Phytochemical and Biological Studies of Cordia Africana Family Boraginaceae Cultivated in Egypt

Phytochemical and Biological Studies of Cordia africana Family Boraginaceae Cultivated in Egypt

A Thesis submitted By Maha Abd El Mohsen Abd El Reheem Mohamed 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

Prof. Dr. Mona El Mahdy El Tantawy Professor of Pharmacognosy, Medicinal Plants Department, Ex. Chairman of National Organization for Drug Control and Research

Pharmacognosy Department Faculty of Pharmacy Cairo University A.R.E 2016

English Abstract This study focus on Cordia africana Lam. cultivated in Egypt. Firstly DNA profiling was done for selective discrimination of this plant. The results of phytochemical screening revealed the presence of different constituents .The chemical composition of the essential oil of flowers was investigated by GC/MS, the percentage of Oxygenated compounds identified in flowers oil (84.16%). Investigation of lipoidal matter of leaves of plant revealed the presence of pentanediol as the major hydrocarbon identified (53.95%). study of carbohydrate content of Cordia africana Lam. Fruits was done .it found that it composed of mannitol (27.60%). Nutritional value of the fruits was evaluated .The fruits are rich in total protein, minerals and vitamins. Qualitatative and Quantitative estimation of polyphenols and flavonoids of some Cordia species in Egypt were performed and comparing between different concentrations of phenolics and flavonoids was done to distinguish between these species. Quantitative determination of polyphenols and flavonoids of leaves, flowers and fruits of Cordia africana Lam. Was carried out. High resolution UPLC-MS analysis of Cordia africana Lam. Leaves and Fruits extracts was done. Twenty one of peaks were tentatively identified in negative mode for each leaves and flowers. Isolation and identification of phytoconstituents of leaves from ethyl acetate and butanol fractions were performed and the compound isolated were Quercetin, Para hydroxy phenyl lactic acid, Quercetin -O-β-D-glucopyranoside, Isorhamnetin -O- rutinosoide, rutin and gallic acid. Biological Study of the different extracts of leaves and fruits of Cordia africana Lam. was done and it was found that the alcoholic extracts of leaves and fruits and aqueous extracts of leaves and fruits have antioxidant, anti-inflammatory and anti- diabetic activities.

Evaluation of cytotoxic activity and antimicrobial activity of different extracts of leaves and fruits and the essential oil of flowers showed activities with different percentage.

(Key Words: Cordia, africana, Boraginaceae, carbohydrate, rutin

INTRODUCTION

Cordia is a genus of flowering plants belong to family Boraginaceae, subfamily Cordioideae. The family Boraginaceae consists of about 2,700 species which are distributed in tropical, subtropical and warmer regions around the world (Gottschling, 2003). It is composed of about 130 genera and six subfamilies: Boraginoideae, Cordioideae, Ehretioideae, Heliotropioideae, Hydrophylloideae, and Lennooideae. The subfamily Cordioideae contains the genus Cordia, which is comprised of evergreen trees and shrubs (Thirupathi et al., 2008); about 300 species of Cordia have been identified worldwide

Many of these species are commonly called Manjack (Quattrocchi, 2000). Many species of Cordia have fragrant showy flowers and are popular in gardens (Bennett, 2003).

The tropical species of Cordia are used widely as anti inflammatory for wound healing (Kuppast & Nayak, 2006), antioxidant (David et al., 2007), for skin diseases, antimicrobial, antifungal, antiviral, astringent, antidiarrhea, digestive, antiulcers, anthelementic (Kuppast & Nayak, 2003), analgesic, hoemostatic, antirhumatic, arthritic and also for treatment of snake-bite, rabies and malaria. Some of the previous uses are proved scientifically and used medicinally and the others are common in folk medicine in their native countries.

Cordia africana Lam have many uses in folk medicine; the wood –ash, mixed with butter is applied to certain skin troubles, leaves decoction are administrated to treat headache, nose bleeding, dizziness and vomiting during pregnancy (Protabase-Plant Resources of Tropical Africa).

The leaves are dried and powdered to sprinkle over wounds. A root decoction is drunk as a treatment for jaundice and schistosomaisis (Burkil, 2004). 3

Fruits of several Cordia species are edible as they have nutrition value due to its carbohydrate content specially mucilage (Verma et al., 1977).

The studies carried about this genus Cordia approved the presence of wide variety of active constituents; Cordia contains a group of chemicals; naphthoquinones derivatives called Cordiachrome A-F & cordiaquinones A-M. Other plant chemicals in Cordia include, cordialin A and B (Velde et al., 1982), Cordianols, A-I. (Kuroyangi et al., 2005) and many flavonoids and phenolic acid as artemetin rutin and robinin (Ficarra et al., 1995).

Although certain species of the genus Cordia had attracted the attention of several authors from the phytochemical point of view, little work appears to have been reported on Cordia africana Lam. It was thus, deemed important to carry out a phytochemical investigation of Cordia africana cultivated in Egypt.

Aim of work Phytochemical investigation of different plant extracts and study of their biological activities. The study includes: I. Taxonomy and DNA Profiling. 1- Taxonomy of plant 2- DNA Profiling of Cordia africana lam. II. Phytochemical study

1. Preliminary phytochemical screening of the Cordia africana 2. Analysis of the essential oil content of flowers 3. Investigation of lipoidal matter of leaves of Cordia africana Lam. 4. Investigations of carbohydrate content of Fruits 5. Nutritional evaluation of fruits. 6. Qualitative and Quantitative estimation of phenolic compounds and flavonoids in some species of Cordia present in Egypt. 7. UPLC/UV/MS analysis of leaves and Fruits extracts 8. Isolation and Identification of some active constituents by different chromatographic and spectroscopic techniques. III. Biological evaluation - Determination of LD50. - Investigation of the anti-inflammatory activity. - Estimation of the antioxidant activity. - Estimation of hypoglycemic activity. - Cytotoxic activity. - Antimicrobial activity of the essential oil and different extracts of leaves and fruits.

REVIEW OF LITERATURE

Reviewing the current literature, genus Cordia contains many chemical constituents viz, volatile constituents as α-Pinene (Oliveria et al., 2007), flavonoides and phenolic derivatives (Marstan et al., 2009), terpenoides compounds (Freitas et al., 2012), Sterols (Geler, 2010), alkaloids (Parks et al., 2010), polysaccharides and free sugars (Benhura & Chidewe, 2002). It was reported also that the plant extracts of different species of Cordia possess different biological and microbiological activities (Emtinan et al., 2015). 5

A- Chemical constituents of genus Cordia Previous studies on different Cordia species revealed the presence of the following chemical constituents: - Volatile constituents: Table (1). - Flavonoids and phenolic compounds: Table (2). - Terpenoid compounds: Table (3).-

1) Volatile constituents: Table (1): Volatile constituents reported in genus Cordia.

Plant Compounds Plant Species Structure Reference Part

Menezes C.trichotoma Heart α-Muurolol et al., Vell. wood (2005(

Menezes et al., C.trichotoma Heart (2001). α-Cadinol Vell. wood Menezes et al., (2005). Menezes et al., Epi-α- C.trichotoma Heart (2005) muurolol Vell. wood

Menezes

C.trichotoma Heart et al., γ-Cadinene Vell. wood (2005)

Continue Table (1): Volatile constituents reported in genus Cordia.

Plant Compounds Plant Species Structure Reference Part

- Menezes

ol - C.trichotoma Heart

11 et al., - Vell. wood (2005)

dien

Guaia 3,10(4)

C.curassavica Stem Oliveria, Jacq. et al., hulenol (2007) Spat

C.salicifolia leaves

Oliveria

- C.globosa Jacq. Stem et al.,

Endo

1 (2007)

bourbonanol

Oliveria C.globosa Jacq Stem et al.,

Linalyl butyrate (2007)

Oliveria

C.globosa Jacq Leaves et al.,

ne - (2007)

C.verbenacea Meccia

Caryophylle – D.C Aerial et al.,

β parts ) 2009(

Continue Table (1): Volatile constituents reported in genus Cordia.

Plant Plant Structure Reference Compounds Species part

H C C. 3 H3C H C CH3 verbenacea 3 Mederios

D.C H C et al., rans 3 T H2C ) 2007(

Caryophllene

C.globosa Leaves Oliveria Jacq. et al., (2007) Mederios et al., (2007).

Passos et al.,

Humulene

- -- (2007).

α C. Fernandes verbenacea et al., D.C (2007). Chaves et al., (2008)

H C H3C 3

C. Oliveria curassavica Stem et al., H C 3 CH

Transe

hydrate 3 Jacq. OH (2007)

Sesquisabinene

2) Flavonoids and phenolic compounds: Table (2): Flavonoids and phenolic compounds reported in genus Cordia.

Plant Plant Compounds Structure Reference species part

CH3 Dominguez 3,4'-Dimethyl C.boissiri HO O Flower et al., kaempferol A.D.C CH3 OH (1973) O

OH 5,7-Dimethoxy- Chauchan C.obliqua H CO O taxifolin-3-O- α-L- Root 3 OH et al., Willd Orhamnose rhamnopyranoside. OCH (1978) 3 O

Chauchan Root et al., OH (1978) Hesperetin-7-O-α-L- C.obliqua O rhamnose O rhamnopyranoside Willd OH Agnihotri O Seed et al., (1987)

3', 5-Dihydroxy-4'- OCH 3

O Tiwari methoxy flavonone C.obliqua Stem rhamnose O OH et al., 7-O-α-L- Willd bark OH O (1979) rhamnopyranoside.

Taxifolin-3-O-α-L- C.obliqua HO O Srivastava Seed OH rhamnopyranoside. Willd (1979) O rhamnose OH O Continue Table (2): Flavonoids and phenolic compounds reported in genus Cordia.

Plant Plant Compounds Structure Reference species part Srivastava&

Srivastava C. OH Taxifolin,3-5- (1979). obliqua Seed HO O H dirhamnoside O Agnihotri Willd O rhamnose et al., orhamnoe O (1987) 5, 6’- HO OCH Dihydroxy-3, C. 3 H CO O Lins 6, 7, 3’, 4’ verbenacea Leaves 3 OCH 3 et al., (1990). H3CO OCH 3 pentamethoxy D.C. OH O flavone.

OH Distylin-3- C.obliqua Srivastava, seed HO O xyloside. Willd (1980) O Xylose OH O Velde et al., C. (1982) verbenacea leaves OCH 3 Sertie D.C. H CO O OCH et al., Artemetin 3 3 (1990) OCH C. H3CO 3 OH --- O Bayeux (2002) curassavica Michielin D.C. et al., (2009)

Continue Table (2): Flavonoids and phenolic compounds reported in genus Cordia.

Plant Plant Compounds Structure Reference species part 4-(2, 4-Dihydroxy- benzoyl)-2, 5-bis (4-

H hydroxy phenyl)- O OH Marstan C.goetzei Stem O tetrahydrofuran3-yl]- et al.,

Guerke bark OH HO O (2,4- O (1988)

dihydroxyphenyl) methanone. [7,9-Dihydroxy OH 2,4bis(4hydroxy HO O phenyl)3,3a,4,9b- Marstan C.goetzei Stem O HO tetrahydro-2H-furo HO et al., Guerke bark O [3,2-C] chromen- OH (1988) 3yl](2,4dihydroxy- OH phenyl)methanone. 1-[3-(2,4-Dihydroxy-

benzoyl)-4,6- HO

O O O dihydroxy-2-(4- CH3 Marstan O HO OH hydroxy-phenyl)- C.goetzei Stem HO O OH OH O et al., benzofuran-7-yl]-3-(4 Guerke bark HO HO O O CH3 (1988) hydroxy-phenyl) HO OH prop-2-en-1-one OH [Cordiagol]

3) Terpenoid compounds: Table (3): Terpenoid compounds reported in genus Cordia. 12

• Terpenoid quinones:

Plant Compounds Plant species Structure Reference part C.alliodora Ruiz &Pav. Moir C.gerascanthus P. et al., Br (1972) C.gharaf (Forsk) Heart Cordiachrome A C.goeldiane wood Huber C.millenii Bak. Moir & C.monoica Roxb. Thomson C.pltythyrsa Bak (1973).

Moir et al., C.goeldiane (1972) O Huber CH Heart 3 Cordiachrome D C.millenii Bak. oMe H

wood H3C CH C.pltythyrsa Bak O 3 Moir &

Thomson (1973).

Continue Table (3): Terpenoid compounds reported in genus Cordia.  Meroterpenoid naphthoquinones:

Compounds Plant species Plant Structure Reference

part

H C 3 CH 3 O Bieber Cordiaquinone C.corymbosa HO Root et al., A G H3C (1990) O Bieber C.corymbosa et al., O G (1990) H C Cordiaquinone 3 H H H Root H C B 3 O Ioset O H CH3 C.linnaei et al., (1998). Bieber

C.corymbosa O H3C et al.,

G O CH3 (1990) Cordiaquinone O CH Root 3 C CH3 H2C Ioset C.linnaei O et al., (1998)

O H C 3 CH3 Ioset Cordiaquinone C.linnaei Root O et al., E H3C O (1998)

HO O H C CH Ioset Cordiaquinone 3 3 C.linnaei Root et al., F OH H3C (1998) O

Continue Table (3): Terpenoid compounds reported in genus Cordia.  Meroterpenoid naphthoquinones:

Compounds Plant species Plant Structure Reference

part

O H C 3 CH3 Ioset Cordiaquinone C. OH Root et al., G linnaei H3C OH (1998). O

O H C 3 Ioset O CH3 Cordiaquinone C. O CH3 Root CH3 et al., H linnaei O H2C (1998) O Ioset C. et al., curassavica CH O 3 (2000) Cordiaquinone o Root Marinho- J O H3C Filho C. CH3 et al., leucocephala (2010)

O CH2 Ioset Cordiaquinone C. Root et al., K multispicata O O (2000) H3C CH3 O Diniz Cordiaquinone C. CH3 OH Root et al., L leucocephala CH3 H2C (2009) O

Continue Table (3): Terpenoid compounds reported in genus Cordia.  Hydroquinone terpenoids: Plant Compounds Plant species Structure Reference part Steven et al., C.elaeagnoides (1973) D.C H3C Alliodorin O Manners

[8(2,5- (1983) Heart H3C dihydroxyphenyl) - 2,6- Manners wood di-methylocta-2,6- HO et al.,

dienal] (1977) C.alliodora Ruiz OH Tortajada & Pav. et al., (1986) C.alliodora Manners Alliodorol Ruiz & Pav OH et al., H3C CH3 CH3 [2-(8-hydroxy-3-7-) Heart CH OH 2 (1977) dimethylocta-2,6- wood C.elaeagnoides OH Manners dienyl]-hydroquinone D.C. (1983) Elaeagin C.alliodora Manners [2-methyl-2-(4-formyl- Ruiz & Pav Heart et al., 4- methyl pent -3- enyl) wood (1977) -2H-chromen- 6-ol] Cordiaquinol C OH CH (5,6,7,8,-tetrahydro- 7- OH 2 Manners C.alliodora D.C Heart isopropenyl -6- methyl - et al., CH3 Ruiz & Pav. wood OH 6- vinyl- naphthalene- OH CH2 (1977) 1,4 diol.

Continue Table (3): Terpenoid compounds reported in genus Cordia.

 Triterpenes: 16

Plant Plant Compounds Structure Reference species part

H3C CH3 Chen 3-Oxo-olean-12- C.alliodora et al., CH3 Leaves CH H C en-27-oic acid. Ruiz & Pav. 3 3 (1983) HOOC O CH H3C 3

H3C CH3 O 3,29-dioxo- Chen C.alliodora CH3 olean-12-en-27- Leaves CH H C et al. , Ruiz & Pav. 3 3 oic acid. HOOC (1983) O CH H3C 3

H3C O 3α-hydroxy-29- Chen C.alliodora CH3 Oxo-olean-12- Leaves CH H C et al., Ruiz & Pav. 3 3 en-27 oic acid. HOOC (1983) H HO CH H3C 3 H C HO 3

3α,29- Chen C.alliodora CH3 CH dihydroxyolean- Leaves 3 H3C et al., Ruiz & Pav. HOOC 12-en-27oic acid. H (1983) HO CH H3C 3

Continue Table (3): Terpenoid compounds reported in genus Cordia.  Triterpenes: Plant Plant Compounds Structure Reference species part

3α-hydroxy H3C HOOC Chen olean-12-ene- C.alliodora CH3 Leaves CH3 H C et al., 27, 29-dioic Ruiz &Pav. 3 HOOC H (1983) HO CH acid. H3C 3

CH3

H2C Agnihotri H Betulin C.obliqua Will Seeds H OH et al., CH3 CH3 H CH3 (1987) HO H H3C CH3 Agnihotri et al., Seeds C.obliqua Will (1987) α-amyrin C.americana leaves Geler

(2010) Cordiaketal A H3C

3β,25-epoxy- H3C OAC Kuroyangi 21β-acetoxy- OH C.multispicata leaves CHO et al., 3α,22β- H3C O CH3 (2001) dihydroxyurs- HO Me Me 12-en-28-al

SUMMARY

Cordia is a genus of flowering plants belong to family Boraginaceae, subfamily Cordioideae. The family Boraginaceae consists of about 130 genera and 2,700 species which are distributed in tropical, subtropical and warmer regions around the world (Gottschling, 2003). It is composed of six subfamilies: Boraginoideae, Cordioideae, Ehretioideae, Heliotropioideae, Hydrophylloideae, and Lennooideae. The subfamily Cordioideae contains the genus Cordia, which is comprised of evergreen trees and shrubs (Thirupathi et al., 2008); About 300 species of Cordia have been identified worldwide. The tropical species of Cordia are used widely as anti inflammatory for wound healing (Kuppast & Nayak, 2006), antioxidant (David et al., 2007), for skin disease, antimicrobial, antifungal, antiviral, astringent, antidiarrhea, digestive, antiulcers, anthelementic (Kuppast & Nayak, 2003), analgesic, hoemostatic, antirhumatic, arthritic and also for treatment of snake-bite, rabies and malaria. Some of the previous uses are proved scientifically and used medicinally and the others are common in folk medicine in their native countries. Although certain species of the genus Cordia had attracted the attention of several authors from the phytochemical point of view, little work appears to have been reported on Cordia africana Lam. It was thus, deemed important to carry out a phytochemical investigation of Cordia africana Lam. cultivated in Egypt.

This study includes: Part l : Taxonomy and DNA profiling Chapter (1): Taxonomy of Cordia africana Lam. Kingdom: plantae Sub kingdom: tracheobionta Super division: spermatophyte Division: Magnoliophyta Class: Magnoliopsida Sub class: Asteridae Family: Boraginaceae Sub family: Cordioideae Genus: Cordia Species: Cordia africana Lam.

Syn.Cordia abyssinica R.Br.

Chapter (2): DNA profiling A DNA sample of the plant was extracted , purified and analyzed by two techniques, Random Amplified Polymorphic DNA (RAPD) technique using six oligonucleotide primers and the other technique was Inter Simple Sequence Repeat Markers (ISSR) using five oligonucleotide primers, the analysis of RAPD and ISSR data achieves the use of A09, B07, C04, C12, C19, Z03, 14A, 44B, HB08, HB09 and HB11 primers for selective discrimination of this plant.

Part II: Phytochemical study of Cordia africana Lam. It contains 8 chapters:

Chapter (1): Phytochemical screening of Cordia africana Lam. A- Preliminary phytochemical screening The results of phytochemical screening revealed The presence of carbohydrates and/or glycosides, tannins, flavonoids, coumarins and Sterols and/or triterpens in all tested organs of the plant. The presence of volatile substances in small amount in the flowers and absence in leaves and fruits. The presence of saponins in trace amount in leaves and flowers and absence in fruits. The absence of Sublimable substance, alkaloids, anthraquinones, cardiac glycosides and cyanophoric glycosides in all organs under investigation.

B- Successive extraction of the powdered Cordia africana Lam. leaves with selective organic solvents and examination of the respective extracts. The percentage yields of different extractives after successive fractionation of the ethanolic extract of Cordia africana Lam. leaves were in descending order: water fraction (10.3%) followed by light petroleum (40-60 °C) (1.5%), butanol (0.48%), ethyl acetate(0.3%) and the least was chloroform fraction (0.22%).

Chapter 2: Investigation of the essential oil content of Cordia africana Lam. flowers Essential oil of flowers was prepared by extraction method. The chemical composition of the oil sample was investigated by GC/MS, the percentage of Oxygenated compounds identified in flowers oil (84.16%), hydrocarbon was (1%), esters (29.75%) was the major class of Oxygenated compounds and eucalyptol (25.8%) was the major compound identified. Chapter 3: Investigation of lipoidal matter of Cordia africana Lam.

Petroleum ether of the leaves was evaporate and the residue was subjected to saponification by (10%) methanolic KOH Then each of the unsaponifiable matter and saponifiable matter were subjected to GC/MS analysis. The percentage of the total lipoidal matter in the leaves of Cordia africana Lam. is 1.93%. The unsaponifiable matter representing (30%) pentanediol was the major hydrocarbon identified (53.95%). While the total fatty acid representing (70%) which contains both saturated fatty acids mainly palmitic acid (28.45%) and un saturated fatty acids mainly linolenic acid (21.94%). TLC for sterols and triterpenes: Unsaponifiable fraction examined for sterols and triterpenes was applied on silica gel chromatogram which detect the presence of β- sitosterol, stigmasterol, β-amyrin, lupeol, oleanolic acid. Chapter 4: Investigations of carbohydrate content of Cordia africana Lam. fruits The Investigations of carbohydrate hydrolysate content of fruits was carried out by extraction of polysaccharides by hot extraction method followed by purification and hydrolysis then HPLC analysis. It may be concluded that the total percentage of polysaccharide hydrolysate identified was (85.2%) & the total non identified sugars were (14.8%). The polysaccharide hydrolysate extracted from the fruits is composed of 27.60% mannitol which is the major sugar followed by glucuronic acid (12.49%) & arabinose (11.63%).

Chapter 5: Nutritional evaluation of Cordia africana Lam. fruits Nutritional value of the fruits including total protein, minerals was evaluated according to (A.O.A.C, 2006) and vitamins by HPLC, total protein content of fruit comprises 5.78 gm/100g dry weight. The fruits are rich in minerals; iron, calcium, sodium & phosphorous, as well as in vitamins A, B6, and C.

Results of evaluation of vitamins Vitamin A: 1063 IU/100g Vitamin C: 8.9 mg/100g Vitamin E: 0 IU/100g Vitamin B2: 0.018 mg/100g Vitamin B6: 0.313 mg/100g Folic acid: 0.055 µg/100g

Results of evaluation of minerals Iron (Fe): 1.8 mg/100g Calcium (Ca): 1.19 mg/100g Sodium(Na): 957.8 mg/100g Phosphorous (P): 95 mg/100g

Chapter 6: Qualitatative and Quantitative estimation of polyphenols and flavonoids of some Cordia species in Egypt. In this chapter comparing between the plant of study ( Cordia africana Lam.) and the some of Cordia species present in Egypt . The Cordia species cultivated in Egypt are: Cordia africana Lam., Cordia boissieri A.D.C., Cordia dichotoma Forst., Cordia lutea Lam., Cordia myxa L. and Cordia sebestena Linn. The only Cordia species growing in Egypt is Cordia sinensis Lam. a) Estimation of total polyphenol content (TP)

Plant extract TP in mg (GAE)/g dry wt Cordia africana Lam. 2.62 Cordia boissieri A.D.C 0.65 Cordia dichotoma Forst. 0.95 Cordia lutea Lam. 0.21 Cordia myxa L. 0.18 Cordia sebestena Linn. 0.70 Cordia sinensis Lam. 0.91

b) Estimation of total flavonoid content: Plant extract RC in mg RE/g dry wt Cordia africana Lam. 8.873 Cordia boissieri A.D.C 0.224 Cordia dichotoma Forst. 1.418 Cordia lutea Lam. 1.302 Cordia myxa L. 0.268 Cordia sebestena Linn. 0.120 Cordia sinensis Lam. 1.215

C) HPLC estimation of phenolics of 70% methanolic extracts of some Cordia species in Egypt: The HPLC analysis of extracts of Cordia species revealed the presence of twenty phenolic compounds present by different concentration in Cordia species.

D) HPLC estimation of flavonoids of 70% methanolic extracts of some Cordia species in Egypt:

The HPLC analysis of extracts of Cordia species revealed the presence of twenty one flavonoids present by different concentration in Cordia species

Quantitative determination of polyphenols and flavonoids of leaves, flowers and fruits of Cordia africana Lam. a- Estimation of phenolic compounds: It was found that the flowers of the plant contain highest amount of phenolics then the leaves and the least amount in fruits. b- Estimation of total flavonoid content: It concluded that Cordia africana lam leaves had the highest flavonoidal content (8.87 mg RE/g) followed by flowers (6.36 mg RE/g) and the least flavonoidal content for fruits (1.42 mg RE/g).

Chapter 7: Identification of phytoconstituents of Cordia africana Lam. Leaves and Fruits using high resolution UPLC-MS analysis From HPLC chromatogram of leaves and fruits forty two peaks were detected. Twenty nine peaks were detected for leaves; twenty one of them were tentatively identified in negative mode based on mass measurements of the [M-H]- as well as the comparison with reported data. Twenty seven peaks were detected for fruits; twenty one of them were tentatively identified in negative mode based on mass measurements of the [M-H]- as well as the comparison with reported data. Chapter 8: Investigation of chemical constituents of Cordia africana Lam. * Investigation of the ethyl acetate fraction of Cordia africana Lam. The ethyl acetate fraction (10.5 gm) is dissolved in least amount of distilled water and then chromatographed on 100 g polyamide in column (5 cm  1 m) using distilled water as the eluent and then decreasing the polarity by increasing methanol part stepwise by 10% till 100% methanol.

A- HPLC qualitative determination of some phenolic acids in the collective fractions from ethyl acetate of Cordia africana Lam.

HPLC qualitative determination of some phenolic acids result in Gallic acid, caffeic acid, Chlorogenic acid, ferulic acid and ellagic acid. B- Isolation of Constituents from ethyl acetate Collected Fractions: Collected fractions of ethyl acetate from polyamide were subjected to different chromatography techniques to isolate and purify the compounds and isolated compounds identified from the physico -chemical properties and NMR.

The isolated compounds from ethyl acetate fraction are: - Quercetin. - Para hydroxy phenyl lactic acid - Isorhamnetin-3-O- rutinoside - Quercetin-3-O-β-D-glucopyranoside. * Investigation of the butanol fraction of Cordia africana Lam. The butanol fraction (17 gm) is dissolved in least amount of distilled water and then chromatographed on 100g polyamide in column (5 cm  1 m) using distilled water as the eluent and then decreasing the polarity by increasing methanol part stepwise by 10% till 100% methanol. Collected fractions of butanol from polyamide were subjected to different chromatography techniques to isolate and purify the compounds and isolated compounds identified from the physico-chemical properties and NMR.

The isolated compounds from butanol fraction are: - Gallic acid - Rutin

All the compounds isolated from the ethyl acetate and butanol fractions were isolated for the first time from Cordia africana Lam.

Part III: Biological Study

Chapter 1: Biological Study of the different extracts of leaves and fruits of Cordia africana Lam.

(1) Acute toxicity study (LD50).

It was found that LD50 for alcoholic leaves 6.2 g/kg, alcoholic fruit 7.4 g/kg, aqueous leaves 6.9 g/kg and aqueous fruit 8.1 g/kg which indicates that all four extracts are safe and non toxic. (2) Antioxidant activity( Determination of blood glutathione level): The alcoholic leaves extract showed the highest antioxidant activity of all tested extracts; since it showed the lowest percentage of change (3.8%) followed by aqueous extract of leaves (5.2%) then the alcoholic extract of fruits (9.6%). The aqueous extract of fruits showed the least antioxidant activity as it showed the highest percentage of change (13.8%). The antioxidant activity of Cordia africana may be attributed to the presence of phenolic acids and flavonoid. (3) Anti-inflammatory activity: Regarding the anti inflammatory activity all the tested extracts of Cordia africana. Induced significant anti inflammatory effect compared with indomethacin and control groups. The alcoholic and aqueous leaves extracts are more potent than the alcoholic &aqueous fruits extracts, respectively; since they showed higher percentage of oedema inhibition (4) Antihyperglycaemic activity: All the tested extractd of Cordia africana. Induced significant antihyperglycemic activity. The alcoholic extract of leaves showed the highest antihyperglycemic activity of all tested extracts. Chapter 2: Evaluation of potential cytotoxicity of Cordia africana Lam. Comparing to doxorubicin as standard, the volatile oil of flowers showed highest cytotoxic activity against all tested cell lines breast cancer, colon cancer and liver cancer

with IC50 = 1.36 μg, 2.44 μg and 2.88 μg, respectively, followed by alcoholic extracts of leaves showed activity against breast and liver with IC50=8.79 µg and 11.7 µg respectively. The others extract water of leaves, alcoholic and water extracts of fruits showed weaker cytotoxic activity against all the tested cell line. Chapter 3: Antimicrobial study of Cordia africana Lam. All the extracts (alcoholic of leaves & fruits, water of leaves & fruits) and essential oil of flowers have antibacterial and antifungal activities. The volatile oil has the highest antibacterial and antifungal activities followed by alcoholic extract of fruits. The alcoholic extract of fruits and leaves are most active against some of the tested fungi than the water extracts of fruits and leaves. Conclusions

 It is highly recommended to increase cultivation of Cordia africana Lam. in Egypt as its fruit is edible, palatable, safe and medicinally used.  Leaves of Cordia africana Lam. has antioxidant, anti inflammatory anti hyperglycemic, antimicrobial and anticancer activity so clinical trials are required to help for incorporation of leaves extract in natural pharmaceutical preparations.  It is highly recommended to study the cytotoxic activity of the total extract of leaves and fruits using more cell lines.  Cordia africana Lam. fruits can be used as a good source of vitamins and minerals and its carbohydrates can be used in food industries as jams, jellies and other food industries.  DNA fingerprinting of the plant firstly done, so it is recommended to be included in the quality control of the plant.  Rutin is the major isolated flavonoid from the plant which may guide to use it as new stable marker for standardization of Cordia africana Lam. leaves cultivated in Egypt.

 It is very interesting to do further study to characterize and differentiate between different Cordia species.