Reports On Bangladeshi Fruits

1.1  Fruits

Due to geographical situation,
Bangladesh does not possess rich mineral resources and consequently the economy
mostly depends on agriculture. The cultivable land is not enough to grow the
required foodstuff to the vast number of people in the country. Plant kingdom
has supported to safeguard the survival of the human being on earth from the very
emergence of the civilization. The four basic needs of human life, i.e. food,
clothing, shelter and medicine are obtained from plant kingdom. A major part of
the global energy requirement is supplied by the plant source as fuel.

In the view of a botanist, a
fruit is the ripened ovary and sometimes the fleshy enlarged floral parts of a
plant or herb. When a fruit is a ripened ovary, it is known as true fruit and
when it is enlarged fleshy floral part, it is known as false fruit. Botanical
or nutritional point of view fruits may or may not be edible. On the other
hand, in general sense, a fruit is the ripened ovary or its associated parts,
which can be eaten raw. Fruits were the first food for human being on the
earth. In ancient times people survived on roasted meats and fresh fruits. In
course of time and with the progress of civilization people have changed their
food habit. Nowadays cooked food as well as fresh fruits is taken as diet.

The fruits because of their
importance in various aspects of human life attract the researchers very much.
Fruits have been used from ancient time to alleviate the suffering of human
beings. At the beginning of nineteenth century when modern chemistry and
pharmacy began to develop, the original impetus to the study of natural
products chemistry utilizing fruits and medicinal plants started to develop. In
the recent decades antibiotics, vitamins and hormones are the follow up results
of researchers in the chemistry of natural products.

Fresh fruits are nutritious and
delicious food all over the world. They are ready source of energy with the
unique capacity of guarding against many diseases caused by deficiency of
nutritional ingredients. This is because fruits are the sources of various
vitamins, carbohydrates, proteins, fats, many essential minerals and enzymes.
They are medicinally very important and easily digestible. Hence, fruits are
extremely beneficial to the human body for their normal growth and healthy
condition.  Fruits in the daily diet have
been strongly associated with reduced risk for some forms of cancer, heart
disease, stroke and other chronic diseases.

Fats and oils are found widely
distributed in nature, in both the plants and animal kingdoms. Vegetables fats
and oils occur predominately in seeds and fruits, but they are also found in
the roots, branches, stems and leaves of plants. In some seeds, as for instance
in most cereals, fat occurs almost exclusively in the germ (embryo).

The formation of fat in the plant
is obscure. The carbohydrate matter which is synthesized by the plant from CO2
and H2O is apparently converted into fat through various metabolic
pathways. In seeds, only a very small amount of fat is present after the fall
of the flower. As the seeds ripen there is an increase in fat and decrease in
carbohydrate. It has also been shown that during the later phase of the
development the instauration of the fat increase while the fatty acid content

1.2 Carbohydrate in


The ultimate source of all
carbohydrates is plants, which built them from carbon dioxide and water, by


 xCO2 + xH2 O
(CH2O) x  + O2

Chlorophyll  Carbohydrate

Carbohydrates constitute one of
the most important groups of natural products. Earlier, Carbohydrates were
defined as compounds containing carbon, hydrogen and oxygen, the latter two
elements being present in the same ratio as in water i.e. they were regarded as
the hydrates of carbon and thus corresponded to the formula Cx(H2O)y,
i.e. glucose C6H12O. But it is found that certain
carbohydrates do not correspond to this formula i.e. rhamnose, C6H12O5;
while several compounds although not carbohydrates, correspond so this formula,
i.e. acetic acid C2H4O2. Now-a-days
carbohydrates are defined as the optically active polyhydroxyaldehydes or
ketones or substances that can be hydrolyzed to either of them.

Carbohydrates exist in plants and
fruits either as polymers or as free sugars. They serve as source of energy
(e.g. sugar) and as store of energy (e.g. starch and glycogen). Certain
carbohydrates (e.g. cellulose) support the plant tissues while some others
(e.g. chitin) form the major constituent of the shells crabs and lobsters.
Sugars make fruits sweet and yield alcohol on fermentation; cellulose materials
such as cotton, linen, jute, straw, grass, wood etc. supply clothes, plastics,
lacquers, paints and explosives; ribose and deoxyribose are components nucleic
acids (RNA & DNA) which determine the human heredity1.

1.3 Free Sugars


Free sugars are white crystalline
carbohydrates that are soluble in water and generally have a sweet taste. In
other words, free sugar is a generic term that includes a class of
water-soluble carbohydrates with various degree of sweetness. Sugars are
classified as monosaccharide & oligosaccharides. The monosaccharides are
polyhydroxyaldehydes or ketones, which cannot be hydrolyzed to simpler sugars,
e.g. glucose, fructose etc. The oligosaccharides yield two to ten
monosaccharide molecule on hydrolysis, e.g. sucrose, maltose etc. Sugars are
further classified as reducing and non-reducing sugars. Reducing sugars are
those carbohydrates, which reduce Fehling solution or Tollens’ reagent. All
monosaccharide and disaccharides other than sucrose are reducing sugars.
Non-reducing sugars are those carbohydrates, which do not react with Fehling
solution or Tollens’ reagent. The non-reducing sucrose must first be inverted
during hydrolysis, i.e. converted into a mixture of the two reducing sugars,
glucose and fructose by dilute acid.

C12H22O11 +   H2O   =
C6H12O6   + C6H12O6

Glucose   Fructose

1.4 Sources of Free


The presence of free sugars in
fruits, vegetables and other plant fractions are known for a long time. Most of
the fruits, especially which are sweet contains substantial amounts of free
sugars. Vegetables are usually not sweet in taste. So these may be considered
to contain only small amounts of free sugars. With the increasing importance of
dietary fiber in human, the fruits and vegetables, the main source of dietary
fiber, have become even more important. The study of dietary fiber will remain
incomplete without proper evaluation the free sugar contents of the
corresponding fruit.  Some representative
and systematic studies of free sugars and dietary fiber analysis have been
reported which discussed here. Studies of the free sugar content of guava,
mango and kamranga, which grow in many tropical countries, had been reported2,3.
Glucose, Fructose and Sucrose were identified and quantified as free sugars in
these fruits.

The free sugar content of the
tropical and sub-tropical fruits like banana, jackfruit, lichi, mango, papaya,
wax-jambu and melon had been reported4. Glucose and Fructose were
identified and quantified in almost equal amounts in most of the cases. Sucrose
was also identified in all cases except in papaya and wax-jambu.

The free sugar content of the
Indian pineapple had been reported5 and glucose, fructose and
sucrose were identified as free sugars by gas chromatographic analysis.

Glucose, fructose, sucrose and
myo-inositol were identified6 in almond, pecan and macadamia nuts.
Traces of sorbitol were also detected in pecans and almonds.

The sugar composition and
components of prunus persica fruit
were identified and analysed7 by HPLC. Sucrose was the major
component of free sugar. The other main sugars were glucose, fructose, sorbitol
and myo-inositol.

The rapeseed meal was extracted
with aqueous 80% ethanol and eleven low-molecular weight carbohydrates were
identified by chromatographic separation8. Identified Carbohydrates
were stachyose, raffinose, di-galactosyl glycerol, melibiose, sucrose,
myo-inositol, glucose, glactitol, galactose and fructose. In this report the
sugars were identified and quantified as their trimethylsilyl derivatives by
means of GLC.

 A comparison of the sugar content of four
fruits (guava, mango, yellow passion fruit and purple passion fruit) was
determined9 by GLC and Nelson-Somogyi’s method. Total sugars as
determined by Nelson-Somogyi’s method were only slightly lower than the total
sugars determined by GLC in the same fruits.

Sugar composition of Carica papaya during fruit development
was determined10 by GLC. Sucrose made up less than 18% of the total
sugar content 110 days after anthesis and increased rapidly to make up 80% of
the sugars about135 days after anthesis.

Several low molecular weight
carbohydrates were determined11 from the seeds of mung bean and
chick bean. During germination, due mainly to their use as an easily available
source of germinating energy, there was a rapid decrease of the raffinose
family oligosaccharides in mung bean and a somewhat slower decrease in chick pea.
Some growing fruits contain substantial amount of starch up to their maturity
but the amount drastically falls below 1- 2 % on ripening12. The
presence of stachyose, raffinose, sucrose and various monosaccharides in
rapeseed meal was reported13 by chromatographic technique. A series
of low molecular weight components were isolated14 form the dehulled
and fat free meal of Brassica Compestris
using chromatography on a carbon celite column, and carbohydrates composition
of Brassica napus was reported15.

The low molecular carbohydrates
from amlaki (Emblica officinalis)
were analyzed. The presence of less than 1% of D-glucose, D–fructose and
myo-inositol in the relative proportion of 1:1:3 was reported16.The
low molecular weight components of seven Bangladeshi fruits such as mango,
pineapple, guava, hogplum, kamranga, latkan and lukluki have been analysed17.
The low-molecular weight sugars were isolated by extraction with aqueous 80%
ethanol. It was found that the fruits contained glucose and fructose as the
major sugar constituents along with myo-inositol.

Free sugar of some common fruits
like, litchi, horbori, amloki, bangi, tarmuj, jamrul, kalojam, jalpai, karamcha
and papaya have been reported18. Glucose and fructose were present
as major sugar constituents including small amounts of sucrose in some of the
fruits. Low molecular weight components were determined19 in stored
tubers of three potato cultivars grown at four localities. Glucose (0.7-1.5%)
and sucrose (0.7-1.2%) were the major components followed by fructose
(0.1-0.8%), and myo-inositol (0.1-0.2%). In some samples negligible amount of
galactose, maltose, melibiose, and raffinose were detected.

Glycerol, erythritol, threitol,
arabinitol, xylitol, glucose, fructose and sucrose were and other free sugars
present in varying amounts in the bank, stem and leaf in pigeon-pen (Cajanus cajon) plant20. Some low-molecular extractives
from the source Pinus Silvestris were
isolated21,22. In the hydrophilic extract, mainly carbohydrates and
related cyclitols were found. The components included glucose, fructose,
sucrose and shikimic acid in a total yield of 1.5-2.5 %.

Low molecular carbohydrates in
the vegetables (E. Bean, L. Finger, Papaya, B. Gourd, Brinjal, W. Gourd and G.
Banana) were analyzed23. Glucose was the main the constituent of the
total polysaccharide of the vegetables but galactose was the major component of
the soluble DF. The total free sugar, reducing sugar and non-reducing sugar of
some Bangladeshi local fruits (Dab, Tarmuj, Komla, Malta, Pineapple, Wax apple,
Blackberry, Burmese grape) were estimated24 by chemical method.

Glucose and fructose were the
only two free sugars identified and quantified25. The total free
sugars of litchi, mango, guava and pineapple were 11.3%, 10.1%, 6.0% and 8.8%
respectively. Free sugars of some local vegetables (potol, karela, fulkopy,
badhakopy, chichinga, gajor, shalgom, chalkumra) were identified and quantified26.
Glucose content in any vegetables was 2-3 times more than the fructose content.

1.5 Fatty Acids


Fats are esters of long chain
fatty acids and alcohols. The ester linkages of the fats are cleaved by NaOH to
yield glycerol and sodium salts of long chain fatty acids (soaps).





Fat   Glycerol

The backbone of these compounds
contains from 4 to more than 20 carbon atoms. Most natural sources of these
compounds have an even number of carbon atoms because the biosynthetic pathway
builds the backbone two carbons at a time. Fatty acid chains may contain one or
more double bonds at specific positions (unsaturated and polyunsaturated), or
they may be fully saturated. The physical and chemical properties of a fat
depend on the composition of the fatty acid mixture. Animal fats tend to have a
larger proportion of long chain saturated acids and are solids at room
temperature. Fats from plant sources contain a higher proportion of unsaturated
acids and are often liquids at room temperature due to hydrogen bonding. These
fatty materials may influence the handling of the plant tissues as well as any
chemical treatment done on it. Therefore, the study of fatty acids and the
major constituent of all fatty matters are important. Poly unsaturated fats are
usually of vegetable origin. Crisco is an example of a vegetable-derived,
unsaturated fatty acid that has been hydrogenated to form a solid material.
Fats are used in cooking because they are very high boiling compounds. The
great numbers of the naturally occurring fatty acid belong to a few homologous
series. The general formula of the fatty acids is CnH2nO2
may represent the series to which stearic acid belongs. As, however,
their functional group is the carboxyl group, ?COOH, they are more conveniently
expressed as CnH2n+1COOH, since these show the nature of
the functional group.

1.6 Sources of Fatty

The fatty acids occur in nature
usually have straight chains and contain even number of carbon atoms. Fatty
acids occur in plants in bound27 form as fats or lipids. Fats are
the triglycerides of fatty acids of the same type or of the different types and
yield fatty acids upon hydrolysis. Lipids are defined by their special
solubility properties and are comprised of different kinds of compounds. These
lipids comprise up to 7%27 of the dry weight in leaves in higher
plants, about 1-5% in stems of green plant and are important as membrane
constructs in the chloroplasts and mitochondria.

Lipids also occur in considerable
amounts in the seeds or fruits of a number of plants. Although numerous fatty
acids are now known in plants, the palmitic
acid (C-16) is the major saturated acid21 in leaf and also
occurs in varying quantities in some seed oils. Stark acid (C-18) is the major
saturated acid in seed fats of a number of plant families27.
Unsaturated acids (mainly C-16 and C-18) are widespread in both leaf
and seed oils. A number of rare fatty acids (e.g.erucic and sterculic acid) are
found in seed oils of a few plants.

Eight vegetables namely, Tricosanthes dioica, Daucus carota, Brassica
campestris var. turnip, Brassica oliracea var. botrytis, Brassica olracea var.
capitata, Momordica charantia, Benincaca cerifera and Trichosanthes anguina

were analyzed28 for fatty acids content and composition. Lauric
acid, myristic acid, palmitic acid, stearic acid, oleic acid, arachidic acid
and behenic acid were present in varying amounts (3.68-34 mg / 100 g in fresh
vegetables) in the lipid part of the vegetables.

The constituent of fatty acids of
some of oils were analyzed29 by gas chromatographic techniques. The
major portions are short as well as long chain-saturated fatty acids like
capric acid, lauric acid, myristic acid, palmotelic acid and palmitic acid. Few
unsaturated fatty acids like arachidic acid, linolenic acid and oleic acid were
also identified in coconut oil. The major proportion of long chain saturated
fatty acid with small proportion of unsaturated one like palmitic acid and
linoleic acid, respectively were identified in palm oil.

1.7 Scientific Classification

Kingdom: Plantae

Division: Magnoliophyta

Class: Eudicotyledoneae

Subclass: Rosidae

(Unranked): Eurosids I

Order: Oxalidales

Family: Elaeocarpaceae



Figure 1:
Elaeocarpus robustus
Genus: Elaeocarpus

Species: Elaeocarpus robustus

Local Name

English Name

Scientific Name



Elaeocarpus aberrans

Elaeocarpus acmosepalus


: India. Endangered.



Elaeocarpus amoenus:
Sri Lanka


Elaeocarpus angustifolius
Blue Marble Tree, Blue Fig, Blue Quandong

Elaeocarpus apiculatus

Elaeocarpus bifidus
– Kalia (O?ahu, Kaua?i)

Elaeocarpus biflorus

Elaeocarpus blascoi

Elaeocarpus bojeri

Elaeocarpus brigittae

Elaeocarpus calomalaanakle,
binting-dalaga, bunsilak


Elaeocarpus ceylanicus

Elaeocarpus colnettianus

: Australia

Elaeocarpus cordifolius

Elaeocarpus coriaceus

Elaeocarpus crassus:
New Guinea

Elaeocarpus cruciatus

: New Guinea


Elaeocarpus dentatusH?nau

Elaeocarpus dinagatensis

Elaeocarpus eriobotryoides

Elaeocarpus eumundi: Australia

Elaeocarpus fraseri


Elaeocarpus ganitrusRudraksha

Elaeocarpus gaussenii

Elaeocarpus gigantifolius

Elaeocarpus glabrescens

Elaeocarpus glandulifer

Elaeocarpus graeffii

: India, Indo-China, Malesia

: Hainan

Elaeocarpus hartleyi:
New Guinea

: Sri Lanka

Elaeocarpus holopetalus: New South
, Victoria (Australia)

Elaeocarpus homalioides

🙁Pokaka) New Zealand.

Elaeocarpus inopinatus

Elaeocarpus integrifolius

: tree up to 15m; Japan, Taiwan, China


Elaeocarpus joga
Merr. – Yoga Tree


Elaeocarpus kirtonii: Australia

: South Asia


Elaeocarpus miegei:
New Guinea, Bismarck Archipelago, Solomon
, Aru Islands and Melville Island.

Elaeocarpus miriensis

Elaeocarpus miratii

Elaeocarpus montanus:
Sri Lanka

Elaeocarpus moratii

Elaeocarpus munronii

Elaeocarpus nanus

: New Guinea, Bismarck Archipelago

Elaeocarpus oblongus

Elaeocarpus obovatus: Australia

Elaeocarpus obtusus


. Ogasawara Islands.

Elaeocarpus prunifolius

Elaeocarpus pseudopaniculatus

Elaeocarpus recurvatus

Elaeocarpus reticosus

Elaeocarpus reticulatusBlueberry

Elaeocarpus robustus:
India,  Bangladesh.

Elaeocarpus royenii

Elaeocarpus rugosus

Elaeocarpus sallehiana

Elaeocarpus sedentarius

Elaeocarpus serratus:
South Asia

: India, Bhutan

Elaeocarpus simaluensis


: Indo-China, Malesia

Elaeocarpus storckii
Seem.: Fiji

Elaeocarpus subvillosus

: tree up to 15m; Japan, Taiwan, China, Indochina.

Elaeocarpus symingtonii

: Sri Lanka.

: New Guinea.


: Southern India.


Elaeocarpus venosus

Elaeocarpus venustus



Elaeocarpus viscosus




2.10.1 Standard
Derivatives of Free Sugars

Paper chromatography

Chromatography jar

No of observation

Weight of samples


Weight of  samples after 72 hours heating in oven (g)

Weight of


% of


Mean % of


















3.3 Determination of
Ash content

Binomial Name –
Elaeocarpus robustus

1: Scientific, English and Local Names of the Native Olive

1.8 General Description
of Elaeocarpus


Elaeocarpus30, 31, 32 is a genus of tropical and
subtropical evergreen
trees and shrubs. The approximately 350 species
are distributed from Madagascar in the west through India, Southeast
, Malaysia, southern China, and Japan, through Australia to New Zealand, Fiji, and Hawaii in the
east. The islands of Borneo and New Guinea have the greatest concentration of
species. These trees are well-known for their attractive, pearl-like fruit
which are often colorful. Many species are threatened,
in particular by habitat loss.

Table-2: Selected
Species of Elaeocarpus

1.9 Investigation of
Native Olive


Olives in our country are not
almost same to the foreign olives. There are many varieties of olives all over
the world. Our native olive is Elaeocarpus robustus. Elaeocarpus
robustus L. (Fam. Elaeocarpaceae) is a well-known
evergreen 25 m tall fruit tree. It is native to Bangladesh and India. The olive
tree has been cultivated for olive oil, fine wood, olive leaf and olive fruits.
The native olive plant is believed to have originated in Australia; however, it
is well grown in Bangladesh. It is cultivated in all districts of Bangladesh
and occurs wild in the evergreen forest of Sylhet and Chittagong. The
importance33 of fleshy sour fruits having citric acid occupies an
important position in tropical countries since they provide needed vitamin-C in

native olive fruit has several uses as food adjuncts for human being. The
fleshy ripe fruit is delicious, which is eaten raw or cooked and pickled. The
plant is also important for its therapeutic uses. Leaves are used in rheumatism
and as an antidote to poison and are considered as a cure for gonorrhea. Fruit
is tonic, emmenagogue, appetizer, useful in biliousness, liver complaints,
scabies, burning of the eyes, carries of the teeth, toothache etc. and
prescribed in dysentery and diarrhea33.

Olives are a naturally bitter
fruit that is typically subjected to fermentation or cured with lye or brine to make it more
palatable. Green olives are typically washed thoroughly in water to remove oleuropein,
a bitter carbohydrate34. Sometimes they are also soaked in a
solution of food grade sodium hydroxide in order to accelerate the
process. The green fruits are eaten fresh and also used in making soup,
chutney, jelly and jams. Elaeocarpus
robustus  tree
produce fine textured, moderately hard and strong wood which takes good finish
and fitting with good working properties. The swan wood has been used better in
parquet flooring. It is also used as suitable wood in making small furniture
and musical instruments. Wood has several important industrial uses as fuel and
to prepare some form of essential equipments such as match splints and boxes,
mathematical instruments, packing cases and boxes

trees like hot weather34 and temperatures below 140 C may
injure even a mature tree. They tolerate drought well. They show a marked preference for calcareous
soils, flourishing best on
slopes and crags, and coastal climate conditions. They grow in any light soil,
even on clay if well drained, but in rich soils, they are predisposed to
disease and produce poorer oil than in poorer soil. They
are commonly grown from seeds, which are recalcitrant and difficult to
germinate even after a short period of storage. The species is predominantly
cross-pollinated leading to high seedling variability. Because of seed
propagation, the plant qualities vary widely among the individuals33.Soup
of the fruit is also given for stimulating secretion from the test buds.
Etanolic extract of leaves are diuretic and cardiovascular stimulant34.

Considerable research supports
the health-giving benefits of consuming olives, olive leaf and olive oil. Olive
leaves are used in medicinal teas. Olives are now being looked at for use as a
renewable energy source, using waste produced from the olive plants as an
energy source that produces 2.5 times the energy generated by burning the same
amount of wood. The smoke released has no negative impact on neighbours or the
environment, and the ash left in the stove can be used for fertilizing gardens
and plants. The process has been patented in the Middle East and the US35.

1.10 Strive of the Work


In the present world, natural
resources are contributing a significant role to the economic upliftment of a
country and thus paved the way for the development in the fields of education
and technology, which ultimately brought the special prosperity. Therefore, it
is inevitable that major efforts should be concentrated on the proper
utilization of natural resources. In general, natural resources may be broadly
classified into agricultural and mineral resources. With a view to achieving
the maximum utilization it is imperative to make a thorough survey of the
agricultural and mineral resources.

Fruits as well as plant kingdom
are directly associated with the lives and livings of human being and a little
known about the chemical composition of these fruits, studies on the isolation,
identification, quantification and characterization of the medicinally active
compounds from them are very important for the well-being of human society.

 Figure 2: Laboratory work during sample

Fruits are the important source
of nutrient and energy. In primitive days people used to take fresh fruits from
their instinct and without knowing their nutritional value but in modern days
with the advancement of food science, people know the nutritional value of
fruits and their uses is on increase. Fruits are delicious and nutritious food
in terms of calories, vitamins, minerals and other nutrients. Nevertheless,
most of fruits of our country are seasonal fruits. Olive is one of them. The
olives that are grown in our country are different from alien olives. Vast
research has not been done in our native olive as to why I took native olive as
my subject of research.

Elaeocarpus robustus locally known as Jalpai, is a well known
fruit that yields during November-December. Green fruits are sour and cooked by
the rural people to make various types of soups,
chutneys, jellies and jams. In addition, it has a medicinal value. The fruit
pulp is rich in vitamin C and citric acid.

are the principal primary metabolite widely distributed in nature. The major
constituent of Elaeocarpus
robustus fruit is carbohydrate. In the
present investigation on the carbohydrates of this fruit was undertaken.

acids, important for various purposes occur in plants, fruits and animals. The
fatty tissues of animal contain large amounts of long chain saturated fatty
acids. Plants contain higher proportion of unsaturated fatty acids. These fatty
acids may influence the handling of the plant tissues as well as any chemical
treatment done on it. Therefore,
the study of fatty acids as well as the major constituents of all fatty matters
is very important.

 According to the data of the nutritional
values, an extended and reliable analysis has been carried out to understand
the composition and presence of several kinds of nutrients. The following
experiments have been done related to this analysis.

Determination of moisture content

Determination of ash content

Analysis of free sugar

Identification of free sugar by PC

Quantification of free sugar by chemical methods

Identification as well as quantification of free sugars by GC

Identification and quantification of fatty acids by GC.

The edible part of olive is skin
and pulp. So all the cases, seeds were omitted for different type of analysis.

Materials and Methods

All the chemicals and reagents
used in the experiments were analytical grade and procured from Sigma, E. Merck
(Germany) and BDH (England).

2.2 Solvents


Solvents used in different
experiments were ethanol, methanol, n-hexane, dichloromethane (DCM), petroleum
ether etc.

2.3 Solid Reagents


Anhydrous sodium sulphate was
freed from interfering organic substances and moisture by heating at 4000
C for at least 4 hours. Pure silica sand was also used.

2.4 Liquid Reagents


Borontrifluoride– Methanol
complex solution of‘Merck Schuchardt’ was kept refrigerator and a definite
amount of the solution was withdrawn for each hydrolysis.

2.5 Standard Reference


Tetramethylsilane, (CH3)4Si,
also called TMS is used universally as standard reference substance for sugar

2.6 Distillation of


Analytical grade solvents
(ethanol, DCM, petroleum ether etc.) were distilled before use. Petroleum ether
(b. p. 400 – 600 C & 600 – 800
C) was obtained by distillation.

2.7 Evaporation


All evaporations were carried out
under reduced pressure at bath temperature not exceeding 400 C to
avoid decomposition of the samples.

2.8 Electric Oven


All glass apparatus were dried
and anhydrous sodium sulphate was stored inside an oven (Memmerat)

2.9 Freeze-Drying


 Freeze drying were carried out by HETOSIC CD
52 (Hetolab Equipment,Denmark)
freeze-dryer. Aqueous extracts and fractions were first frozen in round
bottomed flasks in an ethanol freezer (Hetofrig cd 5, Hetobirkero, Denmark) at
-30 0c to -40 0C and finally the materials were subjected
to freeze-drying operation.

2.10 Standard Solutions


Standard derivative mixtures of
sugar solutions of ‘Sigma’ were prepared. These sugar mixtures were injected in

2.10.2 Standard
Derivatives of Fatty Acids


Standard derivative mixtures of
fatty acid solutions of ‘Sigma’ were prepared. These fatty acid mixtures were
injected in GC as the standard acid mixture.

2.10.3 Preparation of
Methanolic 0.5 M KOH Solution


Methanolic KOH solution was
prepared for hydrolysis of the oil. 7.1311 g KOH was taken in a 250 mL and made
it up to the mark to prepare 0.5 M KOH in MeOH was prepared.

2.11 Refluxing


For the esterification of fatty
acids a refluxing system was employed. The system consists of a condenser and a
pear shaped flask being placed on a hot water bath.

2.12 Determination of
Ash Content

The most widely used methods are
based on the fact that minerals are not destroyed by heating, and that they
have a low volatility compared to other food components.  The three main types of analytical procedure
used to determine the ash content of foods are based on this principle: dry ashing, wet ashing and low temperature plasma dry ashing. The
method chosen for a particular analysis depends on the reason for carrying out
the analysis, the type of food analyzed and the equipment available.

Typically, samples of 1-10g are
used in the analysis of ash content. Solid foods are finely ground and then
carefully mixed to facilitate the choice of a representative sample. Before
carrying out an ash analysis, samples that are high in moisture are often dried
to prevent spattering during ashing. High fat samples are usually defatted by
solvent extraction, as this facilitates the release of the moisture and
prevents spattering. Other possible problems include contamination of samples
by minerals in grinders, glassware or crucibles, which come into contact with
the sample during the analysis. For the same reason, it is recommended to use
deionized water when preparing samples36.

There are a number of different
types of crucible available for ashing food samples, including quartz, Pyrex,
porcelain, steel and platinum. Selection of an appropriate crucible depends on
the sample being analyzed and the furnace temperature used. The most widely
used crucibles are made from porcelain because it is relatively inexpensive to
purchase, can be used up to high temperatures (< 1200oC) and are
easy to clean. Porcelain crucibles are resistant to acids but can be corroded
by alkaline samples, and therefore different types of crucible should be used
to analyze this type of sample. In addition, porcelain crucibles are prone to
cracking if they experience rapid temperature changes. A number of dry ashing
methods have been officially recognized for the determination of the ash
content of various foods (AOAC37 Official Methods of Analysis).
Typically, a sample is held at 500-600 ºC for 24 hours.

2.13 Chromatographic


The present experiments were done
by two types chromatographic methods.

2.13.1 Paper
Chromatography (PC)


Paper chromatography38
is an analytical  technique for separating and identifying
mixtures that are or can be coloured, especially pigments.
This can also be used in secondary or primary colours in ink experiments. This
method has been largely replaced by thin layer chromatography, however it is
still a powerful teaching tool. Two-way paper chromatography, also called two-dimensional chromatography
, involves using two solvents and rotating the paper 90° in between. This is
useful for separating complex mixtures of similar compounds, for example, amino acids.

Figure 3: A paper chromatogram

A small concentrated spot of solution
that contains the sample of the solute is applied to a strip of chromatography paper about two centimetres
away from the base of the plate, usually using a capillary
for maximum precision. This sample is absorbed onto the paper
and may form interactions with it. Any substance that reacts or bonds with the
paper cannot be measured using this technique. The paper is then dipped into a
suitable solvent,
such as ethanol
or water,
taking care that the spot is above the surface of the solvent, and placed in a
sealed container.

The solvent moves up the paper by
, which occurs as a result of the attraction of the solvent
molecules to the paper; this can also be explained as differential adsorption
of the solute components into the solvent. As the solvent rises through the
paper it meets and dissolves the sample mixture, which will then travel up the
paper with the solvent solute sample. Different compounds in the sample mixture
travel at different rates due to differences in solubility in the solvent, and
due to differences in their attraction to the fibres in the paper. The more
soluble the component the further it goes. Paper chromatography takes anywhere
from several minutes to several hours.

In some cases, paper
chromatography does not separate pigments completely; this occurs when two
substances appear to have the same values in a particular solvent. In these
cases, two-way chromatography is used to separate the multiple-pigment spots.

In this method, the solvent is in
pool at the bottom of the vessel in which the paper is supported.

In this method, the solvent is
kept in a trough at the top of the chamber and is allowed to flow down the
paper. The liquid moves down by capillary action as well as by the
gravitational force, thus this method is also known as the gravitational
method. In this case, the flow is more rapid as compared to the ascending
method, and the chromatography is completed more quickly. The apparatus needed
for this case is more sophisticated. The developing solvent is placed in a
trough at the top which is usually made up of an inert material. The paper is
then suspended in the solvent. Substances that cannot be separated by ascending
method can sometimes be separated by the descending method.

(3) Rƒ

Rƒ value may be
defined as the ratio of the distance travelled by the substance to the distance
travelled by the solvent. Rƒ values are usually expressed as
a fraction of two decimal places but it was suggested by Smith that a
percentage figure should be used instead.

(cm) traveled by solute

(cm) traveled by solvent

Usually, the Rf value
is constant for any given compound and it corresponds to a physical property of
that compound.

In the present experiment, descending paper chromatographic method was applied.

2.13.2 Gas


Gas-liquid chromatography38
(GLC), or simply gas chromatography (GC), is a common type of chromatography
used in analytic 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 preparative chromatography, GC can be used
to prepare pure compounds from a mixture. In general, substances that vaporize
below ca. 300 °C (and therefore are stable up to that temperature) can be
measured quantitatively. The samples are also required to be salt-free; they should not
contain ions.
Very minute amounts of a substance can be measured, but it is often required
that the sample must be measured in comparison to a sample containing the pure,
suspected substance.

In gas chromatography, the moving
phase (or “mobile phase”) is a carrier gas, usually an inert gas such as helium or an unreactive 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 function of the stationary
phase in the column is to separate different components, causing each one to
exit the column at a different time (retention time). Other parameters
that can be used to alter the order or time of retention are the carrier gas
flow rate, and the temperature. The instrument used to perform gas chromatography
is called a gas chromatograph (or “aerograph”, “gas

In a GC analysis, a known volume
of gaseous or liquid analyte is injected into the “entrance” (head) of
the column, usually using a micro syringe. As the carrier gas sweeps the
analyte molecules through the column, this motion is inhibited by the adsorption
of the analyte molecules
either onto the column walls or onto packing materials in the column. The rate
at which the molecules progress along the column depends on the strength of adsorption,
which in turn depends on the type of molecule and on the stationary phase
materials. Since each type of molecule has a different rate of progression, the
various components of the analyte mixture are separated as they progress along
the column and reach the end of the column at different times (retention time).

A detector39 is used
to monitor the outlet stream from the column; thus, the time at which each
component reaches the outlet and the amount of that component can be determined.
Generally, substances are identified (qualitatively) by the order in which they
emerge (elute) from the column and by the retention time of the analyte in the
column. Before starting, the detector and chart recorder must be zero.

 Fig 4:
A gas chromatograph with a headspace sampler

The column(s) in a GC are
contained in an oven, the temperature of which is precisely controlled
electronically. The rate at which a sample passes through the column is
directly proportional to the temperature of the column. The higher the column
temperature, the faster the sample moves through the column. However, the
faster a sample moves through the column, the less it interacts with the
stationary phase, and the less the analytes are separated. In general, the column
temperature is selected to compromise between the length of the analysis and
the level of separation.

Generally chromatographic data is
presented as a graph of detector response (y-axis) against retention time
(x-axis), which is called a chromatogram. This provides a spectrum of peaks for
a sample representing the analytes present in a sample eluting from the column at
different times. Retention time can be used to identify analytes if the method
conditions are constant. In addition, the pattern of peaks will be constant for
a sample under constant conditions and can identify complex mixtures of

Quantification is possible in GC
methods by analyzing the peak area or peak height. A larger peak area indicates
a larger amount of analyte present in the sample. The area under a peak is
proportional to the amount of analyte present in the chromatogram. By
calculating the area of the peak using the mathematical function of
integration, the concentration of an analyte in the original sample can be
determined. Concentration can also be calculated using a calibration
created by finding the response for a series of concentrations
of analyte, or by determining the relative response factor
of an analyte. The relative response factor is the expected ratio of an analyte
to an internal standard (or external standard) and is
calculated by finding the response of a known amount of analyte and a constant
amount of internal standard (a chemical added to the sample at a constant
concentration, with a distinct retention time to the analyte). In most modern GC-MS systems, computer software is used to draw and integrate
peaks, and match MS spectra to library spectra.

In the present experiment, free
sugars and fatty acids were analyzed with the help of GC.

3.1 Collection of
Native Olives


Fruits being good quality, green
and fresh were procured from a local market of Dhaka city in 2009. Only fresh
fruits in prime condition can produce a good quality dried product. Wilted ones
were not used. One moldy bean may give a bad flavor to all the lot. Carefully
sorted, discarding any bruised or undesirable products and then washed
carefully and thoroughly in cool water it was kept pieces uniform in size so
they dry in same rate. A fruit slicer or food processor can be used. As many
fruits as can be dried at one time were prepared. Holding fruits, even in the
refrigerator, after washing and preparation for drying will result in loss of
quality and nutrients. Only the edible parts of the fruits were taken for
analytical purposes. In the present work, the seeds of the fruits were manually
removed from the edible parts.

3.2 Determination of
Moisture Content


Fruits can be preserved by
drying. The longer the drying time, the less flavorful and the less tender the
products are. The drying time can be hastened by drying small, uniformly cut
pieces. Fruits are dried until brittle. At this stage, only 10 percent moisture
remains and no microorganisms can grow. Controlled Moisture (CM) in fruits
combine superior performance and concentrated nutrients with vivid color and
exceptional flavor. CM fruits deliver better performance in frozen, fresh and
refrigerated foods because they have less water content. Pizza crusts and dough
formulae stay crisper, sauces, dips and spreads stay true to their original
flavor and consistency; and dishes maintain appealing texture without being
soggy. With lower moisture and higher solid content, CM fruits are
nutritionally dense. In fact, it takes 33% less CM fruits in application to
deliver one fruits serving as compared to fresh and individually quick frozen

A known amount of fresh fruit was
taken in a dry, cleaned and weighed watch glass. Then it was dried at 1050
C in an electric oven.  The watch glass
was cooled in a desicator and weighed. The drying and cooling were repeated
until a constant weight was obtained. The moisture content was calculated and
presented in table 3.

Table 3: Relative Amount of
Moisture Content


Recently, analytical instruments
have been developed to dry ash samples based on microwave heating. These
devices can be programmed to initially remove most of the moisture (using a
relatively low heat) and then convert the sample to ash (using a relatively
high heat). Microwave instruments greatly reduce the time required to carry out
an ash analysis, with the analysis time often being less than an hour. The
major disadvantage is that it is not possible to simultaneously analyze as many
samples as in a muffle furnace40.

The ash content is the percentage
of inorganic residue remaining after ignition of sample. A weighed quantity of
sample of Elaeocarpus robustus was taken in a
crucible. The contents were heated carefully to the ignition point and allowed
to burn spontaneously. When burning was completed, the crucible heated at a
dull–red heat in a muffle- furnace at 6000 C for three hours. The
crucible was then cooled and weighed with accuracy.

3.3.1 Calculation of


% of ash =

M1=Initial weight of sample in g and M2 = Weight of the
ash in g

The results of the determination
of the amount of ash content of the Elaeocarpus robustus
have been calculated and given in the following table 4.

4: The Relative Amount of Ash Content

Weight of empty crucible (g )

Weight of

Crucible +sample ( g )

Weight of


M1 ( g )

Weight of

Crucible + ash

( g )

Weight of ash

M2 ( g )

 %  of







3.4 Extractions


10 pieces of Elaeocarpus
robustus fruits were taken and weighed
that was found to be 303.55 g. After slicing and chopping into
the fruits, the edible parts (259.19 g)
were extracted with aqueous 70 – 80 %
ethanol (900 mL) by refluxing for 45 minutes on a metal heater. The insoluble
residue was then filtered off, air-dried, powered and re-extracted with aqueous
70 – 80 % ethanol (500 mL X 2) by refluxing for 45 minutes on a metal heater.
In each case, the volume of ethanol to be added was calculated by taking into
consideration. The ethanolic extracts were then combined and the ethanol was
removed by evaporation with added water. The resulting aqueous solution was
partitioned with dichloromethane (2 X 200 mL) in a separatory funnel. The
amount of aqueous layer and organic layer were found to be 388 mL and 274 mL
respectively. The aqueous layer was taken and water was liberated under reduced
pressure. This layer contained the low molecular weight carbohydrates. The
aqueous layer was divided into two parts. One i.e. 20 mL was concentrated and
freeze-dried. Another friction i.e. 40 mL was taken into quantitative study for
free sugar analysis by chemical method.

Extraction Scheme of Elaeocarpus

Elaeocarpus robustus


Extracted with C2H5OH
by boiling 45 minutes



  Residue Ethanol extract

Added distilled H2O Water extract

Partitioned with DCM



  Aqueous layer
(60 mL)
Organic layer

   Divided into two parts



  Freeze-dried (20 mL) aqueous part (40 mL)

Scheme 1: Extraction of Elaeocarpus Robustus

3.5 Analysis of Free
3.5.1 Identification
and Detection of Free Sugars by Paper Chromatography Preparation of
Reagents for the Development of Paper Chromatograms


(i). Silver nitrate solution:

Saturated aqueous solution of
silver nitrate (1 mL) was mixed with acetone (200 mL) and water was added drop
wise until the precipitate formed was re-dissolved.

(ii). Sodium hydroxide solution:

Aqueous alcoholic sodium
hydroxide solution (2%) was prepared by dissolving sodium hydroxide (4 g) in
water (100 mL) then diluted with ethanol (200 mL).

(iii). Sodium thiosulphate

Aqueous sodium thiosulphate
solution (10 %) was prepared by dissolving sodium thiosulphate (40 g) in water
(400 mL). Development of
Paper Chromatograms


The irrigated papers were dried
in the air and the sugars were located on paper by dipping in or spraying with
one of the following reagent systems:

(a) The paper was dipped in (i),
dried and soaked with dip (ii).The developed chromatograms were then washed
with dip (iii), followed by water and finally dried in the air.

(b) An alcoholic (0.1 M) solution
of p-anisidine and phthalic acid, followed by heating at 1000 C for
10 minutes.

(c) An alcoholic (1 %) solution
of oxalate followed by heating at 1000C for 10 minutes. Detection of


The dried DCM – extracted aqueous
layer was dissolved in water. It was spotted on Whatmann No. 1 paper
separately, the paper was allowed to run 24 hours in the solvent system
consisting of n- Butanol : ethanol : water (40: 11 : 19). After removing the
papers from the tank, the chromatograms were developed.

Black spots are observed and
marked by encircling with a pencil and labeling them.

+ AgNO3 Ag (s) + HOCH2(CHOH)4COONa
Calculation of Rf Values of Free Sugars by PC

Rf value of Glucose =


Rf value of Galactose

   = 0.45

Rf value of Arabinose

  = 0.50

5: Identification of Sugar Components by Paper Chromatography

Sugar components

Rf value







3.5.2 Quantification of
Free Sugars by Chemical Method


The reagents used in this work
were made by the following procedures: Fehling’s
Solution 1 (Copper Sulphate Solution)


A. R copper pentahydrate (34.64
g) crystals were taken in a beaker and distilled water (containing a few drops
of dilute sulphuric acid) was added to it. The solution was then diluted to 500
mL. Fehling’s
Solution 2 (Alkaline Tartrate Solution)


Pure sodium hydroxide (60 g) and
pure Rochelle salt (sodium potassium tartrate) (173 g) was taken in a beaker
and water was added. Then the solution was filtered through a sintered glass
funnel and made up the filtrate and washings to 500 mL. Then these two
solutions were kept separately in tightly stopper bottles and mixed exactly
equal volumes immediately before use.
0.5 M Sodium Hydroxide Solution


Solid Sodium hydroxide (5.23 g)
was taken in a volumetric flask (250 mL) and made upto the mark with distilled
water.  0.5 M Hydrochloric Acid


The supplied concentrated
hydrochloric acid was 12 M (36 %, density-1.18 g/mL). 4.20 mL of this acid was
taken in a volumetric flask (100 mL) and made upto the mark with distilled
water. Methylene Blue
Indicator Preparation of
Standard Glucose Solution


Dry A.R glucose (1.26 g) was
taken by accurate weighing in a volumetric flask (100 mL). Then the glucose was
dissolved by distilled water and made upto the mark with distilled water. Standardization
of Fehling Solution with Standard Glucose Solution


To standardize the Fehling
solution weighed out accurately 0.5 g of A.R glucose. Then it was dissolved in
water and diluted to 100 ml in a volumetric flask. 10.0 ml of supplied Fehling
solution was transferred in to a 250 ml conical flask and diluted with 100 ml
of water. It was heated to boiling and then added glucose solution from a
burette until the blue colour of the solution just disappeared. This gave an
approximate value of the volume of glucose solution required. To obtain the
exact volume, repeated the titration and added so much of glucose solution that
0.5 to 1.0 ml required to completed the reduction. The liquid was heated to
boiling, maintained the gentle boiling for two minutes added 3 to 5 drops of
ethylene blue indicator. The titration was completed in one minute by adding
the glucose solution drop wise until the methylene blue colour just disappeared
and it was repeated until the constant values were obtained41. The
result was presented in table-6.

6: Standardization of Fehling Solution with Standard Glucose Solution

Weight of A.R glucose was taken =
0.5304 g

No of Observation

Volume of Fehling Solution (mL)

Initial Burette reading (mL)

Final burette

reading  (mL)



Mean (mL)














18.60 Calculation

100 mL standard glucose solution
was contained 0.5304 g glucose (as prepared).

18.6 mL standard glucose solution
?20 mL Fehling solution

0.5304 ×18.60

= g glucose


=0.0986 g glucose.

20 mL Fehling’s solution ? 0.0986
g of glucose. Determination
of Reducing Sugars


The supplied sample was taken in
a burette (50.00 mL). Fehling’s solution (20.0 mL) was taken in a conical flask
(250 ml) and an equal amount of water was added to it. The solution was heated
to boil for about 2 minutes. Then the sample was added slowly to the solution
from burette until the blue colour of the solution just disappeared. The
solution was then boiled for another 2 minutes. Then 2- 4 drops of methylene
blue indicator was added to the solution and titration was continued for the
complete disappearance of the blue color of the solution. The titration was
repeated until consistent values were obtained.

7: Determination of the Reducing Sugars

No. of Observation

Vol. of Fehling Solution /(mL)

Initial Burette Reading/ (mL)

Final burette




Mean (mL)














14.00 Calculation of
Reducing Sugars


20 mL Fehling’s solution = 0.0986
g glucose.

So, 14 mL extract contains 0.0986
g glucose.

100 mL extract contains 0.7043 g

i.e. 40 mL extract contains
0.7043 g glucose.

60 mL extract contains 1.0565 g

So, 259.19 g olive sample
contains 1.0565 g glucose.

100 g olive sample contains
0.4076 g glucose.

Therefore, the amount of reducing
sugar is 0.4076 g / 100 g. Determination
of Total Free Sugars


A known amount (25 mL) of sample
solution was taken in a 100 mL volumetric flask. Diluted the sample with distilled
water and made up to the mark of the volumetric flask. Then exactly 25 mL
solution was taken from this diluted solution and added dilute HCl solution
(0.5 M, 20 mL).  The solution was heated
about half an hour on a boiling water bath. Cooled the solution and neutralized
by adding with dilute NaOH Solution (0.5M). This solution was transferred
quantitatively in 100 mL volumetric flask and made up to the mark with
distilled water.

Measured amount of Fehling
solution (20 mL) was titrated with the sample solution as described in sec. and then the content of free sugar present in the sample was determined
and the result was given in table 8.

8: Determination of the Total Free Sugars Content

No of Observation

Volume of Fehling Solution


Initial Burette Reading


Final burette





Mean (ml)














28.90 Calculation of
Total Free Sugars


28.90 mL hydrolysed extract ?20
mL Fehling’s solution ?0.0986 g glucose

100 mL hydrolysed extract
contains 0.3412g glucose

100 mL hydrolysed extract
contains 25 mL original extract, so 25 mL extract contains 0.3412 g of glucose.

100 mL hydrolysed extract
contains 1.3648 g glucose.

i.e. 100 mL hydrolyzed extract
contains 1.3648 g total sugars.

So, 40 mL olive extract contains
1.3648 g total sugars.

60 mL olive extract contains
2.0472 g total sugars.

So, 259.19 g olive sample
contains 2.0472 g total sugars.

100 g olive sample contains
0.7899 g total sugars.

Reducing sugar obtained 0.4076 g,
so inverted sugar obtained =0.7899 – 0.4076 = 0.3823 g.

Now 360 g inverted sugar = 342 g
non–reducing sugar.

0.3823 g inverted sugar = 0.3632
g non–reducing sugar.

Total sugars = (reducing sugar +
non – reducing sugar)

= 0.4076 g + 0.3632g =
0.7708 g.

Therefore, the amount of total
free sugars is 0.7708 g /100 g.

10 pieces of fruits contain
0.7708 g total free sugars; so a piece fruit contains 0.077 g total free
sugars. Determination
of Non-Reducing Sugars


The value of non-reducing sugar
was determined by subtracting the value of reducing sugar from the total free
sugar and the results of the olive were given in table –9.

9:  Content of Total, Reducing and
Non-Reducing Free Sugars

Fresh weight basis (%)

Total Sugars


Reducing Sugars


Non reducing sugars





3.5.3 Identification
and Quantification of Free Sugars by Gas Liquid Chromatography


All sugars analyses were
performed on GLC following Sweely et.al42. Evaporations of GLC
samples were carried out under reduced pressure at below 400 C. For
gas chromatographic analysis a PYE UNICAM 4500 (FID detector) GLC analyzer
connected with a LKB 2220 recording integrator was used. Separations were
performed on (i) CP Sil 5 WCOT quartz capillary column at 1850 -2100
C, 20 C per minute for trimethylsilyl derivatives and (ii) CP
Sil 88 WCOT quartz capillary column at 1700 – 2200 C, 40
C per minute for alditol acetates. Procedure of
TMS- Derivatives for GC – MS Analysis


(i) Added n-hexane Freeze
dried of Elaeocarpus robustus (from
section 3.4.1)

(ii) 20 µL of filtrate was taken to a
pear shaped flask

(iii) It was evaporated to
dryness with addition of 1 mL MeOH (3 times)

(iv) Added 1mL dry pyridine and
vortex / sonication for 1 minute

(v) It was added silylating
reagent to the dry extract

(vi) It was shaken well or vortex
to mix and incubate at 700 C for 45 minutes

(vii) It was
evaporated into dryness until pyridine free

(viii) 1 mL n–Hexane was added to
dry mass vortex, filtered and then transferred into a vial

(ix) Ready for injection on GC-
MS. Gas
Chromatographs Identification and Determination


1.0 µL
sample was injected through a filter into the injector of GC-MS at the same
condition the sugar standard mixture solutions were injected. Comparing the retention times of
the different peaks in the chromatogram of the sample with those of standards,
different sugars were identified. Identification and
Quantification Scheme of Free Sugars by GC

dried of Elaeocarpus robustus (from
section 3.4.1)

 Added n-hexane

µL was taken in a
pear shaped flask

to dryness with addition of 1 mL MeOH (3 times)

  Added 1mL dry pyridine

 Vortex / sonication for 1 minute

Added silylating reagent to the
dry extract

    Shaken well or vortex

 Incubated at
700 C for 45 minutes

  Evaporation into dryness until
pyridine free

 Added 1 mL n–hexane

and then transferred into a vial

Sent for GC

Scheme 2:
Free Sugars analyses by GC

Table 10: Standard Retention Time
of Different Methyl Esters of Different Sugars from Gas Chromatograms

Standard Retention Times














Glucose Calculation of
the Relative Percentage of Different Type of Sugars

  Area of the peak

% of sugar =   X 100

areas of the peaks

From the Gas Chromatograms three
sugars were identified. They were arabinose, galactose and glucose. From the
analyzed fruits, the relative proportion and relative percentage of sugars are
given below:

Total areas of the peaks of free
sugars = 4572 + 5820 + 23931

  = 34323.

Relative percentage of
arabinose = X 100

= 13.33 %.

Relative percentage of galactose  = X 100

   = 16.95 %.

Relative percentage of
glucose =  X 100

  = 69.72 %.

Relative proportion   =13.33 + 16.95 + 69.72


Table 11: Relative Proportion of
Identified Free Sugars

Retention Times

( min) of sugars

Areas of sugars

Identified sugars

Relative  %













5: Relative Percentage of Identified Free Sugars

3.6 Isolation,
Identification and Quantification of Fatty Acids by GLC

This experiment introduces a
procedure that is used routinely for fat analysis in which non volatile fatty
acids are chemically converted to the corresponding volatile methyl esters. The
resulting volatile mixture can be analyzed by gas chromatography. Any sample
that can be vaporized (or the components could assume a vapor pressure of at
least few mm of Hg) without thermal decomposition at the operating temperature,
could be analyzed by GC. At present due to various reasons, including difficult
instrumentation and lack of high temperatures materials, the separating
temperature is generally limited to about 450oC. Samples that cannot
be vaporized are converted into volatile derivatives (for example, fatty acids
converted into methyl esters) and then subjected to GC analysis.

3.6.1 Analysis of Fatty


The dried DCM extracted-aqueous
layer (100.0mg) of fruit sample was dissolved in hexane (50.0 mL) and extracted
with 5% sodium bicarbonate solution (25.0 mL X 2). The mixture was taken in a
separatory funnel and shaken vigorously and allowed to stand for overnight. Two
layers were obtained. The lower layer (aqueous) was separated and taken for the
analysis of free fatty acid (FFA). The upper layer was separated and taken for
the analysis of bound fatty acid (BFA)43.

3.6.2 Isolation of FFA


The lower part was acidified (pH
2.5) by 2M sulphuric acid. The mixture was then extracted with hexane (25.0 mL
X 3). The hexane fraction was dried over anhydrous sodium sulphate, filtered
and the filtrate was evaporated to dryness. Now the saponified materials
obtained from the hexane extract was taken in a pear shaped flask and 2.0 mL of
borontrifluoride-methanol (BF3-MeOH) complex was added and the
mixture was refluxed on a boiling water bath for 6 to 10 min. The mixture was
then evaporated in a rotavapor to dryness and transferred in a small separatory
funnel containing a little water (6.0 mL). The mixture was shaken vigorously
and then extracted with hexane. The aqueous layer was discarded. The hexane
part containing the methyl esters of fatty acids was made free from water by
adding anhydrous sodium sulphate. The solution was filtered and the filtrate
was concentrated for the analysis of free fatty acids by GLC (Shimadzu 9A,
Column-BP-50, Detector-FID, 170°C-1 min/4°C-270°C-30

3.6.3 Isolation of BFA


The upper part was taken in a
pear shaped flask; methanolic sodium hydroxide (0.5 M, 10.0 mL) was added to it
and shaken well. The mixture was refluxed for 30 min in a boiling water bath.
Then the mixture was evaporated to dryness by means of a rotavapor. A little
water was added to the mixture and transferred to a seperatory funnel to settle
down. The non-saponified materials were separated from the saponified portion
(aqueous layer) by extraction with hexane. The aqueous layer containing fatty
acids (as salts) was acidified by adding sulphuric acid and to pH 2.5. The
mixture was extracted with hexane. The hexane part was taken in a conical flask
and made from water by adding anhydrous sodium sulphate and then filtered. The
filtrate contained saponified materials.

Now the saponified materials
obtained from the hexane extract was taken in a pear shaped flask and 2.0 mL of
borontrifluoride-methanol (BF3-MeOH) complex was added and the
mixture was refluxed on a boiling water bath for 20 min. The mixture was then
evaporated in a rotavapor to dryness and transferred in a small separatory
funnel containing a little water (6.0 mL). The mixture was shaken vigorously
and then extracted with hexane. The aqueous layer was discarded. The hexane
part containing the methyl esters of fatty acids was made free from water by
adding anhydrous sodium sulphate. The solution was filtered and the filtrate
was concentrated for the analysis of bound fatty acids by GLC (Shimadzu 9A,
Column-BP-50, Detector-FID, 170°C-1 min/4°C-270°C-30

3.6.4 Isolation Scheme for Fatty
Acids Analyses

extracted dried aqueous layer of Elaeocarpus robustus

Added 5 % of NaHCO3

  Mixture (shaken vigorously)

Transferred in
separatory funnel


 Organic layer   Aqueous

(Unreacted fatty material)


10 mL of 0.05 M NaOH

Refluxed 30 minutes

of FFA +Glycerol

under reduced pressure with added distilled water


Transferred in a separatory funnel by adding hexane

Aqueous part taken


Added 0.2M H2SO4

in a separatory funnel

Taken Hexane part in a beaker

  Added Na2SO4
& filtered

Filtrate (Dried & weighed which is total fatty

Refluxed 20 minutes &

Added 1 mg of Benzoic acid & 2 mL BF3
– MeOH complex

Transferred in separatory funnel with added 6 mL –
distilled water

Added hexane with shaken & hexane part taken

Added Na2SO4
& filter

Concentrated & transferred into vial

for GLC

part (Aqueous layer)

Aqueous part (Na- salt of fatty acid)

  2 M H2SO4 added to
control pH 2.5

Hexane is
added and shaken

  Transferred in a separatory funnel

hexane part into a beaker

Anhydrous Na2SO4 was added
& filtered

 Transferred filtrate to the
weighed pear shaped flask and dried under reduced pressure

Again weighed which was only FFA

  Added 1 mg of C6H5COOH
& 2 mL of BF3 -MeOH complex

Reflux on boiling water bath, minimum 30 minutes

Dried by rotavapor & transferred in a separatory

   Added 6 mL of distilled
water, shaken & haxen added

Taken hexane part in beaker which is FFA of methyl

Added anhydrous Na2SO4, filter
& concentrated by rotavapor

in a vial & sent for GLC

3: Fatty Acids Analysis

3.6.5 Gas Chromatograms
Identification of Fatty Acids


1.0 µL sample was injected
through a filter into the injector of GC-MS and at the same condition; the
fatty acid standard mixture solutions were injected. Comparing the retention
times of the different peaks in the chromatogram of the sample with those of
standards, different fatty acids were identified.

3.6.6 Calculation of
the Relative Percentage of Different Fatty Acids


  Area of the peak

percentage of fatty acid =  X 100

Total area of the peaks

12: Standard Retention Time (RT) of Different Methyl Esters of Different Fatty
Acids from GC chromatograms.

Standard Retention times (min)

Fatty acids


Caprylic acid


Capric acid


 Myristic acid


Lauric acid


 Palmitic acid


Palmitoleic acid


Oleic acid


 Stearic acid


Arachidic acid


 Behenic acid


Lignoceric acid

3.6.7 Calculation of
Relative Percentage of FFA


From all the analyzed olive
relative proportion and relative percentage of free fatty acids in olive is
shown below-

In the present investigation,
oleic, arachidic, behenic, and lignoceric acids were identified as free fatty

Total areas of all the peaks of
free fatty acids = 3533+2982+2788+1831

 = 11134.

Relative percentage of Oleic acid   =   X 100

 = 31.73%.

Relative percentage of Arachidic
acid =  X 100

= 26.78%.

Relative percentage of Behenic
acid =  X 100

= 25.04%.

Relative percentage of Lignoceric
acid =  X 100

= 16.45%.

Relative proportion   = 31.73+26.78 +25.04+16.45

= 100.

The same calculation procedure
was followed for all.

Table 13: Relative Amounts of
Free Fatty Acids Elaeocarpus Robustus

times (minutes)






Oleic acid




Arachidic acid




Behenic acid




Lignoceric acid




Figure 6: Relative Percentages of
Free Fatty Acids in Elaeocarpus Robustus

Table 14: Relative Proportions of
Bound Fatty Acids in Elaeocarpus Robustus








Myristic acid




Palmitoleic acid




Oleic acid




Stearic acid



7: Relative Percentages of BFAs in Elaeocarpus Robustus

4.1 The Moisture


In the present investigation, the
moisture content of Elaeocarpus robustus was
found to be 84.13%, which is very important for our digestion and all other
mechanistic process of our body. Large moisture content of the fruits indicated
that even after processing of food substantial amount of water is coming to the
body fluid from the fruits. A healthier body needs a sufficient amount of
water. Water refreshes our blood circulation. The relative recommended44
moisture level in fruits helps to digest food properly. This water part has
several minerals and nutrients that help to develop the resistant power of
diseases inside our body.

The edible portions of fresh
fruits are generally juicy and the juiciness in many of the cases characterizes
the fruits themselves, so it is very likely that the water content of the
fruits should be high. These expectations were already been reported18,24
in most of the cases of tropical and subtropical fruits. Olives contain considerable
percentage of water. From the moisture content, it can be said that olive
covers a wide range of water intake inside our body.

Generally the higher the water
content the lower the calorie
45 of each fruit. This high water content can help
replenish fluid balance and make fresh and ready for exercise. It is also hard
to overeat with fruit portions as the large water and fibre content causes the
stomach to fill up quickly. For this reason, it may be a good idea to eat a
piece of fruit before each meal.

Measurement of oil and moisture
in olives provides information that enables maximization of yield through
growth of olive varieties that yield high oil levels, harvesting the crop at
the optimal time, and optimization of the oil extraction process. Since farmers
often receive payment on the basis of moisture and oil content and not simply
weight, both the olive producers and the olive processors profit from being
able to make instantaneous on-line or at-line measurements of these

4.2 The Ash Content


Ash is the inorganic residue
remaining after the water and organic matter have been removed by heating in
the presence of oxidizing agents, which provides a measure of the total amount
of minerals within a food. The amounts of ash content of the olives that are
locally used and conventional dietary intake, taken by the people of Bangladesh
are very important. Ash content measurement has been done to determine the
important minerals in those olives sample. Using ashes later on helped to
proceed on the iron content analysis of those samples. For the analysis of iron
as an important nutrient of human health, the ash content of the sample of
olive showed noticeable presence46. In the present experiment, the
ash content of the sample was found to be 3.72 % that is good agreeable to the
reported value46. Ash contents of fresh foods rarely exceed 5%,
although some processed foods can have ash contents as high as 12 %, e.g., dried
beef 47.

The measurement of ash content
is  the total amount of minerals present
within a food, whereas the mineral content is a measurement of the amount of
specific inorganic components present within a food, such as Ca, Na, K, Fe etc.
Determination of the ash and mineral content of food is important for a number
of reasons:

Nutritional labelling: The
concentration and type of minerals present must often be stipulated on the
label of a food.

Quality: The quality of foods
depends on the concentration and type of minerals they contain, including their
taste, appearance, texture and stability.

Microbiological stability: High
mineral contents are sometimes used to retard the growth of certain

Nutrition: Some minerals are
essential to a healthy diet (e.g. Ca, K, P, Na and Fe) whereas others can be
toxic (e.g. Pb, Hg, Cd and Al)

Processing: It is often important
to know the mineral content of foods during processing because this affects the
physicochemical properties of foods.

Identification of Free Sugars by PC


In the paper chromatograms, the
identified free sugars were found to be glucose,
galactose and arabinose. The Rf values of glucose, galactose and
arabinose were found to be 0.41, 0.45 and 0.50 respectively which are agreeable
to the literature value. If Rƒ value of a solution is zero,
the solute remains in the stationary phase and thus it is immobile. If Rƒ
value = 1 then the solute has no affinity48 for the stationary phase
and travels with the solvent front. The difference in Rf values
required for two substances to be separated depends on the size of the spots
and the length of the solvent flow.

4.4 Determination of
Free Sugars by Chemical Method


Total free sugars of the fruits
normally present the form either of reducing or in the form of non-reducing
sugars. Fruits might be considered as one of the main sources of energy for
human being and other animal due to presence of substantial amount of free
sugars. In present experiment the amount of reducing sugars, non-reducing sugars
and total free sugars were determined by chemical method. The total free
sugars, reducing sugars and non-reducing sugars were given in table-9. 0.77 g
total free sugar per 100 g fruit was found in the present investigation. This
value is somewhat lower than the reported18 value. The above
variations might be due to differences in fruit season, cultivars, storage time
and the procedures of analyses used. In fruits, mainly glucose and fructose
were identified as reducing sugars and in most of the cases, sucrose was
identified, as non-reducing sugar18,24. During hydrolysis
sucrose present in the fruit juice was broken down to equimolecular proportion
of glucose and fructose.

 It was observed that reducing sugar was higher
in proportion than non-reducing sugar. Thus, the fruits that contain higher
proportion of reducing sugar produced energy faster24 than the
fruits, which contain higher proportion of non-reducing sugar. Because the
sugars present in the form of non-reducing sugars like sucrose need to be hydrolyzed
before producing energy.

In the native olive fruit, the
free sugar was found to be less than one in the present investigation.
Therefore, it is recommended that who are facing the problem of excess blood
sugar (problem in diabetics) might take native olives as diet. Excess blood
sugar increases free radicals, which is associated with blocked arteries,
arterial damage, aging and heart disease.

4.5 Identification and Quantification of Free
Sugars by GLC


Glucose, galactose
and arabinose were the three free sugars identified and quantified by GLC. The
relative proportion of glucose, galactose and
arabinose were found to be 69.72 %, 16.95 % and 13.33 % respectively. Other
components from the gas chromatogram were not detected.

As most fruit calories come from
natural sugars, it is great idea to determine sugars for the general people who
can make decision which fruits they take as diet.

4.6 Identification and
Quantification of Fatty Acids


In the present study, oleic acid,
arachidic acid, behenic acid and lignoceric acid were identified and quantified
as free fatty acids. The relative proportions of the oleic acid, arachidic
acid, behenic acid and lignoceric acid were found to be 31.73 %, 26.78 %, 25.04
% and 16.45 % respectively. Myristic acid, palmitoleic acid, oleic acid and
stearic acid were identified and quantified as bound fatty acids. The relative
compositions of bound fatty acids were found to be 50.94 %, 30.03 %, 14.38 %
and 4.35 % respectively.

Fatty acids can be bound or attached to
other molecules, such as in triglycerides or phospholipids.
When they are not attached to other molecules, they are known as free fatty
acids. The uncombined fatty acids49 or free fatty acids may come
from the breakdown of a triglyceride into its components (fatty acids and
glycerol). However as fats are insoluble in water they must be bound to
appropriate regions in the plasma protein
for transport around the body. The levels of free fatty acid
in the blood are limited by the number of albumin binding sites available. Free
fatty acids are an important source of fuel for many tissues since they can
yield relatively large quantities of ATP. Many cell types can use either glucose
or fatty acids for this purpose.

Saturated and unsaturated are the
two types of fatty acids. Saturated50 fatty acid tends to increase
blood cholesterol levels. Most saturated fats tend to be solid at room temperature
with the exception of tropical oils. It is found mostly in meat and dairy
products as well as some vegetable oils. Butter is high in saturated fat, while
margarine tends to have more unsaturated fat. Saturated fats are a major risk
factor for heart attacks and strokes. Diets high in saturated fat50 have
been correlated
with an increased incidence of atherosclerosis
and coronary heart disease. Monounsaturated51
fatty acid tends to lower LDL cholesterol. It is found in both plant and animal
products. Such as olive oil, canola oil, peanut oil and in some plant foods
such as avocado. In the present investigation oleic acid and palmitoleic acid
were identified and quantified as monounsaturated fatty acids.

Poly unsaturated52
fatty acid tends to lower blood cholesterol level. It is found mostly in plant
sources (Safflower, Sunflower, Soya bean, Corn and Cottonseed). Although
polyunsaturated fats are protective against cardiac arrhythmias a study of post
menopausal woman with a relatively low fat intake showed that poly unsaturated
fat was positively associated with progression of coronary atherosclerosis,
where as monounsaturated fat was not. This probably is an indication of the greater
vulnerability of polyunsaturated fats to lipid per oxidation, against which
vitamin E has been shown to be protective. Although unsaturated fats are
healthier than saturated fats, the Food and Drug Administration53recommendations
stated that the amount of unsaturated fat consumed should not exceed 30% of
one’s daily caloric intake.

Fatty acids are essential parts
of all body tissues, where they are a major part of the phospholipid component
of cell membranes. Saturated fatty acids have been suggested to be the
preferred fuel for the heart54. Fatty acids are used as a source of
fuel during energy expenditure, and heavy exercise is associated with decreases
in the plasma concentrations of all free fatty acids. In light exercise, fat
metabolism may be controlled to favor adipose tissue lipolysis and extraction
of free fatty acids from the circulation by muscle, whereas in heavy exercise,
adipose tissue lipolysis is inhibited and hydrolysis of muscle triacylglycerols
may play a more important part55.In the absence of sufficient
dietary fat, the body is apparently capable of synthesizing the saturated fatty
acids that it needs from carbohydrates, and these saturated fatty acids are
principally the same ones that are present in dietary fats of animal origin.

Dietary stearic acid decreases plasma
and liver cholesterol concentrations by reducing intestinal cholesterol
absorption. Recent data from studies with hamsters, which have a lipoprotein
cholesterol response to dietary saturated fat that is similar to that of
humans, suggest that reduced cholesterol absorption by dietary stearic acid is
due, at least in part, to reduced cholesterol solubility and further suggest
that stearic acid may alter the microflora populations that synthesize
secondary bile acids56. Commercially, behenic acid is often used to
make hair conditioners and moisturizers for their smoothing properties. Because
of its low bioavailability and very long chain length compared with other fatty
acids, the effect of dietary behenic acid (behenate) on serum
lipid concentrations in humans is assumed to be neutral. In healthy
subjects, although myristic acid is hypercholesterolemic, it increased both LDL
and HDL cholesterol concentrations compared with oleic acid 57.
Oleic and monounsaturated fatty acid levels in the
membranes of red blood cells have been associated with increased risk of breast cancer.
Oleic acid may be responsible for the hypotensive
(blood pressure reducing) effects ofolive oil. Oleic acid may hinder the
progression of Adrenoleukodystrophy, a fatal disease that affects the brain and
adrenal glands. Oleic acid may help boost memory58.

4.7 Conclusion

The seed-free edible parts of
native olives were taken for the present study where moisture content, ash
content, total free sugars and fatty acids were analyzed by conventional
methods. All estimations were conducted on fresh weight basis considering the
presence of water content of the fruits materials. The water content was found
to be 84.13 % which showed little amounts of dry materials. Due to large
percentage of water the calories present in the fruits usually low. The ash
content was found to be 3.72% which showed46 the presence of
inorganic materials such as Fe, Ca, K, P, Na etc. With the help of ash content
one can analyze the quality of food. Glucose, galactose and arabinose were
identified by paper chromatography and gas liquid chromatography. In GLC the
relative amount of glucose, galactose and arabinose were found to be 69.72%,
16.95% and 13.33% respectively. The total free sugars were estimated by
chemical methods where it was found to be 0.77 g / 100 g. A piece of fruit
contains 0.077 g of free sugars. Portion size can vary between different fruits;
therefore total free sugars were calculated from an average piece of fruit. The
sweetness of fruit depends on the proportion of free sugars present in it.
Higher the free sugars sweeter the fruits are. The total free sugars either in
the form of reducing sugars or in the form of non-reducing sugars presents in
the fruits and these include a class of water soluble carbohydrates with
various degree of sweetness. Reducing sugars as well as non-reducing sugars
were found to be 0.41 % and 0.36 % respectively. Reducing sugars gives energy
quicker24 than non-reducing sugars. As the native olives contained a
little free sugar, it might be a good choice for diabetic patients and other
patients who are suffering from high blood-sugar problems. A number of free fatty
acids and bound fatty acids were detected where the individual fatty acids were
identified and quantified as their methyl esters by GLC. Myristic, oleic,
palmitoleic, arachidic, behenic, lignoceric and stearic acids were identified
and quantified. Among them only oleic acid and palmitoleic acid were found as
monounsaturated acids. Foods containing monounsaturated fatty acids lower low-density lipoprotein (LDL) cholesterol,
while possibly raising high-density lipoprotein (HDL) cholesterol58.
However, their true ability to raise HDL is still in debate. In children, consumption59
of monounsaturated oils is associated with healthier serum lipid profiles. The
relative percentages of the oleic acid, arachidic acid, behenic acid and
lignoceric acid as free fatty acids were found to be 31.73 %, 26.78 %, 25.04 %
and 16.45 % respectively.  The relative
compositions of bound fatty acids i.e. myristic acid, palmitoleic acid, oleic
acid and stearic acid were found to be 50.94 %, 30.03 %, 14.38 % and 4.35 %

8: GLC of Standard Mixture of Free Sugars

9: GLC of Free Sugars

11: GLC of Free Fatty Acids



Stearic acid



Oleic acid




Palmitoleic acid




Myristic acid



12: GLC of Bound Fatty Acids.

Having many varieties and
species, olives in our country are not alike to the alien olives. Our native
olive tree i.e. Elaecarpus robustus L.
(Fam. Elaeocarpaceae),
native to Bangladesh and India is a well-known evergreen 25 metres tall tree
which gives fruits, timber and fuel etc. The fleshy sour fruits having citric
acid and vitamin C are used in making soup, chutney, jelly
and jam which is
very delicious.

In the present study moisture
content, ash content, total free sugars analysis and fatty acids analysis were
carried out and determined. By using analytical grade reagents standard methods
were applied all the experiments. Dry matter and ash content of the fruits were
determined by standard conventional methods. For paper chromatography Whatman
no. 1 papers were used. All sugars analyses were performed on GLC by Sweely et
al. By Morrison et al. fatty acids analyses were performed.

Native olive fruits washing
properly in cool water to free from dirt were carefully sorted and chopped by
using a good fruit slicer which gave uniform pieces in sizes. Pre-weighed
edible portion of fruit samples were dried to constant mass in an electrical
oven at a temperature of 1050 C. The water content was found to be
84.13 %. Sample taken in a crucible was heated at a dull-red heat in a
muffle-furnace at 6000 C. The ash content was found to be 3.72 %.

Extracting with C2H5OH
the sliced edible parts of fruit samples were partitioned with DCM in a
separatory funnel after removing C2H5OH. DCM- extracted
aqueous layer was taken for free sugars analyses and fatty acids analyses. In
paper chromatography, glucose, galactose and arabinose were detected as free
sugars in where Rf values were found to be 0.41, 0.45 and 0.50
respectively. In chemical method the Fehling solution was
standardized with standard glucose solution. The total free sugars were
determined by standard Fehling solution. The total free sugars were found to be
0.77 %. Again, preparing TMS-derivatives the free sugars were identified and
determined by GC. The relative proportion of glucose, galactose
and arabinose were found to be 69.72%, 16.95 % and 13.33 % respectively.

Free fatty acids and bound fatty
acids were identified and quantified by preparing methylesters. The relative
amounts of oleic acid, arachidic acid, behenic acid and lignoceric acid as free
fatty acids were found to be 31.73 %, 26.78 %, 25.04 % and 16.45 %. The relative
proportions of myristic acid, palmitoleic acid, oleic acid and stearic acid as
bound fatty acids were found to be 50.94 %, 30.03 %, 14.38 % and 4.35 %


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