Chapter 5 Isolation, Purification &...

Phytochemical and Pharmacological Profiling of Ficus glomerata Roxb

Department of Biotechnology, Gulbarga University, Kalaburagi 19

Isolation, Purification &

Characterization

Chapter 5


Department of Biotechnology, Gulbarga University, Kalaburagi Page 120

5. ISOLATION, PURIFICATION AND CHARACTERIZATION OF

FLAVONOID FROM FRUITS OF FICUS GLOMERATA ROXB

INTRODUCTION

Identification of bioactive natural compounds for their anti-bacterial, anti-

fungal and anti-cancer capacity by using bioprospecting is one of the important

assignment (Hiil, 2010; Demain and Sanche, 2009). The process of bio prospecting

can be compared to challenge of ‘‘Needle in the Haystack’’ in many facets. As we are

expecting a single successful compound as an outcome from large number of test

extracts (Ebada, 2008).

Repeated research on less effective compounds or earlier identified ones

results in wastage of resources and time. The development of rational screening

methodologies helps to avoid wastage of resources. (Snowden and Green, 2008;

Klinkenberg, 2011).

Influence of different extraction techniques, extraction solvents on the natural

antioxidant compounds was reported by many researchers. Plant materials selected

used for extraction majorly decides the efficiency of solvents used for extraction and

method of extraction employed. Extraction of phenolic compounds from fresh plant

material can be commonly achieved by using solvents, such as methanol, ethanol,

acetone, propanol and ethyl acetate (Grigonisa et al., 2005; Durling et al., 2007;

Alothman etal., 2009).

Variation in total phenolic content (±25% variation) and total antioxidant

capacity (up to 30% variation) of fresh fruits and vegetables can be observed with

varying properties of extracting solvent system. Polarity of solvent plays an important

role in the solubility of phenolics in extraction by using solvent system. Generally

phenolics are found to be with better soluble nature in high polarity solvents. Polar



alcohol based solvents have shown extractive yield up to 22.8%, which is highest

among other solvents. Extraction rate of ethanol can be improved by addition of water

to it. Solvents with more water content results in the lower concentrations of

phenolics and increase yield of other contaminant compounds(Spigno et al., 2009;

Lolita et al., 2012).

Since very long period, plants have become the most important therapeutic

sources available to mankind. From a phytochemical and pharmacological view point

only a relative less percentage of plant species are studied. The plant kingdom is still

remain as unexploited treasure of new molecules with promising remedial concern

(Hostettmann, 1998).

Pharmacognosy research has confirmed that plants can provide a effective

bioactive products. Submitting plant extracts to hyphenated chemical inspection

techniques such as liquid chromatography and mass spectroscopy. These techniques

rapidly provides a number of detailed structural information in order to discover new

bioactive compounds present in plant sources which could become new leads or new

drugs, this shows the way to a partial or a complete on-line structural identification of

interested natural products (Wolfender, 2013; Payal et al., 2013).

Herbs, vegetable and fruits are commonly contain the phenolics as universally

distributed component in them. Phenolics are one of the most diverse groups of

phytochemicals. Approximately 8000 phenolics are have been isolated from natural

sources. These compounds contain at least one phenol moiety (an aromatic ring with

one or more hydroxyl substituent) (Robbins, 2003).

The epidemiological, clinical and laboratory studies assumes that the risk of

chronic diseases such as coronary heart disease and cancer may be reduced by the

consumption of fruits, vegetables and herbs (Middleton et al., 2000; Hertog, 1995).



Antioxidant nature of phenolics supports the beneficial properties of them (Rice-

Evans et al., 1996; Hollman et al., 1998).

It is critical to estimate the exact levels of phenolic compounds present in

plants. Diverse nature among structure of phenolics and their solubility, structural

diversity are important challenges in the extraction and analytical procedures of

phenolics. Preparation of sample in analytical measurement contributes around 30%

of the total errors (Majors, 1995).

Extraction of desired product from target plant material is the first step in any

analytical studies. A variety of parameters such as plant part, particle size, solvent

polarity, extraction procedure and extraction conditions will influence the extraction

of phenolic compounds from plant material. The impact of the extraction of phenolic

compounds on the analysis has often been overlooked as substantial variations found

in the extraction procedures and solvents are documented in the recent literature

(Naczk and Shahidi, 2006).

Several solvents such as methanol, ethanol, acetone, water, ethyl acetate and,

to a lesser extent, propanol, dimethyl formamide, dimethyl sulfoxide and their

combinations have been used for the extraction of different classes of phenolic

compounds (Antolovich et al., 2000; Parejo et al., 2004).

The reasoning for the selection of a particular extraction solvent or solvent

mixture in addition to the extraction procedure is frequently not well studied or not

clearly documented. (Luthria et al., 2006).

Plants produce a diverse range of bioactive molecules making them as rich

source of different types of medicines. Various techniques are employed for their

investigation, which includes bioassays for chemical screening and their evaluation

for proving the biological activities.



Isolation of pure pharmaceutically active constituents from plants remains a

long tedious process. Chemical screening is performed to target isolation of new or

useful type of constituents having potential activities. This procedure enables

recognition of known metabolites present in extracts in the earliest stages of

separation and thus there are economically very important. To characterize the

bioactive compounds several techniques are employed among which chromatographic

techniques were extensively applied.

Phenolic compounds are large and diverse group of molecules, which includes

different families of secondary aromatic compounds in plants (Dai and Mumper,

2011). Around 8000 chemical structures of plant phenolics are studied until now.

These compounds are simple, having low molecular weight, consisting of single

aromatic ring to large and complex-polyphenols (Carlo et al., 1999).

Plant phenolic compounds are primarily derived from phenyl - propanoid and

acetate pathway and commonly found in conjugation with sugars and organic acids.

Phenolics are classified into two groups: Flavonoids and Non- Flavonoids (Mabry et

al., 1970).

Flavonoids

Flavonoids are the largest groups of phenols. More than 5000 chemical

structures of flavonoids have been identified till date.(Middleton and Kandaswami,

1986). The basic structure of flavonoid is comprised of C6-C3-C6 structure forming

the A, B and C rings respectively. These are group of structurally related compounds

with a chromane-type skeleton having phenyl substituent in C2-C3 position. The basic

structural feature of flavonoid is 2-phenyl-benzo-γ-pyrane nucleus consisting of two

benzene rings (A and B) linked through a heterocyclic pyran ring (C) (Sandha et al.,



2011). Based on the backbone structure flavonoids are classified into subgroups:

flavanone, flavone, isoflavone, flavanol and flavonol (Ren et al., 2003).

Biosynthesis of Flavonoids

Two biochemical pathways shikimic acid pathway and acylpolymalonate

pathway together results the biosynthesis of flavonoids. Phenylpropane a derivative of

cinnamic acid, synthesized from shikimic acid, acts as the precursor in a polyketide

synthesis, to this cinnamic acid extra three acetate residues are added into its structure

followed by ring closure. Plants are capable to synthesize various classes of

flavonoids through successive hydroxylations and reductions (Sameulsson, 1993;

Giulia and Nicola, 1999).

Pharmacokinetics of Flavonoids

Due to the limited data available about the extent of absorption of flavonoids it

remains a clearly unsolved question particularly in humans. Flavonoids present in

food are assumed to be non- absorbable as they are attached with sugar molecules as

P-glycosides except catechin. Aglycones which are the flavonoids free from sugar

molecules are thought to be capable of passing through the gut wall. Hydrolysis of

glycone results in the Aglycones i.e. hydrolysis detaches the sugar moiety from

flavonoid and makes it as sugar free flavonoids. 13-glycosidic bonds of flavonoids are

degraded only by microorganism of colon at the same time of degradation of dietary

flavonoids. This is because no enzymes are capable of splitting the bonds present in

flavonoids or the phenolics secrets into the gut. After absorption, the subsequent

metabolism of flavonoids are quite well known from animal studies (Hacket, 1986).

Metabolisms of absorbed flavonoids largely occur in liver which is responsible

for this function. Kidney and intestinal wall are the secondary sites of the flavonoid

metabolism. However, this depends on the various sources of flavonoids: those found



in citrus fruits are poorly metabolized by the intestinal microflora, quercetin is not

absorbed in humans, rutin is poorly absorbed, whereas procyanidolignanes are readily

absorbed in mice. Flavonoids metabolized by intestinal bacteria are converted to

hormone-like compounds with a weak estrogenic and antifecondative activities.

Hydroxy groups are conjugated with glucuronic acid or sulphate and, in addition,

methylation may occur. The glucuronides and sulphates are excreted in the bile

(Hacket, 1986).

Flavonoids, once absorbed may influence various biological functions

including protein synthesis, cell proliferation, differentiation and angiogenesis,

making them beneficial in a variety of human disorders (Giulia et al., 1999).

Biological activities of Flavonoids

Flavonoids have been reported to exert wide range of biological activities.

These include anti-inflammatory, antibacterial, antiviral, antiallergic, cytotoxic

antitumor and vasodilatory actions (Harborne and Williams, 2000) In addition,

flavonoids are known to inhibit lipid-peroxidation, platelet aggregation, capillary

permeability and fragility, cyclo-oxygenase and lipoxygenase enzyme activities (Li et

al., 2007) Flavonoids are capable of modulating the activity of enzymes and affect the

behavior of many cell systems and exerting beneficial effects on body(Gomes et al.,

2008).



MATERIALS AND METHODS

Plant material

Fresh ripe fruits of Ficus glomerata Roxb (Family Moraceae) are collected

from the Gulbarga University Campus. Fruits are washed thoroughly, dried in shade

and grounded to powder and sieved. The fruit powder was stored at room temperature

in airtight container until it is used for extraction.

Extraction

Fruit powder was subjected to hot extraction by using soxhlet extractor. Fruit

powder was defatted with petroleum ether for three times. Further, extracted with

methanol, and resulted methanolic extract was centrifuged at 10,000 rpm for

15minutes; supernatant was collected, dried and used for the purification (Thangavel

and Gupta, 2010).

Qualitative examination of Flavonoids

Shinoda Test: Three pieces of magnesium chips was added to the filtrate followed by

few drops of concentrated hydrochloric acid. A pink, orange, or red to purple

colouration indicates the presence of flavonoids.

Ferric chloride test: To 2 ml of the filtrate, few drops of 10% ferric chloride solution

were added. A green-blue or violet colouration developed indicating the presence of a

phenolic hydroxyl group.

Separation of flavonoids by thin layer chromatography

Sample: Dried methanol extract dissolved in distil water was used for thin layer

chromatographic study.

Loading: Approximately 20µl of the extract was loaded on the activated pre coated

chromatographic plate (Merck, India) with help of capillary tube.



Development: The loaded plate was kept in a saturated chromatographic chamber

containing Toluene –Chloroform – Acetic acid mixture as mobile phase with ratio

equal to (4: 5: 1). After running of about 15 cm the plate was removed from the

chromatographic chamber and dried.

Detection: The developed chromatographic plate was observed under (UV 254 nm)

chamber. Thus, the colour and Rf values of fluorescent bands obtained recorded. The

values of rate of flow (Rf) are calculated for separated flavonoid.

Separation of flavonoids by silica gel column chromatography

Preparation of column

A clean and dry 500 ml capacity column of about 60 cm length was filled

with the slurry of Silica gel-H of mesh size 60-120µ (Himedia, Mumbia) to 45 cm

portion using hexane. Due care was taken to avoid air bubbles while packing the

cloumn with a stationary phase. Then the column was run through twice with the

solvent system containing hexane to make the column air tight and compact one.

Loading

Column chromatography technique was employed for purification of

methanolic extract. A glass column (Merck, India) with 50cm X 2.5cm dimensions

was cleanly washed with distill water and followed by eluting solvent. Column was

packed thoroughly with the Silica gel (60 - 120; Merck, India). 5g of methanol extract

of Ficus glomerata was ground well with a small amount of silica gel and loaded on

to the top of the column. Mixture of methanol-water in various ratios were used as

eluting solvent.

Collection of fraction

Totally 35 fractions with each 100ml were collected, as they came off the

column in a series of conical flasks (100 ml). Thin layer chromatography was done



with collected fractions. Based on the results similar fractions are pooled together and

concentrated in vacuo to isolate the active principle.

Characterization

Characterization of compound was carried out by following techniques.

FTIR spectroscopy

Fourrier–transformation – (FT) – IR spectrum of flavonoid compound isolated

was recorded by using (FT–IR– Bruker, Japan) spectrophotometer for studying the

functional groups. The sample compound was fixed in potassium bromide (KBr) disc

then the spectrum was measured at wave number ranged from (600 – 4000 cm-1). The

various bands are identified related to their wave numbers.

NMR Spectroscopy

5mg-purified compound was dissolved in di methyl sulphoxide and used for

Nuclear magnetic resonance spectroscopic analysis. Sophisticated multinuclear

FTNMR Spectrometer model Avance II (Bruker) is used for the study, with a

cryomagnet of field strength 9.4 T and 1H frequency 400 MHz. The chemical shifts

expressed in ppm are recorded.

MS spectrometry

LC MS instrument (Waters Micromass Q-Tof Micro ) having quadra pole time

of flight, mass equipped with electrospray ionization (ESI) and atmospheric pressure

chemical ionization (APcI) sources having mass Range of 4000 amu in quadruple and

20000 amu in ToF is used to detect the molecular mass and mass by charge ratio of

compound dissolved in DMSO.



RESULTS

Qualitative examination of flavonoid (Table 5.1)

Two preliminary qualitative tests – Shinoda test and Ferric chloride test are

performed to screen the presence of flavonoids in methanol extract of Ficus

glomerata fruits. Appearance of pink colour in Shinoda test confirms the presence of

flavonoids. Phenolic hydroxyl group present in compound was confirmed by the

formation of violet red colour in ferric chloride test.

Thin layer chromatography of Flavonoid (Table 5.2, Fig. 5.1)

Developed thin layer chromatography plates were subjected for iodine vapour

test and appearance of brown colour spots on thin layer chromatography plates when

placed in iodine chamber confirms the presence of flavonoids, and in ferric chloride

test appearance of yellow colour spot after spraying (1%) ethanol ferric chloride

solution also confirms the presence of flavonoids.

FTIR Spectroscopy (Fig 5.2)

Furrier transformation – infra – red (FT – IR) spectrum result of isolated

flavonoid is shown in Fig.5.2. Absorption peaks belonging to functional and / or

structural groups were recorded. Appearance of broad band at 3399.2 cm-1 represents

presence of stretching vibration of phenolic groups containing hydrogen bonding. The

weak band at 2937.30 cm-1 indicates the stretching vibration of aromatic (C-H) group

and also the medium band at 2034.59 cm-1 represents the bending vibration of

aliphatic (C-H) group.

NMR Spectroscopy (Fig 5.3, Table 5.3)

The 1H NMR spectrum of the compound as in Fig. 5.3 demonstrated the

signals of aromatic, pyron ring and prenyl group. The 1H NMR spectrum of the

compound displayed signals at d 6.53 assignable to a phenolic hydroxyl group at C-



40 and a singlet for three protons at d 3.83 was ascribed to a methoxy group at C-7.

The 1H NMR spectrum further displayed a one proton singlet at d 5.35 was attributed

to H-8 proton. The spectral details of the purified compound is tabulated in Table 5.3.

At base 2.00 a chemical shift of 2.0 corresponding to OH group indicates the presence

of alcohol group. Chemical shift of 3.75 at base 3.00 corresponding to OH group

shows the presence of C-OH group. Presence of 1-benzene moiety was confirmed by

a representing node with a chemical shift of 3.36 at base 4.00


Department of Biotechnology, Gulbarga University, Kalaburagi 1

Mass spectrometry (Fig 5.4)

The mass spectrometry data revealed that the mass of purified compound is

353.06 (Fig.5.4), which is the closest value to the flavonoid myricetin (350.03). The

molecular formula is C15H10O10.

Elemental analysis of purified compound found to be:

C- 51.44

H- 2.88

O- 45.68.

From the entire above chemical and spectral test evidences and previous

studies on Ficus species by Li (1998) used to identify the isolated compound as a

flavonol, dihydroxy myricetin [3, 5 dihydroperoxy-7- hydroxyl-2-(3,4,5-

trihydroxyphenyl)-4H-chromen-4-one].



DISCUSSION

When a plant has been identified as a source for new products, the bioactive

compounds present in the relevant fractions in the so called “Bioassay guided

isolation”. In this process the plants are successively extracted with solvents of

increasing polarity and tested by a range of bioassays relevant to the activity of the

compounds (Gedara and Zaghloul, 2005).

It’s a long and tedious process to isolate pure, pharmacologically active

constituents from plants. Thus, it is necessary to have the good methods available

which eliminate unnecessary separation procedures. Chemical screening is thus

performed to allow localization and targeted isolation of new or useful constituents

with potential activities. This procedure enables in recognition of known metabolites

present in extracts or at the earliest stages of separation and is thus economically very

important (Kumara, 2001).

Sample preparation and removal of unwanted substances for quantification of

phenolics is an important method. The extraction procedure is the primary

determinant for the separation and recovery of phenolics. Extraction is generally

influenced by the sample nature, particle size, solvent type as well as extraction

techniques employed. Soxhlet, heated reflux extraction and maceration are the

conventional procedures frequently applied to recover phenolics from solid samples.

The Soxhlet and heated reflux methods are normally performed at 90 °C for

several hours while maceration is performed over days at ambient temperature. These

methods are simple, require relatively cheap apparatus and result in adequately high

phenolic extraction rates. Castro-Vargasa et al., (2010) reported that the highest total

phenolic content of Guava seed extract was achieved by using Soxhlet extraction



techniques. In another study, the phenolic compounds present in seeds of three wild

grapevines were successfully extracted by using Soxhlet technique.

Flavonoids are highly bioactive compounds found in both edible and non-

edible plants. They are often extracted with methanol, ethanol, acetone, water or

mixtures of these solvents using heated reflux extraction methods (Routray and Orsat,

2012).

Biesaga (2011) has extracted flavonoids in maize samples using heated reflux,

microwave-assisted extraction, ultrasonic-assisted extraction and maceration and

compared to the stability of the extracted compounds. The highest stability of the

extracted flavonoids in methanol-water (60:40 v/v) was found in compounds extracted

with traditional heated reflux in a water bath.

In our study we have used a combinatorial solvent methanol –water in a

proportion of 70:30 for extraction of phenolics from fruits of Ficus glomerata. High

extractive value was achieved using this solvent system.

Paper chromatography and thin-layer chromatography are the two partitioning

techniques employed to separate phenolics in foods (Naczk and Shahidi, 2006) paper

chromatography is a simple method and less utilized compared to HPLC and gas

chromatography. In other studies, flavonoids, phenolic acids and glycoflavones have

been separated from three green leafy vegetables using paper chromatography

(Nambiar, 2010).

Thin layer chromatography is more powerful technique than Paper

chromatography to analyze phenolics, especially in crude plant extracts. Phenolics in

crude plant extracts can be separated by a number of thin layer chromatography

techniques, which are cheap and provide a multiple detection on the same thin layer

chromatography plate in a short analysis time (Ignat, 2011). Sajewicz et al (2012)



have indicated that the silica gel thin layer chromatography -based video imaging

method is a valuable complementary fingerprint technique to identify phenolic acids

and flavonoids fractions of different sage species. Oliveira et al., (2012) also utilized

silica gel thin layer chromatography to identify phenolic compounds from Baccharis

trimera extract.

The purified compound was screened to check the presence of flavonoids.

Two generally accepted tests such as Shinoda and Ferric chloride tests are conducted.

The pure compound has responded positively to Shinoda test and Ferric chloride test

confirming the presence of flavonoid.

FTIR has proven to be a valuable tool for the characterization and

identification of compounds or functional groups (chemical bonds) present in an

unknown mixture of plant extracts (Rao,1963).

In our present study the of FTIR spectrum, broad band at 3399.2 cm-1

represents the presence of stretching vibration of phenolic groups containing

hydrogen bonding. Weak band at 2937.30 cm-1 has indicated stretching vibration of

aromatic (C-H) group and also the medium band at 2034.59 cm-1 represents bending

vibration of aliphatic (C-H) group.

NMR is one the most powerful research technique followed to investigate the

structure and some properties of molecules. One of the main applications of NMR in

flavonoid research is the structural elucidation of novel compounds, for which nothing

is known. The proton Nuclear magnetic resonance of purified compound was

analyzed by referring the standard flavonoid NMR spectra (Yamaguchi, 2005)

In the present study, the 1H NMR spectrum of the isolated compound has

displayed signals at d 6.53 assignable to a phenolic hydroxyl group at C-40 and a

singlet for three protons at d 3.83 was ascribed to a methoxy group at C-7. The 1H



NMR spectrum further displayed a one proton singlet at d 5.35 was attributed to H-8

proton.

Mass Spectrometry (MS) has proved to be one of the most effective technique

in biomedical research. The main advantage of MS is its high sensitivity, which

allows analysis of compounds present in the microgram scale, and high specificity, as

it is able to separate molecules of the same molecular weight but different atom

composition (Cuyckens and Claeys, 2005).

The mass spectrometry data of isolated compound in the present study

revealed the mass of 353.06 which is the closest value to the flavonoid myricetin

(350.03). The molecular formula is C15H10O10.Elemental analysis of purified

compound was found to be: C- 51.44, H- 2.88 and O- 45.68.

Myricetin is flavonol, consisting of 3-hydroxyflavone backbone and 6

hydroxyl groups. It is found in several foods such as walnuts, onions, berries, herbs

and red grapes. Myricetin exerts a wide variety of biological effects, including

antioxidant and free radical-scavenging activities (Li and Kuo 1998).

Cereal, vegetable and fruit consumption contributes to improve human health

and lowers the risk of disease. These benefits may depend significantly on the

presence of phenolic content in these foods. The therapeutic benefits of phenolics

have made researchers to discover, modify and utilize techniques for the extraction,

separation and quantification of these compounds from natural sources. The methods

need to be simple, rapid, environmentally friendly and comprehensive.

In our study, we have followed a simple and conventional soxhlet extraction

method for extraction of phenolics using methanol and water mixture as solvent. Thin

layer chromatography was made for separation of compound and silica gel column

chromatography used for purification of flavonoids. Three spectral techniques FT-IR,



NMR and LC-MS techniques applied for characterization and identification of

isolated compound. Hence, our study relies on basic and simple techniques for

isolation of flavonoids from plant source and succeeded in it.

The further biological activity of the isolated flavonoid is needed to screen its

specific therapeutic properties.

Chapter 5 Isolation, Purification &...

Documents

Transcript of Chapter 5 Isolation, Purification &...