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CHAPTER VII

PHYTOCHEMICAL ANALYSIS

Phytochemicals are chemical compounds synthesized during various metabolic

processes. They are naturally synthesized in all parts of the plant body; bark, leaves, stem,

root, flower, fruits, seeds, etc. These chemicals are often called secondary metabolites and

serve as plant defense mechanisms against pathogenic organisms. These are classified as

phenols, quinines, flavonoids, tannins alkaloids, glycosides and polysaccharides (Das et al.,

2010). The quantity and quality of phytochemicals present in plant parts may differ from one

part to another. In fact, there is lack of information on the distribution of the biological

activity in different plant parts essentially related to the difference in distribution of active

compounds which are more frequent in some plant parts than in others.

Phytochemicals have been recognized as the basis for traditional herbal medicine

practiced in the past and currently in vogue. In the search for phytochemicals that may be of

benefit to the pharmaceutical industry, researchers sometimes follow leads provided by local

healers in a region. Following such leads, plant parts are usually screened for phytochemicals

that may be present. The presence of a phytochemical of interest may lead to its further

isolation, purification and characterization. Then it can be used as the basis for a new

pharmaceutical product.

In this section, the methanol extract and ethyl acetate sub fraction of five best plants

viz., Terminalia chebula, Emblica officinalis, Acaia nilotica, Rosa indica and Psidium

guajava, selected out from the earlier tested activities i.e antibacterial, antioxidant, antiurease

and anticollagenase, were subjected to qualitative and quantitative phytochemical analysis.

7.1 Methods

7.1.1 Qualitative phytochemical analysis

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

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

7.1.2 Test for 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.

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7.1.3 Test for 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.

7.1.4 Test for 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.

7.1.5 Test for 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.

7.1.2 Gas Chromatography and Mass Spectroscopy (GC-MS)

GC-MS technique was used in this study to quantitatively identify the volatile

phytocomponents present in extracts. The extracts were analyzed by using Shimadzu Mass

Spectrometer-2010 series. GC-MS analysis of the best bioactive extracts was done using

Shimadzu Mass Spectrometer-2010 series. Methanol extracts of the best five plants and ethyl

acetate fraction of three plants T. chebula, E. officinalis and A. nilotica 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

withhold 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 comparing mass spectra with library of the National

Institute of Standard and Technology (NIST), USA/Wiley.

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7.2 Results and Discussion

7.2.1 Qualitative Phytochemical Analysis

The phytochemical characteristics of medicinal plants tested are summarized in Table

7.1. The results revealed the presence of medically active compounds in the plants screened.

From the table, it could be observed that, tannins, phenols and flavonoids were present in all

the extracts of tested plants. Saponins were absent only from the leaves of A. nilotica.

Alkaloids were present only in the leaves of P. guajava and fruit of E. officinalis.

Present results are in accordance with Dhiman et al., 2011, Manjari et al., 2011, Chang et al.,

2012 and Dahiya et al., 2012.

Phytochemical analysis conducted on the plant extracts revealed the presence of

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

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

activities of these plants. Since 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. 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 (Mason 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. 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 et al., 1996).

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Table 7.1 Phytochemical Analysis of Plant Extracts

Tested Plants

Extracts

T.chebula E. officinalis P.guajava R.indica A. nilotica

Met Et Met Et Met Et Met Et Met Et

Alkaloids

- - + + + + - - - -

Flavanoids

+ + + + + + + + + +

Saponins + + + + + + + + - -

Tannins

+ + + + + + + + + +

Phenols

+ + + + + + + + + +

Met: Methanol extract; Et: Ethyl acetate fraction

7.2.2 Gas Chromatography and Mass Spectroscopy (GC-MS)

There is growing awareness in correlating the phytochemical constituents of a

medicinal plant with its pharmacological activity. The demand for medicinal plant products

has increased considerably because phytocompounds target the biochemical pathway which

makes them safer than synthetic medicines. Lead compounds of the modern medicines are

directly or indirectly obtained from the medicinal plants. So the active principles in

medicinal plants need to be identified.

Gas chromatography and mass spectrometry (GC/MS) are an effective combination

for the analysis of volatile chemicals. Gas chromatography uses a carrier gas to move

analytes through a coated, fused silica capillary. Separation occurs based on differential

partition between the gas phase and the coating inside the capillary. GC/MS requires the

analyte to be vaporized in order for migration through the capillary to occur. Analytes,

therefore, must be volatile or amenable to chemical derivatization to render them volatile.

Certain types of samples are particularly well suited to GC/MS analyses. These include plant

terpenes, alkylsilyl derivatives, eicosanoids, essential oils, esters, perfumes, terpenes, waxes,

volatiles caratenoids, flavenoids and lipids. After derivatization, free fatty acids are also

amenable to GC/MS. The technique is highly sensitive and high chromatographic resolution

of GC permits separation of structurally similar fatty acids.

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After derivatization, free fatty acids are also amenable to GC/MS. The high

chromatographic resolution of GC permits separation of structurally similar fatty acids that

would be very difficult to separate by HPLC. GC/MS provides greater sensitivity than

LC/MS for free fatty acids. Other analytes generally compatible with GC/MS include

steroids, diglycerides, mono-, di- and tri-saccharides and sugar alcohols.

Hence keeping this in context, the present study was also aimed to find out the

bioactive compounds present in the ethyl acetate, methanol and chloroform fractions of E.

officinalis fruit, T. chebula, A. nilotica and methanol extracts of R. indica, P. guajava by

using Gas chromatography and Mass spectroscopy. GC-MS chromatograms of fractions are

shown in Figures 7.1 to 7.11 and compounds are listed in Tables 7.2 to 7.12.

The results pertaining to GC-MS analysis led to the identification of a number of

compounds from extracts of E. officinalis fruit. GC-MS chromatogram of the methanol and

ethyl acetate extracts of E. officinalis fruit showed 28 and 30 peaks indicating the presence of

twenty eight and thirty phytochemical constituents respectively (Figure 7.1 and 7.2). The

active principles of methanol, ethyl acetate and chloroform extracts are exhibited with their

peak no., concentration (peak area %) and retention time (RT) in Table 7.2 and 7.3

respectively. The results revealed that Pyrogallol (23.96%), 2, 6-Xylenol 4 4‟- dimethylene

(16.85%), 5-Hydroximethylfurfural (12.3%), Glucatonic anhydride (6.28%) and Palmitic acid

(5.83%) were found as the major components in methanol extract of E. officinalis. The

present findings are very close to Hasan et al., 2012 and Manikandaselvi et al., 2014, who

also reported Pyrogallol, 5- Hydroxymethylfurfural and Glucatonic anhydride as major

compounds of E. officinalis. Pyrogallol, a phenol is reported to have various biological

activities like allelochemic, antibacterial, abortifacient, aticlastogen, antidermatitic, antilupus,

antimutagenic, antioxidant, antipsoriac, antiseptic, candidicide, cardiovascular, dye, ecbolic,

fungicide, irritant, nephrotoxic, pesticide and prostaglandin synthesis inhibitor (Sangeetha

and Vijayalakshmi, 2011). 5- Hydroxy methyl furfural is an aldehyde possessing the

bioactivities like antimicrobial and preservative (Vanitha and Thangarasu, 2013). It is utilized

to produce pharmaceuticals and plant protection agents and reported in a number of food

products like citrus juices, tomato paste and honey etc. (Kunz, 1993). Furfural has a wide

variety of uses including weed killer and fungicide, affects yeast survival and also affect

biochemical enzyme activities as described by Horvath et al., 2001. 4H-Pyran-4-one, 2,3-

dihydro-3,5-dihydroxy-6-methyl-, a flavonoid, another compound of methanol extract is

reported to have antimicrobial and anti-inflamatory activities (Vadivel and Gopalakrishnan,

2011). Ethyl acetate sub fraction was reported, Oleic acid (26.09%), Docosane (9.25%),

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Sitostenone (5.12%) and Erucylamide (5.00%) compounds with good percentage. Vadivel

and Gopalakrishnan, 2011 reported that Oleic acid was one of the compounds of Mussaenda

frondosa and possess alpha-Reductase-Inhibitor, antialopecic, antiandrogenic,

antiinflammatory, antileukotriene, cancer-preventive, choleretic, dermatitigenic, flavor,

hypocholesterolemic, insectifuge activities, so for present study the highly significant

activities of E. officinalis ethyl acetate extract may be due to the presence of oleic acid.

Antibacterial, antioxidant as well as enzyme inhibitory activities were exhibited by

methanol extract and ethyl acetate sub fraction (Chapter III, V and VI), but in comparison

more significant activities were shown by methanol extract, it may be due to the presence of

5- Hydroxy methyl furfural, pyrogallol and xylenol in respective extract. While the

chloroform sub fraction did not exhibit any activity except antibacterial, it may be due to the

absence of pyrogallol, furfural etc. in the sub fraction (Figure 7.3 and Table 7.4). The major

compounds identified in chloroform fraction were Silane, dimethyl (docosyloxy) butoxy

(29.85%), 3, 5-Di-tert-butylphenol (28.18%), 2, 4-ditert-butylphenol (17.31%) and Stigmast-

5-en-3-ol, oleate (4.87%).

GC-MS chromatogram of methanol and ethyl acetate extract of A. nilotica leaves

showed 21 and 12 peaks (Figure 7.4 and 7.5) respectively. 2, 6-Xylenol 4 4‟- dimethylene

(23.57%), Phosphoric acid (16.67%), 2-Butenthionic acid, 3-(ethyl thio)-, S-(1-methylethyl)

ester (12.30%), Palmitic acid (10.77%), Linolenic acid (7.67%) and Vitamin E (5.20%) were

among the major compounds of methanol extract (Table 7.5), while 2, 6-Xylenol 4 4‟-

dimethylene (29.78%), Z-4-Decenal (14.01%), Methylphosphoric acid (11.13%), Ammonium

lauroyl sarcosinate (10.79%) and Phosphoric acid (9.86%) were observed as the major

phytocompounds of ethyl acetate fraction of A. nilotica (Table 7.6). Palmitic acid possesses

antioxidant, hypocholesterolemic, nematicide, pesticide, flavor, lubricant, antiandrogenic and

hemolytic 5-alpha reductase inhibiting activities (Vadivel and Gopalakrishnan, 2011).

Hemamalini et al., 2013, identified 3-picoline-2-nitro, 1-acetyl beta carboline, hydroxy

citronellal, trans decalone, propionic acid-2-chloroethyl ester, lavandulyl acetate and D-

Glucoronic acid, as major compounds in GC-MS analysis of A. nilotica leaves. These results

are not in accordance with present study. Chloroform fraction of A. nilotica showed the

presence of 63 compounds (Figure 7.6). The major compounds identified were 2, 4 Dimethyl-

butylphenol (22.36%), quercetin (18.28%), linolenic acid (12.40%), palmitic acid (8.70%)

and 2-methylresorcinol acetate (6.30%) (Table 7.7). 2, 4 Dimethyl-butylphenol, an

antioxidant was previously reported by Gupta et al., 2013 in dichloromethane extract of Piper

nigrum. Quercetin, a flavonoid possess antiarthritic, antibacterial, antiageing, antiallergic,

106

antidermatitic, anticarcinomic, antioxidant, antitumor, antiviral, Glucosyl transferase

inhibitor, MMP-9 inhibitor, phospholipase inhibitor, tyrosinase inhibitor etc. according to Dr.

Duke‟s phytochemical and ethnobotanical databases., So the significant antibacterial

activitity of A. nilotica chloroform fraction may be due to the presence of quercetin. As the

chloroform fraction did not inhibit either of enzymes, it may be due to the absence of xylenol,

because xylenol was the major compound of the ethyl acetate and methanol extract, which

inhibited both the enzymes.

The GC-MS analysis of T. chebula fruit extract revealed the presence of 38 and 33

compounds in methanol and ethyl acetate extract respectively (Figure 7.7 and 7.8). Pyrogallol

(36.76%) was the major compound of methanol extract (Table 7.8). Elamparithi et al., 2011

and Singh et al., 2013 reported pyrogallol as the major compound in T. chebula. Gangadhar

et al., 2011 also reported gallic acid from T. bellerica. Pyrogallol is reported as antiseptic,

antioxidant, antidermatitic, fungicide, insecticide, and candidicide (Gopalakrishnan and

Vadivel, 2011). Other compounds in considerable amount were 3-Methylpyridazine (13.94%)

and Palmitic acid (11.78%). Ethyl acetate fraction showed Trans-β-caryophyllene (9.78%),

Cetane (9.51%), Tetradecane (8.90%), β-sitosterol (5.85%) and Octadecane (5.32%) as its

major compounds (Table 7.9). Venkata et al., 2012, reported that Cryophyllene possess

antimicrobial, antioxidant, anti-tumor, analgesic, antibacterial, anti-inflammatory, sedative

and fungicide properties. Terpenes (Cryophyllene) known to be active against a wide variety

of microorganisms, including gram-positive and gram-negative bacteria and fungi (Cowan

1999). Experimental reports already noticed that β-Tocopherol, Neophytadiene, Sitosterol

and Stigmasterol have significant antibacterial activity as major components in the leaves of

E. odoratum (Zhang and Zhou, 2011). Venkata et al., 2012 also claimed that Neophytadiene

acts as antipyretic, analgesic, and anti-inflammatory, antimicrobial and antioxidant agent. He

also reported that sitosterol, a major compound of ethyl acetate fraction, possess anti-diabetic,

anti-angeogenic, anticancer, antimicrobial, anti-inflammatory, antidiarrhoeal, antiviral,

oestrogenic and insecticidal activities. A total of 70 compounds were revealed in GC-MS

analysis of chloroform fration of T. chebula (Figure 7.9). Variety of compounds of different

classes was detected in chloroform fraction viz. acids, alcohol, ketone, flavonoids, terpenes,

fatty acids, hydrocarbons, aldehydes, sterols, phenolic aldehyde, phenylpropanoids etc. The

major compounds observed were Eugenol (5.63%), vanillin (5.15), trans-cinnamic acid

(5.03%), palmitic acid (11.94%), oleic acid (12.11%), octadecanoic acid (5.64%), sesamin

(3.68%) and sylvatesmin (4.44%) (Table 7.10). Miao et al., 2004, repoted the activity of

sylvatesmin in oxidizing free radicals. Other significant compounds detected were 5-

107

hydroxymethylfurfural, thymol, maleic acid, α-bromocapric acid, matairesinol, lansterol etc.

Chloroform fraction exhibited considerable antibacterial and antioxidant activities, the later

may be due to presence of variety of compounds in the fraction, though it inhibited the

enzymes poorly.

GC-MS chromatogram of the methanol extract of R. indica petal showed 12 peaks

indicating the presence of twelve phytochemical constituents (Figure 7.10). The results

revealed that Quinic acid (43.12%), Pyrogallol (21.92%), 5-Hydroxymethylfurfural

(11.52%), 4H-Pyran-4-one,2,3-dihydro-3,5-dihydroxy-6-methyl- (8.31%), and Levoglucosan

(5.69%) were found as the major components in the methanol extract of R. indica petals

(Table 7.11). Quinic acid is a cyclitol, a cyclic polyol, and a cyclohexanecarboxylic acid.

This acid is a versatile chiral starting material for the synthesis of new pharmaceuticals.

Quinic acid based medication for the treatment of influenza A and B strains

called Tamiflu has been successfully developed and launched into the market.

Gopalakrishnan and Vadivel 2011, reported quinic acid as an antimicrobial agent. A recent

report by Inbathamizh and Padmini, 2013 claimed the use of quinic acid as potent drug

candidate in treatment of prostate cancer. 4H-Pyran-4-one, 2, 3-dihydro-3, 5-dihydroxy-6-

methyl exhibit antimicrobial and anti-inflamatory activities (Gopalakrishnan and Vadivel

2011). Hydroxymethylfurfural is reported to have antioxidant, cytoprotective, antitumor and

anti-inflammatory effect (Kim et al., 2011). Other significant compouds detected were 1, 2-

benzenediol, 1, 3, 4-eugenol, isoeugenol and phenylethyl alcohol. The literature search

revealed that there is no previous report on chemical constituents of methanol extract of R.

indica petals.

The GC-MS analysis of P. guajava leaf extract revealed the presence of 23

compounds (Figure 7.11) that could contribute to the medicinal property of the plant. In term

of percentage amount, Pyrogallol (27.64%), Isogeraniol (15.41%), 1-Butanol, 3-methyl

(10.92%) and Cinnamaldehyde (6.72%) were prominent in P. guajava (Table 7.12).

Cinnamaldehyde is reported as antimicrobial and anticancer (Cabello et al., 2009). Previous

studies reported many constituents in guava leaves such as Isopropyl alcohol, Longicyclene,

α-Pinene, β-Pinene, Limonene, Terpenyl acetate, β-Bisabolene, β-Copanene, Farnesene,

Humulene, Selinene, Mallic acids, β-Sitosterol, Ursolic, Quercetin, Avicularin, Eugenol,

Caryophyllene, Guajavolide, Guavenoic acid, and Cryptonine (Shruthi et al., 2013). Kapoor

et al., 2011 identified Methyl 2, 6, 10-trimethyltridecanoate and Methyl octadecanoate in P.

guajava, not in agreement to present study. However, it is noteworthy that the composition of

plant extracts is influenced by several factors, such as local, climatic, seasonal and

experimental conditions (Perry et al., 1999).

108

Thus, the presence of medicinally significant phytocomponents in the extract implies

the pharmaceutical importance of these plants. Further studies are needed on animal model to

evaluate their bioactivity and to ascertain the pharmacological activity of the concerned

compounds.

Figure 7.1 GC-MS chromatogram for methanol extract of E. officinalis

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Table 7.2 GC-MS spectral analysis of methanol extract of E. officinalis

Peak no. Peak Area

(%)

Compound name

Retention Time

1 10.39 Glucatonic anhydride 6.283

2 3.09 4H-pyran-4-one, 2,3-dihydro-3,5-dihydroxy-

6-methyl-

9.655

3 14.91 5-hydroxymethylfurfural 12.300

4 0.52 1-Tridecene 14.932

5 0.24 Pentadecane 15.091

6 0.62 Pyroglutamic acid, methyl ester, L- 15.400

7 16.85 2,6-Xylenol, 4,4'-dimethylene 15.524

8 23.96 Pyrogallol 16.093

9 2.36 Beta-D-Glucopyranose, 1,6-anhydro 17.707

10 0.74 1-Pentadecene 18.996

11 0.26 Tetradecane 19.131

12 0.18 Caprylone 20.576

13 0.66 1-Nonadecene 22.667

14 0.92 Palmitic acid, methyl ester 24.958

15 5.83 Palmitic acid 25.670

16 0.36 1-Nonadecane 25.997

17 0.59 Linoleic acid, methyl ester 27.629

18 0.90 9-Ooctadecenoic acid, mehyl ester 27.714

19 0.84 Heptadecane-(8)-carbonic acid-(1) 28.399

20 1.07 Oleic Acid 28.476

21 1.53 Stearic acid 28.744

22 0.18 1,2-Benzenedicarboxylic acid, 3-nitro- 33.974

23 0.39 Erythrodiol 34.488

24 0.39 Stigmast-5-en-3-ol, (3.beta.)- 41.505

25 2.28 Stigmast-5-en-3-ol, oleat 41.755

26 0.43 γ-sitosterol 43.499

27 9.04 3-Keto-urs-12-ene 43.594

28 0.47 Lupeol acetate 44.012

100

110

Figure 7.2 GC-MS chromatogram for ethyl acetate fraction of E. officinalis

111

Table 7.3 GC-MS spectral analysis of ethyl acetate fraction of E. officinalis

Peak no. Peak Area

(%)

Compound name

Retention Time

1 0.77 Oxalic acid, isobutyl pentyl ester 6.235

2 3.30 Undecane, 3,7-dimethyl- 7.427

3 0.91 2,3,6,7-Tetramethyloctane 8.249

4 4.62 Decane, 3,7-dimethyl- 8.406

5 0.88 3-Tetradecene, (Z)- 10.443

6 0.93 Tridecane 10.625

7 2.85 5-isobutyl nonane 12.433

8 0.75 1-Tridecanol 13.028

9 0.64 Hexadecane 13.327

10 9.25 Docosane 13.457

11 1.55 5-isobutyl nonane 13.671

12 1.35 Heptadecane 15.075

13 1.12 1-Hexadecanol 18.989

14 4.78 Oxalic acid, ethyl neopentyl ester 19.124

15 0.97 Phthalic acid, diisobutyl ester 24.102

16 1.59 Palmitic acid, methyl ester 24.865

17 3.75 Pentadecanoic acid 25.563

18 1.05 Dichloroacetic acid, heptadecyl ester 25.998

19 0.84 9-Tetradecen-1-ol 27.112

20 1.51 9-Octadecenoic acid, methyl ester 27.633

21 1.30 Oleic acid, methyl ester 27.732

22 26.09 Oleic Acid 28.369

23 3.29 Octadecanoic acid 28.629

24 5.00 Erucylamide 28.958

25 3.20 Cholesterol chloroformate 40.986

26 4.67 Gamma-tocopherol 41.437

27 1.03 Acetic acid, (1,2,3,4,5,6,7,8-octahydro-3,8,8-

trimethylnaphth-2-yl)methyl ester

41.852

28 4.14 Vitamin E 41.994

29 2.74 1,3-Cyclopentanedione, 2-bromo- 42.333

30 5.12 Sitostenone 44.696

100

112

Figure 7.3 GC-MS chromatogram for chloroform fraction of E. officinalis

113

Table 7.4 GC-MS spectral analysis of chloroform fraction of E. officinalis

Peak

no.

Area % Compound Retention time

1 0.48 Dihydrocitronellol 7.779

2 0.41 Trichloroacetic acid, 6-ethyl-3-octyl ester 13.053

3 0.72 3-Ethyl-3-methylheptane 13.493

4 0.42 Pentadecane 15.125

5 0.18 Nonane, 5-methyl-5-propyl- 17.077

6 17.31 2,4-ditert-butylphenol 17.631

7 1.12 Cetane 17.981

8 0.46 Pentadecane 19.155

9 0.70 Tricosane 21.177

10 0.29 Undecane, 3,8-dimethyl- 22.795

11 0.65 1,2-Benzenedicarboxylic acid, bis(2-

methylpropyl) ester

24.107

12 3.63 Methyl 12-methyltetradecanoate 24.969

13 1.68 Eicosane 25.543

14 1.57 Palmitic acid, ethyl ester 26.060

15 1.01 Linoleic acid, methyl ester 27.641

16 0.57 Oleic acid, methyl ester 27.726

17 0.68 Stearic acid 28.733

18 0.54 Tetracosane 34.580

19 0.28 Squalene 38.767

20 0.63 Heneicosane 39.896

21 0.75 Cholesterol chloroformate 40.647

22 1.16 Tetrapentacontane 41.181

23 1.67 Stigmast-5-en-3-ol, (3.beta)- 41.499

24 0.21 Cholesta-4,6-dien-3-ol, (3.beta.)- 41.600

25 4.87 Stigmast-5-en-3-ol, oleate 41.751

26 29.85 Silane, dimethyl(docosyloxy)butoxy- 44.187

27 28.18 3,5-Di-tert-butylphenol 46.057

100

114

Figure 7.4 GC-MS chromatogram for methanol extract of A. nilotica

115

Table 7.5 GC-MS spectral analysis of methanol extract of A. nilotica

Peak no. Peak Area

%

Compound name Retention

time

1 12.30 2-Butenethioic acid, 3-(ethylthio)-, S-(1-

methylethyl) ester

10.968

2 23.57 2,6-xylenol, 4,4’-methylenedi 15.541

3 0.82 1-Tridecane 19.031

4 0.99 1-Tetradecanol 22.683

5 0.84 1,3-benzenediol,5-methyl 23.100

6 16.67 Phosphoric acid 23.949

7 4.38 Palmitic acid, methyl ester 24.968

8 10.77 Palmitic acid 25.681

9 0.70 n-Nonadecanol-1 26.004

10 2.83 Linoleic acid, methyl ester 27.633

11 7.67 Linolenic acid, methyl ester 27.738

12 2.26 Stearic acid, methyl ester 28.107

13 1.49 Oleic acid 28.408

14 3.22 Oleic acid 28.489

15 0.47 Arachidic acid methyl ester 30.994

16 0.41 Palmitic acid trimethylsilyl ester 33.022

17 0.55 Arachidic acid methyl ester 33.664

18 2.29 Cholesteryl propionate 34.497

19 0.99 Stigmasterol acetate 41.659

20 1.61 Stigmast-5-en-3-ol, (3.beta.)- 41.765

21 5.20 Vitamin E 42.015

100

116

Figure 7.5 GC-MS chromatogram for ethyl acetate fraction of A. nilotica

117

Table 7.6 GC-MS spectral analysis of ethyl acetate fraction of A. nilotica

Peak

no.

Peak

Area %

Compound Retention time

1 11.13 Methylphosphonic acid 12.291

2 29.78 2,6-xylenol,4,4’-methylenedi 15.636

3 3.76 Phthalic acid, di(3,4-dimethylphenyl) ester 19.540

4 9.86 Phosphoric acid 23.986

5 2.51 Methyl 12-methyltetradecanoate 25.032

6 10.79 Ammonium lauroyl sarcosinate 25.767

7 2.05 Oleic acid, methyl ester 27.746

8 2.81 Stearic acid, methyl ester 28.130

9 14.01 Z-4-Decenal 28.544

10 1.43 1,2-Benzenedicarboxylic acid 34.003

11 6.04 Phenol, 4,4'-methylenebis[2,6-bis(1,1-

dimethylethyl)-

34.505

12 5.83 Cholesterol chloroformate 41.773

100

118

Figure 7.6 GC-MS chromatogram for chloroform fraction of A. nilotica

119

Table 7.7 GC-MS spectral analysis of chloroform fraction of A. nilotica

Peak no. Area % Compound Retention time

1 0.28 Decane, 3,7-dimethyl- 7.273

2 0.36 Dihydrocitronellol 7.790

3 0.60 Pelargonaldehyde 8.459

4 1.02 Undecane 10.616

5 0.34 6-dimethylamine-saccharin 12.178

6 0.39 Nonane, 5-(2-methylpropyl)- 12.442

7 0.48 1-Chlorohexadecane 12.904

8 0.47 Hexadecane 13.477

9 3.59 1,3,4-Eugenol 14.375

10 0.58 Pentadecane 15.085

11 0.51 Phosphoric acid, bis(trimethylsilyl)monomethyl ester 15.500

12 22.36 2,4 Dimethyl-butylphenol 17.608

13 0.51 Heptadecane 17.967

14 0.90 2,6,6-Trimethyl-2-hydroxycyclohexylidene

acetolactone

18.034

15 -21.58 3',5'-Dimethoxyacetophenone 17.608

16 2.40 Fumaric acid, ethyl 2-methylallyl ester 18.972

17 0.32 Phthalic acid 19.206

18 0.61 Megastigmatrienone 19.845

19 0.76 4-(1,5-Dihydroxy-2,6,6-trimethylcyclohex-2-

enyl)but-3-en-2-one

20.083

20 0.34 3-Oxo-.alpha.-ionol 20.300

21 0.73 Cinnamic acid, 3-hydroxy-4-methoxy 21.318

22 0.58 (3-Oxo-2-pent-2-enylcyclopentyl)acetic acid 21.783

23 0.89 Myristic acid 22.325

24 6.30 2-Methylresorcinol, acetate 22.967

25 1.29 Neophytadiene 23.460

26 0.69 Phthalic acid,butyl octyl ester 24.064

27 0.76 Tetracosane 24.846

28 0.23 1,54-Dibromotetrapentacontane 25.342

29 1.21 Eicosane 25.534

30 8.70 Palmitic acid 25.680

31 1.34 Palmitic acid, ethylester 26.031

32 0.81 Isopropyl Palmitate 26.503

120

33 1.06 1,11-Hexadecadiyne 27.389

34 0.34 Linolenic acid, methyl ester 27.713

35 1.05 Cedrane-8,13-diol 27.808

36 0.67 Tetracosane 28.177

37 12.40 Linolenic acid 28.435

38 9.04 Stearic acid 28.741

39 1.61 Stearic acid ethyl ester 29.067

40 0.57 Dotriacontane 29.318

41 0.59 Arachidonic acid 30.408

42 0.77 Dipalmitin 30.560

43 0.80 γ-Linolenic acid 31.036

44 1.92 Octadecane, 3-ethyl-5-(2-ethylbutyl)- 31.635

45 0.97 Tetracosane 31.772

46 0.52 1,3,5-Trisilacyclohexane 32.999

47 0.84 Palmitoyl chloride 33.110

48 0.32 Phenol, 4,4'-methylenebis[2,6-bis(1,1-

dimethylethyl)-

34.471

49 0.36 Tetrapentacontane 34.573

50 0.79 Decyl sulfide 37.567

51 0.60 Hexatriacontane 39.883

52 0.30 Oxirane, hexadecyl- 40.592

53 0.43 Hexatriacontane 40.904

54 0.68 Tetrapentacontane 42.308

55 1.17 (+)-Lariciresinol 42.654

56 1.78 (+)-Lariciresinol 42.794

57 0.37 δ-5-Avenasterol 43.342

58 0.31 3,4,7-trimethylquercetin 44.333

59 1.03 1,3,4-Eugenol 44.709

60 0.24 Lariciresinol 44.875

61 0.73 Phthalic acid 44.960

62 0.67 Terephthalic acid ester of neopentyl glycol cyclic

dimer

45.194

63 18.28 Quercetin 46.061

100

121

Figure 7.7 GC-MS chromatogram for methanol extract of T. chebula

122

Table 7.8 GC-MS spectral analysis of methanol extract of T. chebula

Peak no. Peak Area % Compound name Retention

time

1 13.94 3-Methylpyridazine 6.885

2 0.34 Normanthane 11.540

3 5.43 Pyrocatechol 12.232

4 0.17 1-Heneicosanol 14.399

5 0.96 1-Tetradecene 14.938

6 0.30 Germacrane 15.253

7 2.47 2,6-Xylenol, 4,4‟-methylenedi- 15.523

8 36.76 Pyrogallol 16.217

9 1.71 Cetyl alcohol 18.995

10 0.25 Pentadecane 19.127

11 0.37 1,2-Benzenedicarboxylic acid, mono(2-

ethylhexyl) ester

19.245

12 0.18 n-Nonylcyclohexane 20.203

13 0.32 Caprylone 20.571

14 1.58 1-Nonadecene 22.658

15 0.51 Neophytadiene 23.460

16 0.22 β-Citronellol 23.889

17 0.31 Caprylone 24.106

18 0.16 Neophytadiene 24.186

19 2.14 Palmitic acid 24.946

20 11.78 Palmitic acid 25.655

21 0.83 Behenic alcohol 25.987

22 1.04 Linoleic acid, methyl ester 27.615

23 1.93 Elaidic acid, methyl ester 27.702

24 0.96 Stearid acid, methyl ester 28.088

25 0.91 Linoleic acid 28.308

26 2.42 Elaidinsaeure 28.378

27 3.37 Stearic acid 28.719

28 0.47 9-Hexacosene 29.029

29 0.20 Arachidic acid methyl ester 30.971

30 0.24 Heneicosanoic acid, methyl ester 33.639

31 0.51 3-nitrophthalic acid 33.950

32 0.37 Erythrodiol 34.469

33 0.37 Stigmast-5-en-3-ol (3. beta)- 41.492

34 0.57 Cholesta-4,6-dien-3-ol, (3.beta.)- 41.592

35 1.90 Stigmast-5-en-3-ol, oleate 41.744

36 0.39 Vitamin E 41.997

37 0.23 Tetracosamethyl-cyclododecasiloxane 42.526

38 3.35 γ-sitosterol 43.468

100

123

Figure 7.8 GC-MS chromatogram for ethyl acetate fraction of T. chebula

124

Table 7.9 GC-MS spectral analysis of ethyl acetate fraction of T. chebula

Peak

no.

Area % Compound Retention time

1 4.56 Dodecane 10.619

2 1.07 1-Tridecene 14.917

3 8.90 Tetradecane 15.061

4 1.02 α-Gurjunen 15.333

5 9.78 Trans-β-Caryophyllene 15.557

6 1.86 γ-Muurolen 15.823

7 2.49 Caryophyllene 16.411

8 3.06 2,4-Ditertbutylphenol 17.551

9 1.47 Dodecane, 2,6,11-trimethyl- 17.926

10 3.67 1-Pentadecene 18.967

11 9.51 Cetane 19.091

12 0.79 2-Phenyleicosane 21.116

13 1.59 Octadecane 21.898

14 0.88 Palmityl chloride 22.237

15 4.50 Hexahydroaplotaxene 22.628

16 5.32 Octadecane 22.735

17 4.75 1,2-Benzenedicarboxylic acid, bis (2-

methylpropyl) ester

24.057

18 3.38 Stearic acid methyl ester 24.952

19 0.99 Nonane, 5-(2-methylpropyl)- 25.486

20 0.41 Tridecane, 3-methylene- 25.867

21 3.09 1- Heneicosanol 25.953

22 3.18 Nonadecane 26.044

23 0.66 trans-2-Dodecen-1-ol 27.600

24 1.36 1- Heneicosanol 28.996

25 1.58 Heneicosane 29.069

26 1.95 Heptadecane 31.856

27 1.79 1,2-Benzenedicarboxylic acid 33.942

28 1.34 Tetratriacontane 41.639

29 1.61 Vitamin E 41.990

30 0.63 Stigmasta-5,22-dien-3-ol 42.984

31 5.85 β-Sitosterol 43.443

32 2.30 Oxalic acid, dibutyl ester 44.130

33 4.66 2-tert-butyl-4,6-bis (3,5-di-tert-butyl-4-

hydroxybenzyl) Phenol

45.982

100

125

Figure 7.9 GC-MS chromatogram for chloroform fraction of T. chebula

126

Table 7.10 GC-MS spectral analysis of chloroform fraction of T. chebula

Peak no. Area % Compound Retention time

1 0.50 2-Cyclopenten-1-one, 3-methyl- 5.422

2 3.05 3-Hexen-2-one 5.529

3 1.25 3,5-Hexadien-2-ol 7.209

4 0.18 2-Decenal, (Z)- 7.885

5 0.12 Decane, 3,7-dimethyl- 8.313

6 0.21 Nonanal 8.457

7 0.67 Dehydromevalonic lactone 10.042

8 0.19 Napthalene 10.411

9 0.40 Octadecanoic acid 10.609

10 0.78 5-hydroxymethylfurfural 12.158

11 0.14 5-isobutylnonane 12.439

12 0.39 Thymol 13.068

13 0.13 Diisodecyl ether 13.224

14 0.33 Docosane 13.476

15 5.63 Eugenol 14.341

16 0.35 Pentadecane 15.081

17 5.15 Vanillin 15.448

18 0.55 l-Proline, N-ethoxycarbonyl-, butyl ester 16.198

19 5.03 trans-Cinnamic acid 16.538

20 0.24 Hexadecane 17.064

21 3.60 2,4-Ditertbutylphenol 17.610

22 0.94 Eugenol 17.853

23 0.98 Maleic acid, dibutyl ester 18.020

24 0.39 Octadecane 19.121

25 0.30 Phthalic acid 19.203

26 0.14 1,2-Benzendicarboxylic acid, diethyl

ester

20.307

27 0.99 Isochiapin B %2< 20.517

28 0.94 Benzaldehyde, 4-Hydroxy-3,5-

dimethoxy

20.709

29 0.17 Heneicosane 20.989

30 0.41 Curlone 21.191

31 0.17 Phenol, 2,6-Dimethoxy-4- (2-Propenyl)- 21.318

32 0.49 Eicosane 21.945

33 0.34 Benzene, (2-Decyldodecyl)- 22.110

34 0.86 α-Bromocapric acid 22.312

35 0.20 Heneicosane 22.767

36 0.23 Hexahydrofamesyl alcohol 23.591

127

37 0.23 1,2-Benzendicarboxylic acid, dibutyl

ester or Araldite

24.062

38 0.26 Tetracosane 24.842

39 1.47 Palmitic acid, methyl ester 24.939

40 11.94 Palmitic acid 25.715

41 0.32 Palmitic acid, ethyl ester 26.028

42 0.76 Linoleic acid, methyl ester 27.692

43 1.00 2H-Tetrazole, 5-(3-chlorobenzyl)- 28.110

44 12.11 Oleic Acid 28.427

45 5.64 Octadecanoic acid 28.750

46 0.45 Triacontane, 1-bromo- 31.208

47 0.25 Tetracosane 31.871

48 0.31 Hexatriacontane 33.201

49 0.32 Hexatriacontane 38.023

50 0.72 δ-tocopherol 39.281

51 0.16 Phenol, 2-(1-methyl-2-buthenyl)-4-

methoxy-

40.094

52 0.27 Matairesinol @ Carissanol 40.290

53 0.40 Stigmast-5-en-3-ol, (3.beta)- 40.633

54 0.18 Octadecane 41.125

55 0.52 Cholesta-4,6-dien-3-ol, (3.beta.)- 41.589

56 0.17 Tetracontane 41.644

57 2.25 Stigmast-5-en-3-ol, oleate 41.737

58 0.57 Vitamin E 41.983

59 3.68 Sesamin 42.250

60 0.72 Pluviatilol.gamma. gamma-dimethylallyl

ether

42.513

61 7.70 Paulownin 42.626

62 0.22 (+)-Lariciresinol 42.782

63 4.44 Sylvatesmin 42.967

64 0.17 Hexahydrofamesyl acetone 43.238

65 5.03 γ-sitosterol 43.462

66 0.40 Sisamin 43.662

67 0.18 Chelestanone 43.932

68 0.41 9-Hydroxy-4-fluorenylethylene oxide 44.003

69 0.11 Lanosterol 44.120

70 0.19 Cholesta-3,5-dien-7-one 44.328

100

128

Figure 7.10 GC-MS chromatogram for methanol extract of R. indica

129

Table 7.11 GC-MS spectral analysis of methanol extract of R. indica petals

Peak no. Peak Area

(%)

Compound

Retention Time

1 0.45 Isobutyl chloridocarbonate 7.238

2 4.19 Phenylethyl Alcohol 7.534

3 8.31 4H-Pyran-4-one, 2,3-dihydro-3,5-

dihydroxy-6-methyl-

8.033

4 0.62 1,2-Benzenediol 9.125

5 11.52 Hydroxymethylfurfural 9.335

6 1.72 3-Phenyl-2-propenal 9.658

7 0.58 1,3,4-Eugenol 10.315

8 1.17 1,3,4-Eugenol 10.412

9 21.92 Pyrogallol 11.560

10 0.72 Isoeugenol 12.381

11 5.69 1,6-Anhydro-beta-D-glucopyranose

(Levoglucosan)

12.854

12 43.12 1,3,4,5-Tetrahydroxy-

cyclohexanecarboxylic acid (Quinic

acid)

14.657

100.00

130

Figure 7.11 GC-MS chromatogram for methanol extract of P. guajava

131

Table 7.12 GC-MS spectral analysis of methanol extract of P. guajava leaves

Peak no. Peak Area (%) Compound name

Retention Time

1. 6.72 Cinnamaldehyde 10.021

2. 4.98 Isoeugenol 10.555

3. 2.76 2-Nitro-1-undecene 11.117

4. 27.64 Pyrogallol 11.980

5. 10.92 1-Butanol, 3-methyl 12.837

6. 1.80 2-Butenoic acid, 2-methoxy-, methyl ester 14.328

7. 2.14 1-Ethoxy-2-propyne 15.287

8. 2.52 5-Allyl-2,2-diisopropyltetrahydrofuran 15.525

9. 5.56 2-Butanone,4-(5,5-dimethyl-2-methylene-3-

oxabicyclo[5.1.0]oct-4-ylidene)-

15.722

10. 1.83 Santolina alcohol 16.254

11. 2.36 Ricinoleic acid 16.608

12. 15.41 3-Hydroxy-12-ketobisnorcholanic acid

(Isogeranol)

17.174

13. 2.29 5-Oxatricyclo[8.2.0.0(4,6)]dodecane,

12-trimethyl-9-methylene-,[1R-

(1R*,4R*,6R*,10S*)]-

18.475

14. 0.35 4-Methyl-4-nitro-5-oxoheptanal 26.573

15. 0.54 1-Iodomethoxyprop-2-yne 26.943

16. 1.26 Disulfide, dioctyl 27.409

17. 1.03 1-Iodoheptane 28.105

18. 1.40 2,2-Dimethyl-3-hexanone 28.352

19. 0.89 4-Methyl-4-nitro-5-oxoheptanal 29.011

20. 3.41 Docosane 29.437

21. 1.75 2,5,9-Trimethyldecane 30.383

22. 1.04 1-Iodoheptane 30.712

23. 1.39 Disulfide, dioctyl 32.227

100.00

132

7.4 Conclusion

The plants screened for phytochemical constituents seemed to have the potential to act

as a source of useful drugs and also to improve the health status of the consumers due

to the presence of various compounds that seems to be vital for good health.

The presence of various phenolic compounds, fatty acids, terpenes, flavonoids,

tannins etc. was confirmed in this study. Their presence jusify the good bioactivities

of the samples studied, since these compounds possess various modes of antimicrobial

action, antioxidant mechanisms likewise inactivation of microbial enzymes, cell wall

etc.

Pyrogallol, a phenol was detected as major compound in methnol extracts of

Terminalia chebula, Emblica officinalis, Rosa indica and Psidium guajava. Phenols

are responsible for wide variety of bioactivities e.g. enzyme inhibition, interaction

with proteins, phenolic toxicity to microorganisms etc. So, the good bioactivities of

these plants may be due to its presence.

Comparison of GC-MS chromatogram and biological activities of various fractions of

the same plant reveals that there is correlation between the compounds revealed by

chromatogram and biological activity of the plant samples. Thus, it validate that GC-

MS is an authentic technique to analyze the various components of the given sample.

Samples of the present study confirmed the presence of some compounds with the

already reported work.

The present assignment is successful in revealing a new active compound i.e. 2, 6-

Xylenol 4 4‟-dimethylene in E. officinalis, A. nilotica.

5-Hydroxymethylfurfural, Z-4-Decenal, Trans-β-Caryophyllene, Quinic acid,

Levoglucosan, Isogeraniol and Cinnamaldehyde etc. are first time reported in E.

officinalis, A. nilotica, T. chebula, R. indica and P. guajava.

The present study is the first report on the GC-MS analysis R. indica petals.