Reports On Bangladeshi Fruits

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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 decreases.

1.2 Carbohydrate in Fruits

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


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 Sugars

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 Soap

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 Acids

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

Binomial Name – Elaeocarpus robustus

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

Local Name English Name Scientific Name
Jalpai Olive Elaeocarpus robustus

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 Asia, 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

Elaeocarpus aberrans

Elaeocarpus acmosepalus

Elaeocarpus acrantherus

Elaeocarpus acuminatus: India. Endangered.

Elaeocarpus acutifidus

Elaeocarpus amboinensis

Elaeocarpus amoenus: Sri Lanka

Elaeocarpus amplifolius

Elaeocarpus angustifoliusBlue 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 castanaefolius

Elaeocarpus ceylanicus

Elaeocarpus colnettianus

Elaeocarpus coorangooloo: Australia

Elaeocarpus cordifolius

Elaeocarpus coriaceus

Elaeocarpus crassus: New Guinea

Elaeocarpus cruciatus

Elaeocarpus debruynii: New Guinea

Elaeocarpus decipiens

Elaeocarpus dentatusH?nau

Elaeocarpus dinagatensis

Elaeocarpus eriobotryoides

Elaeocarpus eumundi: Australia

Elaeocarpus fraseri

Elaeocarpus floribundus

Elaeocarpus ganitrusRudraksha Tree

Elaeocarpus gaussenii

Elaeocarpus gigantifolius

Elaeocarpus glabrescens

Elaeocarpus glandulifer

Elaeocarpus graeffii

Elaeocarpus grandiflorus: India, Indo-China, Malesia

Elaeocarpus hainanensis: Hainan

Elaeocarpus hartleyi: New Guinea

Elaeocarpus hedyosmus: Sri Lanka

Elaeocarpus holopetalus: New South Wales, Victoria (Australia)

Elaeocarpus homalioides

Elaeocarpus hookerianus🙁Pokaka) New Zealand.

Elaeocarpus inopinatus

Elaeocarpus integrifolius

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

Elaeocarpus johnsonii

Elaeocarpus joga Merr. – Yoga Tree

Elaeocarpus kaalensis

Elaeocarpus kirtonii: Australia

Elaeocarpus lanceifolius: South Asia

Elaeocarpus mastersii

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

Elaeocarpus miriensis

Elaeocarpus miratii

Elaeocarpus montanus: Sri Lanka

Elaeocarpus moratii

Elaeocarpus munronii

Elaeocarpus nanus

Elaeocarpus neobritannicus: New Guinea, Bismarck Archipelago

Elaeocarpus oblongus

Elaeocarpus obovatus: Australia

Elaeocarpus obtusus

Elaeocarpus petiolatus

Elaeocarpus photiniaefolius. Ogasawara Islands.

Elaeocarpus prunifolius

Elaeocarpus pseudopaniculatus

Elaeocarpus recurvatus

Elaeocarpus reticosus

Elaeocarpus reticulatusBlueberry Ash

Elaeocarpus robustus: India, Bangladesh.

Elaeocarpus royenii

Elaeocarpus rugosus

Elaeocarpus sallehiana

Elaeocarpus sedentarius

Elaeocarpus serratus: South Asia

Elaeocarpus sikkimensis: India, Bhutan

Elaeocarpus simaluensis

Elaeocarpus sphaericus

Elaeocarpus stipularis: Indo-China, Malesia

Elaeocarpus storckii Seem.: Fiji

Elaeocarpus subvillosus

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

Elaeocarpus symingtonii

Elaeocarpus taprobanicus: Sri Lanka.

Elaeocarpus timikensis: New Guinea.

Elaeocarpus tuberculatus

Elaeocarpus variabilis: Southern India.

Elaeocarpus valetonii

Elaeocarpus venosus

Elaeocarpus venustus

Elaeocarpus verruculosus

Elaeocarpus verticellatus

Elaeocarpus viscosus

Elaeocarpus whartonensis

Elaeocarpus xanthodactylus

Elaeocarpus zambalensis

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 robustusL. (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 diets.

The 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 robustustree 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

Olive 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 limestone 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 preparation

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.

Carbohydrates 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.

Fatty 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.

1. Determination of moisture content

2. Determination of ash content

3. Analysis of free sugar

(i) Identification of free sugar by PC

(ii) Quantification of free sugar by chemical methods

(iii) Identification as well as quantification of free sugars by GC

4. 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.

2.1 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 analysis.

2.6 Distillation of Solvents

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

2.10.1 Standard Derivatives of Free Sugars

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

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 dryashing. 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 Method

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.

Paper chromatography
Chromatography jar

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 tube 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 capillary action, 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.

(1) Ascending paper chromatography

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

(2) Descending paper chromatography

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 value

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.

Distance (cm) traveled by solute

Rf =

Distance (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 Chromatography

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