CHAPTER 1
INTRODUCTION
1.1
Background of Study
Plants have always been a rich source of bioactive compounds such as alkaloids,
flavonoids, morphine and cocaine. Many of these compounds are useful drugs in
themselves. The compounds isolated from the plants are known as natural products. A
natural product is a chemical compound or substance produced by living organisms
which are found freely in nature, with a pharmacological or biological function for use
in pharmaceutical drug discovery and design. These small molecules provide the basis of
drug discovery. Recently, investigation into pharmaceutical compounds from natural
products of plants has gained much attention.
Bioactive compounds are usually isolated from plants by a variety of extraction
procedures. The most common extraction methods applied in natural product chemistry
are Soxhlet extraction and meselation process. Thin layer chromatography is used for
qualitative analysis and the collected compounds will be identified and their structure
elucidated by various spectroscopic techniques. Isolation of compounds from plants‘
extracts could be achieved using these procedures, however a long period of time is
required to collect the pure isolates.
2
High performance liquid chromatography (HPLC) was introduced to overcome
this problem. HPLC is a chromatographic technique which is used to separate a mixture
of compounds in analytical chemistry and biochemistry for identifying, quantifying and
purifying the individual components of the mixture. Nowadays, HPLC is extensively
used for natural products analysis. It is an important tool for the analysis of
pharmaceutical drugs, for drug monitoring and for quality assurance. This
chromatographic technique is of two types viz analytical and preparative HPLC.
Analytical chromatography is used to determine the existence and also the concentration
of analytes in a sample while preparative chromatography is used to purify sufficient
quantities of a substance for further use in the chemical and pharmaceutical industry, as
well as in biotechnology and biochemistry. Preparative HPLC is an advanced technique
which does not require traditional purification methods such as distillation,
crystallization or extraction process. By applying preparative HPLC analysis in natural
product chemistry, pure compounds from plant species was successfully isolated in a
shorter time as against the use of the conventional natural product isolation methods.
In this research, separation of natural product was done by applying the
analytical HPLC technique to analyze the distribution of alkaloid and flavonoid in two
plant species which were Glycosmis pentaphylla and Piper porphyrophyllum. The
research only focused on qualitative and quantitative determination of these two
compounds by analytical HPLC.
1.2
Bioactive Compounds in Natural Products
Many plant species have been studied due to their health benefits to human
beings. They can provide vitamins and minerals that help to keep humans healthy.
Nowadays, plants have become exemplary sources of drugs and many of the currently
available drugs have been derived from them. They have many functions of being antidiabetic, anti-cancer, anti-malaria, anti-tumor and many more.
3
The plant kingdom offers a rich source of structural biodiversity in the form of a
variety of natural products (Rahman and Choudhary, 2001). A study of bioactive
compounds from medicinal plants has led to the isolation and structure elucidation of a
number of exciting compounds such as alkaloids and flavonoids.
Alkaloids are a group of naturally occurring chemical compounds which mostly
contain basic nitrogen atoms. More than 10,000 different alkaloids have been discovered
in species from over 300 plant families. It represents a very extensive group of
secondary metabolites, with diverse structures, distribution in nature and important
biological activities (Grycova et al., 2007).
Flavonoids are polyphenols with diphenylpropane skeletons. The four major
classes of flavonoids are 4-oxoflavonoids (flavones, flavonols, etc.), anthocyanins,
isoflavones, and flavan-3-ol derivatives (catechin and tannins) (Miean and Mohamed,
2001). Flavonoids represent a highly diverse class of secondary plant metabolites with
about 9000 structures (Martens and Mithofer, 2005). It has attracted great interest in the
research area, specifically due to its biological and physiological importance (Engida et
al., 2013).
1.2.1 Alkaloids from Genus Glycosmis
Glycosmis is a genus of small trees of forty species from Rutaceae family
commonly found in South East Asia and South China. Fourteen of these species occur in
Peninsular Malaysia and grow on limestone rocks and hills (Rahmani et al., 2010).
Genus Glycosmis is an example of the plant that has various types of alkaloids and
benefit human beings in many ways. G. pentaphylla was traditionally used for the
treatment of fever and liver complaints. A study by Quarder et al. (1999) had reported
that arborinine (1) alkaloid obtained from G. pentaphylla was found to be inhibitory to
crown gall tumors.
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Another Glycosmis species known as G. citrifolia was used in folk medicine to
treat skin itch, scabies, boils and ulcer. Several acridone alkaloid isolated from the root
and stem barks of this plant have shown cell growth inhibition of human promyelocytic
leukaemic cells (Wu et al., 1995). Besides that, glyfoline (2) which has been categorized
as an acridone alkaloid isolated from this species was reported to possess significant
antitumor effects (Su et al., 1991).
G. arborea which was commonly found throughout India has been used to treat
common diseases such as fever, helminthiasis, rheumatism, inflammation, cough and
bronchitis. Besides that, the roots of the plant were used in facial inflammation,
rheumatism, jaundice and anaemia treatment (Silambujanaki et al., 2011). The carbazole
and acridine alkaloids isolated from G. arborea were reported to have inhibitory effects
over Epstein–Barr virus (Ito et al., 2004).
O
O
OH
OCH3
OCH3
N
CH3
OCH3
(1)
OH
HO
H3CO
N
OCH3
CH3 OCH3
(2)
1.2.2 Flavonoids from Genus Piper
Genus Piper has more than 1000 species (Ahmad et al., 2012) distributed in both
hemispheres. The species have high commercial, economical and medicinal importance
(Parmar et al., 1997). It has been used widely in folk medicine as diuretic, febrifuge,
laxative, and anti-inflammatory remedies (Larionova et al., 2010).
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Lago et al. (2012) reported on the isolation of two flavonoid compounds
identified as dihydrooroxylin (3) and pinocembrin (4) and their antifungal activity from
Piper chimonantifolium.
HO
O
R
OH O
(3) R = OCH3
(4) R = H
The ethanol extract of the leaves of Piper porphyrophyllum has been reported to
exhibit antibacterial activity against Staphylococcus aureus and Pseudomonas
aeruginosa. The results showed that the antibacterial activity was increased on
fractionation, especially in the aqueous fractions which has the highest rates of
antibacterial activity (Wiart et al., 2004).
A few years later, another study of Piper porphyrophyllum was investigated by
Ahmad et al. (2011) who reported that the plant species has antibacterial and antiinflammatory potential due to the existence of the bioactive flavonoids in the plant
extract which showed potent antibacterial activity against Staphylococcus aureus a
major human pathogen and Pseudomonas aeruginosa.
1.3
Isolation of Natural Products from Plants
A natural product is a substance or chemical compound produced from living
things that have been used in the pharmaceutical industry. Nowadays, drug discovery
from natural products has found great success in treating diabetes (Grover et al., 2002;
Chien et al., 2009; Tag et al., 2012), cancer (Gao et al., 2011; Toyang et al., 2012; Gu et
al., 2012; Chakravarti et al., 2012) and as anti-oxidant (Kim et al., 2003; Frum et al.,
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2007; Zhang et al., 2007; Rao et al., 2010). The application of drugs from natural
sources for the treatment of various diseases is more advantageous than the use of
synthesized chemicals which has attendant side effects to human health, such as
hematological coma and disturbance of liver and kidney and inappropriate for use during
pregnancy (Suba et al., 2004).
The great enthusiasm in the use of natural product drugs and its discovery has
attracted the curiosity of researchers to isolate more interesting bioactive compounds
from plant species. Isolation of the compounds from plants has been carried out by
natural product chemists using common techniques such as column chromatography
(CC) and thin layer chromatography (TLC). However, these techniques are time and
solvent consuming. Advances in separation technologies have introduced another
technique to isolate the pure compounds from plants‘ extract in a faster and easier way.
Preparative HPLC is widely being used nowadays to overcome the shortcomings of CC.
1.3.1 Column Chromatography (CC)
In natural product chemistry, CC is a technique used to purify a compound from
a mixture of various classes of compounds in plants‘ extract by controlling the polarity
of the mobile phase used. The common stationary phase used in CC is silica gel while
the mobile phase is either one kind of solvent or a mixture of different solvents. The
mobile phase must be chosen by controlling the polarity so that different compounds can
be separated effectively.
Various studies of the isolation of compounds from plant extract by using CC
technique have been reported recently. The isolation of 14 compounds of Anisomeles
indica plant was reported by Rao et al. (2009) using this technique. The constituents
were isolated by applying different polarity of mobile phases which were a combination
of hexane and ethyl acetate, while another combination was chloroform and methanol.
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In addition to using CC technique, six of the isolated compounds were purified by using
HPLC.
Furthermore, a compound identified as 2-(prop-1-ynyl)-5-(5,6-dihydroxyhexa1,3-diynyl)thiophene (5) which possesses anti-amoebic activities against Entamoeba
histolytica was isolated from the methanolic root extract of the plant Pluchea indica. It
was isolated by CC with silica gel using petroleum ether and ethyl acetate mixtures of
increasing polarity as the mobile phase (Biswas et al., 2007).
The same solvent mixture was also used as the mobile phase to isolate anti-tumor
compounds from the crude extract of Zanthoxylum ailanthoides, a traditional Chinese
medicine. In this case, CC was applied as the pre-separation step because there was a
further separation and purification process by high performance counter-current
chromatography (HPCCC) (Cao et al., 2013).
Another study of bioactive compounds from plants reported on the isolation of
anti-oxidant and anti-bacterial compounds from chloroform extract of mango ginger
(Curcuma amada Roxb.) rhizome. A novel compound named ―Amadannulen‖ (6) was
purified repeatedly by silica gel CC using a common traditional natural product
purification process involving fractionation and purification by several solvent system.
The column was eluted stepwise starting from the less polar solvent to more polar
solvent which were hexane, chloroform, ethyl acetate, acetone and methanol. The
purified fraction was then tested for its purity by using HPLC and for the structure
identification and
elucidation,
the compound was
identified using
UV–vis
spectrophotometry (UV-vis), infrared spectroscopy (IR), liquid chromatography–mass
spectrometry (LC–MS) and nuclear magnetic resonance (NMR) (Policegoudra et al.,
2007).
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H3C C C
S
H
C C C C C OH
H2C OH
(5)
HO
O
CH2
C O
CH3
H3C
CH3
(6)
1.3.2 Thin Layer Chromatography (TLC)
TLC is an effective and inexpensive technique used to separate mixtures. TLC is
performed on an adsorbent material; a thin layer of silica gel or alumina coated onto a
piece of glass, aluminium foil or plastic and this is the stationary phase for TLC. It used
to detect the sample which has migrated on the plate with a suitable solvent. From the
TLC profile, the number of components present in the sample mixture can be obtained
and the components can be identified by comparing the Rf value with the Rf value of a
known compound.
TLC requires only small amounts of sample and is ideal for the investigation of
plant constituents, which often occur as complex mixtures (Marston, 2011). This
separation method involves the same principles as CC, but can only be applied to
separate smaller quantities than CC. TLC is still advantageous being a simple method
which can be performed without sophisticated equipment, as the TLC plate is only used
once complicated clean-up procedures do not have to be performed and the visual results
can be easily presented (Ciesla and Hajnos, 2009).
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TLC separation technique has been used by Lynn and Schanderl (1967) on the
investigation of chlorophylls and related plant pigments. Ultraviolet (UV) radiation was
used to detect a very small concentration of these pigments and 16 compounds were
detected. Earlier, Elson et al. (1964) had reported on the identification of plant hormone
known as gibberellins from the seed of Echinocystis macrocarpa greene by TLC. A
study of gibberellin hormones was conducted by Cavell et al. (1967) and seventeen
known gibberellins were separated by TLC and gas chromatography (GC). Since the R f
values of TLC were not reproducible and cannot be standardized, the GC method was
applied to overcome this problem.
Some compounds that are separated by TLC may be colourless, so the spots on
the TLC must be made visible. The most commonly used method is by spraying the
TLC plate with a colouring reagent such as iodine, bromine and potassium
permanganate. Pranitis and Kornfield (1982) reported on the use of Fast Blue B salt
(Diazo Blue B) for visualization in the TLC analysis of cannabinoids in plant extract.
Fast Blue B was not homogeneous even though many compounds related to this
chromogen have been shown to be mutagenicor carcinogenic. In the analysis of
hydrobenzoic acid and hydroxycinnamic acid in plant material, the TLC was sprayed
with titanium (IV) chloride (TiCl4) or iron (III) chloride (FeCl3) solutions and
overspraying with an alkaline diazobenzenesulphonate reagent (Schulz and Herrmann,
1980).
The TLC technique has proven so useful that it was employed in almost every
area of biomedical and pharmaceutical research. Until recently, TLC technique still was
being widely used especially in natural product chemistry. The rapid growth of TLC was
slowed during the 1970‘s with the corresponding rise in popularity of more efficient
separation using HPLC.
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1.3.3 High Performance Liquid Chromatography (HPLC)
One of the chromatographic techniques which have been widely applied to
separate and purify mixture of compounds in various analytical research areas is high
performance liquid chromatography (HPLC). It can be used to carry out the separation
of complex mixtures of compounds and provide qualitative and quantitative information
on the samples that are useful for the identification and determination of sample
components (Jandera, 2000).
1.3.3.1 Preparative HPLC
Preparative (prep) HPLC is used for the isolation and purification of compounds
from a mixture of samples. This method has been widely used for the purification of
natural product compound from plants sample to replace the traditional method as the
process can be accomplished within a short period of time. Due to the high cost of the
instrument, sample pretreatment before the separation is necessary to ensure the column
will not be damaged by impurities from the raw sample.
Rochfort et al. (2006) reported the isolation and purification of glucoraphanin
from broccoli by prep HPLC. Glucoraphanin is the most predominant glucosinolate in
Brassica genus such as broccoli. In this study, the sample was first extracted using solid
phase extraction (SPE) to isolate and concentrate the glucoraphanin in the broccoli seed
before analysis by prep-HPLC.
Prep-HPLC has been combined with many other pre-separation steps to enrich
the chosen target compound. The isolation of anthraquinones from Morinda officinalis
was reported by a combination of two separation modes which were the initial preseparation by high-speed counter-current chromatography (HSCCC) and a further
purification by prep-HPLC (Zhu et al., 2009). Additionally, bioactive components
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known as coumarins were also isolated from Peucedanum praeruptorum by the same
method (Hou et al., 2010). The combination of both separation modes can improve the
separation efficiency as it successfully isolated the complicated natural products.
1.3.3.2 Analytical HPLC
The difference between analytical and prep-HPLC is that the prep-HPLC is able
to isolate pure compounds while the analytical HPLC does not have the ability to do so.
The term analytical HPLC means to analyze and determine the compounds in the sample
qualitatively and quantitatively using an analytical column. Even though the use is
limited to only identifying and quantifying, its application have already been widely
used in many areas such as the pharmaceutical industry (Babu et al., 2009; He and Dahl,
2000), drugs studies (Rao and Nagaraju, 2003; Montgomery et al., 2001), research
(Hayashiba et al., 1989) and natural products analysis (Beek, 2002; Paramapojn et al.,
2008).
There are various modes of operation for analytical HPLC such as reversed
phase chromatography, normal phase chromatography, ion exchange chromatography
and ion pair chromatography. The mechanism of interaction of the solutes with the
stationary phases determines the classification of the mode of liquid chromatography.
For natural product studies, the reversed phase mode is commonly used in
analytical HPLC analysis. The analysis of alkaloids and flavonoids from plant species by
analytical HPLC will be studied in detail in the next chapter.
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1.4
Statement of Problem
The traditional method in natural product chemistry to extract and isolate pure
compounds from plants is very time consuming. It has to undergo various processes
such as soxhlet extraction and repeated CC purification to get the pure compounds. In
addition, the conventional method also uses different solvents in relatively high volumes
which contribute to a lot of waste. There is also an expensive cost associated with the
disposal of such wastes. Thus, the method is not considered a green chemistry
application.
To overcome the problem raised from the conventional method for natural
products chemistry, HPLC is an alternative option in collecting the pure compounds
from plants. This method provides a fast means of analysis. Prep HPLC has been applied
as it can automatically collect the fractions which positively have the respective
compounds. The purity of the collected fractions can be confirmed by NMR.
Because of the unavailability of prep HPLC instrument while carrying out this
research, analytical HPLC will be used to identify the compounds present in the
G. pentaphylla and P. porphyrophyllum plant samples. The quantitative study of the
pure compounds present in both plants will be analyzed using HPLC method. Although
this technique is unable to collect the pure compounds from plants, it aids the natural
product chemist by providing information on the distribution of the studied compounds
based on their origins. This method will provide the profiling data for phytochemical
studies of G. pentaphylla and P. porphyrophyllum in plants from different origins in
Malaysia.
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1.5
Objectives of the Research
The objectives of this research are:
1)
To isolate and characterize marker compound from G. pentaphylla.
2)
To evaluate the efficiency of hexane and ethyl acetate as solvent to extract
marker compound from Glycosmis pentaphylla and Piper porphyrophyllum.
3)
To identify the quantity of the marker compound in two plant species; Glycosmis
pentaphylla and Piper porphyrophyllum collected from several regions in
Malaysia using HPLC.
1.6
Scope of Study
Alkaloid and flavonoid from the leaves of Glycosmis pentaphylla and Piper
porphyrophyllum were extracted by an extraction process using Soxhlet apparatus with
petroleum ether and methanol as the solvents, respectively. The isolated pure alkaloid
and flavonoid were used as the marker in the analysis part. Pure compounds from
previous isolation and identification (Emrizal, 2009) works were used as the
standards/markers.
The analysis of the alkaloids on the leaves of G. pentaphylla and flavonoids from
the leaves of P. porphyrophyllum were carried out using analytical HPLC. Plants to be
analyzed for their alkaloid and flavonoid contents were obtained from different regions
in Malaysia. G. pentaphylla plants were obtained from three different locations in
Terengganu while P. porphyrophyllum was obtained from six different states in
Malaysia. The origins created a variety so that the difference in the quality and quantity
of the studied compounds of each origin can be analyzed.
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1.7
Significance of Study
G. pentaphylla and P. porphyrophyllum are very well known for the alkaloid and
flavonoid contents present in the plants respectively. This study offers a detection and
quantification of arborinine, an alkaloid in G. pentaphylla and three flavonoid known as
3′,4′,5,7-tetramethoxyflavone,
5,7-dimethoxyflavone
and
5-hydroxy-7,2′,4′,6′-
tetramethoxy flavone in P. porphyrophyllum by using HPLC. From this study, the
concentrations of the studied alkaloids and flavonoids present in G. pentaphylla and
P. porphyrophyllum plants respectively from different origins were determined. The
profiling data of the plant species will be used for future studies.
Additionally, this study also provides a suitable HPLC analysis method for the
separation
of
arborinine
tetramethoxyflavone,
flavone.
alkaloid
and
5,7-dimethoxyflavone
three
and
flavonoids
namely
3′,4′,5,7-
5-hydroxy-7,2′,4′,6′-tetramethoxy