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CHAPTER - VII

PHYTOCHEMICAL ANALYSIS OF
MORINGA OLEIFERA SEED POWDER

the defatted seed powder of PKM-1. oleifera seeds found to contain a coagulant protein which affected the purification of drinking water (Ndabigengesere et al.1998). The capacity to coagulate the pollutants has made the Moringa seed powder of great interest in the scientific world. based on the solubility properties of plant proteins. some of the most important phytochemical components in the PKM-1. Hence. The different levels of water purification showed by the different extracts may be due to the variations in the quantity and quality of proteins present in these extracts. such as distilled water. oleifera were extracted in four different solvents. 1995) and hence useful for waste water treatment (Ndabigengesere and Narasiah. PKM-1 (A4+V+N) and wild variety of M.1 Introduction The M. 1% NaCl. Along with this.214 Chapter VII 7.1 Estimation of total protein by Lowry’s method (Lowry et al. 0. PKM-1 (A4+V+N) and wild varieties of M. In the present investigation. the quantity of proteins in each extract and total proteins in the seed powder were analysed and the most effective extract was partially purified by ammonium sulphate precipitation and fractionation of the extract. 7.2.The extracts were centrifuged and the supernatants were used for protein estimation. .1M dibasic sodium phosphate).1M monobasic sodium phosphate and 61 ml of o.2 Materials and methods 7. The fraction showing maximum clarification of synthetic turbid water was separated and its molecular weight was assessed by SDS-PAGE. PKM-1 (A4+V+N) and wild Moringa were estimated by using non-defatted seed powder these varieties of Moringa. oleifera were extracted with 10ml each of phosphate buffer (39ml of 0.1N NaOH and 70% ethanol according to the method suggested by Osborne (1924). 1951) 500 mg of the dried seed sample of PKM-1.

2 Estimation of total protein Pipetted out 0.1 Reagents used i) Alkaline sodium carbonate (Reagent-1): 2 g of Na2CO3 was dissolved in 0. ii) CuSO4-Na-K-tartrate reagent (Reagent-2): 500 mg of CuSO4 was dissolved in 100 ml of 1% Na-K.2.4.2 Estimation of protein in the four extracts of PKM-1. Made up the volume to 1ml in all test tubes. PKM-1 (A4+V+N) and wild Moringa seed powder The seed materials of PKM-1. iii) Alkaline solution: It was freshly prepared each time just before use by mixing 50 ml of reagent 1 and 1ml of reagent 2. 7.2. A standard graph was drawn using the OD value of BSA dilutions and the amount of protein in the sample was calculated by referring the standard curve of BSA.8 and 1ml of the working standard solution of BSA into a series of test tubes and 0. A test tube with 1ml of distilled water served as blank. 1% NaCl.1. iv) Folin-Ciocalteau reagent : The commercial reagent was diluted with equal volume of distilled water just before use. 7.6. 100 mg each of the defatted seed powder was weighed and extracted in 10 ml each of solvents. mixed well and incubated at room temperature in dark for 30 minutes.tartrate solution. 0.Phytochemical Analysis of Moringa oleifera Seed Powder 215 7.1ml of the sample extract in two other test tubes.1 N NaOH and 70% ethanol according to the procedure outlined by Harborne (1973).000 rpm .1.1N NaOH and made up to 100ml using the same solution. The extracts were centrifuged at 5000 rpm for 15 minutes. Results were expressed in mg protein / gm dry weight. 5 ml of alkaline copper solution was added to each test tube including the blank. 0. Optical density was taken at 660 nm with spectrophotometer. 0. such as distilled water. 10 ml of ice cold TCA was added to each of the supernatant and left for 30 minutes at 400C and centrifuged at 10. 0.2.5 ml of Folin-Ciocalteau reagent was added to each tube. PKM-1 (A4+V+N) and wild Moringa were ground into fine powder and defatted using soxhlet apparatus with solvent hexane for 24 hours. Then 0.2. Mixed well and allowed to stand for 10 minutes.

216 Chapter VII for 10 minutes.1 ml of each solution was pipetted out in different test tubes and the protein estimation was done according to the procedure described above for total protein estimation by Lowry’s method. The precipitates were collected and dissolved in 10 ml of phosphate buffer saline (PBS). Then the . Each of the sediments was dissolved in 4 ml of 0.3 Assay of partially purified Moringa oleifera seed protein on turbidity removal from synthetic turbid water 7.3 Assay of partially purified M. These solutions were stored in refrigerator and used for testing their ability to coagulate the contents of synthetic turbid water. A control also was kept. 0.2. 60 and 80% solutions of ammonium sulphate.2N NaOH. Ammonium sulphate fractionation was done in the supernatants by precipitating the proteins using 40.2 Coagulation activity assay in synthetic turbid water The coagulation activity assay was based on the method described by Ghebremichael et al. 7. Turbid water samples of high turbidity with 500 NTU and low turbidity with 50 NTU were prepared from this stock solution by dilution with tap water.3. to which no protein solution was added.3. oleifera seed powder extracts on turbidity removal from synthetic turbid water 7. The extracts were centrifuged at 10.000 rpm for 5 minutes and the supernatants were collected. PKM-1 (A4+V+N) and wild Moringa were extracted in 100 ml each of distilled water and 1% NaCl for 5 minutes and incubated for 24 hours in a shaker at 100 rpm at room temperature.1 Partial purification of Moringa seed extracts by ammonium sulfate fractionation 2 gm each of defatted seed powder of PKM-1.2. (2005). 7. 10g of bentonite clay was mixed with one litre of tap water and stirred with a magnetic stirrer for 30 minutes and allowed to settle down for 24 hours to achieve complete hydration.2. 100 ml each of the two types of the turbid solutions were transferred to 250 ml beakers to which 200 µL of partially purified protein solutions in PBS were added and stirred for 5 minutes and allowed to settle down.2.

3.25 ml of Upper Tris (pH 6.3. .2.3. 7. 1.1 Development of SDS-PAGE system 7. vi) Sample buffer It was prepared by dissolving 150 mg of SDS and 10 mg Bromothymol blue in a mixture of 0. 8 ml of 10% SDS was added to this and made up the mixture to 200 ml with distilled water.34 g Tris HCl was dissolved in distilled water.2 g Acrylamide and 0.3) 3 g Tris HCl.3.1 Filter paper.3.5 ml β-mercaptoethanol. 1 ml of Glycerol. ii) Lower Tris (pH 8.25 ml of distilled water.8) 36.3 Separation of proteins and determination of molecular weight of partially purified protein by SDS-PAGE 7. iv) Ammonium persulphate (APS) It was prepared freshly at the time of use by dissolving 50mg of APS in 500 µl of distilled water. 8ml of 10% SDS was added to this and made up the mixture to 200 ml with distilled water.8) 12. 14. The solution was filtered through Whatman No.Phytochemical Analysis of Moringa oleifera Seed Powder 217 turbidity of the supernatants was measured including the control after 60 and 120 minutes of sedimentation and the NTU values were tabulated.8 g bis acrylamide were dissolved in distilled water and made up the volume to 100 ml with distilled water.1 g Tris HCl was dissolved in distilled water.1 Reagents Required i) Acrylamide stock ( 30%) 29.2.1. iii) Upper Tris (pH 6.4 g glycine and 1g of SDS were dissolved in distilled water and made up to 1000 ml with distilled water. v) Electrophoresis Buffer (pH 8.8) and 7.2.

3.3.1 ml APS : 50 µL TEMED : 7 µL 7.218 Chapter VII 7.3 ml Lower Tris (pH 8.3. Heated it at 1000C for 3 minutes.3. Added 1 cm of water on the top of the separating gel solution and allowed the gel to polymerize for 60 minutes.3. After loading the samples electrophoresis buffer was filled in the top and bottom reservoirs.2. 7.8) : 2.2 Preparation and loading of samples Mixed equal quantities of the protein sample and sample buffer in an eppendorf tube.5 ml Distilled water : 4.1.3. 7.2. PKM- 1(A4+V+N) and wild Moringa into the other wells carefully without air bubbles.3.2. Pipetted out required quantity of this solution into sandwich template carefully by keeping the gel margin uniform at the top. The protein markers were loaded in the first well and the fractionated seed protein samples of PKM-1.8) : 1.5 Preparation and loading of stacking gel Equal quantities of the protein sample and sample buffer were mixed in an eppendorf tube and heated it at 1000C for 3 minutes. Loaded the protein markers in .2.3.1.4 ml APS : 45 µL TEMED : 7 µL 7.5 ml Distilled water : 2.3.3.3 Preparation and loading of separating gel Mixed the components of separating gel just before loading into the sandwich template.4 Composition of Stacking Gel (Final percentage of Acrylamide (10%) Acrylamide : 1 ml Lower Tris (pH 6.2 Composition of separating gel (Final percentage of Acrylamide (10%) Acrylamide : 3.1.1.2.

Then it was rinsed with distilled water.3.3. Then the destaining solution was added into the container and kept the gel in the solution keeping the container closed for 2 hours for destaining.2. .3. Covered the container during staining to avoid evaporation of the staining solution. 1 Requirements i) Staining solution Dissolved 1g of coomassie brillient blue (R-250) in a mixture of 250 ml methanol. Procedure The gel was kept in the staining solution in a flat container with lid and agitated for 5 hours on a slow rotary shaker.4.2.70 ml glacial acetic acid and 630 ml distilled water. After staining the staining solution was poured out and the gel was rinsed with distilled water.3.3 Running of the system Attached the electrode plugs to appropriate electrodes and the power supply was turned on to 60 V until the sample entered into the separating gel and continued at 100V till the end of separating gel.3.4 Staining of proteins in Gels 7. visualized through a luminescent box and the photograph was taken. ii) Gel destainer Prepared a mixture of 300 ml methanol.4. 7.2.Phytochemical Analysis of Moringa oleifera Seed Powder 219 the first well and the protein samples into the other wells carefully without air bubbles.3.2. The migration of the samples continued for about 2 hours.3.3. 7. 7.2. 35 ml glacial acetic acid and 215 ml distilled water. After loading the samples electrophoresis buffer was filled in the top and bottom reservoirs. Then the power supply was turned off and carefully removed spacers from the gel plate and stained the gel with coomassie brilliant blue (R-250).

OD was taken at 725 nm using spectrophotometer. The extracts were then filtered through Whatman No.2. Sealed the flask with cotton plug and covered it with aluminum foil and kept in a shaker for 2 hrs for extraction.5 N HCl and kept in a water bath for hydrolysis and neutralized the samples with solid . 50 µL of each tannin extract was taken in a test tube and made up to 1ml with distilled water.2.4.4.2 Estimation of free amino acids 500 mg of seed samples of PKM-1 and wild Moringa were extracted in 10 ml of 80% ethanol. a mixture of 40 ml of diethyl ether and 1% acetic acid (v/v) was added and mixed thoroughly to remove the pigmented material if any present. 0. centrifuged the contents and the supernatants were collected and concentrated by evaporation. 7. 7.5ml of Folin-Ciocalteau reagent was added and mixed well. To 0.220 Chapter VII 7. By using standard solution of leucine a standard graph was prepared and the free amino acids content in the samples were calculated using the standard graph.1ml of extract 1ml of ninhydrin solution was added and made up the volume to 2ml with distilled water.2.1 Estimation of tannins The seeds of PKM-1 and wild Moringa were dried at 55oC in an oven and ground into fine powder and sieved. Discarded the supernatant after 5 minutes and 20 ml of 70% aqueous acetone was added.3 Estimation of total carbohydrate by phenol sulphuric acid method Extracted 100 mg samples of PKM-1 and wild Moringa in 5ml of 2. Boiled the mixture in a boiling water bath for 20 minutes. To 400 mg of the seed powder in a conical flask.4 Estimation of different phytochemical compounds in Moringa seed 7. After cooling for 15 minutes in room temperature the OD was measured at 570 nm.4. Prepared a standard graph using standard solution of tannin and calculated the amount of tannin in the test sample with the help of standard graph.1 filter paper and the filtrates were kept in refrigerator at 40C until analysis.2. and kept at room temperature for 40 minutes.

The extract was kept on a water bath to evaporate the petroleum ether until no odour of ether remains. OD was measured at 500 nm. Centrifuged and the supernatants were collected for phenol estimation. After 10 minutes shaken the contents in the test tubes and placed in a water bath at 25-300C for 20 minutes.4.5 Estimation of phenol by O-Phenanthroline method 1 gm each of the two varieties of Moringa seed sample was taken and extracted using phosphate buffer saline (PBS) solution. 0.1 ml of each sample extract in two other test tubes.1M FeCl3 and 200 µl of 0. Made up the volume to 1ml in all test tubes and added 1ml of phenol solution to each test tube including one test tube with distilled water serving as the blank. To 100 µl of each sample solution 300 µl of 0. Weighed the oil content in the flask.2.Phytochemical Analysis of Moringa oleifera Seed Powder 221 sodium carbonate until effervescence ceased.2 M sodium acetate (CH3COONa) was added to keep the pH of the solution in between 3 and 4. .2. The percentage of lipid in the seed was calculated by the formula: Weight of lipid % of lipid in the seed sample = × 100 Weight of sample 7.2.4.6. Pipetted out 0. 0. OD was taken at 490 nm.4 Estimation of lipids 100g of the seed powder was extracted with petroleum ether for 6h in a soxhlet apparatus. Cool at room temperature. Then 200 µl of 0.5% O-Phenanthroline solutions were added and incubated for 24 hrs in the dark.4.8 and 1ml of the working standard carbohydrate solution into a series of test tubes along with 0. A standard calibration graph was prepared by using standard phenol solution and the phenol contents in the samples were estimated with the help of standard graph. Made up the volume to 100 ml and then centrifuged. Prepared a standard graph and calculated the amount of total carbohydrate present in the test samples using standard graph. Added 5 ml of 96% sulphuric acid to each test tube including the blank and mixed well. 0. 7.

2. 52.4±0.3±0. oleifera were estimated. The volume was made up to 2 ml with 4 % TCA in all the samples. 7. 1% NaCl.2-1.0 ml was taken in a series of test tubes along with 1 ml each of supernatants of the samples in duplicates were taken in test tubes. Centrifuged the samples at 2000 rpm for 10 minutes. The contents were mixed and incubated at 37°C for 3 hrs resulting in the formation of osazone crystals. Standard ascorbate ranging between 0.2±0. .4±0. Prepared a standard graph and the ascorbate content in the samples were calculated by comparing with the graph readings and the value was converted into percentage. 0. The crystals were dissolved in 2.4. The total protein in the PKM-1 and PKM-1 (A4+V+N) seed powder were 61.2±0.3 % and 61.1).5 ml of dinitrophenyl hydrazine (DNPH) reagent was added in all test tubes followed by 2 drops of 10% thiourea solution.1 % (Table-7. the PKM-1 variety of seed extracts have higher amount of protein in all the samples than the wild variety.4.5 ml of 85% sulphuric acid in cold. The amount of protein in mg/100mg (%) of seed powder in different solvent extracts of PKM-1 were observed as 58.2 % respectively. DNPH reagent and thiourea were added to the blank alone.1 Estimation total protein and the various proteins in different samples The total proteins and the proteins in four different solvent extracts (distilled water.1N NaOH and 70% ethanol) of the defatted seed powder of the PKM-1 and wild varieties of M. 0. When the quantity of proteins in each sample of PKM-1 seed powder was analysed and compared with the seed powder of wild Moringa.1. it was found that. after the addition of sulphuric acid Tubes were cooled in ice and the measured absorbance at 540 nm with spectrophotometer.222 Chapter VII 7.6 Estimation of Ascorbic acid (Vitamin C) 1 g each of two seed samples were extracted using 4% TCA and the volume is made up to 10 ml with the same. A pinch of charcoal was added with the supernatant and stirred vigorously using a cyclomixer and kept it for 5 minutes. while that of wild Moringa was 56.3 Results 7.3. Again centrifuged the mixture to remove the charcoal particles and the supernatant was collected and used for estimation.

1 Quantity of total protein and proteins in the four extracts of the of defatted seed powder of M. 41. 24.1±0. The high and low turbidity solutions of bentonite powder were used for the analysis and the turbidity of non-treated (control) and treated samples were measured.4±0.1 N NaOH extract 32.4±0. Among the Moringa varieties. the NaCl fraction of PKM-1 types were observed to be better than wild Moringa seed powder fractions (Tables-7.2±0. using 40.2 52.3±0.3 7.3.5.1N NaOH and 70% ethanol respectively and the same in PKM-1 (A4+V+N) were 58.2. The different fractions were tested for their purification efficiency using synthetic turbid waters made with bentonite clay.3±0.2&3).2&3). .2 Partial purification of water and NaCl extracted protein and the assay of their turbidity removal efficiency As the water and NaCl extracts were the effective ones in all varieties of Moringa seeds.4±0.3±0.2±0. Whereas the protein content in the defatted seed extracts of wild Moringa was 52.4±0.3±0.3±0.2 56.5 1% NaCl extract 52. Table 7.3 32.6±0.1±0.6±0. 1% NaCl.4±0.1±0.2 and 28.1 41.4 58.1 28.2 and 22.2 24. 60 and 80% ammonium sulphate solutions.4±0.1 52.5±0.1 for distilled water.Phytochemical Analysis of Moringa oleifera Seed Powder 223 32.4±0. an attempt was also made for the partial purification of seed protein of these extracts by ammonium sulphate precipitation and fractionation.2. oleifera varieties PKM-1 PKM-1(A4+V+N) WM % % % Total proteins 61.3. 32. 52.3 22.6±0.3.4±0.5±0.6±0. NaCl fractions were found to be more effective than water fractions.3 and 28. Among the water and NaCl extract fractions. 0.1 Water extract 58.2 0.2 Ethanol extract 28.1±0.3 61.1. It was found that the highest turbidity removal occurred in samples treated with 60% fractions of water and NaCl extract of PKM-1 as well as wild Moringa seeds (Tables-7.

5 ±1.3 49.2 25.5 ±1.1 80% fraction of 30.2 ±3.5 25.1 ±2.2 ±1. 60 min.2 ±2.3 307.5 469.3 ±1.3 480.2 393.5 32.6 37.3 ±1. Control 49.1 ±2.4 30.5 ±1.2 308.3 60% fraction of 307.1 ±2.3 ±1.5 30.3 480.2 352.1 1%NaCl extract ±2.5 40% fraction of 361.3 60% fraction of 20.4 246.6 32.1 ±2.4 325.5 ±2.2 ± 1.1 ±1.2 ±2.2 ± 1.2 47.1 276.2 ±3.9 1%NaCl extract ±1.2 1% NaCl extract ±3.2 333.2 ±1.2 47.2 ±1. oleifera in turbidity removal from highly turbid synthetic turbid water (500 NTU) Turbidity after Turbidity after Turbidity after PKM-1 protein PKM-1 (A4+V+N) W. 120 min.6 18.5 27.4 15.3 ±2.1 ±4.3 ±2.6 26.4 ±2. 120 min.3 ±2.2 ±1.3 40% fraction of 32.1 345.5 ±1.2 ±2. protein treatment protein treatment treatment Protein fractions After After After After After After 60 min.5 37.224 Chapter VII Table 7.6 26.2 295.8 307. 120 min.4 18.3 ±2.3 20.2 ±2.2 ±1.2 34.3 ±2.5 25.2 40% fraction of 34.4 32.3 water extract ±2.2 246.2 ±2. 120 min.2 ±2.2 60% fraction of 24. oleifera in turbidity removal from highly turbid synthetic turbid water (50 NTU) Turbidity after Turbidity after Turbidity after PKM-1 protein PKM-1 (A4+V+N) W.5 ±2.4 321.8 374.2 ±2.3 ±0.2 278. 60 min.1 water extract ±2.3 1% NaCl extract ±2.3 344.3 328.1 ±4.M.3 ±4.8 water extract ±2.3 49.4 29.8 32.6 15. 60 min.2 ±2.1 ±1.2 ±2.3 Table 7.3 ±1.3 1%NaCl extract ±2.1 ±2.1 ±2.5 ±1.2 ±2. 60 min.2 48.2 329.2 ±1.1 325.5 498.5 ±2.1 1% NaCl extract ±2. 120 min.1 ±1.5 ±2.5 ±2.2 ±2. protein treatment protein treatment treatment Protein fractions After After After After After After 60 min.5 295.2 24.4 299.2 ±2.3 ±3.5 ±2.6 45.2 43.2 ± 2.6 306.1 water extract ±2.4 ±2.3 27.1 ±1.6 312.2 Effect of partially purified NaCl extracts of M.1 60% fraction of 278.2 32.2 ±2.3 ±1.3 Effect of partially purified NaCl extracts of M.1 32.M.8 water extract ±2.4 ±1.1 80% fraction of 30.3 80% fraction of 328.2 ±2.3 ±2.2 361.4 ±2.5 ±3. 120 min.3 40% fraction of 346.5 498.5 29. Control 498.5 ±1.4 312.4 385.1 .3 480.1 ± 4.2 water extract ±1.4 80% fraction of 333.8 22.2 ±2.2 ±1.5 457.1 437.2 47.2 ±2.4 306.

3 Isolation and determination of molecular weight of 60% NaCl fraction of protein by SDS-PAGE As the 60% ammonium sulphate fraction of NaCl extract was found efficient among different fractions in all varieties of M.4 kda. . oleifera Lam. 7.4 kda may be present in this fraction and active in the purification. A broad prominent band of protein found to be isolated in the gel at this region. Fig.3. No other bands were isolated in the gel from this fraction.1 SDS-PAGE showing separation of 13. the 60% ammonium sulphate precipitated fraction of NaCl alone was selected for further analysis using SDS-PAGE. oleifera. Hence a single protein or a group of proteins around the molecular weight of 13. The figure 7.1 shows that the seed fractions of all types of Moringa seeds contain the same type of proteins with a molecular weight around 13.4 kda protein from NaCl extracts of wild.Phytochemical Analysis of Moringa oleifera Seed Powder 225 7. PKM-1 and PKM-1(A4+V+N) varieties of M.

03 ±0.3%) than wild Moringa (38.02±2.31±0.02&0.4 Estimation of some components other than proteins in M.90 (A4+V+N) ±2.02&0.31 0.03 and 0.02 ±0. Phenol content was found to be more in PKM-1 and PKM-1 (A4+V+N) (0. in the four solvents studied here were not reported anywhere in literature.2 ±0.7 4. Similarly. oleifera seed powder PKM-1 and wild Moringa seeds were analysed for other components apart from proteins (Table-7.02 ±0.03 7.184±0.03 7.02 PKM-1 6.2).01 ±0.01 ±0.69±0.184 0.2%).2 ±0.03 %) than wild Moringa (0.03 ±0.5 ±0. β carotene content was analysed.31±0.2 ±0.02%). The amount of ascorbic acid was not having much difference (0.55±0. 0.3. oleifera varieties.4).87 WM ±1.02%).68±1. Table 7.90±0.226 Chapter VII 7.05 43.31 0.325±0. the quantity of lipids was found to be higher in PKM-1 and PKM-1 (A4+V+N) (43. The water soluble kernel crude .03%).93±0.05±1. while in wild variety it was less (0.01&3.3 ±0. free amino acids also were higher in wild variety (4.02%).55 0.02 ±0.93 PKM-1 ±1.7±0.02 ±0.316±0. Carbo Free Lipids Tannins Phenol Ascorbic hydrates amino acids % % % acid % % % 6.2 & 42.8 ±0.2%) than PKM-1 and PKM-1 (A4+V+N) (6.316 0.5%&6.29 0.325 0.69 0.69±0.2 3.2 ±0.01%) than PKM-1 and PKM-1 (A4+V+N) (3.02. Carbohydrate content was found to be higher in wild Moringa (7. But there are studies regarding the content of protein in the crude extract and water extract.68 38. but it was found to be insignificant in both the varieties and the result is not included here. On the contrary.4 Discussion Comparative study of the seed protein content of the defatted seed powder of PKM-1 types and wild Moringa extracts.02 ±0.8 3. Tannins also were higher in amount in the PKM-1 and PKM-1 (A4+V+N) (0. PKM-1 (A4+V+N) and wild Moringa respectively) (Table-7.02 42.03% in PKM-1.2±0.29±0.87±0.4).69 0. 4 Quantities of different phytochemical components in the seeds of M.

37±0.5%. fractionation and turbidity reduction analysis showed significant difference in the clarification of turbid water by the different fractions. (2001). The protein content in four different extracts on comparison found that. This difference in the protein content is found to be closely related to the variations exhibited in the water purification and antimicrobial properties of the extracts. NaOH and ethanol extracts in a descending order. But they added that the residues left after water extraction of kernels or meal had crude protein contents of 35. This is an evidence for the presence of other proteins in the kernel and meal (defatted seed powder). Ethanol extracts in higher doses were effective than NaOH extracts.07 g/100 g in the crude kernel. It clearly expressed the fact that there are various types of proteins in the seed extract having different degrees of water purification efficiency. Another observation was that when the proteins were fractionated the degree of clarification of turbid waters was reduced than that by the direct extracts of the seeds without fractionation. Eventhough. Compaore et al. it was observed to be more efficient than water extract. which are not water soluble. The ammonium sulphate precipitation. In antibacterial studies the water and NaCl extracts only were found effective.Phytochemical Analysis of Moringa oleifera Seed Powder 227 protein of wild Moringa was reported as 36. among the studied varieties water extracts contain the highest quantity of protein. . The least content of protein was found to be present in the ethanol extract (Table-7. which is followed by NaCl. oleifera are rich in proteins having 35. Hence it can be inferred that the cumulative effect of all the types of proteins in the extracts will be more effective than the fractions. They also found that the loss of dry matter from kernel and meal (fat free kernel) after water extraction was 20. The extracts having lower quantities of protein were less effective in these processes. The seed material in the present study was the seed meal after oil removal.5 and 41. (2011) observed that he seeds of M. the protein content in the NaCl extracts is lesser than water extract.1).3 and 70.3 % respectively.8% by Foidl et al. in which the amount in the water extract of wild Moringa was 52.8% respectively.

was the prominent one and they suggested that it might be playing major role in removing arsenic from water. et al. Kwaambwa & Maikokera (2008) and Sahni et al. They have the opinion that the active protein is actually composed of two 6. no direct correlation was observed. This proved that there is no change between the protein content of PKM-1 and PKM-1(A4+V+N) seed extracts. Sahni et al (2008) fractionated five prominent cationic protein bands of different intensities in the molecular weight range of 157-158 kDa. (2005) and Gassenschmidt et al. et al. 34-40 kDa and 6-16 kDa from M. Hence. This finding clearly proved . oleifera seed. Eventhough. (1995) described dimeric cationic proteins with 12-14 Kda molecular mass. i. Ndabigengesere. not only the quantity of protein.228 Chapter VII On comparison of the concentration of proteins in water and NaCl extracts and their water clarifying ability. Kwaambwa & Maikokera (2008) also separated a protein having a molecular weight 12-14 after ammonium sulphate precipitation and column chromatography. 97-98 kDa. oleifera seed. it can be concluded that proteins are the main components in coagulating the pollutants. water extract contain higher protein content than NaCl extract. 114-116 kDa.5 KDa. No significant change was observed in the effectiveness of PKM-1 and PKM-1(A4+V+N) seed extracts in the clarification of turbid bentonite clay water. There are reports regarding the isolation and purification of proteins of different molecular weights from M. (1995) studied and purified a flocculating cationic protein with a molecular weight less than 6. The findings of Ndabigengesere. This may be due to the type of protein extracted by the solvents. Ghebremichael et al.4 KDa. but type of protein also is important in the ability of flocculating the impurities in the polluted water or turbid water.e. All the fractions of Ammonium sulphate precipitation showed some degree of clarification of the turbid water. (2008) support the present observation that the most effective protein in the purification of polluted water is the one with a molecular weight of around 13. The protein fractions having molecular weight in the range 6-16 KDa.5 Kda subunits connected with an S-S bond. that is cleaved when protein extraction occurs in reducing conditions. (1995). NaCl protein fractions were found to be superior in the efficiency of turbidity removal.

the best practice would be to purify the protein in the extract showing higher efficiency by removing non-protein substances which may reduce the efficiency of purification. oleifera seed extract are proteins. One fact to remember is that large number of proteins with different molecular weight having lower and higher efficiencies is present in the Moringa seed. The effectiveness of the purification was reduced when they are fractionated into groups based on molecular weight. Hence instead of fractionating the proteins. From the analysis it can be concluded that the proteins in this extract are responsible for its efficiency in the purification of polluted water and antibacterial property. In a single solvent extract. In the present study the 1% NaCl extracts showed better efficiency. .Phytochemical Analysis of Moringa oleifera Seed Powder 229 that the major active components responsible for the water purification and antimicrobial ability of M. proteins of different molecular weight are present. The combined effect of all these proteins is responsible for the water purification and antibacterial properties of seed extracts in different solvents.

230 Chapter VII .