF 0.957 0.966 0.990 1 § ¨ © © ª « ¬ ® ¨ ¯ ® ° ° ® ± ¯ ® ² ® ³ ¬ ¯ ¬ ´ µ ¶ µ · « ª ¸ ª « ¶

135

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136

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137

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138

4.5.2. Correlation analysis between FT-IR estimates and calorimetric data of the Indian green teas

From the infrared vibrational band assignments provided

in Table 4.25, three bands appear to have some relevance to the

antioxidant potentials of the green tea samples. The absorbance (%)

of these three vibrational bands considered for the correlation studies,

are presented in bold numbers in Table 4.32. Since the absorbance

data corresponding to the bands around 1448 and 1113 cm-1 did not

seem to contain any correlation, they were not considered for

correlation studies. � � � � � � � � º � ¹ � Á Â � � � � � � � � � � � � Ã � � � � � � � � � � ¾ £ � � � � � � � � � � � � � ¾ �� ¿ � ¦ � � � � � � � � � � ¥ � � �Absorbance (%)

Vibrational band (cm–1)


3270 ~ 3320* 34.11 32.23 27.56 29.25 31.34 24.20

1629 ~ 1663 22.36 19.18 12.34 12.05 10.68 4.52

1448 ~ 1450 12.39 12.20 15.48 13.52 15.81 15.46

1113 ~ 1148 12.71 13.02 12.32 11.31 9.83 12.10

1014 ~ 1019 23.06 38.71 52.61 52.53 70.67 75.62 Ä Å Æ Ç Æ È É Ê Ë Ì Í É Î Ï Ê Ð Ñ Ò Æ Ñ Ð Î Ò Ð Í Ó Ë Ñ Ô Ë Ñ Ñ Ð Ì Æ Ç È Ë É Æ É Æ Ì Õ Ò È Ò Ö

139

The calorimetric data namely the TPC, TF, DPPH-RSA and

FARP values and the FT-IR estimates of polyphenols, flavonoides

and amines considered for the correlation studies are provided in

Table 4.33. � � � � � � � � � � ¹ � Á Â � � � ¾ � � � � � � � � � � � � ¾ � � � � � � ¿ � ¦ � � � � � � � � � � ¥ � � �FT-IR data (absorbance %) Colorimetric data*

Green tea samples

Polyphenols Flavonoids Alcohols/amines

DPPH-RSA (mg AE g–1)

FRAP (mg AE g–1)

TPC(mg GAE g–1)

TF(mg QE g–1)

MOON 34.13 22.40 24.40 651.00 731.00 267.00 27.40

TAN 32.20 19.10 18.70 610.00 683.00 238.00 24.20

GT 27.50 12.40 52.60 556.00 652.00 219.00 19.60

TET 29.20 12.00 52.50 521.00 424.00 191.00 15.40

KOL 31.30** 10.60 70.60 492.00 381.00 159.00 11.90

ASS 24.20 4.50 75.40 385.00 319.00 135.00 9.80 Ä Å Æ Ç Æ Ó Ñ Ë Ï Æ Î Ç × Ë Ñ Ò Ø Ù Ë Ñ Ú Ô Ë Ï Ï Î É È Ô Æ Ç Ð Í Ð Ì Ò Ð Ù × Ð Ñ Ð ÖÄ Ä Û Æ Ò Ð Ë Ï È Ç Ç Ð Í È É Ô Ë Ñ Ñ Ð Ì Æ Ç È Ë É Ò Ç Î Í È Ð Ò ÖÜ Ò Ô Ë Ñ Ê È Ô Æ Ô È Í Ð Ý Î È Þ Æ Ì Ð É Ç ß Ü à á â Æ Ì Ì È Ô Æ Ô È Í Ð Ý Î È Þ Æ Ì Ð É Ç ß ã Ü à á Ý Î Ð Ñ Ô Ð Ç È É Ð Ý Î È Þ Æ Ì Ð É Ç ß ä à Ö

140

The data presented in Table 4.33 were subjected to

bivariate correlation analysis to establish the mutual correlation

among them. The Pearson’s linear correlation coefficient (R) matrix

thus obtained is presented in Table 4.34. � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � ¡ � � � � � � ¢ � � �¹ � Á Â � � � ¾ � � � � � � � � � � � � ¾ � � � � � ¦ � � � � � � � � � � ¥ � � �FT-IR data Colorimetric data

Variables

Polyphenols Flavonoids Alcohols/ amines

DPPH-RSA FRAP TPC TF

Polyphenols 1

Flavonoids 0.985** 1

FT

-IR

dat

a

Alcohols/ amines

–0.953* –0.940** 1

DPPH-RSA 0.940* 0.977** –0.914* 1

FRAP 0.805 0.886* –0.888* 0.925** 1

TPC 0.924* 0.953** –0.930** 0.974** 0.966** 1

Col

orim

etric

dat

a

TF 0.932* 0.958** –0.954** 0.958** 0.969** 0.991** 1 Ä Ä Û Ë Ñ Ñ Ð Ì Æ Ç È Ë É È Ò Ò È â É È Ó È Ô Æ É Ç Æ Ç å Ö å æ Ì Ð Þ Ð Ì ÖÄ Û Ë Ñ Ñ Ð Ì Æ Ç È Ë É È Ò Ò È â É È Ó È Ô Æ É Ç Æ Ç å Ö å ç Ì Ð Þ Ð Ì Ö

141

4.6. ANTIMICROBIAL POTENTIAL OF INDIAN GREEN TEAS

The antimicrobial activities of the six green teas

against four bacterial species are shown in Plate 4.9. The

diameter of zone of inhibition, measured to estimate the activity

are provided in Table 4.35.

The antifungal potential of the six green tea samples

against four fungal species are shown in Plate 4.10 and the

diameters of the zone of inhibition are presented in Table 4.36.

142

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143

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Green tea samples

Bacillus subtilis Escherichia coli Staphylococcus

aureus Streptococcus

pyrogenes

MOON 12 9 10 10

TAN 11 9 9 10

GT 11 8 11 8

TET 12 8 8 8

KOL 9 9 NA 9

ASS 9 8 8 9 ô õ ö ô ÷ ø ù ú û ü û ú ý

144

þ ÿ � � � � � � � � � � � � � � � ÿ � � � � � � � � � � � � � � � � � � � ÿ � �

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� � ��

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145

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Green tea samples

Candida albicans Aspergillus niger Aspergillus flavus Aspergillus terreus

MOON 10 9 8 9

TAN 9 8 7 9

GT 8 8 8 10

TET 9 8 9 10

KOL 9 9 9 8

ASS 7 7 7 8

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