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CCCChapter hapter hapter hapter 5555

Purification and CharacterisationPurification and CharacterisationPurification and CharacterisationPurification and Characterisation

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Chapter 5 Purification and Characterisation

Nowadays, the traditional knowledge about the therapeutic potential of plants has forced

scientists from all the branches of science such as molecular biology, molecular pharmacology,

biomedicine to lay emphasis on extensive research on the biologically active products from

plants (Sampietro et al. 2009). Biologically active natural products are present in complex

mixtures. They are novel compounds and their isolation therefore becomes necessary.

Isolation and identification of bioactive compounds present in a crude extract sample

serve as the building block for the development of new type of therapeutics with new

mechanisms of action with more potent way to treat various human ailments (Lee et al. 2000).

But isolation and identification of bioactive compounds presents a considerable challenge.

Therefore, sophistication is required for this purpose. More recently, achievements in separation

sciences propose much better solutions for the separation of the complex mixtures than it was

earlier (Servili et al. 1999). Also, the ease with which the active principal can be isolated and

purified depends much on the structure, stability and quantity of the compound. Therefore, by

detailing the structure of the active principal chemically, one may arrive at its significant

contribution for social welfare.

For purification process the sample is subjected to solvents of varied polarity and

chemically characterized by the different spectroscopic experiments (Wang et al. 2003; Jyothy et

al. 2011; Sasidharan et al. 2011). By analyzing the data critically, it is possible to determine its

molecular weight. The molecular interpretation helps to deduce the structure of the compound.

Although many purified compounds are reported with therapeutic potential every year,

rarely a few of them can actually make their way upto product development and pass the scrutiny

of the United States Food and Drug Administration for commercialization.

This chapter deals with purification and chemical chracterisation of the active principal

from Betel leaf stalk (BLS).

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Methodology

Purification of active principal: Purification of the active principal was done following

paper, silica gel column, thin layer separation processes.

Paper chromatography: In the first step, the preparative paper chromatographic technique

was employed for separation of the active principal. The crude extract of BLS was spotted along

a horizontal line on the Whatman No. 1 filter paper (25x15cm) at room temperature. After proper

run of the solvent system, the paper was taken out of the chromatographic chamber and dried it

properly. Activity of each band was detected by bioassay method. In this technique the paper

chromatogram was cut vertically along the length of the paper and further made into pieces. The

pieces were overlayed in the agar plate, previously inoculated with the test organisms

(106CFU/ml) and incubated overnight at 37oC. Next day the results were observed as inhibition

zones around the pieces of paper. The active band was eluted in methanol. The eluted samples

were collected from different chromatogram and pulled together as partially purified compound

and concentrated. Retention factor (Rf) was calculated.

Rf

Silica gel column: The partially purified compound was subjected to column (10x2cm)

packed with 60-120 mesh silica gel washed with petroleum ether. Care was taken to avoid air

bubbles into the column. The silica gel was mixed with the partially purified sample (w/v) and

loaded on the top of the packed column. The adsorbed compound was eluted with petroleum

ether and ethyl acetate in the ratio of 10:1under the flow rate of 3ml/min. Fraction were pooled

together read through spectrophotometer between 200-700nm and also subjected to biological

activity. The targeted fractions were collected in pre-weighed container and concentrated and

was loaded into TLC (silica gel G) plates for further purifications.

Thin layer chromatography: The targeted fraction collected from column

chromatography was subjected to thin layer chromatography for further purification. The TLC

plates were prepared on glass slides and the thickness of the silica gel was 0.1mm and were

activated before use. Sample was carefully spotted on the plates and immeadiately dried with

Distance travelled by the active fraction

Distance travelled by the solvent

=

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hair drier. Different solvent systems were used. Ultimately the desired solvent system

chloroform: methanol 9:1 was used to get the desired spot. The developed chromatogram were

subjected to UV light and Iodine vapour for the detection of any other spot. After analysis the

silica gel was scraped off from the desired location from a reference plate and eluted with

methanol, centrifuged at 8000xg for 30 minutes and analysed through spectrophotometer.

Retention factor (Rf) was calculated.

Physicochemical characterization:

Detection of the chemical nature of the active principal:

For detection of the compound, the sample was loaded to TLC plate and run in the

desired solvent system and was sprayed with 4% (v/v) Folin–Ciocalteu (FC) reagent.

UV-analysis:

The spots obtained from TLC plates were scraped off and dissolved in eluting solvent

and centrifuged. It was analysed by UV- Vis scanning spectrophotometer. UV analysis was done

with a Shimadzu UV-Vis scanning spectrophotometer (Model No. UV-2010 PC)

Infrared analysis

The sample was kept in a vacuum dessicator over solid KOH for 48 hrs and then IR

spectral analysis was done with 1 mg sample by FTIR (Jasco Fourier Transfer Infrared

Spectrophotometer, Model No. FT/IR-460).

NMR

NMR spectral data were obtained by Bruker 400 MHz. The sample, whose spectra was to

be drawn, firstly was dried in a high vacuum pump thoroughly so that no organic solvent or

water remains. After that the sample was dissolved in about 1.5 ml of CDCl3 solvent and

transferred to a NMR standard tube for further assessment.

.

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Results:

Paper chromatography

In the preparative paper chromatographic after proper run of the different solvent system

(Table1), the paper was taken out of the chromatographic chamber and dried properly. Two

visible bands appeared band 4 (Rf 0.76) and band 6 (Rf 0.91) with the solvent system benzene:

hexane in the ratio 3:4. While the presence of any other band if present (1, 2, 3, 5) was detected

by bioassay method. The cut pieces of the paper (1-6) were overlayed in the agar plate,

previously inoculated with the test organism (106 CFU/ml) and incubated overnight at 37oC.

Next day the results were observed as inhibition zones around the pieces of paper. Band 4

showed the antibacterial activity (Fig 1). The active compound was eluted in methanol. The

eluted samples were collected from different chromatogram and pulled together as partially

purified compound and concentrated.

Band Solvent system Ratio Rf

No seperation Ethyl acetate: Hexane 1;1 -

No seperation Benzene: Hexane 3:1 -

Six bands Benzene: Hexane 3:4 0.45, 0.53, 0.67, 0.76,

0.84, 0.91

No seperation Ethyl acetate:Benzene 2:3 -

Table 1: Different solvent systems used and the retention factors of the bands

Chapter 5

Silica Gel Column

The semipurified compound so obtained from paper

column chromatography. The concentrate was passed through the silica gel 60

(Fig 2). Petroleum ether: Ethyl acetate in the ratio of 10:1 was used as the eluting solvent. The

solvent fractions eluted were collected in pre

3ml/min. The fractions (1,

check its purity and also the fraction

found to be bioactive. The target fraction was then subjected to TLC to check its purity.

Fig 1: Shows the paper chromatogram and the bioassay of the band 1

6

55

4

3

2

1

5

Purification

The semipurified compound so obtained from paper chromatography

column chromatography. The concentrate was passed through the silica gel 60

). Petroleum ether: Ethyl acetate in the ratio of 10:1 was used as the eluting solvent. The

solvent fractions eluted were collected in pre-weighed container. The rate of the elution was

3ml/min. The fractions (1, 2, 3) collected were subjected to spectral analysis (200

its purity and also the fractions were subjected to antibacterial testing.

active. The target fraction was then subjected to TLC to check its purity.

Shows the paper chromatogram and the bioassay of the band 1

4

5

6

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Purification and Characterisation

chromatography was subjected to

column chromatography. The concentrate was passed through the silica gel 60-120 mesh column

). Petroleum ether: Ethyl acetate in the ratio of 10:1 was used as the eluting solvent. The

eighed container. The rate of the elution was

3) collected were subjected to spectral analysis (200-700nm) to

were subjected to antibacterial testing. Fraction 3 was

active. The target fraction was then subjected to TLC to check its purity.

Shows the paper chromatogram and the bioassay of the band 1

1

2

3

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3

2

2

1

31

12

3

Thin layer chromatography

The fraction 3 as found positive was subjected to TLC which were exposed to UV light

and iodine vapour for detection of any other spot. But no other spot developed other than at Rf

0.80 thus confirming its purity (Fig 3). The purified compound was then used for

physicochemical analysis.

Iodine vapour 254 nm

366 nm

356nm 254nm Iodine vapour

Fig 2: Column chromatography and the bioassay of the fractions

Fig 3: TLC plate exposed to UV analysis and Iodine vapour

Chapter 5

Physicochemical analysis

Detection of the nature of the compound

For detection of

desired solvent system and was sprayed with

developed a purple spot (R

the active principal as phenolic

Fig 4: Chromatogram for the detection of chemical nature

Purification

Physicochemical analysis

Detection of the nature of the compound

For detection of the compound, the sample was loaded to TLC plate and run in the

and was sprayed with 4% (v/v) Folin–Ciocalteu (FC) reagent

rple spot (Rf 0.80) against a purple colour background identifying th

as phenolic (Fig 4).

Chromatogram for the detection of chemical nature

Spot appeared

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loaded to TLC plate and run in the

Ciocalteu (FC) reagent. The reagent

against a purple colour background identifying the nature of

Chromatogram for the detection of chemical nature of the spot

Spot appeared

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UV analysis

The UV scanning was performed in the range between 200-300nm wavelength. Fig 5a

represents the absorption spectrum of the crude sample. The purified compound showed a sharp

peak near 270nm (Fig 5b).

3 0 0 4 0 0 5 0 0

0 .0

0 .5

1 .0

1 .5

2 .0

2 .5

3 .0

3 .5

Abs

orba

nce

(a.u

.)

W a v e le n g t h ( n m )

Fig 5a: Visible absorption spectra of crude sample

Fig 5b: Visible absorption spectra of purified sample

Chapter 5

IR analysis of the purified compound

The sample was kept in a vacuum dessicator over solid KOH for 48 hrs and then IR

spectral analysis was done w

The IR (Fig 6) spectrum of the purified compound showed peaks at number: 3463 – For OH stretchingtrans vinyl C=C double bond,1211 – C-O bending.

Fig 6

Purification

IR analysis of the purified compound:

sample was kept in a vacuum dessicator over solid KOH for 48 hrs and then IR

spectral analysis was done with 1 mg sample.

The IR (Fig 6) spectrum of the purified compound showed peaks at OH stretching, 3064- For aromatic C-H stretching,1616

C=C double bond, 1600 – For aromatic ring C=C, 1506

Fig 6: IR analysis of the purified compound

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sample was kept in a vacuum dessicator over solid KOH for 48 hrs and then IR

The IR (Fig 6) spectrum of the purified compound showed peaks at following wave H stretching,1616 – Strong peak for

1506 - Aromatic C=C bending,

Chapter 5

NMR analysis:

1H NMR (400 MHz, CDCl

3.90 (s, 3H); 3.42 (d, J = 5.6 Hz, 2H)

13C NMR (100 MHz, CDCl

39.6.T (Fig 7b).

Purification

H NMR (400 MHz, CDCl3): δ 6.99-6.95 (m, 1H); 6.79 (s, 2H); 6.06 ( s, 1H); 5.21

= 5.6 Hz, 2H) (Fig 7a)

CDCl3): δ 146.3, 143.6, 137.6, 131.6, 120.9, 115.2, 114.2, 111.0, 55.5,

Fig 7a: 1H NMR peak of the purified compound

Fig 7b: 13C NMR peak of the purified compound

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6.95 (m, 1H); 6.79 (s, 2H); 6.06 ( s, 1H); 5.21-5.16 (m, 2H),

146.3, 143.6, 137.6, 131.6, 120.9, 115.2, 114.2, 111.0, 55.5,

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The probable molecular formula C10H12O2 and the molecular structure (Fig

7c).Therefore by IR and NMR data it can be concluded that the compound is eugenol as reported

(Yong 2009).

O

OH

Fig 7c: The molecular structure of the active principal

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Discussion:

A very large number of phenolic compounds occur in nature; therefore no single

procedure may be valid for their separation. Each class of phenolic compound, in relation to their

chemical structure, spectral characteristics, polarity or glycosidic linkages, needs an appropriate

analytical separation and identification technique (Miniati 2007). Analytical separation

techniques for phenolic compounds have passed from classic paper, thin layer chromatography

(TLC) and gas chromatography (GC) to HPLC coupled with spectrometry to HPLC coupled with

Mass spectrometry (MS) and nuclear magnetic resonance spectrometry (NMR) according to

advances in technology in the recent years (Thorngate 2006).

Spectral properties are generally utilized for the identification of the phenolics. For

chromatographic analyses, usually the simplest way is the use of a UV-Vis detector. Almost all

phenolic compounds absorb with a maximum in the UV region at 270-280 nm. This is very

much true with the findings of Larrauri et al. (1997). Additional spectra/shifts occur in the 220-

230 nm band e.g., for catechins, hydroxytyrosol or in the 330 region as in the case of

hydroxycinnamic acids and flavonoids. Anthocyanins alone absorb in the visible region showing

a maximum at 520-550 nm (Miniati 2007).

To define them fully, purification is very important and largely depends on the

physicochemical properties as discussed. Here in this investigation purification was done using

paper chromatography, column chromatography and thin layer chromatography. In paper

chromatography the solvent system benzene:hexane in the ratio 3:4 was taken. Then the

antibacterial activity of each band was detected by bioassay method. The active band was

subjected to column chromatography which yielded purified compound as determined both by

spectral analysis and TLC. Soberon et al. (2009) used paper chromatography, LH-20 column

chromatography and reverse phase HPLC to purify phenolics from Tripodanthus acutifolius

leaves. In some cases workers use only single method to purify phenolics. Lin et al. (2013) used

silica gel column to purify phenolics from roots of Piper betle.

Further information regarding the characterisation, can be obtained by collecting

the IR spectra MS spectra and the NMR spectra of the compound. It is particularly evident in the

case when the phenolic compounds involved may be numerous and unknown or partly unknown

(Matern and Magera 2003).

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ReferenceReferenceReferenceReferencessss::::

Jothy S L, Zakaria Z, Chen Y, Lau Y L, Latha L Y, Shin L N, Sasidharan S (2011). Bioassay-directed isolation of active compounds with antiyeast activity from a Cassia fistula seed extract. Molecules. 16: 7583-7592.

Larrauri J A, Ruperez P, Saura-Calixto F (1997). Effect of drying temperature on the stability of polyphenols and antioxidant activity of red grape pomace peels. Journal of Agricultural and Food Chemistry. 45:1390–1393.

Lee M J, Prabhu S, Meng X, Li C, Yang C S (2000). An improved method for the determination of green and black tea polyphenols in biomatrices by high performance liquid chromatography with coulometric array detection. Analytical Biochemistry. 279: 164-169.

Lin C F, Hwang T L, Chien C C, Tu H Y, Lay H L (2013). A new hydroxychavicol dimer from the roots of Piper betle . Molecules.18: 2563-2570.

Matern D, Magera M J (2003) Mass spectrometry methods for metabolic and health assessment. Journal of Nutrition. 131: 1615S- 1620S.

Miniati E (2007). Assessment of phenolic compounds in biological samples. Ann Ist Super Sanità. 43: 362-368.

Sampietro D A, Catalan C A N, Vattuone M A (2009). Isolation, identification and characterization of allelochemicals/natural products. Science publishers. Taylor and Francis group. CRC press.pp 1-555.

Sasidharan S, Chen Y, Saravanan D, Sundram K M, Yoga Latha L (2011). Extraction, isolation and characterization of bioactive compounds from plants' extracts. African Journal of Traditional, Complementary and Alternative Medicine. 8: 1–10.

Servili M, Baldioli M, Miniati E, Selvaggini R, Macchioni A, Montedoro G F (1999 ). High-Performance liquid chromatography evaluation of phenols in olive fruit, virgin olive oil, vegetation waters, pomace and 1D- and 2D-Nuclear Magnetic Resonance characterization. Journal of the American Oil Chemists’ Society.76: 873-882.

Soberón J R, Sgariglia M A, Sampietro D A, Quiroga E N, Sierra M G, Vattuone M A (2009). Purification and identification of antibacterial phenolics from Tripodanthus acutifolius leaves. Journal of Applied Microbiology.108: 1757-1768.

Thorngate J H III (2006). Methods for analyzing phenolics in research. American Journal of Enology and Viticulture. 57: 269-279.

Wang X, Kapoor V, Smythe G A (2003). Extraction and chromatography-mass spectrometric analysis of the active principles from selected Chinese herbs and other medicinal plants. American Journal of Chinese Medicine. 31: 927-944.

Yong P K (2009) Drying and Solid-Liquid Extraction of Hydroxychavicol and Eugenol from Betel Leaves (Piper Betle L.). PhD thesis, Universiti Putra Malaysia.

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