الفهرس 
ABSTRACTOnly 14 pages are availabe for public view
 |  | from | 222 |  |  |
| | | from | 222 | | |
Abstract
Cycadaceae family contains only one genus, Cycas. The latter includes nine species in Egypt (Moawad et al., 2010); Cycas armstrongii Miq., Cycas revoluta Thunb., Cycas circinalis L., Cycas litoralis K.D. Hill, Cycas thouarsii R.Br., Cycas media R.Br, Cycas tansachana K.D. Hill, Cycas rumphii Miq. and Cycas pectinata Griff. The most common species are Cycas revoluta Thunb. and Cycas circinalis L. on which several chemical and biological studies were carried out.
In the present study, three closely related species were selected; C. armstrongii Miq., C. revoluta Thunb. and C. circinalis L. for comparative chemical characterization and evaluation of their biological activities. Rare reports on C. armstrongii Miq. encouraged the phytochemical study explore its active chemical constituents and main therapeutic purposes.
Part I: Comparative Phytochemical Study
Chapter I: Preliminary phytochemical screening
Results of the phytochemical screening of total ethanol 70% of aerial parts of Cycas armstrongii Miq., C. revoluta Thunb. and C. circinalis L., revealed the followings :
• Carbohydrates and/or glycosides, tannins, flavonoids, sterols and/or triterpenes in all the samples under investigation .
• Saponins in all the tested species except C. revoluta.
• Alkaloids, anthraquinones and cardenolides were absent from all of the investigated samples.
Chapter II: Determination of phenolic content:
Results revealed that amounts of phenolics and flavonoids content are the highest in C.circinalis followed by C.armstrongii then C.revoluta, while amount of tannins in C.armstrongii exceeded those of C.circinalis and C.revoluta.
Part II: Comparative Metabolomic Study
Results of metabolomic profiling of the secondary metabolites of the aerial parts of three Cycas species under investigation, using LC‒HR‒ESI‒MS for dereplication purposes, showed a variety of constituents. Among these conistituents, flavonoids and phenolic acids predominated. Forty five metabolites were detected and identified using macros and algorithms that coupled MZmine with online and in-house databases (METLIN and DNP databases for plant natural products).
Part III: Comparative Biological Study
Chapter I: In vitro biological studies:
1. Evaluation of antimicrobial activity
2. Evaluation of antimalarial activity
3. Evaluation of antileshmanial ant antitrypanosomal activity
4. Evaluation of free radical scavenging activity
Results of all biological activities showed the most effective total 70% ethanolic extract was C. circinalis then C.armstrongii then C. revoluta except in antiprotozoal activity which showed best activity for C.armstrongii then C. circinalis then C. revoluta.
Chapter II: In vivo biological studies
1. Determination of LD50
Results showed the safety of all the total 70% ethanol extracts of C. armstrongii, C. revoluta and C. circinalis up to 0.5g/kg b.w.
2. Effeect of different extracts of Cycas species extract on γ radiation toxicity in brain and pancreas of albino rats
Results showed ameliorative role of the three tested species on γ radiation toxicity in brain and pancreas of albino rats.
2.a. Biochemical analysis
Results of several biochemical analysis done as follows:
• Liver and kidney functions
- ALT serum activities reduction: significant decrease in activity in the following order: (C.armstrongii (61.8%) > C.revoluta(61%) > C.circinalis(56%))
- AST serum activities reduction: significant decrease in activity in the following order: (C.revoluta (57.7 %)> C.circinalis (56.6%) >C.armstrongii (56.3%))
- Blood urea and creatinine concentration: significant decrease in concentration back to normal values and serum concentrations of creatinine activity in the following order:
(C.armstrongii (60%) > C.revoluta(51%) > C.circinalis(46%)).
• Glucose and insulin levels
Results compared to the control group showed that γ-radiation induced a significant increase in serum glucose by 67.6% (p<0.001). On the other hand, γ-radiation caused a significant reduction in serum insulin level by 47.2 %.
Results compared to γ- radiation group showed that:
-Serum glucose significant reduction for both C.armstrongii and C.revoluta as equal (29%) but better than C.circinalis (22.5%).
- Elevation of insulin level (C.revoluta (80%) > C.circinalis (53%) >C.armstrongii (49%)
• Platelet count assay
Results revealed that C.circinalis and C.revoluta are equal (31%) but better than C.armstrongii (26.8%) in restoring platelet count.
• White blood cell count assays
Results revealed that C.revoluta (157%) possessed the most activity followed by C.armstrongii (142%) then C.circinalis (101%).
• IL-18, Amyloid A
Compared to the control group, γ-radiation induced a significant increase in serum IL-18 and amyloid A by 153% and 677 respectively (p<0.001). On the other hand a significant reduction in
- serum IL-18: (C.revoluta (50.4%)>C.armstrongii (38.7%)> C.circinalis (25.7%)).
-amyloid A (C.revoluta (68.5%)>C.armstrongii (68%)> C.circinalis (55%)).
• Oxidative stress parameters
-MDA decrease in brain (C.circinalis (62.6%)>C.revoluta (56.7%)>C.armstrongii (46.7%))
-GPx increase in brain (C.armstrongii (138%)>C.circinalis (137%)>C. revoluta (131%))
-TAC increase in brain (C.revoluta (169%)>C.armstrongii (165%)>C.circinalis (137%))
-MDA decrease in pancreas (C.revoluta (58%)>C.armstrongii (50%)>C.circinalis (49%))
- GPx increase in pancreas (C.armstrongii = C.revoluta (58.8%)> C.circinalis(43%))
-TAC increase in pancreas (C.revoluta (209%) > C.circinalis (148%) > C.armstrongii (107%)).
• Detection of brain Injury (NFkb, Cox2, MCP-1, GFAP)
- The decrease in NFkb (C.revoluta(69%)>C.armstrongii (62%)> C.circinalis (53%))
-The decrease in Cox2 (C.armstrongii (60%)>C.revoluta(55%)> C.circinalis (52%))
-The decrease in GFAP (C.armstrongii (42%)>C. circinalis (36%)>C. revoluta (34%))
-The decrease in MCP-1 (C.revoluta(38.5%)>C.armstrongii(37%)>C.circinalis (34%))
• Detection of pancreatic injury (amylase, lipase, microRNA216a)
- MicroRNA216a regulation: Treatment with the three extracts exhibited anti-inflammatory effects in pancreas by significantly down regulating microRNA216a expression compared to γ-radiation group (C. revoluta(59%)>C. armstrongii (58%)> C.circinalis(50%)).
- Serum amylase levels reduction. (C. revoluta(62%)>C. armstrongii(60.9%)> C.circinalis(57%)).
-Lipase level reduction (C. revoluta(57%)>C. armstrongii (54%)> C. circinalis(49%)).
2.b. Histopathological examination (Ameliorative role of Cycas species extract on γ-radiation toxicity)
• Brain tissue of albino rats (neurons in the cerebral cortex, subiculum and fascia dentate, striatum and cerebellum).
• Pancreatic tissue of albino rats (the islands of Langerhans cells).
Results: Cycas extracts, significantly ameliorate radiation induced hyperglycemia. This effect confirmed by reliving the radiation induced pancreatic damage. The effect was more potent in extracts of Cycas armstrongii then Cycas revoluta.
Part IV: Phytochemical Study of Cycas armstrongii Miq.
Based on results of biological studies and the rare chemical reports on Cycas armstrongii Miq.; it was selected for detailed chemical investigation.
Chapter I: Extraction and isolation
- Investigation of n-hexane fraction
Study of lipoidal matters of the aerial parts extract of C. aremstongeii Miq.10 gm of n-hexane fraction was fractionated into unsaponifiable and saponifiable fractions (3.7 gm, 2.75 gm respectively) which were qualitatively and quantitatively analyzed using GC apparatus and results are summarized in the followings:
1. GC analysis of the unsaponifiable matter resulted in identification of 24 components, n-eicosane was the major hydrocarbon followed by n-nonadecane, while GC analysis of the saponifiable matter resulted in identification of 12 components of fatty acids. The major saturated fatty acid was identified as hexadecanoic acid methyl ester and 7-octadecenoic acid methyl ester as the major unsaturated fatty acid.
2. USM portion (2.5 g) of C. armstrongii was subjected to a silica gel VLC using eluents of increasing polarity, where two compounds were isolated and identified as compounds β-sitosterol and stigmasterol using co-TLC with authentic sample, mass,1H NMR and 13C NMR.
- Investigation of dichloromethane fraction
The dichloromethane extract (3.5g) was subjected to VLC using eluents of increasing polarity. Six compounds were isolated as compounds; naringenin, kaempferol, dihydroamentoflavone, dihhydrohinokiflavone, amentoflavone and β-sitosterol glucoside. The isolated compounds were further purified using different chromatographic techniques and identified using HR-ES-IMS, 1HNMR and 13CNMR, DEPTQ, HMQC and HMBC.
- Investigation of ethyl acetate fraction
The ethyl aetate extract (4.2g) was subjected to CC using eluents of increasing polarity and three compounds were isolated and identified as compounds; isoginkgetin, pruinin and vitexin using HR-ESI-MS, 1HNMR and 13CNMR, DEPTQ, HMQC and HMBC.
- Investigation of n-butanol fraction
The n-butanol fraction (7g) was subjected to CC using eluents of increasing polarity and two compounds were isolated and identified as; naringin and vitexin-2″-rhamnoside.
- Investigation of water fraction
• Total phenols extraction and investigation of its constituents
The total phenols were extracted from the water left after n-butanol (20g) by acid - base extraction method resulted in isolation and identification of two compounds; vanillic acid and p- Coumaric acid using HR-ESI-MS, 1HNMR and 13CNMR, DEPTQ, HMQC and HMBC.
• Column chromatography
A portion of the water extract was subjected to CC followed by RP-HPLC to afford four compounds; Caffeine, catechin, epicatechin and epigallo-catechin-3-gallate.
Chapter II: Identification of Isolated Compounds
Table (32) summarizes all of the isolated and identified chemical compounds from different fractions of aerial parts of Cycas armstrongii Miq. The structures of the isolated compounds are reported in figure (114).
Table 32. List of chemical compounds isolated from different fractions of the aerial parts of Cycas armstrongii Miq.
Compound no. (amount) Fraction Structure name Detection Note
1(14 mg) Hexane β-sitosterol CO-TLC, 1H NMR, DEPTQ
First report
from C.armstrongii
2(11 mg) Hexane Stigmasterol CO-TLC, HR-ESI-MS, 1H NMR, 13CNMR
3(12 mg) Dichloromethane Naringenin HR-ESI-MS, 1H NMR, DEPTQ, HMQC, HMBC.
4(5 mg) Dichloromethane Kaempferol HR-ESI-MS, 1H NMR
5(22 mg) Dichloromethane 2,3-Dihydroamentoflavone 1H NMR
6 (12mg) Dichloromethane 2,3-Dihydrohinokiflavone 1H NMR
7 (295 mg) Dichloromethane Amentoflavone HR-ESI-MS, 1H NMR, 13C NMR, HMBC
8(10 mg) Dichloromethane β-sitosterol glucoside CO-TLC
9 (8 mg) Ethyl acetate Isoginkgetin HR-ESI-MS, 1H NMR, DEPTQ, HMQC, HMBC
10(16 mg) Ethyl acetate Pruinin HR-ESI-MS, 1H NMR, 13C NMR, HMQC
11(10mg) Ethyl acetate Vitexin HR-ESI-MS, 1H NMR, DEPTQ, HMQC, HMBC
12(15 mg) n-butanol Naringin HR-ESI-MS, 1H NMR,13C NMR, HMQC, HMBC
13 (10 mg) n-butanol Vitexin-2″-rhamnoside HR-ESI-MS, 1H NMR, DEPTQ, HMQC, HMBC
14 (12 mg) Water Vanillic acid HR-ESI-MS, 1H NMR, 13C NMR, HMQC, HMBC
15(17 mg) Water P- Coumaric acid HR-ESI-MS, 1H NMR, 13C NMR, HMQC
16(3 mg) Water Caffeine HR-ESI-MS, 1H NMR First report
from Cycadaceae family
17 (15 mg) Water Catechin HR-ESI-MS, 1H NMR, 13C NMR, DEPTQ, HMQC, NOESY First report
from C.armstrongii
18 (18 mg) Water Epicatechin
19 (20 mg) Water Epigallo-catechin 3-gallate First report
from Cycas genus
1 2 3
4 5 6
7 8 9
10 11
12 13
14 15 16
17 18 19
Figure 114. Structures of isolated chemical compounds from different fractions of aerial parts of Cycas armstrongii Miq.