Studies on the Chemical Constituents of Carum roxburghianum Benth. (Radhuni) Seeds for its Essential Oil, Fatty Oil, Minerals and other Valuable Nutrients.

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Studies on the Chemical Constituents of Carum roxburghianum Benth. (Radhuni) Seeds for its Essential Oil, Fatty Oil, Minerals and other Valuable Nutrients.

1.1 General discussion:

From the prehistoric period the practical aspects of food, shelter, clothing and heat were of primary importance for these men depends on green plants. So man migrated to those areas where such facilities could be obtained readily. Plants are generally differentiated by human by their ability to manufacture food, the presence of cell walls, and their unlimited type of growth. The basic food for all organisms is produced by green plants. In this process of food manufacture, oxygen gas is liberated. This oxygen, which we obtain from air as we breathe in, is essential to life. The only source of food and oxygen is plant; no animal or man alone can supply these. Natural products are traditionally the cornerstones of drug discovery and research in this field continues to provide tremendous variety of lead structures, which are used as templates for the development of new drugs. Despite tremendous advances witnessed in modern medicine through synthesis, similarly aromatic plants are continually getting importance in healing of diseases and comfort of man. Medicinal and aromatic plants continue to hold an important part of pharmacopoeias in both the developed as well as developing countries. Medicine is a very ancient art and drugs have been used in days of antiquity as far back as history can take us. In particular higher plants have been the source of medicinal agents since earliest times and today they continue to play a dominant role in the primary health care of about 80% of more than 6 billion in habitants of the world rely chiefly on traditional medicines for their primary health care needs and it can be safely be presumed that a major part of the traditional therapy involves the use of plants, extracts or their active principles. It is impossible to think of medicine as something not connected with treatment and drugs have formed an integral part of treatment from the commencement of human memory. Increasing human population and increasing pressures on plants are leading to shrinkage in the world’s natural floral resources and having deleterious impacts on species and ecosystems. Recent estimates from the International Union for Conservation of Nature and Natural Resources (IUCN) has given grim statistics that over 12.5% of the worlds. Vascular plants are threatened at the global scale (Walter and Gillett 1998).Natural products and medicinal agents derived there from are also an essential feature in the health care systems of the remaining 20% of the population residing mainly in developed countries, with more than 50% of all drugs. Clinical use having natural product origin1. This is because of various reasons:1) There is historical legacy of folklore, ethno medicinal uses and well- documented indigenous system of medicine using natural resources for the treatment of diseases;2) They are believed to be comparatively safer than drugs modern medicine; 3) They are economical;4) They are environ mentally better suitable to local conditions; 5) They possess curatives/preventives for certain disorders for which modern medicine has nothing to provide 2. Bangladesh is endowed with rich plant resources on which we depend directly or indirectly not only for food and essential requirements but also for our very existence. This plant resource is impoverished by continuous loss and degradation of the plant diversity. To halt the decline in this priceless heritage, urgent action is needed at local, national and international levels.No concrete steps have been taken to arrest the process. The first and foremost step in this direction is to identify the threatened species and to assess their status in the wild. 1.2 Medicinal importance of the plant materials:From ancient times plants have been used as a source of medicines that form the backbone of human health care. The use of medicinal plants as a source for relief from illnesses can be traced back over five millennia to written documents of the early civilization in China, India and the nearest but it is doubtless an art as old as mankind 3.In the modern age, the scientists are endeavoring to isolate different chemical compounds from plants having biological properties. On the other hand while hunting well known lead compounds, many useful chemicals can be isolated from plant which are important for human comfort and health care 4, 7. 1.3 Chemical constituents of medicinal plants6:Plants produce within their cells and tissues, through metabolic activities, not only food materials but also the life sustaining oxygen gas and other substances, such as glycosides, steroids, terpenoids, alkaloids, flavones, tannins, pigments etc. There are usually called secondary metabolites that are responsible for their various, therapeutic properties and pharmacological actions. The compounds stored in their bodies may exert both harmful and useful effects on human beings. Traditional healers have been using toxic as well as nontoxic materials for treatment of various diseases. But unfortunately the traditional practitioners did not document the active principles of these natural products, so researchers have been trying to find out the active ingredient(s) of toxic and nontoxic plants 6.

The active principles of both the medicinal and poisonous plants are often definite chemical substances but in other cases. They are complicated mixtures. Briefly the following group of substances occurs in plants and is responsible for their medicinal as well as their toxic properties:

1) The first classes of these substances with medicinal and toxic properties are vegetable basis which include amines and alkaloids. As a class these bodies are characterized by their profound physiological action and in many case their intensely poisonous nature. Some of the amines give a footed odor to some weeds and to some mushrooms their poisonous characters. The alkaloids as a rule give a bitter taste to a plant in which. They naturally occur and that in itself is frequently a sufficient protection against livestock eating it, except in unusual cases of hunger. Examples of alkaloids are strychnine from nix, vomica, aconitum from aconites, atropine and allied alkaloids from belladonna, nicotine from tobacco, morphine from poppy, etc.

2) Another Class of these substances are represented by glycosides which form a large group and are much wider in occurrence than alkaloids many are non-toxic but quite a large number of them are intensely poisonous. They have generally a bitter taste and occur in many of the plant extracts used in medicine. Well known examples of toxic glycosides are those occurring in the oleander family (Apocynaceae) and Digitalis (Serophulariaceae).

3) The third group of active substances is furnished by essential or volatile oils which give characteristic odors to plants. These bodies are characterized by their insecticidal and insect-repellent properties, while in man and livestock they produce toxic effects by gastro-intestinal irritation. Common examples are those occurring in eucalyptus, in absinth which produces convulsions by its action on the nervous system.

4) The fourth group of toxic substances is known as to albumins which occur in castor, croton and abrus seeds; these are essentially blood-poisons and are responsible for frequent losses among livestock.

5) The fifth group of substances are called resins such as those occurring in podophyllum, bitters such as are found in wild members of the cucumber family for example colocynth, phenolic compounds such as those found in many members of the cashew family.

6) The sixth group of active substances occurring in plants is the antibiotics. It is well-known that some of the most powerful antibiotics now in use such as penicillin, streptomycin, etc are derived from the lower from of vegetable life, i.e. fungi 5.1.4 Herbaceous medicinal plant is extensively used as Spices and Condiments:Most of the medicinal plants are still a minor forest products and their population is decreasing day by day. Urbanization and deforestation, has led to alteration in the ecosystem with considerably decreased forest area. The only alternative appears to be large scale commercial cultivation of medicinal plants, particularly of those which are in high demand. It is rather significant that the intensive development on the earth of annual herbs that is of small, no woody plants that flower and produce seed in one reason had its beginnings in this early period and that during this same epoch man was emerging from the primitive stage. In fact this epoch has been called the age of man and herbs. Herbaceous annual plants were an important factor in human development, not only in the early period but also throughout man’s entire history. In ancient times, spices ranked with precious stones in the inventory of royal possessions and were monopolized by the few. The determined the wealth and policies of nations and also played an important role in ancient medicine. Besides, they also provided an incentive for the discovery of new water ways and new continents. The term ‘spices and condiments’ applies to “Such natural plant or vegetable products or mixtures thereof, in whole or ground form as are used for imparting flavor aroma and piquancy to and for seasoning of foods”.There are over 80 spices grown in different parts of the world and 50 spices are grown in India and Bangladesh. It constitutes an important group of agricultural commodities which are virtually indispensable in the culinary art. They also play a significant role in our national economy and so also in the national economics of several spice producing, exporting and importing countries. The people of those times used spices, as we do today, to enhance or vary the flavors of their foods, spices were also flavor disguisers, masking the taste of the fainted food that was still nutritious but would if unspiced have to be thrown away. Some spices were also used for preserving food. Such was the economic importance of spices in those days8?10.

1.5 General Description of Carum roxburghianum. Benth. (Radhuni):The family umbelliferae or Apiaceae consists of about 270 genera and 3,000 species which are cosmopolitan but chiefly temperate, especially in the northern hemisphere. Characteristic of the family11:This family of nearly exclusively herbaceous species is characterized by hermaphrodite flowers in a double umbel (Figure-1.1); note that the closely related Araliaceous have a simple umbel. Typical for the family are the furrowed stems and hollow internodes, leaveswith a sheathing base and generally a much divided lamina. The flowers are relatively inconspicuous, with two pistils, an inferior gynaecium with two carpels, a small calyx and generally a white to greenish corolla, with free petals and sepals.Chemical characteristics of the family11: Unlike the Araliaceae, members of this family are often rich in essential oil, which is one of the main reasons for the pharmaceutical importance for many of the apiaceous drugs. Also common are 17-carbon skeleton polyacetylenes, which are sometimes poisonous, and coumarins, which are responsible for phototoxic effects (e.g. in Heracleum mantegazzianum, Sommier and Levier, hogweed). Some species accumulate alkaloids (e.g. the toxic coniine from hemlock, Conium maculatum).? Genus Carum having three species:1) Carum roxburghianum Benth. (syn. Trachyspermum roxburghianum (DC.) 2) Carum copticum Heirn (syn. Trachyspermum ammi Linn.)3) Carum carviCarum roxburghianum Benth. (Syn. Trachyspermum roxburghianum (DC.) Craib.; Athamantha roxburghianum (Benth.) Wall. T. involucratum Marie) belongs to the family Apiaceae native to tropical Asia and is cultivated in Bangladesh, India and Indo-China. 1.5.1 Scientific Classification of Carum roxburghianum. Benth. Kingdom : Plantae Division : Magnoliophyta Class : Magnoliopsida Order : Apiales Family : Apiaceae (Umbelliferae) Genus : Trachyspermum (Carum) Species : T. roxburghianum (C. roxburghianum) Binomial name : Trachyspermum roxburghianum (DC.) CraibSynonyms: T. involucratum Wolff non Marie, Carum roxburghianum Benth Seeds of Trachyspermum roxburghianum (also known as Carum roxburghianum)1.5.2 Various names12, 13:Local name : Radhuni, Randhuni, Randhuni Soj, Choto Soj, Choto Dhonia English name : Celery, Bishops weed168 Other name : Marathi: Ova, Gujrati: Ajamoda, Hindi: Ajamoda, Tamila: Ashamtavoram, Punjabi: Kernauli, Tamil: Ajmoda, Bengali: Bandhuri, Chanur, Malayalam: Ayamodakam, Nepali Name: Ajmoda, Spanish : Aipo, French : Celeri, German : Sellerie, Swedish : Selleri, Arabic : Karafs, Dutch : Selderij, Italian : Sedano, Portuguese : Apio, Russian : Syel’derey, Japanese : Serorii, Chinese : Chin, Thai : Phak chi lom.1.5.3 Description of the herb Carum roxburghianum. Benth12:It is an annual or biennial herb. It is often cultivated as cold weather crop. Cultivated, adventives on forest margins and in rural areas. And also cultivated as a spice throughout the Indian subcontinent, SE Asia, and Indonesia. Stem: Plants annual, 20.100 cm. Leaves petiolate, petioles slen-der, 1.2 cm; blade ovate in outline, 3.8 × 2.12 cm, 2-pinnate or ternate-pinnate; ultimate segments narrowly oblong, 5.20 × 2.3 mm, base cuneate. Stem 1-3 feet (90 cm) high, erect, slightly branched, cylindrical, finely striated, smooth, pale green.Leaves:Leaves reduced upwards, ultimate seg-ments becoming linear-lanceolate. Umbels 2.4 cm across; peduncles 5.9 cm; bracts and bracteoles few, linear-subulate or ciliate, 3.5 mm; rays 4.12, 1.3 cm, filiform, unequal, hirsutu-lous or glabrescent; umbellules 12.20-flowered; pedicels 1.5 mm, unequal, hirsutulous. Flower:Flowers rather small, in terminal or axillary, compounds umbles; white or greenish white; numerous. On longish slender Pedicels. Calyx, teeth wanting. Petals, roundish, entire, with an involute obtuse apex, shining. Filaments, short, in curved stylopod?flat, over lapping the base of the petals; somewhat to bed. Styles are very short.Fruit:Fruit ovoid, Fruit about 1/5 inch (1.5-3.0 mm) long, broadly oval (ovid) in outline, apex contracted forming a very short neck, densely hirsutulous or glabrescent. Flower and fruits at Feb-July, rounded at both ends, smooth, tipped with the small stylopod; mericarps readily separable, much dorsal ally compressed, yellow to yellowish green when ripe; vittae solitary in each groove, broad, and two in the commissural.Root: Tap roof branched Used part: Seeds are mainly used as spice but it has a great use in medicine and its Herb can also use as same. ? The different parts of Carum roxburghianum (Radhuni) are shown in Figure–1.2, 1.3 & 1.4

Figure–1.2: View of the leaf and blooming flowering plant of Carum roxburghianum Benth. (Radhuni)Figure–1.3: View of the leaf of Carum roxburghianum Benth. (Radhuni)

Figure–1.4: View of the seeds of Carum roxburghianum Benth. (Radhuni)1.5.4 Habitat and range 5, 11, 14?16:Carum roxburghianum Benth. (Radhuni) is one of the 3 species of the genus Carum belonging to the family umbelliferae or Apiaceae. It is an annual herb, as a wild plant this is not unfrequented among corn and other crops throughout southern Europe, extending from Spain to the Caucasus and Persia and southward into Egypt and Ethiopia. It also occurs more rarely as a cornfield weed or casual straggles in Northern Europe, It is indigenous to the countries bordering the Mediterranean Sea, but also cultivated in the south of France, Xony and Russia. It has long been cultivated as a garden plant, and was grown by the Greeks and Romans. It was introduced to England in 1570 12, 13, 17, 18. It is also found to grow in the sub tropical region of India and Bangladesh. The plant is cultivated faintly extensively in the northern part of Bangladesh. In Bangladesh radhuni is grown in Dhaka, Natore, Rajshahi, Naogaon, Bogra, Dinajpur, Rangpur, Gaibandha, Jaipurhat, Sirajgonj, and middle part in Dhaka (Keranigonj) and Faridpur. Gajipur. And it is also grown in experimental field of Spice research center (BARI) Plant History:C. roxburghianum regarded as a Radhuni or Randhuni soj in Bangladesh, Indian counterparts of is a cold weather crop, largely cultivated for culinary and medicinal purposes and also for fodder C. roxburghianum is grown mostly for its herb oil and for its seed oil. This species very closely resembles and is probably a cultivated from of T. stictocarpum (C.B. Clarke) wolff syn. C. stictocarpum C.B. Clarke, Which is found wild from the lower Himalayas to South India. There are, minute difference in the fruit due to cultivation12, 21. The fruit ajmod like those of ajowan are subjected to the attack of spice-beetle or drugstore beetle in store house22. C. roxburghianum, often used in Indian cuisine, are a kind of very strong spice with characteristic smell similar to parsley. A couple of pinches can easily overpower a curry23. In Bengali cuisine the seeds are used in whole, quickly fried in very hot oil until they crackle. They are part of a local panch phoran (Bengali five spice) mixture, where they replace the more commonly used mustard seed; the other ingredients are white cumin seed, fenugreek seed, fennel seed and kalongi24. The main component of seed oil was limonene. Other notable constituents were sabinene, terpinen-4-ol, (Z) – ligustilide and ?-terpinene26. Fruit contains Bergapten, essential oil comprises of ? and ?- pinenes, sabinene, terpenine, ? and ?- phellandrene, linalool, ?- terpineol, thymol, carvacrol, ?- cyclovandulic acid and serelin11.1.5.6 Planting and cultivation:Radhuni is a cold-season (Rabi) crop and is fairly tolerant to frost and very low temperature. Under irrigation, the crop can be grown as pot-herb throughout the year. It is best suited to tracts of moderate or low rain fall20.Radhuni grows well in any good garden soil, a fertile, prepared, sandy loam being most suitable, light sand or heavy clay should be avoided. It seeds are small size; the soil must be smooth and free of clods. Care should be taken not to fertilize directly beneath radhuni, because this would favor uneven ripening and development of weeds. Although a native of Mediterranean countries, Radhuni is quite a hardy plant and may thrive in much cooler climates, provided it finds a warm situation and well-drained soil12, 20. For good early growth of the crop and better yield the field should be well prepared. The land should be plowed in the fall, or as early in the winter as the weather permits. The usual procedure is to sow the seed, November-December (very early in winter), about 1/2 inch deep, with specially constructed drills. The rows are spaced at 14 to 18 inches the drills 1ft apart. Depending on the method of cultivation for the control of weeds, the rows are sometimes spaced as far as 3ft apart. The planted seed needs but a thin covering of soil. It has been claimed that the seed crop is better when the plants are not crowded too much. For the same reason, the plants should be thinned at the proper time, so that their distance in the rows is not less than 6 to 15 inchs20. One–half ounce of seed suffices for 150ft of drill; at this rate, 1 lb. should sow one acre. Goods result has been obtained by sowing late in the fall; the seed then germinates in the winter as soon as conditions become favorable. This is often much earlier than the ground can be prepared and the seed planted, in the case of winter sowing. Radhuni herb seems to be more susceptible to weather hazards than most crops. If heavy hail hits the plants during the flowering period, they may be injured so much that the yield of oil is practically nil. The same might happen through damage by strong winds or driving rains. Extreme heat during the critical period of maturity is apt to blight the herb, with similar results in regard to yield of oil. Nitrogen and phosphorus as urea and superphosphate was applied uniformly in the plots for yield eve crop 80kg N/ha and 30kg P/ha were found best for fruit yield and 40kg N/ha and 30kg N/ha and 30kg P/ha for essential oil. The combined application of same doses of N and P gave much more pronounce results. The combination of N80 P30 and N40 P45 gave highest fruit yield and essential oil content; respectively 80kgN, 30kg P and The combination of N80P30 gave 53, 20.2 and 96.8% more fruit yield over the respective controls8.1.5.7 Harvesting:Proper timing of the harvest is very important, the quality of oil depending mainly upon the state of maturity of herb and seed the harvest takes place in early fall. For the production of Radhuni herb oil, the plant should be harvested immediately after the blooming period, when the seed has just started to ripen but is not yet fully developed. This period is very short, lasts not longer than two weeks, and usually falls sometime between the 1¬¬¬st¬ ¬week of March and the end of April20. To obtain good, typical herb oil, distillation has to be speeded up and a great amount of plant material must be distilled within a short period of time. It is advisable to harvest only as much as can be processed in the distillery during one day. After very short drying in the fields, the herb is hauled to the distillery. Drying for many hours in the field would result in considerable loss of oil by evaporation, especially of the more volatile terpenes consequently, the oil distilled from dried herb would be relatively high in carvone. The herb should be distilled as fresh as possible; otherwise the seed attached to the stacked up plant material continues to ripen, and the oil thus obtained approaches the undesired seed oil character.From the middle of April to mid May the seeds mature fully and must be harvested very carefully when the seeds have lost their green colour. For this purpose the plants are cut with scythes or machines, then bundled and stacked up in the field until all seeds become fully ripe and dry. Subsequently the threshed seed is dried in barns in order to prevent the formation of mold. Finally the seed is winnowed. The main November?December sown crop is ready for harvesting during March–April and January–sown crop is ready in April?May. The herb is not affected by many diseases and pests. However, collar and root- pot, caused by Sclerotium rolfsii Sacc has been recorded. It has been also reported that Radhuni treated with a number of common fungicides18, 20, and 25. 1.5.8 Yield:Yields are variable, depending upon whether the crop is grown as a dry crop or as an irrigated crop, mixed or pure and on the number of cuttings before seed-setting. The yield of seeds varies considerably. The yield of seed per acre in North America ranges from 500-700 Ib and herb oil varies from 20-30 Ib per acre; it may be higher or lower in exceptional years. In India Radhuni is grown in about 10,000 hectors producing nearly 5000 MT. of seeds19. In Hungary produces 1000 to 2000 kg of green Radhuni herb per 1.422acres and yield of herb oil ranges from 0.29 to 1.50 percent, yield of Radhuni seed 1.422 acres produces about 300 to 700 kg and yields oil from 2.30 to 2.50 percent12. In England one acre yields about 700 Ib of Radhuni seed. In Bangladesh Radhuni is grown in about 10000 hac producing nearly 5000Mt of seeds and yield of essential oil from seeds ranges from 1.20 to 2.5 percent20.1.5.9 Storage:Fresh Radhuni for vegetable does not store well and should, therefore be disposed of soon after harvesting. The well-dried leaves however can be stored for one year the seed can be stored for two years10, Use of Carum roxburghianum herb and its seeds:In Medicine:C. roxburghianum plant oils and extracts have been used for a wide variety of purposes for many thousands of years27. The seeds are well known for their medicinal properties, mostly due to the essential oil in them both seeds and oil enter into the composition of various indigenous medicinal preparations. The essential oil, Radhuni oil, or its emulsion in water, are the main constituents of “ grip water ” and considered to be aromatic, carminative specially useful in flatulence, collie pain, Anti-diarrheal, Anti-tumor, Anti-oxidant, CNS, Diuretic, Cardiovascular, Hypotensive, vomiting, Spasmolytic and hiccups of infants and children the seeds are useful in hiccup, vomiting and pain in the bladder13. They form ingredients of carminative and stimulant preparations and are very useful in dyspepsia and flatulence28. Seeds ketonic compound showed antispasmodic activity particularly on smooth muscle of rabbit gut 29. Essential oil and crystalline substance lowered blood pressure in dogs and rats due to direct action on blood vessels30. Fruits left after extraction of essential oil showed marked cardio tonic activity30 and it also shows antibacterial activity31. In particular, the antimicrobial activity of plant oils and extracts has formed the basis of many applications, e.g. in raw and processed food preservation, pharmaceuticals, alternative medicine and natural therapies32, 33. Pharmacological studies have been carried out on different fractions of drug. The crystalline ketonic substance (C13H12O3; m.p., 117-18?C) exhibited powerful antispasmodic activity34: the action was particularly marked on the smooth muscle of the rabbit’s gut. The essential oil and crystalline substance were found to lower blood pressure in dogs and rats35, 36; the effects were due to the direct action on the blood–vessel. The oil produced marked diuretic effect in rabbits. The fruits left after the extraction of the essential oil showed pronounced cardio tonic activity12.As a food flavoring37: Radhuni seeds are used, both whole and ground, as a condiment in soups, salads, processed meats, sausages, spicy, table sauces, salads, sauerkraut and particularly in Radhuni pickling. Radhuni stems and blossom heads are used for Radhuni pickles and for flavoring soups, Ground seed is an ingredient of seasonings. Sometimes, it is used as a substitute for caraway12. Radhuni oils are used as soap perfume and n food industry for flavoring and seasoning8, 10. The oil has a strong odor, and an unctuous farinaceous taste with slight bitterness38, 39. Herb is also used as a fodder for cattle40, 41.Other uses:The essential oil of the seed and its fractions possess toxicity and insect repellent properties against pesticides of stored grain insect, especially during the rainy season. The insect repellent property investigated against Tribolium castaneum herbs38, 39. The oil and its fractions, applied topically or impregnated on filter papers, were shown highly toxic to larvae and adult beetles of Tribolium castaneum. At the highest dosage of 100?l/ml killed all the adult beetles and larvae within 48h the overall repellency in the range 45-95%. The seeds extracts of Radhuni products can improve glucose metabolism and the overall condition of persons with diabetes not only by direct hypoglycemic effects but also by improving lipid metabolism, antioxidant status and capillary function40, 41. Use of essential oils is not under regulatory control in many countries, although very little is known about their acute toxicity. Only a few papers contain data on their “latent” toxicities such as teratogenesis, carcinogenesis and mutagenesis8, 10. 1.6 Objective of the Research Program:Bangladesh is flourished with plants, herbs and trees. Various herbaceous medicinal and aromatic plant have been used for medicinal, spices and flavoring purpose in Bangladesh. Carum roxburghianum (Radhuni) is a popular pot herb. It has been cultivated and consumed mostly in the northern part of Bangladesh. The composition of plant based product depends on the agro climatic condition (climate and soil) of the country. The chemical composition of the Radhuni herbs varies with the agro climatic conditions of the part of the country where it is grown.Many Investigations have been carried out but no systematic research has been carried out on Carum roxburghianum (Radhuni) in Bangladesh. Some disagreement about the presence of its constituents was observed. Therefore the present work was undertaken to carry out a complete investigation of the Carum roxburghianum (Radhuni) seeds. The followings are the aims and objective of the study: 1) To carry out the complete study and to get information for consumption and utilization as well as industrial application of plant and its product. 2) To observe its popularity consumption and availability as a pot herb and condiment (its seed) as well as to explore export market to the foreign countries. 3) Characterization quantification and identification of the constituent compounds of the essential oil isolated by steam distillation from its seed by GC-MS (gas chromatography and mass spectroscopy). 4) Characterization of the fatty oil isolated from the seeds by TLC and GLC. 5) To investigate the mineral contents including toxic heavy metals and nutrients which remained after extraction of essential oil as well as fatty oil of seeds by XRF Spectrometer and Atomic absorption spectrometer and flame photometer (AAS). 6) A comparative study on the composition of Carum roxburghianum (Radhuni) seeds of different region of Bangladesh. 7) To carry out the complete study on the nutrients of Carum roxburghianum (Radhuni) seeds.1.7 Scheme of the work:Carum roxburghianum (Radhuni) seeds were collected from different parts of Bangladesh. Following experiment were undertaken for complete investigation on Radhuni seeds. 1. Essential oil part: a. Extraction of Essential oil by means of steam distillation. b. Determination of Physical and Chemical properties of the essential oils. c. Analysis of the oils by GC?MS for identification and quantification of its constituent compounds as well as their structures. 2. Fatty oil part: a. Extraction of fatty oil. b. Determination of Physical and Chemical properties of the fatty oils. c. Methylation of the extracted fatty oil for having methyl ester of fatty acids for TLC and GLC experiment. d. Determination of approximate number of fractions of methylated fatty oils by TLC. e. Determination of fatty acids methylated constituents of the fatty oils for identified and quantified by GLC. 3. Residual part: a. Determination of moisture, ash, crude fiber, nitrogen, i.e., protein carbohydrates content and food energy of the seed and flower. b. Determination of mineral content of seeds of the herb by XRF spectrometry and AAS and flame photometry. c. Identification of toxic and useful trace elements contained in the seeds.

Scheme–1.1: Scheme of the research work1.5 Collection of sample:Carum roxburghianum regarded as a Radhuni or Randhuni soj in Bangladesh, Indian counterparts of is a cold weather crop, largely cultivated for culinary and medicinal purposes in Bangladesh. According to the aim of our research work we collected the sample from three different district of Bangladesh. These are the following region of sample collection:Sample-1: Mature seeds are collected from Regional Spice Research Center Joydebpur, Gajipur, in the month of March, 2009 which mentioned as sample ID: C. ROX-1.

Sample-2: Mature seeds are collected from field of cultivation of Keranigonj, Dhaka, in the month of March, 2009 which mentioned as sample ID: C. ROX?2.

Sample-3: Mature seeds are collected from Regional Spice Research Center Faridpur, in the month of August, 2008 which mentioned as sample ID: C. ROX?3.

The collected sample were kept in airtight plastic bags, sealed and transported to the laboratory. The sample was cleaned to separate dirt and sun dried.

Figure–1.5: Area of sample collection from different parts of BangladeshLiterature survey

Carum roxburghianum Benth. is a species of umbelliferae or Apiaceae and genus carum. Genus carum having three species:1) Carum roxburghianum Benth. (Syn. Trachyspermum roxburghianum (DC.) 2) Carum copticum Heirn (Syn. Trachyspermum ammi Linn.)3) Carum carviCarum roxburghianum is grown is the sub-tropical and temperate regions of India and Bangladesh. It is a popular herb in Bangladesh mainly in the North–Eastern part of Bangladesh. People of this region use the green herb as a pot–herb and as a flavoring agent especially in the winter season. The green herb is also used as a flavoring agent in soups, table sauces and salads. Seed has got a very limited use as condiment but it has got an export possibility.

The herb and its seeds are used as folkloric medicine e.g. aromatic carminative especially useful in flatulence, colic and hiccups of infants and children.The popularity of the herb is primarily due to the characteristic flavor, which is responsible to the essential oil present in the herb as well as in its seed. Many important elements which are present in the herb and its seed may have important metabolic and nutritional activity for human being.No systematic study has been carried out so far in Bangladesh to ascertain the usefulness of the herb and its seed. The following are the reported information found in the literature.

Thomas, Allahad Fmr22 reported, this species very closely resembles and is probably a cultivated from of T. stictocarpum (C.B. Clarke) wolff syn. C. stictocarpum C.B. Clarke, Which is found wild from the lower Himalayas to South India. There are, minute difference in the fruit due to cultivation. The fruit ajmod like those of ajowan are subjected to the attack of spice-beetle or drugstore beetle in store house.

Ashraf, M.; Aziz, J.; Bhatty, M.K42 investigated on the essential oils of the Pakistani species of the family Umbelliferae.V. Carum roxburghianum seed oil. And reported that Yield, physico-chemical properties and chemical composition of essential oil from fresh mature and 1 yr-old unripe seeds of C. roxburghianum were compared. Tabulated results showed that compared with 1 yr old unripe seeds, fresh mature seed oil had lower optical rotation (+19 degree 12′ vs. +35 degree 14′) and higher acid value (3.1-3.8 vs. 1.42-1.50) lower total hydrocarbons (44.2 vs. 55.0%), monoterpenes (20.9 vs. 26.0%), limonene (15.1 vs. 20.8%), terpinene (1.9 vs. 2.6%) and sesquiterpene (23.3 vs. 29.0%) contents, and higher oxygenated compounds (55.8 vs. 45.0%) and a novel unidentified ketonic acid (mol. wt. 168, C10H16O2, previously found in Carum roxburghianum essential oil).

K. M. Braj and D. Sikhibhushan34 & S. Choudhury, A. Rajkhowa, S. Dutta, et al26 reported that Terpinen-4-ol, (Z)-ligustilide and ?-terpinene, which have been reported as major constituents in the fruits essential oil of Indian origin.

Chowdhury, Bhuiyan, and Begum43, reported that Essential oil from the leaves and fruits of Carum roxburghianum Benth. is analyzed by gas chromatography-mass spectrometry (GC-MS). The oil leaf is dominated by apiol (20.81%), 2(3H)-furanone,dihydro-5-pentyl- (17.21%), 1,3-benzodioxole, 4-methoxy-6-(2-propenyl)- (9.51%) and citronellol (6.62%). The fruit oil is rich in 2-cyclohexen-1-one, 2-methyl-5-(1-methylethenyl)- (40.03%), and other components that follow are apiol (18.71%), limonene (17.11%), myristicin (12.30%), dihydrocarvone (7.89%) and eugenol (1.68%). The compositions of both oils vary qualitatively and quantitatively.

Malavya & Dutta45 reported that the Carum roxburghianum fruits yield an essential oil (up to 2.5 %), a fixed oil (4.5 %). The essential oil obtained by steam distillation is greenish yellow in colour and has following characteristics sp. Gr.20?C, 0.9488; [?]D33?,+25.5?; n20?C,1.4880; acid value, 4.9; sap. val., 49.1; sap. value after acetylation, 74.2. The oil contains: d-linalool, 4.7; d-limonene, 35.1; ?- terpinene, 19.4; dl-pipertone,5.7; thymohydroquinone 0.2: thymol 1.7; dl- piperitone, 13.6; cuminic acid,0.4; cumminaldehyde, traces; an unidentified ketone (C10H14O3).1.0; unidentified esters ,5.9; And d- limonene and dipentene mixture, 2.5 %.

M.L. Gujral et al.46 reported that Seeds of C. roxburghianum yielded 1.8-2.0% of an essential oil, 4.4-4.5% of fatty oil (n2D 1.4914, 98.4, Sapon. no. 189.5), and 0.08-0.09 % of a ketonic cryst. Compd.(?); 2,4 dinitro phenyl hydrazone m. 95-6?C, p-nitro phenylhydrazone m. 204-5?C,has spasmolytic activity.

Mahmud, Shahid ; Saleem, Muhammad ; Yamin, Muhammad ; Khan, Muhammad Naeem47 reported that Total lipids extracted from the powdered seeds of Carum roxburghianum were fractionated into hydrocarbons (0.30%), wax esters (0.30%), sterol esters (1.35%), triacylglycerols (72.41%), free fatty acids (6.06%), 1,3-diacylglycerols (4.60%), 1,2- diacylglycerols (0.64%), glycolipids (5.10%), sterols (1.20%), 2-monoacylgylcerols (3.18%), 1-monoacylglycerols (1.46%), phosphatidylethanolamines (1.08%) phosphatidylcholines (0.40%), lysophosphatidylethanolamines (1.48%) and phosphatidylinositols (0.44%) with the help of TLC. The fatty acid composition of all the lipid fractions was determined after converting them into their methyl esters with BF3–methanol reagent and then analyzing them by GC. Oleic acid was found as a major component in all the lipid classes, whereas palmitic, linoleic and linolenic acids were present in lesser quantities. Arachidic acid was identified as a minor component in only seven out of twelve lipid classes.

Tanveer Ahmad Chaudri, Ijaz Ahmad, M. Yamin and Shahid Mahmud52 reported that the triacylglycerols were first sepd. from the seed oil of Carum roxburghianum by plain TLC and then fractioned into groups differing in unsatn. by argentation. The position of fatty acids at 1, 2 and 3 carbons of triacylglycerol mols. in each group were detd. by lipolytic hydrolysis with pancreatic lipase and GLC by methyl esters. The position of these glycerol’s was found to be occupied by the unsatd. acids preferentially. The level of unsatn. detn. the distribution of the fatty acids. The results of the study allow possibility of predicting the distribution pattern of fatty acids in different triacylglycerol fractions.

Shiojir;MasatoshI; Ito; Hisatomi53 reported that Carum plant seed exts. From Carum copticam, C. bulbocastum and C. roxburghianum are claimed as NGF formation promoters and health foods for treatment and prevention of memory and learning disorders Alzheimer’s disease and diabetic neuropathy. Formulation examples of health foods and health drinks were given.Mahmud, Shahid; Wahid, Amran; Khnum Razia54 were reported that the lipase and phospholipase activities of C. roxburghianum were studied at different temps. Solvents and pH. Both the enzymes showed the max. activities at 40?C and in n-heptane used as solvent. However the max. activities of two different pH one at pH 5 (acidic) and other at pH 8 (alk). Whereas phospholipase showed only one pH optimum at pH 8. During the course of germination the lipase showed an increase whereas reverse was the case with phospholipase.

Chandhuri. Tanweer Ahmed; Ahmed, Ijaj; Muhammad, Din; Ahmad, Manjoor55 reported in the family Umbelliferae octadecenoic acid (C18:1) is a phylogenitic character having distribution on the different positions of triacylglycerols mols. It has a role as a key intermediate in fat metab. during seed germination which created an interest to investigate further to find out its positional isomers in the oils of Carum species. Therefore, the octadecenoic acid sepd. from the seed oil of Carum copticum, Carum carvi and Carum roxburghianum of umbelliferae family was oxidized septd. By von Rudloff’s, reagents. The liberated mono and difunctional fatty acids were sepd. and identifiedby the application of thin layer and gas liq. Chromatog. to det. positional isomers. The positional isomers detd. among these three species were cis-6-octadecenoic acid, cis-9-octadecenoic acid. The percentage of these isomers varied from 4.5 to 46.0%.Proximate Analysis

3.1 Determination of moisture content and dry matter 56?59, 155:Procedure112:At first Petri dish was dried and weighed in an electronic balance. Then definite amount of sample was taken in the Petri dish and weighed accurately. The Petri dish with sample was placed in an oven at 110°C for 6 hrs. The oven dried Petri dish was taken in desiccators. After 30 min the Petri dish was removed from desiccators and weighed. In this way the Petri dish was weighed repeatedly after heating and cooling in the same way until the constant weight was obtained. The percentage of moisture content was calculated by the following formula:% of moisture content = Where,Weight of moisture = the wt. of sample before drying – the wt. of sample after drying. % of dry matter = 100?moisture %Calculation method: Before drying,Weight of Petri dish = w1 gmWeight of Petri dish + sample = w2 gmWeight of sample = (w2?w1) gm After drying,Weight of Petri dish + sample = w3 gmWeight of moisture = (w3?w2) gm% of moisture = = % of dry matter = 100 ? moisture %Table–3.1: Moisture and dry mater contents of C. roxburghianum seeds:

Sample collectedfrom Weight ofSample, (w2- w1) gm Weightof moisture, (w3- w2) gm Moisture %, Dry matter %, 100 – moisture %.Joydebpur, Gajipur 7.2428 0.7099 9.8014 90.1985Keranigonj, Dhaka 7.5459 0.8081 10.7091 89.2909Faridpur 5.8469 0.6783 11.6010 88.3990

? Each value represents the average value from three experiments.3.2 Determination of ash content (the destruction of organic matter) 56-59:

Plant sample contains organic matter which must be destroyed prior to the estimation of minerals. Dry ashing is generally used for the destruction of organic matter. The choice of procedure depends on the nature of organic material, the nature of any inorganic constituent present, the metal to be determined and the sensitivity of the method used for the determination. The ash remaining following ignition of medicinal plant materials is determined by three different methods which measure total ash, acid-insoluble ash and water-soluble ash.The total ash method is designed to measure the total amount of material remaining after ignition. This includes both “physiological ash”, which is derived from the plant tissue itself, and “non-physiological” ash, which is the residue of the extraneous matter (e.g. sand and soil) adhering to the plant surface.Acid-insoluble ash is the residue obtained after boiling the total ash with dilute hydrochloric acid, and igniting the remaining insoluble matter. This measures the amount of silica present, especially as sand and siliceous earth.Water-soluble ash is the difference in weight between the total ash and the residue after treatment of the total ash with water.Apparatus:? Crucible (porcelain or nickel)? Bunsen burner? Muffle furnace? Ash less filter-paperReagents:? Potassium dichromate? Sulphuric acid? Dil. HCl

3.2.1 Cleaning the crucible and it prepared for ash62:At first nickel or porcelain crucible were taken. For cleaning the crucible chromic acid was prepared. For this purposes potassium dichromate was taken in a beaker and sulfuric acid was poured to the beaker. For better cleaning chromic acid was kept on crucibles for several hours. Washing the crucibles with clean water and finally rinsed with distilled water. Crucibles were kept in oven at 110?C for drying. Then blank crucibles were heated in a muffle furnace at 650?C for 6 hrs and cooled in desiccators and weighed. The process of heating and cooling was continued till a constant weight was obtained.3.2.2 Determination of Total ash 62, 121: A certain amount of moisture less sample was taken in a crucible and it was weighed (which was previously been cleaned, heated at 650?C and cooled and weighed). To remove the organic matter as much as possible, the crucible with sample was fired with Bunsen burner. Then the crucible heated to a temperature controlled muffle furnace at 650?C for 6 hrs. Then the crucible was removed from the furnace and allowed to cool in desiccators and weighed. If charred matter still remains, add small amount of ethanol, break up the ash with a glass rod, wash the glass rod with small amount of ethanol, and evaporate carefully the ethanol. Then, proceed as directed previously and weigh accurately. The process of heating and cooling was repeated till a constant weight was obtained and the ash is almost white or grayish white in colour.Calculation method: Before ignition,Weight of crucible = w1 gmWeight of crucible + sample = w2 gmWeight of sample = (w2 – w1) gm

After ignition,Weight of crucible + ash = w3 gmWeight of ash = (w3- w1) gm% of ash = % of organic matter = 100 – ash %Sample collected from Weight ofSample,(w1- w2)gm Weight ofAsh,(w3- w1)gm % of ash , % of organic matter, (100 – ash %)Joydebpur, Gajipur 5.0277 0.4521 8.9920 91.008Keranigonj, Dhaka 5.3340 0.4807 9.0124 90.9876Faridpur 3.7628 0.3102 8.2431 91.7569

Table?3.2: Total ash contents of Carum roxburghianum (Radhuni) seeds:

? Each value represents the average value from three experiments.3.2.3 Determination of Acid soluble and insoluble ash100, 121:The Acid-insoluble ash limit Test is designed to measure the amount of ash insoluble to diluted hydrochloric acid.Procedure: Add carefully 25 ml of dilute hydrochloric acid to the 1 gm of ash (obtained as directed under the Ash Limit Test or total ash), boil gently for 5 minutes, collect the insoluble matter on an ash less filter-paper for quantitative analysis, wash thoroughly with hot water, and dry the residue together with the filter paper. Ignite it for 1 hour in a crucible of platinum, quartz, or porcelain, which has been prepared as directed in the Ash Limit Test and whose weight is already known. Cool it in desiccators (silica gel) and weigh accurately. If the measured amount is larger than the specified value, ignite until a constant weight is obtained.Calculation method: Weight of ash sample = w gmWeight of crucible = w1 gmWeight of crucible + insoluble ash = w2 gmWeight of acid insoluble ash = (w2 – w1) gm% of acid insoluble ash = % of acid soluble ash = 100 – % of acid insoluble ashTable?3.3: Acid soluble and insoluble ash content of C. roxburghianum seeds:Sample collected from Weight of ash sample, w gm Weight of acid insoluble ash, (w2 – w1) gm % of acid insoluble ash, % of acid soluble ash, 100 – % of acid insoluble ashJoydebpur, Gajipur 1.1344 0.4534 39.9682 60.0318Keranigonj,Dhaka 1.1514 0.5236 45.4750 54.5249Faridpur 1.1929 0.6035 50.5924 49.409

? Each value represents the average value from three experiments.3.2.4 Determination of Water soluble and insoluble ash 91, 100:The water soluble ash limit Test is designed to measure the amount of ash soluble to hot water.Procedure: Add carefully 25 ml of distilled water to the 1 gm of ash (obtained as directed under the Ash Limit Test or total ash), and boil for 5 minutes. Collect the insoluble matter in a sintered-glass crucible or on an ash less filter-paper. Wash with hot water and ignite in a crucible for 15 minutes at a temperature not exceeding 450°C. Subtract the weight of this residue in gm from the weight of total ash. Calculate the content of water soluble ash in per gm of air dried material.Calculation method: Weight of ash sample = w gmWeight of crucible = w1 gmWeight of crucible + insoluble ash = w2 gmWeight of water insoluble ash = (w2 – w1) gm

% of water insoluble ash = % of water soluble ash = 100 – % of water insoluble ash

Table?3.4: Water soluble and insoluble ash contents of Carum Sample collected from Weight of ash sample, w gm Weight of water insoluble ash, (w2 – w1) gm % of water insoluble ash, % of water soluble ash, 100 – % of water insoluble ashJoydebpur, Gajipur 1.0860 0.8748 80.5525 19.4475Keranigonj,Dhaka 1.0461 0.8027 76.7326 23.2674Faridpur 1.0006 0.7028 70.2345 29.7655 roxburghianum (Radhuni) seeds:

? Each value represents the average value from three experiments.3.3 Estimation of nitrogen content: Kjeldahl’s method 57, 65, 100:This process is widely employed in industrial and research laboratories. The principle of the method is the conversion of the nitrogen of the nitrogenous substances into ammonia by boiling with concentrated sulfuric acid which was fixed by the excess of acid as ammonium sulfate. The latter is determined by adding an excess of caustic alkali to the solution after digestion with the acid and distilling off the liberated ammonia into standard acid. Simple digestion with concentrated sulfuric acid is, however, a slow process and various modifications have been suggested to increase the speed of the reaction. This includes the addition of potassium sulfate, which raises the boiling point of the acid (Kjeldahl-Gunning process), and of catalysts, such as mercury, mercuric oxide etc. the simple process works well for nitrogen determinations in proteins and also in amines and amides, but is not applicable to nitro, azo, hydrazo and cyano compounds without modification. In the presence of nitrates there is a danger of loss of nitric acid, salicylic acid is then added to the sulfuric acid, which fixes the nitric acid as nitro-salicylic acid. Upon the addition of zinc dust or of anhydrous sodium thiosulfate, the nitro-salicylic acid is reduced to the amino compound, which can then be estimated by the Kjeldahl process.

Reagents: ? 0.1 N H2SO4solution? 0.1N Na2CO3 solution? 60% NaOH solution? 2% Boric acid solution? Digestion mixture? Concentrated sulfuric acid A. R.? Methyl red indicator? Methylene blue indicator? Methyl orange indicator (0.1%)? Phenolphthalein indicator (1%)

3.3.1 Reagent Preparation: 1) 0.1 N H2SO4 Solution :0.7 ml conc. H2SO4 (36N) was taken in 250 ml volumetric flask and it was diluted up to the mark with distilled water.2) 0.1 N Na2CO3 solution:2.65 g Na2CO3 was taken in 50 ml volumetric flask and it was diluted up to the mark.The strength of Na2CO3 = = 0.10006 N3) 60% NaOH solution: 60g of NaOH was taken in 100 ml water.4) Digestion mixture:The mixture of 98 parts K2SO4 and 2 parts CuSO4.5) Methyl red indicator:0.1g of Methyl red dissolved in 60 ml of alcohol and water added to make to 100 ml.6) Methyl orange (0.1%): 0.1g Methyl orange dissolves in 100 ml distilled water.7) Phenolphthalein indicator (1%):1g Phenolphthalein dissolves in 100ml alcohol8) Methylene blue indicator:1 gm Methylene blue in100ml water9) Preparation of 2% Boric acid solution:5g of Boric acid was taken in 250 ml volumetric flash and diluted up to the mark with distilled water.10) Preparation of mixed indicator: Mixed indicator was prepared freshly by mixing 2 parts of Methyl red and 1 part of Methylene blue in a Stoppard test tube.3.3.2 Procedure for determination of nitrogen content: A) Digestion of the residue:0.05–0.5 g of the sample was taken into dry Kjeldahl flask, (a round bottomed flask with a long narrow neck) and 5ml concentrated H2SO4 and 0.5g of the digestion mixture were taken into it. Glass beads ware added to prevent bumping. Two Kjeldahl flasks were taken. One of them for sample and another one were for blank determination. In the blank determination flask samples were absent the flasks were shaking for well mixing. The flasks were supported on a hole (4.5 cm in diameter) in a piece of asbestos board and the necks were inclined at an angle of 60º. The flasks were closed with loosely fitting glass stopper elongated to a point and having a balloon-shaped top. The flasks were heated with a small flame in a fume cup-board. When foaming had ceased the flame was increased until the mixture boiled gently. The mixture digested by heating for 4 to 5 hours. The heating was continued for 30/60 minutes after the solution became colourless or clear (Usually 90-120 minutes) and the solutions were allowed to cool. B) Standardization of H2SO4 with 0.1N Na2CO3 solution:10 ml of Na2CO3 was taken in a conical flask with the help of pipette, a few drops of methyl orange were added to the solution and the solution becomes pink colour. H2SO4 solution was taken in a burette. H2SO4 solution was added drop wise into Na2CO3 solution. At the end of titration the colour of the solution became yellowish.Titration was carried out exactly three times the results are tabulated as follows: Table–3.5: Titration data for determining the strength of H2SO4 acid Solution:Observe. no. Na2CO3 solution (ml) Burette reading , Volume of H2SO4 solution (ml) IBR FBR Difference Average volume1. 10 0.0 9.5 9.5 9.5

2. 10 9.7 19.2 9.5 3. 10 0.0 9.5 9.5

Calculation: As we know, V1S1 = V2S2 Here, V1 = Volume of Na2CO3 solution = 10 ml S1 = Strength of Na2CO3 solution = 0.10006 N V2 = Volume of H2SO4 solution = 9.5mlStrength of H2SO4 solution, S2 = V1S1V2 = = 0.1059 N C) Distillation of ammonia in Kjeldahl distillation unit:5 ml of 2% boric acid solution was taken in a 100ml conical flask and it was placed in such a way that the tip of the condenser outlet dipped below the surface of the boric acid solution. 2 to 3 drops of mixed indicator were added to the conical flask. The digested sample was transferred completely by means of rapid ringing to chamber of steam distillation apparatus. Which was previously been cleared of any contaminating ammonia by repeated washing. The enough 60% NaOH (nearly 11-16 ml) was added to the digest in the chamber to more than neutralized the amount of acid present, which is ensure by means of phenolphthalein indicator. Than the distillation set was made air tight and steam generation in the boiler was started. The sample was steam distilled until 20-40 ml of distillate collect in the receiving flask (in about 20 minutes of distillation). Than the receiving flask is lowered and the steam was stopped side of the condenser outlet tube was rinsed into the receiving flask with a little water. The colour of the content of the receiving flask would have changed during the distillation. D) Titration:Ammonia in the receiving flask was titrated against 0.1 N Sulfuric acids, the end point being the reversion to the original greenish blue colour. E) Blank determination:Blank was determined by digesting only the sulfuric acid and distilling the same after addition of 60% NaOH and enough water and titrated the ammonia liberated against the standard acid.The titrate value of blank was subtracted from the titter value of the sample, which gives the amount of 0.1N H2SO4 needed to neutralize the ammonia evolved from 0.05g sample. 1 ml of 0.1N H2SO4 ? 0.014 g Nitrogen ? 14 mg Nitrogen.Calculation method:The percentage of nitrogen in the solid residue was calculated as flows:% of Nitrogen = Where, s = Volume (ml) of standard acid used in the sample titration. b = Volume (ml) of standard acid used in the blank titration. N = Strength of the H2SO4 acid (N). W = Weight (gm) of the sample. Strength of H2SO4, N = 0.1059 N

Table – 3.6: (Blank Titration with 0.1 N H2SO4 solution) Observe no Weight of Digestion mixture, gm Vol. of Conc. H2SO4,ml Vol. of standard acid used in blank titration, s ml1. 0.1896 5 0.052. 0.1998 5 0.05

So, the Volume of standard acid used in the blank titration, b = 0.05 ml

Table-3.7: Titration data for determination of nitrogen content of Carum roxburghianum seeds:

a) For the sample of Joydebpur, Gajipur:Observe. no Wt. of Digestion mixture, gm Wt. of sample, w gm Vol. of Conc. H2SO4,ml Vol. of acid used in sample titration, s ml % of Nitrogen, Mean % of Nitrogen

1. 0.1938 0.1160 5 2.85 3.5787 3.57872. 1.8560 0.1159 5 2.85 3.5786 3. 1.8350 0.1139 5 2.80 3.5788

Result: The Nitrogen content of Radhuni seeds from Joydebpur, Gajipur 3.5787 Observe. no Wt. of Digestion mixture, gm Wt. of sample, w gm Vol. of Conc. H2SO4,ml Vol. of acid used in sample titration, s ml % of Nitrogen, Mean % of Nitrogen

1. 0.1720 0.1360 5 2.80 2.9978 2.96982. 0.1640 0.1390 5 2.85 2.9856 3. 0.1865 0.1419 5 2.85 2.926 b) For the sample of Keranigonj, Dhaka:

Result: The Nitrogen content of Radhuni seeds from Keranigonj, Dhaka 2.9698c) For the sample of Faridpur:Observe. no Wt. of Digestion mixture, gm Wt. of sample w mg Vol. of Conc. H2SO4,ml Vol. of acid used in sample titration, s ml % of Nitrogen, Mean % of Nitrogen

1. 0.1942 0.1227 5 2.75 3.2624 3.26242. 0.1356 0.1212 5 2.70 3.2424 3. 0.1455 0.1242 5 2.8 3.2829

Result: The Nitrogen content of Radhuni seeds from Faridpur 3.26243.4 Determination of protein content 57, 65, and 93:Proteins (also known as polypeptides) are organic compounds made of amino acids arranged in a linear chain and folded into a globular form. The amino acids in a polymer chain are joined together by the peptide bonds between the carboxyl and amino groups of adjacent amino acid residues. The sequence of amino acids in a protein is defined by the sequence of a gene, which is encoded in the genetic code. In general, the genetic code specifies 20 standard amino acids. Like other biological macromolecules such as polysaccharides and nucleic acids, proteins are essential parts of organisms and participate in virtually every process within cells. Many proteins are enzymes that catalyze biochemical reactions and are vital to metabolism.

The protein content of a foodstuff is obtained by estimating the nitrogen content of the material and multiplying the nitrogen value by 6.25(a factor) this referred to as crude protein content since the non protein nitrogen(NPN)present in the material is not taken into consideration57. So, the protein content is = N2 content × 6.25

Table –3.8: Protein content of Carum roxburghinum (Radhuni) seeds: Sample collectedfrom % of Nitrogen % of Protein,% of Nitrogen × 6.25Joydebpur, Gajipur 3.5787 22.3663Keranigonj, Dhaka 2.9698 18.5618Faridpur 3.2624 20.3899

? Each value represents the average value from three experiments.3.5 Determination of crude fiber 57, 66, 70:Crude fiber is made up primarily of plant structural carbohydrates such as cellulose and hemicelluloses and lignin, which is a highly indigestible material. Often there is much confusion about the difference between dietary fiber (soluble fiber) and crude fiber, or what is now referred to as insoluble fiber. Most crude fiber contains one-seventh to one-half dietary fiber. Crude fiber is determined by laboratory analysis and is mainly composed of lignin, which is found in the tissues of plants and cellulose basically a plant’s skeleton. In layman’s terms, analysis of crude fiber in the laboratory involves oven-drying the fiber to be analyzed after exposing it to a series of sulfuric acid and sodium hydroxide solutions. Crude fiber left a mixture of insoluble fibers that have no nutritional value.

Insoluble or crude fiber is expelled by the body and aids in maintaining regular intestinal peristalsis (bowel) movements. In short, most people need some crude fiber in their diets. Excellent sources of crude fiber, or insoluble fiber, include: vegetables like leafy greens, whole grains like whole wheat and rye, and beans such as kidney beans and black beans. While most doctors and nutritionists recommend eating a diet that is high in both soluble and insoluble fiber, many suggest that those suffering from irritable bowel syndrome not eat insoluble fiber on an empty stomach. Reagents: ? 0.255 N H2SO4 ? 0.313 N NaOH Solutions. ? Distilled water ? Alcohol and diethyl ether. ? BaCl2 solution? Phenolphthalein indicator 3.5.1 Reagent Preparation: 1) 0.255 N H2SO4 Solution: 3.6 ml Concentrated H2SO4 (36N) was taken in 500ml volumetric flask and flask was made up to the mark with distilled water. 2) 0.313 N NaOH Solution:6.26 g NaOH pellet was taken in 500ml volumetric flask and flask was made up to the mark with distilled water. 3.5.2 Procedure for determination of crude fiber 57:An amount of 2-5 g moisture and fat free sample was weight into a 500ml R.B. flask than 200ml boiling 0.255 N H2SO4 was added. The mixture was heated exactly 30 minutes. A water condenser attached with R.B flask for keeping the volume constant. The mixture was filtered through linen cloth. The residue was washed repeatedly with boiled water to free the residue form acid. 3 or 4 drops of the filtrate collected in a test tube and a drop or two of BaCl2 solution was added for testing the presence SO42– ion. The process was continued precipitate of SO42– appeared. Then the residue has free from acid. The charge was again started in another R.B. flask with 200ml 0.313 N NaOH solution after boiling for 30 minutes (keeping the volume constant as before). The mixture was again filtered through linen cloth. The residue is washed with hot water till free from alkali. 2/3 drops of filtrate taken in a test tube and 1 drop of phenolphthalein solution was added to it. If there was no pink colour appeared. It can be conclude the alkali was removed from the residue. Then filtrate was washed initially with alcohol and secondly with diethyl ether. The residue over the cloth was then transferred in porcelain crucible which is previously cleaned and weighted. It was dried at 1100c in oven and weighted until a constant weight. The difference of weight of the crucible gave the weight of crude fiber. The crucible was than heated in a muffle furnace at 6000C for 3 hour. The crucible was allowed to cool and weighted again. The difference in the weights present the weight would gave the amount of ash present in crude fiber.

Thus the crude fiber percentage can be calculate with this formula, % of crude Fiber = Calculation method:Weight of moisture & fat free sample (taken) =m1 gmWeight of empty crucible = m2 gmWeight of dried fiber + crucible = m3 gmWeight of dried fiber, w1 = (m3 ? m2) gmWeight of crucible with ash after firing at 600?C = m4Weight of ash, w2 = (m4 ? m2) gmMoisture content = M Fat content = F % of crude Fiber = Table–3.9: Crude fiber content of Carum roxburghianum (Radhuni) seeds:a) For the sample of Joydebpur, Gajipur:? Moisture content, M = 9.8014 %? Fat content, F = 15.3137 %No of observe. Weight of sample, m1 gm Weight of dried fiber, w1 gm Weight of ash,W2gm % of Fiber, Mean % of fiber1 5.5695 1.6769 0.1224 20.9011 20.90112 5.5240 1.7778 0.1342 22.2810 3 7.0457 2.0050 0.1683 19.5212

Result: The crude fiber content of Radhuni seeds from Joydebpur, Gajipur 20.9011 %b) For the sample of Keranigonj, Dhaka:? Moisture content, M = 10.7091? Fat content, F = 20.3176 %No of observe. Weight of sample, m1 gm Weight of dried fiber, w1 gm Weight of ash,W2gm % of Fiber, Mean % of fiber1 5.2449 1.7495 0.1012 21.6602 21.66022 5.7287 1.8292 0.1132 20.6605 3 5.6259 2.0015 0.1532 22.6599

Result: The crude fiber content of Radhuni seeds from Keranigonj, Dhaka 21.6602 %c) For the sample of Faridpur:? Moisture content, M =11.6010 %? Fat content, F = 20.2304 %No of observe. Weight of sample, m1 gm Weight of dried fiber, w1 gm Weight of ash,W2gm % of Fiber, Mean % of fiber1 5.3364 2.0328 0.1608 23.9262 23.92622 7.4124 3.0280 0.208 25.9345 3 7.0029 2.4508 0.1992 21.9179

Result: The crude fiber content of Radhuni seeds from Faridpur 23.9262 %3.6 Determination of Carbohydrates content 57:Carbohydrates or saccharides are the most abundant of the four major classes of biomolecules. They fill numerous roles in living things, such as the storage and transport of energy (e.g., starch, glycogen) and structural components (e.g., cellulose in plants and chitin in arthropods). In addition, carbohydrates and their derivatives play major roles in the working process of the immune system, fertilization, pathogenesis, blood clotting, and development60. Carbohydrates are simple organic compounds that are aldehydes or ketones with many hydroxyl groups added, usually one on each carbon atom that is not part of the aldehyde or ketone functional group. The basic carbohydrate units are called monosaccharides; examples are glucose, galactose, and fructose. The general stoichiometric formula of an unmodified monosaccharide is (CH2O)n, where n is any number of three or greater; however, not all carbohydrates conform to this precise stoichiometric definition (e.g., uronic acids, deoxy-sugars such as fructose), nor are all chemicals that do conform to this definition automatically classified as carbohydrates 61.

The content of the available carbohydrates is determined by difference, i.e., by subtracting from the sum of the values (per 100 gms) for moisture, protein, ether extractives, ash, and crude fiber.

So, the carbohydrates content = 100 – (moisture % + ash % + protein % + Crude fiber % + ether extractives %)Table – 3.10: Carbohydrates content of Carum roxburghianum seeds:Sample collected from Moisture content% Ash content% Protein content% Crude fiber% Ether extract.% Carbohy-drates content%Joydebpur, Gajipur 9.8014 8.9920 22.3663 20.9011 15.3137 22.6255Keranigonj, Dhaka 10.7091 9.0124 18.5618 21.6602 20.3176 19.7389Faridpur 11.6010 8.2431 20.3898 23.9