CHARACTERIZATION OF DESERT DATE (Balanites aegyptiaca)

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the various tissues of the B. aegyptiaca grown in Israel. For this purpose, liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography-mass spectrometry /mass spectrometry (LC-MS n ) techniques were employed for the identification of the saponins in the fruit mesocarp, seed kernel, and root extracts of Balanites, and high field nuclear magnetic resonance (NMR) was used for structural elucidation of the major saponins in support of the LC-MS. The second aim of this study was to characterize the biological activities of the saponins from Balanites tissues. Various saponins and mostly saponin-rich extracts (SREs) were evaluated for the antifungal and larvicidal activity using some of the most prevalent phytofungi, and mosquito larvae. To increase the understanding of the interaction of saponin molecules with the plant membrane, another part of the study focused on the use of SREs as delivery adjuvants across isolated leaf cuticle membranes (CMs). A comparative study of saponin content in seed kernels and its correlation among five Israeli, as well as five international, provenances and the possibility of production of diosgenin (sapogenin) in callus culture in Balanites was also carried out. The LC-ESI-MS n characterization of the methanol extracts of fruit mesocarp of B. aegyptiaca grown in Israel found five major saponins with main one as1064 Da (ca. 42% of the total saponins). Other saponins found in the fruit mesocarp were: 1078, 1196, 1210, and 1046 Da. The structures elucidated by 800 MHz NMR reveled that the main saponin (1064 Da) of fruit mesocarp was 26-(O-β-D-glucopyranosyl)-3β,22,26- trihydroxyfurost-5-ene3-O-β-D-glucopyranosyl-(1→4)-[α-L-rhamnopyranosyl-(1→2)]- β-D-glucopyranoside (1). Subsequently, the second (1210 Da) and third (1078 Da) major saponins in the fruit mesocarp were 26-(O-β-D-glucopyranosyl)-22-O-methylfurost- 5ene.3β,26-diol 3-O-β-D-xylopyranosyl-(1→3)- β-D-glucopyranosyl-(1→4)-[α-L- rhamnopyranosyl-(1→2)]-β-D-glucopyranoside (2) and 26-(O-β-D-glucopyranosyl)-22- O-methylfurost-5ene.3β,26-diol 3-O-β-D- glucopyranosyl-(1→4)-[α-L- rhamnopyranosyl-(1→2)]-β-D-glucopyranoside (3), respectively. The LC-ESI-MS n analysis of the methanol extract of the kernel detected six saponins with main one being 1210 Da (ca. 36% of total saponin). Similarly, nine saponins, with main one 1196 Da (ca. 52.6% of the total saponins), were found in root extracts. In all these tissues, diosgenin was found only the sole aglycone for saponins. The chemical characterization experiment iii
of various Balanites tissues suggested that LC-ESI-MS n can effectively be used for rapid identification of saponins in Balanites using minimum manipulations and chromatographic separation. All SREs of B. aegyptiaca including fruit mesocarp (ME), kernel (KE), and root extracts (RE) were found highly larvicidal against the vector of Denuge (Aëdes aegypti) and West Nile Virus (Culex pipiens). Among the SREs, the highest larvicidal effects were found in KE followed by RE and ME. The larvicidal experiments with pure isolated saponins (1), (2), and (3) revealed that saponins with a methyl group in the C-22 position was found to be highly larvicidal against mosquito larvae, compared with the saponin lacking the methyl group at this position. The presence of a xylose sugar unit at the sugar chain was found to be less active compared to saponons lacking this molecule. Regarding the fungicidal activity, fungal and/or dose specific inhibition of SREs was achieved. Moderate and high inhibition of SRE of fruit mesocparp (ME) was found against the growth of Alternaria solani and Pithium ultimum, respectively, whereas no inhibition was found against the growth of Fusarium oxysporum, Verticillium dahliae, and Colletrotrichum coccodes. SREs of fruit mesocarp, kernel, and root of Balanites were also found to accelerate the delivery of 2,4-D ( 14 C) through isolated CMs. Among the three SREs, mesocarp extract showed better performance. The transmission electron microscope (TEM) and dynamic light scattering (DLS) characterization showed that Balanites saponins produce nano size vesicles and these nano vesicles could have played a vital role in 2,4-D delivery together with the association of saponin molecule with the phytosterol present in the CMs. Among the tested Balanites provenances, the highest sapogenin (2.74 % DW) level in kernels was found in Bet-Shean provenance (Israel) whereas the lowest sapogenin level (1.41% DW) was found in Rajasthan provenance (India). A strong positive correlation (R 2 = 0.849) between sapogenin level and oil content in seed kernels was also observed. In vitro study of callus cultures and subsequent determination of the total sapogenin accumulation, revealed that shoot-derived callus produced the highest level of total sapogenin in vitro when raised on MS basal media supplemented with 1.0 mg l -1 2,4-D alone or in combination with 0.5 mg l -1 BAP, compared to the root, hypocotyl, and epicotyl derived callus. However, the total sapogenin was found to be significantly affected by the iv
Page 1 and 2: CHARACTERIZATION OF DESERT DATE (Ba
Page 3 and 4: This work was carried out under the
Page 5: Abstract The desert date (Balanites
Page 9 and 10: Contents Acknowledgements i Abstrac
Page 11 and 12: Materials and methods 38 - Sample c
Page 13 and 14: - Effect of the saponin source on p
Page 15 and 16: Figure 2.9 1 H NMR spectrum showing
Page 17 and 18: List of Tables Table 2.1 Major HPLC
Page 19 and 20: Glossary CID collision induced diss
Page 21 and 22: Thesis organization This thesis is
Page 23 and 24: fruits have been found in Pharaohs'
Page 25 and 26: Fig 1.3. Map showing the Balanites
Page 27 and 28: themselves provide shade and shelte
Page 29 and 30: should be no surprise that the name
Page 31 and 32: Association with membranes Most bio
Page 33 and 34: the African or Indian samples. No w
Page 35 and 36: Objective and Hypothesis Objectives
Page 37 and 38: CHAPTER 2 Characterization of the f
Page 39 and 40: Extraction and separation For the e
Page 41 and 42: and analysed with an ELS detector (
Page 43 and 44: c d a Figure 2.2. Electrospray mass
Page 45 and 46: corresponds with the claims of earl
Page 47 and 48: Table 2.2. MS n Electrospray mass s
Page 49 and 50: 1209, and could be isomers of these
Page 51 and 52: compound as 26-(O-β-D-glucopyranos
Page 53 and 54: In the 1 H NMR spectrum, resonances
Page 55 and 56: Figure 2.11. Structure of the sapon
Page 57 and 58:
Conclusions From this study we can
Page 59 and 60:
that a study of the saponin profile
Page 61 and 62:
HPLC pump and a refractive index (R
Page 63 and 64:
Table 3.1 Peaks, retention time, am
Page 65 and 66:
(a) (b) (c) (d) (e) Figure 3.2 ESI-
Page 67 and 68:
Table 3.2 The LC-ESI-MS and corresp
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quoted here also are integers obtai
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(a) (b) (c) (d) (e) (f) (g) (i) Fig
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considered cleavage behavior, from
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Table 3.4 The LC-ESI-MS and corresp
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The results of this study also show
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ich extracts of Agave sisalana (Piz
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food preparation based on baby food
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Table 4.3 LC50 and LC90 (ppm) of sa
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1 2 3 Figure 4.1 Structure of the t
Page 87 and 88:
When the images of dead larvae were
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CHAPTER 5 Antifungal activity of sa
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visually and microscopically, and s
Page 93 and 94:
Effects of the SREs on in vitro gro
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Table 5.2 Effect of saponin rich ex
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(Table 5.3). In the case of C. cocc
Page 99 and 100:
CHAPTER 6 Effect of saponins on del
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astomatous cuticles from the upper
Page 103 and 104:
Results Effect of the saponin sourc
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Effect of humidity and temperature
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Table 6.3 Effect of humidity and te
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Discussion and conclusions Agro-mat
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solutions used in our studies has s
Page 113 and 114:
CHAPTER 7 General conclusions and s
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as a delivery adjuvant, not only in
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Botanic Gardens, Kew. 960 pp. Bushw
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Ganzera, M., Bedir, E., Khan ,I.A.
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Keukens, E.A.J., de Vrije, T., van
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Morrissey, J.P., Osbourn, A.E. (199
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Schonherr, J., Riederer, M. (1986).
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Appendices A1 - A4: Articles publis
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new and alternative sources of thes
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first dissolved in 1 ml methanol an
Page 133 and 134:
In Table 1, the total sapogenin as
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114 5. Beneytout JL, Nappez C, Lebo
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Appendix: A2 Natural Product Commun
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min, and finally rinsed three times
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level was significantly increased f
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Table1. Effect of plant growth regu
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A B C D Fig 1. Different callus typ
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Appendix: A3 African Journal of Bio
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The data obtained were statisticall
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Table 4. Mortality of Culex pipiens
Page 153 and 154:
Appendix: A4 Dengue Bulletin 2005;
Page 155 and 156:
of the Institutes for Applied Resea
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References 136 1. Halstead SB. Glob
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112 H20a-H21-H23b-H24bb 23a 31.5 1.
Page 161 and 162:
112.0 H15 β -H17a-H20a-H21 23a 31.
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112 H20b-H24aa-H24bb 23a 31.4 1.64
Page 165 and 166:
םהיתויוליעפו םהיתו
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. םיינגוזה םורדו תח
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םוקאזה ישרושב ירקי
Page 171:
םיטביה לש הנבהה היי
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CHARACTERIZATION OF DESERT DATE (Balanites aegyptiaca)

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