A new ellagic acid rhamnoside and other biologically active compounds from roots and stems of Synadenium Glaucescens pax
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Date
2023-05
Authors
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Publisher
Sokoine University of Agriculture
Abstract
Medicinal plants have been used as a source of drugs and
drug templates. The extracts of Synadenium glaucescens
Pax (SG) from roots and stems contain bioactive secondary
metabolites against bacterial and viral infections. However,
majority of its pure compounds were uncharacterized and
their phytochemical profiles in different phytogeographical
regions were unknown. Therefore, this study aimed at
characterization of more compounds which are promising
drug leads. Furthermore, the study investigated the
phytochemical profiles of SG from three regions of Tanzania.
Samples of SG were collected from Njombe, Morogoro and
Tanga
regions
as
representatives
of
three
phytogeographical zones of Tanzania. All sample were dried
in a cold and dark room between 15 to 18 oC at the Tanzania
Tree Seed Agency-Morogoro. Samples were pulverized
using a laboratory milling machine (Christy Hunt Engineering
Ltd, England) to afford approximately 1 mm particles’ size.
The isolation of pure compounds involved root barks and
stem barks of SG from Njombe. Phytochemical profiling
involved five parts (root barks, root wood, stem barks, stem
wood and leaves). Sample extraction was done by total
maceration using either methanol or ethanol. Fractionation
of the crude extracts was done through vacuum Liquid
chromatography in solvent gradient systems from Hexane,
ethyl acetate/ dichloromethane and finally methanol/
ethanol. Concentration of extracts was done using rotary
evaporator at <60 oC. Isolation of pure compounds was done
by column chromatography in a solvent gradient system. A
single spot on the thin layer chromatography plate in both
treatments was an indicator for a pure compound. Structures
of compounds were elucidated through (1D and 2D) of a 600
MHz Bruker Avance III HD nuclear magnetic resonance
(NMR) machine. Confirmation of structures involved LC/GCMS, FTMS-ESI, NIST library and in comparison, to
the literature. The SciFinder database was used for
reference of a new compound. The processing NMR
spectral data was performed using Bruker TopSpin 3.6.2
while compound structures were drawn in ChemDraw
Professional 16.0.
Biological activities involved cytotoxicity and antibacterial
tests of pure compounds. Cytotoxicity studies were done
using Brine shrimp assay involving Artemia salina cysts
which were hatched and used within 24 hours of incubation.
The varying concentrations (2400- ȝJ PO LQ P/ RI
% v/v DMSO were employed in duplicate. 7KH FRPSRXQGV¶
concentration killing 50 % of the nauplii larvae was
determined. An In vitro antibacterial efficacy of pure
compounds was done by using microtitre plate assay. Four
standard strains of bacteria were used (Staphylococcus
aureus ATCC 25923, Enterococcus faecalis ATCC51559,
Escherichia coli ATCC25922 and Pseudomonas aeruginosa
ATCC 27853. The activities of the compounds were
evaluated using the observed MIC values.
Determination of the phytochemical profiles on the thin layer
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mg/mL. The number of compounds were recorded at
254nm, 365nm of an ultraviolet lamp and after colour
reaction. Data from the thin layer chromatography were
analysed in JMP statistical software.
A total of fifteen (15) compounds were isolated and
elucidated. A new ellagic acid rhamnoside 3',4'-di-O-
methylellagic acid-4-Į-L-rhamnopyranoside, 28 mg (32) was
isolated as greyish powder. it showed high res. FTMS-ESI-
of 476.0921 and melting point of 251.0~ 252.7 oC. Other
known phenolics 3,4,3'-tri-O-methylellagic acid, 14 mg (33),
hemicosanylferulate,65 mg (34) and octacosylferulate, 19
mg (35) were also isolated. The study also led to isolation of two hydrocarbons from the root barks: 1-nonacosene, 11 mg
(38), and hexacosane,173 mg (39) and one fatty acid from
the stem barks; hexacosanoic acid, 26 mg (40). The isolated
triterpenoids include Lupeol, 65 mg (18) from the stem
barks, euphol, 1855.5 mg (28) from both roots and stem
barks, epifriedelanol, 167 mg (36) from both roots and stem
barks and a steroid ȕ-sitosterol 64 mg (30) from the root
barks.
Cytotoxicity tests indicated that all the tested compounds
were non-toxic (LC50 ! ȝJ P/) up to a maximum tested
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Octacosylferulate
demonstrated strong activity against S. aureus (MIC= 0.125
mg/mL)
Phytochemical screening by TLC indicated a significant
variation in concentration and distribution of compounds in
SG among the three regions (p< 0.0001). Njombe registered
the highest number of compounds (mean= 12.4000) while
Morogoro had the least of all (mean= 9.7000). Euphol, a
triterpenoid in root barks was detected to be at the highest
concentration in Tanga and the lowest in Morogoro.
Derivatives of ellagic acid were detected in root barks at all
ages and locations. The results also indicated continuous
disappearance of red fluorescing compounds in stems barks
and leaves as the plant age increased. The number of
compounds increased with age while the optimal age for a
maximum number of phytochemicals was 3 to 4.5 years.
Furthermore, the stem barks registered the highest number
of phytochemicals (>15).
A new compound: 3',4'-di-O-methylellagic acid-4-Į-L-
rhamnopyranoside along with 14 other known compounds
were isolated from root and stem barks of SG. The isolated
compounds during this study represent six major groups of
secondary metabolites of pharmacological importance
which are Phenolics, triterpenoids, steroids, long chain
hydrocarbons, long chain amine and Fatty acids. This new
compound is suggested to serve as a marker for standardization and quality control of any formulation from
the root barks of S. glaucescens Pax.
Description
Thesis
Keywords
Ellagic acid rhamnoside, Biologically active, Compounds from roots, Stems synadenium, Glaucescens pax