(D. Don Endl) and Taxodium Distichum (L. Rich) Cultivated in Egypt

Comparative Pharmacognostical Study and Tissue Culture of Sequoia sempervirens (D. Don Endl) and Taxodium distichum (L. Rich) Cultivated in Egypt

A thesis submitted by: Mahmoud Anter Mohamed Arafat Demonstrator of Pharmacognosy Department, Faculty of Pharmacy, Nahda University

For the partial fulfillment of the M.Sc. in Pharmaceutical Sciences (Pharmacognosy)

Under the supervision of:

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

Assistant Prof. Mohamed Abd El-Atty Rabeh Assistant Professor of Pharmacognosy, Department of Pharmacognosy, Faculty of Pharmacy, Cairo University

Assistant Prof. Essam M. Abd El-Kadder Assistant Professor at Horticulture Research Institute Agricultural Research Center

Pharmacognosy Department Faculty of Pharmacy Cairo University 2018

Abstract: Aiming to identify and authenticate Sequoia sempervirens (D.Don Endl) and Taxodium distichum (L.Rich) cultivated in Egypt. RAPD technique was employed to identify unique DNA markers and establish a typical fingerprint for S. sempervirens and T. distichum. The amplification profile of S. sempervirens was screened and a total of 18 different RAPD fragments had been recorded while the amplification profile of T. distichum was screened and a total of 15 different RAPD fragments had been recorded. Macromorphological and micromorphological studies of S. sempervirens and T. distichum had been done. The results of preliminary phytochemical screening of both plants showed the absence of alkaloids, anthraquinones, catechol tannins or cardiac glycosides in non-flowering parts of S. sempervirens and T. distichum. Sterols and/or triterpenoids, pyrogallol tannins, carbohydrate and/or glycosides and flavonoids were detected in all of the tested parts of the plants. Volatile substances were detected in very low amounts in non-flowering parts of both plants. GC/MS analysis of hexane extract of non-flowering parts of S. sempervirens and T. distichum revealed that unsaturated fatty acids were present in higher percentage than saturated fatty acids in both S. sempervirens and T. distichum. 8β-hydroxypimar-15-en- 19-oic acid had been isolated from hexane fraction of T. distichum. Apigenin, luteolin and tricetin had been isolated from ethyl acetate fraction of S. sempervirens and hyperoside and catechin had been from ethyl acetate fraction of T. distichum. Total phenolics and flavonoids content were measured by Folin–Ciocalteu and AlCl3 methods respectively in non- flowering parts of S. sempervirens and T. distichum. S. sempervirens contained higher amount of phenolics and flavonoids than T. distichum. HPLC analysis of phenolic and flavonoid compounds in leaves and stems of S. sempervirens

and T. distichum identified four flavonoids (apigenin, kaempferol, quercetin and luteolin). A tissue culture study was conducted to investigate the possibility of in vitro propagation of the plants and enhance its production of secondary metabolites. Different parts of the plant (young and fully grown leaves and stem nodes) and different growth regulators were used for callus production and all treatments produced callus in case of S. sempervirens but no callus was formed in case of T.distichum. The formed callus was treated with different physical and chemical elicitors. Total phenolics and flavonoids contents with their HPLC analysis were estimated for callus formed. Antimicrobial, anti-arthritic, anti-diabetic and hepatoprotective studies had been done for ethanolic extract of non-flowering parts of S. sempervirens and T. distichum.

Introduction:

I. Sequoia sempervirens (D. Don Endl):

Genus Sequoia includes one species sempervirens which belongs to the gymenosperms, class Coniferophytes, order Coniferales, sub-order Abietales, family Taxodiaceae. Redwoods Sequoia sempervirens (D. Don Endl) are one of the most productive timber types in the United States and provide an important supply of high quality wood fiber. Several other tree species occured in the middle and lower strata and among them evergreen broad leaf trees were most important. Average tree height of the upper stratum is 87.6 m. The range of Sequoia sempervirens extends along the Pacific Coast of the United States from the extreme southwestern corner of Oregon southward to Salmon Creek Canyon in the Santa Lucia Mountains of southern Monterey

County. The tree is characterized by a thick, fibrous, and deeply furrowed bark along with fire-resistant reddish-brown heartwood. Leaves of S. sempervirens 3

are dimorphic, including linear and scale-like leaves. The linear leaves are spirally arranged or occasionally sub-opposite.

II. Taxodium distichum (L. Rich):

Taxodium distichum Rich (Taxodiaceae), commonly known as “bald” or “swamp” cypress, is well known as an extant deciduous, living fossil conifer indigenous to the southern part of North America. Its location is primarily in the South East United States. Taxus is a conifer and grows in similar habitats to bald cypress. Both genera are evergreens with linear two- ranked leaves and both prefer damp environments. Its heartwood is used for building materials because of its resistance to attack from subterranean termites. It is growing over 25 m in height and over 300 cm d.b.h. (diameter at breast height). The leaves are small, 5–20 mm long, green to yellow-green and appearing two-ranked. Young trees have a pyramid shape but eventually form an irregular flattened canopy. The fruits are cones and are composed of pellate scales forming a woody, brown sphere with rough surface 1.5 to 4 cm in diameter. The cones disintegrate into irregular seeds.

Aim of work: This work is designed to provide the basic pharmacognostical characteristics of S. sempervirens (D.Don End) and T. distichum (L. Rich) introduced in Egypt through: - Characterization of the S. sempervirens (D. Don Endl) and T. distichum (L. Rich) including DNA fingerprinting and botanical study. - Phytochemical investigation, isolation and identification of the major secondary metabolites from S. sempervirens (D. Don Endl) and T. distichum (L. Rich) non-flowering parts.

- Exploring possible bioactivities of S. sempervirens (D. Don Endl) and T. distichum (L. Rich) extracts from non-flowering parts. The objectives of tissue culture study were:- - To develop biotechnological methods for culturing plant cells and tissue to provide a new mean of conserving and rapidly propagating the plant. - Establish a protocol for callus culture (initiation and growth) from S. sempervirens (D. Don Endl) and T. distichum (L. Rich). - Study the effect of different chemical and physical elicitors on growth and secondary metabolites production from callus cultures.

Review: A- Constituents review: I. Sequoia sempervirens (D. Don Endl): (Son et al. 2005) isolated abietane diterpene called 20-hydroxyferruginol, ferruginol, 18-hydroxyferruginol, sugiol, and 6-hydroxysugiol from ethanolic extract of cones using preparative reverse phase HPLC. (Zhang et al. 2004) isolated norlignane called sequosempervirin A from acetone extract of branches and leaves. (Zhang et al. 2005) isolated norlignanes called sequosempervirins B-E, agatharesinol and agatharesinol acetonide from acetone extract of branches and leaves. Other norlignanes called sequosempervirins F-G and sugiresinol were isolated from methanolic extract of branches and leaves. (Balogh & Anderson 1965) isolated and identified three phenolic compounds called sequirins A, B, and C from the extractives of redwood heartwood (bark). (Begley et al. 1978) isolated and identified phenolic compound called sequirins D from the extractives of redwood heartwood (bark) and this compound was determined by X-ray analysis. (Taha & El Shakour 2017) identified and quantified twelve phenolic acids

called caffeic acid, sinapic acid, cinnamic acid, ferulic acid, coumaric acid, protocatechuic acid, gentisic acid, chlorogenic acid, salicylic acid, syringic acid and hydroxybenzoic acid from ethyl acetate fraction of the leaves using high performance liquid chromatography (HPLC). (Anderson et al. 1968) isolated four cyclitols compounds called myo-inositol, (+)-inositol, sequoyitol and pinitol from the extractives of redwood heartwood using cellulose-column chromatography. (Riffer et al. 1969) obtained essential oils from Sequoia sempervirens and analyzed by gas-liquid chromatography (GLC). The main compounds were α-pinene (77%), β-pinene (1%), myrcene (1%), and Limonene (21%). (Okamoto et al. 1981) isolated about thirty-five volatile components from the leaves by steam distillation and pentane extraction. These components were characterized by GC-MS and infrared spectra. Four resin acids were obtained from Sequoia sempervirens and analyzed by gas- liquid chromatography (GLC) by (Riffer et al. 1969) . The main compounds were levopimaric acid, isopimaric, a small amount of sandaracopimaric, and a trace of pimaric acid. (Stafford & Lester 1986) isolated four flavonoids called catechin, epicatechin, gallocatechin and epigallocatechin from aqueous methanolic extract of the leaves. (Taha & El Shakour 2017) identified and quantified fifteen flavonoids called luteolin-7-O-glucoside, vicenin-II, mangiferin, quercetin-3,7-di-O-glucoside, isovitexin, kaempferol, vitexin, quercetin, luteolin, apigenin, orientin, isoorientin, rutin, apigenin-7-O- rutinoside and epicatchin from ethyl acetate fraction of the leaves using high performance liquid chromatography (HPLC).

II. Taxodium distichum (L. Rich):

(Morris et al. 1969) isolated two diterpenoids structures called taxodone and taxodione from chloroform fraction of aqueous ethanol extract of the ground seeds. (Hirasawa et al. 2007) isolated two abietane-type diterpenes 6

called taxodistines A and B from dichloromethane fraction of the cones after extraction with methanol. (Kusumoto et al. 2008) isolated abietane-type diterpene called taxodal 1 from hexane fraction of the cones. The structure was estimated on the basis of NMR spectral data and confirmed by X-ray crystallographic analysis. (Kusumoto & Ashitani 2009) isolated eight abietane-type diterpenes from hexane extract from cones of Taxodium distichum. They were identified as 6,7- dehydroroyleanone, taxodal, taxodione, salvinolone, 14-deoxycoleon U, 5,6-dehydrosugiol, sandaracopimaric acid, and xanthoperol. (Kusumoto & Ashitani 2010) isolated twelve diterpenoids including components from the hexane extract from cones of Taxodium distichum. They were identified as 6,7- dehydroferruginol, ferruginol, 6,7-dehydroroyleanone, sandaracopimaric acid, taxodione, taxodal, taxodone, sugiol, xanthoperol, salvinolone, 5,6- dehydrosugiol and 14-deoxycoleon U. (Su et al. 2013) isolated diterpene compound called 8β-hydroxypimar-15-en-19-oic acid from cones and leaves and flavonoid compound called (Son et al. 2005) revealed that abietane diterpene called 20-hydroxyferruginol, ferruginol, sugiol, and 6-hydroxysugiol isolated from ethanolic extract of the cones had antitumor activity against colon, lung, and breast human tumors. Growth inhibitory activity of 20- hydroxyferruginol and 6-hydroxysugiol is more than 10 times stronger than that of ferruginol and sugiol. B- Biological review: I- Sequoia sempervirens (D. Don Endl): (Zhang et al. 2005) isolated norlignane called agatharesinol acetonide showed anticancer activity on lung cancer cell line with IC50= 27.1 mM using taxol as positive control. (Zhang et al. 2005) explained that acetone extract of branches and leaves showed antifungal activity on candida glabrata with an

IC50 =15.98 µg/ml using fluconazole as positive control. (Zhang et al. 2005) revealed that acetone and methanolic extracts showed activity toward cathepsin B with IC50 = 4.58 and 5.49 µg/ml respectively, using leupeptin as positive control. (Davies et al. 2014) explained that acetone extract and ethanolic extract of S. sempervirens reduced the growth of two fungi Gloeophyllum trabeum and Trametes versicolor.

II- Taxodium distichum (L. Rich):

(Morris et al. 1969) isolated two diterpenoids structures called taxodone and taxodione from chloroform fraction of aqueous ethanol extract of the ground seeds showed significant activity in vivo against the Walker intramuscular carcinosarcoma 256 in rats and in vitro against cells derived from human carcinoma of the nasopharynx (KB). (Ogunwande et al. 2007) observed that the essential oils from fruits and leaves of T. distichum exhibited pronounced cytotoxic activities against PC-3 (prostatic carcinoma cells), Hep G2 (hepatocellular carcinoma cells), and Hs 578T( breast ductal carcinoma cells) human tumor cell lines at tested concentrations. (Hirasawa et al. 2007) confirmed two abietane-type diterpenes called taxodistines A and B isolated from the cones had antimitotic effect against murine lymphoma P388 cells at

IC50 0.43 and 6.5 µg/ mL respectively, by inhibition of tubulin polymerization. (Ogunwande et al. 2007) showed that Fruit oil had a promising antifungal activity against Aspergillus niger with a minimum inhibitory concentration of 19.5 mg/ml using Amphotericin B as a positive control. (Kusumoto et al. 2010) revealed that taxodione , taxodal , taxodone , sugiol , xanthoperol , salvinolone , 5,6 dehydrosugiol , and 14-deoxycoleon U are diterpenoids isolated from cones of Taxodium distichum which have antifungal activities against two wood decay fungi, Trametes versicolor (white-rot) and Fomitopsis

palustris (brown-rot). (Tantawy et al. 1999) said that the fruit oil had a promising antifungal activity against Candida albicans using nystatin as standard. This activity is due to high α-pinene content. Both the leaves and fruit oils had weak antibacterial activities against Gram-positive bacteria (Bacillus cereus and Staphylococcus aureus) and Gram-negative bacteria (Pseudomonas aeruginosa and Escherichia coli) using Gentamicin as a positive antibiotic control and DMSO as a negative control. Minimum inhibitory concentrations (MICs) were determined using the micro broth Dilution technique (Ogunwande et al. 2007). (Tantawy et al. 1999) showed that fruit oil had antibacterial activities against the selected Gram positive bacteria (Bacillus subtilis and Staphylococcus aureus). Also the oil had a remarkable activity against the tested Gram negative bacteria, (Proteus mirabtlis and Klebsiella pneumonia). These activities are due to high α-pinene content. (Kusumoto et al. 2009) evaluated the antitermitic (termicidal and antifeedant) activities of 6,7-dehydroroyleanone, taxodal, taxodione, salvinolone, 14-deoxycoleon U,5,6-dehydrosugiol, sandaracopimaric acid and xanthoperol against the subterranean termite Reticulitermes speratus Kolbe. Compounds 6,7-dehydroroyleanone, taxodal and taxodione showed potent termicidal activity, 14-deoxycoleon U and xanthoperol showed potent antifeedant activity. (El Tantawy et al. 1999) revealed that the oil had a powerful antispasmodic effect as it causes relaxation to rabbit intestine using rabbits of 1-1.5 kg body weight. (El Tantawy et al. 1999) revealed that the oil had a strong anti-inflammatory effect compared with the standard diclofenac sodium using fifteen mature rats (weighing from 200-220 g) divided into three equal groups and subjected to pedal inflammation test. One group was kept as a control and another one was injected subcutaneously with diclofenac sodium 20 mg/kg and the third group was injected with different concentrations of the

tested oil (oil in Tween 80). (Kushwaha et al. 2016) revealed that ethanolic extract of aerial parts of the plant T. distichum had potent antifilarial activity. (Naman et al. 2016) isolated two new compounds, namely, a para- benzoquinone ring-containing abietane and a para-benzoquinone ring containing 7,8-seco-abietane, and 14 other known highly oxidized abietane diterpenoids which were tested in vivo using Leishmania donovani-infected mice and was found to dose dependently reduce the parasite burden in the murine livers after I.V. administration of this crude mixture at 5.6 and 11.1 mg/kg.

C- Tissue culture review:

1-Effect of mercuric chloride concentrations and duration on disinfection and survival percentages of different plants explants

(Bhatt & Dhar 2004) sterilized the shoot segments of Myrica

esculenta with different concentrations of HgCl2 (0.05% or 0.1 %).

They found that 0.1 % HgCl2 for 10 min treatment gave the most

aseptic culture. However, lower concentration of HgCl 2 (0.05 %) was found less effective. They mentioned also that duration of treatments exceeding 10 min was found deleterious. (Hussein; 2013) investigated

the effect of HgCl2 at 0.0, 0.1, 0.2 or 0.3 % for 5 min followed by commercial bleach (Clorox) at 30, 40 or 50% for 20 min on three

species of Aglaonema. The data revealed that HgCl2 at 0.3 % for 5 min followed by Clorox 50% for 20 min was the most effective sterilization treatment. The highest survival rates of the sterilized explant were (60-

80%) when the treatment of HgCl2 0.3% for 5 min followed by Clorox for 20 min was applied.

2-Effects of plant growth regulators on callus formation

In vitro callus culture techniques were successfully applied in different fields of applications such as genetic improvement of plants organogenesis, embryogenesis of plant, production of secondary metabolites, etc… (Zelcer et al. 1989, Youssef; 1997, Hussein; 2013). (Abdellatef & Khalafalla 2010) studied the effect of 2,4 dichlorophenoxy acetic acid at different concentrations 0.1 – 3 mg/L on Moringa oleifera callus induction from leaves and hypocotyl explants. It was found that the explants failed to produce callus on MS medium alone. Among the 2,4 – D concentrations tested, using 0.1 , 0.5 , 1 and 2 mg /L gave callusing for leaves explants, while using 2 mg/L 2,4 – D resulted in 100% callusing for hypocotyl explants. Increasing 2,4 – D level up to 3 mg /L significantly reduced the callusing response to 84% for both types of explants. According to (Yadav & Singh 2011) internodal stem segments of Aegle marmelos were cultured on MS medium supplemented with different concentrations (0.25 – 4 mg / L) of BAP and 2,4–D in an individual or combination forms. The data showed that BAP was more effective than 2,4–D for callus formation. MS medium fortified with 2 mg /L BAP resulted in the maximum callusing rate (80%). Moreover using combination of BAP (1 mg /L) and 2,4–D (0.5 mg/L) or BAP (2 mg/L) and 2,4–D (0.5 mg/L) resulted in callusing rate of 70 and 80% respectively. (Rawat et al. 2013) incubated three types of explants (leaf, shoot tip and nodal segment) of Aconitum violaceum on MS medium supplemented with 0.5 to 10 µM 2,4 – D and 0.25 to 2 µM Kin for callus induction. It was observed that a greater proportion of explants on medium with a combination of 5µM 2,4-D plus 0.5 µM Kin initiated the callus at the

highest rates (83.1% for leaves, 24.1%, for shoot tips and 39% for nodal segments) than other growth regulators treatments tested.

3-Effects of plant growth regulators on callus growth

(Jeong et al. 2007) studied the effects of various concentrations of different types of Auxins IBA (0 to 5 mg/L), NAA (0 to 2mg/L) and IAA (0 to 10 mg/L) and cytokinins BA (0 to 1mg/L), Kin (0 to 1 mg/L) on callus formation in Panax ginseng hairy roots. It was found that the greatest callus formation was observed in cultures amended with BA and Kin. (Rateb et al. 2007) investigated the fresh weight of the formed callus tissue in Tanacetum parthenium as affected by various plant growth regulators 2,4-D, NAA or BAP at 0, 0.5 or 1 mg/L). It was found that using 1mg/L NAA plus 1mg/L BAP resulted in the heaviest callus fresh weight and this might be due to that such combination of high concentrations of auxin NAA plus cytokinin BAP had a pronounced effect on increasing the growth of callus in terms of fresh weight. (Abdellatef & Khalafalla 2010) Examined the effect of 2,4-D concentrations (0.1 to 3 mg/L) on callus growth rate in Moringa oleifera Lam. The most suitable 2,4-D concentration was 2 mg/L for hypocotyl explants and 1 mg/L for leaf explants.

4-Effects of ultraviolet irradiation on secondary metabolites production.

(Mahdavian et al. 2008) Studied the effect of ultraviolet radiation on pigments content in Capsicum annum. They attributed significant increase in flavonoid and anthocyanin content in UV-B (280-315nm) treated plants to their requirements for synthesizing additional pigments to protect them from the UV-B damage. A study

by (Mahdavian et al. 2008) on Capsicum annum, showed an increase in flavonoids under UV-B (280-315nm) and UV-C (254nm) treatments that probably means either that the increment in flavonoids allows for protection of oxidative stress in plants, or prevents the penetration of the destructive bands of UV light to the most sensitive tissues.The effect of UV-B and UV-C at various doses on callus of bladder campiom (Silene vulgaris) were studied by (Günter et al. 2009) and results revealed that UV irradiation resulted in decreasing the arabinose and galactose residues in the silenon-the pectin fraction isolated from callus. At the irradiation of callus with UV, the antioxidant activity of the silenan and the relative content of phenolic compounds in it, increased, the highest increase was observed after irradiation of callus by UV-B. UV-B irradiation induced a rise in nitric oxide production and phenylalanine ammonia layase (leading to flavonoids synthesis) as well as flavonoid level in Ginkgo biloba callus (Hao et al. 2009).(Balouchi et al. 2009) investigated the changes in photosynthetic pigments and other physiological and biochemical trials of durum wheat exposed to UV-C radiation. The results showed that anthocyanins, flavonoids and proline content increased significantly by UV-C as compared to control. UV-C light exposture of sterile cultures of Scenedesmus quadricauda over one hour did not influence the total soluble phenol and flavonoids according to (Kováčik et al. 2010). Selected flavonol (quercetin and kaempferol) were not detected after UV-C treatment. The authors concluded that the exposture time to UV light was not sufficient to stimulate more considerable changes of phenolic metabolites in tissue cultures. (Sun et al. 2010) examined the effect of different radiation

time applied by device with UV-B intensity on flavonoid concentration in freshly collected Ginkgo biloba leaves. It was reported that young leaves and moderate irradiation time of 120 min UV-B significantly increased the content of quercetin, kaempferol and isorhamnetin as well as total flavonoids concentration. Global metabolites profiling of UV-induced changes also showed that phenylpropanoid-related metabolites shikimic acid, quinic acid and phenylalanine were remarkably higher in Melissa officinalis exposed to UV-B for two hours as compared to control (Kim et al. 2012). (Ning et al. 2012) studied the effect of UV-A radiation on secondary metabolites in freshly collected flower buds of the medicinal plant Lonicera japonica. Level of one secoiridoid glycoside and three derivatives of chlorogenic acids were reported to rise after UV application. (Eichholz et al. 2012) irradiated Asparagus officinalis spears with UV-B (0.54 or 1.08 KJm-2) and showed that concentration of quercetin-4-O-monoglucoside with UV-B, was accompanied with rise in activity of polyphenol related enzymes (phenylalanine ammonia layase and peroxidase).

5-Effect of silver nitrate on secondary metabolites production

(Jayalakshmi et al. 2012) comparatively studied change in growth pattern and anthocyanin production with various induced stress in Malva sylvestris L. Exogenous administration of 0.1 mM of abscisic acid, silver nitrate as foliar spray for five successive days and supplemental thirty minutes exposure dose of UV-B for continuous three days significantly induced variation in the growth pattern and anthocyanin content. The abscisic acid and silver nitrate treatment

increased anthocyanin content up to five folds and three folds accompanied by morphological variations by increasing height, leaf mass, leaf number, fresh and dry weight in comparison to untreated plants. (Vildová et al. 2016) studied the use of silver nitrate as elicitor to enhance flavonolignans and flavonoid taxifolin production in suspension culture of Sylibum marianum (L.). Silver nitrate in various concentrations (5.887.10-3 mol/L, 5.887.10-4 mol/L, 5.887.10-5 mol/L) was used as elicitor. The content of secondary metabolites in cell suspension cultures was determined by high performance liquid chromatography. Samples were taken after 6, 12, 24, 48, 72 and 168 hours of treatment. The highest content of taxifolin production (2.2 mg/g) in cell suspension culture of Silybum marianum (L.) was detected after silver nitrate (5.887.10-4 mol/L) treatment and 72 hours application.

6-Effect of phenylalanine on secondary metabolites production

To increase the production of flavonoids from callus tissue of Hydrocotyle bonariensis, (Masoumian et al. 2011) used different precursors. The results showed that 4 mg/L proline produced the highest flavonoids yield (10.77 ± 0.25 mg/g DW). The increase in proline concentration did not significantly increase the production of flavonoids. The highest flavonoids yield (10.59 ± 0.18 mg/g DW) was produced in 1 mg/l of glutamine. No significant increase was attained in the flavonoids yield in callus treated with 2, 3 mg/L compared with the control. Phenylalanine at the concentration of 3 mg/L, successfully triggered the production of flavonoid (11.43 ± 0.12 mg/g DW), which was 23% higher than the control. (Bemani et al. 2013) investigated

the effects of different concentrations of phenylalanine on the production of Taxol, antioxidant activity, and cytotoxic effects of extracts of suspension-cultured hazel cells. The cells were treated with different concentrations of phenylalanine on day 7 of subculture and were harvested on day 14. The results showed that the amounts of Taxol and antioxidant activity were increased by increasing the phenylalanine supply.The effects of methyl jasmonate (MeJA), spermine (Spm), epibrassinolide (EBL) and L-phenylalanine on sweet basil (Ocimum basilicum L.) were studied by (Koca & Karaman 2015) to determine the amount of phenolic compounds and enzymatic activity of phenylalanine ammonia-lyase (PAL). Total phenolic and total flavonoids contents of sweet basil were determined by a spectrophotometer, and individual phenolic compounds and activity of PAL were analysed by HPLC/UV. The highest total phenolic (6.72 mg GAE/g) and total flavonoids contents (0.92 mg QE/g) were obtained from 1.0 mM Spm+MeJA application. Rosmarinic acid and caffeic acid contents were significantly enhanced after the applications but no much differences were observed in chicoric acid content or PAL activity. Rosmarinic acid was the main phenolic acid in all samples and its concentration varied from 1.04 to 2.70 mg/g of fresh weight.

7-Tissue culture studies on Sequoia sempervirens (D. Don Endl).

(Bourgkard & Favre 1988) used seeds of Sequoia sempervirens in vitro for somatic embryo production using Murashige and Skoog mineral solution complemented with 6-benzylaminopurine (2 µM), kinetin (2 µM) and 2,4-dichlorophenoxyacetic acid (2.5 . µM(. (Sul & Korban 1994) used different cytokinins for axillary shoot proliferation and elongation of Sequoia

sempervirens using stem segments. Cytokinins used were called benzyladenine (BA), BA plus adenine hemisulfate, N-benzyl-9(2 tetrahydropyranyl) adenine (BPA), N6-[2-Isopentenyl] adenine (2ip), kinetin, thidiazuron (TDZ), and zeatin using Wolter and Skoog (WS) medium. Zeatin used resulted in the highest number of shoots and the longest shoots. (Blažková et al. 1997) produced roots in vitro using branch segments of Sequoia sempervirens after treatment with indole-3-butyric acid (IBA). The presence of 25 µM IBA in the medium resulted in the appearance of roots at the base of the cuttings using Murashige and Skoog medium (MS(. (Mihaljević et al. 1999) demonstrated hairy root technique in Sequoia sempervirens using an agrobacterium-mediated gene transfer system. Agrobacterium rhizogenes wild strain 8196 and the pRi A4 transconjugant strain of Agrobacterium tumefaciens were used as bacterial strains. Callus cultures derived from putative transformed roots were established on medium supplemented with 1.13 µM 2,4-D and 2.22 µM BA. (Liu et al. 2006) used needles of Sequoia sempervirens in vitro for somatic embryo production. The results showed that embryogenic callus was induced and proliferated on Schenk and Hildebrandt (SH) medium with BA (0.5 mg/l), KT (0.5 mg/l) and IBA (1.0 mg/l).SH medium containing BA (0.5 mg/l), KT (0.2 mg/l) and IBA (0.2 mg/l) effectively promoted adventitious bud regeneration. The highest frequency (66.3%) of direct somatic embryogenesis was obtained in the combination of BA (0.5 mg/l) and IBA (0.5 mg/l).

8-Tissue culture studies on Taxodium distichum (L. Rich).

(Abou Dahab et al. 2007) produced Callus in vitro by using of shootlet segments using MS or LS nutrient media supplemented with different concentrations of 2,4-D, BA and Kin. Using of LS medium supplemented with 3.0 mg/l 2,4-D + 1.0 mg/l BA showed high significant of fresh, dry weight and 17

dry matter content (%) of mass callus production during the 3rd week of cultivation. The comparative study showed that calli extracts have lower cytotoxicity activity than in vivo extracted plants.

Summary:

Aiming to identify and authenticate Sequoia sempervirens (D. Don Endl) and Taxodium distichum (L. Rich) cultivated in Egypt, this work was designed to study the botanical, genetic, phytochemical and pharmacological profiles of the plants. In addition, a tissue culture study was conducted to investigate the possibility of in vitro propagation of the plants and enhancing its production of secondary metabolites. The study was conducted in 4 main parts as follows:

Part 1: Botanical study of non-flowering parts of Sequoia sempervirens (D. Don Endl) and Taxodium distichum (L. Rich)

Chapter 1: DNA fingerprinting of Sequoia sempervirens (D. Don Endl) and Taxodium distichum (L. Rich)

RAPD technique was employed for this study, in an attempt to identify unique DNA markers and establish a typical genetic fingerprint for S. sempervirens and T. distichum.

The amplification profile of S. sempervirens was screened and a total of 18 different RAPD fragments had been recorded showing 3 bands by OPA-09 ranging from (0.471 to 0.581 Kbp), 1 band by OPA-16 at (0.29 Kbp), 5 bands by each of OPB-01 and OPC-05 ranging from (0.189 to 0.63 Kbp) and (0.118 to 0.581 Kbp) respectively, and 4 bands by OPB-18 ranging from (0.238 to 0.43 Kbp).

The amplification profile of T. distichum was screened and a total of 15 different RAPD fragments had been recorded showing 2 bands by each of OPA-09 at (0.433 and 0.591 Kbp), OPA-16 at (0.201 and 0.513 Kbp), and OPB-01 at (0.233 and 0.353 Kbp), 5 bands by OPB-18 ranging from (0.144 to 0.735 Kbp), and 4 bands by OPC-05 ranging from (0.227 to 0.497 Kbp).

Chapter 2: Macromorphological study of Sequoia sempervirens (D. Don Endl) and Taxodium distichum (L. Rich)

S. sempervirens is an evergreen tree native to coastal California and the southwestern corner of coastal Oregon within the United States. This tree is found in El-Orman garden, Giza, Egypt with rough trunk, a diameter of 1 m, reaching heights of 8 m and a roundish crown.

Leaves of S. sempervirens are variable, being less than 10 cm long, flat on young trees and shaded shoots in the lower crown of old trees. They are paripinnate compound leaves. Leaflets are sessile, needle-like or scale-like. They are dark green in color. They are smooth, alternate and linear to linear- lanceolate in shape. They have an entire margin, acute apex and decurrent base.

T. distichum is an evergreen tree native to southern part of North America from southeastern Virginia to southeastern Louisiana. This tree is found in El-Orman garden, Giza, Egypt with rough trunk, a diameter of 2– 3 m and reaching heights of 20 m tall.

Leaves of T. distichum are dark green in color, with flat blades borne on the twig that are spirally arranged on the stem, but twisted at the base to lie in two horizontal ranks and being less than 10 cm long. They are paripinnate compound leaves. Leaflets are sessile, smooth, alternate and linear to linear-

lanceolate in shape. They have an entire margin, acute apex and decurrent base. It is deciduous, losing its leaves in the winter months.

Chapter 3: Micromorphological study of Sequoia sempervirens (D. Don Endl) and Taxodium distichum (L. Rich)

1- The leaf: A transverse section in the leaf showed one-layered of epidermis with polygonal isodiametric cells which consist of thick-walled cells (especially the outer walls). Below the epidermis there is the one-layered hypodermis which is isodiametric parenchymatous cells with cellulosic walls and narrow intracellular spaces. The palisade appears in one row beneath the upper epidermis. The mesophyll is slightly divided into spongy tissue. Abaxially, there is one resin duct placed between the vascular bundle and epidermis. Spongy parenchyma usually consists of circular or oval cells with intercellular space.

2- The stem: A transverse section in the stem is almost circular in outline. It is formed an outer cork (in case of T. distichum only) followed by the cortex, which is formed of parenchymatous cells. The pericycle is formed of patches of lignified fibres interrupted by parenchymatous cells. The vascular tissue is associated with abundant parenchyma in curved tangential bands and is traversed by broad medullary rays. The central pith is somewhat and formed of parenchyma.

The vascular tissue:

The phloem: consists of soft tissue, devoid of fibers, formed mainly of thin walled parenchymatous cells, sieve tubes and companion cells.

The xylem: is formed of radially arranged elements. The vessels are mainly spiral thickenings. Medullary rays are formed of uniserriate or biserriate rectangular cells with pitted walls.

The pith: Consists of more or less rounded parenchymatous cells.

Part 2: Phytochemical study of non-flowering parts of Sequoia sempervirens (D. Don Endl) and Taxodium distichum (L. Rich)

Chapter 4: preliminary phytochemical screening of Sequoia sempervirens (D. Don Endl) and Taxodium distichum (L. Rich)

The results of this preliminary phytochemical screening of both plants showed the absence of alkaloids, anthraquinones, catechol tannins or cardiac glycosides in non-flowering parts of S. sempervirens and T. distichum. Sterols and/or triterpenoids, pyrogallol tannins, carbohydrate and/or glycosides and flavonoids were detected in all of the tested parts of the plants. Volatile substances were detected in very low amounts in non-flowering parts of both plants.

Chapter 5: Study of lipid content of Sequoia sempervirens (D. Don Endl) and Taxodium distichum (L. Rich)

GC/MS analysis of hexane extract of non-flowering parts of S. sempervirens and T. distichum revealed that there are twenty nine compounds were identified in unsaponifiable matters of non-flowering parts of both S. sempervirens and T. distichum. Sequoia contains high quantity of α-Eudesmol (10.96%) in non-flowering parts of unsaponifiable matter while T. distichum (L.) Endl contains high quantity of Widdrene (9.25%).

Unsaturated fatty acids were present in higher percentage than saturated fatty acids in both S. sempervirens (27.72%) and T. distichum (34.77%). Oleic acid was the major fatty acid in S. sempervirens at 27.72%, while linolenic acid was the major fatty acid in T. distichum at 28.85%.

Chapter 6: Investigation of the hexane fraction from non-flowering parts extract of Taxodium distichum (L. Rich).

Five grams of hexane fraction was saponified by refluxing with (10%) alcoholic KOH for 4 hours then cooled and filtered. The saponifiable solution (filtrate) was evaporated till dryness. The residue mixed with 100 ml distilled water, then transferred to a separating funnel, then shaked with successive portions of diethyl ether (3x500 ml). Two g of residue was obtained after evaporation of diethyl ether.

Part of the hexane extract (1 g) was subjected to silica gel column (110 cm x 2 cm) using silica gel (50 g). Elution started with Elution started with hexane, followed by gradient addition of DCM in 10% increment starting with 10% and up to 100%, followed by gradient addition of ethyl acetate starting with 10% and up to 100%, and finally washing the column by addition of MeOH 100%.

Fractionation and purification of hexane fraction afforded the isolation of one compound:

Compound H1: 8 β-hydroxypimar-15-en-19-oic acid (14 mg, White crystal powder).

Chapter 7: Investigation of the ethyl acetate fraction from non-flowering parts extract of Sequoia sempervirens (D. Don Endl) and Taxodium distichum (L. Rich).

Six grams of the ethyl acetate fraction was chromatographed over a column (90 cm x 5 cm) using polyamide (100 g). The elution started with deionized water followed by gradient addition of methanol (at 10% increments) to 100% methanol. The obtained fractions were separately subjected to re chromatography on Sephadex using with methanol (80%) and deionized water (20%) for further purification. Fractionation and purification of ethyl acetate fraction afforded the isolation of three compounds:

Compound E1: apigenin (13.5 mg, yellow powder)

Compound E2: luteolin (14.8 mg, yellow powder)

Compound E3: tricetin (5.0 mg, yellow powder)

Four grams of the ethyl acetate fraction was chromatographed over a column (90 cm x 5 cm) using polyamide (100 g). The elution started with deionized water (90%) and methanol (10%) followed by gradient addition of methanol (at 5% increments) to 60% methanol followed by gradient addition of methanol (at 10% increments) to 100% methanol. The obtained fractions were separately subjected to re chromatography on Sephadex LH-20 using with methanol (80%) and deionized water (20%) for further purification. Fractionation and purification of ethyl acetate fraction afforded the isolation of two compounds:

Compound E4: hyperin (27.6 mg, yellow powder)

Compound E5: catechin (20.0 mg, orange powder)

Chapter 8: Standardization of extracts

A- Determination of total phenolic and flavonoid contents:

Total phenolics and flavonoids content were measured by Folin–

Ciocalteu and AlCl3 methods respectively in non-flowering parts of S. sempervirens and T. distichum. S. sempervirens contained higher amount of phenolics and flavonoids than T. distichum with values at 1074.3 and 746.6 mg GAE/100g dry powder total phenolics in S. sempervirens and T. distichum, respectively and 214.9 and 149.3 mg KE/100g dry powder total flavonoids in S. sempervirens and T. distichum, respectively.

B- HPLC determination of some flavonoids HPLC analysis of phenolic and flavonoid compounds in non-flowering part of S. sempervirens and T. distichum identified four flavonoids (apigenin, kaempferol, quercetin and luteolin). These compounds were quantified and luteolin was recognized as the major compound at 0.096 and 0.325 mg/100g dry powder in S. sempervirens and T. distichum respectively. All flavonoids showed lower concentration in non-flowering parts of S. sempervirens than that in T. distichum.

Part 3: Tissue culture study of different parts

1- Sequoia sempervirens (D. Don Endl) Different parts of the plant (young and fully grown leaves and stem nodes) were used as explants for callus production in 12 different MS medium compositions. All treatments produced callus but the most successful supplements produced callus which was the MS medium supplemented with P-chloro-phenoxy acetic acid 4 mg/l and P-chloro-phenoxy acetic acid 6 mg/l. The green in color callus was formed from all the explants (young and fully grown leaves as well as the stem internodes).

In vitro secondary metabolites production

The formed callus was treated with different physical and chemical elicitors and precursors including phenylalanine, silver nitrate and UV irradiation in different concentrations and strengths for the purpose of increasing secondary metabolites concentrations.

Effects of physical and chemical elicitors on total phenolics and total flavonoids content of callus culture of S. sempervirens (D. Don Endl)

The highest phenolics and flavonoids contents were estimated in callus treated with phenylalanine 200 mg/l at 1186.4 mg/100 g and 237.3 mg/100 g, respectively.

Determination of secondary metabolites in calli

Results of HPLC analysis of samples showed that treatment containing silver nitrate at 200 mg/l stimulated the production of the highest concentration of apigenin when compared to mother plant and control callus sample. Treatment containing combination of Phenylalanine 100 mg/l and exposure to UV light at 15W for 60 min showed higher concentration of kaempferol compared to mother plant but lower than control sample. Treatment containing Phenylalanine 100 mg/l and exposure to UV light at 15W for 60 min stimulated the production of the highest concentration of quercetin when compared to mother plant and control callus sample. Treatment containing silvernitrate 100 mg/l and exposure to UV light at 15W for 60 min stimulated the production of the highest concentration of luteolin when compared to mother plant and control callus sample.

2- Taxodium distichum (L. Rich)

Different parts of the plant (young and fully grown leaves and stem nodes) were used as explants for callus production in 27 different MS medium compositions. No callus formation was obtained although different trials were carried out but propagation was produced (buds formation).

Part 4: Biological study of non-flowering parts extract of Sequoia sempervirens (D. Don Endl) and Taxodium distichum (L. Rich).

Chapter 9: In vitro studies

Antimicrobial screening of S. sempervirens (D. Don Endl) and T. distichum (L. Rich) non-flowering parts extracts

Ethanolic extract of non-flowering parts S. sempervirens has more antimicrobial activity than that of T. distichum. The most potent antibacterial activity of non-flowering parts ethanolic extracts of S. sempervirens and T. distichum were obtained against E.coli with MIC of 3.9 µg/ml and 125 µg/ml respectively, whereas both plant extracts showed no activity against P. aeruginosa. The most potent antifungal activity of non-flowering parts ethanolic extracts were obtained against A.fumigatus with MIC of 3.9 µg/ml for S. sempervirens and 125 µg/ml for T. distichum.

Chapter 10: In vivo studies

A- Acute toxicity study No clinical signs of toxicity and mortality appeared in mice treated with the ethanolic extracts of S. sempervirens and T. distichum non-flowering parts even after administration of 5000 mg/kg dose.

B- In vivo evaluation of anti- arthritic activity

Body weight in Complete Freund’s Adjuvant (CFA) treated rats was significantly reduced compared to normal control. The body weight reduction in S. sempervirens and T. distichum extracts was less than that of CFA treated rats. However, the body weight increased in both extracts was lower than normal rats. Swelling of paws was measured using a plethysmometer this edema was reduced after injection with S. sempervirens and T. distichum extracts. S. sempervirens extract reduced edema more than T. distichum extract. The hotplate indicates the sensitivity and the reaction to pain stimulus, in which the CFA group rats responded faster than the control group. Treatment of the CFA treated rats with S. sempervirens and T. distichum extracts normalized the response compared to the control group.

C- In vivo evaluation of anti-diabetic activity Type 2 diabetes mellitus was induced in rats by the intraperitoneal injection of freshly prepared streptozotocin (STZ). Oral administration of S. sempervirens and T. distichum extracts resulted in significant decreases in the levels of glucose, TG, TC and LDL compared to the diabetic group and increased HDL-C concentration compared to non-treated diabetic group.

D- In vivo evaluation of hepatoprotective activity Induction of liver injury with injection with thioacetamide (TAA) increased the serum levels of ALT, AST, total bilirubin and a decrease in serum albumin level compared to normal values. Treatment of TAA-treated rats with tested S. sempervirens and T. distichum extracts for two weeks reduced serum levels of ALT, AST, total bilirubin and an increase in serum albumin levels compared to the TAA-control group. Liver histopathological findings showed that administration of ethanolic extract of S. sempervirens was more potent hepatoprotective than ethanolic extract of T. distichum.

Conclusion:

 Unsaturated fatty acids were present in higher percentage than saturated fatty acids in both S. sempervirens (27.72%) and T. distichum (34.77%) and the major are oleic acid and linolenic acid, respectively.  Flavonoid (tricetin) was isolated for the first time from non-flowering parts of S. sempervirens.  Ethanolic extracts of non-flowering parts of S. sempervirens and T. distichum are considered safe in dose up to 5 g/kg body weight.  The ethanolic extract of non-flowering parts of S. sempervirens showed the best hepatoprotective, anti-arthritic and anti-diabetic activities at concentration of (200 mg/kg).