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Family Scrophulariaceae is a large family with almost 87 genera and 4800 species. Eremophila (Scrophulariaceae) is an endemic Australian genus with 214 species, which is commonly known as Fuchsia bush, Emu bush or Poverty bush. Plants of this genus played an important role for the Australian Aborigines who used them widely for medicinal and ceremonial purposes.
The genus Eremophila possesses many biological and pharmacological activities such as antimicrobial, antiviral, antiproliferative, anti-inflammatory and immunomodulatory activities.
Although, many studies have been carried out on many species of this genus and have generated immense data about the chemical composition and corresponding biological activity of extracts and their isolated secondary metabolites, there are still a lot to be explored. To the best of our knowledge, little biological and phytochemical studies were found in the available literature for Eremophila maculata (Ker Gawl.) F. Muell.
This study was divided into four main chapters:
Chapter (1): An in-depth comparative macro- and micromorphological study of the stems and leaves of two Eremophila species, viz E. maculata and E. purpurascens was done. Moreover, a comparative genetic study was done, in which the DNA of both plants was extracted from leaf samples and analyzed using 10 decamer random primers which will help in further standardization and quality control studies.
Chapter (2): A preliminary biological screening of Eremophila maculata methanol extract was carried out including the cytotoxicity, antimicrobial, antiviral, anti-allergic, in vitro antioxidant and hepatoprotective activity. The latter was further supported by an in vivo study together with an in vivo study of its antidiabetic activity.
Chapter (3): A preliminary phytochemical screening of Eremophila maculata leaves followed by an in-depth phytochemical study of its methanol extract was done. Further chromatographic separation was carried out to characterize and isolate the relevant compounds that may be responsible for the biological activity. Moreover, LC-MS run was done on the methanol extract with the aim to tentatively identify its chief active constituents.
Chapter (4): A comprehensive study was performed on the essential oils of Eremophila maculata leaf, flower, fresh and air-dried stems with the target to explore their chemical constituents. Moreover, the antimicrobial activity of both the flower and leaf oils was thoroughly studied on different Gram positive, Gram negative, MRSA isolates as well as fungi.
Botanical and Genetic characteristics of Eremophila maculata (Ker Gawl.) F. Muell and Eremophila purpurascens Chinnock (Scrophulariaceae)
a) Botanical Study of Leaves and Stems and of Eremophila maculata and Eremophila purpurascens
1- Macromorphological study of the plant
The results of this study revealed that macromorphological features of E. maculata and E. purpurascens are highly similar to those of the family Scrophulariaceae, order Myoporea. They occur as a perennial evergreen shrub of less than 2 m tall, a shrub or small tree less than 5 m tall or a tree 5-10 m tall in case of E. maculata and about 0.3-1.5 m high for E. purpurascens.
They are characterized by the presence of erect, cylindrical branches. The young branches are light brown to green in colour while the old ones are brown showing longitudinal fine striations, carrying alternate and sessile leaves. The leaf lamina is ovate lanceolate in shape with an acute apex and entire margin. The base of the leaf is symmetric. The leaf shows pinnate reticulate venation which is anastamosing near the margin. The upper and lower surfaces are glabrous. Flowers are solitary, actinomorphic, hermaphrodite and pentamerous with distinct calyx and corolla.
Morphologically, they could be differentiated by the colour of the flower, being yellow in E. maculata and hairy spotted purplish in E. purpurascens. They also differ in the fracture of the stem and the dimensions of the leaf being larger in the former and smaller in the later.
2- Micromorphological study of the plant
Micromorphological study of E. maculata different organs showed the following features:
i) The Leaf
A transverse section in the leaf shows upper and lower epidermises and a lamina with isobilateral mesophyll with scattered starch granules and calcium oxalate rosettes. The palisade is continuous over the midrib only in the upper surface and exists in two rows, while on the lower side; it is present in a single row and is shorter in length. The spongy tissue consists of several rows of more or less rounded parenchyma cells with wide intercellular space traversed by secretory schizolysigenous cavities lined with epithelium and secreting oily material observed in the lamina. Crystals are present in the form of small rosettes of calcium oxalate.
The epidermal cells of both surfaces are nearly similar in shape. They are polygonal, isodiametric with beaded anticlinal walls. The upper and lower epidermises are covered with thin striated cuticle. The stomata are found on both sides of the leaf mostly of anomocytic type but more on the lower surface.
The midrib shows two rows of palisade beneath the upper surface followed by 3-4 layers of thin walled parenchymatous cells. The cortical tissue of the midrib consists of 4 or 5 rows of collenchymatous cells abutting the lower epidermis. The pericycle is collenchymatous in the form of an arc shape above and below the vascular bundles in the vein.
The vascular tissue consists of crescent shaped collateral vascular bundle consisting of phloem and xylem and traversed by uni-to biserriate non-lignified medullary rays.
ii) The Stem
A transverse section in the young stem branch is almost rounded in outline. The epidermis consists of polygonal, axially elongated lignified cells with beaded anticlinal walls and covered with striated cuticle. Stomata are rare. Nearly all the epidermal cells are filled with rosettes crystals of calcium oxalate.
The cortex is formed of 4–6 rows of thin walled oval parenchyma cells containing calcium oxalate rosettes. In the structure of the cortex, there are large subepidermal rectangular cells in some regions. The endodermis is distinct and filled with starch showing a starch sheath. The pericycle is sclerenchymatous consisting of small isolated groups of lignified fibers.
The vascular tissue consists of a complete ring of collateral vascular bundles, and traversed by uni- to biseriate medullary rays filled with starch followed by the pith which is comparatively wide consisting of lignified large rounded pitted parenchymatous cells, containing numerous rosettes crystals of calcium oxalate.
Concerning the old stem, it differs from the young stem branches in the presence of atypical lignified cork (rhytidoma) dead tissues formed of tangentially elongated brown cells with lignified thick walls, the presence of irregular rectangular cells beneath the cork. In surface view, the cells appear polygonal; some cells contain prisms of calcium oxalate. It possess narrow secondary cortex consisting of 3 or 4 layers of polygonal cells, interrupted by patches of fibers and large patches of sclereids nearly reaching the pericyclic region. The vessels are arranged in radial rows. The pith is narrower containing lignified pitted parenchyma.
• The micromorphological structure of the leaf and stem of E. purpurascens is more or less similar to their respective ones in E.maculata but could be differentiated by:
i) The leaf
It is dorsiventral with epidermal cells that are more beaded and striated. The upper epidermis is slightly wavy, while the lower is straight. The lower surface is less prominent. The schizolysigenous oil glands and stomata are less in number. The pericycle is parenchymatous. The leaves contain no starch. Rosettes of calcium oxalate are larger in size.
ii) The stem
There are also many differences in the anatomical structure of the young stem branch such as the presence of non- glandular, multicellular, uniseriate hairs covered with warty cuticle. The number of stomata is more. The long lignified tracheids and the rosettes of calcium oxalate are smaller in size than that of E. maculata. The medullary rays contain less starch granules.
Moreover, many variations exist between the 2 species concerning the microscopical characters of the old stem branch being harder and more lignified in E. purpurascens. The cork is narrower and less lignified with cork cells smaller in size. The absence of calcium oxalate prisms beneath the cork. The sclereids and fibers interrupting the cortex are less in number.
b) DNA Profiling of E. maculata and E. purpurascens Using Random Amplified Polymorphic-DNA Polymerase Chain Reaction Technique (RAPD-PCR)
On the genetic level, it can be concluded that the most relevant fragment resulting from the successful combination of template and primer was that produced by OPB-17 and OPA-07 RAPD primers. Such primers could be used to discriminate between the two Eremophila species depending on their low values of similarity coefficients and high level of polymorphism.
However, the other estimated RAPD primers, which produce high values of similarity coefficient and low levels of polymorphism, could be used in the identification of different Eremophila species especially OPC-12 RAPD primer which produces 100% similarity coefficient.CHAPTER 2
Biological Screening of the Leaf Methanol Extract of Eremophila maculata (Ker Gawl.) F. Muell
This chapter involves screening the following activities:
The cytotoxicity of EMM was evaluated using thirteen different cancer cell lines. It exhibited weak cytotoxic effect against A549, PC3 and HepG2 cancer cells after 72 hrs incubation using SRB assay. It showed more cytotoxicity to PC3 as compared to A549 and HepG2 with IC50 values of 280.86, 527.48 and above 600 μg/ml for the PC3, A549 and HepG2 cell lines, respectively.
Also, EMM showed moderate cytotoxic effect on Vero cells (CCL-81) cell line with IC50 of 92.87 μg/ml.
Furthermore, initial cytotoxic screening was done on EMM using nine different human cancer cell lines namely, HeLa, DLD-1, HCT 116, T47D, MCF-7, MDA-231, K562, Molt4 and RBL-2H3 using the MTT assay. EMM exhibited low to no cytotoxicity across the cell lines tested with IC50 above 600 μg /ml.
• Antimicrobial and antiviral activity
a) The antimicrobial activity
The antimicrobial activity of the EMM was assessed by the agar well diffusion method and MICs were determined. In general, the extract showed inhibition of standard Gram-positive and Gram-negative bacterial growth in the diffusion method. Moderate inhibition was observed for almost all the tested fungi except for Syncephalastrum racemosum which showed very weak inhibition.
All tested Gram-positive and Gram-negative bacteria were susceptible to EMM with MIC values ranging between 1.95 – 7.8 μg/ml. Staphylococcus aureus, Bacillus subtilis and Aspergillus fumigatus were highly susceptible to the antimicrobial effect of EMM with MIC value of 1.95 μg/ml for all the three previously mentioned organisms. Escherichia coli was the most susceptible Gram-negative bacteria for EMM with MIC value of 3.9 μg/ml. EMM moderately inhibited Pseudomonas aeruginosa, Geotrichum candidum and Candida albicans with MIC value of 7.8 μg/ml. Syncephalastrum racemosum was less sensitive to the total EMM antimicrobial activity showing MIC value of 31.25 μg/ml.b) The antiviral activity
Concerning the antiviral activity, EMM showed a potent antiviral activity against VSV and HSV- 1 in a dose dependent manner. VSV was most sensitive to the antiviral activity of the EMM than HSV-1 showing 73.45 and 84.17% inhibition of the viral replication for the VSV and HSV-1, respectively at a dose of 50 μg/ml.
• Anti-allergic activity
EMM exhibited very week or even no anti-allergic activity showing neither inhibition in either A23187 or IgE-induced degranulation in sensitized RBL-2H3 cells nor reduced β-hexosaminidase release.
• In vitro antioxidant and hepatoprotective activity
a) 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity assay
EMM exerted a moderate antioxidant activity as indicated by reduction of the DPPH• radicals attaining the 50% scavenging activity of DPPH• by 0.32 mg/ml.
b) HepG2 cells
Treatment of HepG2 cells with EMM at concentrations of 1.0, 0.1, 0.01 mg/ml showed significant concentration-related amelioration of CCl4-induced damage in cell membrane as evidenced from normalized values of AST and ALT. The effect of the tested sample on the hepatocyte membrane integrity was comparable to that of the reference standard silymarin.
Pretreatment of HepG2 cells with EMM at 1, 0.1, 0.01 mg/ml significantly replenished the levels of GSH, SOD and TAC in a concentration-related manner, compared to cells treated with CCl4 only.
• In vivo hepatoprotective activity
This activity was assessed through the determination of different parameters including:
a) Serum enzymes
Serum ALT and AST in rats treated with TAM were significantly elevated by (164.8% and 90%) respectively, as compared to the corresponding control group. Treatment with EMM exhibited an observable protective effect with regard to the assessed enzymes and significantly inhibited the rise in ALT and AST by 45.23 and 45.79%, respectively, as compared to the corresponding TAM group values.Oxidative status
TAM treatment significantly elevated TBARS (55.56%) as compared with the corresponding control group. Treatment with EMM significantly ameliorated TAM-induced oxidative stress reducing TBARS by 28.57% as compared with the corresponding TAM-treated group.
c) Effect on TNF-α
Treatment of rats with TAM significantly increased the TNF- α level (337.5%) when compared with control group. EMM treatment in the combination group significantly decreased the TAM-induced elevation in TNF- α level by 54.29%.
• In vivo antidiabetic activity
Injection of STZ into rats exhibited significant elevations in FBG level by 278% as compared to normal control rats. These changes were concomitant with significant declines in serum insulin level by 52.63% as compared to normal rats. Administration of GLB to STZ-diabetic rats produced significant declines in FBG by 28.57% associated with a marked increase in serum insulin level by 22.22%, as compared to STZ-diabetic rats. Administration of EMM to STZ-diabetic rats elicited a significant increase in serum insulin (88.89%) accompanied by a significant decrease in FBG level by 65.60% as compared to STZ-diabetic rats. However, it produced a significant antidiabetic activity exceeding the effect of the oral antidiabetic agent GLB.
Chemical Composition of the Methanol Leaf Extract of Eremophila maculata (Ker Gawl.) F. Muell
a) Phytochemical Screening of E. maculata leaves
Preliminary phytochemical screening revealed the presence of essential oil, flavonoids, sterols and / or triterpenes, carbohydrates and / or glycosides which is typical to this genus; traces of saponins with absence of anthraquinones, alkaloids, cardiac glycosides and tannins were also detected.b) Phytochemical study of E. maculata leaves
E. maculata methanol extract was prepared and fractionated with n-hexane, dichlormethane and ethyl acetate, respectively. Both the n-hexane and ethyl acetate were subjected to further chromatographic fractionation.
These fractions were manipulated through column chromatography for several times leading to the isolation of the individual chemical constituents which were further purified using preparative thin layer chromatography.
The phytochemical investigation of the tested fractions resulted in the isolation and structural elucidation of 12 compounds belonging mainly to lignans and phenylpropanoids. Compounds were identified by 1H-NMR, APT and 2D-NMR including 1H -1H COSY, HSQC and HMBC spectroscopic data after comparison with previously reported data.
The isolated compounds include;
Compound (1): 1a: β-Sitosterol
Compound (2): (+)-Epieudesmin
Compound (3): Phillygenin
Compound (4): Prunasin
which were isolated from the n- hexane fraction.
Compound (5): Pinoresinol-4-β-D-glucopyranoside
Compound (6): Leucoseptoside A
Compound (7): Martynoside
Compound (8): Verbascoside
Compound (9): Campneoside II
Compound (10): Phillyrin
Compound (11): Pinoresinol-4-O-[6’’-O-(E)-feruloyl]-β-D-glucopyranoside
which were isolated from the ethyl acetate fraction.
from the isolated compounds, pinoresinol-4-O-[6’’-O-(E)-feruloyl]-β-D-glucopyranoside is a rare compound and second to be isolated in nature. Moreover, stigmasterol, leucoseptoside A, campneoside II, martynoside and (+)-epieudesmin were first to be isolated from the genus, while verbascoside, phillyrin and phillygenin were first to be isolated from the species.Concerning, pinoresinol-4-β-D-glucopyranoside and prunasin, they were previously isolated from the species.
Moreover, tentative identification of other five compounds from EMM using the LC-MS analysis was done which are:
• Epipinoresinol-4-O-β-D- glucoypranoside
which were previously isolated from genus Eremophila.
• Syringaresinol-4-O-β-D- glucoypranoside
which was previously isolated from family Scrophulariaceae but not from genus Eremophila.
Chemical composition and biological activity of essential oil of E. maculata
a) GC analysis of the oil
The essential oils of E. maculata were subjected to high-resolution gas chromatographic analyses in order to determine the variation in the volatile constituents of leaves, flowers and both fresh and air-dried stems. All the oils are yellow with a characteristic aromatic odour. About 0.089% v/w yield was obtained for the leaf oil, while the flower essential oil does not exceed 0.026% v/w. Both fresh and dried stems contained traces of volatile oil constituents. Altogether 180 components were identified by GLC-MS representing 98.88, 97.81, 95.07 and 92.49 of the total components from leaf, flower, fresh and air-dried stem oils, respectively. The chemical composition of these oils was documented according to retention indices.
Sabinene represented the major component of the flower oil (46.36%) which consisted of 63 identified components. In addition, limonene (12.63%), α-pinene (9.15%), α-guaiene (3.76%), camphene (3.03%), citroviol (2.54%), germacrene D (2.35%) and (Z)-β-ocimene (2.33%) are major flower oil components.In the leaf oil, 61 components were identified; the major constituents were sabinene (57.93%), limonene (12.87%), (Z)-β-ocimene (3.72%), spathulenol (3.00%), α-pinene (2.69%), α-guaiene (2.48%), α-gurjunene (2.29), germacrene D (2.18%) and trans-phytol (2.10%).
Concerning the fresh stems, 148 components were identified. The major constituents were benzaldehyde (18.57%), α-pinene (18.48%), palmitic acid (13.15%), β-myrcene (10.53%), limonene (7.40%), 9,12-linoleic acid (6.26%) and spathulenol (6.02%). from the air-dried stem, 141 components were detected in which standard fatty acids, such as palmitic acid constituted the major components (20.93%), but SM such benzaldehyde (18.33%), spathulenol (10.02%), lauric acid (6.92%), myristic acid (5.97%), cis-9-octadecanoic acid (3.80%), and nonal (2.47%) were also present in high concentrations.
The monoterpene hydrocarbons from leaf, flower and fresh stems oils accounted for 78.07, 74.66 and 37.01 %, respectively; this is likely due to the high amount of sabinene and limonene in the formers and α-pinene and β-myrcene in the latter. The overall amount of oxygenated monoterpenes is very low; being 0.37, 4.21 and 0.94% in leaf, flower and fresh stems oils, respectively. Furthermore, α-guaiene constituted the most abundant sesquiterpene hydrocarbon, which accounted for 3.78% in the flower oil, while in the leaf oil, it represented 2.48%. In the fresh stem oil τ-cadinene is the major sesquiterpene (1.06%). However, oxygenated sesquiterpenes are present in higher concentration than sesquiterpenes in the fresh and air-dried stem oil accounting for 9.04 and 12.78%, respectively with spathulenol being the most abundant one in both. The air-dried stem oil contains small percentages of monoterpenes (2.55%), oxygenated monoterpenes (2.38 %) and sesquiterpenes (0.81%) but very high levels of fatty acids and others (73.37%).
Variation in the chemical composition of the essential oil in different organs of a plant is very common. The high percentage of fatty acids in the air-dried stems rather than in flower and leaves was previously reported. The significant differences found in the components and relative contents of the essential oils of flowers, stem and leaves can be used as a chemical fingerprint to determine the authenticity of the supplied oils.
b) Antimicrobial activity
The leaf and flower oils were tested against four Gram-positive, four Gram-negative standard bacterial strains, three MRSA and two fungi. The essential oil of both the leaf and flower showed some antimicrobial activity against all tested organisms with MIC values ranging between 0.25-4 mg/ml. However, their effects are more pronounced against Gram positive than Gram negative bacteria. The flower oil exhibited nearly similar activity as that of the leaf against all the tested organisms except for Pseudomonas aeruginosa.
The antimicrobial activity of the oil is likely due to its high content of monoterpenes mainly sabinene and limonene that were previously reported to successfully inhibit bacterial strains, fungi and yeasts.