A Pharmacognostical Study of capensis (Thunb.) lindl. Family Cultivated in Egypt

A Thesis Submitted By

Mariam Gamal Abd El Nasser Alex For the Degree of Master in Pharmaceutical Sciences (Pharmacognosy)

Under The Supervision of

Prof. Dr. Seham Salah El Din El-Hawary Professor of Pharmacognosy, Faculty of Pharmacy, Cairo University

Dr. Amira Safwat El- Senousy Dr. Hanan Samir Marzouk

Lecturer of Pharmacognosy, Lecturer of Pharmacognosy, Faculty of Pharmacy, Cairo Faculty of Pharmacy and drug University Manufacturing, Pharos

University – Alexandria

Pharmacognosy Department

Faculty of Pharmacy Cairo University A.R.E

2019 Thesis Summary

Abstract

Family Bignoniaceae includes 120 genera with 800 mainly distributed within tropical floras of the world. It consists mainly of woody trees, and lianas. Some species are used worldwide as ornamentals. The eight species and cultivars of Tecoma were collected from different places like El-Orman, Mazhar, Antonyades and St.Mina's botanical gardens, to carry on phytochemical investigation of the most active species. The were first subjected to thorough botanical study and DNA fingerprinting to aid their authentication and identification in any future study.Preliminary phytochemical screening showed promising results with T. capensis (Thunb.) lindl. The HPLC analysis of the total methanolic extract of eight Tecoma species and cultivars showed that T. capensis (Thunb.) lindl. had the highest phenolic content among the eight plants and chemometric study was done to display the similarity between the eight plants. The LC-MS/MS of the total methanolic leaf extract of eight Tecoma species and cultivars lead to identification of 53 compounds and showed that T.capensis (Thunb.) lindl. was the richest extract of metabolite among the eight plants. As a result, T. capensis (Thunb.) lindl. was considered the most active and subjected to further phytochemical investigation. This included investigation of lipoid content using GC-MS, LC-MS/MS of different organs (fruits and seeds) and different fractions (Chloroform, ethyl acetate and butanol) which showed that ethyl acetate was the most active fraction so, isolation and identification of compounds was done on ethyl acetate fraction which lead to isolation of 2 iridoids. Anti-microbial, anti-oxidant and anti-Alzheimer's biological activity was done on the eight Tecoma species and cultivars to evaluate the most active in treating these diseases. This study is the first Anti-alzheimer's disease to be done on genus Tecoma and also the first comparative botanical study to be done on eight Tecoma species and cultivars. Keywords: Bignoniaceae, Tecoma capensis, harmony, red, pink, yellow, T. radicans, T, grandiflora, cultivars, DNA fingerprinting, LC-MS/MS, HPLC, iridoids, Chemometrics, metabolomics, anti-alzheimer's, anti-oxidant, anti-microbial.

Introduction

Family Bignoniaceae includes 120 genera with 800 species mainly distributed within tropical floras of the world. It consists mainly of woody trees, shrubs and lianas. Some species are used worldwide as ornamentals. Bignoniaceae commonly called the ‘trumpet vine’ or ‘trumpet creeper family’ is a widespread family named after the genus Bignonia. The well known medicinally important members of this family are Tecoma, Catalpa, Tabebuia and Jacaranda, while other ornamental species have large spectacular flowers. A striking feature of Bignoniaceae is the assortment of colorful showy flowers. Tecoma capensis (Thunb.) Lindl also known as ‘Cape-honey suckle’ is a climbing grown as an ornamental plant in gardens. Traditionally the leaves were used to treat pneumonia, enteritis, diarrhoea, tonic and analgesic antimicrobial, anti fungal, antipyretic and antioxidant activity

The Aim of the Work

The objective of this work was targeted to compare between the different eight Tecoma species and cultivars cultivated in Egypt. A comparative study between the eight selected species included the following:

1. Reviewing current literature (Chemical and Biological activities) of the genus Tecoma.

2. Collection, identification & authentication of the plant material.

3. Comparative interspecies profiling (botanical, DNA fingerprinting and phytochemical screening).

4. Comparative LC-HR-ESI(QTOF)-MS/MS metabolomic profiling of the total methanol extracts of the leaves & flowers of the eight selected species and different extracts and organs of Tecoma capensis.

5. Qualitative and quantitative analyses of the total methanolic extracts of the eight selected species via appropriate procedures (spectrophotometric and HPLC) & chemometric analysis of the data.

6. Investigation of lipoidal constituents present in Tecoma capensis (Thunb.) lindl.

7. Isolation, identification & structure elucidation of the main constituents using recent chromatographic & spectrometric techniques.

8. Comparative evaluation of anti-microbial activities of the eight selected species and/or cultivars.

Summary

Chapter I: Comparative DNA fingerprinting of Eight Tecoma Species and Cultivars

1. The extracted DNA of the eight Tecoma species and cultivars was amplified using 10 primers to reveal RAPD fragments. Each of the 10 primers successfully directed the amplification of a genome-specific fingerprint of DNA fragment. 2. Different RAPD fragments were produced by PCR, Each primer was subjected to the eight species and results are recorded for each primer. The M.W. bp if it has (0) it stands for absent and if it has (1) it stands for present. If a certain M.W bp is present in all eight Tecoma species then it is a monomorphic band. If the M.W bp present in some species and absent in others then it is a polymorphic band. If M.W bp present in only one species then it is a unique band for this species. 3. A dendrogram was done using average linkage (between groups) to know which species have similarity/close to others and which have genetic diversity. 4. Each primer showed monomorphic bands, polymorphic bands and unique bands. This bands was calculated by comparing the results of each primer. 5. The eight species of the genus Tecoma were subjected to RAPD assay of their genomic DNA. 6. The dendogram showed that there is a high degree of similarity between 3 cultivars of Tecoma capensis: T. capensis var. yellow , T. capensis var. red and T. capensis var. pink indicating that these three species are very closely related. 7. The next species which is related to pink, red and yellow cultivars is T. grandiflora (Thunb.) loisel, T. radicans (L.) Juss., T. smithii Wil. Wats. , T. capensis (Thunb.) lindl and finally T. capensis var. harmony. 8. The B2, C4 and K2 primers were found to be the most effective in generating polymorphic bands on application the RAPD technique to the eight Tecoma species. 9. Such primers could be used to discriminate between the eight investigated species based on the high level of polymorphism between the produced fragments. However, the other estimated RAPD primers, which showed similarity, could be used in the identification, characterization and authentication of different Tecoma species and cultivars

Chapter II: Comparative Macromorphology of Eight Tecoma Species and Cultivars

The comparative macromorphological study of the eight species and cultivars was done on the compound leaves, leaflet, the flower for each species. The leaves and flowers are the most important features for identification of the plant morphologically especially the flower.

From this study the leaves of the different species were observed and found to be very closely related.

A. The Leaves

1. Condition All species were observed and photographed fresh.

2. Colour: The colour of the leaves is generally dark green almost all the year with the upper surface darker in colour than the lower surface.

3. Size and dimensions

T.capensis and its four cultivars is small shrub reaching 1 to 1.5 meter sometimes reaches 2 meters long. T.capensis (Thunb.) lindl, its four cultivars and T.smithii Wil. Wats. compound leaves ranges from 5.5 to 9.5 or 12 cm long, 3-5 cm width. T.grandiflora (Thunb.) loisel. and T.radicans (L.) Juss. is tall trees reaching 2.5 to 3 meters. compound leaves length about 20 to 22 cm long, 5-6 width. Each leaf carries 11-15 leaflets.

4. Insertion: All are cauline. 5. The phyllotaxis are opposite decussate in all eight Tecoma species.

6. Leaf base: All are exstipulated with no sheath present. 7. Petiole: All are short petiolated and show a groove on their upper surface, short petiolated with large groove on the upper surface (small in T. smithii Wil. Wats. and connected in T. grandiflora (Thunb.) loisel. Sometimes, they are nearly sessile.

8. Lamina

- composition: Compound, odd-pinnately, usually 4-10 pairs of foliateleaflets.

-Shape: the leaflets elliptic to sub-orbicular. Sometimes, obovate and usually the terminal leaflet is elliptic. -The venation of all eight species are pinnated veins.

-Margin: All leaves with serrate margins except T.capensis var.harmony shows crenate margin. -Apex: The leaf apex of all eight Tecoma species are acute to obtuseexcept in both T.grandiflora (Thunb.) loisel. and T.radicans (L.) Juss. are acuminate apex. - Base: All leaflets with symmetric base except in T.capensis var. pink shows unsymmetrical base. -Size of leaflet: T.grandiflora (Thunb.) loisel. and T.radicans (L.) Juss. the leaflet measures 5-8 cm length and 1.2 - 1.7 cm width. T x smithii Wil. Wats. and T.capensis (Thunb.) lindl. with its four cultivars leaflet 1.5 to 5 cm long and 0.5 – 1.2 cm width.

B. The Flowers The flowers are zygomorphic, hermaphrodite (bisexual) and have no characteristic odour, however, flowers are very attractive to insects which have long bell shaped five-lobed symsepalus calyx,and trumpet like bilabiate sympetalus corollas of different colors yellow in Tecoma capensis var.yellow and T.smithii, pink in T.capensis var. pink, orange in Tecoma capensis (Thunb.) lindl, T.radicans (L.) Juss. and T.grandiflora (Thunb.) loisel. (orange to red), or red as in T.capensis var. red.

The flower of T.capensis cultivars generally smaller in size than the original. There is a big difference in size between flower of T.radicans (L.) Juss. or T.grandiflora (Thunb.) loisel. and any other T.capensis flower. The flower of T.radicans (L.) Juss. is much bigger in size than T.capensis (Thunb.) lindl.

Chapter III: Comparative Micromorphology of Eight Tecoma Species and Cultivars

I. The leaf:

The leaves of the eight plants were subjected to detailed micromorphological study through microscopical examination of transverse sections and powdered samples.

A. Transverse sections of the eight Tecoma species and cultivars

1. The lamina:

A transverse section of the leaflet through the lamina of eight Tecoma species and cultivars shows upper and lower epidermis enclosing a dorsiventral mesophyll except in T. capensis var. red shows isobilateral T.S. The palisade is formed of one row (except with Tecoma capensis and Tecoma smithii two rows). The midrib appeared more prominent on the lower surface. The vascular tissue of the midrib consists of an arc of crescent shaped collateral vascular bundle and a smaller inverted arc towards the upper side, enclosing in-between narrow pith. a.The epidermis:

The upper epidermal cells are polygonal, nearly isodiametric with wavy walls except in Tecoma grandiflora and Tecoma radicans shows straight walls. The lower epidermal cells similar to those of the upper epidermis, but they have more wavy walls and showing more frequent stomata and trichomes.

Trichomes are very numerous on both surfaces. Branched non glandular, unicellular and multicellular non glandular trichomes are present. b.The mesophyll

The mesophyll is dorsiventral and differentiated into palisade tissue and spongy parenchyma. The palisade tissue is formed of a closely packed layer of one row (two rows in T.capensis) of radially elongated columner cells that show straight anticlinal walls and contain chloroplasts. They are discontinued in the midrib region by collenchymatous tissue. The spongy tissue is formed of irregular round shaped parenchyma cells with wide intercellular spaces. small vascular bundles of the lateral veins are embedded within the spongy tissue. c.The midrib:

The cortex: consists of upper and lower cortical tissue. The upper cortical tissue consists of 4-5 rows of collenchyma cells followed by 3-4 rows of large, thin walled round parenchyma cells, while, the lowe rcortical tissue consists of 2-3 rows of collenchyma cells followed by 4-6 rows of large, thin walled rounded parenchyma cells. Theendodermis is indistinct.

The pericycle: consists of parenchymatous cells surrounding the vascular bundle. They are small, polygonal to rounded cells with thick cellulosic walls.

The vascular bundle very small in size, open collateral arranged in crescent shaped groups forming of almost continuous ring, showing xylem upwards and the phloem downwards.

The phloem is comparatively narrow and consists of thin-walled cellulosic phloem elements, sieve tubes, companion cells and phloem parenchyma with no fibers.

The xylem is composed of xylem vessels. The xylem vessels are lignified, arranged in rows, with annular and spiral thickenings.The parenchyma cells are rectangular in shape having pitted lignified walls.

Medullary rays are uni- to biseriate, formed of radially elongated cells with thin cellulosic non-lignified walls separating the vessels.

The cambium is almost indistinguishable.

B. Isolated elements of eight Tecoma species and cultivars leaves

A.Upper and lower epidermis

The cuticle is smooth in all the eight plants. The upper and lower epidermis is strongly wavy in T. capensis (Thunb.) lindl., T. capensis var. pink and T. capensis var. red

The upper and lower epidermis is slightly wavy in T. capensis var. harmony and T. grandiflora (Thunb.) loisel.

The upper epidermis of Tecoma x smithii Wil. Wats. and T. radicans (L.) Juss. is straight while, their lower epidermis is slightly wavy.

The eight Tecoma species and cultivars in general characterized by presence of stomata in the lower epidermis much more than the upper epidermlis.

Stomata were present in all lower epidermis and observed to be anomocytic stomata with four or five subsidiary cells with no definite shape.

Stomata were present in the upper epidermis only in T. capensis (Thunb.) lindl. and T. capensis var. red.

Calcuim oxalate cluster appeared only in T.capensis (Thunb.) lindl

Calcium oxalate Prisms were found in all eight species except Tecoma x smithii Wil. Wats.

B.Trichomes

1. Unicellular non glandular hair was present in all eight Tecoma species and cultivars

2. Branchedmulticellular non glandular hairs present in T.capensis (Thunb.) lindl, its four cultivars and T.radicans(L.) Juss.

3.T.capensis var.yellow showed least number of branched hairs while, T.capensis var.red showed maximum number of branched hairs in both upper and lower epidermis than the other T.capensis cultivars.

4. Collapsed multicellular non glandular hair present in T.capensis var. yellow, T.capensis var. red and T.capensis (Thunb.) lindl.

5. Bent non glandular hair was found only in T.capensis (Thunb.) lindl and T.capensis var. red.

6.Glandular hair with unicellular head and unicellular stalk was found in T.capensis var.pink , T.capensis var.harmony, T.radicans and Tecoma x smithii Wil. Wats.

C. Stomatal index, Palisade ratio and vein islet number

The stomatal index was calculated through the following equation: Stomatal index= S / S+E x 100 in one millimeter. The palisade ratio is estimated by observing how many palisade cells present in one epidermal cell. While, the vein islet number estimated by measuring how many blocks divided by the veins present in one millimeter square. Those three parameters was measured in the eight Tecoma species and cultivars. T. radicans (L.) Juss. showed maximum number of vein islet number. Part II: Comparative Phytochemical Study of Eight Tecoma Species and Cultivars

Chapter I: Comparative preliminary phytochemical screening of eight Tecoma species and cultivars

The air dried powdered of aerial parts of eight Tecoma species and cultivars under investigation viz., Tecoma capensis (Thunb.) lindl, T.capensis var. harmony, T.capensis var. yellow, T.capensis var. pink, T.capensis var. red, Tecoma x smithii Wil. Wats, T.radicans (L.) Juss., T.grandiflora (Thunb.) loisel. were, separately, subjected to preliminary phytochemical screening for the presence of different types of plant metabolites.

A Similarity in response to the applied tests was noticed between the eight species and cultivars suggesting that they are nearly similar in composition.

All the eight species and cultivars gave positive results with carbohydrates and/or glycosides, sterols and/or triterpenes, phenols and iridoids.

Whereas, Some of them gave positive or/ negative results with alkaloids, tannins, volatile oils , flavonoids and anthraquinones .

T. capensis (Thunb.) lindl showed positive results with all the tests than the other species and cultivars.

ChapterII: Comparative LC-HR-ESI(QTOF)-MS/MS Metabolomic Profiling of the Total Methanolic Extracts of the Leaves & Flowers of Eight Tecoma Species and Cultivars

21 samples of Tecoma species and cultivars were subjected to metabolomic analysis using LC-QTOF-MS/MS.8 total methanolic leaf extracts, 8 total methanolic flower extract, 3 fractions of T. capensis (Thunb.) lindl.[Chloroform, ethyl acetate and butanol]and 2 organs of T. capensis (Thunb.) lindl [Seeds and Fruits].

The crude methanolic extract of eightTecoma species and cultivars leaves and flowers were subjected to metabolomic analysis using analytical techniques of LC/HR/ESI/MS. The ESI spectra obtained using: Synapt G2- Si QTOF mass spectrometer (Waters company, Germany). Briefly, thetotal extract (1 mg/ml in MeOH) wasuploaded on an Accela HPLC (Thermo Fisher Scientific, Bremen, Germany) combined with Accela UV/VIS and Exactive (Orbitrap) mass spectrometer from Thermo Fisher Scientific (Bremen, Germany). The mobile phase composed of purified water (A) and acetonitrile (B) with 0.1% formic acid in each solvent. The gradient elution started at a flow rate of 300 µL/min with 10% B linearly increased to 100% B within 30 min and remained isocratic for the next 5 min before linearly decreasing back to 10% B for the following 1 min. The mobile phase was then equilibrated for 9 min before the next injection. The mass range was set fromm/z (mass-to-charge ratio) 100-2000 for ESI-MS using in-source CID (collision-induced dissociation) mechanism and m/z 50-1000 for MS/MS using untargeted HCD (high energy collision dissociation).In MZmine 2.12, a framework for differential analysis of mass spectrometry data, the raw data were imported. Chromatogram deconvolution was then performed followed by peaks deisotoping.For chromatographic alignment and gap-filling, the retention time normalizer was applied. Excel macros were used to combine positive and negative ionization mode data files generated by MZmine. Peaks produced from the sample were extracted. The Excel macro was used to dereplicate each m/z ion peak with compounds in the customized database (using RT and m/z threshold of ±5 ppm), which provided details on the putative identities of all metabolites in the total extract. The macro was then utilized to identify the top 20 features (ranked by peak intensity) and the corresponding putative identities by creating a list for the extract. Identification of compounds done by using program MZ-mine version 2.28. Fragmentation MS/MS were compared by the NIST database (National institute of standards and technology) fragmentation pattern mass spectrum and fragmentation of previous literature reports.

A. Chemical profiling of the secondary metabolites of eight Tecoma species and cultivars leaves

53 compounds were identified: 13 flavonoids, 11 Alkaloids, 9 Iridoids , 4phenolic acids, 3 phenyl ethanoids, 3 quinones, 1 coumarin , 4 carboxylic acids, 4 Fatty acids and 1 sterol. From comparing LC-MS/MS of 8 leaf extracts; 37 compounds were detected in positive mode. While, 16 compounds were detected by negative mode and the major compounds were found to be flavonoid (rutin and tiliroside), alkaloids (N- nor methyl skytanthine and tecostanine) and phenolic acids (chlorogenic acid and sinapic acid). The order of the richest extract in metabolite was: T. capensis (Thunb.) lindl comes in the first place (50 out of 53 identified metabolites) then, Tecoma x smithii Wil. Wats. (43 out of 53) and T. capensis var. harmony (42 out of 53) inthe third place.

B. Chemical profiling of the secondary metabolites of eight Tecoma species and cultivars flowers

The crude methanolic extracts of eight Tecoma species and cultivars flowers were subjected to metabolomic analysis using analytical techniques of LC/HR/ESI/MS. The ESI spectra obtained using: Synapt G2- Si QTOF mass spectrometer (Waters Company, Germany). All apllied conditions were the same as LC-MS/MS of leaves.

18 compounds were identified: 7 alkaloids, 4fatty acids, 2 flavonoids, 2 carboxylic acids, 1 iridoid, 1 phenolic acid and 1 phenylthanoid. 11 compounds were detected in the positive mode while, 7 compounds were detected in the negative mode. The major compounds were apigenin and caffeic acid which present only in the eight flower extracts however, it was absent from the leaf extracts.

Since, all identified compounds in the flower extracts already existed in the leaf extract.

The use of leaves is recommended as it contains more metabolites than the flowers.

The presence of caffeic acid only in flower extract confirms the HPLC which gave negative result to the eight leaf extracts.

Chapter III: Qualitative and Quantitative determination of phenolic acids and flavonoids in the aerial parts (non-flowering) of eight Tecoma species and cultivars using HPLC analysis

HPLC fingerprinting was performed in view to evaluate the degree of similarity and/or variability in composition between the eight Tecoma species under investigation. The phenolic composition was taken as a measure for discrimination between the eight species and cultivars.

1. In fact, HPLC analysis revealed both qualitative and quantitative variability among the phenolic composition of the eight species.

2. Quantitative results revealed the prescence of rutin and chlorogenic acid in all Tecomaspecies with highest concentrations in T.smithii=0.192 mg/g chlorogenic acid and 0.828 mg/g rutin.

3. Phenolic acids were prevalent in all eight samples such as sinapic acid was found in all species except T.smithii. While, Caffeic acid was absent in all species.

4. gallic acid was only found in T.smithii with amount of 0.28 (mg/g of sample)

5. Among flavonoids, quercetrin appeared to be major in T.capensis and its 4 cultivars.

6. Tecoma capensis species showed promising results in estimation of total Phenolic contentand since chlorogenic acid was present in all species there is probability of being with antioxidant activity.

Chemometric analysis of eight Tecoma species and cultivars using multivariate statistical analysis (MSA) An exploratory analysis of the data was carried out using Principal component analysis (PCA) and hierarchical clustering analysis (HCA), which were used to display the similarity of the HPLC profiles of the samples. The data were analyzed using The Unscrambler v9.7 software (CAMO Software AS, Oslo,Norway) and SIMCA(Umetrics AB, Umea, Sweden). The normalized areas of the HPLC peaks detected in all the studied Tecoma species and cultivars were subjected initially to PCA analysis to outline the compositional differences among them. The PCA score plot (Fig.71-A) shows a clear separation between T. radicans, T.grandiflora &T. smithii, which were distributed to the right of the plot (positive PC1 values). However, the rest of the Tecoma species are clustered closely to each other at the left side of the vertical line representing PC1 (negative PC1 values), which accounted for 65% of the variance. The most influential metabolites for this separation were rutin, catechin and quercetin as implied by their high factor loadings regarding PC1 (Fig.71-B).

The undecoded similarities between the different samples can then be worked out by Hierarchical cluster analysis (HCA) using Wards algorithm, which resulted in dendrogram (Fig.2) sorting the samples into 12 component-variability-dependent. Both T. smithii and T. radicans were clustered intotwo groups exhibiting a long distance compared with the other samples on the cluster scale.

T. capensis (Thunb.) lindl. and its four cultivars are very close to each other while, Tecoma x smithii Wil. Wats., T. grandiflora (Thunb.) loisel. and T. radicans (L.) Juss.

Chapter IV: Quantitative determination of total flavonoid and phenolics of the aerial parts (non-flowering) of eight Tecoma species and cultivars using Colorimetric assays

A. Quantitative determination of total phenolic content by Folin- ciocalteu method

The polyphenolic content of the aerial parts of the eight Tecoma species, estimated as gallic acid equivalent, was deduced from the following equation: Y= 0.0041x – 0.016, R2=1

Where Y=absorbance, x= corresponding concentration (µg/ml) and R2= correlation coefficient

The absorbances recorded for the extracts of the aerial parts of the eight Tecoma species and cultivars showed that the total polyphenolic content of T. capensis (Thunb.) lindl was the highest absorbance then the four cultivars except the harmony showed lower absorbances than T.capensis. Then, T.radicans, T.capensis var. harmony and T.grandiflora lower absorbances in compare to T.capensis. while, Tecoma smithii showed the least absorbance of polyphenolic content.

Tecoma capensis aerial parts indicated that total polyphenolic is about twice that present in Tecoma smithii.

The following eight species represents the absorbances of polyphenolic content in an ascending order (from high to low):

Tecoma capensis>T.capensis var. pink > T.capensis var. yellow > T.capensis var. red > T.radicans > T.capensis var. harmony > T.grandiflora > T.smithii B. Quantitative determination of total flavonoid content by Aluminium Chloride Colorinetric method

The flavonoid content of the aerial parts of the eight Tecoma species, estimated as quercetin equivalent, was deduced from the following equation: Y= 0.0094x + 0.0181, R2=0.9964 Where Y=absorbance, x=corresponding concentration (mg/g) and R2=correlation coefficient

The recorded absorbances for the eight Tecoma species and cultivars showed that T.grandiflora have the highest concentration of flavonoid content expressed as quercetin equivalent. While, T.capensis (Thunb.) lindl showed the least concentration of total flavonoid.

The following eight Tecoma species represents the absorbances of flavonoid content in an ascending order (from high to low):

Tecoma grandiflora > T.capensis var. red > T.capensis var. yellow (aurea) > T.capensis var. pink > T.radicans > T.capensis var. harmony >T.smithii > T.capensis (Thunb.) lindl.

T.grandiflora (Thunb.) loisel. demonstrated the highest total flavonoid content (3.541 mg/g)

T. capensis (Thunb.) lindl showed the highest total phenolic content (8.831 mg/g) comparing with the other investigated species.

Part III: Phytochemical investigation of Tecoma capensis (Thunb.) lindl.

Chapter I: Comparative LC-HR-ESI(QTOF)-MS/MS metabolomic profiling of the seeds and fruits extracts of T. capensis (Thunb.) lindl T. capensis (Thunb.) lindl showed highest number of metabolites in the total methanolic leaf extracts. So, the aim of this study is to focus on T. capensis organs (fruits and seeds).

A. Seeds

Ten compounds were identified in the seeds of T. capensis (Thunb.) lindl.as the following: 6 flavonoids, 2 alkaloids, 1 phenolic acid and 1 quinone. These results indicate that seeds are rich in flavonoids.

B. Fruits

Six compounds were identified in the fruits of T. capensis (Thunb.) lindl.as the following: four iridoids, 1 alkaloid and 1 fatty acid. These results indicate that fruits are rich in iridoids.

The use of leaves is recommended as it is richer in compounds than the flowers, fruits and seeds Chapter II: Comparative LC-HR-ESI(QTOF)-MS/MS metabolomic profiling of the chloroform, ethyl acetate and butanol fractions of T. capensis (Thunb.) lindl

A. Chloroform fraction of T. capensis (Thunb.) lindl.

Five compounds were identified in the chloroform fraction of T. capensis (Thunb.) lindl.as the following: 3 alkaloids and 2 fatty acids. So, chloroform fraction was not rich in compounds

B. Ethyl acetate fraction of T. capensis (Thunb.) lindl.

Ninteen compounds were identified in the ethyl acetate fraction ofT. capensis (Thunb.) lindl.as the following: 6 iridoids ,4 flavonoids , 3 alkaloids, 2 phenolic acids and 2 fatty acids. This fraction was too rich in compounds especially in iridoids and the major compound was the iridoid Tecomoside. Ethyl acetate is the fraction of choice for further investigation and isolation.

C. n-Butanol fraction of T. capensis (Thunb.) lindl.

Eleven compounds were identified in the butanol fraction of T. capensis (Thunb.) lindl.varies between four iridoids, 2 alkaloids,2 flavonoids, quinine, anthranilic acid and phenyl ethanoid. The results showed that the compounds identified in the butanol fraction characterized all by its high molecular weight.

Chapter III: Analysis of Lipoidal content of Tecoma capensis (Thunb.) lindl.

Results of GC/MS analysis of the unsaponifiable matters (USM) of the hexane extract of the aerial parts of T.capensis revealed that

a) The total hydrocarbons constituted (26.42%) of the unsaponifiable matter: six compounds were identified among which tetracosane (4.55%) is the major of them. b) Two sterols; β-Sitosterol and γ-Sitosterol, were identified in the sample, they constituted (57.87%) and (11.11%) respectively. c) One diterpene; phytol constituted (9.54%). d) One triterpene; β-Amyrin constituted (2.90%).

The results of GC analysis of the fatty acid methyl ester (FAME) of the hexane extract revealed that:

a) The total identified fatty acids from the GC analysis were thirteen compounds with percentage (98.77%) , from which the total identified unsaturated and saturated fatty acids were 22.79% and 75.97% respectively. b) Nine saturated fatty acids were identified by GC analysis. Palmitic acid had the highest concentration of saturated fatty acid presented in Tecoma capensis(37.13 %). c) Four unsaturated fatty acids were identified by GC analysis. Linolenic acidn−3 omega fatty acid had the highest concentration of unsaturated fatty acid presented in Tecoma capensis (21.09%). Chapter IV: Isolation and Identification of the Constituents of Ethyl Acetate Fraction of Tecoma capensis (Thunb.) lindl aerial parts

• The ethyl acetate fraction (10 gm) was chromatographed on Silica gel G-60 chromatography column (CC.) (80 L x 3 D cm) packed with 260 gm silica gel G-60. Gradient elution was performed starting with chloroform 100% then CHCl3: MeoH 90:10, 80:20 and so on until reaching absolute methanol yielding 85 fractions; Fractions with similar chromatographic patterns were pooled together as following: Fr. (1-14) pooled together at 100% chloroform, Fr. (15-22) at CHCl3: MeOH (90:10), Fr.(23-30) at CHCl3: MeOH (80:20), Fr. (31-38) at CHCl3: MeOH (70:30), Fr. (39- 47) at CHCl3: MeOH (60: 40), Fr. (48-55) at CHCl3: MeOH (50:50), Fr. (56-60) at CHCl3: MeOH (40:60) and so on until reaching Fr. (83 -85) absolute MeOH. Then, Fr. (23-30) (250 mg) at CHCl3: MeOH (80:20) was subjected to another silica gel G-60 chromatography column (CC.) (80 L x 2D) packed with 133 gm silica gel. Isocratic elution was performed using S2 solvent system (ethyl acetate: methanol 4.5:0.5).Fractions (20 ml, each) were collected and monitored by TLC using solvent system S2, yielding 5 fractions numbered from 100 to 104. The third fraction, number 102 (130 mg), was subjected to CC. Sephadex LH- 20 ( 30 L x 1 D cm, 25 gm) MeOH, Fraction 20 ml, to give finally two compounds E1 and E2 with pink spots on TLC with spraying with p- anisaldehyde /H2SO4 and no fluorescence under UV light by preparative TLC. Compound E1 H-NMR showed the most characteristic signals of iridoid as in table (59).

Compound E2 is very similar to E1 in 1H-NMR,UV and IR .The only difference noticed was in 1H-NMR is the presence of extra signal at 0.9 ppm which indicates the presence of extra methyl substitution on H-8 CH3 Part IV: Comparative Antimicrobial Study of Eight Tecoma species and cultivars Chapter I: Comparative antimicrobial activity of total methanolic extracts of eight Tecoma species and cultivars Antimicrobial activity of total methanolic extracts of eight Tecoma species and cultivars against three gram positive bacteria, two gram negative bacteria, one yeast and one fungi were evaluated by measuring the diameter of zones and the result was tabulated in (Table 58).  All extracts showed activity against gram positive, gram negative and antifungal except Tecoma x smithii Wil. Wats. and T. radicans (L.) Juss.  T. capensis var yellow showed highest activity against gram positive bacteria  Tecoma x smithii Wil. Wats. showed highest activity against gram negative bacteria with no antifungal activity.  T. capensis var. red showed highest antifungal activity.

This indicates that Tecoma species and cultivars have strong antimicrobial activity which can be used as antibiotics.

 T. capensis var. yellow showed the highest antimicrobial activity.  T. capensis (Thunb.) lindl had a higher phenolic content however less active.  The antimicrobial activity prevailed is attributed not only to phenolic content but as a synergistic effect of its other phytoconstituents.

Chapter II: Comparative In-vitro evaluation of Al-zheimer's disease using anticholinesterase model of eight Tecoma species and cultivars

This is the first study of Alzheimer's disease to be done on Tecoma plant using anticholinesterase model (donepezil). All of the eight Tecoma extracts revealed a potent ACHE inhibition percentage shown in (Table 59). While, the minimum concentration that gives 50% inhibition (IC50) showed that the strongest AChE inhibition activity were exhibited by T. capensis var. red with (IC 50 = 42.04±1.77 ng/ml) followed by T. capensis (Thunb.) lindl. with (IC50= 195 .89±9.26 g/ml) when compared to donepezil (IC 50= 34.88±1.39 ng/ml) as shown in (Table 60).

From the present investigation it was concluded that all the eight total methanolic extracts of Tecoma have shown promising Anticholinesterase inhibitor activity but T. capensis (Thunb.) lindl. and T. capensis var. red showed the most significant ACHE inhibition. This indicates that Tecoma has wide margin of medicinal value even between its cultivars and also has capabilities for the production of novel drugs for the treatment of Alzheimer’s disease and also for other neurodegenerative disorders.

Chapter III: Comparative In-vitro evaluation of anti-oxidant activity of eight Tecoma species and cultivars

This is the first comparative study of anti-oxidant activity to be done on eight Tecoma plants and the IC50 concentrations showed that the eight Tecoma extracts have potent anti-oxidant activity compared to ascorbic acid but T. capensis (Thunb.) lindl., T. capensis var. yellow and T. capensis var. red showed the most potent antioxidant activity among the eight extracts as shown in (Figure 104).

In light of the results of the present investigation, we can conclude that the crude extracts of Tecoma even if there is variation between Tecoma cultivars in the anti- oxidant effect. Tecoma could be a potential source of natural antioxidants that could have great importance as therapeutic agents in preventing or slowing the progress of aging and associated oxidative stress-related degenerative diseases. It also might have an impact to produce antioxidants for food industries as preservatives. However, further studies are needed to understand the underlying mechanisms of antioxidant action and to isolate the compound(s) responsible for such activity.

General Conclusion and Recommendations

 This study offers the first botanical study and genetic profiling for the 8 investigated Tecoma species and cultivars introduced to Egypt.  The preliminary phytochemical screening showed that T. capensis (Thunb.) lindl showed positive results with almost all the tests.  Determination of the total phenolic and flavonoids was done using colorimetric assay showed that T. capensis (Thunb.) lindl had the best total phenolic content. While, T. grandiflora (Thunb.) loisel. Showed the best total flavonoid content.  LC-HR-ESI(QTOF)-MS/MS showed that the use of total extract of T. capensis (Thunb.) lindl is recommended due to the almost similar composition of different fractions.  The use of leaves is recommended as it contains more rich in metabolites than the flowers, seeds and fruits. However, seeds are rich in flavonoids.  The LC-HR-ESI(QTOF)-MS/MS confirms the HPLC analysis results in the absence of caffeic acid in the leaf extracts and presence of quercetin only T.capensis and its four cultivars.  The Chemometric analysis of the data using PCA and HCA showed that T. capensis and its four cultivars are closely related while, the other species were slightly different in composition.  While, the three fractions of T. capensis (Thunb.) lindl: chloroform, ethyl acetate and butanol, the ethyl acetate fraction, was the best fraction among them containing a lot of metabolites than the others. So, it was considered as the fraction of choice for further investigation.  Two compounds were isolated and identified via physicochemical characterization and spectroscopic analysis from the ethyl acetate fraction of T. capensis (Thunb.) lindl namely: 7-O-methyl cinnamoyl Tecomoside and 8-dimethyl-7-O-methyl cinnamoyl Tecomoside.  T. capensis var. yellow showed the highest antimicrobial activity.  T. capensis (Thunb.) lindl had a higher phenolic content however less active.  The antimicrobial activity prevailed is attributed not only to phenolic content but as a synergistic effect of its other phytoconstituents.  Nowadays, the demand for herbal medicine is continuously increasing due to the serious side effects of synthetic drugs.  In this respect, T. capensis var. yellow could be recommended as antibiotic and antifungal due to its high antimicrobial activity.  This is the first study of Alzheimer's disease to be done on Tecoma plant using anticholinesterase model (donepezil).  The eight total methanolic extracts of Tecoma have shown promising Anticholinesterase inhibitor activity but T. capensis (Thunb.) lindl. and T. capensis var. red showed the most significant ACHE inhibition. This indicates that Tecoma has wide margin of medicinal value even between its cultivars and also has capabilities for the production of novel drugs for the treatment of Alzheimer’s disease and also for other neurodegenerative disorders.

 This is the first comparative study of anti-oxidant activity to be done on eight Tecoma plants and the IC50 concentrations showed that the eight Tecoma extracts have potent anti-oxidant activity compared to ascorbic acid but T. capensis (Thunb.) lindl., T. capensis var. yellow and T. capensis var. red showed the most potent antioxidant activity among the eight extracts.  Tecoma could be a potential source of natural antioxidants that could have great importance as therapeutic agents in preventing or slowing the progress of aging and associated oxidative stress-related degenerative diseases. It also might have an impact to produce antioxidants for food industries as preservatives. However, further studies are needed to understand the underlying mechanisms of antioxidant action and to isolate the compound(s) responsible for such activity. However, extensive clinical trials must be performed in order to support this biological activity and further investigation is also needed