EXPERIMENTAL RESULTS

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! " #!! $!!! % & % ' ( % ( % ( ) * + *, * - # S. No. Phytochemical Result (Qualitative) 1 Tannin --- 2 Saponin --- 3 Flavonoids ++ 4 Steroids +++ 5 Terpenoids ++ 6 Alkaloids +++ 7 Amino acids +++ 8 Polyphenol +++ 9 Glycoside +++ 10 Protein ++

68

. / 69

70 0 1 2 3 2 Peak# R. Time Area Area% Name 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

H F5 G G M5 IL 8 M8 K L H 7 C J E I5 7 5 B 8 F D E B C @ A? = > < ; : 9 : 67 8 4 5 Compound name Nature/Type Uses Mol. Wt. Formula Area (%) Peak# R. Time 3 4.650 5.94 C6H8O4 144 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl Flavonoid fraction Antimicrobial, Anti-inflammatory 4 5.485 2.86 C5H12O5 152 1-Deoxy-d-arabital Unknown 10 11.212 11.31 C8H16O6 208 Ethyl-alpha-d-glucopuranoside Sugar moiety Preservative Antialopecic, Lipotropic, antineuropathic, Sweetner anticirrhotic, Cheolesterolytic 11 12.714 30.83 C7H14O6 194 MOME INOSITOL Inositol Both, pro and and antioxidant anticarcenogenic, bronchodialator 12 13.413 8.77 C8H10N4O2 194 Caffeine Alkaloid Both pro and antioxidant vasodilator, diuretic 13 13.708 1.06 C7H8N4O2 180 1-H-PURINE-2,6-DIONE, 3,7-DIHYDRO-3,7-DIMETHYL Xanthine alkaloid Antioxidant, nematicide pesticide, flavor antiandrogenic 15 14.350 14.27 C16H32O2 256 n-hexadecanoic acid Palmitic acid 17 16.198 3.82 C18H36O2 284 Octadecanoic acid Fatty acid (Stearic acid) Skin care Antioxidant, nematicide, pesticide, hypocholesterolemic, hemolytic 21 19.150 1.06 C18H36O2 284 Hexadecanoic acid ethyl ester Palmitic acid ethyl ester Antimicrobial, antioxidant antiinflammatory, antiartheritic, diuretic antiashma, anti HIV reserve transcriptase 23 28.676 1.16 C29H48O 412 Stigmasterol Steroid Antimicrobial, antioxidant antiinflammatory, antiartheritic, diuretic antiashma, anti HIV reserve transcriptase 24 30.027 2.23 C29H50O 414 STIGMAST-5-EN-3-OL, (3, BETA) Steroid 71

/ 2 2 N O P Q Q R 72

73

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 (a) (b) (c) (d) 0 N R S T N R 2 T N R 2 2 U 2 N 2 R 3 V W

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 Z Z 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

77 z b c { i \ [ \ [ w x y f b e b l i g i e a d m n Z o p a Peak 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 ƒ ƒ ƒ ƒ ƒ ƒ ˆ ƒ 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

X Y Z [ \ [ [ X z ` Š x a f i j g e b d m b Œ 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 Œ a q h g r i a Y s i t n Z o p a 79

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 ` Š x a f i j g e k l d m Z e i i h Z e b l a f e d k g a b l f { i Absorption band (cm 1 ) Vibrational group and mode Chemical compound References Vanaja et al. (2013) 3398* O H stretch (hydroxyl) Phenols Awwad et al. (2013) Rajathi and Sridhar (2013) N H stretch Amino group Awwad et al. (2013) 2929 C H stretch Alkanes Vanaja et al. (2013) Rajathi and Sridhar (2013) Aldehydes Umashankari et al. (2012) O H stretch Carboxylic acid Vanaja et al. (2013) 2349 N H stretch Amino acids Umashankari et al. (2012) 1641 C=O (carbonyl) Polyphenols 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) 1408 C O stretch Amino acids Theivasanthi and Alagar (2013) C C stretch Aromatics Mallikarjuna et al. (2012) 1249 C O C stretch Glycoside linkages Raghavandra et al. (2013) 1081 C N stretch (carbonyl) Amide-I in proteins Awwad et al. (2013) Aliphatic amines Vanaja et al. (2013) C O stretch Carboxylic acid 669 C H bend Phenyl ring substituents Ž š œ ž Ÿ 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.

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 Œ ª «± ² ³ µ ¹ º ²» ¼ ± ½ ¹ ¾ À Á Á  ± ² µ ³ µ º à Á Ä Å Å Æ Ç Æ À û È À» Æ À Ã Ç Ã Ä È À É Ê Ë Ì Ë È É Ä Ã Ä Í Æ» È À Ã Ç È Î 82

83 Ï Î Æ Ð Ê Ñ Ê Ò À à Ļ 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 û 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) Label Bacteria Concentrations ( g ml 1 ) Ag NPs P 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 í î 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

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 à Á Ä Å Å Æ Ç Æ À û È À» Æ À Ã Ç Ã Ä È À É Ê Ë Ì Ë È É Ä Ã Ä Í Æ» È À Ã Ç È Î 84

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 û 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) Label Fungi Concentrations ( g ml 1 ) Ag NPs P 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 í î 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.

88! " # $! " " % " " " & ' & ( ) & ) & ) * +, + - +. $ 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 0 : ; < = 4 >? 4 4 6 @ 6 > 6 A B C C D > 7 8 9

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

0 0 E 3 F E G 4 5 6 7 8 9 92

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.

0 H 0 3 I < 4 > 6 > 5 6 7 8 9 94

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.

: J K 7 8 9 > L M N O P Q R S P T U O V W 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 s t 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 96

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 ƒ i y t r 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 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 r Zone of inhibition (mm) Label Bacteria Concentration of ZnO NPs ( g ml 1 ) 5 10 20 P Gram-negative a K. pneumoniae 10 b P. aeruginosa 3 c E. coli Gram-positive d S. aureus 2 5 ˆ Š Œ Ž Œ Ž Ž š œ ž Ÿ

t w h l o s s i l m n i z h w h d l m v m l q n ƒ 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 V d 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 w y u y o z h l m v o d q i l p q w 98

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 ƒ i y t r 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 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 r Zone of inhibition (mm) Label Fungi Concentration of ZnO NPs ( g ml 1 ) 5 10 20 P a A. fumigatus 5 3 b Penicillium sp. 7 2 c A. flavus 3 3 d A. niger 3 2 ˆ Š Œ Ž Œ Œ Ž š œ ž Ÿ 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.

p q h l q z p h q n ª y l o h 100

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 9.644 97341 0.13 Nonane, 5-(2-methylpropyl) 10.150 32840 0.04 Nonane, 5-(2-methylpropyl) 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 11.958 66129 0.09 1,3,4,5-TETRAHYDROXY-CYCLOHEXANECARBOXYLI 12.098 81645 0.11 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 13.746 2274908 3.00 1H-PURINE-2,6-DIONE, 3,7-DIHYDRO-3,7-DIMETHYL 13.908 225958 0.30 Hexadecanoic acid, methyl ester 14.299 2194161 2.90 n-hexadecanoic acid 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) 16.178 630108 0.83 Octadecanoic acid 18.835 298639 0.39 Cyclohexanol, 4-[(trimethylsilyl)oxy]-, cis 18.961 154801 0.20 PREGNANE, SILANE DERIV. 19.158 629245 0.83 Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester 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

p q h l q z p h q n { y l o h 102

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 r Peak# 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 5.549 143309 0.29 2-(HYDROXYMETHYL)-2-METHYL-1-PYRROLIDINECA 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 9.094 112476 0.23 Nonane, 5-(2-methylpropyl) 9.207 38178 0.08 1-CHLOROHEXADECANE 9.592 47723 0.10 Benzoic acid, 4-ethoxy-, ethyl ester 9.645 89819 0.18 Nonane, 5-(2-methylpropyl) 10.261 88262 0.18 1-Tridecene 11.022 4708214 9.64 1,3,4,5-TETRAHYDROXY-CYCLOHEXANECARBOXYLI 11.486 43687 0.09 1,2,4-BUTANTRIOL, 4-O-OCTYL 11.622 66031 0.14 Nonane, 5-(2-methylpropyl) 12.097 88232 0.18 Dodecane, 2,6,11-trimethyl 12.514 44508 0.09 Cyclotetradecane 13.489 27927732 57.17 Caffeine 13.698 1587815 3.25 1H-PURINE-2,6-DIONE, 3,7-DIHYDRO-3,7-DIMETHYL 13.905 302487 0.62 Hexadecanoic acid, methyl ester 14.289 747434 1.53 n-hexadecanoic acid 14.547 35770 0.07 n-heptadecanol-1 15.560 90492 0.19 9,12-Octadecadienoic acid (Z,Z) 15.628 300686 0.62 9,12,15-Octadecatrienoic acid, methyl ester, (Z,Z,Z) 15.739 164577 0.34 2-HEXADECEN-1-OL, 3,7,11,15-TETRAMETHYL-, [R-[R* 15.823 103436 0.21 HEXADECANOIC ACID, METHYL ESTER 16.012 1393467 2.85 16.169 270303 0.55 9-OCTADECENOIC ACID (Z) 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 19.144 579389 1.19 Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester 19.436 55082 0.11 1,2-BENZENEDICARBOXYLIC ACID, DIISOOCTYL EST 19.708 76496 0.16 Heptadecane, 2,6,10,15-tetramethyl 20.386 60506 0.12 Cyclohexanol, 4-[(trimethylsilyl)oxy]-, cis 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

± ² ³ ² ± µ ³ ² 104

105 ² ¹ º» ¼ ½» ¼ ¾ ² À ³ µ ³ ² Á ± À Â Ã Ä Å Peak# R.Time Area Area% Name 3.188 208322 0.23 5-METHYL-5,6-DIHYDRO-2(1H)-PYRIDINONE 4.194 275556 0.31 1-(N,N-DIETHYL)AMINOPROPYNE 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 9.093 107767 0.12 Nonane, 5-(2-methylpropyl) 9.595 66539 0.07 Benzoic acid, 4-ethoxy-, ethyl ester 10.260 71745 0.08 1-Tridecene 11.139 1504502 1.67 1,3,4,5-TETRAHYDROXY-CYCLOHEXANECARBOXYLI 11.621 70162 0.08 Nonane, 5-(2-methylpropyl) 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 13.745 3905943 4.33 1H-PURINE-2,6-DIONE, 3,7-DIHYDRO-3,7-DIMETHYL 14.299 1906147 2.11 n-hexadecanoic acid 15.630 285566 0.32 9,12,15-Octadecatrienoic acid, methyl ester, (Z,Z,Z) 15.737 773773 0.86 Phytol 15.950 471711 0.52 9,12-Octadecadienoic acid (Z,Z) 16.024 1864530 2.07 9,12,15-Octadecatrienoic acid, (Z,Z,Z) 16.172 365893 0.41 Octadecanoic acid 18.950 75521 0.08 Butanedioic acid, 2-hydroxy-2-methyl-, dimethyl ester 19.134 924854 1.03 Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester 19.436 42798 0.05 1,2-BENZENEDICARBOXYLIC ACID, DIISOOCTYL EST 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

± ² ³ ² ± µ Æ ³ ² 106

107 ² ¹ º» ¼ ½ Ç ¼ ¾ ² À ³ µ Æ ³ ² Á ± À Â Ã Ä Å Peak# 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 9.595 66451 0.08 Benzoic acid, 4-ethoxy-, ethyl ester 9.647 82996 0.10 Nonane, 5-(2-methylpropyl) 10.263 67130 0.08 1-Undecanol 10.344 143181 0.18 Tetradecane 11.080 6698033 8.35 1,3,4,5-TETRAHYDROXY-CYCLOHEXANECARBOXYLI 11.489 45360 0.06 2-Undecene, 5-methyl 11.623 71890 0.09 Nonane, 5-(2-methylpropyl) 12.098 67844 0.08 Nonane, 5-(2-methylpropyl) 12.568 166863 0.21 5-ISOPROPENYL-2-METHYL-7-OXA-BICYCLO[4.1.0]HE 13.552 46119250 57.52 1,3,7-TRIMETHYL-3,7-DIHYDRO-1H-PURINE-2,6-DIONE 13.730 2862414 3.57 1H-PURINE-2,6-DIONE, 3,7-DIHYDRO-3,7-DIMETHYL 13.909 182457 0.23 HEXADECANOIC ACID, METHYL ESTER 14.293 946305 1.18 n-hexadecanoic acid 15.562 68154 0.08 9,12-Octadecadienoic acid (Z,Z) 15.628 231995 0.29 9,12,15-Octadecatrienoic acid, methyl ester, (Z,Z,Z) 15.740 155362 0.19 2-HEXADECEN-1-OL, 3,7,11,15-TETRAMETHYL-, [R-[R* 15.824 75304 0.09 HEXADECANOIC ACID, METHYL ESTER 16.014 1599955 2.00 cis,cis,cis-7,10,13-hexadecatrienal 16.171 273535 0.34 Octadecanoic acid 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 19.147 332192 0.41 Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester 19.439 40752 0.05 1,2-BENZENEDICARBOXYLIC ACID, DIISOOCTYL EST 80184244 100.00

± ² ³ ² ± µ È É Ê ³ ² 108

109 ² ¹ º» ¼ ½ Ë ¼ ¾ ² À ³ µ È É Ê ³ ² Á ± À Â Ã Ä Å Peak# R.Time Area Area% Name 3.194 192821 0.39 6-Azabicyclo[3.2.1]octane 4.196 232131 0.48 1-(N,N-DIETHYL)AMINOPROPYNE 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 9.094 118548 0.24 Nonane, 5-(2-methylpropyl) 9.597 55523 0.11 Benzoic acid, 4-ethoxy-, ethyl ester 9.645 91032 0.19 Nonane, 5-(2-methylpropyl) 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 11.024 4932019 10.10 1,3,4,5-TETRAHYDROXY-CYCLOHEXANECARBOXYLI 11.489 40592 0.08 Hexadecane 11.623 72194 0.15 Dodecane, 2,6,11-trimethyl 12.098 93455 0.19 Dodecane, 4,6-dimethyl 12.512 60180 0.12 7-Heptadecene, 1-chloro 13.490 29197331 59.79 1,3,7-TRIMETHYL-3,7-DIHYDRO-1H-PURINE-2,6-DIONE 13.700 1090341 2.23 1H-PURINE-2,6-DIONE, 3,7-DIHYDRO-3,7-DIMETHYL 13.908 233863 0.48 HEXADECANOIC ACID, METHYL ESTER 14.291 568908 1.17 n-hexadecanoic acid 15.564 69821 0.14 9,12-Octadecadienoic acid (Z,Z) 15.631 242346 0.50 9,12,15-Octadecatrienoic acid, methyl ester, (Z,Z,Z) 15.825 93050 0.19 HEXADECANOIC ACID, METHYL ESTER 16.016 1173311 2.40 cis,cis,cis-7,10,13-hexadecatrienal 16.174 216055 0.44 9-OCTADECENOIC ACID (Z) 18.834 98067 0.20 6-Ethyl-3-trimethylsilyloxydecane 18.957 93835 0.19 Tridecanoic acid, 3-hydroxy-, ethyl ester 19.156 242002 0.50 Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester 20.390 51071 0.10 Cyclohexanol, 4-[(trimethylsilyl)oxy]-, cis 48833185 100.00

± ² ³ ² ± µ Ì Í Í ³ ² 110

111 ² ¹ º» ¼ ½ Î ¼ ¾ ² À ³ µ Ì Í Í ³ ² Á ± À Â Ã Ä Å Peak# R. Time Area Area% Name 3.189 2207529 2.13 5-METHYL-5,6-DIHYDRO-2(1H)-PYRIDINONE 3.771 794821 0.77 Cyclopentane, 1-acetyl-1,2-epoxy- 4.185 2044516 1.98 1-(N,N-DIETHYL)AMINOPROPYNE 4.640 2198879 2.12 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl 5.551 612518 0.59 2-(HYDROXYMETHYL)-2-METHYL-1-PYRROLIDINECA 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 9.092 96870 0.09 Nonane, 5-(2-methylpropyl) 9.645 268760 0.26 Nonane, 5-butyl 11.441 32558968 31.46 1,3,4,5-TETRAHYDROXY-CYCLOHEXANECARBOXYLI 12.095 127518 0.12 Dodecane, 2,6,11-trimethyl 12.226 620335 0.60 1,3,4,5-TETRAHYDROXY-CYCLOHEXANECARBOXYLI 12.568 141270 0.14 2(4H)-BENZOFURANONE, 5,6,7,7A-TETRAHYDRO-6-HY 13.547 44239019 42.75 1,3,7-TRIMETHYL-3,7-DIHYDRO-1H-PURINE-2,6-DIONE 13.849 4308457 4.16 1H-PURINE-2,6-DIONE, 3,7-DIHYDRO-3,7-DIMETHYL 14.306 3708333 3.58 n-hexadecanoic acid 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 15.950 734099 0.71 9,12-Octadecadienoic acid (Z,Z) 16.026 1555646 1.50 9,12,15-Octadecatrienoic acid, (Z,Z,Z) 16.175 553048 0.53 Octadecanoic acid 19.136 950152 0.92 Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester 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 à ³ ² Õ ½ Ö µ Ð É É ³ ² Peak R.T. Area (%) Molecular formula M. wt. Name of the compound Type Therapeutic use 8 8.135 18.40 C6H6O3 126.11 1,2,3-Benzenetriol Pyrogallol Antioxidant Antiseptic fungicide, Antidermatitic insecticide, candidicide 15 11.158 13.96 C7H12O6 192.16 1,3,4,5-Tetrahydroxycyclohexanecarbonyl 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) Xanthine alkaloide Vasodilaton both pro-and antioxidant natural diuretic diuretic 27 14.299 2.90 C16H32O2 256.42 n-hexadecanoic acid Palmitic acid Antioxidant Nematicide pesticide hemolytic anti-inflammatory hemolytic 30 15.742 1.34 C20H48O 296.53 Phytol Diterpene Antioxidant Antimicrobial, 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 à ³ ² Õ ½ Ö µ Ì ³ ² Peak R.T. Area (%) Molecular formula 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-Tetrahydroxycyclohexanecarbonyl 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 à ³ ² Õ ½ Ö µ ³ ² Peak R.T. Area (%) Molecular formula M. wt. Compound name Type/nature Therapeutic use 7 8.141 28.36 * * 1,2,3-Benzenetriol Pyrogallol * 11 11.139 1.67 * * 14 13.564 56.48 * * Caffine 1,3,4,5-Tetrahydroxycyclohexanecarboxyl 1,3,7-Trimethyl-3,7- dihydro-1h-purine-2,6- dione Quinic acid * Alkaloid * 15 13.745 4.33 * * 1H-Purine-2,6-dione, 3,7-Dihydro-3,7-Dimethyl Xanthine, Alkaloid * 16 14.299 2.11 * * n-hexadecanoic acid Palmitic acid * 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 à ³ ² Õ ½ Ö µ Æ ³ ² Peak R.T. Area (%) Molecular formula M. wt. Compound name Type/nature Therapeutic use 7 7.859 1.62 * * Tetradecane Alkane * 8 8.146 21.55 * * 1,2,3-Benzenetriol Pyrogallol * 14 11.080 8.35 * * 19 13.552 57.52 * * Caffine 1,3,4,5-Tetrahydroxycyclohexanecarboxyl 1,3,7-Trimethyl-3,7- dihydro-1h-purine-2,6- dione Quinic acid * 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 C 6H 26O 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 à ³ ² Õ ½ Ö µ È É Ê ³ ² Peak R.T. Area (%) Molecular formula M. wt. Compound name Type/nature Therapeutic use 9 8.128 17.41 * * 1,2,3-Benzenetriol Pyrogallol * 17 11.024 10.10 * * 22 13.490 59.79 * * 1,3,4,5- Tetrahydroxycyclohexanecarboxyl 1,3,7-Trimethyl-3,7- dihydro-1h-purine- 2,6-dione Quinic acid * Caffeine * 23 13.70 2.23 * * 1H-Purine-2,6-dione, 3,7-Dihydro-3,7- Dimethyl Xanthine, Alkaloid * 25 14.291 1.17 * * n-hexadecanoic acid Palmitic acid * 29 16.016 2.40 C 6H 26O 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 à ³ ² Õ ½ Ö µ Ì Í Í ³ ² Peak R.T. Area (%) Molecular formula M. wt. Compound name Type/nature Therapeutic use 1 3.189 2.13 3 4.185 1.98 5-Methyl-5,6-dihydro- 2(1H)-pyridinone 1-(N,N- Dimethyl)aminoporpyne Unknown 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,-tetrahydroxycyclohexanecarboxyl Quinic acid * 16 13.547 42.75 * * 1,3,7,-trimethyl-3,7- dihydro-1h-purine-2,6- dione Coffeine alkaloid * 17 13.849 4.16 * * 1H-Purine-26-Dione, 3,7- Dihydro-3,7-dimethyl Xanthine, Alkaloid * 18 14.306 3.58 * * n-hexadecanoic acid Palmitic acid * 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 à µ Ó Á Ó ² º À Ò ³ Á ± Ó Á ² º Å µ à ³ ² Å ² ± À º Å ² à ² º Ò ö Ä ¹ Ò Ð Í Compounds Peak area (%) of green tea samples MOON TAN GT TET KOL ASS ø ù ø Þ ß Ý ú á ß Û Ý ú á Þ ß á ß û á ß Þ 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

± 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 119

120

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).

122

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 à µ ² Ä Ó ¹ ² ³ Ó Ã ² º ¹ ² Ã Ä Å µ à ³ ² Å ² ± À º Å Wavenumber (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) 2946 C H stretch (asym.) Alkanes Vanaja et al. (2013) O H stretch Carboxylic acid Sharmin et al. (2013) 2833 C H stretch (sym.) Alkanes Umashankari et al. (2012) Arokiyaraj et al. (2013) 1629 ~ 1663 C=O stretch (carbonyls) Flavonoids Heneczkowski et al. (2001) Polyphenols, catechins Rajathi and Sridhar (2013) C=C stretch Aromatics Kong and Yu (2007) 1449 C C stretch (in ring) Aromatics 1239 C N stretch Aliphatic amines Umashankari et al. (2012) Heneczkowski et al. (2001) Mallikarjuna et al. (2012) Heneczkowski et al. (2001) Nagajyothi et al. (2013) 1113 C O stretch Alcohols, esters, carboxylic acids Mallikarjuna et al. (2012) 1014 ~ 1019 C O stretch Alcohols, esters, carboxylic acids Rajathi and Sridhar (2013) Mallikarjuna et al. (2012) C N stretch Aliphatic amines Nagajyothi et al. (2013) 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  ŠÀ Å Ã ³ Ó Ã ³ ³ ² º À à º Ó Á Á à ³ à ³ Å µ à ³ ² Å ² Ã Ä ² º Ó Á ² Á Ó Ä µ ² Ó Â Å Á à Á à ³ ² ³ Ó Ã Å Optical density values Concentration of extracts g ml -1 Gallic acid (standard) Green tea samples MOON TAN GT TET KOL ASS 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 E C D >8 A B ; @? 7 ; <=> 6 7 8 9:! " # $ % & & ' ( ) * + ), -., / ( ) 0 % 3 4 5 1 2 F G H I J I K L I M N O P Q R P O S N T O P U V R W P O N X Y N Y Z [ S \ P T N [ G U U N T Y P T Y O N X H R P P T Y P Z O Z ] S [ P O

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 \ P H 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 O Optical density values Concentration of extracts g ml -1 Green tea samples MOON TAN GT TET KOL ASS 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 E C A B ; D >8 @? 7 ; <=> 6 7 8 9: d! " # $ % & & ' ( ) * + ), -., / ( ) 0 % 3 4 5 1 2 F G H I J I K e I M N O P Q R P O S N T O P U V R W P O N X Y N Y Z [ X [ Z W N T N G b O N X H R P P T Y P Z O Z ] S [ P O

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 O Concentration of extracts g ml -1 Absorbance (%) Ascorbic Green tea samples 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

oe 6? n m 130 i d j * ( - k / *. * / l! " # $ % & & d i d ' ( ) * + ), -., / ( ) 0 % 3 4 5 1 2 F G H I J I K p I M N O P Q R P O S N T O P U V R W P O N X 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 r s Z T b Y \ P O Y Z T b Z R b Z O U N R _ G U Z U G b

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 O Absorbance (%) Concentration of extracts g ml -1 Ascorbic acid (standard) Green tea samples 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 E C A B ; D >8 @? 7 ; <=> 6 7 8 9: d j * ( - k / *. * / l! " # $ % & & d v ' ( ) * + ), -., / ( ) 0 % 3 4 5 1 2 F G H I J I ` w I M N O P Q R P O S N T O P U V R W P O N X F t u f Z O O Z c N X Y \ P H R P P T Y P Z O Z ] S [ P O Z T b Y \ P O Y Z T b Z R b Z O U N R _ G U Z U G b

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 Y S 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 s N X Y \ P H R P P T Y P Z O Z ] S [ P O S. 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 ~ ƒ ƒ ˆ ƒ ˆ

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 Š Œ Ž Š Œ Š š š š œ ž Ÿ Š š Š Š Œ š Š š Š Š Ÿ Š Š Ÿ Œ Variables DPPH-RSA FRAP TPC TF ª «± ² ³ µ µ «ª ª «DPPH-RSA 1 FRAP 0.925 1 TPC 0.974 0.966 1 TF 0.957 0.966 0.990 1

¹ š Ž º» Œ Š š ¼ ½ Š ¾ š Š Œ Š š Š š š š œ Š ž» Š ¾ œ ž ¹ Š Š Ÿ Œ 135

¹ š Ž º º» Œ Š š ¹ À Š š š š œ Š ž» Š ¾ œ ž ¹ Š Š Ÿ Œ 136

¹ š Ž º» Œ Š š ¼ ½ Š ¾ ¹ À Š Š 137

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. Š Œ Ž º ¹ Á  Š Š œ à ž Œ ¾ š Š š Š Œ Š ¾ Š Š Ÿ Œ Vibrational band (cm 1 ) Absorbance (%) MOON TAN GT TET KOL ASS 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. Š Œ Ž ¹ Á  Š ¾ Œ š Ÿ š ¾ Š Š Š Š Ÿ Œ 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 Ö Ä 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 Ö Ü 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. Š Œ Ž Ž Š Œ š Š Œ Š š š š œ ž Ÿ Š š Š Š Œ š ¹ Á  Š ¾ Œ š Ÿ š ¾ Š Š Š Š Ÿ Œ FT-IR data Colorimetric data Variables Polyphenols Flavonoids Alcohols/ amines DPPH-RSA FRAP TPC TF Polyphenols 1 FT-IR data Flavonoids 0.985** 1 Alcohols/ amines 0.953* 0.940** 1 DPPH-RSA 0.940* 0.977** 0.914* 1 Colorimetric data FRAP 0.805 0.886* 0.888* 0.925** 1 TPC 0.924* 0.953** 0.930** 0.974** 0.966** 1 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 Ö 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 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 í ò ò í ò ò ð ê ñ é ê ê ë ð ê ñ é ê ê ë ì ï ì î ì ì í ë ì ï ì î ì ì í ë ð ê ñ í ò ò é ê ê ë ì ï ì ð ê ñ ì í ë î ì í ò ò ì ï ì î ì ì í ë é ê ê ë Œ Š Ž è À š Š š Š Œ Š š š š Š Š Ÿ Œ

143 Š Œ Ž ó À š Š š Š Œ Š š š š Š Š Ÿ Œ Green tea samples Bacillus subtilis Diameter of zone of inhibition (mm) Volume of sample (30 L) 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

145 ÿ! ÿ " ÿ Green tea samples Diameter of zone of inhibition (mm) Volume of sample (30 L) 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