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Research Article CODEN: IJPRNK ISSN: 2277-8713 Bhanwar Lal Jat, IJPRBS, 2015; Volume 4(4): 1-23 IJPRBS

Available Online at www.ijprbs.com 1

PHYTOCHEMICAL ANALYSIS OF METHANOLIC EXTRACTS OF MURRAYA KOENIGII THROUGH HPLC TECHNIQUES

BHANWAR LAL JAT1, RAMESH JOSHI2, DILIP GENA3, RAAZ K MAHESHWARI4

1 Department of Botany, SBRM Govt. PG College, Nagaur, Rajasthan. 2 Department of Botany, SPC Government College Ajmer, Rajasthan. 3 Department of Botany, SPC Government College Ajmer, Rajasthan. 4 Department of Chemistry, SBRM Govt. PG College, Nagaur, Rajasthan.

Accepted Date: 03/06/2015; Published Date: 27/08/2015

Abstract: HPLC analysis for quantitative analysis of phenols and flavonoids was conducted in order to separate the antioxidative components from different parts of in vivo plantlets of Murraya koenigii and compared them with the peaks of available commercial standard antioxidant compounds such as Citric acid, Ascorbic acid, BHT and Quercetin. In addition the study was also conducted of compounds in each part of plant like root and leaves under in vivo condition and also to analyze the availability or synthesis of bioactive compounds in different stages of biological development of fruits and seeds of field growing Murraya koenigii. In present investigation correlation was established between the Spectrophotometric estimation of antioxidant activity using DPPH free radical of plant extracts and the HPLC resolution of extracts of plant parts and their comparison with the peaks of available standard antioxidative compounds. In the HPLC chromatograms the antioxidative compounds were identified by their Retention time by spiking with standards (Citric acid, Ascorbic acid, BHT and Quercetin) under the same conditions. The biochemical investigation of different plant parts showed maximum quantity of phenols and flavonoids in dried fruit (DF) and than ripened fruit (RF) with highest antioxidant capacity discoloration of DPPH. The scavenging potential of dried fruit (DF) and ripened fruit (RF) was also maximum as compared to other plant parts and it was further confirmed by HPLC analysis ripened fruit (RF) and dried fruit (DF) that the none of the standard antioxidative compounds was available in DF sample as observed in HPLC peaks where as only BHT was identified in the RF sample. The HPLC chromatogram also showed that total 17 compounds were separated from the extract but available standard compounds was not found in DF sample which clearly indicates that in DF extract which showed highest antioxidant activity in Spectrophotometric analysis but the antioxidant of DF sample may be due to the presence of antioxidative compound other than standards. The RF sample showed antioxidant activity and in this sample the BHT was found as an antioxidative compound. In root ®, HRF and DS two standard antioxidative compounds were identified but their antioxidant activity is poorer than DF and RF. The strongest antioxidant activity was exhibited by freshly prepared extract of ripened fruit (RF) and dry weight of fruit (DF). Keywords: Murraya koenigii, Aromatic plant, flavonoid, antioxidant, Phytochemicals, Citric acid, Ascorbic acid, BHT and

Quercetin

INTERNATIONAL JOURNAL OF

PHARMACEUTICAL RESEARCH AND BIO-SCIENCE

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Corresponding Author: DR. BHANWAR LAL JAT

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How to Cite This Article:

Bhanwar Lal Jat, IJPRBS, 2015; Volume 4(4): 1-23



INTRODUCTION

The term "phytochemicals" refers to a wide variety of compounds produced by plants. They are

found in fruits, vegetables, beans, grains and other plants. They are non nutritive and have

protective or disease preventive properties. The plants of medicinal values are major sources of

natural products used as pharmaceuticals, flavor and fragrance ingredients and food additives,

(Balandrin and Klocke, 1988). Though many phytochemicals have been identified and there

significance and mode of action have been reported time to time. Public interest in plant based

medicine coupled with rapid expansion of pharmaceutical industries and their dependence on

wild population of these medicinal plants for the supply of raw materials for extraction of

medicinally important compounds has necessitated an increased demand for medicinal plants.

The search for new plant derived chemicals especially secondary metabolites should thus be a

priority in current and future efforts toward sustainable conservation and rational utilization of

biodiversity (Phillipson, 1990), for production of drugs for human welfare. Phyto chemicals are

often classified as either primary or secondary metabolites (proteins and nucleic acids are

generally excluded from this classification). Primary metabolites are substances widely

distributed in nature, occurring in one form or another in virtually all organisms. In higher

plants such compounds are often concentrated in seeds and vegetative storage organs because

of their role in basic cell metabolism they are needed for physiological development of plant. As

a general rule, primary metabolites obtained from higher plants for commercial use are high

volume low value bulk chemicals. Phenolic derivatives represent the largest group known as

‘secondary plant products’ synthesized by higher plants. Many of these phenolic compounds

are essential to plant life, e.g., by providing defense against microbial attacks and by making

food unpalatable to herbivorous predators. Although a precise chemical definition may be given

for plant phenolics, it would inevitably include other structurally similar compounds such as the

terpenoid sex hormones. Therefore, an operational definition of metabolic origin is preferable

and thus the plant phenols being regarded as those substances derived from the shikimate

pathway and phenylpropanoids metabolism, following the phosphoenolpyruvate →

phenylalanine → cinnamate → 4-coumarate course, leading to chalcone, flavanone,

dihydroflavonol and anthocyanin. Significant antioxidant, antitumoral, antiviral and antibiotic

activities are frequently reported for plant phenols. They have often been identified as active

principles of numerous medicines. In recent years, the regular intake of fruits and vegetables

has been highly recommended, because the plant phenols and polyphenols, they play

important roles in long term health and reduction in the risk of chronic and degenerative

diseases. Flavonoids may have existed in nature for over one billion years and thus have

interacted with evolving organisms over the eons. Clearly, the flavonoids possess some

important purposes in nature, having survived in vascular plants throughout evolution (Swain,

1975). The very long association of plant flavonoids with various animal species and other



organisms throughout evolution may account for the extraordinary range of biochemical and

pharmacological activities of these chemicals in mammalian and other biological systems.

Antioxidants are gaining a lot of importance as to cure a number of diseases like aging, cancer,

diabetes, cardiovascular and other degenerative diseases etc. owing to our sedentary way of

life and stressful existence. Added to these are the deleterious effects of pollution and

exposure to harmful chemicals. All the above can cause accumulation of harmful free radicals.

Free radicals are types of Reactive oxygen species (ROS), which include all highly reactive,

oxygen containing molecules. Types of ROS include the hydroxyl radical, the super oxide anion

radical, hydrogen peroxide, singlet oxygen, nitric oxide radical, hypochlorite radical and various

lipid peroxides. All these are capable of reacting with membrane lipids, nucleic acids, proteins

and enzymes and other small molecules, resulting in cellular damage. In living organisms

various ROSs can be formed in different ways, including normal aerobic respiration, stimulated

polymorphonuclear leukocytes, macrophages and peroxisomes. These appear to be the main

endogenous sources of most of the oxidants produced by cells, where as exogenous sources of

free radicals include tobacco smoke, ionizing radiation, certain pollutants, organic solvents and

pesticides. Free radicals may be defined as chemical species associated with an odd or unpaired

electron. They are neutral, short lived, unstable and highly reactive to pair up the odd electron

and finally achieve stable configuration. They are capable of attacking the healthy cells of the

body, causing them to lose their structure and function. Cell damage caused by free radicals

appears to be a major contributor to aging and degenerative diseases of aging such as cancer,

cardiovascular disease, cataracts, immune system decline, liver diseases, diabetes mellitus,

inflammation, renal failure, brain dysfunction and stress among others. To protect the cells and

organ systems of the body against reactive oxygen species, humans have evolved a highly

sophisticated and complex antioxidant protection system, that functions interactively and

synergistically to neutralize free radicals. Thus, antioxidants are capable of stabilizing or

deactivating free radicals before they attack cells. Naturally there is a dynamic balance between

the amount of free radicals produced in the body and antioxidants to scavenge or quench them

to protect the body against deleterious effects. The amount of antioxidant principles present

under normal physiological conditions may be insufficient to neutralize free radicals generated.

Therefore, it is obvious to enrich our diet with antioxidants to protect against harmful diseases.

Hence there has been an increased interest in the food industry and in preventive medicine in

the development of "Natural antioxidants" from plant materials. The plants with antioxidant

properties are becoming more and more popular all over the world. Considering the

importance of this area, we have listed some of the important medicinal plants having potent

antioxidant property, which are traditionally used in the Indian subcontinent for various

disorders where free radicals are thought to be involved. The biochemical analysis especially

evaluation of total phenols and flavonoids and antioxidant activity of different parts of Murraya

koenigii would quantify their latent chemical constituent to ensure their scientific utility. Highly

http://www.herbs2000.com/h_menu/oxygen.htm

http://www.herbs2000.com/h_menu/hydrogen_peroxide.htm

http://www.herbs2000.com/h_menu/lipids.htm

http://www.herbs2000.com/h_menu/nucleic_acids.htm

http://www.herbs2000.com/h_menu/enzymes.htm



reactive free radicals such as superoxide radicals, hydroxyl radicals, non free radical species and

oxygen species are present in biological systems from a wide variety of sources such as

environmental pollutions, chemical toxins and physical stress which are associated with cellular

and metabolic injury. The free radicals may oxidize nucleic acids, proteins and lipids and cause

depletion of immune system, change in gene expression and induce abnormal proteins

accelerating aging gastritis, cancers cardiovascular and neuro degenerative

MATERIALS AND METHODS

HPLC Analysis

A duplicate of 15mg of freeze-dried sample was extracted in 15ml absolute methanol for one

hour at room temperature. The filtrated samples were stored at –23 °C and used for HPLC

analyses by KNAUER · ASI · Advanced Scientific Instruments, Germany. High-performance liquid

chromatography (HPLC) fingerprint is a powerful approach for the rapid identification of

phytochemical constituents in botanical extracts, and it can be used to avoid the time-

consuming isolation of all the compounds to be identified. Analytical studies for the

determination of different compounds in different extracts of M. koenigii the chromatographic

method HPLC was used because individual compounds can be separated and determined in one

assay procedure. Using these analytical methods, the selection and modification for the

chromatographic conditions were focused on the stationary phase, mobile phase, and the

detector. C-18 or ODS column were used for analysis of extracts. Computer-based optimization

programs have been followed in the wake of microcomputers. The concentration of the

polymer peaks was quantified in mg QE g-1 DW by comparison to peaks of some standard

antioxidant compounds such as Gallic acid, BHT, Ascorbic acid, Qurcetine, and citric acid.

Methanolic extracts of leaves, fruits, and roots of field growing plants and leaves and roots of

tissue-cultured plants were prepared and 100μl extract was injected. Injection valves have been

used successfully for sample delivery valves in HPLC. They ensure precise sample delivery in

both partial and full sample loop-filling modes. In the LOAD position, the sample loop is filled;

when switched to the INJECT position, the sample is injected. The eluent passes through the

sample loop in the opposite direction to minimize band broadening. This is especially

advantageous when sample loops are partially filled. When using partial loop filling, the highest

degree of accuracy is obtained by filling less than 50% of the volume of the sample loop. Upon

switching the valve into the INJECT position, the resulting reversal of the flow direction within

the loop ensures that the sample is transferred completely onto the column. When using full

loop injection technique however, the highest degree of accuracy can be achieved by overfilling

the loop three times. In this case, the eluent is completely pushed out of the loop by the sample

and the reproducibility of the injection volume no longer depends on factors such as peak

broadening or dispersion. Sample loops are available for the analytical field in volume sizes



ranging from 2 - 2500μl in stainless steel and PEEK. For preparative HPLC applications, sample

loops are also available in stainless steel and PEEK in volumes up to 45 ml. The Pronto SIL C18

ace-EPS belongs to the new group of stationary RP-supports with Polar embedded groups. The

packing is very stable over a wide pH range (pH 1-10). In addition, it offers a maximum of

hydrophobicity combined with a maximum of polar selectivity. The silanophilic activity of the

support is very low. Ultra strong basic compounds such as amitriptyline can be eluted from the

column at neutral pH values with excellent symmetrical peak shapes. The main application area

of these packings is the pharmaceutical industry, where analytes often have basic or acidic

groups. For the separation of these compounds these supports exhibit an enhanced polar

selectivity. That means: In comparison to a classical bonded C18 column acidic compounds

show a higher retention whereas basic compounds show a slight decrease of retention on an

embedded polar column. The C18 ace-EPS - bonding type is available in several particle and

pore sizes.

High performance liquid chromatography (HPLC) Analysis

The methanolic extracts of different plant materials were run in HPLC (KNAUR Advanced

Scientific Instrument Germany) using the Pronto SIL C18 ace EPS Column for qualitative and

quantitative analysis of different methanolic extracts such as roots (R), Leaf (L), un-ripened



(green colored) fruit (URF) and its seed (URS), half ripened fruit (HRF) and its seed (HRS),

ripened fruit (RF), its seed (RS) flower bud (FB),dried fruit (DF) and its seed (DS).

In present investigation the 100µl extracts was passed through the C-18 Column of HPLC which

allowed the separation of many compound present in the sample. Characteristic HPLC profiles

of methanolic extracts of some standard phenol (Gallic acid), flavonoid (Quercetin) and other

compound (Citric acid, Ascorbic acid and BHT) having well known antioxidant activities were

observed. The retention time, areas of peak and percentage amount of compound available in

the sample are shown in Table-1. The Peak of chromatogram for Ascorbic acid (fig-1.1), BHT

(fig-1.2), Citric acid (fig-1.3), Quercetin (fig-1.4) and Gallic acid (fig-1.5) are shown. The data of

chromatogram revealed that the absorption of 2498.68mAU (Ascorbic acid), 385.63mAU (BHT),

219.47mAU (Citric acid), 16.11mAU (Quercetin) and 20.91mAU (Gallic acid). The retention time,

start time and area of peaks of fractioned compounds are shown in Table-1.

-500

0

500

1000

1500

2000

2500

3000

0.02

1.02

2.01

3.01

4.01

5.01

6.01

7.01

8.01

9.01 10

Run time

Absor

ption

(mAU

)

1

Ascorbic acid

-50

0

50

100

150

200

250

300

350

400

450

0.02 1.02 2.01 3.01 4.01 5.01 6.01 7.01 8.01 9.01 10

Run time

Absorp

tion (m

AU) 1

BHT

0

50

100

150

200

250

0.02

1.02

2.01

3.01

4.01

5.01

6.01

7.01

8.01

9.01 10

Run time

Absor

ption

(mAU

) 1

Citric acid



Table-1. Standards of antioxidants

HPLC analysis of leaf extract:-

The methanolic extract of fresh leaf of M. Koenigii was injected in HPLC unit. Thirteen peaks

were observed in HPLC chromatogram (fig-2). The Retention time, start time and peak area of

the compound in HPLC column were calculate and shown in Table-2. The start time of leaf

extracts were compared with the peaks of standards solutions. The chromatogram showed that

leaf extracts that the start time of peak no.2 is 2.38min.Which has 260.25mAU absorption and

compound at peak no.7 has 67.53mAU absorption which was nearly similar to the Quercetin

and BHT respectively (Table-2). The percentage area of each peak represents the amount of

each compound separated in HPLC in µgs/400µg fresh weight. The data of area of all the peaks

are given in Table-2.

-100

0

100

200

300

400

500

600

700

1 61 121

181

241

301

361

421

481

541

601

Run time

Ab

sorp

tion

(m

AU

)

1

2

10

11 12

13

7

6

4

98

5

3

Leaf

Standards Ret. time [min]

Start [min]

End [min]

Area [m AU* min]

Height [m AU]

% Area

Ascorbic acid

2.32 1.50 2.77 481.38 2498.68 100

BHT 3.85 3.74 4.44 38.33 385.63 100 Citric acid 2.28 1.43 3.06 80.95 219.47 100 Quercetin 2.53 2.35 3.06 3.26 16.11 100 Gallic acid 2.57 2.47 2.75 1.46 20.91 100



Table-2 (Leaf)

Peak No.

Ret. time [min]

Start [min]

End [min]

Area [m AU*min]

Height [m AU]

% Area Qunt. Of Compound µg/400µg

Compound Mg/gm Fresh weight

1 2.33 2.05 2.38 33.79 261.23 10.09 40.37 100.93 2 2.43 2.38 2.75 44.10 260.25 13.17 52.69 131.72 3 3.01 2.75 3.06 24.40 180.44 7.28 29.15 72.89 4 3.13 3.06 3.45 38.47 273.22 11.49 45.97 114.92 5 3.33 3.28 3.43 1.85 25.37 0.55 2.22 05.55 6 3.56 3.45 4.22 65.17 386.85 19.46 77.86 194.66 7 3.76 3.70 3.90 7.50 67.53 2.24 8.97 22.42 8 4.02 3.95 4.18 2.35 20.79 0.70 2.81 07.04 9 4.35 4.22 4.47 1.37 12.65 0.41 1.64 04.10 10 4.73 4.47 5.45 90.10 639.05 26.91 107.64 269.11 11 5.18 5.08 5.43 5.92 31.93 1.76 7.07 17.68 12 6.70 6.52 6.80 3.76 24.20 1.12 4.49 11.23 13 6.95 6.80 7.32 15.96 68.83 4.76 19.07 47.68

HPLC analysis of root extract:-The freshly prepared methanolic extract of root of M. Koenigii

was analyzed by HPLC and it was found that the eight peaks were obtained (fig-3). The start

time of peak no.1 (1.46891min.) is nearly similar with the start time of citric acid (1.43471 min)

which has 261.23mAU absorption. The percentage area of individual peak (1 to 8) is presented

in Table-3 which represents the amount of the particular compound in µg.

0

200

400

600

800

1000

1200

1400

1600

1800

1

61

12

1

18

1

24

1

30

1

36

1

42

1

48

1

54

1

60

1

Run time

Ab

sorp

tio

n (

mA

U)

1

Root

6

7

8

34

52



Table-3 (Root)

Peak No.

Ret. time [min]

Start [min]

End [min]

Area [m AU*min]

Height [m AU]

% Area Qunt. of Compound µg/400µg

Compound mg/gm Fresh weight

1 2.22 1.46 2.73 94.46 322.78 20.06 80.25 200.64 2 2.67 2.57 2.72 1.95 16.96 0.41 1.65 04.14 3 2.90 2.73 3.00 37.34 193.43 7.93 31.73 79.32 4 3.07 3.00 3.25 28.68 148.93 6.09 24.37 60.93 5 3.30 3.25 3.38 12.13 110.02 2.57 10.31 25.77 6 3.50 3.38 3.68 171.69 1622.07 36.46 145.87 364.67 7 3.80 3.68 4.33 83.26 757.67 17.68 70.73 176.84 8 4.79 4.47 5.45 41.26 333.61 8.76 35.05 87.64

HPLC analysis of un-ripened fruit (URF) extract:-The fruit of M. Koenigii were analyzed in their

different stages of biological development and ripeness. Samples of methanolic extracts of un-

ripened fruit (URF) were analysed and the results of retention time and start time are shown in

Table-4. The HPLC chromatogram of URF showed that total twelve peaks of URF sample did not

match with any of the peaks of standard samples. The compound showed in peak no. 8 was

found in maximum quantity (47.82%) and its retention time is 4.72 min. The compound in peak

no. 10 was observed to be present in minimum quantity (0.13%) in the URF extract.

-500

0

500

1000

1500

2000

2500

3000

3500

1 61 121

181

241

301

361

421

481

541

601

Run time

Abs

orpt

ion

(mA

U)

8

1

1211109

2

45

3

6 7

URF

Table-4 (URF)

Peak No.

Ret. time [min]

Start [min]

End [min]

Area [m AU*min]

Height [m AU]

% Area

Qunt. of Compound µg/400µg




1 2.20 1.83 2.68 216.28 858.61 17.13 68.52 171.32 2 2.93 2.70 3.00 58.66 430.62 4.64 18.58 46.46 3 3.06 3.00 3.31 58.27 380.47 4.61 18.46 46.16 4 3.50 3.31 3.56 128.55 1005.93 10.18 40.73 101.83 5 3.60 3.56 4.45 133.79 909.05 10.59 42.39 105.97 6 3.92 3.85 4.07 4.49 41.22 0.35 1.42 03.55 7 4.35 4.17 4.43 6.56 58.95 0.51 2.07 05.19 8 4.72 4.45 5.77 603.81 2862.39 47.82 191.31 478.28 9 5.17 5.08 5.45 8.28 38.38 0.65 2.62 06.56 10 6.32 6.22 6.55 1.72 9.39 0.13 0.54 01.36 11 6.70 6.58 6.82 2.53 18.05 0.20 0.80 02.00 12 6.98 6.82 7.59 39.47 174.68 3.12 12.50 31.26

HPLC analysis of half-ripened fruit (HRF) extract:-The extract of half ripened fruit (HRF) of M.

Koenigii was also analysed by HPLC and chromatogram (fig-5) showed that total eleven peaks

were observed. The peak no.1 with start time 1.55 min. Which has 519.75mAU absorption and

whereas peak no.2 has 57.05mAU absorption with start time 2.35 min. resembles with the

peaks of standards ascorbic acid and quercetin respectively. The results of chromatogram (fig-

5) showed that the compound separated in peak no. 8 was found to be present in maximum

quantity 45.74%. Whereas the compound of peak no.7 was found in minimum (0.05%) quantity

in the extract injected for HPLC.

0

500

1000

1500

2000

2500

3000

1

61

12

1

18

1

24

1

30

1

36

1

42

1

Run time

Ab

sorp

tio

n (

mA

U)

8

111097

56

34

2

1

HRF

Table-5 (HRF)



HPLC analysis of ripened fruit (RF) extract:-The methanolic extract of ripened fruit (RF) was

analysed. The chromatogram (fig-6) of RF extract showed that eight peaks were detected and

the peak no, 7 showed the maximum (51.87%) percent whereas the compound of peak no. first

was present in minimum (0.21%) quantity. The retention time and start time of the compound

in HPLC is presented in Table-6. Amongst the eight compounds separated in RF extract, the

start time of compound of peak no.5 (3.75 min.) with 703.57mAU absorption resembles with

the peak of standard BHT. The percentage area of this compound of peak no.5 is 24.11% which

has the retention time 3.85 min.

-200

0

200

400

600

800

1000

1200

1400

1600

1 61 121

181

241

301

361

421

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541

601

Run time

Abs

orpt

ion

(mA

U)

5

4

3

6

7

8

2

1

RF

Table-6 (RF)

Peak No.

Ret. time [min]

Start [min]

End [min]

Area [m AU*min]

Height [m AU]

% Area



1 2.23 1.55 2.73 166.47 519.75 18.03 72.13 180.33 2 2.38 2.35 2.50 5.18 57.05 0.56 2.24 05.62 3 2.98 2.73 3.05 43.57 282.17 4.71 18.87 47.19 4 3.13 3.05 3.38 55.56 340.59 6.01 24.07 60.19 5 3.57 3.38 4.52 192.08 598.87 20.80 83.23 208.07 6 3.97 3.93 4.18 2.32 15.29 0.25 1.00 02.51 7 4.40 4.18 4.50 0.51 32.21 0.05 0.28 00.56 8 4.78 4.52 5.87 422.29 2697.40 45.74 182.97 457.44 9 5.23 5.12 5.52 6.64 31.42 0.71 2.87 07.19 10 6.79 6.62 6.89 2.57 17.34 0.27 1.11 02.79 11 7.05 6.89 7.62 25.90 115.09 2.80 11.22 28.05



Peak No.

Ret. time [min]

Start [min]

End [min]

Area [m AU*min]

Height [m AU]

% Area



1 1.88 1.83 2.06 1.26 16.86 0.21 0.86 02.1612 2 3.13 2.61 3.23 35.22 164.81 6.02 24.09 60.2277 3 3.31 3.23 3.53 34.41 143.17 5.88 23.53 58.8364 4 3.70 3.53 3.75 52.25 430.70 8.93 35.73 89.3365 5 3.85 3.75 4.80 141.06 703.57 24.11 96.46 241.17 6 4.57 4.43 4.75 7.01 48.84 1.19 4.79 11.9921 7 5.15 4.80 6.67 303.42 1461.79 51.87 207.50 518.762 8 7.80 7.37 8.40 10.24 29.66 1.75 7.00 17.5142

HPLC analysis of un-ripened seed (URS) extract:-The HPLC analysis of seeds of different stages

of biological development was also carried out. The methanolic extract of un-ripened seed

(URS) was analysed and chromatogram (fig-7) showed five peaks. The area of individual peak

was varied from 0.35% to 39.79% in the extract of URS and the start time 1.40 min of

compound on peak no.-1 with 434.55mAU absorption resemble with the start time (1.43 min.)

of standard citric acid which has 1.96 min, retention time. The chromatogram also showed that

the compound separated at peak no.4 was present in minimum quantity (0.35%) with retention

time 4.18 min. The 20 µl extract of URS took 4.95 min. to run completely in HPLC column.

-200

0

200

400

600

800

1000

0.01

67

1.01

58

2.01

5

3.01

42

4.01

33

5.01

25

6.01

17

7.01

08

8.01

9.00

92

10.0

08

Run time

Abs

orpt

ion

(mA

U)

1

5

3

2

4

URS

Table-7 (URS)



Peak No.

Ret. time [min]

Start [min]

End [min]

Area [m AU*min]

Height [m AU]

% Area



1 1.96 1.40 3.15 121.73 434.55 39.79 159.19 397.98 2 2.83 2.61 3.00 10.03 46.87 3.28 13.12 32.82 3 3.35 3.15 3.71 61.00 322.44 19.94 79.77 199.42 4 4.18 4.01 4.28 1.08 10.24 0.35 1.41 03.54 5 4.57 4.30 4.95 112.02 897.22 36.62 146.48 366.22

HPLC analysis of half-ripened seed (HRS) extract:-The chromatogram of HRS presented in fig-8

revealed that total eleven peaks were found and the area of peak ranging ‘between’ 0.12% to

51.36% (Table-8). The compound separated at peak no.11 was presented in maximum

(51.36%) quantity in the extracts of half-ripened seed whereas the compound no.10 (peak

no.10) was found in minimum quantity (0.12%). The results of chromatogram showed that the

start time 2.33 min with 28.00mAU absorption of compound no.4 resembles with the start time

of standard compound Quercetin (Table-8). The retention time of no.-10 compound was 2.40

min which is present in poor quantity (4.45%) in the HRS extract.

-500

0

500

1000

1500

2000

0.02

1.02

2.01

3.01

4.01

5.01

6.01

7.01

8.01

9.01 10

Run time

Abso

rptio

n (m

AU)

11

11

10

9

87

6

5432

HRS

Table-8 (HRS)



Peak

No.

Ret.

time

[min]

Start

[min]

End

[min]

Area

[m

AU*min]

Height

[m AU]

% Area Qunt. of

Compound

µg/400µg

Compound

mg/gm

Fresh

weight

1 2.03 1.76 2.08 8.98 86.86 1.90 7.63 19.08

2 2.15 2.08 2.18 8.99 102.74 1.90 7.63 19.09

3 2.25 2.18 2.33 12.75 92.55 2.70 10.83 27.08

4 2.40 2.33 2.72 20.95 119.68 4.45 17.80 44.50

5 2.90 2.73 2.98 15.43 150.87 3.27 13.11 32.78

6 3.07 2.98 3.30 22.51 151.17 4.78 19.12 47.80

7 3.55 3.32 3.60 69.94 676.32 14.85 59.41 148.52

8 3.63 3.60 3.90 67.58 600.30 14.35 57.40 143.52

9 3.98 3.92 4.24 1.29 7.80 0.27 1.10 02.75

10 4.39 4.24 4.49 0.57 28.00 0.12 0.49 01.23

11 4.77 4.49 5.44 241.85 1863.71 51.36 205.44 513.60

HPLC analysis of Ripened seed (RS) extract:-The chromatogram of Ripened seed (RF) of M.

Koenigii showed total eight peaks (fig-9). The compound no.1 was found in maximum quantity

(39.38%) in the RS extract which has start time 0.06 min. and retention time 1.53min. The

compound separated at peak no.2 was found 0.30% in the extract which has the retention time

2.51 min. at peak no.-1. The compound was found to be of maximum quantity (39.38).

-200

0

200

400

600

800

1000

1 61 121

181

241

301

361

421

481

541

Run time

Abs

orpt

ion

(mA

U)

1

32

4

5

6

7

8

RS

Table-9 (RS)



Peak

No.

Ret.

time

[min]

Start

[min]

End

[min]

Area

[m

AU*min]

Height

[m AU]

% Area Qunt. of

Compound

µg/400µg

Compound

mg/gm

Fresh

weight

1 1.53 0.06 1.58 169.71 357.98 39.38 157.53 393.83

2 2.51 2.05 2.58 1.30 15.77 0.30 1.21 03.03

3 2.93 2.83 3.15 3.07 14.45 0.71 2.85 07.12

4 3.28 3.15 3.48 10.76 59.35 2.49 9.98 24.97

5 3.62 3.48 3.67 16.35 153.84 3.79 15.18 37.95

6 3.77 3.67 4.20 74.86 492.26 17.37 69.49 173.73

7 4.45 4.20 4.65 4.39 30.12 1.01 4.07 10.19

8 5.00 4.67 6.10 150.45 778.60 34.91 139.65 349.14

HPLC analysis of Flower bud (FB) extract:-The methanolic extracts of whole flower bud was

analysed by HPLC and the chromatogram (fig-10) showed that total five peaks were resolved

and the five compounds separated from the FB extract showed that the compound no.1 which

has start time 1.55 min. was resemble with start time of standard ascorbic acid. The compound

separated in peak no.1 retained for 2.13 min. which has 679.14mAU absorption. The

chromatogram also showed that the peak no.2 resembled with the peak of standard quercetin.

The compound at peak no.2 had retention time 2.43 min. The five compound separated from

the extracts of FB were present in varied quantities ranging between 0.51% to 55.19%. The

compound separated at peak no.1 which resemble with Ascorbic acid was present in maximum

quantity (55.19%) in the extract of FB whereas compound on peak no.2 which resembles with

quercetin is present in poor quantity (1.22%) in the sample extract of FB.

-100

0

100

200

300

400

500

600

700

800

1 61 121

181

241

301

361

421

481

541

601

Run time

Abso

rptio

n (m

AU)

1

2

FB

3

4

5

Table-10 (FB)



Peak No.

Ret. time [min]

Start [min]

End [min]

Area [m AU*min]

Height [m AU]



1 2.13 1.55 2.75 174.63 679.14 55.19 220.78 551.95 2 2.43 2.37 2.55 3.87 44.60 1.22 4.89 12.23 3 3.67 3.37 4.18 58.45 322.63 18.47 73.90 184.76 4 4.40 4.25 4.50 1.61 14.29 0.51 2.04 05.10 5 4.79 4.50 5.64 77.81 608.59 24.59 98.37 245.94

HPLC analysis of dried fruit (DF) extract:-The fully ripened fruit of Murraya koenigii were dried

and methanolic extract of pulp of dried fruit was analysed by HPLC. The picture of

chromatogram (fig-11) showed seventeen peaks of different compounds. The retention time

and quantity of individual compound in percentage is presented in Table-11. The data showed

that the retention time for all the seventeen compounds separated from the extract of DF

ranging between 2.13 min. to 7.87 min. whereas the quantity of all the compounds separated

at different peaks were ranging ‘between’ 0.10% to 44.03%. The compound on peak no.8 was

found in maximum (44.03%) quantity in the extract of DF (Table-11). The data also revealed

that the peak no.2 of the chromatogram (fig-11) resembles with the peak of standard

compound quercetin. The compound on peak no.2 was found in less quantity (1.61%) in the

extract as compared to the peak no.8. the retention time of the compound of peak no.2 was

2.45 min with 2.31 min. start time. The compound on peak no.2 has the absorption of

142.98mAU.

-500

0

500

1000

1500

2000

2500

3000

3500

1 61 121

181

241

301

361

421

481

541

601

Run time

Ab

sorp

tion

(m

AU

)

12

DF

12

13

11

10

9

3

4

5

14

15 1617

6 7

8

Table-11(DF)



Peak No.

Ret. time [min]

Start [min]

End [min]

Area [m AU*min]

Height [m AU]



1 2.13 1.66 2.31 32.96 231.80 1.47 5.88 14.70 2 2.45 2.31 2.68 36.21 142.98 1.61 6.46 16.16 3 2.78 2.68 2.81 12.77 112.51 0.57 2.28 05.70 4 3.06 2.81 3.41 239.13 867.31 10.67 42.68 106.70 5 3.56 3.41 4.50 694.36 2541.08 30.98 123.93 309.83 6 4.00 3.91 4.15 21.75 197.56 0.97 3.88 09.70 7 4.38 4.20 4.48 28.73 252.59 1.28 5.12 12.82 8 4.75 4.50 8.69 986.80 2931.74 44.03 176.12 440.31 9 5.22 5.12 5.28 4.87 43.52 0.21 0.86 02.17 10 5.32 5.28 5.40 5.18 50.08 0.23 0.92 02.31 11 5.53 5.40 5.68 22.04 140.24 0.98 3.93 09.83 12 5.75 5.72 5.92 2.33 5.53 0.10 0.41 01.04 13 6.05 5.92 6.25 5.35 -0.51 0.23 0.95 02.38 14 6.43 6.25 6.58 3.67 44.28 0.16 0.65 01.63 15 6.75 6.58 6.90 41.57 255.75 1.85 7.42 18.55 16 7.03 6.90 7.65 52.55 263.93 2.34 9.38 23.45 17 7.87 7.65 8.50 50.78 210.20 2.26 9.06 22.65

HPLC analysis of dried seed (DS) extract:-The seed of dried fruit (DS) were also analysed by

HPLC. The methanolic extract of dried seed was injected in HPLC unit and the chromatogram

(fig.11) showed that total sixteen peaks were observed and the retention time, quantity of each

separated compound and absorption (m AU) are presented in Table-12. The data of

chromatogram revealed that the absorption of 2916.3mAU. The chromatogram also revealed

that the peak no.2 and peak no.3 resembles with the peaks of standard compound Quercetin

and Gallic acid respectively. The compound resembled with Quercetin at peak no.2 has

175.21mAU absorption whereas compound at peak no.2 has 170.39mAU absorption. The

compound no.2 and 3 were present in the quantities of 1.36% and 1.58% respectively.



-500

0

500

1000

1500

2000

2500

3000

3500

1 61 121

181

241

301

361

421

481

541

601

Run time

Abs

orpt

ion

(mA

U)

8

DS

12

11

1413

1516

6

54

7

321

10

Table-12 (DS)

Peak No.

Ret. time [min]

Start [min]

End [min]

Area [m AU*min]

Height [m AU]



1 2.21 1.93 2.30 21.58 173.13 1.27 5.08 12.71 2 2.40 2.30 2.46 23.09 175.21 1.36 5.44 13.60 3 2.53 2.46 2.68 26.97 170.39 1.58 6.35 15.88 4 2.78 2.68 2.83 12.59 101.26 0.74 2.96 07.41 5 2.93 2.83 2.98 21.97 188.62 1.29 5.17 12.93 6 3.05 2.98 3.15 26.55 179.37 1.56 6.25 15.63 7 3.30 3.15 3.41 46.92 213.46 2.76 11.05 27.63 8 3.65 3.41 4.50 632.39 2895.56 37.24 148.97 37.24 9 4.38 4.23 4.48 26.07 254.62 1.53 6.14 15.35 10 4.75 4.50 5.92 712.66 2916.30 41.97 167.88 419.70 11 5.33 5.23 5.43 6.05 40.14 0.35 1.42 03.56 12 5.53 5.43 5.68 11.96 74.55 0.70 2.81 07.04 13 6.43 6.15 6.58 7.13 41.47 0.41 1.67 04.19 14 6.77 6.58 6.90 32.85 187.52 1.93 7.73 19.34 15 7.05 6.90 7.62 57.71 260.49 3.39 13.59 33.98 16 7.87 7.63 8.68 31.47 115.05 1.85 7.41 18.53



DISCUSSION & CONCLUSION:-

Potential sources of antioxidant compounds have been searched from several types of plant

materials (Ramarathnam, et al., 1997) using a number of methods. Phenols, flavonoids and

other plant phenolics are common in leaves, flowering tissues fruits, seeds and woody parts

such as the stem and bark (Larson, 1998). The antioxidant activity of phenolics is mainly due to

their redox properties which allow them to act as reducing agents, hydrogen donors and singlet

2

1

0

2

1 1 1

0

2

1

2

0

0.5

1

1.5

2

2.5

Com

arat

ive

HP

LC

R

HR

F

UR

S

RS DF

Different extracts of Murraya koenigii

Num

ber o

f com

poun

d re

sem

ble

with

stand

arad

com

poun

ds

Series1

Comparative HPLC analysis of extracts of Murraya koenigii

1

8

12 11

8

5

11

8

5

17 16

0

2

4

6

8

10

12

14

16

18

L R URF HRF RF URS HRS RS FB DF DS

Different extracts of Murraya koenigii

Series1



oxygen quenchers (Rice-evans, et al., 1995). Dasgupta and De, (2004) have reported a

comparative account on antioxidant activity of some leafy vegetable of India. A large body of

literature is available on components having antioxidant activities from a variety of medicinal

plants, fruits, and vegetable and spice plants (Lacikovo, et al., 2007; Jose, et al., 2007: Sharma et

al., 2007). Poly phenols are most abundant antioxidant in the diet. Their total dietary intake is

higher than that of all other classes of phytochemicals and known dietary antioxidants. For

perspective this is 10 times higher then the intake of Vitamin E and carotenoids (Manach, et al.,

2004: Scalbert and Williamson, 2000). Current evidences strongly support a contribution of

polyphends to the prevention of neurogenerative diseases and diabetes mellitus (Scalbest, et

al., 2005). However our knowledge still appears too limited for formulation of

recommendations for the general population or for particular population at risk of specific

diseases. Present investigation highlights a comprehensive profile of antioxidant activity of

extracts of different plant parts and fruits/seeds of field growing aromatic and spice plant

Murraya koenigii with respect to its containment of phenols and flavonoids. The investigation

provides the HPLC analysis of all the samples and established a correlation between

quantitative analysis of antioxidant activity of extracts of different plant parts and identification

of compounds having antioxidant by using known antioxidant compounds in HPLC analysis.

HPLC analysis to identify the specific compound present in the extract having antioxidant

activity with the help of chromatogram of known standard compounds run in the HPLC unit. In

the early days of High Performance Liquid Chromatography (HPLC) it was stated that the liquid

chromatography gives accurate and specific results, it is slow relative to total phenol assay

procedures requires expensive equipments and specialized skills. The introduction of enhanced

resolution and increased automation has resulted in HPLC becoming the most popular analysis

method for plant phenolics (Robards, et al., 1997; Waksmundzka-Hajnos, 1998). The phenolic

compounds of natural origin have the positive property to being solution in polar solvents. This

leads to possibility of using HPLC in their analysis sufficient retention being achieved. Present

study describes and compares the HPLC profiles detected at 254nm of fresh weight of

methanolic extracts of different parts of fresh weight of methanolic extracts of different parts

of Murraya koenigii. In addition to antioxidative compounds (Citric acid, Ascorbic acid

Quercetin and BHT) many other peaks were simultaneously separated and main components

identified by comparing the peaks of unknown samples with available commercial standards

such as citric acid, Ascorbic acid, BHT and Quercetin. Potential sources of antioxidant

compounds have been searched from several types of plant materials (Ramarathnam, et al.,

1997) using different methods. In present investigation the quantitative analysis of total

phenols, flavonoids and antioxidant activities of extracts were carried out and a modified

gradient HPLC analysis of same samples for identification and quantification of phenols and

flavonoids in the plant samples was used. The study was conducted on 15 samples representing

plant tissues of various parts of Murraya koenigii and their different stages of biological



development. Although reports are available on quantitative analysis of antioxidant vitamins

such as lutein ά tocopherol and β carotene from fresh curry leaves using Reversed phase HPLC

(Palaniswany, et al., 2003) but in this study the HPLC analysis for quantitative analysis of

phenols and flavonoids was conducted in order to separate the antioxidative components from

different parts of in vivo plantlets of Murraya koenigii and compared them with the peaks of

available commercial standard antioxidant compounds such as Citric acid, Ascorbic acid, BHT

and Quercetin. In addition the study was also conducted of compounds in each part of plant

like root and leaves under in vivo conditions and also to analyze the availability or synthesis of

bioactive compounds in different stages of biological development of fruits and seeds of field

growing Murraya koenigii. In Present investigation correlation was established between the

Spectrophotometric estimation of antioxidant activity using DPPH free radical of plant extracts

and the HPLC resolution of plant parts and their comparison with the peaks of available

standard antioxidative compounds. In the HPLC chromatograms the antioxidative compounds

were identified by their Retention time by spiking with standards (Citric acid, Ascorbic acid, BHT

and Quercetin) under the same conditions. The biochemical investigation of different plant

parts showed maximum quantity of phenols and flavonoids in dried fruit (DF) and than ripened

fruit (RF) with highest antioxidant capacity discoloration of DPPH. The scavenging potential of

dried fruit (DF) and ripened fruit (RF) was also compared to other plant parts and it was further

confirmed by HPLC analysis of ripened fruits (RF) and dried fruits (DF) that the none of the

standard antioxidative compounds was available in DF sample as observed in HPLC peaks where

as only BHT was identified in the RF sample. The HPLC chromatogram also showed that total 17

compounds were separated from the extract but available standard compounds was not found

in DF sample which clearly indicates that in DF extract which showed highest antioxidant

activity in Spectrophotometric analysis but the antioxidant activity of DF sample maybe due to

the presence of antioxidative compound other than standards. The RF sample showed

antioxidant activity and in this sample the BHT was found as an antioxidative compound. In root

(R), HRF and DS two standard antioxidative compounds were identified but their antioxidant

activity is poorer than DF and RF. The strongest antioxidant activity was exhibited by freshly

prepared extract of ripened fruit (RF) and dry weight of fruit (DF). Present study reports first on

Spectrophotometric analysis of methanolic extracts of different parts of in vivo plants of

Murraya koenigii and their simultaneous HPLC analysis to confirm the identification of

antioxidative compounds in the plant extract by comparing with the peaks of known and

unknown samples. During HPLC analysis five available standard compounds (Ascorbic acid, BHT,

Citric acid, Quercetin and Gallic acid) were used as standard samples and their peaks were

compared with the peaks of different extracts the results obtained suggest that the ascorbic

acid was found in the leaf and it appeared up to half repined stage (HRF) of fruit but it was not

appeared in extract of the plant which confirms that the poor antioxidant activity exhibited by

leaf and HRF may be due to the presence of ascorbic acid in them. The BHT was again found in



root and it appeared only in ripened fruit (RF). The antioxidant activity of RF was strongest

among all the fresh materials of Murraya koenigii. The strongest antioxidant activity may be

due to the presence of BHT which is a very strong antioxidative compound or in other words

the antioxidant activity of RF may be the cumulative presence of a number of antioxidant

compound and BHT in RF. The important observation of the results of present investigation

regarding presence of citric acid; however the Murraya koenigii is a member of citrus group

which normally contains citric acid. The similar observation was found in present investigation

that citric acid was identified in most of the plant materials of Murraya koenigii. It was found in

root, URF, HRF, HRS and even in the extract of dried seed (DS) but more importantly that the

citric acid is not identified in fresh sample of ripened fruit (RF) and extract of dried fruit (DF) but

both the materials have exhibited highest scavenging activity with DPPH and our observation

suggest that in DF which showed highest antioxidant activity without the presence of any of the

standard antioxidant compounds as observed in HPLC analysis but total phenols and flavonoid

contents measured during Spectrophotometric analysis on the basis of significantly contribute

to the antioxidant potential of DF and RF. In present study only a comparative account of

antioxidant activities of different extracts of Murraya koenigii were carried out by comparing

with limited commercial available standard compounds which are known to have antioxidant

potential. Further experimentation should be carried out with isolated constituents in order to

identify the compounds responsible for antioxidant activity detected in crud extracts. The

preparative RP-HPLC separation should be carried out with computer aided optimization in

order to isolate the compounds in Murraya koenigii and other plants of interest and to

determine the bioactivity of the compounds using the high through put testing approach.

ACKNOWLEDGEMENT The authors express deep sense of gratitude & heartfelt thanks to Dr.

Vinod Joshi Deputy Director of Desert Medicine Research Center (DMRC) Jodhpur for providing

lab facility. Sincere thanks are extended to Prof Bhagirath Singh, Former Vice-Chancellor of

M.D.S University Ajmer Rajasthan, whose continuous encouragement and constant help carry

out this research work.

REFERENCES

1. Balandrin, M.J. and Klocke, J.A. (1988). Medicinal aromatic and industrial materials from

plants. In Y.P.S. Bajaj (Ed.); Biotechnology in Agriculture and Forestry. Medicinal and Aromatic

Plant, Springer-Verlag, 4:pp.1-36.

2. Dasgupta, N., and De, B. (2004). Antioxidant activity of Piper betel L. leaf extract in vitro.

Food Chemistry, 88:219-224.

3. Lacikova, L., Muselik, J., Masterova, I. and Grancai, D. (2007). Antioxidant activity and total

phenols in different extracts of four Staphylea L. Species Molecules, 12:98-102.



4. Larson, R.A. (1998). The antioxidant of higher plants. Phytochemistry, 27: 969-978.

5. Manach, C., Scalbert, A., Morand, C., Remesy, C., and Jimenez, L. (2004). Polyphenols: food

sources and bioavailability. Am. J Clin. Nutr, 79:727-747.

6. Palaniswany, U.R., Caporuscio, C. and Stuart, J.D., (2003). Chemical analysis of antioxidant

vitamins in fresh leaf (Murraya koenigii) by reversed phase HPLC with UV detection. Acta Hort.,

(ISHS) 620:475-478.

7. Phillipson, J.D. (1990). Plant as source of valuable products. In B.V. Char wood and M.J.C.

Rhodes (eds), Secondary Products from Plant Tissue Culture. Oxford Clarendon Press, pp.1-21.

8. Ramarathnam, N., Ochi, H. and Takeuchi. M. (1997). Antioxidative defense system in

vegetable extracts. In: Natural antioxidants: Chemistry, health effects, and applications. Shahidi,

F. Ed. AOCS Press, Champaign, 76-87.

9. Rice-Evans, C.A., Miller, N.J., Bolwell, P.G., Bramley, P.M. and Pridham, J.B. (1995). The

relative antioxidant activities of plant-derived polyphenolic flavonoids. Free Rad. Res, 22:375-

383.

10. Robards, K., Li, X., Antolovich, M. and Boyd, S. (1997). Characterisation of citrus by

chromatographic analysis of flavonoids. J. Sci. Food Agric., 75:87-101.

11. Scalbert, A., and Williamson, G. (2000). Dietary intake and bioavailability of polyphenols. J

Nutr., 130:2073S-2085S.

12. Scalbert, A., Johnson-Ian, T. and Saltmarsh, M. (2005). Dietary polyphenols and health

proceeding of the 1st international conference on polyphenols and health. American Journal of

Clinical Nutrition, 81:215S-217S.

13. Sharma, N.K., Dey, S. and Prasad, R. (2007). In vitro antioxidant potential evaluation of

Euphorbia hirta L. Pharmacology online, 1:91-98.

14. Swain, T. (1975). Evolution of flavonoid compounds, in The Flavonoids (harborne JB, Mabry

TJ and Mabry H eds); Chapman and Hall, Ltd., London, pp 109-1129.

15. Waksmundzka-Hajnos, M. (1998). Chromatographic separation of aromatic carboxylic acids.

J. Chromatogr. B., 717:93-118.

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