4.1 HPTLC Fingerprint 4.1.1...

Experimental technique

58

4.1 HPTLC Fingerprint

4.1.1 Introduction

Separation methods commonly known as chromatography have

grown in significance and popularity to become a leading type of

analysis in instrumental analytical chemistry1,2,3

. Today

chromatographic technique have little to do with the separation of color

(the techniques name evolved from the earliest work of separating dyes

or plant pigments on paper), but do involve the separation of

components in a sample mixture. A number of types of separation

methods have developed over the years to accommodate the various

physical and chemical states of the many a sample mixtures; one may be

interested in separation and analyzing. This chromatography is

classifieds into six types mainly thin layer chromatography (TLC), Gas

chromatography (GC), Liquid chromatography (LC), Capillary

electrophoresis (CE), Supercritical fluid chromatography (SFC) and ion

chromatography (IC).

Chromatography is process where in a mobile phase transports

the sample mixture across or through a stationary phase medium. As the

sample comes in contract with the stationary phase, interactions occur.

A partitioning or separation of the components in the mixture results

from the differential affinity of each component with the stationary

phase,

Components with the least affinity for the stationary phase pass

through more quickly than the components with greater affinity for the

stationary phase. As the separated components emerge or elute, a

detector responds with signal change that is plotted against time, thus

producing a chromatogram. In thin layer chromatography, components

don not emerge from the stationary phase bed. Here they are detached

on the surface of the layer after the chromatographic process has been

interrupted and the mobile phase is evaporated.


59

The mobile phase is either a liquid or a gas and the stationary

phase is a solid or liquid. The theoretical explanations for the

partitioning or separating phenomenon for the various techniques can be

fairly complex. Intermolecular forces involving mechanical, physical or

chemical processes are responsible for the differential distribution of the

components and resulting chromatogram. More rigorous treatment of

partition theory can be found in text books4,5,6

.

The basic TLC Procedure has largely remained unchanged over

the last 50 years. It involves the use of thin, even sorbent layer, usually

about 0.10 to 0.25 mm thick, applied to a firm backing of glass,

aluminum or plastic sheet to act as a support.

Fingerprint analysis approach using chromatography has become

the most potent tools for quality control of herbal medicines because of

its simplicity and reliability. It can serve as a tool for identification,

authentication and quality control of herbal drugs.

The construction of chromatographic fingerprints plays an

important role in the quality control of complex herbal medicines7.

Chemical fingerprints obtained by chromatographic techniques are

strongly recommended for the purpose of quality control of herbal

medicines, since they might represent appropriately the “chemical

integrities” of the herbal medicines and therefore can be used for

authentication and identification of the herbal products. Based on the

concept of phyto-equivalence, the chromatographic fingerprints of

herbal medicines could be utilized for addressing the problem of quality

control of herbal medicines8.

By definition a chromatographic fingerprint of a herbal medicine

is, in practice, a chromatographic pattern of pharmacologically active

and or chemically characteristic constituents present in the extract9,10,11

.

This chromatographic profile should be featured by the fundamental


60

attributions of “integrity” and “ fuzziness” or “ sameness” and

“differences” so as to chemically represent the herbal medicines

investigated. This suggest that chromatographic fingerprint can

successfully demonstrate both “sameness” and “differences” between

various samples and the authentication and identification of herbal

medicines can be accurately conducted even if the number and

concentration of chemically characteristic constituents are not very

similar in different samples of herbal medicine. Thus chromatographic

fingerprint should be considered evaluate the quality of herbal medicines

globally considering multiple constituents present in the herbal

medicines.

4.1.2 Need for development of chromatographic fingerprint

Herbal medicines have a long therapeutic history and are still

serving many of the health needs of a large population of the world. But

the quality control and quality assurance still remains a challenge

because of the high variability of chemical components involves. Herbal

drugs singularly and in combinations, contain a myriad of compounds in

complex matrices in which no single active constituent is responsible for

the overall efficacy. This creates a challenge in establishing quality

control standards for raw material and standardization of finished herbal

drug1.

Traditionally only a few markers of pharmacologically active

constituents were employed to assess the quality and authenticity of

complex herbal medicines. However, the therapeutic effects of the

herbal medicines are based on the complex interaction of numerous

ingredients in combination, which are totally different from those of

chemical drugs. Thus many kinds of chemical fingerprint analysis

methods to control the quality of herbal drugs have gradually come into

being, such as thin layer chromatography, gas chromatography, high

performance liquid chromatography etc.


61

Chromatographic fingerprint analysis of herbal drugs represents

comprehensive qualitative approach for the purpose of species

authentication, evaluation of quality and ensuring the consistency and

stability of herbal drugs and their related products. The entire pattern of

compounds can then be evaluated to determine not only the presence or

absence of desired markers or active constituents but the complete set of

ratios of all detecatable analytes. The chemical fingerprints obtained by

chromatographic and electrophoresis techniques, especially by

hyphenated chromatography are strongly recommended for the purpose

of quality control of herbal medicines, since they might represent

appropriately the “chemical integrities” of herbal medicines and

therefore be used for authentication and identification of the herbal

products.

4.1.3 Chromatographic techniques for fingerprint analysis of herbal

material

Herbal materials are concerned there are always number of

components and many of them are in too low amounts. On other hand,

usually exists variability within the different and even the same herbal

materials. Consequently, to obtain reliable chromatographic fingerprints

that represent pharmacologically active and chemically characteristic

components is not a trivial task. The performance of a chromatographic

fingerprint obtained is closely dependent on the chromatographic

separation degrees and concentration distribution of all chemical

components in the herbal material is investigated. Fortunately,

chromatography offers very powerful separation ability, such that the

complex chemical components in herbal extracts can be separated in to

many relatively simple sub fractions. Furthermore, the recent

approaches of applying hyphenated chromatography and spectroscopy

such as High-Performance Liquid Chromatography-Diode Array

Detection (HPLC-DAD), Gas Chromatography-Mass Spectroscopy


62

(GC-MS), Capillary Electophoresis-Diode Array Detection (CE-DAD),

HPLC-MS and HPLC-NMR , could provide the additional spectral

information , which will be very helpful for the qualitative analysis and

even for the on-line structural elucidation. With the help of the spectral

information the hyphenated instruments show greatly improved

performances in terms of the elimination of instrumental interferences,

retention time shift correction, selectivity, chromatographic separation

abilities and measurement precision. If hyphenated chromatography is

further combined with chemometric approaches, clear pictures might be

developed for chromatographic fingerprints obtained. A chemical

fingerprint obtained by hyphenated chromatography will become the

primary tool for quality control of herbal materials12,13

.

4.1.4 Chromatographic technique

Thin layer chromatography used for fingerprint analysis of herbal

materials:

Thin layer chromatography (TLC) is frequently used for the

analysis of herbal materials since various pharmacopoeias such as

American Herbal Pharmacopoeia (AHP), Chinese Drug Monographs

and analysis; Pharmacopoeia of People’s Republic of China etc. Still use

TLC to provide first characteristic fingerprints of herbs. Rather, TLC is

used an easier method of initial screening with a semi quantitative

evaluation together with other chromatographic techniques. High

performance TLC has the advantages of many fold possibilities of

detection in analyzing herbal medicines. In addition, HPTLC analysis is

rather simple and can be employed for multiple sample analysis. With

the help of CAMAG video store system (CAMAG Switzerland). It is

possible to get useful qualitative and quantitative information from the

developed TLC plate14

. The advantage of using TLC to construct the

fingerprints of herbal medicines are its simplicity, versatility, high

velocity, specific sensitivity and simple sample preparation. Thus, TLC


63

is a convenient method of determining the quality and possible

adulteration of herbal products15

.

4.1.5 Difficulties in development of chromatographic fingerprints of

herbal materials.

When herbal drugs are considered for analysis, a large number of

chemical components are involved and many of them are in low

concentration. Chromatographic instruments and experimental condition

are difficult to reproduce during real analysis. Thus the baseline and

retention time shifts surely will be in existence from one chromatogram

to another. Many other problems associated with chromatographic

fingerprints such as the occurrence of abnormal chromatograms form

outlying herbal samples or experiments inevitably will be encountered16

.

4.1.6 Traditional medicines and chromatographic fingerprints

Due to pharmacological activity, traditional medicine, containing

mainly of herbal drugs, has been popularly and extensively used for

many centuries. However, it is not easy to conduct quality control and

assurance of herbal medicines because of high variability of chemical

components involved. Thus, as pointed out by World Health

Organization (WHO), Since the quality and quantity of safety and

efficacy data on traditional medicine are far from sufficient to meet the

criteria needed to support its use worldwide and there is still a lack of

adequate or accepted research methodology for evaluating traditional

medicine up to now, traditional medicine has not been officially

recognized in the most countries. Under this situation, the U.S. FDA

does not definitely emphasize the developments of very clear pictures of

all chemical pictures of all chemical components from herbal medicines,

and thus the fingerprinting approach has been recently recognized and

accepted for quality assessment of traditional medicines. The concept of

phytoequivalence was developed in Germany in order to ensure


64

consistency of herbal products. According to this concept, a chemical

profile, such as chromatographic fingerprint, for a herbal product should

be constructed and compared with the profile of a clinically proven

reference product. Chinese State Food and Drug Administration have

said to regulate the compositions of liquid injections with herbal

ingredients using stringent quality procedures such as chemical assay

and standardization. Fingerprint of herbal medicinal liquid injections are

compulsorily carried out for this purpose. In addition, among the various

experimental techniques, chromatographic method is highly

recommended for finding out fingerprints of herbal products because of

the high separation ability of chromatography.

4.1.7 Chemometric approaches and data processing for chromatographic

fingerprint

Due to complexity of the chromatographic fingerprint and the

irreproducibility of chromatographic instruments and experimental

conditions, several chemometric approaches such as variance analysis,

peak alignment, correlation analysis were employed to deal with the

chromatographic fingerprint. Many mathematical algorithms are used

for data processing in chemometric approaches. The basis principles for

this approach are variation determination of common peaks/regions and

similarity comparison with similarity index and linear correlation

coefficient. Similarity index and linear correlation coefficient can be use

to compare common pattern of the chromatographic fingerprints

obtained. In general the mean or median of the chromatographic

fingerprints under study is taken as the target and both are considered to

be reliable. Data loading, removing cutting, smoothing, compressing,

background and retention time shift correction, normalization, peak

identification and matching, variation determination of common

peaks/regions, similarity comparison, sample classification and other


65

data processes associated with the chromatographic fingerprint can be

investigated with this winCAT software.

The problem of quality assurance of herbal medicines has been

solved to a great extent with the help of chromatographic fingerprint

analysis. The variation determination of common peaks/regions in a set

of chromatographic fingerprints could provide useful qualitative and

quantitative information on the characteristic components of herbal

medicines investigated. On the other hand, whether the real sample was

identified as the herbs with the same quality grade could be determined

successfully by way of comparing the chromatographic fingerprints with

the similarity index and linear correlation analysis17

. Thus

chromatographic fingerprint analysis serves as a promising quality

control tool for herbal medicines. Hence, we can conclude that the most

useful and simple technique for the fingerprint of herbal analysis is

HPTLC. In current research work the same technique i.e; HPTLC

techniques was adopted for deriving the fingerprint patterns of both the

plant materials.

4.1.8 Preparation of plant extract for HPTLC fingerprint

Procedure

Argemone mexicana linn. was collected from patan district. It

was cleaned and washed under tap water and dried at room temperature

for two days and then it was dried. The dried materials were powdered

and sieved through ASTM sieve (85/BS sieve) and was kept in airtight

container. Sample for HPTLC fingerprinting were prepared by using

optimized condition for extraction.

Step 1: Accurately weighed 250mg of Argemone mexicana linn. leaf, stem and

rood powder separately in 20ml test tubes and 12.5ml methanol were

added in each test tubes. The test tubes were kept on test tube shaker for


66

90 minutes. The contents were filter through Whatman filter paper no.

41.

Step 2: The residue obtained in step 1 were subjected for further extraction

using 12.5ml of methanol for 90 minutes and filtered through Whatman

filter paper no. 41.

Step 3: Extraction procedure was repeated again like step2. (The optimized

condition number of extraction was observed to be three in the case of

Argemone mexicana linn.

Step 4: The filtrated obtained in Step 1,2 and 3 were pooled together

concentrated to the half volume and were used for further analysis.

4.1.9 High performance thin layer chromatography

High Performance Thin Layer Chromatography was used to

establish the fingerprint of the plant extracts. In TLC(>90%) silica is

used mainly for the separation of pharmaceuticals and drugs. The

stationary phase used was silica gel 60 F254. A mobile phase was

developed in such a way that the maximum separation was achieved on

a TLC Plate useful for phytochemical analysis.

Optimized chromatographic condition for Argemone mexicana

linn. leaf, stem and root extracts for establishment of HPTLC fingerprint

are given in below table.


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Table-1 Optimized condition for HPTLC fingerprint

PARAMETERS DESCRIPTION

Instrument CAMAG HPTLC

Stationary Phase Silica gel 60 F254 HPTLC pre coated plates.

Sample Applicator CAMAG LINOMAT V

Band Width 8.0mm

Application Volume 20µl

Syringe CAMAG Linomat Syring (100 µl)

Mobile Phase Toluene:Chloroform:Ethanol(8:8:2)

Volume of Mobile Phase 18ml

Development Mode CAMAG Twin Trough Chamber

Development Distance 80mm

Chamber Saturation Time 20mins.

Densitometer Scanner 4

Scanning Wavelength 254nm, 366nm,540nm.

Software WinCATS Version 1.4.6

Lamp D2,Hg,W

Measurements Mode Absorbance & Fluorescence

Photo Documentation CAMAG TLC Visualizer


68

Sample Application

Linomat 5 application parameters

Spray gas: Inert gas(N2)

Sample solvent type :Methanol

Dosage speed : 150 nl/s

Predosage volume : 0.2 ul

Sequence

Syringe size: 100 μl

Number of tracks: 12

Application position Y : 8.0 mm

Band length : 8.0 mm

Table -2 Sample Application Parameter

Sr. No. Appl. position Appl. volume Vial Sample ID

1 20.0mm 2.0µl 1 Leaf

2 34.5mm 4.0 µl 1 Leaf

3 49.0mm 8.0 µl 1 Leaf

4 63.5 mm 10.0 µl 1 Leaf

5 78.0 mm 2.0 µl 2 Stem

6 92.5 mm 4.0 µl 2 Stem

7 107.0 mm 8.0 µl 2 Stem

8 121.5 mm 10.0 µl 2 Stem

9 136.0 mm 2.0 µl 3 Root

10 150.5 mm 4.0 µl 3 Root

11 165.0 mm 8.0 µl 3 Root

12 179.5 mm 10.0 µl 3 Root

Detection

Information

Application position 8.0 mm

Solvent front position 80.0 mm

Instrument: CAMAG TLC Scanner

Number of tracks 12

Position of first track X 23.9 mm

Distance between tracks 14.5 mm

Scan start pos. Y 5.0 mm

Scan end pos. Y 85.0 mm

Slit dimensions 6.00 x 0.45 mm,

Scanning speed: 20 mm/s


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Measurement Table

Wavelength 254

Lamp D2

Measurement Type Remission

Measurement Mode Absorption

Detector mode Automatic

Figure 1 Image at 254nm

Figure 2 Image at 366nm before derivatization


70

Figure 3 Image at 366nm after derivatization

4.2 Qualitative analysis phytochemical

Extraction of plant sample involves the separation of medicinally

active component from complex matrix plant using selective solvent in

standard extraction procedures. The plant has large number of

compounds like alkaloids, flavonoids, phenols, seponins, sterols,

steroids, tannins, glycosides etc that exert a physiological and

therapeutic effect. The compounds that are responsible for medicinal

property of the plant are usually secondary metabolites. The plant

material is subjected to preliminary phytochemical test for detection of

various plant constituents18

.

4.2.1 Alkaloids

About 1gm dry powder of methanolic extract Argemone

mexicana linn. was dissolved in 10ml 2 N HCl. The mixture was

filtered and the filtrate was divided into 3 equal portions.

i) Mayer’s Test

To a 1 ml of filtrate, two drops of Mayer’s reagent was added

along the side of the test tube. Formation of white or creamy precipitate

indicates the presence of alkaloids.


71

ii) Dragendroff’s test

To a 2 ml of filtrate, 1 or 2 ml of Dragendroff’s reagent was

added. A Prominent reddish brown precipitate indicates the presence of

alkaloids.

iii) Wagner’s test

To a few ml of filtrate, few drops of Wagner’s reagent were added

along the sides of the test tube. Formation of reddish brown precipitate

confirms the test as positive19

.

4.2.2 Flavonoids

i) Shinoda test.

About 0.5gm dry powder of methanolic extract Argemone

mexicana linn. was treated with 2ml of concentrated HCl and

magnesium metal powder. The appearance of pink or red colour within

few minutes indicated the presence of flavonoids20

.

ii) Alkaline test

The dry powder of methanolic extract of Argemone mexicana

linn. was treated with few drops of diluted sodium hydroxide (NaOH)

separately. Formation of intense yellow color which turned colorless on

addition of few drops of diluted HCl indicated presence of flavonoids.

4.2.3 Phenolic Compounds

i) Ferric chloride test

About 0.5gm of extract was dissolved in 2ml of distilled water

and added few drops of neutral 5% ferric chloride solution. Formation of

blue/green/violet color indicates the presence of phenolic compounds21

.

ii) Lead Acetate Test

About 0.5gm of extract was dissolved in 5ml distilled water and

added 3 ml of 10% lead acetate solution. A bulky white precipitate

indicates the presence of phenolic compounds.


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4.2.4 Saponins

The presence of saponins was determined by Frothing test. 2gm

extract was vigorously shaken with 20ml distilled water and was

allowed to stand for 10 minutes and classified for saponin content as

follows: no froth indicates absence of saponins and stable froth for more

than 1.5 cm indicated the presence of saponins22

.

4.2.5 Sterols/Steroids

Liebermann-Burchard reaction was performed for the presence of

steroids. About 1gm methanolic extract was treated with 2ml acetic

anhydride and few drops of concentrated H2SO4 were added down the

sides of test tube. A blue green ring indicated the presence of steroids.

4.2.6 Tannins

About 0.5 g of the extract was boiled in 10ml of water in a test

tube and then filtered. A few drops of 0.1% ferric chloride was added

and observed for brown ish green or a blue black coloration. indicated

the presence of tannins23

.

4.2.7 Glycosides

This test perfomed by Keller-kiliani test about 0.5gm powder of

methanolic extract was treated with 1ml mixture of 5% FeCl3 and glacial

acetic acid (1:99 v/v). To this solution, few drops of concentrated H2SO4

were added. Appearance of greenish blue color within few minutes

indicated the presence of glycosides24

.

4.2.8 Class of Compound by HPTLC

A comparative phytochemical analysis was performed to detect

the presence of different class of compounds in various part of plant

(Leaves, stem, root) of selected plants. Plants extracts were spotted on

the HPTLC plates and developed with different mobile phases. For each


73

class of compounds and chromatogram for the class was obtained25

.

Different mobile phases used for qualitative analysis of compounds

Table-3 Class of compound by HPTLC

Class of

compound

Mobile phase Derivatization

reagent

Alkaloids Toluene: Ethyl Acetate: Diethyl Amine

(7:2:1)

Dragendroff Reagent

Flavonoids Ethyl Acetate:Formic Acid: Glacial

Acetic Acid: Water (10:0.5:0.5:1.3)

Anisaldehyde

Sulphuric Acid

Phenols Cyclohexane:Ethyl Acetate: Formic Acid

(4:6:1)

Anisaldehyde

Sulphuric Acid

Seponins Chloroform: Acetic Acid: Methanol:

Water ( 6.4: 3.2: 1.3: 0.8)

Anisaldehyde

Sulphuric Acid

Sterols Chloroforms: Ethyl Acetate (4:6) Anisaldehyde

Sulphuric Acid

Steroids n-butanol: Methanol: Water (3:1:1) Anisaldehyde

Sulphuric Acid

Tannins Toluene: Ethyl Acetate: Formic Acid

(6:4:0.3)

Ferric Chloride

Regent

Glycosides Ethyl Acetate: Methanol: Water

(20:2.8:2)

Alcoholic KoH


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Sample Application


Spray gas : Inert gas(N2)

Sample solvent type : Methanol



Sequence

Syringe size : 100 μl

Number of tracks : 9





1 20.0mm 2.0µl 1 Leaf

2 34.5mm 4.0 µl 1 Leaf

3 49.0mm 8.0 µl 1 Leaf

5 78.0 mm 2.0 µl 2 Stem

6 92.5 mm 4.0 µl 2 Stem

7 107.0 mm 8.0 µl 2 Stem

9 136.0 mm 2.0 µl 3 Root

10 150.5 mm 4.0 µl 3 Root

11 165.0 mm 8.0 µl 3 Root

Detection

Information




Number of tracks 9


Distance between tracks 20 mm






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4.2.9a Alkaloids

Figure 4 Image at 254nm Figure 5 Image at 254nm after

Derivatization

4.2.9b Flavonoids

Figure 6 Image at 254 nm Figure 7 Image at 366 nm

Figure 8 Image at 366nm after derivatization by Anisaldehyde Sulphuric acid

reagent


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4.2.9c Phenols

Figure 9 Image at 254nm Figure 10 Image at 366nm

4.2.9d Saponins


Figure 13 Image at 366nm after derivatization by Anisaldehyde Sulphuric Acid


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4.2.9e Sterols

Figure 14 Image at 254 nm Figure 15 Image at 366nm

Figure 16 Image at 366nm after derivatization by Anisaldehyde Sulphuric Acid

4.2.9f Steriods



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Figure 19 Image at 366nm after derivatization by anisaldehyde sulphuric acid

4.2.9g Tannins


4.2.9h Glycosides



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4.3 Quantitative analysis of active principle

4.3.1 Identification and Quantification of Ferulic Acid

The identification and quantification of active constituents in the

plant material and have come into play a very important role in

standardization of herbal formulations. HPTLC offers many advantages

beside then the other chromatographic technique such as flexibility of

change of mobile phase, stationary phase and choice of detection of

wavelength thus HPTLC is most widely used for routine analysis of

herbal medicine. Ferulic acid is one of the reported components in seed

of plant26

. For the quantitative analysis sample extract of plant parts (i.e.

leaf, stem, root and seed) were prepared. The reference standard of

ferulic acid (purity 98%) was obtained from Sigma-Aldrich.

As a matter of analytical interest, it has to be watched that all

components in an analytes should be detected. Identification in a

qualitative analysis can be done by use of retention time/migration

distance of the corresponding substance by the position of peak

maximum in chromatogram. The height and area of the individual peaks

are quantitatively evaluated27-34

.

Sample Preparation

Take 500mg dry powder of Leaf, Stem, Root and Seed powder

were dissolved in 10ml methanol in 10ml volumetric flask. Then

sonicated the sample on ultrasonic bath for 30min (conc. 50µg/µl).

A standard stock solution of ferulic acid (1mg/ml) was prepared

by dissolving 10mg of accurately weighed ferulic acid in methanol and

diluting it 10ml with methanol. Further dilution was made with 10ml

methanol to obtain working standard of ferulic acid (conc. 0.1µg/µl).

From the prepared working standard of ferulic acid (0.1µg/µl),

were applied on HPTLC plate. Along with the standards, plant extracts


80

were applied on same plate. After drying of spots, the plates were

developed to a distance of 80mm in a Camag twin-trough chamber,

previously saturated with respective mobile phase vapour for 20min.

Table 5 Optimized condition for quantification

Parameters Description

Instrument CAMAG HPTLC

Stationary Phase Silica gel 60 F254 HPTLC pre coated plates.

Sample Applicator CAMAG LINOMAT V

Band Width 8.0mm

Syringe CAMAG Linomat Syring (100 µl)

Mobile Phase Toluene: Ethyl Acetate: Formic Acid (14:8:0.15)

Volume of Mobile Phase 22ml

Development Mode CAMAG Twin Trough Chamber

Development Distance 80mm

Chamber Saturation Time 20mins

Densitometer Scanner 4

Scanning Wavelength 254nm, 366nm,540nm

Software WinCATS Version 1.4.6

Lamp D2,Hg,W

Measurements Mode Absorbance & Fluorescence

Photo Documentation CAMAG TLC Visualizer


81

After development the plates were dried then densitometric evaluation

of plate was performed at 254nm and 366nm with Camag Scanner III in

conjugation with winCATS software for quantification2. Peak height and peak

areas were integrated for the entire track. Under this condition a proper

separated peak and retention factor (Rf) value for ferulic acid was obtained.

Sample application


Spray gas : Inert gas(N2)

Sample solvent type : Methanol



Sequence

Syringe size : 100 μl

Number of tracks : 17



For Identification



1 15.0mm 5.0 µl 1 Ferulic Acid

2 25.6 mm 20.0 µl 2 Leaf

3 36.2 mm 20.0 µl 2 Leaf

4 46.8 mm 20.0 µl 2 Leaf

5 57.4 mm 5.0 µl 1 Ferulic Acid

6 68.0 mm 20.0 µl 3 Stem

7 78.6 mm 20.0 µl 3 Stem

8 89.2 mm 20.0 µl 3 Stem


10 110.4 mm 20.0 µl 4 Root

11 121.0 mm 20.0 µl 4 Root

12 131.6 mm 20.0 µl 4 Root


14 152.8 mm 20.0 µl 5 Seed

15 163.4 mm 20.0 µl 5 Seed

16 170.0 mm 20.0 µl 5 Seed



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For Quantification



1 15.0mm 5.0 µl 1 Ferulic Acid

2 28.0 1.0 µl 1 Ferulic Acid








10 132.0 30 µl 1 Leaf

11 145.0 30 µl 1 Leaf

12 158.0 30 µl 1 Leaf

13 171.0 30 µl 1 Leaf

14 184.0 30 µl 1 Leaf

Detection

Information




Number of tracks 17


Distance between tracks 10.6 mm






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Figure 24 Image at 254nm for Figure 25 Image at 366nmfor

Identification Identification

Figure 26 Image at 254 for Figure 27 Image at 366nm for

Quantification Quantification

4.4 Isolation of phytochemical using Sepbox 2D 2000

The sepbox has been developed especially for separation and

purification of highly complex mixture. This system can fractionate one

sample of complex mixture within 24 hour. The sepbox concept is based

on a patented combination of high performance liquid chromatography

(HPLC) and solid phase extraction (SPE) reliable and proven techniques

that provide a universal platform suitable for processing large number of

sample35

.

4.4.1 Separation of phytochemical by sepbox 2D-2000

The Sepbox 2D-2000 has been developed especially for the

preparative separation and purification of highly complex mixture. The


84

purification of compounds from natural sources which usually takes

weeks or months with conventional method. This system can fractionate

one sample of complex mixture within 24 hours36

. The Sepbox concept

is based on a patented combination of high performance liquid

chromatography (HPLC) and solid phase extraction (SPE) reliable and

proven techniques that provide a universal platform suitable for

processing large sample number. Sepbox produces high qualitative and

reproducible result, has possibility to inject up to 2000mg of a natural

extract and has the unique concept of totally automated preparative

separation of compound.

4.4.2 Functional principle of HPLC

High-performance liquid chromatography (High-pressure liquid

chromatography), is a technique in analytic chemistry used to separate

the components in a mixture, It relies on pumps to pass a pressurized

liquid solvent containing the sample mixture through a column filled

with a solid adsorbent material. Each component in the sample interacts

slightly differently with the adsorbent material, causing different flow

rates for the different components and leading to the separation of the

components as they flow out the column. HPLC has been used for

separating the components of a complex biological sample, synthetic

drugs, Herbal drugs and many ways in manufacturing purpose.

4.4.3 Reverse phase high performance liquid chromatography

In reverse phase HPLC, the mixture to be separated is added to a

mobile phase (micture of methanol and water) which is forced through a

column containing a stationary phase (chemically modified silica). The

separtion of the mixture is based on the difference in strength of the

interaction of the components of the sample with the mobile phase and

the stationary phase. Some components of the mixture will be strongly

retained by the stationary phase and have a relatively weak interation


85

with the mobile phase: these compounds will exhibit a long retention

time(i.e.they are slowly eluted from the colum). Other components will

be weakly retained by the column and have a stronger interaticion with

the moble phase; these compounds will exhibit a short retention time

(that is, they are rapidly eluted from the column, some physical property

of the mobile phase is monitored.

The sepbox is supplied with a UV detector and an ELSD. The

UV detector records the change in UV absorption when a compund is

passing through its flow cell. The ELSD detects the change in light

scattering of the mobile phase caused by a compound.

4.4.4 Functional principle of solid phase extraction

Solid phase extraction (SPE) is used to collect, accumulate and

isolate compounds in high dilution, The sepbox uses reversed phase

extraction technology. In revesed phase extraction the column is packed

with a non-polar adsobent The polarity of the mobile phase cab ne

increased by addition of water, so that the dissolved non-polar

compunds are adsorbed on the non-polar stationar phase. The thin layer

adsorbed compoud on the SPE column can be rinsed with wter to

remove buffer and other additives. Afterwards the compound can be

desobed with an organic compounds.

Figure 28 Principle diagram of sepbox


86

4.4.5 Functional principle of the sepbox 2D-2000

The sample is introduced either as solid via injection or liquid via

injection loop and separated at separation column 1 using a low pressure

gradient. The polarity of the eluent form separtion column 1 is increased

by the addition of the water to such an extent that the fractions eluted

from separation column 1 are adsorbed onto the 15 non polar trap

columns. These trapped fractions are then passed through the separation

columns 2 where final separation is completed. The individual

components eluted are flushed into the fraction collector where they are

colledcted into vials and cab be further processed on screening systems.

Table 8 Instument Parameter of Sepbox 2D-2000

Gradient Pump 100ml head, 400bar, ternary gradient

Water Pump 100ml Head, 400bar

Valves 13 electrically actuated valves

Injection Up to 2000mg of extract with injection column

technique or liquid using a loop.

1st separation column 1 column,150x32mm

2nd

separation column 6 column, 250x16mm

Trap column 18 column, 30x32mm

UV Detector 10mm flow cell

ELSD Detector Splitted flow for ELSD

Fraction Collector 12 racks, 48 vials each, max. capacity

60ml/vial, ma. 576 fraction

Dimentions 266x65x89cm (WxDxH)


87

Figure29Sepbox instrument with UV, ELSD detector and fraction colletor

4.4.6 Preparation of Sample for Separation

Take the methanolic extract of Argemone mexicana Linn. whole

plant powder then wash it by hexane using separating funnel for

removing of oil part form extract then dry the extract and make fine

powder. 2gm of fine extract powder with 4gm pure silica was mixed.

The extract were absorbed onto RP material (Silica Gel) was transferred

carefully into the column in following manner.

Column was filled with silica during this process, kncok carefully

against the column to allow the material to be setteled and get firmly

packed. Remaining portion of the column was filled up to the groove

with pure silica while knocking carefully against the column to allow

that material to settle37

.That would act as solvent filter and flow

distributor through the column.

Solvent used for a Sample Separation

Water: 8.8 Ltr Approx. (HPLC Grade)

Methanol: 2.2 Ltr. Approx. (HPLC Grade)

Acetonitrile: 3.8 Ltr. Approx. (HPLC Grade)


88

4.4.7 Trap Column Conditioning and Equilibration

During the trap column equilibration, the gradient pump

equlibrates the trap column to prepare them for the collection of the

fraction during the main separation. The gradient that are used for this

step are set in the method.

4.4.8 Main Column Eqilibration

During the main column equibration, the main column

equilibrated by the gradient pump accrding to the setting in the method.

Since the eluent is flowing through the detector it can be observed

throgh chroamatogram and previous separation is flushed from the

column during the equilibration of system.

4.4.9 Main Separation

To start the separation with the injection column is introduced in

to the flow path by switching from position 1 to 2. Thereby the sample

is carried by the eluent onto the main column for the first separation.

To collect the substances eluted rom the main separation column

the trap column are swithced in to the flow path according to the

collection pattern programmed in the method. The water pump is used to

add water to trap substances on the trap columns.

4.4.10 Second Step Separation

Here the trap column works as the injection column which is

switched in the flow path before the second step separation column. Due

to sophisticated valve connections the detectors can now alos be used to

gather chromatograms. After the detectors the flow is led into the

fraction collector for collection of isolate comounds. The collection of

fractions can be done by peak detection or time based. If the right vial

volume is entered in the system setup, the software automaticaly

switches to the next vial before it is to full.


89

The fractions collected by sepbox were stored at very low

temperature for the further analysis.

4.5 Identification of phytochemical using GCMS

The fraction collected from sepbox were lyophilized in order to

concentrate them and identified by GCMS. Like a good combination

both GC can separate the volatile compound with great resolution and

MS can provide detailed structural information on most compound such

that can be identified38

.

4.5.1 Gas chromatography

In 1952, James and Martin study on gas chromatography (GC)

began the era of modern, high performance chromatography. Since that

time, GC has become the premier technique for the separation and

analysis of volatile compounds, and gas chromatographs have been the

most widely used analytical instrument in the world. GC can be used up

to 3500 C corresponding to an upper molecular weight of 600 D.

However, high temperature GC work has been done to 4500 C, and there

are some alternative ways of handling non volatiles by GC39

.

4.5.2 Principle of gas chromatography

Gas chromatography (GC), is a common type of chromatography

used in analytical chemistry for separating and analyzing compounds

that can be vaporized without decomposition. Typical uses of GC

include testing the purity of a particular substance, or separating the

different components of a mixture (the relative amounts of such

components can also be determined). In some situations, GC may help

in identifying a compound. In gas chromatography, the mobile phase is a

carrier gas, usually an inert gas such as helium or an un reactive gas

such as nitrogen. The stationary phase is a microscopic layer of liquid or

polymer on an inert solid support, inside a piece of glass or metal tubing

called a column. The gaseous compounds being analyzed interact with

the walls of the column, which is coated with a stationary phase. This


90

causes each compound to elute at a different time, known as the

retention time of the compound. The comparison of retention times is

what gives GC its analytical usefulness. Gas chromatography is in

principle similar to column chromatography but has several notable

differences. Firstly, the process of separating the compounds in a

mixture is carried out between a liquid stationary phase and a gas mobile

phase, whereas in column chromatography the stationary phase is a solid

and the mobile phase is a liquid. Secondly, the column through which

the gas phase passes is located in an oven where the temperature of the

gas can be controlled, whereas column chromatography (typically) has

no such temperature control. Thirdly, the concentration of a compound

in the gas phase is solely a function of the vapor pressure of the gas. Gas

chromatography is also sometimes known as vapor-phase

chromatography (VPC), or gas–liquid partition chromatography

(GLPC).

4.5.3 Mass Spectroscopy

Mass spectrometry is a powerul analytical technique used to

identify unknown compounds and elucidate the structure and chemcal

properties of different molecures. The use of a mass spectrophotometer

for chromatographic detection offers several special advantages. The

most important is that can provide unique informaton about the chemical

composition of analyte. It truly provide a second dimentsion of

infromation to the chromatographic analysis. Furthermor, mass

spectrophotometers show high sensitivity for volatile compounds

because they are mass flow sensitive, the detector response can be used

for quantitative purposes40

.

4.5.4 Principle of Mass Spectroscopy

Mass spectrometry (MS) is an analytical techniue that separates

ionized particles such as atoms, molecules and clusters by using


91

differences in the ratio of their charges to their respective masses

(mass/charge; m/z), and can be used to determine the molecualr weight

of the particles.

The commonly use technique for obtaining the mass spectrum of

an organic compound is electron impact (EI) technique. Other

techniques are chemical ionization (CI), the field desorption and fast

atom bombardment (FAB) are used for organic compounds, while

electron spray ionization (ESI) and matrix assisted laser desorption

ionization (MALDI) are used for large biopolymers.

4.5.5 Ionization of Molecule by Electron Impact (EI)

A netural sample of molecule is bombarded with high energy

electrons. Energy of an electron is around 70 (eV), Which is greater than

the ionization energy for most compunds. On electron impact, the

molecules are energized sufficiently to eject an electron. This process

lead to the formaton of a redical cation symbolized more accurately by

(M+) but often written as M

+ for simlification which is called the

molecular ion.The Molecular ion represents the intact molecure which

has the same weight as the original molecule (M), because weight loss

of an electron is negligible.

M + e-

M+ + 2e

-

Netural Molecual High energy Molecular ion

electron


92

Table 9 Instument Parameter of GCMS

Model Shimadzu GC-2010Plus(Plate:3.3)

Column Rtx-5 MS, 30m x 0.25mm

Oven Up to 4500 C

Injector port temperature Up to 4500

C

AFC Pressure range 0 to 970 kPa

Column oven temperature Up to 4500 C

GCMS Interface Direct connection with capillary

column

Temperature 50 to 3500 C

Ion Source EI (Standard),PCI (option),and NCI

with NCI sorce option

Ion source temperature 140 to 3000 C

Mass Analyzer Metal quadrupose mass filter with

pre-rod

Mass range m/z 1.5-1900

Software Scan,SIM,FASST

Mass Spectrum Libraries NIST,Wiley, Pesticide Library,

FFNSC Library (Flavor and

Fragrance), Drug Library

Procedure for GCMS analysis

The fraction colleceted from sepbox were lyophilized in order to

concentrate them then compounds that had been got after lyophilized were

dissoved in very low amount in respective solvent. Then GCMS parameter


93

were set as per the requirement.

2µl of unkown sample was injected to GC and observation was being

made. Method was set at below parameter.

Sample Application

[GC-2010]

Sample Volume : 2µl

Column Oven Temp. : 80.0 °

C Injection Temp. : 280.00 °C

Injection Mode : Split

Flow Control Mode : Pressure

Pressure : 65.9 kPa

Total Flow : 14.0 mL/min

Column Flow : 1.00 mL/min

Linear Velocity : 36.8 cm/sec

Purge Flow : 3.0 mL/min

Split Ratio : 10.0

High Pressure Injection : OFF

Carrier Gas Saver : OFF

Splitter Hold : OFF

Oven Temp. Program

Rate Temperature(°C) Hold Time(min)

- 80.0 0.00

10.00 270.0 0.00

Equilibrium Time : 3.0 min

[GCMS-QP2010 Plus]

IonSourceTemp : 230.00 °C

Interface Temp : 290.00 °C

Solvent Cut Time : 2.00 min

Detector Gain Mode : Relative

Detector Gain : 0.00 kV


94

Threshold : 1000

[MS Table]

Start Time : 2.40min

End Time : 19.00min

ACQ Mod : Scan

Event Time : 0.25sec

Scan Speed : 5000

Start m/z : 50.00

End m/z : 1000.00

The data was analyzed after completion of sample run; the desire

peak was subjected to MS libraries for identification.

4.6 Biological activity evaluation

Antibacterial and Antifungal potentialities of medicinal plants

parts such as Leaf,stem and roothave been well noted for some of the

medicinal plants for the past two decades41

. Medicinal plants and their

substances are luxuriant antibacterial compounds can fight against

bacterial ailments42

. The plant is purgative, destroy worms, bitter cure

itching leprosy, different skin disorders, bilious fever and inflammation.

Argemone mexicana linn. (Papaveraceae), commonly known as Prickly

Poppy found in Mexico and now has widely naturalised in the India,

Bangladesh and Many other countries. It occurs as wasteland weed in

almost every part of India43-44

.In Mexico, the seeds have been used as an

antidote to snake poisoning45

. In India, the smoke of the seeds is used to

relieve toothache. The fresh yellow, milky seed extract contains protein-

dissolving substances effective in the treatment of diuretic, anti-

inflammatory, malarial fever, leprosy, scorpion sting, warts, cold sores,

wound healing, skin diseases, itches, jaundice and an antidote to various

poisons46-48

.The seeds are purgative and sedative useful in skin diseases

and leu coderma49

.The fresh juice of the leaves and the latex both are


95

reported to be used externally as a disinfectant for open wounds and

cuts50

.The seeds are used in cough, asthma, nauseant, emetic, demulcent,

expectorant and laxative. The root is useful in anthelminticand treatment

of antibacterial, antimicrobial, cytotoxicity, wound healing, antioxidant

and antifungal properties51-52

.

The present research work is evaluate the antibacterial and

antifungal potentiality of Argemone mexicana linn. leaf, root and stem

against two fungi(Aspergillus niger and Candida albicans), two gram

positive(Staphylococcus aureus and Bacillus subtilis) and two gram

negative(Escherichia coli and Pseudomonas aeruginosa) bacteria for

use as a antimicrobial agent in industries.

Extraction of plant material for Biological evaluation

Fresh plant parts viz. leaf,stem and root were collected and

cleaned separately using normal and distilled water. The leaf, stem and

root were dried at room temperature 35 ± 10 C. The dried leaf, stem and

root part were grind well into a fine coarse powder in grinder. The

powder was stored in airtight bottles at room temperature53

. 10gm of

dried leaf, stem and root powder was taken in round bottom flask for

extracting the phytochemicals in 250ml of ethyl acetate, methanol and

water separately for 24h. After the completion of extraction each extract

were filter through whatman filter paper no.1.All filtrate obtained were

concentrated on water bath. Each concentrated sticky mass was stored in

Dimethyl Sulfoxide (DMSO) at -40C till use. Then the extracts were

used for antimicrobial potentiality.


96

4.6.1 Antimicrobial Assay

Antifungal and antibacterial activity were screened of Argemone

mexicana linn. leaf, stem and root extract by agar well diffusion

method54

.Two fungi (Aspergillus niger, Candidaalbicans) and four

bacteria, two Gram positive (Staphylococcus aureus, Bacillus subtilis)

and two Gram negative (Escherichia coli, Pseudomonas aeruginosa)

were used for current research work. The fungi strain were grown on

Czepek dox agar and bacterial strain were grown on Muller hilton agar

medium. Wells of 6mm diameters were expurgated from the medium

and extracted 50 µl solution was added (100 mg/ml) into the well. Next

day zoneof inhibition was measured in mm. Fluconazole and

Ciprofloxacin (10µg/ml) were taken as a control.

4.6.2 DPPH free radical scavenging assay

The free radical scavenging activity of the methanolic extracts of

leaf, stem and root of Argemone mexicana linn. were estimated by using

DPPH method55

. Solution of DPPH (0.1 mM) in methanol was prepared

by dissolving 1.9 mg of DPPH in methanol and volume was made up to

100 ml with methanol. The solution was kept in dark for 30 min to

complete the reaction. 1 ml of DPPH solution was added to 1 ml of

different (250, 500, 750 and 1000 µg/ml) concentrations of extract and

allowed to stand at room temperature for 30 min. The mixture was

measured at 517nm by spectrophotometer (UV-1800, UV-VIS

spectrophotometer, Shimadzu). The free radical scavenging activity was

calculated as using below formula and Ascorbic acid was used as

standard.

Inhibition (%) = (A0 – Ai / A0) × 100

where, A0 is the absorbance of standard and Ai is the absorbance

of sample. A standard of ascorbic acid was measured using same

concentrations as that of extracts56-57

.


97

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