A Pharmacognostical study of certain and Haworthia species, cultivated in Egypt

A Thesis submitted by

Heba Ahmed Fahmy

Assistant lecturer -Faculty of Pharmacy Modern University for Technology and Information

For the degree of Doctor of Philosophy in Pharmaceutical Sciences (Pharmacognosy)

Under the Supervision of:

Prof. Dr. Ali Mohamed Prof. Dr. Seham Salah El

ElShamy Din El Hawary

Professor of Pharmacognosy, Professor of Pharmacognosy,

Faculty of Pharmacy, Faculty of Pharmacy,

Cairo University Cairo University

Pharmacognosy Department Prof.Faculty Dr. of Shahira Pharmacy Ezzat Prof. Dr. Sanaa Ahmed Ali

Professor of Pharmacognosy, Professor of Biochemistry, Faculty of Pharmacy, Therapeutic Chemistry Departement,

Cairo University National Research Centre

Pharmacognosy Department Faculty of Pharmacy Cairo University 2019

English abstract Haworthia limifolia Marloth, (Mill) Duval and Gasteria minima Poelln. are small succulent which belong to family according to APG IV classification system. The DNA fingerprinting using RAPD-PCR analysis was made using 10 primers and it revealed that primers OPB-07 and OPB-01, recording high percentage of polymorphism, can be used to differentiate between the examined species. Meanwhile, primers OPB-01, OPB- 04, OPB-07 and OPB-08, generating fragments with wide molecular size, can be used for their identification. Macromorphological and micromorphological botanical features of the leaves and stems of the three species under investigation were illustrated using the entire specimens, transverse sections and powdered . The preliminary phytochemical screening of their aerial parts revealed the presence of carbohydrates and/or glycosides, condensed tannins, free and combined flavonoids, sterols and/or triterpenes, and free and combined anthraquinones and the absence of essential oil, hydrolysable tannins, saponins, alkaloids and/or nitrogenous bases, cardiac glycosides and oxidase enzyme. Using LC-MS/MS analysis, several compounds belonging to different classes as phenolic acids, flavonoids, anthraquinones and chromones were identified in the investigated species. Vanillic acid hexoside, naringin, 5-hydroxyaloin, aloesaponarin I and 7-methylether of 2`feruloylaloesin were among the compounds identified in the three-examined species. Other compounds were identified in only one species as aloeresin F which was detected in Gasteria minima Poelln. and orientin which was detected in Gasteria carinata (Mill) Duval, thus they could be considered as chemotaxonomic markers for the investigated species. Concerning the total phenolic contents, the study revealed that Gasteria minima Poelln. has the highest concentration (29.27 mg/ml) followed by that of Gasteria carinata (Mill) Duval (27.4 mg/ml) and it was the lowest in Haworthia limifolia Marloth (22.05 mg/ml). While the concentration of tannins was the highest in Haworthia limifolia Marloth (8.04%) then in Gasteria minima Poelln. (2.58%) and it was the lowest in Gasteria carinata (Mill) Duval (1.02%). The determination of the phenolic and flavonoid content using HPLC analysis using external phenolic and flavonoid standards revealed that the percentage of total identified flavonoids in aerial parts of Gasteria carinata (Mill) Duval surpassed its percentage in Haworthia limifolia Marloth and it was the least in Gasteria minima Poelln. . Quercetrin represented the major identified flavonoid in Haworthia limifolia Marloth, while Hesperetin is the major identified one in Gasteria carinata (Mill) Duval and catechin is the major identified one in Gasteria minima Poelln. . On the other hand, the percentage of total identified phenolic compounds was higher in aerial parts of Gasteria carinata (Mill) Duval followed by Gasteria minima Poelln. and it was the least in Haworthia limifolia Marloth. Salicylic acid was the highest identified phenolic compounds in Haworthia limifolia Marloth while vanillic acid was the highest identified one in Gasteria carinata (Mill) Duval and Gasteria minima Poelln. .

The results of the GC/MS analysis of the unsaponifiable matter of their aerial parts revealed the presence of seventy-five compounds. The major identified compounds in H. limifolia were the hydrocarbons benzene-1-butylheptyl (16.71%), benzene-1-pentylheptyl (9.55%) and benzene-1-butyloctyl (8.75%), while the major compounds identified in G. carinata were sitosterol (13.98%), phytol (13.58 %) and benzene 1-butylheptyl (10.9%). The major compounds in G. minima were sitosterol (16.89%), phytol (13.06 %) and 9,19-cyclolanost-24- en-3-ol, acetate (6.82%).

The results of GC/MS analysis of the saponifiable matter revealed the presence of 28 compounds. Eight compounds were identified as methyl esters of unsaturated fatty acids (49.66, 45.24 and 64.27%) in H. limifolia Marloth, G. carinata (Mill) Duval and G. minima Poelln. respectively and the other identified twenty compounds are methyl esters of saturated fatty acids (50.34, 54.76 and 35.73%) in H. limifolia Marloth, G. carinata (Mill) Duval and G. minima Poelln. respectively. Methyl palmitate was the major identified fatty acid methyl ester in H. limifolia Marloth and G. carinata (Mill) Duval, followed by oleic acid methyl ester then linoleic acid methyl ester. While the major one in G. minima Poelln. was linoleic acid methyl ester, followed by methyl palmitate then linolenic acid methyl ester. The results of the analysis of mucilage content using HPLC revealed that the investigated species were rich in polysaccharides; Gasteria minima Poelln. had the highest polysaccharide content, followed by Haworthia limifolia Marloth and Gasteria carinata (Mill) Duval 1.01%, 0.541% and 0.44%, respectively. The monosaccharides glucose, galactose, rhamnose and arabinose were detected. The disaccharide, sucrose was also detected. The oligosaccharide, stachyose, was the highest detected sugar in the three- investigated species. Concerning the sugar acids, galacturonic acid was detected in the three- investigated species in higher concentration than glucuronic acid which was still present in high concentration. The results of the present study were compared with another study carried by (El Sayed et al, 2016) on the HPLC of the mucilage content of eight Aloe species where the results revealed that both mucilages are qualitatively and quantitatively different, specially the absence of galactose and rhamnose in Haworthia limifolia Marloth and Gasteria minima Poelln. and the absence of glucose in Gasteria minima Poelln. and the absence of arabinose in Haworthia limifolia Marloth and Gasteria carinata (Mill) Duval. On the other hand, they differ greatly quantitatively in the ratios of the different sugars. Concerning the isolation and identification of different compounds from Haworthia limifolia Marloth, six compounds were isolated from the hexane and ethyl acetate fractions. The new dihydroanthracenone, 4,6,9-trihydroxy,2,8-dimethyl-3,4-dihydroanthracene-1(2H)-one, the new anthraquinone-C-glycosides, 5-Hydroxyaloin A-2′-O-sinapoyl-6′- O-acetate, 5- Hydroxyaloin A 6`- O-sinapoyl were isolated. β-sitosterol, Diheptyl phthalate and 5- Hydroxyaloin A 6`- O-acetate were isolated for the first time from Haworthia limifolia Marloth. The methanolic extract of the aerial parts of Haworthia limifolia Marloth and Gasteria carinata (Mill) Duval displayed In vitro radical scavenging activites compared to the reference antioxidant ascorbic acid, showing relatively low IC50 values, where the activity of Haworthia limifolia Marloth (IC50 = 143.6 µg/ml) supperpassed that of Gasteria carinata

(Mill) Duval (IC50 = 249.1 µg/ml). While Gasteria minima Poelln. showed weak antioxidant activity IC50 ≥ 3000 µg/ml under these experimental conditions. The COX-2 inhibitory activities of Haworthia limifolia Marloth was very potent with a very close IC50 (19.15µg/ml) to that of ibuprofen (18.5 µg/ml), followed by G. minima Poelln. (113.7 µg/ml) then G. carinata (Mill) Duval (122.9 µg/ml). The antimicrobial screening showed that the methanolic extracts of the aerial parts of Haworthia limifolia Marloth, Gasteria carinata (Mill) Duval and Gasteria minima Poelln. have broad spectrum activity against Gram-positive and Gram-negative bacteria. In addition, they have also antifungal effect except against candida albicans. Gasteria carinata (Mill) Duval extract was the most potent followed by Haworthia limifolia Marloth extract followed by Gasteria minima Poelln. extract Gasteria carinata (Mill) Duval & Haworthia limifolia Marloth extracts have a very potent antibacterial effect against Gram-negative bacteria specially against K. pneumoniae, E. cloacae, E. coli and against the tested Gram-Positive bacteria specially B. subtilus, E. faecalis and S. aureus. Considering their antifungal effect, they showed a very potent activity against Aspergillus fumigatus, Syncephalastrum racemosum and Geotricum candidum. While they showed no activity against Candida albicans. Gasteria minima Poelln. extract showed less potent antibacterial and antifungal activities. It showed a very high effect against the tested Gram-negative bacteria which are K.pneumoniae, E. cloacae and E. coli compared to Gentamycin and against the tested Gram positive bacteria (B. subtilis, E. faecalis and S. aureus) compared to Ampicillin. While it showed activity against the tested mould and yeast which are Aspergillus fumigatus, Syncephalastrum racemosum and Geotricum candidum compared to Amphotericin B. The methanolic extract of their aerial parts displayed In vitro cytotoxic activity against three tested cell lines which are: Human hepatocellular carcinoma (HepG2), Breast carcinoma (MCF-7) and colon carcinoma (HCT-116). Where Gasteria carinata (Mill) Duval showed the most potent activity against (HepG2), (MCF-7) and (HCT-116) cell lines with IC50 (40.2, 46.6 and 39.3 µg/well respectively) followed by Gasteria minima Poelln. with IC50 (45.9, 79.3 and 44.2 µg/well respectively) while Haworthia limifolia Marloth showed the least potent inhibitory activity with IC50 (72, >100 and >100 µg/well respectively). Acute toxicity study of their methanolic extracts (5.0 g/kg, p.o.) produced no sign of acute toxicity or death in the treated animals. No significant changes in food and water intake or body weight were observed during the fourteen days of observation. Therefore, the LD50 could not be estimated and it is possibly higher than 5.0 g/kg. The acute toxicity study of their fresh aerial parts did not show any symptoms of toxicity, changes in behavior or mortality. Also the body weights, liver and kidney weights showed no significant difference from the normal group. Furthermore, the liver and kidney function tests were in the normal range. Therefore, it can be concluded that the fresh aerial parts of of H.limifolia Marloth, G. carinata (Mill) Duval and G. minima Poelln. are non-toxic. Assessment of antihepatotoxic activity demonstrated the safety of the adminstration of the methanolic extracts as all the tested biochemical parameters,as well as the histopathological results showed non-significant change from the normal group. The present study also revealed that, the treatment of the CCl4-intoxicated rats with the methanolic extracts of the aerial parts of the plants under investigation caused significant serum levels of ALT, AST,

albumin and total protein amelioration. Treatment with the extract of the aerial parts of H. limifolia Marloth showed the best improvement in the liver function and antioxidant parameters followed by the extracts of G. carinata (Mill) Duval , followed by that of G. minima Poelln. and this was validated by the histopathological results. The results of the docking studies of different identified compounds in H. limifolia Marloth, G. carinata (Mill) Duval and G. minima Poelln. on COX-2 enzyme revealed that the affinity of the compounds identified in H. limifolia Marloth, Gasteria carinata (Mill.) Duval and Gasteria minima poelln. to the COX-2 receptor may explain their anti-inflammatory activity specially H. limifolia Marloth which is the most potent one due to the presence of 5- Hydroxyaloin A 6`- O-sinapoyl, 5-Hydroxyaloin A-2′-O-sinapoyl-6′-O-acetate, 5- hydroxyaloin and 5-Hydroxyaloin A 6`- O-acetate which have the highest affinity to COX-2 enzyme. Introduction Plants have provided man with all his needs in terms of shelter, clothing, food, flavours, fragrances and medicines. Plants have formed the basis of sophisticated traditional medicine systems that have been in existence for thousands of years and continue to provide mankind with new remedies. The past decade has witnessed a tremendous resurgence in the interest and use of medicinal plant products (Briskin, 2000). A high percentage of the world's population still relies upon plants for primary health care; even today in Western medicine, and despite progress in synthetic chemistry, about 25% of prescription medicines are still derived either directly or indirectly from plants. Nearly 50,000 species of higher plants have been used for medicinal purposes (Barboza et al., 2009). Current research in drug discovery from medicinal plants involves a complex approach combining botanical, phytochemical, biological, and molecular techniques (Barboza et al., 2009). Haworthia and Gasteria (Haw.) (Duval, 1809) are small succulent plants native to South Africa and they are usually grown for ornamental purposes. Haworthia limifolia Marloth has been extensively used by traditional healers for the treatment of a wide variety of ailments especially gastro-intestinal ailments (Fawole et al., 2009). H. limifolia Marloth is often used by traditional healers as a spiritual remedy and as a treatment as blood purifiers and cures against coughs, skin rashes, sun burns, burns, treatment of sores and to promote pregnancy in women (Coopoosamy and Naidoo, 2011) (Coopoosamy and Naidoo, 2012). The antifungal and antibacterial activities were tested by (Coopoosamy and Naidoo, 2011), while the anti-tumor, anti-inflammatory and wound healing activities were assessed by (Coopoosamy and Naidoo, 2012). The use of Gasteria bicolor in traditional medicine as treatment of secondary fungal infections in HIV/AIDS patients and its antioxidant properties was validated (Otang et al., 2012). Three new dihydroanthracenones, (gasteriacenone A), (gasteriacenone B) and (gasteriacenone C), were isolated from the leaves of Gasteria bicolor (Dagne et al., 1996), while two new isocoumarin glucosides, haworforbins A and B, and a new chromone, haworforbin C were isolated from Haworthia cymbiformis (Yamasaki et al., 2013). Few studies have been made on both genera and no phytochemical studies were made on the two-selected species of Gasteria although they have promising pharmacological effects. And being closely related to Aloes, which provide a fascinating subject for research from a taxonomic, horticultural, economic, chemical, biochemical and pharmaceutical, point of view, Haworthia and Gasteria deserve a great attention and more studies should be done concerning mainly their phytochemical constituents and pharmacological effects.

Aim of work So, our aim of work is to carry out a phytochemical as well as pharmacological investigation on Haworthia limifolia Marloth, Gasteria carinata (Mill) Duval and Gasteria minima Poelln. This work comprises: 1- Genetic Profiling of H. limifolia Marloth, G. carinata (Mill) Duval and G. minima Poelln. 2- Botanical study of the non-flowering aerial parts of H. limifolia Marloth, G. carinata (Mill) Duval and G. minima Poelln. 3- Phytochemical study of non-flowering the aerial parts of H. limifolia Marloth, G. carinata (Mill) Duval and G. minima Poelln. including: A- Preliminary phytochemical screening B- UPLC-MS/MS profiling C- Qualitative and quantitative determination of the phenolic content D- Investigation of the lipid content E- Analysis of mucilage content using HPLC F-Isolation and identification of different compounds in Haworthia limifolia Marloth 4- Biological study of the non-flowering aerial parts of H. limifolia Marloth, G. carinata (Mill) Duval and G. minima Poelln., which included: A- Determination of in vitro antioxidant activity using DPPH assay. B- Determination of in vitro anti-inflammatory activity using Cox-2 inhibitor screening assay C- Antimicrobial screening D- Determination of cytotoxic activity E- Acute toxicity study F- Assessment of antihepatotoxic activity Review of literature Genetic study Treutlein et al. (2003) made molecular studies to explore phylogenetic relationships in the family Asphodelaceae. Genomic fingerprinting by ISSR (Inter Simple Sequence Repeats) analysis was compared to sequence data of the chloroplast genes matK and rbcL. Molecular data indicate that some long-established taxonomic concepts would have to be re-evaluated. Several Alooideae genera, including Aloe and Haworthia, are apparently not monophyletic. From a molecular point of view, Haworthia can be divided into two distinct groups: a monophyletic group including species of subgenus Haworthia, and a second polyphyletic group with the subgenera Hexangulares and Robustipedunculares. Ramdhani et al. (2011) showed that the issue of a polyphyletic Haworthia is more complicated than previously reported, using considerably expanded datasets of both chloroplast (trnL-trnF and psbA-trnH intergenic spacers) and nuclear (ITS1) markers

Botanical study Beaumont et al. (1985) The presence of thin-walled parenchymatous cells in the inner bundle sheath of species of the liliaceous genera Aloe, Chamaealoe, Astroloba, Lomatophyllum, Gasteria, Haworthia, Asphodeline, Asphodelus, Bulbine, Eremurus and Trachyandra was observed and described. Some species, however, have lignified sclerenchymatous cells in this position and this is general in Kniphofia. Cells in this region produce a secretion in the form of exudate in many species of Aloe, or much scattered contents in the case of related genera. The leaves of most species contain small amounts of anthraquinones, while the related anthrone-C-glycosides accumulate in others. It is not certain if synthesis occurs in the thin-walled bundle sheath cells or if these have only a storage function.

Otang et al. (2014) studied the epidermal cells of Gasteria bicolor and found that they are either hexagonal or pentagonal in form and are compactly arranged. The epidermal cell width was approximately 50 µm. Stomata apertures are elliptical, and the upper epidermis of the leaf has paracytic stomata which are slightly raised above the epidermal surface with 4 to 5 subsidiary cells surrounding each stoma. Based on the microanalysis, the mineral crystals present at the level of the mesophyll of G. bicolor were probably mixtures of calcium oxalate, calcium sulphate and silica. Nuzhyna and Gaydarzhy (2015) investigated the comparative anatomical and morphological characteristics of plants of two subgenera: Haworthia and Hexangularis. The study revealed two different strategies of adaptation to arid conditions of the growth of different subgenera of the genus Haworthia. Plants of the subgenus Haworthia adapted to arid conditions by increasing the accumulation of water, the presence of “windows”, a smaller stoma size, and a thinner outer wall of the epidermis cells. On the other hand, plants of the subgenus Hexangularis adapted to arid conditions by reducing overheating and transpiration as well as by the presence of papillae and a thickened outer wall of the epidermis cells. Active constituents I-Reviewing the current literature, a few phytochemical studies was found about Haworthia and Gasteria genera which is given as follows: Dagne et al. (1996a) isolated three new dihydroanthracenones from the aerial parts of Gasteria bicolor namely 3,4-dihydro-2,6,9-trihydroxy-8-methyl-1(2H)-anthracenone (t A), 3,4-dihydro-2,4,9-trihydroxy-6-methoxy-8-methyl-1(2H)-anthracenone (gasteriacenone B) and 3,4-dihydro-4,6,9-trihydroxy-7-carbomethoxy-8-methyl-1(2H)-anthracenone (gasteriacenone C). Their structures were elucidated by spectroscopic methods including 2D NMR techniques. Otang et al. (2012b) investigated the chemical contents of the extracts of Gasteria bicolor and Pittosporum viridiflorum and found that there was no significant differences in the flavonoid and proanthocyanidins contents between the leaves and bark extracts of Gasteria bicolor and Pittosporum viridiflorum respectively, while the total phenolic content of the bark extract of P. viridiflorum was significantly higher than that of G. bicolor leaf. Coopoosamy and Naidoo (2012) investigated the presence of lectins or lectin like derivatives in H. limifolia Marloth. Yamasaki et al. (2013) isolated two new isocoumarin glucosides, haworforbins A and B, and a new chromone, haworforbin C, from Haworthia cymbiformis. Their structures were determined on the basis of NMR. Folk medicine I- Haworthia and Gasteria genera Coopoosamy and Naidoo (2012) Harworthia limifolia Marloth is currently used by indigenous people for sun burns, burns, sores as well as a systemic remedy and spiritual benefits. Otang et al. (2012b) G. bicolor is traditionally used in South Africa for the management of opportunistic fungal infections in HIV patients. Biological activities I- Haworthia and Gasteria genera Antimicrobial Coopoosamy and Naidoo (2011) validated the use of H. limifolia Marloth crude extracts from the leaves was successful against five Gram-positive and four Gram-negative

bacteria. Extracts from the leaves also exhibited antimicrobial activities in all treatments against the selected bacterial and fungal strains. Otang et al. (2012a) investigated the antifungal activity of A. arctotoides and G. bicolor against opportunistic fungi common in HIV/AIDS patients using agar diffusion and micro-dilution methods. All the hexane and acetone extracts were active against at least one of the fungi, while none of the aqueous extracts was active against any of the fungi. The inhibitory activity of the active extracts, based on the overall mean inhibition diameters, was in the order: A. arctotoides (hexane) > A. arctotoides (acetone) > G. bicolor (hexane) > G. bicolor (acetone). This study validates the use of these plants in traditional medicine in the treatment of secondary fungal infections in HIV/AIDS patients. Antioxidant Otang et al. (2012b) indicated that the aerial parts extracts of Gasteria bicolor and Pittosporum viridiflorum possess antioxidant properties and could serve as free radical inhibitors, acting possibly as primary antioxidants. Since reactive oxygen species are thought to be associated with the pathogenesis of AIDS, and HIV-infected individuals often have impaired antioxidant defenses, the inhibitory effect of the extracts on free radicals may partially justify the traditional use of these plants in the management of OFIs in HIV patients in South Africa. Anti-tumor and wound healing Coopoosamy and Naidoo (2012) investigated the presence of lectins or lectin like derivatives in H. limifolia Marloth. The derivatives were tested against rat, rabbit and human serum, and a positive reaction with human alpha -2-macroglobulin was observed. Furthermore, anti-tumor and wound healing properties have been validated. General Summary

Haworthia and Gasteria genera are small succulent plants native to South Africa which are usually grown for ornamental purposes. Haworthia genus comprises approximately 61 species, while Gasteria genus includes 23 species. Their classification has changed a lot throughout the different taxonomic classification systems and they belong to family Asphodelaceae according to APG IV classification system. Haworthia limifolia Marloth is often used by traditional healers as blood purifiers, it also used to cure cough, skin rashes, sun burns, burns, treatment of sores and to promote pregnancy in women and for the treatment of gastro-intestinal ailment. While Gasteria is always used in cases of fungal infections specially in the treatment of secondary fungal infections in HIV/AIDS patients. Little studies have been made on both genera and no phytochemical studies were made on the two-selected species of Gasteria although they have promising pharmacological effects. Being closely related to Aloes, which provide a fascinating subject for research from a taxonomic, horticultural, economic, chemical, biochemical and pharmaceutical, point of view, Haworthia and Gasteria genera deserve great attention and more studies should be done concerning mainly their phytochemical constituents and pharmacological effects. Our objective was to set identifying parameters to authenticate and differentiate between H. limifolia Marloth, G. carinata (Mill) Duval (Mill.) Duval and G. minima Poelln. using the DNA fingerprinting as well as macro and micromorphological characters and to investigate their phytochemical constituents and pharmacological activities. This study includes five main parts: 1- Genetic Profiling of Haworthia limifolia Marloth, Gasteria carinata (Mill) Duval and Gasteria minima Poelln. .

2- Botanical study of the aerial parts of Haworthia limifolia Marloth, Gasteria carinata (Mill) Duval and Gasteria minima Poelln. . 3-Phytochemical Investigation of the aerial parts of Haworthia limifolia Marloth, Gasteria carinata (Mill) Duval and Gasteria minima Poelln. . 4-Biological study of the aerial parts of Haworthia limifolia Marloth, Gasteria carinata (Mill) Duval and Gasteria minima Poelln. 5-Docking study of the main identified compounds in the three species under investigation on COX-2 enzyme

Part I: Genetic Profiling of Haworthia limifolia Marloth, Gasteria carinata (Mill) Duval and Gasteria minima Poelln. . DNA fingerprinting of H. limifolia Marloth, G. carinata (Mill) Duval and G. minima Poelln. RAPD technique was employed for the current study, in an attempt to address the following objectives: (1) To estimate the genetic polymorphism among Haworthia limifolia Marloth, Gasteria carinata (Mill) Duval (Mill.) Duval and Gasteria minima Poelln. , (2) To assess the genetic relationships between them and (3) To establish a typical fingerprint for them. The results implicate that the ten decamer oligonucleotide primers used had induced successive amplifications with a wide range of molecular sizes. The analysis of the amplified fragments generated by RAPD reactions revealed that the primers OPB-01, OPB-04, OPB-07 and OPB-08 can be used for the identification of the three examined species since they generated fragments with wide molecular size. The RAPD primers used revealed a total of 38.8 % polymorphism among the examined plants. The primer OPB-07 recorded the highest percentage of polymorphism (78.6%), followed by OPB-01 with 70.6% polymorphism. So, they can be used to discriminate between the examined species. It can be concluded from the UPGMA analysis of genetic diversity based on RAPD analysis that G. carinata (Mill) Duval and G. minima Poelln. are closely related using all the stated primers except OPB-02 which shows that G.minima Poelln. and H. limifolia Marloth are more closely related, while primer OPB-010 shows that G. carinata (Mill) Duval and H. limifolia Marloth are more closely related. All the used primers can differentiate between H. limifolia Marloth and the two Gasteria species except OPB-02 which can discriminate G. carinata (Mill) Duval from G. minima Poelln. and H. limifolia Marloth, while primer OPB-010 can distinguish G.minima Poelln. from G. carinata (Mill) Duval and H. limifolia Marloth.

Part II: Botanical study of the aerial parts of H. limifolia Marloth, G. carinata (Mill) Duval and G. minima Poelln.

Chapter I: Macromorphological study H. limifolia Marloth, G. carinata (Mill) Duval (Mill.) Duval and G. minima Poelln. are green fleshy plant. They flourish in Egypt. Flowers early starts in February till June, at the end of this period they develop into fruits The leaf: H. limifolia Marloth leaves are densely rosette usually from 15-20 leaves in number. While G. carinata (Mill) Duval and G. minima Poelln. leaves are distichous usually from 6-14 leaves in number. The leaf is simple, exstipulate, sessile and succulent. They are bright green in colour and they turn into red when exposed to sun or to arid conditions. H. limifolia Marloth leaf surface has very attractive sculptures, while G. carinata (Mill) Duval leaf has oval white patches on the upper and lower sides, whereas, G. minima Poelln. leaf has white, circular patches on both the upper and lower surfaces.

The stem in the three plants is short, fleshy and thick. It is cylindrical, solid and unbranched. The stem of H. limifolia Marloth and G. minima Poelln. are pinkish white in colour, while that of G. carinata (Mill) Duval has a white colour. The flower of the three investigated species is raceme type inflorescence, with a simple, cylindrical peduncle which is straight in H. limifolia Marloth and inclined in G. carinata (Mill) Duval and G. minima Poelln. , bearing 15-25 flowers. The flowers of H. limifolia Marloth are small tubular, curved with 2/3 of petals fused, its perianth is whitish, more or less bilabiate. The flowers of G. carinata (Mill) Duval and G. minima Poelln. are gasteriform, curved and flask-shaped, they are reddish in colour with greenish tips and are borne on an inclined raceme, their perianth is tubular, with an inflated basal portion and a cylindric end portion. Being an Alooideae member, the tepals are fused. The stamens are included in the perianth tube with inserted anthers. The style is straight and also included. The fruit in the three species are dehiscent capsules with oblong seeds.

Chapter II: Micromorphological study The leaf The transverse sections through H. limifolia Marloth, G. carinata (Mill) Duval (Mill.) Duval and G. minima Poelln. resemble a crescent or boat-like (cymbiform), like most species of Alooideae. This outline is an obvious adaptation to aridity. They consist of epidermis, chlorenchyma, vascular bundle and central aquiferous tissue. The epidermis consist of one layer of papillosed polygonal cells with straight anticlinal walls and covered with thick cuticle. Anomocytic stomata are present on the upper and lower sides and are more abundant on the lower one. Chlorenchyma isn’t differentiated into spongy and palisade tissues. It consists of more or less isodiametric and sometimes elongated cells with thin walls. It contains scattered idioblasts with calcium oxalate crystals. The leaf centre is occupied with aquiferous tissue that consists of large parenchymatous cells, filled with water and mucilage. It occupies about 70, 40 and 40 % of the volume of the leaves in cross sections measured in the middle part of the leaves of H. limifolia Marloth, G. carinata (Mill) Duval, and G. minima Poelln. , respectively. Vascular bundles are arranged in a ring which is located at the boundary between the chlorenchyma and the aquiferous tissues. Each vascular bundle is surrounded by one layer of thin parenchymatous sheath and a cap of parenchymatous cells. It is formed of circular collateral vascular strand with phloem directed outwards. 2-The stem The transverse sections in the stem of H. limifolia Marloth, G. carinata (Mill) Duval , and G. minima Poelln. are circular in outline, where the layers of a monocot stem appear. Epidermis is the first layer which is replaced by cork in the older stages, followed by wide cortex containing idioblasts with crystals of calcium oxalate, after that a layer of cambium appears followed by numerous scattered closed vascular bundles, which are more crowded near the periphery than in the centre. The central part is parenchymatous.

Part III: Phytochemical Investigation of the aerial parts of H. limifolia Marloth, G. carinata (Mill) Duval and G. minima Poelln.

Chapter I: Preliminary phytochemical screening The obtained results of preliminary phytochemical screening of the aerial parts of Haworthia limifolia Marloth, Gasteria carinata (Mill) Duval and Gasteria minima Poelln. revealed the presence of carbohydrates and/or glycosides, condensed tannins, free and combined flavonoids, sterols and/or triterpenes, and free and combined anthraquinones and the absence of essential oil, hydrolysable tannins, saponins, alkaloids and/or nitrogenous bases,

cardiac glycosides and oxidase enzyme in the aerial parts of the three plants under investigation.

Chapter II: UPLC-MS/MS profiling The present study attempts to analyze the secondary metabolites of the methanolic extracts of the aerial parts of the three plants under investigations using UPLC-PDA-MS/MS in negative ion mode. Several compounds belonging to different classes as phenolic acids, flavonoids, anthraquinones, chromones and coumarins were identified in the investigated species. Many compounds were identified in the three-examined species as vanillic acid hexoside, naringin, 5-hydroxyaloin, aloesaponarin I and 7-methylether of 2`feruloylaloesin. Others could be considered as chemotaxonomic markers for the investigated species as they were identified in only one species of them like, aloeresin F, acetyl aloin and orientin.

Chapter III: Qualitative and quantitative determination of the phenolic content (A): Quantitative determination of total phenolic and tannin content The determination of total phenolic contents of Haworthia limifolia Marloth, Gasteria minima Poelln. and Gasteria carinata (Mill) Duval revealed that the concentration of phenolic compounds was the highest in Gasteria minima Poelln. (29.27 mg/ml) then in Gasteria carinata (Mill) Duval (27.4 mg/ml) and it was lowest in Haworthia limifolia Marloth (22.05 mg/ml). While the concentration of tannins was the highest in Haworthia limifolia Marloth (8.04%) then in Gasteria minima Poelln. (2.58%) and it was lowest in Gasteria carinata (Mill) Duval (1.02%). (B): Qualitative and quantitative determination of the phenolic and flavonoid content using HPLC analysis The HPLC investigation of Haworthia limifolia Marloth, Gasteria minima Poelln. and Gasteria carinata (Mill) Duval using external phenolic and flavonoid standards revealed that the percentage of total identified flavonoids in aerial parts of Gasteria carinata (Mill) Duval (12.02%) surpassed its percentage in Haworthia limifolia Marloth (11.3%) and it was the least in Gasteria minima Poelln. (8.71%). Catechin, luteolin, naringin, rutin, hesperidin, quercitrin, quercetin, hesperetin, kaempferol and apigenin were identified in them. Quercitrin represented the major identified flavonoid in Haworthia limifolia Marloth (8876.15 ppm), while hesperetin is the major identified one in Gasteria carinata (Mill) Duval (2270.42 ppm) and catechin (1382.05 ppm) is the major identified one in Gasteria minima Poelln. . On the other hand, the percentage of total identified phenolic compounds was higher in aerial parts of Gasteria carinata (Mill) Duval (22.3%) followed by Gasteria minima Poelln. (20%) and it was the least in Haworthia limifolia Marloth (16.6%). Salicylic acid and vanillic acid are the major identified phenolic acids followed by catechol, pyrogallol, 3-OH Tyrosol, rosmarinic and chlorogenic acid. Salicylic acid is the highest identified phenolic acids in Haworthia limifolia Marloth (56666.76 ppm), while vanillic acid is the highest one in Gasteria carinata (Mill) Duval and Gasteria minima Poelln. (20009.76 and 11395.41ppm respectively). The major identified phenolic compounds in Haworthia limifolia Marloth are salicylic acid, vanillic acid, α- coumaric, and rosmarinic acids (56666.76, 17308.363, 1616.29, and 982.23 ppm respectively). While the major identified phenolic compounds in Gasteria carinata (Mill) Duval are vanillic acid, salicylic acid and rosmarinic acid (20009.76, 2427.69 and 1273.76, respectively). The major identified phenolic compounds in Gasteria minima Poelln. are vanillic acid, salicylic acid, catechol and 3-OH Tyrosol (11395.41, 3020.74, 1700.94 and 1279.34 ppm, respectively).

Chapter IV: Investigation of the lipid content The results of the GC/MS analysis of the unsaponifiable matter of aerial parts of H. limifolia, G. carinata and G. minima revealed the presence of seventy-five compounds representing 99.82, 98.93 and 99.84% of the unsaponifiable matter in H. limifolia, G. carinata and G. minima, respectively. The major identified compounds in H. limifolia were the hydrocarbons benzene-1- butylheptyl (16.71%), benzene-1-pentylheptyl (9.55%) and benzene-1-butyloctyl (8.75%), while the major compounds identified in G. carinata were sitosterol (13.98%), phytol (13.58 %) and benzene 1-butylheptyl (10.9%). The major compounds in G. minima were sitosterol (16.89%), phytol (13.06 %) and 9,19-cyclolanost-24-en-3-ol, acetate (6.82%). The results of GC/MS analysis of the saponifiable matter of H. limifolia Marloth, G. carinata (Mill) Duval and G. minima Poelln. aerial parts revealed the presence of 28 compounds representing 100% identification for the three plants under investigation. Eight compounds were identified as methyl esters of unsaturated fatty acids (49.66, 45.24 and 64.27%) in H. limifolia Marloth, G. carinata (Mill) Duval and G. minima Poelln. respectively and the other identified twenty compounds are methyl esters of saturated fatty acids (50.34, 54.76 and 35.73%) in H. limifolia Marloth, G. carinata (Mill) Duval and G. minima Poelln. respectively. Methyl palmitate was the major identified fatty acid methyl ester in H. limifolia Marloth and G. carinata (Mill) Duval (39.02 and 44.2% respectively), followed by oleic acid methyl ester (27.14 and 23.94% respectively) then linoleic acid methyl ester (21.36 and 21.03%). While the major one in G. minima Poelln. was linoleic acid methyl ester (45.81%), followed by methyl palmitate (28.81%) then linolenic acid methyl ester (17.9%). Methyl stearate represented 3.87, 4.09 and 2.69% of the saponifiable matter in H. limifolia Marloth, G. carinata (Mill) Duval and G. minima Poelln. . The present study including the investigation of the unsaponifiable and saponifiable matters of H. limifolia Marloth, G. carinata (Mill) Duval and G. minima Poelln. aerial parts by GC/MS, could be helpful in their authentication. This is the first record for the GC/MS analysis of any of them.

Chapter V: Analysis of mucilage content using HPLC The results revealed that the investigated species were rich in polysaccharides; Gasteria minima Poelln. had the highest polysaccharide content, followed by Haworthia limifolia Marloth and Gasteria carinata (Mill) Duval 1.01%, 0.541% and 0.44%, respectively. The monosaccharides glucose, galactose, rhamnose and arabinose were detected. Glucose was detected in Haworthia limifolia Marloth and Gasteria carinata (Mill) Duval at a concentration of 0.02 and 0.04 g%. Galactose and rhamnose, having the same retention time, were detected either together or one of them only in Gasteria carinata (Mill) Duval (0.005 g%). While arabinose was detected only in Gasteria minima Poelln. (0.03 g%). The disaccharide, sucrose, was detected in Gasteria carinata (Mill) Duval, Gasteria minima Poelln. and Haworthia limifolia Marloth (0.07, 0.067 and 0.01g %, respectively). The oligosaccharide, stachyose, was the highest detected sugar in the three-investigated species (3.59, 2.76 and 0.6 g % in Gasteria carinata (Mill) Duval, Haworthia limifolia Marloth and Gasteria minima Poelln., respectively). Concerning the sugar acids, galacturonic acid was detected in the three- investigated species in higher concentration than glucuronic acid which was still present in high concentration. Galacturonic acid was detected in Gasteria carinata (Mill) Duval, Haworthia limifolia Marloth and Gasteria minima Poelln. at concentration of 1.6, 1.37 and 0.47 g %, respectively. While glucuronic acid concentration determined was 1.2, 0.91 and 0.35 g % in Gasteria carinata (Mill) Duval, Haworthia limifolia Marloth and Gasteria minima Poelln.

The results of the present study were compared with another study carried by (El Sayed et al, 2016) of the HPLC of the mucilage content of eight Aloe species which are Aloe arborescens Mill., Aloe eru, Aloe grandidentata Salm-Dyck, Aloe perfoliata L., Aloe brevifolia Mill., Aloe saponaria L. and Aloe ferox Mill. HPLC analysis of the saccharides of the Aloe, Haworthia and Gasteria species revealed that their composition is more or less similar qualitatively using the prementioned reference sugars, except for the absence of galactose and rhamnose in Haworthia limifolia Marlothand Gasteria minima Poelln. and the absence of glucose in Gasteria minima Poelln. and the absence of arabinose in Haworthia limifolia Marloth and Gasteria carinata (Mill) Duval. On the other hand, they differ greatly quantitatively in the ratios of the different sugars. Quantitative HPLC analysis revealed that glucuronic acid was the dominant sugar in all Aloe species investigated ranging from 0.05 to 2 g% except Aloe perfoliate which resembles Gasteria carinata (Mill) Duval, Haworthia limifolia Marloth and Gasteria minima Poelln. in their major sugar content as galacturonic acid as the major sugar (0.3, 1.6, 1.37 and 0.47g %, respectively). other quantitatively important saccharides were stachyose, sucrose, rhamnose, galactose and glucose.

Chapter VI: Isolation and identification of different compounds in Haworthia limifolia Marloth Hexane extract (6 g) was fractionated on a silica gel column using gradient elution of different solvents. The obtained fractions were subjected to re-chromatography on sephadex LH-20 and silica gel (using several solvent systems) for further purification. Fractionation and purification of the hexane extract of Haworthia limifolia Marloth afforded the isolation of two compounds: Compound I: β-sitosterol (white crystalline needles, 35 mg). Compound II: 4,6,9-trihydroxy,2,8-dimethyl-3,4-dihydroanthracene-1(2H)-one (orange needle crystals, 11 mg) (new compound). Ethyl acetate fraction (10.6 g) was subjected to VLC silica column using gradient elution of different solvents. The obtained fractions were separately subjected to re- chromatography on sephadex LH-20 and silica gel (using several solvent systems) for further purification. Fractionation and purification of the ethyl acetate extract of Haworthia limifolia Marloth afforded the isolation of four compounds: compound III: Diheptyl phthalate (yellowish viscous liquid, 10 mg). compound IV: 5-Hydroxyaloin A-2′-O-sinapoyl-6′- O-acetate (brown amorphous powder, 35 mg) (new compound). compound (V): 5-Hydroxyaloin A 6`- O-sinapoyl (yellowish brown amorphous solid, 20 mg) (new compound). compound (VI): 5-Hydroxyaloin A 6`- O-acetate (yellowish brown amorphous solid, 25 mg).

Part IV: Biological study of the aerial parts of H. limifolia Marloth, G. carinata (Mill) Duval and G. minima Poelln.

Chapter I: In vitro pharmacological screening (A) Determination of In vitro antioxidant activity using DPPH assay The methanolic extract of the aerial parts of Haworthia limifolia Marloth and Gasteria carinata (Mill) Duval displayed In vitro radical scavenging activities compared to the reference antioxidant ascorbic acid, showing relatively low IC50 values, where the activity of Haworthia limifolia Marloth (IC50 = 143.6 µg/ml) supper passed that of Gasteria carinata (Mill) Duval (IC50 = 249.1 µg/ml). While Gasteria minima Poelln. showed weak antioxidant activity IC50 ≥ 3000 µg/ml under these experimental conditions

(B) Determination of In vitro anti-inflammatory activity using COX-2 inhibitory assay The COX-2 inhibitory activities of Haworthia limifolia Marloth is very potent with IC50 (19.15µg/ml) very close to that of ibuprofen (18.5 µg/ml), followed by G. minima Poelln. (113.7 µg/ml) then G. carinata (Mill) Duval (122.9 µg/ml). (C) Antimicrobial screening The methanolic extract of the aerial parts of Haworthia limifolia Marloth, Gasteria carinata (Mill) Duval and Gasteria minima Poelln. have broad spectrum activity against Gram-positive and Gram-negative bacteria. In addition, they have also antifungal effect except against candida albicans. Gasteria carinata (Mill) Duval extract is the most potent followed by Haworthia limifolia Marloth extract followed by Gasteria minima Poelln. extract. Gasteria carinata (Mill) Duval & Haworthia limifolia Marloth extracts have a very potent antibacterial effect against Gram-negative bacteria specially against K. pneumoniae, where their effect exceeds that of Gentamycin (105.9% and 100.5%, respectively). Also, their effect was very close to that of Gentamycin against E. cloacae (96.2% and 94.1%) and they exerted a potent effect against E. coli (75.8% and 72.5%, respectively). Their activity is very close to that of Ampicillin against the tested Gram-Positive bacteria specially B. subtilus (91.7% and 90.2%, respectively), E. faecalis (90.1% and 87.8% respectively) and to a less extent S. aureus (77.2% and 74.1%, respectively). Considering their antifungal effect, they showed a very potent activity against Aspergillus fumigatus (89.5% and 86.9% respectively), Syncephalastrum racemosum (80.6% and 76.6%, respectively) and Geotricum candidum (71.5% and 63.7% respectively). While they showed no activity against Candida albicans. Gasteria minima Poelln. extract showed less potent antibacterial and antifungal activities, yet it is still considered very strong. It showed a very high effect against the tested Gram-negative bacteria which are K.pneumoniae, E. cloacae and E. coli (78.2, 68.1 and 66.7% potency respectively) compared to Gentamycin and aginst the tested Gram positive bacteria (B. subtilis, E. faecalis and S. aureus) with potency % (69.3, 68.8 and 56.1% respectively) compared to Ampicillin. While it showed activity against the tested mould and yeast which are Aspergillus fumigatus, Syncephalastrum racemosum and Geotricum candidum (76.8, 68 and 55.5% respectively) compared to Amphotericin B. (D) Determination of cytotoxic activity The methanolic extract of the aerial parts of Gasteria carinata (Mill) Duval and Gasteria minima Poelln. and Haworthia limifolia Marloth displayed In vitro cytotoxic activity against three tested cell lines which are: Human hepatocellular carcinoma (HepG2), Breast carcinoma (MCF-7) and colon carcinoma (HCT-116). Where Gasteria carinata (Mill) Duval showed the most potent activity against (HepG2), (MCF-7) and (HCT-116) cell lines with IC50 (40.2, 46.6 and 39.3 µg/well respectively) followed by Gasteria minima Poelln. with IC50 (45.9, 79.3 and 44.2 µg/well respectively) while Haworthia limifolia Marloth showed the least potent inhibitory activity with IC50 (72, >100 and >100 µg/well respectively). The methanolic extract of the aerial parts of Gasteria carinata (Mill) Duval displayed the most potent inhibitory activity against (HCT-116) with IC50 39.3 µg/well, followed by (HepG2) with IC5040.2 µg/well then (MCF-7) with IC5046.6 µg/well. While the methanolic extract of the aerial parts of Gasteria minima Poelln. displayed the most potent inhibitory activity against (HCT-116) with IC50 44.2 µg/well, followed by (HepG2) with IC5045.9 µg/well then (MCF-7) with IC5079.3 µg/well. On the other hand, the methanolic extract of the aerial parts of Haworthia limifolia Marloth showed an inhibitory activity against (HepG2) with IC50 72 µg/well while it exhibited a weak inhibitory activity against both (MCF-7) and (HCT-116) cell lines with IC50 >100 µg/well.

Chapter II: In vivo pharmacological screening (A) Acute toxicity study Acute toxicity of the methanolic extracts It was observed that the methanolic extracts of H.limifolia Marloth, G. carinata (Mill) Duval and G. minima Poelln. (5.0 g/kg, p.o.) produced no sign of acute toxicity or death in the treated animals. No significant changes in food and water intake or body weight were observed during the fourteen days of observation. Therefore, the LD50 could not be estimated and it is possibly higher than 5.0 g/kg. Acute toxicity of the fresh aerial parts of H.limifolia Marloth, G. carinata (Mill) Duval and G. minima Poelln. The acute toxicity study did not show any symptoms of toxicity, changes in behavior or mortality when adminstering the fresh aerial parts of H. limifolia Marloth, G. carinata (Mill) Duval and G. minima Poelln. as 10% w/w of the total daily diet for 30 days. Also the body weights, liver and kidney weights showed no significant difference from the normal group. Furthermore, the liver and kidney function tests were in the normal range. Therefore, it can be concluded that the fresh aerial parts of of H.limifolia Marloth, G. carinata (Mill) Duval and G. minima Poelln. are non-toxic. (B) Assessment of antihepatotoxic activity The present study demonstrates the safety of the adminstration of the methanolic extracts as all the tested biochemical parameters show non significant change from the normal group. Histopathological results also demonstrate the safety of the adminstration of either H. limifolia Marloth or G. carinata (Mill) Duval extracts (Group 3 and 4, respectively), where they both showed grade 0 of hepatic histological damage i.e. similar to the normal group. While that of G. minma extract only caused mild swelling of hepatocytes (Grade I). The remarkable elevation AST, ALT and liver MDA together with a decrease in total protein and albumin and liver GSH in CCl4 administered rats in this study is only a confirmation of previous reports on the hepatotoxicity of CCl4. The present study also revealed that, administration of the methanolic extracts of the aerial parts of the plants under investigation causes significant serum levels of ALT, AST, albumin and total protein amelioration after CCl4-intoxication. Treatment with the extract of the aerial parts of H. limifolia Marloth showed the best improvement in the liver function and antioxidant parameters followed by G. carinata (Mill) Duval , followed by G. minima Poelln. and this was validated by the histopathological results.

Part V: Docking studies of different identified compounds in H. limifolia Marloth, G. carinata (Mill) Duval and G. minima Poelln. on COX-2 enzyme The results revealed that the affinity of those compounds identified in H. limifolia Marloth, Gasteria carinata (Mill.) Duval and Gasteria minima poelln. to the COX-2 receptor may explain their anti-inflammatory activity specially H. limifolia Marloth which is the most potent one due to the presence of 5-Hydroxyaloin A 6`- O-sinapoyl, 5-Hydroxyaloin A-2′-O- sinapoyl-6′-O-acetate, 5-hydroxyaloin and 5-Hydroxyaloin A 6`- O-acetate which have the highest affinity to COX-2 enzyme.

Conclusion The studies carried on the aerial parts of Haworthia limifolia Marloth, Gasteria carinata (Mill) Duval and Gasteria minima Poelln. showed that: -DNA fingerprinting, macromorphological and micromorphological botanical features could be used to identify the plants under investigation and differentiate between them.

- Phytochemical screening, HPLC of the phenolic compounds and mucilage, UPLC-MS/MS and colorimetric assays revealed that the plants under investigation are rich in polysaccharides, phenolics, flavonoids and anthraquinones. -Lipid profile has been estimated using GC-MS. -They show promising pharmacological effects as antioxidant, anti-inflammatory, antimicrobial, cytotoxic and antihepatotoxic effect. -The acute toxicity studies showed that the plants under investigation are safe. -After the encouraging phytochemical and biological results, we recommend the establishment of a program to cultivate genera Haworthia and Gasteria in Egypt both in the wild and in specialized gardens, specially that their cultivation is very easy with minimum demands. -Further chemical and biological studies on these rich, promising, poorly investigated species are recommended. -Also, those studies should include other members of both genera. -Subclinical and clinical trials should be done to support all the accomplished investigations.