Pharmacognostical Investigation of some () A Thesis submitted By

M.Sc. Samar Mohamed Bassam Moaz

For The Degree of Doctor of Philosophy in Pharmaceutical Sciences (Pharmacognosy)

Under the Supervision of

Prof. Dr. Seham Salah El Din El-Hawary

Professor of Pharmacognosy, Faculty of Pharmacy, Cairo University

Assistant Prof. Dr. Abeer Mohamed Ali El-Sayed

Assisstant Professor of Pharmacognosy, Faculty of Pharmacy, Cairo University

Assistant Prof. Dr. El-Moataz Bellah Ali El-Naggar Assisstant Professor of Pharmacognosy, Faculty of Pharmacy, Damnhour University

Dr. Hanan Samir Marzouk Lecturer of Pharmacognosy, Faculty of Pharmacy, Pharos University in Alexandria

Pharmacognosy Department Faculty of Pharmacy Cairo University

A.R.E

2019

1

Abstract Yucca is a of woody perennial shrubs and , native to Central America northward to southernmost . This genus is highly reputed for its economic importance as a source of fibers and saponins for rope and soap industries, respectively.

Yucca aloifolia L., and its variety variegata L., L. and Yucca elephantipes Regel were collected from Orman Botanical Garden, to investigate their biological activities and 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.

Biological investigation of the total alcoholic extracts of the leaves of the 4 plants, lead to the conclusion of the excellent hepatoprotective effect of the 4 plants using in-vivo CCl4-induced hepatotoxicity model. In-vitro cytotoxicity investigation proved the outstanding cytotoxic activity of Y. aloifolia variegata L., especially against hepatocellular carcinoma and breast cancer cell lines. However, antimicrobial activity screening showed the lack of activity in both water and alcoholic extracts of the 4 plants.

As a result, Y. aloifolia variegata L. was considered the most active and was subjected to further phytochemical investigation. This included preliminary phytochemical screening, determination of total phenolic content, quantitative and qualitative determination of flavonoids and phenolic acids in fraction using HPLC, investigation of lipoid content using GC-MS for both saponifibale and unsaponifiable fractions, and finally LC-MS/MS of the total extract and some fractions. LC-MS/MS was used to for identification of 10 isolated compounds.

These investigations lead to the identification of 129 compounds in Y. aloifolia variegata L., among which saponins, flavonoids, phenolic acids, stilbenes, hydrocarbons and fatty acids.

This study is the first to investigate Y. aloifolia variegata L. botanically, genetically and phytochemically.

Molecular docking in-silico experiments were done for some of the major compounds against possible protein targets in cytotoxic activity. The docking scores were good for all the investigated compounds, indicating the holistic action of the alcoholic extract.

Keywords: Yucca aloifolia, Yucca aloifolia variegata, Yucca filamentosa, Yucca elephantipes, LC-MS, HPLC, saponins, flavonoids, phenolics, DNA fingerprinting, molecular docking, hepatoprotective, cytotoxic 2

Introduction

Yucca is a genus of woody perennial shrubs and trees. It is a terrestrial , stems are absent, short or thick and arborescent, then usually more or less branched. Its species are notable for their rosettes of evergreen, with numerous sword-shaped leaves which are more or less ensiform. These leaves are nearly linear, thin or flexible maybe somewhat thicker and very rigid having horny or smooth margins, which are often desintegrate into fibres and terminal spines often present (Szabo K. , 2011).

Genus Yucca was first classified under family Liliaceae (Pai and Patil, 2011), tribe Yuccae, due to its superior ovary (Szabo K. , 2011). However, later it was moved to family Agavaceae (Oleszek and Bialy, 2006). Agavaceae is a monocot family of flowering plants in the , more recently reduced to the subfamily of the family Asparagaceae(Chase et al., 2009). The family includes many well-known desert and dry zone types. It comprises about 550-600 species in around 24 genera, widespread in the tropical, subtropical and warm temperate regions of the world.

The genus Yucca has been estimated to conservatively comprise 35–40 species within its native range from Central America northward to southernmost Canada. Historically, three sections have been defined based on differences in morphology. Two sections contain species with indehiscent fruit, including the fleshy-fruited Sarcocarpa and the spongy-fruited Clistocarpa, and the remainder belonging to the section Chaenocarpa with dry, dehiscent . Within the Chaenocarpa, there is also consensus in historical, morphology-based treatments for a subset referred to as the Rupicolae. Clistocarpa is monotypic, while the two other sections have roughly equal numbers of taxa. Relationships among these sections remain unsettled (Olle P., 2007)

These flowering plants generally thrive in arid parts of Southwestern US and , namely Mojave, Sonoran, Colorado and Chihuahuan Deserts, of coastal , sand dunes and Baja California.The majority are xeromorphic and succulent rosette plants of desertic regions. However, the species Yucca aloifolia L. (Spanish bayonet) grows in Southeastern USA. Yucca whipplei ( yucca) is prevalent in the south California chaparral. (Joshua ) is the signature plant of . (Mojave yucca) is prevalent across Mojave and Sonoran Deserts and regarded highly for its pharmaceutical values. (Soap tree yucca) is another distinct species in Sonoran and deserts. Yucca elephantipes (Giant yucca) is often planted for landscape purpose in urban area (Patel, 2012).

3

Yucca is widely recognized for its mutualism with yucca moths. Pollination of Yucca depends entirely on moths of the genera Tegeticula and Parategeticula ().These moths have coevolved as obligate mutualists with their yucca hosts, and their whole life cycle is intimately associated with the host plant. Yucca moths mate within host , and the mated female uses unique mouth parts to collect pollen from anthers (Svensson et al., 2011).

The main application of yucca products is in animal nutrition, in particular as a food additive to reduce ammonia and fecal odors in animal excreta. The positive effects of dietary supplementation with yucca products on the growth rates, feed efficiency, and health of livestock seem to be due not only to the saponin constituents but also to other constituents (Paola Montoro, 2008).

Yucca gloriosa were used historically to remove gall bladder stones. Crushed roots are taken every morning and night (Mohammed Rahmatullah, 2010).

Aim of the work

Very few studies were done to investigate different yucca species introduced to Egypt. Therefore, this study aims at investigating the chemical composition and the biological activities of three yucca species cultivated in Egypt; Yucca aloifolia and its variety Yucca aloifolia variegata, Yuuccafilamentosa and Yucca elephantipes.

This work includes the following:

1. Review of the current literature. 2. Botanical study and DNA fingerprinting of the plant. 3. Investigation and evaluation of biological activities of different extracts and fractions. 4. Preliminary phytochemical screening of different organs of the different plant species. 5. Biologically-guided phytochemical investigation of the most active species. 6. Identification of major chemical constituents of the active species.

4

Review of Literature

Chemistry of Genus Yucca

Review of literature illustrated that genus Yucca is rich in saponins, which represents the major chemical constituent. Other constituents reported to be isolated from yucca include, sterols and phenolic constituents.

1- Saponins:

Steroidal saponins, both spirostanol type and furostanol type have been isolated from different species of Yucca.

Yokosuka et al. (2014) succeeded to isolate six steroidal glycosides and 14 known compounds from the underground parts of . Their structures were determined from extensive spectroscopic analysis, including two-dimensional NMR data, and chemical transformations. The isolated saponins were both of spirostan and furostan types. The compounds were also evaluated for cytotoxic activities against HL-60 human leukemia cells and A549 human lung adenocarcinoma cells.

Ibrahem and Awad (2013) performed a study to detect the presence of the most important steroidal sapogenin “Tigogenin” in the leaves of Yucca aloifolia widely cultivated in Iraq. The identification of isolated Tigogenin was carried out using melting point (M.P.), thin layer chromatography (TLC), infrared spectroscopy (IR) and High performance liquid chromatography (HPLC).

2- Phenolic compounds and Flavonoids (Table 3 and Table 4) Many phenolic compounds have been isolated or identified from Yucca species. Juarez-Trujillo et al. (2018) investigated the methanol crude extract of Yucca elephantipes Regel flowers from Mexico using LC-MS analysis. The analysis led to the tentative identification of a considerable number of flavonoids and phenolics. Nakashima et al. (2016) isolated three new phenolic compounds, yuccalides A–C from the roots of Y. gloriosa. The structures of the new compounds were established by extensive NMR spectroscopic analyses.

5

3- Sterols and hydrocarbons (Table 5): Several sterols (Blunden and Hardman, 1969) and hydrocarbons (Tröger et al., 2019) have been identified from leaves and floral scents of some Yucca species, respectively. B. Biological studies on genus Yucca:

 In vivo anticoccidial activity

Djezzar et al. (2013) and Rambozzi et al. (2011) proved that anticoccidial- based natural extract of "Yucca schidigera L.. Alfaro et al. (2007) studied the addition of the Y. schidigera extract to the diet of coccidiosis vaccinated birds and found that it improved average daily gain and feed conversion rate compared with the other treatments.

 Anti-inflammatory and anti-arthritic effects

Cheeke et al. (2006) proved that Yucca polyphenolics may have several roles in anti-arthritic activity.

Marzocco et al. (2004) demonstrated that Yuccaol C from Yucca schidigera roezl reduced inducible nitric oxide synthase iNOS protein expression via the transcription factor NF-nB.

Backer et al. (1972) investigated the saponin-containing fraction of the leaves of Yucca schottii Engelm.And showed that the fraction possesses anti-inflammatory properties against carrageenin-induced edema in rats.

 Antimicrobial activity

Bononi et al. (2013) demonstrated that Y. filamentosa extracts displayed specific growth inhibitory activity against Saccharomyces cerevisiae.

Thamizhavanan et al. (2012) and Sobia et al. (2012) investigated the antimicrobial activity of petroleum ether extract of Y. gloriosa L. and Y. aloifolia, respectively and were found to exhibit good antibacterial activity against both Gram positive and Gram negative organisms.

Jacobs et al. (2010) demonstrated the antimicrobial activity of n-butanol fraction of leaves and stems of Y. glauca.

6

Jin et al. (2007) investigated the extract of the leaves of Yucca smalliana and showed its potential antimicrobial activity.

Qingmin et al. (2007) investigated the antimicrobial effects of Y. gloriosa ethanol, butyl alcohol and water extracts on Penicillium dititatum, Botrytisallii Munn and Botrytis cinerea Pers isolated from plants. The antimicrobial effect of ethanol extraction was the best.

Favel et al. (2005) reported the antifungal activity of a crude steroidal glycoside extract from Y. gloriosa flowers, named alexin.

Miyakoshi et al. (2000) reported that the saponin fraction from the stems of Y. schidigera exhibited potent yeast growth-inhibitory activities.

Killen et al. (1998) studied the benefits of supplementing livestock diets with extracts from Y. schidigera. These benefits have been attributed to inhibition of selected gut microbes.

 Anthelmintic activity

Thamizhavanan et al. (2012) proved that the petroleum ether extract of L. showed comparable activity with that of standard piperazine citrate, a drug widely used as anthelmintic.

 Antioxidant activity

Sun et al. (2019) tested the effects of Y. shidigera extract on growth performance of broilers, its antioxidative enzyme activities and corresponding gene expressions in the small intestine. The extract showed improved growth performance results of broilers.

Ince et al. (2013) studied the effects of dietary supplementation with Y. schidigera. The results demonstrate that Y. shidigera especiallyhigh-dose supplementation in diet decreases arsenic-induced oxidative stress and enhances the antioxidant defence mechanism and regeneration of tissues in Swiss albino mice.

Sobia et al. (2012) proved that Y. aloifolia leaves extract showed considerable antioxidant activity but were solvent-dependent.

Cigerci et al. (2009) proved that Y. shidigera treatment was found to decrease methemoglobin, blood and tissue malondialdehyde, and tissue nitric oxide concentrations, and to increase the glutathione in blood and various tissues. 7

Olas et al. (2008) proved that the polyphenol rich extract of Y. schidigera bark had antioxidant and anti-platelet activities better than that of resveratrol alone.

Enginar et al. (2006) studied the effects of dietary Y. schidigera extract supplementation on lipid peroxidation in rabbits exposed to γ-radiations. The results suggested that the extract exhibits some antioxidant properties.

Piacente et al. (2004) demonstrated the significant antioxidant activities exhibited by the phenolic fraction and its constituents which show the potential use of Y. schidigera as a source of antioxidant principles.

Torres et al. (2003) studied the compounds 4,4'-dihydroxstilbene, resveratrol and 3,3',5,5'-tetrahydroxy-4-methoxystilbene,isolated from , and demonstrated their scavenging properties toward 2,2- diphenyl-1-picrylhydrazyl in TLC autographic and spectrophotometric assays.

 Pesticidal activity

Patricio Torres et al. (2003) demonstrated that the methanol extract from the bark of Yucca periculosa F. Baker had growth regulatory activity against the Fall Army worm Spodoptera frugiperda J.E. Smith, an insect pest of corn.  Anticancer effect

Balestrieri et al. (2006) results indicated that Yuccaols were more effective than resveratrol in inhibiting the VEGF-induced KS cell proliferation. This study provided evidence of the anti-tumor properties of Y. schidigera phenolic compounds.

Uenobe et al. (1997) proved that the methanol extract of Y. schidigera had a suppressive effect on mutation induced by Trp-P-1. In Salmonella typhimurium. The active compound was identified as 3,4',5- trihydroxystilbene.

Sharma et al. (1978) reported that the aqueous alcoholic extract of fresh flowers of Y. glauca Nutt. showed striking antitumor activity against B16 melanoma in mice. Systematic fractionation of the extract by means of solvent extraction and gel permeation chromatography led to separation of two galactose containing polysaccharide fractions with marked inhibitory activity against B16 melanoma in mice.

8

 Hepatoprotective activity The petroleum ether extract of whole plant of Yucca gloriosa L. was found to possess hepatoprotective activity against carbon tetrachloride induced hepatotoxicity in rats. The extract at the doses of 200 and 400mg/kg inhibited CCl4 induced liver toxicity in Wistar albino rats as assessed by the biochemical changes and histopathological studies (Rani and Lakshmi, 2012).  Antigiardial Activity High-extract concentrations of Y. baccata significantly reduced the trophozoite counts similar to metronidazole. Extracts of Y. baccata may represent an effective and natural therapeutic alternative for human giardiasis (Luis Quihui-Cota, 2014).

 Photoprotective activity Some flavonoids, stilbenes and phenylpropanoids were isolated from Y. periculosa and showed photoprotective activity against UV-B (García- Bores et al., 2013).

C. Non-Biological activities

Surfactant activity

Ralla et al. (2018) examined the Yucca shidigera L. saponin extract containing steroidal saponins for its interfacial and emulsifying properties. The results indicate Yucca saponin extract as a new potential natural surfactant that may be used to replace synthetic surfactants in the food and beverage industry for selected applications.

The present work includes the following;

Part I: Genetic profiling and botanical study of Yucca aloifolia L., Yucca aloifolia variegata L., Yuucca filamentosa L. and Yucca elephantipes Regel.

Part II: Biological investigation of the extracts of the leaves of Yucca aloifolia L., Yucca aloifolia variegata L., Yucca filamentosa L. and Yucca elephantipes Regel to determine the most active species

Part III: Phytochemical study of the extracts of the leaves of Yucca aloifolia variegata L. • Chapter I : Preliminary Phytochemical Screening • Chapter II: Quantitative Determination Of Total Phenolic Content

9

• Chapter III: Qualitative and Quantitative Determination of Flavonoids and Phenolic Acids • Chapter IV. Investigation of the lipoid content • Chapter V: LC-MS/MS of the Total Extract

Part IV: Molecular Docking study of major constituents against PIM-1 kinase and EGFR. Part I: Genetic profiling and botanical study of Yucca aloifolia L., Yucca aloifolia variegata L., Yucca filamentosa L. and Yucca elephantipes Regel.

Chapter I: DNA fingerprinting of Yucca aloifolia L, Yucca aloifolia variegata L., Yucca filamentosa L. and Yucca elephantipes Regel DNA sequencing: DNA was extracted from the four plants, amplified using PCR and DNA sequencing was performed. These DNA sequences were investigated using Basic Local Alignment Search Tool (BLAST). Comparison showed a 95-99 % similarity between the studied Yucca species and other species of Yucca registered on National Center for Biotechnology Information, U.S. National Library of Medicine (NCBI) database. However, genomic DNA sequences for the exact species studied have not been found on the database. Comparing DNA sequences of Yucca aloifiolia variegata to those of Yucca aloifolia by the tool Global Alignment resulted in 99% similarity confirming their common origin. RAPD analysis: The extracted DNA was also amplified 10 decamer primers to reveal RAPD fragments. RAPD was performed to ensure the taxonomic correlation between Y. aloifolia and Y. aloifolia variegata. Results of RAPD confirmed the close relationship between the two samples. Primers 1, 4, 5, 8 and 10 showed the best results. This study suggests that primers; A04, B10, S4, S15 and S20 are the most suitable for identification and authentication of Yucca species under investigation.

10

Chapter 2: Macromorphology of the leaves of Yucca aloifolia L., Yucca aloifolia variegata L., Yucca filamentosa L. and Yucca elephantipes Regel

This study aimed at providing information for the identification and differentiation of the leaves of the four species of Yucca under investigation. The four species of Yucca exist as woody perennial shrubs or trees. It is a terrestrial plant, stems are very short or absent in young plant. In adult plant they form a bearing woody shoot. Yucca aloifolia L. leaf: The leaf is simple, sessile and dark green in colour. The leaf blade is linear sword -shaped (about 60 cm length and 4.4 cm width at the widest part) with a terminal 5 mm spine emerging from the acuminate apex. The margin is finely serrate. The leaf is slightly thick and succulent. Yucca aloifolia variegata L. leaf: The leaf is almost similar to that of Yucca aloifolia except for the creamy white colour at the margins. It is also simple, sessile, linear, sword-shaped, thick and succulent, and with a sharp serrate margin. Fully grown leaf has the same dimensions as Yucca aloifolia. Yucca filamentosa L. leaf: The leaf is simple, sessile and very long. The leaf blade is linear lanceolate measuring about 77 cm in length and 6.4 cm in width at the widest part. The leaf is bright green, with the lower surface lighter in colour than the upper. The margin is finely serrate and the apex is acuminate. Yucca elephantipes Regel leaf: The leaf is simple, sessile, linear and dark green. Its length is about 43 cm with a width of 4.8 cm at the widest part. Apex is acuminate and margin is finely serrate. Chapter 3: Micromorphology of the leaves of Yucca aloifolia L., Yucca aloifolia variegata L., Yucca filamentosa L. and Yucca elephantipes Regel

Transverse sections of the leaves A transverse section in the leaf is almost rectangular, due to linear shaped lamina. a. The epidermis is almost identical in the 4 leaves, except that Y. elephantipes Regel shows a less thick layer of cuticle.

 The upper epidermis It is formed of tubular cells with thickened outer walls. The epidermis is covered with a thick layer of cuticle, a feature reported for xerophytic plants. The stomata are sunken, with an outer vestibule on the surface side and deep substomatal chambers.  The lower epidermis

11

It is also formed of tubular cells with thickened outer walls. The epidermis is covered with a thick layer of cuticle and show more abundant sunken stomata. b. The mesophyll Mesophyll shows one row of palisade cells and many layers of spongy tissues in Y. aloifolia L. and Y. aloifolia variegata L., Mesophyll consists of round parenchymatous cells containing calcium oxalate raphides and prisms in random arrangement. The mesophyll is occasionally interrupted by scattered closed vascular bundles and bundles of fibers. The vascular bundle is surrounded by a schlerenchyma cap of fibers from above and below. Both vascular bundles and fibers run in two directions, a feature known as three- dimensional venation. The transverse section shows many cavities and vacuoles. The former may be traced back to the fact that bundles of fibers tore through tissues during cutting paraffin sections using microtome. The latter may be due to vacuolar crystal formation system previously reported for Yucca.

Powdered drugs The microscopical examination of the powdered drug elements of the four plants, indicated they were close in structure, but with few differences; mainly in Y. elephantipes Regel, which shows more abundant prisms of calcium oxalate and dentate fibers.

Table 13: Main microscopical differences between the 4 plants: Y. aloifolia Y. filamentosa Y. elephantipes Y. aloifolia L. variegata L. L. Regel Cuticle Thick Thick thick thin sunken anomocytic stomata with an outer vestibule on the surface side and Stomata deep substomatal chambers Caox Raphides, More Raphides and prisms Crystals abundant prisms Fibers Straight wall Dentate wall Vascular Vascular bundles are closed, wrapped by schelerenchyma cap Bundles Mesophyll parenchyma cells containg CaOX crystals Stomatal 4.40 4.76 5.00 5.55 Index Palisade 1.875 1.5 2.375 1.75 Ratio

12

Part II: Biological investigation of the extracts of the leaves of Yucca aloifolia L., Yucca aloifolia variegata L., Yucca filamentosa L. and Yucca elephantipes Regel

Chapter 1: In Vivo Investigation of the Hepatoprotective Activity

Acute toxicity study The sub-acute toxicity of the ethanolic extracts of the leaves of the four plants was investigated on 45 adult male albino rats. The dried extracts were dissolved in DMSO, diluted with saline, and given orally at a dose of 100 mg/kg body weight. This dose was administered to the rats daily for 15 successive days. Body weight, blood urea nitrogen (BUN), creatinine level, aspartate aminotransferase (AST) level, and alanine aminotransferase (ALT) level were checked at the end of the experiment to observe the effect of the extracts on the rats. The ethanolic extracts of the four species showed no mortality nor any significant effect on serum biochemical parameters in normal rats indicating that the plants are safe for use.

In vivo hepatoprotective activity investigation Assessment of hepatoprotective activity was done at different dose levels against CCl4-induced hepatotoxicty in rats for 15 days. Rats were divided into 14 groups, each comprising 3 rats; the negative control group received saline only, the hepatotoxicity control group received 1 ml/ kg bodyweight CCl4 daily, groups assigned to extracts were given doses of 10, 20 and 40 mg/ kg body weight.

This study showed that CCl4 intoxication significantly elevated the transaminases (AST and ALT) activity as compared to the normal control group. Course treatment with the extracts significantly decreased serum ALT, AST in rats with CCl4 induced hepatotoxicity; moreover, the extracts significantly decreased the level of transforming growth factor beta (TGF-β) and tumour necrosis factor alpha (TNF-α). These results were close for all the species and attest to the hepatoprotective properties of these plants. Liver index and oxidative stress parameters, namely superoxide dismutase (SOD), glutathione (GSH), malondialdehyde (MDA) levels and total antioxidant capacity (TOAC) showed the better activity of Y. aloifolia variegata. Histopathology proved that treatment with ethanolic extract of the four plants, especially Y. aloifolia variegata, at a dose of 40 mg/kg body weight for 14 days, showed good activity in protecting the liver cells from CCl4-injury as evidenced by preserving hepatic architecture and reducing necrotic cells, inflammatory cells infiltration and fibrotic changes as well.

13

Chapter 2: In Vitro Investigation of The Cytotoxic Activity of Yucca aloifolia, Yucca filamentosa, Yucca elephantipes and Yucca aloifolia variegata The potential cytotoxicity of the ethanolic extracts of the leaves of Yucca aloifolia, Y. aloifolia variegata, Y. elephantipes and Y. filamentosa were evaluated on four types of human cancer cell lines; lung cancer A549 cell line, liver carcinoma HEPG-2 cell line, breast cancer MCF-7 cell line and colon cancer Caco-2 cell line. The assessment was performed using the Microculturetetrazolium (MTT) assay method. Results of MTT assay indicated that Y. aloifolia variegata ethanolic extract was the most powerful against four types of cancer cell lines namely, lung cancer A549, liver cancer HEPG-2, colon cancer Caco-2 and breast cancer MCF-7 with IC50s 229.8, 36.06, 162.5 and 45.5 μg/mL, respectively. Y. aloifolia was next in activity with IC50s 271.5, 61.55, 871.5 and 1584 μg/ mL, respectively. Y. filamentosa showed IC50s of 247.7, 37.42, 1412.5 and 631 μg/mL, respectively, and Y. elephantipes showed values of 153.256, 449.818, 0.7985 and 0.398 ng/mL.

Chapter III. Antimicrobial Activity Screening Screening of antimicrobial activity of both water and alcoholic extracts of the 4 plants was done using the Disc Diffusion Method, using 5 standard microorganisms. Inhibition zones were measured in mm, for the extracts, the standard antibiotics and the negative control.

Results of screening of antimicrobial activity were compared with those obtained by standard antibiotic Ciprofloxacin and standard antifungal Clotrimazole. A negative control containing the solvent only was also included in the experiment.

Despite the reports of antimicrobial activities of different species of Yucca in literature, the four investigated extracts showed no antimicrobial activities against the tested organisms. Both the water extract and the methanolic extract showed no considerable inhibition zones. This may lead to the conclusion that either the species cultivated in Egypt has no antimicrobial constituents or that these constituents exist in the non-polar fraction of the extract.

14

Part III: Phytochemical Investigation of leaves of Yucca aloifolia L., Yucca aloifolia variegata L., Yucca filamentosa L. and Yucca elephantipes Regel

Chapter I. Preliminary phytochemical screening

The air dried leaves, stems and roots of the four investigated Yucca species; Y. aloifolia, Y. aloifolia variegata, Y. filamentosa and Y. elephantipes were air dried, finely ground and subjected to preliminary phytochemical tests in order to determine the constituents present in these organs.

The leaves of the four species showed close results in terms of the constituents that were detected in the plants, namely; carbohydrates and/or glycosides, sterols and/or triterpenes, flavonoids and saponins. Coumarins were weakly detected in Yucca aloifolia and Yucca aloifolia variegata leaves. Although stems and roots were positive for these constituents, the results were weaker insinuating that constituents were present in lower amounts. Based on this phytochemical screening and due to their better availability; leaves were chosen as the organ to be further investigated in the four species. According to biological activity investigation results, Y. aloifolia variegata was determined to be the most active.

Extraction and examination of extractives of Y. aloifolia variegata L.

The air dried powdered leaves of Y. aloifolia variegata (2 kg) were subjected to exhaustive cold maceration in 70% ethanol. The extract was later collected and concentrated under vacuum at 40OC. The residue left after evaporation of ethanol weighed 47.181 gm. This residue was then suspended in the least amount of distilled water and then fractionated by shaking with n-hexane, chloroform, ethyl acetate and n-butanol saturated with water, successively. The extracting solvents, in each case, were removed under vacuum.

The different dried extractives were weighed and the corresponding percentage yields calculated relative to the dried plant material. The physicochemical properties of the extractives were also recorded.

The preliminary phytochemical screening showed that the 70% ethanol extracts were strongly positive for carbohydrates and/or glycosides, flavonoids, sterols and/ or triterpenes and saponins. Saponins appeared to be the main components of n-butanol and water extracts. Flavonoids seemed to be the main components of chloroform and ethyl acetate extracts.

15

Chapter II: Quantitative Determination of Total Phenolics of The Extracts of the leaves Yucca aloifolia, Yucca filamentosa, Yucca elephantipes and Yucca aloifolia variegata using Colorimetric Assay Total phenolic content was evaluated using a modified colorimetric method described previously by A.O.A.C method (2007) that involves the reduction of Folin Ciocalteau reagent. The measurement was compared to a standard curve prepared with gallic acid solution. The total phenolic content was expressed as milligrams of gallic acid equivalents per gram of fresh weight (mg GAE g-1 FW). Total phenolic content was higher in Y. aloifolia, followed by Y. aloifolia variegata, Y. elephantipes and Y. filamentosa with values of 180.429, 92.698, 52.119 and 28.342 mgGAE/gm, respectively.

As a result of the high phenolic content of Y. aloifolia variegata and its superior biological activity, it was presumed necessary to carry on a qualitative and a quanitative determination of its content of flavonoids and phenolic acids using HPLC.

Chapter III: Qualitative and Quantitative Determination of Flavonoids and Phenolic Acids in The Alcoholic Extract of Yucca aloifolia variegata L. leaves From Cairo Using HPLC Analysis

Phenolic compounds and flavonoids were determined by HPLC according to the method of Goupy et al.HPLC analysis of the different fractions of Y. aloifolia variegata L. allowed the identification and quantification of a number of flavonoids and phenolic acids. The richest fraction was that of ethyl acetate; showing 14 flavonoids and 18 phenolic acids with percentages of 4.2 and 2.15%, respectively. The butanol fraction comprised 18 flavonoids, in addition to the same 18 phenolic acids with percentages of 1.53 and 0.77%, respectively. The chloroformic fraction showed the presence of 16 flavonoids and 18 phenolic acids with percentages of 3.21 and 1.25%, respectively. The hexane fraction showed the presence of 17 flavonoids and 19 phenolic acids with percentages of 0.8 and 0.41%, respectively. The major flavonoid in butanol and ethyl acetate fractions was hesperidin; while in hexane and chloroform fractions, it was kaemp-3-(2-p-coumaroyl) glucose. On the other hand, the major phenolic acid in the other fractions was gallic acid; while in hexane fraction, it was protocatechuic acid.

16

Chapter IV: Investigation of lipoid content (Hexane fraction) of the most active species "Yucca aloifolia variegata L."

Saponification of the hexane fraction

The dried hexane extract (2 g) was refluxed with alcoholic potassium hydroxide (10%) for 5 hours. The reaction mixture is diluted with distilled water. The unsaponifiable matter is then extracted with petroleum ether through shaking in separating funnel. The petroleum ether extracts are combined and washed with distilled water till free from alkalinity. The unsaponifiable fraction is finally dried under vacuum using rotary evaporator, to be ready for GC/MS analysis. The remaining aqueous mother liquor left after removal of petroleum ether was acidified with 10% HCl to liberate free fatty acids. The mixture is extracted with petroleum ether. Petroleum ether is evaporated under pressure till having a constant weight of the saponifiable matter. The extract was then methylated and subjected to GC/MS analysis. Results of GC-MS analysis of the unsaponifiable fraction illustrated that:

1. 82% of the components of the unsaponifiable fraction were identified 2. The number of identified hydrocarbons was 21 representing only 39.64 % of the composition of the unsaponifiable fraction. 3. Only three sterols were detected. However, γ-stiosterol was the major constituting 19.92 % of the unsaponifiable fraction. Sterols represented 42.36 % of the fraction. Results of GC-MS analysis of the saponifiable fraction illustrated that: 1- The number of identified fatty acids was 26 2- Saturated fatty acids constituted 64.643 % of the identified fatty acids, while unsaturated fatty acids constituted 32.509 %. 3- The major components of the saponifiable fraction were palmitic acid, palmitoleic acid and caproic acid representing 58.28 %, 30.989 % and 4.717 % of the sample, respectively.

Chapter V. Investigation of the total alcoholic extracts using Liquid chromatography-Mass spectrometric analysis Two analyses have been carried out, using different instruments •First analysis: LC-MS/MS ion trap (Institute of Marine Sciences, Alexandria University) •Second analysis: Triple TOF 5600+ (57357 hospital)

17

First analysis: Investigation of the chemical composition of the total alcoholic extract of Yucca aloifolia variegata L. using LC-ESI- MS analysis (135 eV) Mass spectra obtained from the first analysis, allowed the tentative identification of 14 compounds. Saponins comprised the majority of the identified compounds; precisely 10 saponin peaks were identified. Identified saponins belonged to two groups, as revealed by their aglycone peaks. Second analysis: Investigation of the chemical composition of the total alcoholic extract of Yucca aloifolia variegata L. using Nano- LC-ESI-MS analysis (35 eV) Analysis of the total alcoholic extract of Yucca aloifolia variegata L. was performed using electrospray ionization at a relatively low fragmentation energy; equal to 35 eV. Analysis in the Negative-ion Mode This relatively low energy had a good impact on the analysis and lead to better identification of flavonoids and phenolic acids and their glycosides. Mass spectra obtained from the analysis, allowed the tentative identification of 41 compounds. However, a total of 5332 unknown peaks were detected, reflecting the highly complex nature of the extract. Identified compounds can be classified into 10 phenolic acids and their derivatives, , 21 flavonoids, represented by compounds. The analysis also lead to the tentative identification of 5 saponin derivatives. Other compounds, such as coumarins and stilbene derivatives could be identified.

Analysis in the Positive-ion Mode A total of 34 compounds were identified. However, a total of 3706 unknown peaks were detected, confirming the highly complex nature of the extract. The analysis in the positive mode proved to be better in identification of saponins, giving more explanatory fragmentation patterns. However, other classes of compounds were also identified, including phenolic acids, flavonoids and other compounds of steroidal nature.

18

LC-MS/MS comparison of the 4 plant extracts The total ion chromatograms of the four investigated species; Y. aloifolia L., Y.aloifolia vatriegata L., Y. filamentosa L. and Y. elephantipes Regel. were superimposed to illustrate the differences between the different extracts. The most remarkable observation was the higher concentration of constituents in Y.aloifolia vatriegta L., proven by the higher intensity of most peaks compared to same peaks in other extracts. This high concentration may explain the better biological activity of Y.aloifolia vatriegta L.. Y. aloifolia L. showed a similar total ion chromatogram to that of Y.aloifolia vatriegata L. in terms of compounds present at the same retention times and having the same fragmentation patterns. However, the intensities of all compounds were less than in Y.aloifolia vatriegta L. Y. filamentosa L. showed a somewhat different profile. Saponin-rhamnosyl- hexosyl, hecogenin rhamnoside, spirostanol-dihexosides and spirostan-diol- dihexosides were absent. Y. elephantipes showed also a slightly different chromatogram. Sapon- rhamnosyl-hexosyl was not detected, in addition to the presence of spirostan-diol- monohexosides instead of spirostan-diol-dihexosides present in Y.aloifolia vatriegata L. Y. aloifolia L. These results imply that Y.aloifolia vatriegata L extract was the richest extract, which was apparent in its superior biological activity. Chapter VI: Investigation of the butanol fraction of the ethanolic extract of Yucca aloifolia variegata Gravimetric determination of saponin fraction: Butanol fraction wass redisolved in methanol and then saponins are precipitated by addition of acetone. The residue is then separated by filtaraion using filter paper. % saponin in butanol fraction = 47.5 % The remaining acetone fraction was subjected to chromatographic separation. Chapter VI .Investigation of the remaining fraction of butanol after precipitation of saponins Separation was done on silica gel 60 (70g, 80*2.5cm). Gradient elution was carried out starting with 100% ethyl acetate followed by gradual increments of methanol and then distilled water.

19

Investigations of fractions using LC-MS/MS lead to the identification of 10 compounds.

Analysis of saponin fraction S2 using MALDI –TOF (Matrix Assissted Laser Desorption/Ionization – Time Of Flight) The butanol fraction was treated with acetone, three successive times in order to precipitate saponins. Residue was also washed with acetone. An amount of 10 mg of the saponin residue was mixed with the matrix and MALDI-TOF analysis was performed. Compounds were identified by comparison of their exact masses to data recorded on the dictionary of natural products. The precipitated saponin fraction was fractionated on Sephadex LH-20 column using gradient MeOH: H2O as mobile phase. The fractionations lead to saponins being separated into 2 fractions only; S1 and S2, even after several repetitions. This was later explained by the LC-MS/MS analysis which proved the presence of many saponin isomers having the same molecular weight. MALDI-TOF analysis of Fraction S2 lead to the detection of 9 peaks, 4 of them were identified by comparison of their exact masses to those recorded in the Dictionary of Natural Products, CRC. Identified compounds were 1 steroid, 2 spirostanol saponin glycosides and 1 triterpenoidal saponin.

Part V. Molecular docking Hesperidin , Gallic acid, yucca spirostanosides B1 & B2 and hecogenin are some of the major components of Y. aloifolia variegata L. extract were subjected to molecular docking simulation experiment to investigate PIM-1 kinase and EGFR as a possible target for their cytotoxic activity. Both Gallic acid and Hesperidin showed good fitting scores to the active sites of PIM-1 Kinase, indicating their contribution to cytotoxicity activity and possibility of using PIM-1 Kinase as their target. Hesperidin, yucca spirostanosides B1 and B2 showed good fitting scores with EGFR binding site.

20

Conclusion

 The use of total extract of Yucca aloifolia variegata L. is recommended due to the almost similar composition of different fractions

 Y. aloifolia variegata L. showed significant hepatoprotective and cytotoxic activities, using in-vivo and in-vitro models, respectively.

 The activity prevailed to Y. aloifolia variegata L. is attributed not only to phenolic content but as a synergistic effect of its other phytoconstituents.

 Y. aloifolia L. had a higher phenolic content however less active.

 The chemical compositionof the alcoholic extract of Y. aloifolia variegata L. was investigated using different MS-techniques.

 Analysis proved the main constituents of Y. aloifolia variegata L.were flavonoids, phenolic acids and saponins.

 LC-MS/MS is a complementary technique, elution order on liquid chromatography column plays a role in identification of structural isomers e.g discriminating hexoses.

 Many saponins existed as isomers of the same molecular weight, suggesting only different sugar or site of glycosidation.

 Variations in results of LC-MS analyses are mainly due to differences in techniques used.

 The lower the collision energy, the better was the identification of compounds.

 Flavonoids and phenolics are better detected in negative mode of ESI- MS/MS.

 Saponins are better detected in positive mode of ESI-MS/MS.

 MALDI-TOF analysis is suitable for high Mwt compounds in presence of sufficient exact mass data

 Molecular docking simulation study suggested PIM-1 kinase and EGFR as possible targets for extract components belonging to different classes.

 Y. aloifolia variegata L. is a good candidate for hepatoprotective and cytotoxic drugs of natural origin. Further, clinical investigation is recommended.

21