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CHAPTER - V

PHYTOCHEMICAL ANALYSIS

Phytochemicals are chemical compounds synthesized during the various

metabolic processes. These chemicals are often called secondary metabolites and these

are found to have antimicrobial activity and serve as plant defense mechanisms against

pathogenic organisms. These compounds are classified as phenols, quinines, flavonoids,

tannins alkaloids, glycosides and polysaccharides (Das et al., 2010). Phytochemical

analysis involves testing of different classes of compounds. Methanol extracts of four

best plants i.e Terminalia chebula, Syzygium aromaticum, Rosa indica and Psidium

guajava were subjected to qualitative tests.

5.1 Material and Methods

5.1.1 Qualitative phytochemical analysis

The extracts were tested for the presence of bioactive compounds by using

standard methods (Sofowra, 1993, Trease and Evans, 1989 and Harborn, 1973).

Flavonoids

Extract was mixed with few fragments of magnesium turnings. Concentrated HCl

was added drop wise. Pink scarlet colour appeared after few minutes which indicated the

presence of flavonoids.

Phenols and Tannins

The sample was mixed with 2ml of 2% solution of FeCl3. A blue-green or black

coloration indicated the presence of phenols and tannins.

76

Saponins

5ml of distilled water was mixed with extract in a test tube and was shaken

vigorously. The formation of stable foam was taken as an indication for the presence of

saponins.

Alkaloids

2ml of 1% HCl was mixed with crude extract and heated gently. Mayer’s and

Wagner’s reagent was added to the mixture. Turbidity of the resulting precipitate was

taken as evidence for the presence of alkaloids.

5.1.2 Gas Chromatography and Mass Spectroscopy (GC-MS)

GC-MS analysis of the best plant extracts and essential oils was done using

Shimadzu Mass Spectrometer-2010 series. Methanol extracts of four plants Terminalia

chebula, Rosa indica, Psidium guajava and Syzygium aromaticum and six essential oils

Thymus vularis, Melaleuca alternifolia, Cinnamomum zeylanicum, Syzygium

aromaticum, Eucalyptus globulus and Valerian officinalis were analyzed using this

technique.

1 µl of sample was injected in GC-MS equipped with a split injector and a PE

Auto system XL gas chromatograph interfaced with a Turbo-mass spectrometric mass

selective detector system. The MS was operated in the EI mode (70 eV). Helium was

employed as the carrier gas and its flow rate was adjusted to 1.2 ml/min. The analytical

column connected to the system was an Rtx-5 capillary column (length-60m × 0.25mm

i.d., 0.25 µm film thickness). The column head pressure was adjusted to 196.6 kPa.

Column temperature programmed from 100˚C (2 min) to 200˚C at 10˚C/min and from

200˚ to 300˚C at 15˚C/min with hold time 5 and 22 min respectively. A solvent delay of 6

min was selected. The injector temperature was set at 270°C. The GC-MS interface was

maintained at 280°C. The MS was operated in the ACQ mode scanning from m/z 40 to

600.0. In the full scan mode, electron ionization (EI) mass spectra in the range of 40–600

(m/z) were recorded at electron energy of 70 eV. Compounds were identified by

77

comparing mass spectra with library of the National Institute of Standard and Technology

(NIST), USA/Wiley.

5.1.3 High Performance Liquid Chromatography (HPLC)

HPLC analysis of the most potent antibacterial agent of the study i.e. T.chebula

was performed. The HPLC system (Shimadzu Corporation, Kyoto, Japan) was equipped

with two Shimadzu LC-10 ATVP reciprocating pumps, a variable Shimadzu SPD-10

AVP UV-VIS detector and a Rheodyne Model 7725 injector with a loop size of 20 µl.

The peak area was calculated with a Winchrom integrator. Reverse-phase

chromatographic analysis was carried out in isocratic conditions using a C-18 reverse

phase column (250 x 4.6 mm i.d., particle size 5 µm, Luna 5µ C-18(2); phenomenex,

Torrance, CA, USA) at 25°C. Running conditions included: injection volume, 5µl;

mobile phase, methanol: Water (60: 40 v/v); flow rate, 1 ml/min; and detection at 290

mm. Samples were filtered through an ultra membrane filter (pore size 0.45 µm; E-

Merck, Darmstadt, Germany) prior to injection in the sample loop. Pyrogallol was used

as standard. Compounds present in each sample were identified by comparing

chromatographic peaks with the retention time (Rt) of standard and further confirmed by

co-injection with isolated standards. The amount of each phenolic acid was expressed as

mg per gram of fresh weight unless otherwise stated.

5.2 Results and Discussion

5.2.1 Qualitative Phytochemical Analysis

The phytochemical characteristics of four medicinal plants tested are summarized

in table 5.1. The results revealed the presence of medically active compounds in the four

plants screened. From the table, it could be seen that, tannins, phenols and flavonoids

were present in all the plants. Saponins were absent only from the flower buds of S.

aromaticum. Alkaloids were present only in the leaves of P. guajava. Our results were in

accordance with Dhiman et al., 2011, Manjari et al., 2011, Dahiya et al., 2012 and Chang

et al., 2012.

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Phytochemical analysis conducted on the plant extracts revealed the presence of

constituents which are known to exhibit medicinal as well as physiological activities

(Sofowra, 1993). Analysis of the plant extracts revealed the presence of phytochemicals

such as phenols, tannins, flavonoids, saponins, and alkaloids. It is evident from the results

that phenols, tannins and flavonoids present in the plant samples tested could be

responsible for the antibacterial activity of these plants. The phenolic compounds are one

of the largest and most ubiquitous groups of plant metabolites (Singh et al., 2007). They

possess biological properties such as antiapoptosis, antiaging, anticarcinogen,

antiinflammation, antiatherosclerosis, cardiovascular protection and improvement of

endothelial function, as well as inhibition of angiogenesis and cell proliferation activities

(Han et al., 2007). Several studies have described the antioxidant properties of medicinal

plants which are rich in phenolic compounds (Krings and Burger, 2001). The site and the

number of hydroxyl groups on the phenol group are thought to be related to their relative

toxicity to microorganisms, with evidence that increased hydroxylation results in

increased toxicity (Geissman, 1963). The mechanisms thought to be responsible for

phenolic toxicity to microorganisms include enzyme inhibition by the oxidized

compounds, possibly through reaction with sulfhydryl groups or through more

nonspecific interaction with proteins (Masson and Wasserman, 1987).

Tannin is a general descriptive name for a group of polymeric phenolic

substances. Their mode of antimicrobial action, may be related to their ability to

inactivate microbial adhesions, enzymes, cell envelope, transport-proteins etc. They also

complex with polysaccharide (Ya et al., 1988). A number of studies indicated that tannins

can be toxic to filamentous fungi, yeasts and bacteria (Scalbert, 1991).

Flavonoids are hydroxylated phenolic substances and occur as a C6- C3 unit

linked to an aromatic ring. Their activity is probably due to their ability to complex with

extracellular soluble proteins and with bacterial cell walls. Lipophilic flavonoids may

also disrupt microbial membranes (Tsuchiya, 1996).

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TABLE 5.1

Phytochemical Analysis of Plant Extracts

Plant Alkaloids Flavanoids Saponins Tannins Phenols

T.chebula _ + + + +

S.aromaticum _ + _ + +

P.guajava + + + + +

R.indica _ + + + +

5.2.2 Gas Chromatography and Mass Spectroscopy (GC-MS)

GC-MS chromatogram of methanol extracts of four plants: R. indica, T. chebula,

P. guajava and T. chebula and six essential oils: M. alternifolia, T. vulgaris, C.

zeylanicum, E. globulus, S. aromaticum and V. offficinalis are shown in figures 5.1 to

5.10 and compounds are listed in tables 5.2 to 5.11.

GC-MS analysis of plant extracts showed that eugenol (69.61%) was most

abundant compound in S. aromaticum extract. Our findings are close to Nassar et al.,

2007 with 71.56% eugenol presence. R. indica possessed n-capric acid (43.12).

Pyrogallol (79.12%) was most prevalent in T. chebula. Gangadgar et al., 2011 reported

gallic acid from T. bellerica. While P. guajava contained Isolongifolol (27.78%) and D-

torvlosol (22.24%). Kapoor et al., 2011 identified Methyl 2, 6, 10-trimethyltridecanoate

and Methyl octadecanoate in P. guajava, these were not in agreement to our studies.

In essential oils trans cinnamaldehyde (80.10%) was most abundant compound in

C. zeylanicum. Gupta et al., 2008 Oussalah et al., 2006 reported C. zeylanicum bark is

rich in cinnamaldehyde (50.5%), active against many pathogenic gram-positive and

80

gram-negative bacteria. Ali et al., 2005 reported cinnamaldehyde as the active agent to

inhibit the growth of both antibiotic-sensitive and resistant strains of Helicobacter pylori.

T. vulgaris contained α-Terpine (42.29%), Thymol (30.06%) and β –Cymene

(22.19%). While Imelouane, 2009 reported presence of camphor (38.54%), camphene

(17.19%), α-pinene (9.35%), 1, 8-cineole (5.44%), borneol (4.91%) and β-pinene (3.90%)

as the major oil components in T. vulgaris.

Aspiral (79.89%) was present in maximum amount in V. officinalis while reports

revealed patchoulol (16.75%), α-pinene (14.81%), and β-humulene (8.19%) as the major

compounds identified in the oil of V. officinalis by Wang et al., 2010, these results were

not in accordance to our findings.

M. alternifolia possessed α-Pinene (32.59%) and Cymene (27.30%). Tea tree oil

is characterized by a high proportion of terpinen-4-ol and c-terpinene, and moderate

levels of 1,8 cineole, p-cymene, a-terpinene, terpinolene and a-terpineol (Shellie et al.,

2003).The presence of terpenes seems to be present in all studies.

E. globulus contained eucalyptol (79.02%). The antibacterial activity of

Eucalyptus extracts may be due to the presence of compounds such as 1,8 cineole,

citronellal, citronellol (Nezhad et al., 2009). Our results are also supported by the

findings of Vratnica et al., 2011 who also reported 1,8 cineole in E.globulus oil to be

responsible for antibacterial activity.

S. aromaticum oil possessed eugenol (72.88%). Eugenone was the major

component identified in S. aromaticum oil by Joseph and Sujatha, 2011. Our findings are

close to to that of Dorman, 2000 and Lakshmi, 2010 who reported that S. aromaticum oil

contains high (75%) eugenol, and the antibacterial activity of S. aromaticum is attributed

to this compound.

81

FIG 5.1

GC-MS Chromatogram of S. aromaticum Extract

82

TABLE 5.2

Chemical Composition of S. aromaticum Extract

Peak Compound Retention Time

Peak Area (%)

1 Linalool 6.667 0.07

2 β-Citronella 8.320 0.07

3 Cinnamaldehyde 9.237 0.35

4 Citronella acetate 9.784 0.22

5 Rhodinol 9.932 3.40

6 Eugenol 10.291 69.61

7 Dihydropinene 10.940 0.36

8 Citronellyl propionate 11.088 8.49

9 Myrcenol 11.638 0.18

10 Eugenyl acetate 12.298 13.97

11 Heptyl propionate 12.751 2.74

12 3-Acetoxydodecane 13.249 0.03

13 4-Tert butylcyclohexene 15.793 0.51

100.00

83

FIG 5.2

GC- MS Chromatogram of R. indica Extract

84

TABLE 5.3

Chemical Composition of R. indica Extract

Peak Compound Retention Time Peak Area (%)

1 1-Caprylaldehyde 7.238 0.45

2 Benzyl hydrazine 7.534 4.19

3 2- Butene epoxide 8.033 8.31

4 Pyrocatechol 9.125 0.62

5 Tripropyl borane 9.335 11.52

6 Trans- Cinnamaldehyde 9.658 1.72

7 Eugenol 10.315 0.58

8 Eugenol acetate 10.412 1.17

9 Isonicotinic acid 11.560 21.92

10 P-Allyl guaiocol 12.381 0.72

11 3,4- Altrosan 12.854 5.69

12 n- Capric acid 14.657 43.12

100.00

85

FIG 5.3

GC-MS Chromatogram of T. Chebula Extract

86

TABLE 5.4

Chemical Composition of T. Chebula Extract

Peak Compound Retention Time Peak Area(%)

1 Phenyl carbamate 6.303 12.44

2 Hytrol 6.759 0.23

3 Dihydropyran 7.015 0.09

4 Itaconic anhydride 7.293 1.53

5 Levoglucosenone 8.017 0.51

6 2,3 Dimethyl oxirane 8.392 0.50

7 Pyrocatechol 9.197 0.43

8 Isosorbide 9.577 0.35

9 Chloroacetic acid 9.836 0.48

10 Eugenol 10.369 0.26

11 Eugenol acetate 10.524 0.16

12 Pyrogallol 11.221 79.21

100.00

87

FIG 5.4

GC-MS Chromatogram of P. guajava Extract

88

TABLE 5.5

Chemical Composition of P. guajava Extract

Peak Compound R. Time Peak area (%)

1 Copaene 10.059 0.24

2 Caryophyllene 10.690 0.62

3 Aromadendrene 10.956 0.29

4 Isoledene 11.924 2.24

5 Ethylallyl Phthalate 12.882 13.47

6 Isolongifolol 16.156 27.78

7 Caryophyllene oxide 16.706 8.56

8 D-torvlosol 16.973 22.24

9 Palmitic acid methyl ester 18.017 0.82

10 Methyl linolelaidate 20.517 1.07

11 Methyl-9-octadecenoate 20.569 1.31

12 Aromadendrenoxid 22.333 0.09

13 Alloaromadendrine oxide 22.383 0.16

14 Androst-7-ene 22.464 0.43

15 Bicycloelemene 22.666 0.39

16 Methyl steviol 22.862 0.25

17 5-alpha-Androst-7-ene 22.918 0.62

18 13,13-Dimethylpodocarp-7-en-3-ol 23.083 6.30

19 Dehydroabietic acid 23.248 1.33

20 Abiet-7-en-18-oic acid 23.421 0.09

21 Methyl abietate 23.640 0.18

22 Benzophenone anil 23.828 2.39

23 Heneicosane 24.053 0.49

89

FIG 5.5 GC-MS Chromatogram of C. zeylanicum Essential Oil

90

TABLE 5.6

Chemical Composition of C. zeylanicum Essential Oil

Peak. Compound R. Time Peak area (%) 1 α-Pinene 6.556 0.27 2 P-Cymene 8.293 0.74 3 D-Limonene 8.389 0.87 4 1-8-Cineole 8.461 0.48 5 γ-Terpinene 9.048 0.04 6 Linalool 10.013 3.28 7 Terpeniol 12.495 0.57 8 Terpinyl isobutyrate 12.650 0.06 9 trans- Cinnamaldehyde 15.488 80.10 10 Eugenol 17.039 2.09 11 Caryophyllene 18.575 5.91 100

91

FIG 5.6

GC-MS Chromatogram OF T. vulgaris Essential Oil

92

TABLE 5.7

Chemical Composition of T. vulgaris Essential Oil

Peak Compound Retention Time Peak Area (%)

1 β-Ketopropane 3.615 0.27

2 Trichloro methane- 1,4- hexadiene 3.842 0.17

3 4-Methyl-3- methylene 6.455 0.01

4 1- Cyclooctyne 6.623 0.06

5 β –Pinene 7.554 0.93

6 β –Myrcene 7.709 0.04

7 β –Cymene 8.774 22.19

8 Dimethylsuccinate 8.961 0.02

9 α-Terpine 9.798 42.29

10 2-Nonynoic acid 13.452 0.25

11 Durenol 18.484 3.70

12 Thymol 18.831 30.06

93

FIG 5.7

GC-MS Chromatogram of V. officinalis Essential Oil

94

TABLE 5.8

Chemical Composition of V. officinalis Essential Oil

Peak Compound Retention Time Peak Area(%) 1. 3-Furaldehyde 2.299 1.68 2. Aspiral 3.664 79.89 3. 2- Methyl furan 3.908 0.78 4. Enanthic acid 4.799 5.01 5. Amyl bromide 7.210 0.13 6. 1- Bromopropane 13.704 0.10 7. trans-α- Bergamotene 17.670 1.24 8. Chloro isobutylene oxide 18.703 0.07 9. Myrcene 19.051 0.29 10. Germacrene D 19.493 1.26 11. 1- Bromo- adamantine 19.701 0.14 12. β –Myrcene 20.057 0.54 13. Gamma-Elemene 20.159 0.76 14. 5,9- Tetradecadiyne 20.424 1.51 15. α-Farnescene 20.653 0.51 16. Farnescene 20.768 0.19 17. 3,6 Dimethyl-1,7-octadiene 22.306 0.67 18. 2,5 octadiene 22.928 0.25 19. α- Bisabolene epoxide 24.876 3.27 20. Terpineol 26.256 0.29 21. Longipinene epoxide 28.527 1.09 22. 2-Acetyl -2H- tetraazole 30.128 0.19

100.00

95

FIG 5.8

GC-MS Chromatogram of M. alternifolia Essential Oil

96

TABLE 5.9

Chemical Composition of M. alternifolia Essential Oil

Peak Compound Retention Time Peak Area(%)

1 Trichloromethane 3.842 0.14

2 1,8-Nonadiyne 6.458 0.29

3 α-Pinene 6.676 32.59

4 1- Cyclooctyne 7.419 0.09

5 β –Myrcene 7.569 3.03

6 2-Propynylcyclopentane 7.704 0.10

7 Cymene 8.814 27.30

8 α-Limonene 8.909 11.02

9 1,8- Cineole 9.968 2.48

10 α-phellandrene 9.697 6.54

11 Artemesiatriene 10.575 1.03

12 6-Methyl-3-heptyne 12.530 0.28

13 Linalool 13.989 12.77

14 α-Terpineol 14.424 1.97

15 Nitropentane 16.818 0.30

16 3- Heptene 23.362 0.06

97

FIG 5.9

GC-MS Chromatogram of E. globulus Essential Oil

98

TABLE 5.10

Chemical Composition of E. globulus Essential Oil

Peak Compound Retention Time Peak Area (%)

1 α-Pinene 6.566 3.57

2 Camphene 6.848 0.12

3 1,3,8-p-Menthtriene 7.255 0.10

4 Nopinen 7.369 0.75

5 β –Myrcene 7.488 0.28

6 3-Carene 8.065 13.66

7 Eucalyptol 8.747 79.02

8 Fenchyl alcohol 10.497 0.04

9 Camphor 11.295 0.09

10 Linalool 12.134 0.11

11 Trimethyl benzylalcohol 12.400 0.06

12 Terpineol 12.499 0.32

13 D- Vebenone 13.344 0.07

14 Camphenol 15.140 0.10

15 4- Carene 15.228 0.09

16 Berbenone 15.850 0.23

17 α-Bisbolene epoxide 16.292 0.13

18 δ-3-Carene 16.433 0.30

19 3-Carene-2-ol 16.653 0.19

20 Farnesene 17.211 0.78

99

FIG 5.10

GC-MS Chromatogram of S. aromaticum Essential Oil.

100

TABLE5.11

Chemical Composition of S. aromaticum Essential Oil

Peak Compound Retention Time Peak Area(%) 1 Isoeugenol 10.625 0.52 2 Carvol 15.295 0.22 3 Eugenol 20.491 72.88 4 Eugenyl methyl ether 20.825 0.12 5 Caryophyllene 21.401 15.40 6 Aromadendrene 21.975 0.05 7 Humulene 22.254 4.02 8 Isoeugenyl acetate 24.386 6.33 9 Caryophyllene oxide 25.990 0.41 10 Limonene oxide 26.695 0.04 100

101

5.2.3 HPLC Analysis

The examination of HPLC chromatograms revealed the presence of several

compounds. The Fig. 5.11 chromatogram indicated the control run pyrogallol at retention

time 3.99 having an area percent of 95.20. The results in further figures indicated the

qualitative composition of phenol (pyrogallol) in the various samples of T. chebula .

Two major products of methanol extract with area percent of 70.88 and 29.12

were eluted at 3.80 and 5.26 minutes respectively (Fig. 5.12). The spectrum showed

maxima at 270 nm. The results of the hexane sub fraction (Fig. 5.13) showed three

products. The first one with a relative area of peak of 70.74 for which the retention time

was 3.97 minutes. The second product had a retention time of 4.80 minutes for which the

relative area was 27.41. The third compound had a relative area of peak of 1.84 with the

retention time 8.20 minutes.The extracts of chloroform subfractions contained four

compounds which were eluted at 3.82, 4.30, and 5.05 and 7.34 minutes with a relative

area of 21.48, 9.81, 5.66 and 63.05 respectively (Fig. 5.14).The results of the ethyl

acetate sub fraction (Fig. 5.15) showed four products. The first one with a relative area of

peak of 82.14 for which the retention time was 3.80 minutes. The second product had a

retention time of 5.06 minutes followed by 7.45 and 10.26 with the relative area 2.54,

5.29 and 10.04 respectively. A RP-HPLC revealed fourteen components (gallic acid,

chebulic acid, 1,6-di-O-galloyl-D-glucose, punicalagin, 3,4,6-tri-O-galloyl-D-glucose,

casuarinin, chebulanin, corilagin, neochebulinic acid, terchebulin, ellagic acid, chebulagic

acid, chebulinic acid, and 1,2,3,4,6-penta-O-galloyl-D-glucose) in the fruit of Terminalia

chebula Retz.. The separation was achieved within 80 min using a binary gradient with

mobile phases (Juang et al., 2004). Pyrogallol has been reported to be an effective

antimicrobial agent and its toxicity is attributed to the three hydroxyl groups present in its

structure (Singh and Kumar 2013). The differences in the change of absorption spectra of

many compounds depend on the chemical change: hydroxylation, methylation,

glycosylation and condensation with other phenolic molecules or not (Macheix et al.,

2005).

102

FIG 5.11

HPLC Chromatogram of Pyrogallol (Control)

103

FIG 5.12

HPLC Chromatogram of Methanol Extract (T. chebula)

104

FIG 5.13 HPLC Chromatogram of Hexane Sub Fraction (T. chebula)

105

FIG 5.14 HPLC Chromatogram of Chloroform Sub Fraction (T. chebula)

106

FIG 5.15 HPLC Chromatogram of Ethyl Acetate Sub Fraction (T. chebula)

107

5.3 Conclusions

GC-MS is an authentic technique to analyze the various components of the given

sample.

Our samples confirmed the presence of some compounds with the already

reported work.

The present assignment is successful in revealing new active compounds found in

some of the plants like rose, guava and valerian.

Antibacterial studies and GC-MS studies correlate role of some phenols like

Pyrogallol, eugenol and terpines etc. responsible to inhibit the growth of various

epidermally significant bacteria.

HPLC technique confirmed the presence of an active principle compound

pyrogallol in various antibacterial active samples of T. chebula.

The highest percentage of pyrogallol (82.14) was found in ethyl acetate fraction

of T. chebula. Objective 2 concluded the highest antibacterial activity of ethyl

acetate fraction of T. chebula. The two reports help in concluding the antibacterial

activity of pyrogallol.