PhD Thesis
Browse
15 results
Search Results
Item Phytochemical and Biological Investigation of the Plants Moringa Oleifera Lam. and Duranta Repens Linn(University of Rajshahi, 2005) Nikkon, Farjana; Haque, M. Ekramul; Rahman, M. HabiburThe plant Moringa oleifera Lam. belongs to the family Moringaceae is locally known as "Sajna" (Eng. Drumstick) found to grow every., here in Bangladesh, India, Pakistan and other tropical and subtropical countries. It has various folk medicinal uses and effective against dysentery, diarrhea, cholera, pneumonia, scabies, relieves the pain of gout and acute rheumatism· etc. Isolation of two alkaloids, meringue and Moring nine and an antibacterial substance, pterygospermin have been reported. For chemical investigation, ethanolic extract of the dried powdered root barks of Moringa oleifera Lam. was successively fractionated with petroleum ether (40-60°C) and chloroform. The chloroform soluble fraction was subjected to a column of silica gel, eluted with n-hexane and ethyl acetate and their mixtures and re chromatography of the suitable portion afforded compound-I (31.4 mg) as needles, from petroleum ether, MP. 58-60°C. Compound-I was identified as deoxy-niazimicin (N-benzyl, S-ethyl thiocarbamate), a novel compound by analysis of its spectral data (IR, Mass and NMR) …………………….Item Chemical and Microbiological Investigations of Cassia fistula Linn(University of Rajshahi, 2002) Ali, Md. Abbas; Sayeed, Md. Abu; Absar, NurulThree lectins were extracted and purified from Cassia fistula seeds in biologically active form by gel filtration of I 00 % ammonium sulfate saturated crude protein extract on Sephadex G-50 followed by ion-exchange chromatography on DEAE celluiose and then affinity chromatography on Sepharose 4B. The lectins were found to be homogeneous justified by polyacrylamide disc gel electrophoresis. The molecular weights of the lectins CSL-I, CSL-2 and CSL-3, determined by gel filtration on Sephadex G-75 column were 37,000, 42,400 and 46,000, and by SDS gel electrophoresis were 37500, 42000 and 46500, respectively. The lectins agglutinated rat red blood cells and the agglutination was inhibited specifically by galactose and galactose containing saccharide. The neutral sugar contents of the lectins, CSL:-1 , CSL-2 and CSL-3 were estimated to be 3.5, 3.1 and 2.0 % respectively. The sugar composition of the lectins was found to be galactose for CSL-I, galactose and glucose for CSL-2, and galactose and mannose for CSL-3. The lectins displayed strong cytotoxic effect in brine shrimp lethality bioassay and among the lectins purified, CSL-2 was found to be highly toxic followed by CSLI and then CSL-3. The purified lectins in aqueous solution gave absorption maxima around 274-278 run and minima around 243-248 run. Biological activities of the lectins CSL-I, CSL-2 and CSL-3, were investigated after various physico-chemical treatments. Biological activities were highly affected with the changes of pH and temperature, and the lectins exhibited maximum hemagglutinating activities around pH 7.2 to 7.5 and at temperature 20° to 35 °C. Biological activities of the lectins were abolished sequentially with the increase in concentration of acetic acid and denaturant solutions such as urea and guanidine-HCI.-----Item Comparative Studies on the Nutrient Content of Five Varieties of Rice Brans and Purification, Characterization and Structure Function Analysis of Proteins from the Best Quality Rice Bran(University of Rajshahi, 2007) Rahman, Md. Lutfor; Absar, NurulFive varieties of rice bran were analyzed to obtain comparative data on their chemical compositions and nutritive values. Rice bran oils were also characterized with respect to their physical and chemical properties. The results indicated that moisture, ash, total soluble solid and crude fiber contents of the different varieties of rice bran were in the ranges of 9.98-10.90%, 9.45-11.12%, 10.06-11.25% and 9.18-10.35% respectively. The ranges in the values of total protein, water soluble protein, dry matter, polysaccharide, total sugar, reducing and non-reducing sugar compositions of rice brans were betweenl2.45- 14.06%, 3.20-4.71 %, 89.10-90.02%, 10.95-12.86%, 4.35-5.04%, 1.08-1.45%, and 3.10-3.96% respectively. Some minerals such as potassium, calcium, magnesium, sodium, phosphorus, Sulphur, iron, zinc and manganese are available in all the varieties of experimental rice brans. Vitamin B1 and vitamin B2 contents of rice brans were found to be ranged from 2.98-3.81 and 0.61-0.95 mg/100 g respectively. Twelve amino acids were detected in all the five varieties of rice bran in the total form by paper chromatography. Among the variety IRRI-28 contained relatively higher amount of amino acids. Rice bran contained about 14.95-16.16% oil. The bran oils were also characterized with respect to their physico-chemical properties such as specific gravity, refractive index, smoke point, flash point, fire point, saponification value, iodine value, peroxide value, acid value, %FFA and unsaponifiable matter. Triglyceride diglyceride monoglyceride, and non-glyceride are the constituents of bran oils were found to be varied from 91.76-93.08%. l.51-2.30%, 1.45-1.96% and 3.63-5.24% respectively. Rice bran oils contained mostly unsaturated fatty acids which was varied from 7 l.06-74.95%. The total lipids were fractionated into lipid classes by silicic acid column chromatography and the bran oil contained triglyceride, hydrocarbon, free sterols, glycolipid, phospholipid arid partial glyceride. GLC analysis of the oil indicated palmitic, stearic, oleic and linoleic acid, as a major fatty acid in all the varieties of rice bran oil. On the other hand, linolenic, myristic and arachidic acid were also detected in small amount.Item An Analysis of the Physico-Chemical Characteristics of Hilsa (Hilsa Ilisha) Fish, and Purification and Characterization of Two Lipases From its Dorsal Part(University of Rajshahi, 2004) Hossain, A. K. M. Motahar; Absar, Nurul; Haque, Md. EkramulA comparative analysis on the nutrient contents of six different parts of Hilsa fish was performed. Most of the nutrient contents of different parts were varied remarkably. The moisture and ash content of the different parts of hilsa fish were found in the ranges of 13.00-54.87% and 1.7-3.8% respectively. The ranges in the values of oil, protein, free sugar and polysaccharide composition of hilsa fish were 9.61-22.78%, 5.5-23.18%, 0.053-0.09%, and 0.03-0.065% respectively. The minerals such as iron, calcium and phosphorus were found to be varied from 199-212 mg%, I 60-180 mg% and 259-278 mg% while the vitamins such as BI and 82 contents were varied in the ranges of 0.35-0.48 mg% and 2.44-2.98 mg% respectively. The oils extracted from different parts of hilsa fish were also characterized with respect to its physico-chemical prope11ies such as specific gravity, refractive index, saponification value, iodine value, peroxide value and acid value. The specific gravity and refractive index of oil were varied from 0.920 to 0.932 and 1.4700 to 1.4722 respectively. The saponification value, iodine value, peroxide value and acid value of the oils were found to be ranged from 180.28 to 194, 80. 70 to l 26.40, 7 to 10 and 4.16 to 12. 00, respectively. The oils, extracted from different parts of hilsa fish were stored at temperatures -10°C, 0°C and 25°C for 120 days. The hydrolytic deterioration of the oils was more pronounced at 25°C as compared to that from 0°C and -l 0°C.Item Purification, Characterization and Structure-Function Analysis of Proteins from Pota,Nogeton Nodosus Root Stocks(University of Rajshahi, 2005) Pervin, Farzana; Absar, NurulPotamogeton nodosus rootstock contained ash, moisture considerably but significantly higher amount of protein. Rootstock contained smaller amount of lipid as compared to and seeds fruits. It contains carbohydrate such as reducing sugar, non reducing sugar and starch. The root stock oil was also characterized with respect to its physicochemical properties such as specific gravity, refractive index, smoke point, flash point, fire point, cloud point, solidification point, pour point, saponification value, sapanification equivalent acid value, % FFA, and iodine value. -- Six lectins were purified from the root stock of Potamogeton nodosus, locally name as Ghechu. The method was accomplished by gel filtration of 100% ammonium sulphate saturated fraction from the crude extract on Sephadex G-50 followed by ion-exchange chromatography on DEAE-cellulose and on CM-cellulose. The purity of lectins were judged by polyacrylamide slab gel electrophoresis. The molecular weight of PNL-1, PNL-2, PNL-3, PNL-4, PNL-5 and PNL-6 as determined by Sephadex G-150 and polyacrylamide slab gel electrophoresis method was estimated to be about 98,000, 91,000, 77500, 68,500; 62,500 and 38,500 and 97,500, 83,000; 77,000; 67,500; 62,000 and 38,100 respectively. -----Item Analysis on the Nutrient compositions of Two Varieties of Sajna (Moringa Oleifera L.) Leaves and Purification, Characterization and Structure-function Analysis of Sajna Leaves Protein(University of Rajshahi, 2004) Khatun, Shahanaz; Absar, NurulTwo types of Sajna leaves, small and large sized were analysed to obtained a comparative date on their chemical composition and nutritive values at different maturity levels. The large sized leaves contained higher amount of TT A, dry matter, total chlorophyll, chlorophyll-a, chlorophyll-b, water soluble protein, lipid, total sugar, sucrose, starch, vitamin-C, vitamin-B2 and iron while the small sized leaves contained higher amount of specific gravity, moisture, ash, total protein, total phenol, reducing sugar, f3-carotene, vitamin-B1, calcium and phosphorus .The nutrient compositions of Sajna leaves were found to be changed at different maturity levels. The specific gravity, dry matter, ash, lipid, total sugar, reducing sugar, sucrose, f3-carotene, vitamin-B2 and phosphorus content increased while TT A, moisture, starch, calcium and iron content decreased with the advancement of maturity in both type of leaves. The total chlorophyll, chlorophyll-a, chlorophyll-b, total protein, water soluble protein, total phenol, vitamin-B 1 and vitamin-C content were increased upto mature stage and then decreased in ripen stage. The activities of protease and peroxidase enzymes increased with the changes of maturity, whereas amylase, cellulase, invertase, ascorbic acid oxidase and Polyphenoloxidase increased upto mature stage and then decreased drastically in ripen stage. Fresh Sajna leaves contained significantly higher amount of oxidative enzymes as compared to the hydrolytic enzymes. Polyphenoloxidase, a highly active enzyme in large sized Sajna leaves at mature stage was purified and characterized. The purification was accomplished by ion-exchange chromatography of crude enzyme extract on DEAE-cellulose followed by CM-cellulose chromatography. The purified enzyme gave single band on polyacrylamide gel indicating its homogeneity. The molecular weight of enzyme, as indicated by gel filtration and SDSpolyacrylamide gel electrophoresis were estimated to be 56,000 and 55,500 respectively and the enzyme contained only one subunit. The enzyme gave maximum activities in the pH range of 6.0-6.4 and at temperature 32°C. ----------Item Studies on some Enzymes from Germinating oil and Cereal Seeds(University of Rajshahi, 2004) Sana, Niranjan Kumar; Huque, Entazul M.Degradation of three varieties of Brassica and wheat seed storage substances at different periods of germination have been studied to have a comparative data on their chemical composition. Free sugar contents of the three varieties of Brassica and wheat seeds differ slightly. Wheat seeds contain a little higher amount of free sugar than that of Brassica seeds. Reducing sugar contents also vary in Brassica and wheat seeds. During germination, the degradation of free sugar and reducing sugar in Brassica seeds is faster than in wheat seeds. Wheat seeds contain a larger amount of starch than that of Brassica seeds. Among the three varieties of wheat seeds, Akbar variety has the highest percentage of starch. The starch contents of both seeds decrease gradually dming germination. Total protein and water-oluble protein content of the three varieties of Brassica seeds is slightly higher than that of wheat seeds. During germination, protein depletion starts after initial imbibition, and is completed in between 96 to 120 hours. Brassica seeds contain a significant amounts of lipid. Among Brassica seeds, napus variety contains the highest amount of lipid, followed by campestries and jtmcia varieties. Lipid degradation starts after 24 hours of ( germination and is completed within 72 to 96 hours. The results suggest that degradation of seed reserve nutrients accelerate the development of seedling growth during germination. Lipid degrading enzyme lipase was identified in germinating Brassica napus L. The enzyme was purified to homogeneity by solvent extraction, followed by Sephadex G- 75, DEAE and CM-cellulose ion exchange chromatography. The enzyme was purified to 68 fold with the final specific activity of 39-mmol min·1 mg·1 at 37°C using olive oil as a substrate. ---------Item Studies on the Physicochemical Properties and Shelf Life of Postharvest Mangifera indica during Storage Environments(University of Rajshahi, 2008) Islam, Md.Khairul; Islam, A.K.M. Rafikul; Absar, NurulAn investigation was conducted for obtaining information on the reduction of postharvest losses, pattern of physical, biochemical and mineral content changes, storability as well as shelf life of postharvest mango. The trials included four different experiments involving two factors where variety was a factor comprising of Langra and Khirshapat. These varieties were treated with different postharvest treatments viz., control, paraffin coating, perforated polyethylene cover, unperforated polyethylene cover, hot water (55 ± 1 )°C, low temperature in refrigerator (4±1)°C (experiment 1); different doses of Maleic hydrazide solution namely, control, 200,400 and 600 ppm (experiment 2); different doses of GA3 solution such as, control, 100, 200 and 400 ppm (experiment 3), and different doses of Bavistin DF solution viz., control, 250, 500 and 750 ppm (experiment 4 ).These experiments were laid out in Randomized Complete Block Design with three replicates. Data obtained from various biochemical analyses in terms of physicochemical properties and shelf life of postharvest mango, were recorded and statistically analyzed for comparison among the mean values using DMRT and LSD. The res1,.1lts of the experiments exhibited that only the single effect of varieties was found to be significant in most of the parameters studied. Variety Langra performe<;I better in accumulating higher quantity of edible portion, pulp to peel ratio, dry matter, ash, vitamin c, tltratable acidity, TSS (after 3 rd day In case of experiment 2 and 4 as well as 6th day in case of experiment 3), crude fiber, lipid, water soluble protein, phosphorus, potassium, magnesium, iron and manganese content in all four experiments over Khirshapat. On the other hand, Khirshapat showed better performance in achieving higher quantity of moisture, progressively lost physiological weight, increased pulp pH, TSS (in terms of experiment 1, initial to 3rd dc;Jy for experiment 2 and 4 as well as initial to 6th day for experiment 3), produced more quantity of sugar (total, reducing and non reducing), calcium, copper and zinc content as well as extended shelf life and delayed skin color changes than Langra at all the storage duration. Different postharvest treatments subjected to the investigation demonstrated significant variation in most of the physicochemical properties and shelf life of mango at different c;lays after storage. The results explored that some physicochemical properties viz., edible portion, pulp to peel ratio, physiological weight loss, moisture content, pulp pH, TSS, sugar (total, reducing and non reducing), lipid, water soluble protein, phosphorus, potassium, calci1,.1m, magnesium, iron and manganese content were rapidly increased as well as skin color, dry matter, ash, vitamin C, titratable acidity, crude fiber, copper and zinc content along with shelf life drastically decreased from untreated mangoes, but, low temperature in refrigerator caused delaying of these changes except physiological weight loss. The unperforated polyethylene cover was found to be the best method for reduction of physiological weight loss. Low temperature in refrigerator was the best inhibitor of ethylene synthesis that delayed ripening and prolongation of shelf life. In case of other experiments, Malle hydrazlde, GA3, and Bavlstln DF at the doses of 600, 400 and 750 ppm, respectively showed better results in delaying the changes in physicochemical properties and extended shelf life.Item Nutritional, Phytochemical & Antidiabetic Properties of Oyster Mushroom (Pleurotus ostreatus)(University of Rajshahi, 2013) Karim, Md. Rezaul; Hossain, Md. TofazzalPlants are often condemned as non-scientific and inactive. But various phytochemical and biological investigations have proved its therapeutic utility and functional properties. Now-a-days, the use of plant constituents as remedy for diseases is very promising. According to folk medicine the mushrooms Pleurotus ostreatus (Family: Tricholomataceae) is used in protein deficiency diseases, diabetes, anemia, cardiac diseases, typhoid fever, and diseases of blood. Pleurotus ostreatus is locally known as Oyster mushroom and consumed by the local people as foods and vegetables. In many countries various types of foods are produced from mushrooms like burger, bread, sandwich, curry etc. In our country too, people started preparation of such types of foods. The present study included nutritional analysis, insecticidal and repellent activity against Tribolium castaneum adult, cytotoxicity against Brine shrimp nauplii, larvicidal activity against mosquito larvae, antimicrobial activity of some gram (+) and gram (-) pathogenic bacteria, antidiabetic, and hepatoprotective activity in long evan rats of Pleurotus ostreatus. The physico-chemical and nutritional parameters changes in the three different (immature, mature and over mature) stages of Oyster mushroom (Pleurotus ostreatus) were studied to obtain a comparable data on their nutritional qualities. The physico-chemical parameters such as pH and moisture, contents are increased and ash and crude fiber contents are decreased with its maturity. The amount of total sugar, reducing sugar and sucrose was higher at over mature stage than immature and mature stage. Protein content was higher at mature stage than immature and over mature stage. The mentionable case is that the protein content was higher than the other nutrients. So, Oyster mushroom can be considered as good source of protein. In this study, phytochemical investigation of fruiting body of Pleurotus ostreatus was performed. The extracts of fruiting body of the Oyster mushroom were obtained from its powder using methanol, ethyl acetate, chloroform, pet-ether and hot water. In the insecticidal activity of the crude extracts dose mortality results against T. castaneum were found promising. Methanol extracts of Pleurotus ostreatus showed high mortality of the beetles and the results have been presented in table 4.5. The LD50 values for methanol extract were 0.480, 0.142, and 0.106, 8.49E-02 mg/cm2 for 12, 24, 36 and 48 hrs. of exposure respectively. The LD50 values for Chloroform extract were 0.992, 0.944, 0.719, 0.452 mg/cm2 for 12, 24, 36 and 48 hrs. Of exposure respectively. In the same way LD50 values for Ethyl acetate, pet-ether and hot water were 5.614, 3.322, 2.495, 2.199 mg/cm2; 3.681, 3.225, 3.322, 2.722 mg/cm2; and 3.77, 3.41, 2.981, 2.785 mg/cm2 respectively. All the extracts of Pleurotus ostreatus showed promising repellent activity against the adults beetles of T. castaneum. The P values of methanol, ethyl acetate, chloroform, pet-ether and hot water were 0.017, 0.01, 0.009, 9.38E-05 and 0.013 respectively. The extracts ethyl acetate and pet-ether showed repellent activity against T. castaneum adults at (p<0.01) level, while other extracts showed at (p<0.05) level. The larvicidal activity of the crude extracts dose mortality results against clues mosquito larvae were found promising. The chloroform extract showed highest larvicidal activity and the results have been shown in table 4.7. The LC50 of the extract were 1224.768, 902.232, 617.195, 434.093ppm for 12, 18, 24 and 30 hrs. Of exposure respectively………………………………………………….Item Phytochemical and Biological Studies on the Plants Calotropis gigantea (Linn) and Amoora rohituka Roxb(University of Rajshahi, 2013) Habib, M. Rowshanul; Karim, M. RezaulThe universal role of medicinal plants in the treatment of diseases is established by their employment in all important systems of medicine. Many active drugs have been derived from different medicinal plants, and the process is going on. As a preliminary approach along this direction, this study was designed to carry out phytochemical and biological studies on the two important medicinal plants, Calotropis gigantea (Linn.) and Amoora rohituka (Roxb.). The plant Calotropis gigantea (Linn.) (Bengali Name: Boro Akanda) belonging to the Asclepiadaceous family has wide folk medicinal uses and various parts of this plant possess different pharmacological properties. As a part of phytochemical investigation of this study, the root bark and flower powder of Calotropis gigantea was extracted with methanol and ethyl acetate, respectively, at room temperature to get methanol (ME) and ethyl acetate (EECF) extracts. I was fractionated with petroleum ether and chloroform successively to yield petroleum ether (PEF) and chloroform (CF) soluble fractions, respectively. EECF was applied on silica gel column chromatography using n-hexane with a gradient of ethyl acetate. Fractions 21~30 afforded white crystals as compound-2 whereas fractions 40~48 were combined and subjected on preparative thin layer chromatography (PTLC) to find colorless oily liquid as compound-1. Based on TLC profile, fractions 7~13 were combined and rechromatographed on silica gel column eluting with n-hexane and ethyl acetate (19 : 1) and three compounds were purified as compound-3 (white crystals), compound-4 (white amorphous powder) and compound-5 (colorless crystals) from different fractions of this second column. Amoora rohituka (Roxb) (Bengali Name: Pithraj) another plant of this study, belongs to the Meliaceae family and according to ayurvedic classical texts, the stem bark of Amoora ruhituka is being prescribed in liver and spleen diseases, oedema, anaemia, intestinal worms, urinary disorders, internal tumours and abdominal complaints. As a part of phytochemical study of this research work, the stem bark powder of Amoora rohituka was successively extracted with ethyl acetate and dichloromethane at room temperature to have ethyl acetate (EAEAR) and dichloromethane (DMEAR) extracts. Preparative thin layer chromatography (PTLC) was then applied on DMEAR and an UV active band afforded orange oily liquid as compound-6. Finally based on analysis and comparison of spectroscopic evidences (Mass, IR, 1H- and 13C-NMR data) of each compound with the literature, compound-1, -2, -3, -4, -5 and -6 were identified as di-(2-ethylhexyl) phthalate, anhydrosophoradiol-3-acetate, taraxasteryl acetate, lup-12,20(29)-dien-3β,28-diol, β-boswellic acid and 2-methoxy-14-calamenenone, respectively. Except taraxasteryl acetate, all of the five isolated compounds are reported here for the first time from the corresponding plant. In brine shrimp lethality bioassay, the cytotoxicity exhibited by EECF, compound-1, compound-2, compound-5 and DMEAR was promising with the LC50 values of 14.61, 9.19, 15.55, 15.26 and 17.67 µg/mL, respectively. But in comparison to ampicillin trihydrate (LC50: 7.21 µg/mL), compound-3, compound-4 and EAEAR demonstrated moderate activity with the LC50 values of 45.46, 30.58 and 26.59 µg/mL, respectively. EECF and compound-1 showed a better broad spectrum of antibacterial activity against pathogenic bacteria In vitro than the other isolated compounds and extracts. The intensity of antibacterial activity was found in the order of EECF > compound-1 > EAEAR > compound-5 > DMEAR > compound-2 > compound-4. The zone of inhibition produced by these extracts and compounds was found in the range 06 to 24 mm whereas the lowest minimum inhibitory concentration (MIC) values for these samples were in the range 16 to 64 μg/ml. In antifungal activity test, only EECF and compound-1 exhibited activity against the test fungi and produced zone of inhibition between 07 to 15 mm. In vivo antineoplastic effect of EECF (50, 100 and 200 mg/kg), compound-1 (10, 20 and 40 mg/kg), compound-2 (10 and 20 mg/kg), ME (10 and 20 mg/kg), PEF (40 and 80 mg/kg), CF (20 and 40 mg/kg), EAEAR (20 and 40 mg/kg) and DMEAR (20 and 40 mg/kg) was assessed by evaluating the viable tumour cell count, survival time, body weight gain due to tumour burden, heamatological (hemoglobin content, RBC and WBC count) and biochemical (Glucose, cholesterol, triglyceride, blood urea, SALP, SGPT and SGOT) parameters of Ehrlich ascites carcinoma (EAC) bearing mice. Treatment with the all above samples dose dependently and significantly (P < 0.05; P< 0.01 and P < 0.001) decreased the viable EAC cells and body weight gain thereby increasing the life span of EAC bearing mice and also brought back the altered hematological (Hb, total RBC and total WBC) and biochemical parameters more or less to normal level. Among the test samples, EECF, CF and DMEAR and compound-2 showed prominent antineoplastic activity as compared with standard drug bleomycin. In addition, treatment of normal mice with EECF did not cause any extreme abnormality at the three doses used in this study. In vitro cytotoxicity assay against A431 cell line (human vulval-derived epidermoid carcinoma), compound-1 (IC50: 0.34 μg/mL) and compound-5 (IC50: 0.36 μg/mL) showed strong cytotoxic effect than the others in comparison with doxorubicin (IC50: 0.31 μg/mL). In insecticidal activity against both larvae and adults of Tribolium castaneum (Herbst), EECF caused the highest mortality of the 1st instars larvae in comparison with other larval instars indicating high susceptibility to the newly hatched larvae with lowest LD50 value (0.134 mg/cm2) and less susceptibility to the adult (after 72 hr exposure) with highest LD50 value (1.371 mg/cm2). From the overall results of this study, it is concluded that extracts and purified compounds from flower of Calotropis gigantea and stem bark of Amoora rohituka have noteworthy antibacterial, cytotoxic and antineoplastic effects that might be a source of herbal drugs in respective therapeutic area.