THE PREPARATION OF NANOPARTICULATE

Doctoıral

Dissertation

.••

"EVALUATION OF AMPH!PHILIC

~-CYCLODEXfRINS AS NOVEL EXCIPIBNTS IN

THE PREPARATION OF NANOPARTICULATE

DELIVERY

EREM MEM!şoCLU Co-tutele Ph. D. Thesis

Hacettepe Universily (Turkey)-Universite de Paris-Sud (France)

Pharmaceulical Teclınology Programme

Supervisors: Prof Dr, A. A._tilla Hıncal (Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Technology, Ankara, Turkey) and Prof. Dr.

Dominique Duchene (Universite de Paris-Sud, Faculte de Pharmacie, Laboratoire de Physicochimie, Biopharmacie et Ph._ırrrı.acotechnie, UNIR CNRS 8612, Chatenay-J\t1alo;.bry, France)

Date of exar.ninatic.ın: February 8, 2002

ln this study, amphiplülic ~-cyclodextrins rnodified on the primary or secondary faces were synthesized, chemically characterized by ¹H NMR, FAB MS, DSC, FT IR spectroscopy and elemental analysis to ensure their purity and structural integrity. Their aınphiphilic properties and behaviour in oil/'ivater and air/water systen1s were demonstrated by surface pression and surface tension experiments. Inclusion forn:üng capacities of these CD derivatives were alsa evaluated to demonstrate the formation of inclusion complexes by the co-lyophilization technique. Amphiphilic 1)-cyclodextrins; modified by acyl chains of different 1ength (C6 and C14) and structures (linear and branched) on different faces (primary and secondary) \Vith different bonds (ester or anüde) yielded nanocapsules and nanospheres by the nanoprecipitation technique '>Vithout using surfactants. Nanocapsule size and tirne-dependent p~ysical stability are infiuenced by the structure and concentration of an1phiphilic 13-cyclodextrin.

Inclusion forming capabilities of amphiphilic p-cyclodextrins •vere demonstrated with three model drugs; bifonazole, clotrimazole and progesterone.

Nanospheres prepared from these pre-formed inclusion coınplexes exhibited significantly higher loading and considerable ieduction of burst effect. Hemolytic activity of amphiphilic ~-cyclodextrins are significantl.y lower than nahual ~-cyclodextrin. Moreover, nıinirnun1 inhibitory concentration of bifonazole and clotrirnazole against Candida albicans have been significantly reduced by association to amphiphiJic ~-cyclodextrin nanospheres.

KEYWORDS: Amphiphilic ~-cyciodextdns, cha- racterization, inch.ısion complex, nanocapsule, nanosphere, loading, release, bifonazole, clotri.rnazole,. progesterone

PHYTOCHEMICAL AND B!OLOGICAL STLDIES ON SOME TURKISH MEDICINAL PLANTS

Şebnem Haıput

Supervisor: Prof. Dr. Y. Ogihara (Nagoya City University, Faculty of Pharmaceutical Sciences, Nagoya-Japan), Pro.f. Dr. L Saracoglu (f-Iacettepe University, Faculty of Pharmacy, Ankara, Turkey),

Date of examioalion: March, 2002.

The use of herbal medicine is a traditional way to treat illness and herbal medicine has been applied for more than millennia in several civilizations. The major challenge today is the discovery of plants with promising activities

aı'ld the isolation of active principles. In .our continuous re- search on Turkish medicinal plants, We focused on Scrop- hulariaceae and Lamiaceae families. In this dissertation, chemical con1ponents of Veronica species and Phlomis spe- cies have been evaluated together vvith their biological ac- tivities since they are widely distributed in Turkish flora and used c..s a folk medicine in Turkey.

Anti-inflammatory and cytotoxic activities :~: E"i.ve Ve- Tonica species, V, pectinata var. glandulosa/ \/. r :__-rsica/ V.

hederifolia/ V. polita and V. cymbalaria vvere evaluated.

Their methanol extrac:ts showed both the inhibitory activity of nitric oxide (NO) production in lipopolysaccharide- stimulated macrophages and cytotoxic activity against KB and B16 cells. rv1ethanol extracts were fractionated between water and chlorofonn. Wlüle water fractions significantly inhibited NO production '>Vithout any cytotoxicity exp- laining the usage of these plants for the inflammatory di- seases, chloroform fractions showed cytotoxicity dose- dependently. Concerning the bioactivity results, phytoc- hemical studies ı;-vere performed on the vvater fractions of

V. ~oectinata var. glandulosa/ \/. persica and V. hederifolia.

The use of chrornatographical techniques and extensive 1D and 2D N'MH.. spectroscopy led to the isolation and struc- ture determination of 26 compounds including eleven iri- doid glucosides, eight phenylethanoid glycosides and four flavonoid glycosides as well as one cyanogiucoside, one megastigman glucoside and one hexitoL Seven of them were isolated from the nature for the first time in this study.

In a continuation of the systematic studies on Turkish Phlomis species, antioxidant activity of P. pungens var.

pungens vvas tested. The aqueous extract, phenylethanoid and iridoid fraction, major components of the pheny- lethanoid fraction exhibited protective effect against free ra- dical-induced impairment of endothelium dependent re- laxation in isolated rat aorta. In addition, phytochemical studies on P. puııgens var. pungens/ P. lycia,. P. sieheana/ P.

grandi.flora var. fimbn.lligera and P. fruticosa \.Vere resulted to the isolation of 18 compounds including four iridoid glu- cosides, ten phenylethanoid glycosides one of which vvas nevv, two lignan glycosides together with one caffeic acid ester and one monoterpene glucoside.

Key words: Veronica species, anti-iıillammatory ac- tivity, cytotoxic activity, free radical scavengers, Phlomis species/ phenylethanoid glycoside, iridoid glycoside and flavonoid glycoside,

Doctıoral Dissertatiiıon

Abstracts •••

INVESTIGATION OF SOME PLANTS GROWING IN TURKEY FRO THA VlEW POINT OF

ACETYLCHOLTh.'ESTERASE INHIBITORY ACTIVfTY

İlkay ORHAN

Supervisor: Prof. Dr. Bilge ŞENER

Date of Examination: June, 28, 2002

In this study, it was aimed to evaluate the acetykholinesterase inhibitory activity of soıne plants gro•ving in Turkey. ·

Acetykholinesterase inhibitors are the compotmds vdUch play role in the treatn1ent of Alzheimer's Disease as well as myasthenia gravis, glaucoma and helminthiazis by preventing reduction of acetylcholine amount via inhibiting acetylcholinesterase enzyme that hydrolyze the neuron1ediator called acetylcholine 1n the ne- uronal end froın which it is released and İn the mechanism of ac- tion of insecticide drugs.

According to the literature survey, the plant species, on which no research have been done previously, from Lycopodiaceae (L.

annotinnın/ L. alpinum, L. clavatum, L. comp}anatum subsp. cha-

nıaesiparissus and L. selago), Arnaryllidaceae (Galanthus elıvesii,

G. ikariae, Narcissus tazetta subsp. tazetta, Leucojum aestivum and Pancratium marHimum), Fumar.ioideae (Fumaria asepala, F.

capreolata, F. ôlicica, F. densiflora, F. judaica, F. kralikii, F. ınac­

rocaıpa, F. parviflora, F. petteri subsp. thuretti, F. vaillantii and Carydalis solida subsp. solida) and Buxaceae (Buxus sem- pervirens) families as well as Zygophyllaceae family (Tribuhıs ter- restris and Zygophyllıım fabago) considering their use in the folk medicine with Fabaceae (Vicia !aba), were investigated from the view point of their acetylcholinesterase activity. Activ.ity de- terminations were carried out by Ellman method which is a spect- rophotometric, in vitro robotic screening method. Bioactivity- directed fractionation and isolation shldies were carried on Lyco- podium clavatum, Galanthııs ikariae, Nardssus tazetta subsp. ta- zetta and Fun1aria vaillantii which showed 50 "/o and more in- hibitory activity in the prescreening program.

a-Onocerin (L-1), a triterpenoid isolated by preparative TLC from Lycopodium c]avahım, was found ta be the responsible com-

poımd for the activity.

In total, six Amaryllidaceae type alkaloids called lycorine (G- 1), tazettine (G-2), crinine (G-3), galanthaınine (G-4), 3-epi-

hydrcixybulbisperınine (G·.S) and 2-demethoxymontanine (G-6) from the active fractions of Galanthus ikariae were obtained by co- lumn chromatography.

In addition, lycorine (N-1), tazettine (N-2), N-nor- galanthamine (N-3), haemantamin (N-4) and 3-epi- hydroxybulbispermine (N-5) ıvere isolated from the active frac- tions of Nardssus tazetta subsp. ta.zetta as the Amaryllidaceae al- kaloids.

The isoquinoline alkaloids called canadine (F-1), hydrastine (F- 2), ophiocarpine (F-3), bulbocapnine (F-4), fumarophycine (F-5),

corydaldirıe (F-6), ophiocarpine-N-oxide (F-7), protopine (F-8),

P-

allocryptopine (F-9) and berberine (F-10) were obtained froın the active fractions of Fumaria vaillantii.

Although G. ikariae and N. tazetta subsp. tazetta extracts sho- wed 75.56 °/o and 46.62 °/o inhlbition, respectively; it rnade us con- sidered that the activity of the extracts lower than 50 'Yu resulted from the synergistic interaction between the alkaloids isolated. it was established that the compounds responsible for the inhibitor activity of F. vaillantii extract (94.23 °/o) were ophiocarpine, op- hiocarpine-N-oxide, protopine, P-allocryptopine and berberine and there was alsa synergism in the interaction behveen the al- kaloids.

PHARMACOGNOSTICAL STUDIES ON GLOBULARIASPECJES

Hasan KlRM!ZlllEKMEZ

Supervisor: Prof. Dr. İhsan ç,~LIŞ, t·Iacettepe University, Faculty of Pharmacy, Dept. of Pharmacognosy, 06100, Sıh­

hıye, Ankara, Turkey.

Date of Examination: july 12, 2002

In this study, G. trichosantha, G. orientalis, G. da- visiana, G. cordifolia and G. dumulosa (Globulariaceac) 1vere investigated from t.he point of view of the isolation of their secondary n1etabolites, structure elucidation and ra- dical scavenging (antioxidant) properties. The plant ma- terials used in this study ıvere extracted vvith either rnet- hanol or ethanol. After evaporation, the extracts 1.-vere dissolved in vvater and extracted with chlorofonn in order to get rid of the lipophilic contents. The vvater-soluble parts of the crude extracts were used far isolation studies, Pref- ractionation and purification studies on the extracts were performed by using chromatographic rnethods (VLC, OCC, and A1PLC). Consequently, 11 compounds, two of which were new, from the underground parts of G. trichosantha;

12 compounds from the aerial parts as well as 6 corn- pounds, one of which vvas neıv, from the underground parts of G. orientaliş; 17 compounds, one of which was new, from the aerial parts of G. davisiana; 19 cornpounds, three of which were new, from the underground parts of G.

cordifolia; and 15 compounds, two of which \Vere new, from the aerial parts of G. dumulosa were obtained. The structures of the com.pounds were elucidated by spect- roscopic (UV, IR, 1D and 2D NMR and MS) and chernical (partial methylation, alkaline hydrolysis, reduction and acetylation) methods. Totally, 80 compounds, cor- responded to 49 different structures were isolated and ca- tegorized under 7 main groups: Iridoids, phenylethanoid glycosides, sugar esters, lignan glycosides, flavon glyco- sides, acetophenone glycoside and sterols. The free radical scavenging (antioxidant) properties of the extracts and ob- tained phenolic cornpounds were detennined by reduction of DPPH radical rnethod.

Keywords: Globularia trichosantha; Globularia ori- entalis, Globularia davisiana; Globulaıia cordifolia, Glo- bularia dumulosa, Globulariaceae, iridoids, phenylethanoid glycosides, sugar esters, lignan glycosides, flavon glyco- sides, acetophenone glycoside, sterols, free radical sca- venging property.

Doctoral Dissertation

•.•

PHARMACOGNOSTICAL RESEARCHES ON SOME PHLOMIS SPECIES

Funda Nuray Yalçın

Supervisor : Prof. Dr. Tayfun Ersöz, Department of Phar- macognosy, Faculty of Pharmacy, Hacettepe University, 06100, Ankara, Turkey.

Date of Examinalion: July 29, 2002

The genus Phlomis is represented by 34 species in Tur- kish flora. The genus is classified into three groups (Group A, Group B and Group C) in this study, three Phlomis spe- cies, each belongs to a different group have been searched frorn the point of vievv of their chemical constituents by means of phytochemical investigations on their overg- round parts.

The overground parts of the plants were extracted vvith methanol. The crude extract was dissolved in water and extracted vvith hexane, chloroform and n-BuOH, suc- cesively. The same extraction method has been applied for three species. By means of a serial chromatographic studies on the n-BuOH extracts, shanzhiside rnethylester, rnart- ynoside, 4"-0-acetyl rnartynoside, sarnioside, 2,6- dimethoxy-4-hydroxyphenol-1-0-P-D-glucopyranoside, uridine and phlornuroside, 1-rnethyl-f)-D-gluco pyranoside from Phlomis samia; larniide, ipolarniide, verbascoside, forsythoside B, alyssonoside and syringaresinol-4'-0-f)-D- glucopyranoside, from P. monocephala, and verbascoside¹ syringin, dihydrosyringin, coniferin, 216-dimethoxy-4- hydroxyphenol-1-0-P-D-glucopyranoside picein, be- tulalbuside A, 8-hydroxylinaloyl-3-0-P-D-glucopyranoside and 1-methyl-f)-D-glucopyranoside from P. carica were iso- lated.

Radical scavenging activity of the water and n-BuOH extracts prepared from the plant specimens and the com- pourtds isolated were tested towards the 2,2,-diphenyl-1- pirylhydrazyl (DPPH) radical in TLC system.

Key Words: Phlomis samia, Plılomis monocephala, Phlo.mis carica, Lamiaceae, iridoid glucoside, pheny- lethanoid glycoside, lignan glucoside, phenolic glucoside, acetophenone glucoside, monoterpene glucoside, me- gastigrnane glucoside.

PHYTOCHEMICAL AND BIOLOGICAL INVESTIGATIONS ONA TURKISH AJUGA SPECIES, AJUGA SALICIFOLlA

PınarAKBAY

Supervisor: Prof. Dr. Otto Sticher*, Coexaminers: Prof. Dr.

Ihsan Çalış'*, Prof. Dr. August Schubiger*, Dr. jörg He- ilmann*, * ETH-Zürich (Swiss Federal Institute of Tech- nology), Dept. of Applied Biosciences, Inst. of Phar- maceutical Sciences, Winterthurerstr. 190, 8057 Zurich, Switzerland. **Hacettepe University, Faculty of Pharmacy, Dept. of Pharmacognosy, 06100, Sıhluye, Ankara, Turkey.

Date ofExaminalion: September 18, 2002

ln the flora of Turkey, the genus Ajuga L., commonly named as bugle, is represented by 11 species some of which are traditionally used in wound healing, as diuretic, as well as against diarrhea and high fever.

Ajuga salicifolia was collected near Ankara-Beytepe in july 1998. Air-dried and powdered aerial parts (1 kg) were extracted successively with PE, DCM, EtOAc, MeOH, and Me0H-H₂0 (1:1). After a TLC control, the DCM and EtOAc extracts were combined. Tite fractionation of the DCM/

EtOAc and MeOH extracts by means of various chro- matographic methods (VLC, CC, MPLC and HPLC) led to the isolation of 18 compounds, 10 of which were new to the literature. Structures of the isolates were established by ex- tensive use of lD- and 2D-NMR (COSY, HSQC, HSQC- TOCSY, HMBC) experiments. Additional information was gathered by mass spectrometry (F AB-, ESi- and HRMAL- DIMS) UV spectroscopy, physical ([a]₀ and chemical (acid hydrolysis) methods. The relative stereochemistry was de- termined from the results of 2D ROESY or NOESY ex- periments.

The majority (nine) of the isolated compounds were new stigmastane-type sterols, showing a high structure va- riability. Two of them were novel sterol glycosides, disp- laying three epoxidations on the stigmastan-7-en skeleton, which enabled the presence of three additional (fi.ve, six and eight mernbered) ring systems. The other secondary metabolites were ionone, iridoid and phenylethanoid glycosides. The ionone glycosides were reported for the first time from Ajuga species. üne of thern was new to the literature. Except the ubiquitous compounds harpagide and 8-0-acetylharpagide, the isolated iridoid and pheny- lethanoids were new for A. salicifolia. Furthermore, this is the first report of 8-0-acetylrnioporoside from the family, Larniaceae.

Ali the isolated compounds were tested in in-house as- says. Some of the sterols showed significant cytotoxic ac- livity against KB (Hela) or jurkat T cells. The cytotoxicity of ajugasalicigenin against KB cells was remarkable (IC50 = 1.9 µNI). Ajugasalioside C was the most active sterol glyco- side against jurkat T cells (!Cso = 3 µM) followed by the novel compound ajugasalicioside A (IC50 = 6 µM).

Ajugasalicioside A induced cell-cell contacts in Jurkat T celi populations similar to phorbol 12-myristate 13-acetate (PMA). To follow up this effect, the possible modulation of ajugasalicioside A on PMA-induced rnRNA profiles in Jur- kat T cells with reverse transcription real time PCR (RT-!t- PCR) was measured. The results suggested

a

dependent induclion of cyclin Dl by ajugasaliciside A which indicates that this compound may stimulate dif- ferentiation processes.