2.

1 Introduction

The biggest human use of the natural world is represented by the use of

medicinal plants. The number and diversity of medicinal plants differ greatly in various

regions, countries, and continents. For human welfare and healthcare herbal medicines

are being globally recognized on the basis of their enormous curative responses. One

major obstacle that limits the potential use of the medicinal plant as “medicine of

choice” is the lack of standardization. The advancement and recognition in the field of

chemical markers assure herbal medicines a challenging era.

Moreover, in the current years, significant interest has been revealed for

utilization of native drugs in the management of various diseases. Several members of

the Rutaceae family are being used traditionally for a wide array of ethnomedical

properties. From the beginning of human civilization plant and plant, products have

been exploited to be used as medicines. Plants continue to be a major source of

commercially consumed drugs. One of the main resources of commercially consumed

drugs is plants. Various synthetic drugs have their source from natural plant products. In

recent years the tendency of using natural products has increased and new drug

discoveries have taken place by using the active plant extracts (Ncube et al., 2008).

There are a large number of medicinal plants whose scientific value has not been

explored. For conventional as well as modern medicines, plants have served as the

richest source of raw materials globally but predominantly in Africa and Asia

(Miemanang et al., 2006). Knowledge acquired by ancient people was transmitted from

generation to generation and new knowledge added to it by the next generation.

61
Gradually, a group of people in each generation started specializing in collecting and

processing medicinal plants and using them against various diseases even though many

of them had not been recognized scientifically. Diseases are best controlled through

pharmacotherapy. Since the majority of the drugs are being used as medicines, the study

of their chemical composition and structure is very important for their synthesis (Ghani,

1990).

The various phytoconstituents from different parts of Aegle marmelos tree have

already been investigated and isolated. Different phytochemicals have been isolated

from the stem bark of Aegle marmelos such as marmesin–1”- α -L - rhamnopyranoside

and 1, 5 -dihydroxy - 6 - methoxy -2 - methyl anthraquinone along with lupeol and β-

sitosterol (Nema & Srivastava, 1991). The leaves of Aegle marmelos also yielded other

chemical compounds such as Aegeline (De Smet, 1997), Lupeol (Shu, 1998), Cineol

(Biswas et al., 2002), Citral (Chatopadhyay et al., 2004) and Eugenol. Moreover,

Marmelosin (Swarnakar et al., 2005), Luvangetin (Badam et al., 2002) and Marmelide

(Jagetia et al., 2005) have been reported and isolated from fruits of Aegle marmelos. The

study is aimed at identifying the bioactive compounds in the methanolic and aqueous

extracts of Bael leaves both quantitatively as well as qualitatively and its GC-MS

analysis.

2.2 Materials and Methods

2.2.1 Collection and Identification of Plant Material

62
The fresh leaves of Aegle marmelos from 18 varieties/accessions were collected from

the orchard of Narendra Deva University of Agriculture and Technology, Kumarganj,

Faizabad, India (Table 2.1). The taxonomy of the plant was authenticated. Finely

powdered and air shade-dried plant material was taken for experiments.

Table 2.1: Morphological presentation of sampled Bael (Aegle marmelos) leaves

S. No. List of Bael leaf varieties/accessions Morphological

characteristics

1. AM-1

2. AM-2

3. AM-3

63
4. AM-4

5. AM-6

6. AM-7

7. AM-8

8. NB-1

64
9. NB-4

10. NB-5

11. NB-7

12. NB-9

13. NB-16

65
14. NB-17

15. Pant Aparna

16. Pant Sujata

17. Pant Shivani

18. Kaghzi

66
2.2.2 Chemicals

Methanol, Ferric chloride (FeCl3), Hydrochloric acid (HCl), Dragendroff reagent,

glacial acetic acid (GAA), sulphuric acid (H2SO4), chloroform (CHCl3), acetic

anhydride, ammonia solution, Fehling’s solution A and B, ammonium hydroxide

(NH4OH), ethyl acetate, Folin-Denis reagent, sodium carbonate (Na2CO3), isobutyl

alcohol, magnesium carbonate (MgCO3), ether, pentanol, gallic acid, Folin- Ciocalteu

reagent, aluminium chloride (AlCl3), potassium acetate, rutin and all other chemicals

used were from Merck and Himedia.

The UV spectrophotometer (Genesys 10S UV-VIS Spectrophotometer, Thermo

scientific, USA) was used for the measurement of absorbance of different extracts under

study.

2.2.3 Extract preparation

The leaves of the plant were properly washed in tap water and rinsed in distilled

water. The rinsed leaves were hot air dried for 3 days. The dried leaves of each plant

were pulverized using pestle mortar to obtain a powdered form which was stored in

airtight glass containers at 4°C until used. 10 gm of powdered sample was soaked in

distilled water and methanol (200 ml and 100 ml) separately for 12 hrs at room

temperature. The extracts were then filtered and concentrated to a final volume of 50 ml

and subjected to phytochemical analysis.

67
2.2.4 Phytochemical Analysis

Qualitative phytochemical analyses of both the extracts were performed by

following the protocol of Adetuyi (Adetuyi & Popoola, 2001) Trease and Evans (Trease

& Evans, 1989), Sofowora (Sofowora, 1982).

Tannins: 200 mg of dried extract was boiled in 10 ml distilled water and few drops of

FeCl3 was added to the filtrate, a blue-black precipitate indicated the presence of

Tannins.

Alkaloids: 200 mg plant sample was boiled in 10 ml methanol and filtered. 1% HCl was

added followed by 6 drops of Dragendroff reagent, and the brownish-red precipitate was

taken as evidence for the presence of alkaloids.

Saponins: (Frothing test): 5 ml distilled water was added to 200 mg dried plant

material. 0.5 ml filtrate was diluted to 5 ml with distilled water and shaken vigorously

for 2 minutes. Formation of stable foam indicated the presence of saponins.

Cardiac glycosides (Keller-Kiliani test): 2 ml filtrate was treated with 1 ml glacial

acetic acid containing few drops of FeCl3.Conc. H2SO4 was added to the above mixture,

green-blue colour depicted the positive results for the presence of cardiac glycosides.

Steroids (Liebermann-Burchard reaction): 200 mg powdered dried leaves was added in

10 ml chloroform. Acetic anhydride was added in the ratio of 1:1 which resulted in the

formation of blue-green ring pointing towards the presence of steroids.

Terpenoids (Salkowski test): To 200 mg specimen sample 2ml of chloroform (CHCl3)

and 3 ml of concentrated H2SO4 was carefully added. A reddish brown colouration

signified the presence of terpenoids.

68
Flavonoids: To the aqueous filtrate 5ml of dilute ammonia solution was added,

followed by concentrated H2SO4. A yellow colouration indicated the presence of

flavonoids.

Phlobatannins: The deposition of a red precipitate denoted the presence of

phlobatannins when 200 mg of plant material was dissolved in 10 ml of aqueous extract

and few drops of 1% HCl were added in the boiling tube.

Anthraquinones: 500 mg of dried plant leaves were boiled in 10% HCl for 5 mins and

the filtrate was allowed to cool. An equal volume of CHCl3 with few drops of 10% NH3

was added to the 2ml filtrate. The formation of rose-pink colour implies the presence of

anthraquinones.

Reducing sugars: To the 10 ml of aqueous extract a few drops of Fehling’s solution A

and B were added, an orange-red precipitate suggested the presence of reducing sugars.

2.2.5 Quantitative estimation of phytochemicals

2.2.5.1 Determination of Alkaloids

Alkaloids content was measured by following the protocol described by

Harborne (Harborne, 1973). A suspension was prepared by dispersing 5 gm of the dried

leaves in 10% acetic acid solution in ethanol and kept at 28C for 4hrs which was

further filtered through Whatman No. 42. Thereafter alkaloid was precipitated by

concentrating the filtrate to one-quarter of its original volume and drops of conc.

aqueous NH4OH were added. Finally, the precipitate was washed with 1% ammonia

69
solution and dried at 80°C in the oven. The content of alkaloid was calculated and

expressed as mg/g of sample.

2.2.5.2 Determination of Flavonoids

The flavonoids content was also determined by Harborne (Harborne, 1973) method.

Briefly, 5 gm of leaves were boiled in 2M HCl for 30 min under reflux and filtered after

cooling. An equal volume of ethyl acetate was then added dropwise to the filtrate. The

weight of precipitated flavonoid was determined and reported as mg/g.

2.2.5.3 Determination of Tannins

The quantitative estimation of tannins was performed by the method of Swain

(Swain, 1979) with minor modifications. The finely powdered leaves of Aegle marmelos

were kept in a beaker containing 20 ml of 50% methanol covered with parafilm and then

heated at 80oC in a water bath for 1 hr with continuous stirring. The extract was

quantitatively filtered using a double layered Whatman No.1 filter paper and rinsed with

50% methanol. 1 ml of sample extract was treated with 20 ml distilled water, 2.5 ml

Folin-Denis reagent and 10 ml of 17% Na2CO3 for the development of a bluish-green

colour and was allowed to stand for 20 mins. The absorbance was measured at 760 nm

and the amount of tannin was calculated by comparing it with a standard curve prepared

in the range of 0-10 ppm.

70
2.2.5.4 Determination of Saponins

Saponin analysis was performed according to the method described by Brunner

(Brunner, 1984). 100 ml Isobutyl alcohol was added to 1 gm of the finely powdered

sample and stirred for 5 hrs. 20 ml of 40% saturated solution of Magnesium carbonate

was added to the mixture and filtered. 2 ml of 5% FeCl3 solution and 50ml volume of

distilled water was added to 1ml of colourless solution and kept for 30 mins for colour

(blood red) development The absorbance of the samples as along with the standard were

read at 380 nm and calculated in mg/g. Standard saponin solution was prepared in the

reference range of 0-10 ppm.

2.2.5.5 Determination of total phenols

Five gms of the powdered leaves were boiled with 50 ml of ether for 15 mins and

distributed in the ratio 1:2 (extract: distilled water). 2ml of ammonium hydroxide

followed with 5ml of pentanol was added to it and incubated at the room temperature for

30mins. The absorbance was read at 505 nm wavelength Obadoni and Ochuko (Obodoni

& Ochuko, 2001).

2.2.6 Quantitative analysis of phytochemical constituent

2.2.6.1 Preparation of aqueous extract of plant sample

The aqueous extract of each plant sample was prepared by soaking 10gm of the

powdered sample in 200 ml of distilled water for 12 hrs. The extracts were then filtered

71
using filter paper. The extracts were then concentrated to ¼ of the original extracts i.e.

50 ml.

2.2.6.2 Preparation of methanol extract of plant sample

The methanolic extract of each plant was prepared by soaking 10 gm of

powdered samples in 100 ml of methanol for the same 12 hrs. The extracts were then

filtered using filter paper. The extracts were then concentrated to 50 ml of the extracts

sample and stored in airtight container.

2.2.6.3 Total Phenolics determination:

The amount of total phenolics in extracts was determined by the Folin–Ciocalteu

method (Folin & Ciocalteu, 1927). Gallic acid was used as a standard by using different

concentrations of (20-200µg) from which the total phenol content in the extract was

expressed in terms of gallic acid equivalent (mg GAE /gm) extract. Different aliquots of

0.1 to 1.0 ml of plant extract were also prepared in methanol and 0.5 ml of each sample

were introduced into test tubes and mixed with 2.5 ml of a 10-fold dilute Folin-

Ciocalteu reagent and 2 ml of 7.5% sodium carbonate. The mixture was allowed to

stand for 30 mins at room temperature. Phenols react with the phosphomolybdic acid in

Folin- Ciocalteau reagent in alkaline medium and produce blue coloured complex

(Molybdenum blue). The absorbance of the resulting solutions was measured at 760 nm

against reagent blank. A standard calibration curve was prepared by plotting absorbance

against concentration and it was found to be linear over this concentration range. The

72
concentration of total phenol in the test sample was determined from the calibration

graph. The assay was carried out in triplicate and the mean values with ± SD are

presented.

2.2.6.4 Total Flavonoids determination:

The aluminium chloride colorimetric method was used for flavonoids

determination (Chang et al., 2002). Each plant extracts (0.5 ml of 1:10 gm ml-1) in

methanol were separately mixed with 1.5 ml of methanol, 0.1 ml of 10% aluminium

chloride, 0.1 ml of 1 M potassium acetate and 2.8 ml of distilled water. It was kept at

room temperature for 30 min; the absorbance of the reaction mixture was measured at

418 nm. The percentage of total flavonoids were calculated from the calibration curve of

Rutin (200-1000μg) plotted by using the same procedure and total flavonoids was

expressed as Rutin equivalents in mg per gm sample.

2.3 Gas Chromatography-Mass Spectrometry analysis

The Gas chromatography-Mass spectrometry (GC-MS) analysis of a methanolic

extract of leaves of Aegle marmelos (var. Pant Aparna) was performed using a GC-MS

(Model; QP 2010 Plus, Shimadzu, Tokyo, Japan) equipped with a VF-5ms fused silica

capillary column of 30m length, 0.25mm diameter, and 0.25μm film thickness. The

column oven temperature was programmed from 80°C to 310°C for 2°C min-1.

Ionization of the sample components was performed in electron impact mode (EI, 70

eV). The temperature of the injector was fixed to 270°C and one of the detectors to

73
230°C. Helium (99.9995% purity) was the carrier gas fixed with a flow rate of 1.21 ml

min-1. The mass range from 40-650 m/z was scanned at a rate of 3.0 scans/s. 2.0 μl of

the methanolic extract of Aegle marmelos was injected with a Hamilton syringe to the

GC-MS manually for total ion chromatographic analysis in split injection technique.

Total running time of GC-MS was 56 mins. The relative percentage of the each extract

constituents was expressed as a percentage with peak area normalization.

The bioactive compounds of methanol extract were identified by comparing their

retention indices and patterns of mass spectra with reference to Wiley Registry of Mass

Spectral Data’s, New York (Wiley 8) and Fatty Acid Methyl Esters Library version 1.0

(FAME library) sources.

2.4 Result and Discussion

2.4.1 Phytochemical profiling of aqueous and methanolic extracts of Aegle

marmelos

The present study was carried on aqueous and methanolic extracts of Aegle

marmelos to investigate the presence of medicinally important phytochemicals in the

leaves of different varieties/accessions. Both the extracts revealed the presence of

various phytochemicals such as tannins, saponins, flavonoids, alkaloids, terpenoids,

carotenoids, cardiac glycosides and reducing sugars in all the varieties and accessions

while phlobatannins and anthocyanins were absent (Table 2.2). The presence of

different phytochemicals and the antimicrobial activity of ethanolic, petroleum ether,

chloroform and methanolic extract of a single unidentified variety of Aegle marmelos

74
has been previously reported (Venkatesan et al., 2009; Kothari et al., 2011) however,

our study is first ever report to the best of our knowledge on qualitative and quantitative

comparative analysis of various varieties/accessions available in India.

Table 2.2: Qualitative Analysis of Phytochemicals in aqueous and methanol extract

of different varieties/accessions of Bael (Aegle marmelos) leaves
a b c d e f g h i j
Diff. Bael TA PHL SAP TER FLA CAR ANTH CAR RED ALK
variety/
accessions

NB-17 + - + + + + - + + +

Pant + - + + + + - + + +
Aparna

NB-9 + - + + + + - + + +

NB-5 + - + + + + - + + +

AM-4 + - + + + + - + + +

NB-7 + - + + + + - + + +

AM-7 + - + + + + - + + +

AM-3 + - + + + + - + + +

NB-1 + - + + + + - + + +

Kaghzi + - + + + + - + + +

NB-4 + - + + + + - + + +

NB-16 + - + + + + - + + +

Pant + - + + + + - + + +
Shivani

Pant + - + + + + - + + +
Sujata

75
 AM-1 + - + + + + - + + +

AM-2 + - + + + + - + + +

AM-6 + - + + + + - + + +

AM-8 + - + + + + - + + +
a b c d
TA=tannins PHL=phlobatannins SAP=saponins TER = terpenoids
e
FLA=flavonoids fCAR =cardiac glycosides gANTH=combined anthraquinones hCAR
= carotenoids iRED = Reducing sugar, jALK = alkaloids , + = Present, - = Absent
The quantitative phytochemical estimation specifies that the leaves of all

varieties contain a significant amount of alkaloid, flavonoids, phenolic, saponins and

tannin content. However, the variety called Pant Aparna contains the highest amount of

alkaloids, flavonoids, and phenol (Table 2.3). The alkaloids content was quantitatively

estimated and was found in the range of 3.78±0.15- 16.08±0.05 mg/g in different

varieties of Bael leaves but the maximum content was observed in Pant Aparna

(16.08±0.05 mg/g). Similarly, all the varieties exhibited good quantity of flavonoids and

phenols starting from 10.4±0.047mg/gm and 5.8±0.085mg/gm respectively in variety

NB-1, reaching up to 63.9±0.061 mg/gm and 29.4±0.004 mg/gm in Pant Aparna. An

earlier report on quantitative yield also revealed that Aegle marmelos contained the

highest quantity of alkaloids, flavonoids, and tannins as compared to other medicinal

plants (Dhandapani & Sabna, 2008). The highest content of saponins was found in

accession AM-7 (13.40±0.30 mg/gm) followed by Pant Aparna and AM-6. Among all

the varieties analyzed, Pant Aparna was found the most promising one, which prompted

us to project Pant Aparna for further studies.

76
Table 2.3: Quantitative estimation of phytochemicals (mg/gm)

Different Alkaloids Flavonoids Phenols Saponins Tannins

Bael
variety/
accessions

NB-17 8.62±0.10 26.2±0.065 22.8±0.004 7.65±0.14 4.53±0.15

Pant 16.08±0.05** 63.9±0.061** 29.4±0.004** 11.98±0.20 8.32±0.40

Aparna

NB-9 10.7±0.15 19.8±0.058 10.22±0.032 5.36±0.15 3.78±0.35

NB-5 9.56±0.10 23.2±0.055 12.6±0.005 6.57±0.25 6.43±0.05

AM-4 11.6±0.14 23.9±0.053 11.4±0.007 4.23±0.44 4.97±0.10

NB-7 6.56±0.25 18.5±0.051 10.0±0.005 4.65±0.10 3.45±0.25

AM-7 4.28±0.30 39.1±0.049 19.6±0.045 13.40±0.30 5.76±0.35

AM-3 14.34±0.20 28.9±0.048 17.78±0.079 12.76±0.20 7.24±0.15

NB-1 3.78±0.15 10.4±0.047 5.8±0.085 4.21±0.45 3.20±0.25

Kaghzi 5.94±0.15 17.4±0.046 11.4±0.004 5.85±0.35 5.31±0.18

NB-4 10.57±0.30 12.4±0.043 9.6±0.004 7.50±0.24 3.60±0.42

NB-16 5.80±0.25 10.4±0.007 7.6±0.007 3.20±0.15 4.35±0.36

Pant 7.45±0.45 16.4±0.070 15.82±0.007 6.02±0.30 6.62±0.28

Shivani

Pant 11.98±0.25 23.9±0.088 13.64±0.008 8.53±0.25 6.43±0.08

Sujata

AM-1 12.79±0.20 23.5±0.225 15.22±0.082 7.89±0.55 4.05±0.24

AM-2 8.43±0.35 12.4±0.045 8.6±0.084 5.9±0.34 5.32±0.31

AM-6 15.03±0.40 26.9±0.108 20.22±0.010 10.94±0.28 10.54±0.05

AM-8 13.36±0.25 26.6±0.078 18.38±0.078 9.25±0.55 7.29±0.18

Data represent mean± SD of three measurements

77
2.4.2 Total flavonoid content (TFC)

Flavonoids are water-soluble polyphenolic compounds which are extremely

common and widespread in the plant kingdom as their glycosides. The flavonoids act

through scavenging or chelating process (Kessler et al., 2003; Cook & Samman, 1996).

The isolation of flavonoids from biological samples is important for the

quantitative analysis. Because of the complexity of the biological matrix, other

components could interfere in the analysis. The most common procedure used to

evaluate the total flavonoid content is a spectrophotometric assay, based on the

formation of a complex between the aluminium ion and the carbonyl and hydroxyl

groups of the flavonoids. Some works have demonstrated that this procedure has

different responses depending on the flavonoid structure (Popova et al., 2004).

Total flavonoids were measured by aluminium chloride method and are

expressed in terms of rutin equivalent (RE) the standard curve equation: y= 0.686x-

0.246, R2=0.927. The data is appended in Table 2.4. The results obtained demonstrated

that the total flavonoid content of 18 varieties/accessions of Bael was found in the range

of 68.2 mg rutin/gm for Pant Aparna to 14.3 mg rutin/gm for NB-1 in methanol extract

whereas in aqueous extract the flavonoids content ranged from as high as 50.2 mg

rutin/gm for Pant Aparna to 10.4 mg rutin/gm for NB-1. Previous studies have shown

that total flavonoids content varied from 1.087 ± 0.002 mg kg-1 in roots to 1.400 ± 0.029

mg kg-1 and 8.248 ± 0.029 mg kg-1 in the stem and leaves respectively.

78
Table 2.4: Comparative analysis of total flavonoid content in methanol and
aqueous extract

S. No. Bael Total flavonoid content Total flavonoid content

varieties/accessions (mg rutin equivalent (mg rutin equivalent
(RE) /gm) (methanol) (RE) /gm) (aqueous)

1. AM-1 31.3± 0.084 32.3± 0.011

2. AM-2 22.8± 0.062 19.5± 0.018

3. AM-3 39.4± 0.054 25.9± 0.094

4. AM-4 29.5± 0.065 17.4± 0.019

5. AM-6 33.7± 0.062 37.1± 0.02

6. AM-7 47.8± 0.078 31.3± 0.068

7. AM-8 35.8± 0.051 22.0± 0.042

8. NB-1 14.3±0.06 10.4± 0.021

9. NB-4 18.5±0.048 16.1± 0.036

10. NB-5 28.6±0.059 19.2± 0.054

11. NB-7 21.8±0.046 12.4± 0.025

12. NB-9 23.4±0.057 12.7± 0.018

13. NB-16 20.4±0.08 14.0± 0.042

14. NB-17 32.8±0.048 28.6± 0.047

15. Pant Aparna 68.2±0.142 50.2± 0.093

16. Pant Sujata 27.5±0.062 22.9± 0.096

17. Pant Shivani 26.3±0.048 16.8± 0.051

18. Kaghzi 30.6±0.073 15.1± 0.043

79
 Total flavonoid content
80
Flavonoid content (mg rutin

70
equivalent (RE)/gm

60 fla (m)
50 fla (aq)

40
30
20
10
0

Pt rna

K ni
hi a

i
1
4
5
7

N 9
-1
-2
-3
-4
-6
-7
-8

N 6
.A 7

hz
Pt ujat
1
Pt - 1
B-
B-
B-
B-
B-

va
M
M
M
M
M
M
M

B-

ag
pa
B
N
N
N
N
N
A
A
A
A
A
A
A

.S
.S
Bael accessions/varieties

Figure 2.1 Total flavonoid content in eighteen Bael varieties and accessions. Data
represent mean± SD of three measurements

2.4.3 Total phenolic content (TPC)

The polyphenol compounds are important plant constituents because of their free

radical scavenging ability, facilitated by their hydroxyl groups. Total polyphenolic

content (TPC) was estimated and expressed using the Folin–Ciocalteu method. Gallic

acid was used as standard and the results (as gallic acid equivalent, mg/gm of extract)

were expressed as means ± standard deviation of triplicate analysis as depicted in Table

2.5

80
Table 2.5: Comparative analysis of total phenolic content in methanol and aqueous
extract

S. No. Bael Total phenolic content Total phenolic content

varieties/accessions (mg GAE/gm) (mg GAE/gm) (aqueous)
(methanol)
1. AM-1 17.2± 0.066 29.2± 0.053

2. AM-2 10.7± 0.062 20.8± 0.045

3. AM-3 22.1± 0.049 29.6± 0.065

4. AM-4 12.3± 0.038 23.0± 0.028

5. AM-6 25.6± 0.062 41.4± 0.086

6. AM-7 27.4± 0.053 22.8± 0.234

7. AM-8 20.8± 0.058 33.2± 0.123

8. NB-1 10.9± 0.032 13.6± 0.069

9. NB-4 8.8± 0.046 24.0± 0.159

10. NB-5 14.6± 0.047 22.4± 0.088

11. NB-7 8.4± 0.051 18.4± 0.129

12. NB-9 11.6± 0.040 17.8± 0.046

13. NB-16 6.1± 0.041 18.2± 0.045

14. NB-17 30.2± 0.068 35.6± 0.074

15. Pant Aparna 35.5± 0.085 40.4± 0.105

16. Pant Sujata 17.8± 0.074 29.6± 0.027

17. Pant Shivani 15.7± 0.036 25.4± 0.076

18. Kaghzi 12.1± 0.042 10.8± 0.145

81
 The phenolic content expressed as gallic acid equivalent per gram (GAE/gm)

(standard curve equation: y = 0.347x- 0.150 R2= 0.999) was found to be highest in the

aqueous extract of AM-6 (41.4 mg GAE/gm) followed by Pant Aparna (40.4 mg

GAE/gm) while in methanolic extract the phenolic content was less as compared to

aqueous extract. The phenolic content was found to be high in Pant Aparna (35.5 mg

GAE/gm) followed by NB-17 (30.2 mg GAE/gm). The total phenol contents (Gallic

acid equivalents, mg/gm) in methanol extract and aqueous extract of Aegle marmelos

seeds were calculated as 65.20±0.2 and 27.12±0.6 mg/gm, respectively (Sharma et al.,

2011) Similarly, the quantitative estimation of previous studies have shown that total

phenolic content varied from 1.7281 ± 0.049 to 9.8367 ± 0.02335 mg kg-1 in different

parts of Aegle marmelos. The total phenol in methanolic extract of the leaves (9.8367 ±

0.0235 mg kg-1) and in stem extract (7.4693 ± 0.047 mg kg-1) was higher than that in the

extracts of the root (1.7281 ± 0.049 mg kg-1) as reported by Siddique et al., 2010.

The findings conclude that amongst 18 varieties and accessions, the variety Pant

Aparna was found to be the best. The methanolic extract was further confirmed by

performing GC-MS which revealed the presence of different phytochemicals.

82
 Total Phenolic content
Phenolic content (mg 45
40 phe (m)
35 phe (aq)
GAE/gm)
30
25
20
15
10
5
0

.S a

K ni
.S ta

i
1

N 9
-1

-2

-3

-4

-6

A 7
-8

N 6
.A 7

hz
Pt rn
-

1
Pt - 1
B-

B-

B-

B-

B-

Pt uja
va
M

M
M

B-

ag
pa
B
N

hi
A

A
Bael accessions/varieties

Figure 2.2 Total phenolic content in eighteen Bael varieties and accessions.
Experimental results were mean ± S.D of three parallel measurements

2.5 GC-MS analysis of methanolic extract of Bael leaves

GC-MS chromatogram of the methanolic extract of variety Pant Aparna is

shown in Fig. 2.3. On comparison of the mass spectra of the constituents with the Wiley

8 and FAME library sources, thirty-four peaks of phytoconstituents were obtained

which were characterized and identified (Table 2.6). In all thirty-four, compounds were

identified from the GC-MS analysis of a methanolic extract of Bael leaves exhibiting

various phytochemical activities and might be responsible for various biological

activities. The retention time and percentage peak of various bioactive compounds are

presented in Table 2.6

83
Figure 2.3 GC-MS chromatogram of methanolic Pant Aparna extract of Bael
(Aegle marmelos)
The major phyto-constituent present in the leaf extract were 1-Dodecanol (4.83),

4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl-(1.11),

2,3Dioxabicyclo[2.2.2]oct-5-ene,1-Methyl-4-(1-Methylethyl)- (Limonene dioxide

84
1)occupying two peak areas i.e. (0.53) and (0.40)., Bicyclo[3.1.1] heptane-2,3-diol,

2,6,6-trimethyl (2,3-Pinanediol )(0.97), 2-Cyclohexen-1-one, 4-hydroxy-3-methyl-6-(1-

methylethyl)-(0.63,0.41,0.17), Phenol, 2,6-bis(1,1-dimethylethyl)-4-methyl-( BHT)

(0.87), Tetradecanoic acid (Myristic acid ) (2.00), 2(4H)-Benzofuranone 5,6,7,7A-

Tetrahydro-6-hydroxy-4,4,7a-trimethyl,1,3-cyclohexadiene,2-methyl-5-(1methylethyl)-

(1-Phellandrene), 2-Propenoic acid, 3-(4-hydroxy-3-methoxyphenyl)-, methyl ester

(Cinnamic acid, 4-hydroxy-3-methoxy-, methyl ester ), 3,7,11,15-Tetramethyl-2-

hexadecen-1-ol commonly known as phytol a diterpene has significant antimicrobial

properties against many bacterial strains (Bharathy et al., 2012). 9,12,15-

Octadecatrienoic acid, methyl ester (Linolenic acid, methyl ester) showing antibacterial

and anticandidal activity, 2-Hexadecen-1-ol, 3,7,11,15-Tetramethyl (Phytol isomer)

(6.37), Octadecanoic acid (Stearic acid) (4.07), Benzene, 1,2-dimethoxy-4-[[(4

methylphenyl)sulfonyl]methyl (10.76), fatty alcohols such as Ergost-5-en-3-ol, (3.beta.)

(campesterol), Stigmasta-5, 22-dien-3-ol, Stigmast-5-en-3-ol, (3.beta.)- may be

synergistically responsible for the antimicrobial activity.

85
Table 2.6: Activity of the phyto-components identified from methanolic leaf extract
of Aegle marmelos
S. R.T Compound name Molecular MW Peak Compound **Activity
No. formula Area
% Nature

1. 7.178 4H-Pyran-4-one, C6H8O4 144 1.11 Flavonoid Antimicrobial,

2,3-dihydro-3,5- fraction Anti
dihydroxy-6- inflammatory,
methyl- antiproliferative

2. 6.555 1-Butanol, 3- C7H14O2 130 5.32 Alcoholic Antimicrobial

methyl-, acetate compound

3. 12.09 2,3 C10H16O2 168 0.53 Terpene Antimicrobial

Dioxabicyclo[2.2.2] activity
oct-5-ene, 1-methyl-
4-(1-methylethyl)-
(Limonene dioxide
1)

4. 13.385 Bicyclo[3.1.1]hepta C10H18O2 170 0.97 Terpene antimicrobial

ne-2,3-diol, 2,6,6- activity
trimethyl (2,3-
Pinanediol)

5. 13.762 2-Cyclohexen-1- C10H16O2 168 0.63 Antibacterial

one, 4-hydroxy-3-
methyl-6-(1-
methylethyl)-

6. 15.203 1-Dodecanol C12H26O 186 4.83 long-chain Antibacterial

fatty alcohol

7. 16.143 Phenol, 2,6-bis(1,1- C15H24O 220 0.87 Antimicrobial,

dimethylethyl)-4- antioxidant
methyl (BHT) activity

8. 16.457 Benzoic acid, 4- C11H14O3 194 0.33 Aromatic Antimicrobial

ethoxy-, ethyl ester acid ester Preservative

9. 18.101 2-Propanol, 1,1'-[(1- C9H20O4 192 0.87 Antimicrobial

methyl-1,2- activity
ethanediyl)bis(oxy)]
bis-(Tripropylene
glycol)

10. 20.189 1-Tetradecanol, C17H32O2 268 1.46 Fatty acid Anti-

acrylate esters inflammatory,
Antimicrobial

86
11. 20.523 1,3,4,5 C7H12O6 192 0.39 Aromatic antimicrobial
Tetrahydroxy- acid activity, anti-
cyclohexanecarboxy inflammatory
lic acid (Quinic
acid)

12. 21.839 Tetradecanoic acid C14H28O2 228 2.00 Fatty acid Antifungal,
(Myristic acid) Antioxidant,
cancer
preventive,
nematicide,
hypercholesterol
emic, Lubricant

13. 22.070 2(4H)- C11H16O3 196 0.26 Triterpene Antimicrobial

Benzofuranone
5,6,7,7a-tetrahydro-
6-hydroxy-4,4,7a-
trimethyl

14. 22.262 1-Heptadecanol C17H36O 256 0.33 Aliphatic Antimalarial,

alcohol antifungal,
(1-Eicosanol) Antioxidant

15. 22.409 1,3-Cyclohexadiene, C10H16 136 0.23 Monoterpene Antibacterial

2-methyl-5-(1-
methylethyl)-(1-
Phellandrene)

16. 22.736 1,6-Octadiene, 7- C10H16 136 0.13 Monoterpene Antibacterial

methyl-3-methylene
(beta.-myrcene)

17. 23.404 2-Propenoic acid, 3- C11H12O4 208 0.66 Aromatic Antimicrobial,

(4-hydroxy-3- methyl esters antioxidant,
methoxyphenyl)-, antiviral
methyl ester
(Cinnamic acid, 4-
hydroxy-3-methoxy-
, methyl ester )

18. 23.819 Pentadecanoic acid C15H30O2 242 0.08 Fatty acid Antibacterial

19. 24.897 3,7,11,15- C20H40O 296 0.32 Diterpene Antimicrobial,

Tetramethyl-2- anticancer, anti-
hexadecen-1-ol inflammatory,
(Phytol) anti-diuretic,

87
20. 24.986 hexadecanoic acid, C17H34O2 270 0.72 Fatty acid Antioxidant,
methyl ester methyl ester hypocholesterole
(Palmitic acid mic, nematicide,
methyl ester) pesticide, anti
androgenic
flavor,
hemolytic, 5-
Alpha reductase
inhibitor

21. 24.951 Pentadecanoic acid C15H30O2 242 7.99 Saturated Antimicrobial

fatty acid

22. 27.266 9-Octadecenoic acid C18H34O2 282 0.31 Unsaturated Antibacterial

fatty acid

23. 27.664 Heptadecanoic acid C17H34O2 270 0.39 Saturated Antimicrobial

fatty acid

24. 28.298 9,12,15- C19H32O2 292 1.07 Fatty acid Antibacterial,

Octadecatrienoic methyl ester anticandidal,
acid, methyl ester antiinflammatory
(Linolenic acid, ,
methyl ester) Hypocholesterol
emic, Cancer
preventive,
Hepatoprotective
, Nematicide,
Insectifuge
Antihistaminic,
Antiarthritic,
Anticoronary,
Antieczemic
Antiacne, 5-
Alpha reductase
inhibitor
Antiandrogenic

25. 28.554 2-Hexadecen-1-ol, C20H40O 296 6.37 Diterpene Antimicrobial

3,7,11,15- Anti-
tetramethyl (Phytol inflammatory
isomer) Anti cancer
Diuretic

26. 29.227 Cis-9-Hexadecenal C16H30O 238 11.40 Aldehyde Antimicrobial

27. 29.573 Octadecanoic acid C18H36O2 284 4.06 Fatty acid Antimicrobial
(Stearic acid)

28. 41.240 Benzene, 1,2- C16H18O4S 306 10.76 Aromatic Antimicrobial

dimethoxy-4-[[(4 sulfur
methylphenyl)sulfon

88
 yl]methyl compound

29. 44.155 Cholest-5-en-3-ol C27H46O 386 0.24 Steroidal Antibacterial

(3.beta.)-

30. 45.238 Ergost-5-en-3-ol, C27H46O 386 0.50 Steroidal Antimicrobial,

(3.beta.)- anti-
inflammatory
effects

31. 45.509 Stigmasta-5,22- C29H48O 412 0.63 Steroidal Antioxidant,

dien-3-ol antibacterial
activity,
antiinflammatory
,antiarthritic
antiasthma,
diuretic

32. 46.038 Stigmast-5-en-3-ol, C29H50O 414 3.08 Steroidal Antimicrobial,

(3.beta.)- antioxidant,
antiinflammatory
, antiarthritic,
antiasthma,
Diuretic

33. 47.033 Vitamin E C29H50O2 430 0.58 Fat soluble Antioxidant and
Antimicrobial
activity,
Analgesic,
Antidiabetic
Antiinflammator
y,
Antidermatitic,
Antileukemic,
Antitumor,
Anticancer,
Hepatoprotective
, Antispasmodic

34. 44.454 alpha -Tocopherol C29H50O2 430 0.26 Anti-

inflammatory,
antioxidant,
antimicrobial,
radical
scavenging,
antispasmodic

**Source: Dr. Duke’s: Phytochemical and Ethnobotanical Databases

89
 The different phyto compounds responsible for bioactivity have been identified

and characterized in different medicinal plants. 4H-Pyran-4-one, 2, 3-dihydro-3, 5-

dihydroxy-6-methyl- a potent anti-inflammatory and antioxidant compound possessed

antibacterial activity in Barleria prionitis (Linn.) rhizome. A long-chain fatty alcohol, 1-

Dodecanol was reported with highest antibacterial activity against Staphylococcus

aureus (Togashi et al., 2007). Phenol, 2,6-bis(1,1-dimethylethyl)-4-methyl commonly

known as Butylated hydroxytoluene (BHT), an antioxidant has also demonstrated

marked antimicrobial activity inhibiting or decreasing the growth of Gram-positive

bacteria at a higher degree than the Gram-negative bacteria belonging to the family

Enterobacteriaceae (Turcotte & Saheb, 1978). Derivatives of cinnamic acid such as

esters, amides, acids, hydrazides have been reported to have antibacterial, antifungal and

antiviral properties (Sova, 2012). 2(4H)-Benzofuranone, 5,6,7,7a-tetrahydro-4,4,7a-

trimethyl, a bioactive compound possessing the properties such as analgesic,

antidiabetic, antibacterial, and antifungal was identified from the methanolic fractions of

the Azadirachta indica (Moorthy & Boominathan, 2011). The extract contained (0.23%)

of 1,3-Cyclohexadiene, 2-methyl-5-(1-methylethyl)- commonly known as 1-

phellandrene. Stigmasterol has been reported earlier as a strong antioxidant having

antibacterial activity against multi-drug resistant mycobacteria (Hamdan et al., 2011;

Navarro-Garc´ıa et al., 2011).

Several bioactive compounds viz. Marmin (Nugroho et al., 2011) and

Marmelosin coumarin derivatives (Ram et al., 2012) and Aegeline an alkaloid have

been previously reported from Aegle marmelos (Nugroho et al., 2011). However, the

90
flavonoids and phenolics present in significant amount in this plant are still unexplored.

The preferential quantity of these compounds in the methanolic extract of Pant Aparna

as revealed by the present study directed to focus towards the purification and

characterization of the potential compound from this variety.

2.6 Conclusion

Medicines derived from plants have made an immense contribution towards the

betterment of human health and acts as a source of inspiration for novel drug

compounds. From the above research, it can be concluded that this plant has immense

potential to be used in the area of pharmacology and as a prospective source of valuable

drugs. Due to the presence of various compounds that are essential for good health, it

can also be used to improve the health status of society.

A spectrum of compounds showing strong antibacterial, antioxidant and anti-

inflammatory activities was revealed by the GC-MS analysis of the methanolic extract

of Aegle marmelos.

Antimicrobials derived from plants possess vast curative properties since they

have fewer side effects as compared to synthetic antimicrobials drugs. It is of utmost

importance for ethnobotanical purposes, and it has been placed on the priority list of

thirty-two medicinal plants by The National Medicinal Plants Board of Govt. of India

(Kala, 2006). The present study contributes to the current knowledge of the presence of

various phytochemical active compounds in 18 varieties/accessions of Aegle marmelos

which are responsible for wide range of biological activities. Further, fractionation and

91
purification will elucidate the potential compound, which is a pressing need because of

the upcoming resistance of the currently available antibiotics.

92