AN IN VITRO CULTURE PROTOCOL, ANALYSIS OF SAPONIN AND BIOASSAY OF BIOLOGICAL ACTIVITY OF VERBASCUM THAPSUS L., A MEDICINAL PLANT ” 123492 ARZU UCAR TURKER THESIS SUBMITTED TO THE GRADUATE SCHOOL OF NATURAL AND APPLIED SCIENCES OF THE ABANT IZZET BAYSAL UNIVERSITY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN THE DEPARTMENT OF BIOLOGY voxsexOcRerion KOROLY % Ce NARDSYON werkt a! JUNE 2002Approval of the Graduate School of Natural Sciences Prof. Dr. Nihat CELEBI Director | certify that this dissertation satisfies all the requirements for the degree of Doctor of Philosophy. Prof. Dr. M.Tekin BABAG > Head of Department This is to certify that we have read this dissertation and that in our opinion it is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. = Cuan Wo Assoc. Prof. Dr. Ekrem GUREL Supervisor Examining Committee Members 4. Prof. Dr. Ismail TURKAN 2. Prof. Dr. M.Tekin BABAG 3. Assoc. Prof. Dr. Sebahattin OZCAN 4. Assoc. Prof. Dr. Ekrem GUREL 5. Assist. Prof. Emel USLUABSTRACT AN IN VITRO CULTURE PROTOCOL, ANALYSIS OF SAPONIN AND BIOASSAY OF BIOLOGICAL ACTIVITY OF VERBASCUM THAPSUS L., A MEDICINAL PLANT Turker Ucar, Arzu Ph.D., Department of Biology Supervisor: Assoc. Prof. Dr. Ekrem Garel June 2002, 127 pages Verbascum thapsus L. (common mullein) is a medicinal plant that has been used to treat pulmonary problems, inflammatory diseases, asthma, spasmodic coughs, diarrhea and migraine headaches. A reliable in vitro culture protocol for common mullein was established. Explants (leaf discs, petioles and roots) were cultured on Murashige and Skoog minimal organics (MSMO) medium with benzyladenine (BA) or kinetin (KIN). Best shoot proliferation was obtained from leaf discs and petiole explants with 3 mg/l BA. Leaf discs were cultured on MSMO medium with 3 mg/ BA in combination with naphthalene acetic acid ~(NAA) or 2,4- dichlorophenoxyacetic acid (2,4-D). More shoot development was obtained with 3 mg/l BA and 1 mg/l NAA. Shoots were transferred to rooting media containing different levels of NAA or 2,4-D. Majority of shoots rooted on medium with 1 mg/l NAA. 2c. VUMSEROCHETIN KORY ‘DOKUMANTASYONAn extraction and analytical protocol for saponins was established for Verbascum thapsus L. Four different kinds of plant sample were analyzed Commercially obtained leaves had a higher concentration of saponin (0.215 mg/g tissue) than jin vitro cultured and field-grown leaves (0.081 and 0.198 mg/g tissue, respectively) or the capsule samples (0.016 mg/g tissue) Biological activities of mullein extracts and commercial mullein products were assessed using selected bench-top bioassays. Bioassays included antibacterial, antitumor, and two toxicity assays (i.e., brine shrimp and radish seed bioassay). In general, aqueous extracts were the most effective and showed antibacterial activity against K. pneumoniae and S. aureus. Commercial products and purified saponins showed little or no antibacterial activity. Agrobacterium tumefaciens-induced tumors in potato tissue were inhibited by all mullein extracts, commercial products and purified saponins. Methanolic extracts of in vitro-grown leaves and commercially obtained leaves inhibited tumor formation better than the other extracts. Results of brine shrimp bioassay showed that extracts of mullein were toxic at higher doses (around 1,000 mg/l). Among aqueous extractions, decoctions had more toxicity (LCso < 1,000 mg/l) than the infusion extracts In radish seed bioassay, at high doses (10,000 mg/l, all extracts reduced the root length. However, at 1,000 mg/l, extracts did not affect the root length as much. Seed germination was also inhibited by mullein extracts at 7,500 mg/l. Low doses (1,000 mg/l) affected germination, but not as much ashigher doses. Ethanolic extracts inhibited seed germination more than other types of extracts Keywords: Verbascum thapsus L., common mullein, in vitro culture, HPLC, saponin, bioassay, antibacterial, antitumor, toxic, brine shrimp bioassay, radish seed bioassay.OZET TIBBI BIR BITKi OLAN VERBASCUM THAPSUS L.’UN iN VITRO KULTUR PROTOKOLU, SAPONIN ANALIiZi VE BIYOLOJIK AKTIVITESININ DEGERLENDIRILMESi Turker Ugar, Arzu Ph.D., Biyoloji Bélima Tez Danismant: Dog. Dr. Ekrem Girrel Haziran 2002, 127 Sayfa Verbascum thapsus L. (sigir kuyrugu) akciger problemleri, iitihablr hastaliklar, astim, oksiruk, ishal ve migren agnilarinda kullanilan tibbi bir bitkidir. V. thapsus L. igin givenilir bir in vitro cogaltim protokold _gelistirilmistir Eksplantlar (yaprak diskleri, petiyoller ve kékler) benziladenin (BA) veya kinetinin (KIN) bulundugu Murashige ve Skoog minimal organik (MSMO) ortami igerisinde kulture alinmisiardir. Yaprak diskleri ve petiyollerden en iyi govde olusumu 3 mg/l BA igeren ortamlarda elde edilmistir. Ayrica yaprak diskleri 3 mg/l BA’nin naftelen asetik asit (NAA) veya 2,4-diklorofenoksi asetik asit (2,4-D) ile kombinasyonunu igeren ortamlarda kiltire alinmig ve en fazla govde olusumu 3 mg/l BA ve 1 mg/l NAA kombinasyonunda elde edilmistir. Govdeler vifarkli duzeylerde NAA veya 2,4-D igeren kéklendirme ortamlarina gecirilmistir. En fazla kok olugumu 1 mg/l NAA igeren ortamda gézlenmistir. V. thapsus L. bitkisinde bulunan saponinler igin bir ekstraksiyon ve analiz metodu gelistiriimistir. Dért gesit bitki Srnegi analiz edilmistir. Sifal bitki olarak satilan yapraklardan tespit edilen saponin miktari (0.215 mg/g doku) in vitro kultura yapilmig ve arazide yetismig bitkinin yapraklarindan (0.081 ve 0.198 mg/g doku) veya kapsiillerde (0.016 mg/g doku) bulunan miktardan daha fazla olmustur. Laboratuvarda elde edilen sigir kuyrugu ekstraktinin ve ticari olarak satilan bu bitkinin drinlerinin biyolojik aktiviteleri_gesitli_biyolojik testler kullanilarak degerlendirilmistir.Antibakteriyal, antitumor ve iki farkli toksisite testi (brine shrimp ve turp tohumu biyolojik testleri). Genel olarak su ile hazirlanmis ekstraklar daha etkili olmug ve K. pneumoniae and S. aureus’a karsi_ antibakteriyal etki géstermislerdir. Bitkinin ticari Urunleri ve diger test edilen saponinler bu bakterilere kari cok az antibakteriyal etki gostermisler veya hig géstermemislerdir. Butan sigir kuyrugu ekstraklan, ticari Urinleri ve test edilen saponinler patates dokusunda Agrobacterium tumefaciens tarafindan olusturulan timérleri inhibe etmistir. In vitro yetistirilmig ve satin alinmis yapraklarin metanol ile yapilan ekstraksiyonu tumdr olugumunu diger metanolik, etanolik ve suyla yapilan ekstraklardan daha fazia inhibe etmistir. Brine shrimp vilbiyolojik testinin sonuglan sigir kuyrugu ekstraklarinin yuksek doziarda (1,000 mg/l civarinda) toksik oldugunu géstermistir. Su ile yapilan ekstraklar arasinda, kaynatilarak hazirlanan ekstrakin (LCso < 1,000 mg/l) kaynamig suya batinip demlenme ile hazirlanan ekstrakta oranla cok daha fazla toksik oldugu gézlenmistir. Turp tohumu biyolojik testinde yuksek dozlarda (10,000 mgjl) botiin ekstraklar turp kok uzunlugunu azaltmasina ragmen, dsik dozlarda (1,000 mg/l) ekstraklar kdk olusumunu ok fazla etkilememistir. 7,500 mg/l dozunda turp tohumu gimlenmesi bittun ekstraklar tarafindan inhibe edilmistir. Disk dozlar (1,000 mg/l) cimlenmeyi azaltmistir fakat bu yUksek doziardaki kadar degildir. Etanolik ekstraklar tohum gimlenmesini diger ekstraklardan daha fazla inhibe etmistir. Anahtar Kelimeler: Verbascum thapsus L., sigir kuyrugu, in vitro kultir, HPLC, saponin, biyolojik test, antibakteriyal, antitlimér, toksik, brine shrimp biyolojik testi, turp tohumu biyolojik testi viiiDEDICATION | dedicate this dissertation to my husband, Hakan Turker; my daughter, Damla Nur Tirker; my mother and father, Nurhayat and Necati Ucar; and my mother-in-law, Arife Tirker for their love and encouragement.ACKNOWLEDGEMENTS | wish to acknowledge the indispensable assistance afforded me by so many people throughout my life and this experience. | would like to express my appreciation and gratitude to my supervisor Dr. Ekrem Gurel for his guidance, support, patience and encouragement. | would like to thank to Dr. N. Dwight Camper, with my deepest appreciation, for giving me the opportunity to work as. a visitor scientist under his guidance and his help and support during my time at Clemson University in USA. | would like to thank my committee members Dr. ismail Turkan of Ege University, Dr. Sebahattin Ozcan of Ankara University and, Dr. M. Tekin Babag and Dr. Emel Uslu of Abant Izzet Baysal University for their __ advice and review of this manuscript. Thanks are due to Dr. Melissa B. Riley for her valuable advice during the course of this work. | am grateful to Dr. Ihsan Gali, Faculty of Pharmacy, Hacettepe University, Turkey for his kind gift of ilwensisaponin A and other saponins used as standard. | would like to express my gratitude to the entire Plant Pathology and Physiology faculty, staff, and student body for making my work at Clemson University very enjoyable and beneficial. Appreciation is due to all members of Department of Biology, Abant izzet Baysal University in Turkey. This research would not be completed without reference to my fellow grad students in Dr. Camper's lab. | am grateful to my lab-mate, Dr. TharathornBoonkaew without whose assistance and conversation in the lab would have made this work unbearable. She has contributed so much work and time to this research. She was a great friend, thank you very much for making me smile. Thanks goes for the assistance | have received from my other lab-mates; Karen Carlson Hall, Dr. Aranya T. Pimmongkol, Joe-Ann Helen McCoy, David Hooper and Pamela Cooker. How nice to be in a place where ideas and frustrations could be shared. My special thanks go to my wonderful husband, Hakan Turker. His love, patience, support, and help enabled me to achieve this goal. | also thank to my daughter, Damla Nur for giving me a good mood with her existence during this work. My appreciation is extended to my mother, my father and my mother-in-law for their love, patience, encouragement and help for the completion of this research. | would also like to thank my friend Selma Merdun and Filiz Dik for sharing the stressful moments with me and for helping me whenever I needed. Finally, | apologize to those not mentioned-they are, however, not forgotten xiTABLE OF CONTENTS Page TITLE PAGE ..... i ABSTRACT ii OzET vi DEDICATION... ix ACKNOWLEDGEMENTS .........0..000000000+ x TABLE OF CONTENTS....scscssssaisieininctittttiiineneienenenenen Xl LIST OF TABLES... xv LIST OF FIGURES. ........... xviii LIST OF ABBREVIATIONS .....cccssssstsinstssntntintintinssieieetentea xxi CHAPTERS, 4, INTRODUCTION AND LITERATURE REVIEW... 1 1.1. Introduction 41 1.2. Literature Review 6 1.21. BOtany wscssssnieiniatntenentetetnieeenes 6 1.2.2. Habitat...... seeeneteneeeeneneee 9 1.2.3. Biology and ecology... seitititatnenenenene 10 1.2.4. Medicinal or historical use... oscteneunene 14 1.2.5. In vitro culture of V. thapsus L. and of related species . 18 xiiTable of Contents (Continued) Page 1.2.6. Active ingredients of V. thapsus L. 20 1.2.7. Saponins of Verbascum species, extraction and analysis........... 24 1.2.8. BIOASSAYS... ecccecccteeseeseenstene ceceeeteeeineee 24 2. IN VITRO CULTURE OF COMMON MULLEIN (VERBASCUM THAPSUS L.) . 27 2.1. Introduction .....-.e-eesseeseee . - cose a 27 2.2. Materials and Methods .. fn... a... sone 29 2.3. Results and Discussion 33 3. HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC DETERMINATION OF A. SAPONIN FROM VERBASCUM THAPSUS L. ee a 43 3.1. IMtrOdUCtiOn .....ccceeesssesstesereneseeetene sevens 43 3.2. Materials and Methods 45 3.2.1. Plant material . see wee 45 3.2.2. Sample preparation . . 46 3.2.3. HPLC analysis . see ceceeeeneeseeeee see 49 3.3, Results and Discussion sestennstusetesssteenstinstenseeisseesnssens 52 4, BIOLOGICAL ACTIVITY OF COMMON MULLEIN 58 4.1. Introduction ... 58 4.2. Materials and Methods........... - 62 4.2.1, Plant material and extraction ........cccceeseenseenetee 62 4.2.2. Antibacterial assay.......--cesccseeeee sess 64 xiii ‘tc YOKSEKOCRETIM KUROLU DOKUMANTASYONTable of Contents (Continued) Page 4.2.3. ANtiMUMOF ASSAY... .cacscsssctesstestnsteeentee . 65 4.2.4. Brine shrimp bioassay.......... secs 67 4.2.5. Radish seed bioassay.......... seseeteee 70 4.3. Results and Discussion . 71 5. CONCLUSIONS ....... oes essessseeeeeeeee 97 REFERENCES... APPENDICES ....c.:sccsssesssestensecsenes 119 A. Zone diameter interpretive chart used in antimicrobial assay — aa . 120 B. Bacteria used in antibacterial assay and their representative diseases a. aa oe . 124 C. Antibacterial activity against Agrobacterium tumefaciens... 124 CURRICULUM VITAE ..... xivTable 24 2.2 2.3 3.4 3.2 3.3 44 LIST OF TABLES Page Shoot formation from leaf discs, petiole and root segment explants from in vitro grown seedlings of common MUIlEIN.....-sccseweeenenn 34 Shoot development from leaf discs from greenhouse-grown plants incubated on media containing combinations of BA with NAA or 2,4 Du... Effects of NAA and 2,4-D on root formation from shoots after 5 WeeKS INCUBATION... cesses eesseecseeeseseseesseesneenseeenecenee see 441 HPLC gradient solvent system. Composition: A, acetonitrile with 0.1% orthophosphoric acid; B, water with 0.1% orthophosphoric acid .... 49 Retention times of digitoxin, ilwensisaponin A and saponin-1 in leaves (field-grown, in vitro cultured and commercially obtained) and capsules (from field-grown plants) of V. thapsus L. 55 Content of saponin-1 in leaves (field-grown, in vitro cultured and commercially obtained) and capsules (from field-grown plants) of V. thapsUs L. .eecsesessecsesee 55 Designation of extracts and different saponins used in bioassays and their extraction procedures econectinistitistisietetsieee 62List of Tables (Continued) Table 4.2 4.3 44 45 46 47 48 Page Antibacterial activity of mullein extracts, selected saponins and reference antibiotics. Data presented as zone of inhibition of bacterial growth in mm. ....... 71 Mean number of tumors observed with mullein extracts, selected saponins, and controls (water and camptothecin) ....... 76 LCs0 (lethal concentration for 50% mortality after 24 hr exposure) values for mullein extracts, different saponins and MS-222 (a Positive COMMON) .....sscssrstcsesteststetse 80 Effects of water (control), various Mullein extracts and commercial products on radish seediing root elongation (at 10,000 mg/l)... 83 Effects of water (control), various mullein extracts, commercial products and different saponins on radish seedling root elongation (at 1,000 mg/l) Effects of different saponins on radish seedling root elongation at 100 MgM..sscrecessessteseersessese 87 Effects of mullein extracts, commercial products and control (water) on radish seed germination (at 7,500 mg/l). 89List of Tables (Continued) Table Page 4.9 Effects of mullein extracts, commercial products, control (water) and different saponins on radish seed germination (at 1,000 mg/l) 90 4.10 Effects of different saponins on radish seed germination at 100 mgfl.. 93 A.1___ Interpretation of inhibition zones of antibiotics used in antimicrobial assay for Kirby-Bauer antibiotic susceptibility testing (Atlas, 1988)...... 120 B.1 Bacteria used in antibacterial assay and their representative GiSCASES 0... esseseeseen 121 xviiFigure 11 12 13 21 2.2a 2.2b 2.2¢ 2.2d 2.2e 2.2f 3.1 3.2 3.3 3.4 LIST OF FIGURES. Common mullein plants ....:cieucusen Leaf and flower morphology of V. thapSUs L s.r: Commercial mullein products Protocol for in vitro culture of Verbascum thapsus L. (common mullein) Shoot formation from petiole explant with 3 mg/l BA Shoot formation from root segment explant with 3 mg/l BA ........ Shoot formation from leaf disc with 3 mg/l BA + 1 mg/l NAA.............2. Root formation from shoots after 5 weeks with 1 mg/l NAA. Regenerated plants after 3 weeks in Magenta containers ...... Regenerated plants transferred to pots 3 weeks later. Chemical structure of digitoxin used as an internal standard ...... Chemical structure of ilwensisaponin A used as an external standard (Calis et al., 1991) Standard curve of external standard ilwensisaponin A. Standard curve of internal standard digitoxin ........... xviii 32 37 37 38 38 39 39 47 48 51 51List of Figures (Continued) Figure 3.5 41 42 43 44 45 HPLC profiles: (1) of digitoxin and ilwensisaponin A; (2) of field- grown leaves; (3) of in vitro cultured leaves; (4) of commercially obtained leaves; (5) of capsules from field-grown plants....... Brine shrimp egg (right) and mature nauplii (left) used in the experiment Antibacterial activity of mullein extracts, commercial products, different saponins and controls [water, carbenicillin (CB-100) and erithromycin (E-15)] against K. pneumonia... Antibacterial activity of mullein extracts, commercial products, different saponins and controls [water, chloramphenicol (C-30) and erithromycin (E-15)] against S. aUreUS «ees Inoculated petri dishes with K. pneumonia shows an inhibition zone with CL-Dec water extraction (decoction) from commercial leaves (right) and quality control, erithromycin (E-15) (left)... Inoculated petri dishes with S. aureus shows an inhibition zone with FL-Dec (below right), IL-Dec and CL-Dec (below left) [water extraction (decoction) from field, in vitro cultured and commercial leaves] and quality controls, chloramphenicol (C- 30) (above left) and erithromycin (E-15) (above right) .......:sceeee. Page xix 53 69 73 74 75 75List of Figures (Continued) Figure Page 4.6 Mean number of tumors observed with mullein extracts, commercial products, different saponins, and controls (water and camptothecin)... 78 4.7 Tumors produced by Agrobacterium tumefaciens with distilled water (above) and with IL-MeOH (methanolic extract from in vitro cultured leaves) (below). 79 4.8 LCs0 (lethal concentration for 50% mortality after 24 hr exposure) values for mullein extracts, commercial products, different saponins and MS-222 (a positive COntPOl) accesses BZ 4.9 Effects of mullein extracts (10,000 mg/l - above; 1,000 mg/l - below) on radish seediing root length.. 86 4.10 Radish seedling root elongation with CL-EtOH (ethanolic extraction from commercial leaves) (left) and water (right) at 10,000 mg/l after 3 days... 4.11. Effects of mullein extracts (7,500 mg/l — above;1,000 mg/l - below) and control (water) on radish seed germination as a function of incubation time (days) 92 4.12 Radish seed germination with water (left) and CL-EtOH (ethanolic extraction from commercial leaves) (right) at 7,500 mgfl after 1 day... ccc 95 xxAbbreviations 2,4-D AE-Com ANOVA BA Cre c-30 CB-100 CF-30 CHCls CL-Dec CL-EtOH CL-MeOH Dccc. E15 EtOH FC-MeOH FDA FL-Dec FL-EtOH FL-Inf FL-MeOH FO-Com G25 GA3 6c Gly LIST OF ABBREVIATIONS Full Name 2,4-dichlorophenoxyacetic acid Commercial mullein extract in alcohol Analysis of variance Benzyladenine Octadecyl Chloramphenicol (30 4g) Carbenicillin (100 ug) Cephalothin (30 yg) Chloroform Water extract-decoction from commercially obtained leaves Ethanolic extract from commercially obtained leaves Methanolic extract from commercially obtained leaves Droplet counter current chromatography Erythromycin (15 yg) Ethanol Methanolic extract from field capsules Food and Drug Administration Water extract-decoction from field leaves Ethanolic extract from field leaves Water extract-infusion from field leaves Methanolic extract from field leaves Commercial mullein flower oil Sulfisoxazole (2.0 mg) Giberellic acid Gas chromatography Glycyrrhizic acid (Glycyrrhizin) xxi Teri Ruut YON RnAbbreviations HPLC IL-Dec IL-EtOH IL-MeOH LCs0 MeOH MS-222 MSMO M-H NAA NCCLS. PBS Sap IL Sap P Sap Q SC-Com SPE TB-Com TLC TSA TSB UV-Vis YEM Full Name High pressure liquid chromatography Water extract-decoction from in vitro cultured leaves Ethanolic extract from in vitro cultured leaves Methanolic extract from in vitro cultured leaves Lethal concentration for 50% mortality after 24 hr exposure Methanol Tricaine methane sulfonate Murashige and Skoog Minimal Organic Mueller-Hinton Naphthalene acetic acid National Committee for Clinical Laboratory Standards Phosphate buffered saline llwensisaponin A from Scrophularia ilwensi C. Koch Pure saponin Saponin from Ouillaja bark Commercial mullein swallow capsule Solid phase extraction Commercial mullein tea bag Thin layer chromatography Tryptic soy agar Tryptic soy broth Ultraviolet visible Yeast extract media xxiiCHAPTER ONE Introduction and Literature Review 4.1. Introduction Verbascum thapsus L. (common mullein), a biennial plant of Scrophulariaceae family, produces a low vegetative rosette in the first year which flowers with a stout stem (0.3-2.0 m tall) in the next growing season (Gross and Werner, 1978) (Figure 1.1 and 1.2). Common mullein is native to Europe and Asia (Semenza et al., 1978) and was probably introduced into North America several times as a medicinal herb. Mullein leaves and flowers have expectorant and demulcent properties which are used to treat respiratory problems such as bronchitis, dry coughs, whooping cough, tuberculosis, asthma, and hoarseness (Grieve, 1981; Mabey, 1988; Tyler, 1993; 1994). The plant is mildly diuretic and has a soothing and anti-inflammatory effect on the urinary tract, and also acts as a mild sedative (Mabey, 1988). Mucilaginous constituents are primarily responsible for the soothing actions on mucous membranes, and the saponins are responsible for the expectorant actions’ of mullein (Tyler, 1993). Saponins also have anti-inflammatory, analgesic and cytotoxic/anti-tumour activities (Marston and Hostettmann, 1991). Although common mullein has been used medicinally since ancient times, the popularity of this medicinal plant has been increasing commercially for the last few years. Today, dried leaves and flowers, swallow capsules,Figure 1.1 Common mullein plants.Figure 1.2 Leaf and flower morphology of V. thapsus L.alcoholic extracts, and flower oil of this plant can easily be found in health stores in United States (Figure 1.3). Native populations of common mullein are easily found in pastures, abandoned fields and along roadsides, but they are threatened by chemical (herbicides) and biological (insects and pathogens) controls. Sustainable organic agriculture allows for conscientious and chemical- free planting, cultivating and processing. Organic products are grown in harmony with nature, protecting our air and water, and nurturing the soil Organic products also benefit the health and well being of human and animal life. Common mullein is easily out competed in areas with a densely vegetated ground cover, but readily grows in disturbed sites. Because of its low dispersal rate, the establishment of V. thapsus L. in a particular site depends primarily on the presence of dormant seeds in the soils. It is an ephemeral plant, which is eventually displaced by other plants in undisturbed sites (Gross and Werner, 1978). Manual removal of plants before flowering, the establishment of a dense vegetative cover, and minimizing the availability of bare soil are probably adequate to destroy mullein populations. For medicinal purposes, it is worthy of propagation to compensate for the steady demand by sufferers of pulmonary diseases. The overall goal of this research was to obtain in vitro cultured plants that are free of any pesticide residues or disease infestation and bulk material for future physiological and biochemical studies, and to support the biological activity of this medicinal plant with scientific data. The specific objectives weresjonpoid uje|inus jeroseWW0D €") esnB Lyto i) establish an experimental protocol for in vitro culture of Verbascum thapsus L., ii) develop extraction and analysis procedure for the biologically active ingredient saponin and iii) assess biological activity using specific bioassays (anti-bacterial, anti-tumor and toxicity assessments) 1.2. Literature Review 1.2.1. Botany The Scrophulariaceae family is an important family of plants comprising over 200 genera and about 2500 species. They occur mostly in temperate and sub-tropical regions, and many of them produce flowers of great beauty in either a garden setting or as roadside ‘weeds’. The family includes Mimulus, Penstemon, Digitalis, Veronica and Verbascum (Grieve, 1981). A number of the Scrophulariaceae are, or have been, valued for their curative properties and are widely employed both in domestic and regular medicine. At least 250 species of Verbascum are known. Of the species formerly used in medicine, the most important is Verbascum thapsus L. Common names include mullein, common mullein, great mullein, wooly mullein, candlewick plant, velvet plant, blanket leaf, white mans footsteps, Aaron's rod, Jacob's staff, hedge taper, high taper, old man’s flannel, lady's foxglove and sigir kuyrugu (in Turkish) (Strange, 1977). * The generic name of this plant, Verbascum, is believed to be a corruption of barbascum, from the Latin barba, meaning a beard, referring to the shaggy appearance of the genus, while thapsus, its specific name, may refer to the Greek island of that name, where the species originally thrived. The word “mullein” comes from the Middle English moleyne and the Old French moleine,and originally from the Latin mollis, meaning ‘soft’ and referring to the leaves (Strange, 1977) Verbascum thapsus L. is a biennial, or rarely an annual plant, with a deep tap root. In its first year, it produces a low vegetative rosette up to 60 cm in diameter, which over winters and is followed in the succeeding growing season by a stout flowering stem 0.3-2.0 m tall. The basal leaves are oblong-obovate to obovate-lanceolate and 10-40 cm long including the petiole (Figure 1.2). The flower stem is longitudinally ridged by the bases of decurrent leaves and is densely woolly with branched hairs. Cauline leaves are elliptic-lanceolate, decurrent, and gradually reduced up the stem (Milspaugh, 1974). The leaf system is so arranged that the smaller leaves above drop the rain upon the larger ones below, which direct the water to the roots. This is a necessary arrangement since the mullein grows mostly on dry soils. The stellately branched hairs, which cover the leaves so thickly, act as a protective coat, thus reducing moisture loss, and also providing a defense. They prevent attacks of creeping insects and set up an intense irritation in the mucous membrane of any grazing animals that may attempt to browse upon them. The hairs are not confined to the leaves alone, but are also on every part of the stem, on the calyces and on the outside of the corollas, so that the whole plant appears whitish or gray (Gross and Werner, 1978; Whitson et al., 1992). Muzik (1970) reported that these epidermal hairs protect the species from aqueous solution. of 2,4-D because the droplets are held away from the leaf surface. Ice crystals are held away in the same manner. The homely but valuable “mullein tea’, aremedy of the greatest antiquity for coughs and colds, must always be strained through fine muslin to remove any hairs that may be floating in the hot water, which was poured over the flowers or leaves. They can cause intolerable itching in the mouth (Grieve, 1981) The inflorescence is a spike-like raceme 20-50 cm long and approximately 3 cm in diameter. It is usually very dense; rare axillary racemes may arise from the upper leaves. The sessile flowers are usually one per axil with pedicels less than 2 mm and slightly irregular with rotate corollas (Whitson et al., 1992). Individual flowers of mullein are ephemeral, opening before dawn and closing before midaftemoon of the same day. They are protogynous, the style maturing first and then bending downward once the anthers appear. Flowers are also autogamous, selt-pollination occurring at the end of the day if cross-pollination has not occurred. The style retums to its original position and the corolla closes, pushing the still receptive stigma against the anthers. The calyx consists of five lanceolate or ovate sepals, 7-9 mm long with caudate tips. The corolla is 20-25 mm broad consisting of five yellow (rarely white) petals. Stamens are irregular and attached to corolla, three upper filaments are shorter and densely white-villous, lower two are longer and glabrous. Anthers are larger and colored. The ovary is superior and two-celled. The fruit is an ovoid, stellate-pubescent, capsule 3-6 mm long, longer than calyx and splits into two valves at maturity. There are numerous brown seeds, 0.5-1.0 mm long which are six-sided and have angular lateral surfaces with rows of pits (Abrams, 1951; Davis, 1965-1985; Munz and Keck, 1973; Gross and Werner 1978; Radford et BC. YORSEKOGRETIM KURDLUS DOKUMANTASYON MEREal., 1968). Chromosome counts from plants collected in Ottawa and British Colombia gave 2n=36 (Packer, 1964; Mulligan, 1961). Other counts from European material have given 2n=34 and 2n=36 (Darlington and Wylie, 1955; Love and Love, 1961) and n= 9, 11, 17 (Love and Love, 1961). 1.2.2. Habitat V. thapsus L. is found growing in neglected meadows and pasture lands, along fence rows and roadsides, on waste ground, more especially on gravel, sand or chalk and in industrial areas throughout North America (Semenza et al., 1978). It occurs in areas where the mean annual precipitation is 50-150 cm and the growing season is at least 140 days. Dry sandy soils are preferred, but it is common in the chalk and limestone districts of England. In Canada, it grows abundantly in, but is not restricted to, pastures with well-drained soils and a pH of 6.5-7.8 (Gross and Werner, 1978). In Turkey, common mullein is distributed in Black Sea region (Kastamonu, Ordu, Trabzon, Rize, Coruh) and commonly found in riversides, forests, Corylus and Quercus scrub and volcanic tuff (Davis, 1965-1985). V. thapsus L. is native to Europe and Asia. It was probably introduced into North America several times as a medicinal herb. It was introduced in the mid-1700 to Virginia as a piscicide (fish poison) and spread rapidly (Semenza et al., 1978). It quickly became so well established that an 1818 flora of the East Coast described it as a native. By 1939 it had spread as far as Michigan andbecame widely naturalized on the Pacific Coast by 1876 (Gross and Werner, 1978) Although extracts from mullein can inhibit growth of wheat seedlings, it is not a serious agricultural weed, since it can be controlled by cultivation. In overgrazed or poor pastures, the presence of common mullein represents a further degradation of the pasture because grazing animals avoid eating mullein. This species is not allelopathic, allergenic or poisonous to humans (Gross and Werner, 1978). 1.2.3. Biology and ecology Williams and Kemp (1976) have shown that seedlings of V. thapsus L. collected from a range of cold to warm temperature habitats (based on altitude and latitude) exhibit similar rates of photosynthesis within a temperature range of 20-35 °C. Only at the highest temperature tested, 40 °C, the seedlings from the warmest habitat (low altitude and latitude) exhibit higher photosynthetic rates than those from the coolest habitat. They concluded that the ability of an individual plant to photosynthesize over a broad range of temperature has contributed to mullein's success across a diversity of habitats. Williams et al. (1975) reported a CO, compensation point of 58 vpm CO2 for V. thapsus L. and on this basis concluded that the species had a Cs photosynthetic pathway. Wuenscher (1970) shaved the dense trichomes of the leaves of V. thapsus L. and found that the unshaved half of the leaf was consistently warmer than the shaved half. The hairs must, then, affect leaf energy exchange, since two halves of the same leaf, differing only in thepresence of hairs, reached different equilibrium temperatures. Convection and the latent heat loss are reduced by the hairs although the hairs have little effect on radiation absorption. Transpiration rate is reduced in hairy leaves. All of these effects are explained by an increase in boundary layer thickness caused by the hairs. The boundary layer forms above the surface of the hair coating rather than directly over the leaf surface. This thickens the boundary layer by the distance to which the hairs extend from the leaf surface. Increased transpiration resistance has obvious ecological significance for V. thapsus L., which grows on dry, exposed sites. The dense hair coating is an efficient water- conserving mechanism. Parkhurst (1976), evaluating the work of Wuenscher (1970), showed by calculation that the main effect of the trichomes was to increase stomatal resistance with only a slight increase in boundary layer resistance. Lortie and Aarssen (1997) demonstrated that clipping the shoot apex of V. thapsus L. resulted in significantly more branches and branching intensity could not be increased by greater resource levels in mullein when the apical meristem was intact. Branching was stimulated by the addition of nutrients only when the shoot apex was damaged. These results indicated that nutritional status does not solely determine the degree of branching expressed in mullein. Glier and Caruso (1973) demonstrated that decreasing temperatures were observed to induce starch degradation in roots of mullein. Virtually no starch remains in the roots when the acclimation period of decreasing temperature was followed by an extended period of exposure to 4 °C. Thebreakdown of starch which was presumed to occur in late autumn under field conditions may provide cryoprotective chemicals for the over wintering rosette. ‘Such chemicals might also serve as an energy source for the bolting process, which occurs during the following spring or early summer. Later, they showed that starch content in roots of V. thapsus L. was reduced when rosettes were exposed to low temperatures because of the increased activities of starch degradative enzymes (Glier and Caruso, 1974). They also (Glier and Caruso, 1977b) reported that two nonspecific enzymes, namely, acid and alkaline phosphatase increased their activities in Verbascum roots exposed to decreasing temperatures; alkaline phosphatase was of more importance than acid phosphatase in over wintering rosettes. It was known that mullein had a cold-requirement for bolting and that applied gibberellin substituted for this requirement (Caruso and Glier, 1970). Glier and Caruso (1977a) also described the influence of gibberellin on activities of starch degradative enzymes and phosphatase. There was a sharp decrease in starch in the roots of Verbascum as a result of treating rosettes with GAs and all three starch degradative enzymes revealed increases in their activities. Also, application of GAs resulted in an increase in the activity of alkaline phosphatase in roots and rosettes showed an early response to applied gibberellin. Gross (1981) showed that the rosette sizes of V. thapsus L. gives a reliable estimate of an individual's fate the following year. A minimum size must be reached before a plant is capable of flowering and the probability of flowering increases steadily with rosette size. For mullein, all rosettes with a diameter 12greater than 41 cm flower the subsequent year and rosettes less than 9 cm in diameter do not flower. Conversely, the probability of death decreases with increasing rosette size Seeds of V. thapsus L. are contained in a capsule with two cells. Salisbury (1942) reported the mean number of capsules per explant as 226442 (SD; n=37), with an average of 596430 (SD; n=16) seeds per capsule. This gives an approximate average of 100,00-180,000 seeds per individual plant, each seed averaging 0.067 mg (Gross, 1980; Gross and Werner, 1982). The seeds posses no specialized morphological adaptations for dispersal by wind or animals. The capsules split open along its longitudinal axis when mature, and movement of the stalk by wind or a large animal is required to release the seeds from the parent. Seeds are dispersed as far as 11 m, although 93% of them fall within 5 m and 75% of them fall within 1 m of the parent plant. Seeds may remain viable for over 100 years and viable seeds have been found in soil samples archaeologically dated from A.D. 1300 (Gross and Werner, 1978). Mullein seeds may germinate under a wide variety of environmental conditions. Germination is completely inhibited below 10 °C and at constant temperatures above 40 °C (Semenza et al., 1978). Chilling during the winter lowers the temperature requirement for germination; thus, seeds brought to the surface in the autumn by soil disturbance are able to germinate early the next spring (Baskin and Baskin, 1981). Sernenza et al. (1978) found that only 35% of seeds germinated in the dark, compared to 93% germination in the light. This light sensitivity varies seasonally, but on the whole, only those seeds, which lie 43at, or near the soil surface (0.5 cm or less) will be able to germinate. If seed burial occurs due to subsequent disturbance or heavy rains rapidly sifting the seeds below the soil surface, germination may be reduced or prevented through light deprivation (Gross, 1980) . There are some natural enemies (insects and pathogens) of common mullein such as curculionid weevil (Gymnaetron tetrum Fab.) which is specific to V. thapsus L. and was introduced to North America from Europe (Burcham, 1937). The larvae mature in the capsules (Sleeper, 1954) and destroy up to 50% of the seeds. The other insect, the mullein moth (Cucullia verbasci) feeds and develops on mullein species. There are some pathogens, which cause disease in common mullein such as Erysiphe cichoracearum (powdery mildew) and Phymatotrichum omnivorum (root rot) (Gross and Wermer, 1978). 1.2.4, Medicinal or historical uses Historically, mullein has been used as a remedy for the respiratory tract, particularly in cases of irritating coughs with bronchial congestion (Hoffman, 1988). Some herbal texts extend the therapeutic use to pneumonia and asthma (Grieve, 1981). The leaves, roots and the flowers are anodyne, anti-inflammatory, antiseptic, antispasmodic, astringent, demulcent, diuretic, _ emollient, expectorant, nervine and vulnerary. Some of the uses like analgesic, antihistaminic, anti-inflammatory, anticancer, antioxidant, antiviral, bacteristat,cardiodepressant, estrogenic, fungicide, hypnotic, sedative and pesticide are also valid (Lucas, 1969; Harris, 1972; Null and Null, 1972; Grieve, 1981) Demulcent and emollient properties come from the polysaccharide mucilage and gums that soothe the irritated tissue. The expectorant property is the result of saponins that stimulate fluid production. The anti-inflammatory property is due to of iridoid glycosides and flavonoids that decrease inflammation (Grieve, 1981). The mullein combines the expectorant action of its saponins with the soothing effect of its mucilage, making this a most useful herb for the treatment of hoarseness, tight coughs, bronchitis, asthma and whooping cough (Mabey, 1988). The whole plant seems to possess slightly sedative and narcotic properties. The dried leaves are sometimes smoked in an ordinary tobacco pipe to relieve the irritation of the respiratory mucus membrane, and will completely control the hacking cough of consumption. The leaves are employed with equal benefit when made into cigarettes, for asthma and spasmodic coughs. It is also diuretic, helping to reduce inflammation of the urinary system and counter the irritating effect of acid urine (Ambasta, 1986; Tyler, 1993; 1994). The flowers placed in a bottle and set in the sunshine are said to yield a fatty matter valuable as a cure for haemorrhoids. Fomentations and poultices of the leaves have been found serviceable in haemorthoidal complaints. Mullein is said to be of much value in diarrhoea, from its combination of demulcent with astringent properties, by this combination strengthening the bowels at the same time (Grieve, 1981; Mabey, 1988). In Europe, a sweetened infusion of the flowers strained in order to separate therough hairs is used as a domestic remedy in mild catarrhs and colic. A conserve of the flowers has also been employed on the Continent against ringworm, and a distilled water of the flowers was long reputed a cure for burns and erysipelas (Millspaugh, 1974; Grieve, 1981). Decoction of leaves was used as a hearth stimulant. Roots febrifuge; their decoction is used to alleviate toothache and also relieve cramps, convulsions and migraines. The juice of the plant and powder made from the dried roots is said to quickly remove rough warts when rubbed on them (Tyler 1993; 1994). An oil produced by macerating mullein flowers in olive oil, stored in a corked bottle during prolonged exposure to the sun, or by keeping it near the fire for several days, is used as a local application in country districts in Germany for piles and other mucus membrane inflammations, and also for frost bites and bruises. Mullein oil is recommended for earache and discharge from the ear, and for any eczema of the external ear and its canal (Mabey, 1988; Yarnell, 1997). Mullein oil is a valuable destroyer of disease germs (Chopra et al., 1956; Milspaugh, 1974). The fresh flowers, steeped for 21 days in olive oil, are said to make an admirable bactericide. An alcoholic tincture is prepared by homoeopathic chemists, from the fresh herb with spirits of wine, which has proved beneficial for migraines or sick headaches of long standing, with oppression of the ear (Bianchini and Corbetta, 1977; Lewis and Elvin-Lewis, 1977). The seeds of mullein are said to be toxic and should not be used in any of these preparations (Berk, 1996). The seeds when thrown into the water are said to intoxicate fish, and are used by poachers for that purpose, being slightly narcotic. To be effective, a piscicide plant must bereadily available for use, must have great solubility and rapid diffusion in water, and must have such an effect that the fish do not have a toxic quality when they are eaten by humans. The common mullein satisfies these piscicide requirements. Major toxic elements found in fish poisons include saponin, rotenone and glycoside. These substances affect the circulatory, respiratory and central nervous systems of the fish. The common mullein causes fish to have difficulty in breathing (Wilhelm, 1974). Sweetish settlers called it wild tobacco and tied the leaves around their feet and arms when they had the ague. Some prepared a tea from the leaves for dysentery. A decoction of the roots was injected into the wounds of cattle when afflicted with worms, which caused them to die and fall out. Also some American Indian tribes (Mohegans, Penobscots, Catawbas, Chochtaws, Creeks, Forest Potawatomis and Menominees) used common mullein as a medicinal herb. The Mohegans smoked them to relieve asthma and sore throat, and the Penobscots smoked the dried and powdered leaves for asthma. The Catawbas boiled the root and sweetened it to make syrup for croup in children. The leaves were mashed and applied as a poultice for pain and swelling, sprains, bruises and wounds. The Chochtaws put the leaves on the head as a headache poultice. The Creeks boiled the roots with those of Button Willow for a drink used for coughs. The leaves were also boiled and the patient bathed in the infusion while it was hot. The Forest Potawatomis smoked the dried leaves for asthma, but it is not certain whether they learned the practice from the whites or vice versa. A smoke smudge was made of the leaves and the fumes inhaled forcatarth and to revive an unconscious patient. The Menominees smoked the root for pulmonary diseases. Whites smoked the leaves for asthma and bronchitis and that the flowers were believed to be diuretic and had been used for tuberculosis (Moerman, 1986; Vogel, 1990) The flowering stem was used dried by Greeks and Romans as taper dipped in tallow for light. Mullein torches were said to repel witches. There is evidence that at one time it was a ‘magical plant” of the ancients. Agrippa, a general and minister under Caesar Augustus, claimed that the scent from the leaves had an overpowering effect on demons. Mullein was thought to be an ingredient in brews and love potions, and mentioned in incantations used by witches during the Middle Ages. The women of Rome also infused the flowers and mixed the resulting liquid with lye, using it as a wash to turn their hair golden yellow (Strange, 197) 1.2.5. In vitro culture of V. thapsus L. and of related species There is no indication of previous attempts to initiate tissue cultures of V. thapsus L. Caruso (1971) showed that excised internodal segments of flowering specimens of V. thapsus L. with vascular tissues grew and formed numerous buds on a simple nutrient medium which lacked added growth regulator. Pith explants without vascular tissues became brown in a matter of 2 to 3 weeks with no visible sign of growth. Endogenous growth regulators supplied by vascular tissues were believed to be major factors in bud formation in excised internodal segments of this species. 18Several species of Scrophulariaceae have been regenerated. Torenia fournieri has been studied extensively (Bajaj, 1972). Shoots were initiated in numerous hormone treatments for both leaf (Bajaj, 1972) and internode (Kamada and Harada, 1979) explants. Five different cytokinins were used to initiate shoot formation in T. fournieri. In most cases 4.4 uM (1 mg/l) 6-BA, 4.6 LM (1 mg/l) zeatin, or 7.3 UM (1 mg/l) 4-phenylurea was sufficient to initiate shoot formation (Kamada and Harada, 1979), whereas numerous combinations of cytokinin and auxin also have been successful (Bajaj, 1972). Shoot formation was induced from internode segments when cultured in 0.5 UM (0.1 mg/l) NAA with either 3.35 uM (1 mg/l) SD8339, a cytokinin that has been used with Nicotiana tabacum (Nitsch et al., 1967), or 4.7 pM (1 mg/l) kinetin. Organogenesis of Torenia also could be regulated by application of amino acids; shoot formation was observed in cultures with glutamic acid and aspartic acid added to the medium (Kamada and Harada, 1979). Sangwan et al. (1976) demonstrated the action of exogenously applied hormones in the induction of morphogenesis in Limnophila chinensis (Osb.) Mert. tissue culture. Various levels of kinetin, gibberellic acid and indole-3-acetic acid were tested on stem explants. The formation of normal shoots and roots was stimulated by treatment with kinetin. GA 3 treatment stimulated bud differentiation, but inhibited the root initiation. Arrebola et al. (1997) showed the optimal micropropagation procedure for Isoplexis canariensis (L.) G. Don, using nodal segments with two axillary buds as explants. A concentration of 0.5 uM (0.1 mg/l) kinetin in MS (Murashige and 19‘Skoog, 1962) liquid basal medium was found to be optimal for micropropagation and rooting in vitro was unnecessary for ex vitro survival Raste and Ganapathy (1970) cultured the internodal segments of the inflorescence of Mazus pumilus on Murashige and Skoog's revised medium (Murashige and Skoog, 1962). Indole-acetic acid, kinetin, gibberellic acid and adenine sulphate were used as growth regulators. The inflorescence segments produced calli, which differentiated into either roots or leafy shoots, which subsequently flowered, or only flower buds. Dietrich et al. (1990) established the optimum conditions for the tegeneration of Digitalis Janata from shoot tips, for daughter shoot formation and rooting as well as for the adaptation of the regenerated plants to the open ground. Gene banks of valuable clones were built by keeping shoots at 4 °C on media with high sucrose concentration or by growing juvenile clone plants in the greenhouse at temperatures preventing the induction of flowering 1.2.6. Active ingredients of V. thapsus L. The constituents of V. thapsus L. include polysaccharides; iridoid glycosides including harpagoside; harpagide and aucubin (especially in the leaf); flavonoids, including 3'-methylguercitin, hesperedin and verbascoside; saponins and volatile oils (Pascual Teresa et al., 1978a; 1978b; 1980; Hattori and Hatanaka 1958; Khuroo et al., 1988; Mehrotra et al., 1989; Warashina et al., 1991; 1992) Pascual Teresa et al. (1978a) isolated veratric acid and a-spinasterol 20from the benzene extract of capsules of V. thapsus L. From the hydrolyzed ethanol extract, the triterpene A, saikogenin A, benzyl alcohol and methylfurfural were isolated. They also showed that the oil from mullein seed (benzene extract) had the following components: Fatty acids: palmitic, steriac, oleic, linoleic, linolenic, arachic and behenic. Unsaponifiable matter: B-sitosterol and ergosta-7-en-3-f-ol (Pascual Teresa et al., 1978b). Phenylethanoid and lignan glycosides (Warashina et al., 1992), sterones, iridoid glycosides and sesquiterpene acid (Khuroo et al., 1988; Warashina et al. 1991) and verbacoside, a new luteolin glycoside (Mehrotra et al., 1989) were obtained from whole plants of V. thapsus L. Bourquelot and Bridel (1910) discovered the verbascose, an oligosaccharide in the root of the mullein. Hattori and Hatanaka (1958) demonstrated the oligosaccharides in V. thapsus L. and distribution of mono- and oligosacharides in various organs of the plant in various stages was studied. Saponins of Verbascum species, extraction and analysis Hartleb and Seifert (1994; 1995) isolated songarosaponin D, E, F and buddlejasaponin | from the aerial parts of V. songaricum Schrenk. A combination of chromatographic separation on silica gel (CC and TLC) and RP- 8 (HPLC) of the methanolic extract of the 160 g dried and powdered aerial parts resulted in 3 mg songarosaponin E, 2 mg songarosaponin F, 1 mg buddlejasaponin | and 14 mg songarosaponin D. Klimek et al. (1992) isolated two triterpene saponins from the methanol 21extract of the inflorescences of V. nigrum L. From 400 g dried powdered plant material, crude saponin (240 mg) was obtained as a brown amorphous powder which was chromatographed on alumina. The crystalline mixture of saponin 1 and 2 was finally resolved by chromatography on silica gel; yields were 19 mg of saponin 1 and 14 mg of saponin 2. Anil (1980) also isolated two triterpenoid saponins from the leaves of V. nigrum L. Fresh 18 kg leaves were extracted with methanol three times. After chromatographic separation on silica gel, 3.7 g saponin | and 2.1 g saponin Il were obtained. Klimek (1996) obtained a new saponin in addition to two known ones, and identified as desrhamnosylverbascosaponin from the flowers of V. phlomoides. Air-dried and powdered petals (170 g) were extracted with ethanol, and it was fractionated between diethyl ether, n-butanol and water. The n- butanol-soluble fraction was subjected to repeated column chromatography on polyamide, followed by silica gel and sephadex to obtain 12 mg of verbascosaponin, 30 mg of verbascosaponin A and 5 mg of desrhamnosylverbascosaponin Pascual Teresa et al. (1980) isolated four saponins from the capsules of V. thapsus L, thapsuine A, thapsuine B, hydroxythapsuine A and hydroxythapsuine B with a chromatographic separation on silica gel (TLC) Crushed capsules (5 kg) were extracted with benzene and ethanol and subjected to chromatography on silica gel and sephadex. They obtained 300 mg thapsuine A and 460 mg thapsuine B. A range of methods has been employed for the qualitative and 22quantitative analysis of saponins: haemolysis, piscicidal activity, gravimetry, spectrophotometry, TLC (thin layer chromatography), GC (gas chromatography), HPLC (high pressure liquid chromatography), DCCC (droplet counter current chromatography), etc. (Price et al., 1987). For TLC analysis of crude saponin extracts, either silica gel plates with a CHCLIs-MeOH-H:0 solvent systems (65:35:10, lower phase or 58:35:, for example), or reversed phase (octyl-or octadecylsilica) plates with MeOH-H20 or CHsCN-H20 solvent systems can be used. The saponins may be detected by means of the Godin reagent (vanillin-sulphuric acid) (Marston and Hostettmann, 1991). HPLC analysis of crude extracts of Phytolacca dodecandra has been achieved by Domon et al. (1984). Reversed-phase (RP-8) and polar-bonded supports (Diol), with eluents containing methanol-water or acetonitrile-water mixtures were employed. Detection was affected at 206 nm. The use of elution gradients was possible with acetonitrile as it has a relatively weak absorbent at this wavelength. Under these conditions mono- and bidesmosidic saponins could be separated. The problem with the HPLC analysis of saponins is the lack of a suitable chromophore in many of the gylcosides. One of the methods, however, of overcoming this disadvantage is to derivatize the saponin with a chromophoric reagent, which allows suitable UV detection. Attaching a 4- bromophenacyl group at position Czs of the aglycone is one method of achieving this aim (Slacanin et al., 1988; Marston and Hostettmann, 1991). Many papers concerning the separation of saponins by HPLC have been published. The reversed phase method is the most often described and the 23solvent generally used is a mixture of water and acetonitrile either in the isocratic mode or with an elution gradient (Domon et al., 1984; Slacanin et al., 1988; Kazanawa et al., 1990; Crespin et al., 1993). Acids (orthophosphoric acid and trifluoroacetic acid) have been added to the mobile phase to improve the separation of monodesmosidic saponins or saponin including glucuronic acid (Burnouf-Radosevich and Delfel, 1986). Saponins were detected at 210 nm (Crespin et al., 1993; Reznicek et al., 1996). Reznicek et al. (1996) also showed that digitoxin turned out to be a good intemal standard for the determination of the saponins. Using the HPLC on RP-8, the standard was eluted between the saponins at ta=39.6 min. 1.2.8. Bioassays Generally, the disc diffusion assay has been used to screen for antibiotic activity (Barry and Thornsberry, 1985). McCutcheon et al. (1992) used the disc diffusion method to show the antibiotic activity of common mullein leaves and demonstrated that methanolic extract of leaves had antibacterial activity against Escherichia coli {inhibition zone (iz); 8.0-10.0 mm], Mycobacter phlei (iz; 8.0- 10.0 mm) and Staphylococcus aureus methicillin-resistant (iz; 10.1-15.0 mm).” Methanolic extract of common mullein leaves was found to partially inhibit the cytopathic effects of bovine herpesvirus type 1, two double-stranded DNA viruses, which causes respiratory, genital, conjunctival or encephalitic infections which become latent in the trigeminal ganglion (McCutcheon et al., 1995). ‘TC. YOKSEKOGRETIM KURULD DOKDMANTASYON MERKEREMoreover, this extract had antifungal activity against Microsporum cookeril (iz; 8.0-10.0 mm) and M. gypseum (iz; 8.0-10.0 mm) (McCutcheon et al., 1994) Ferrigini et al. (1982) modified and evaluated the potato disc assay for its potential use as a prescreen and fractionation monitor of plant extracts for 3PS. (in vivo, mouse leukemia) activity. The modified assay was performed on a series of natural compounds and plant extracts (known to have 3PS activity) and on extracts of 41 Euphorbiaceae species and results showed that the potato disc assay was a safe, simple, rapid, in-house, low cost, prescreen for 3PS antitumor activity. No potato disc assay was performed on common mullein extracts. A procedure for general toxicity screening that does not require much specialization is essential as a preliminary stage in the study of bioactive compounds. A simple animal that has been used for this purpose is the brine shrimp, Artemia salina Leach (Sam, 1993). The first report of the use of the brine shrimp as a test organism appeared in 1956 (Michael et al., 1956). Since then there have been many reports on the use of this animal for environmental studies, screening for natural toxins, and as a general screening for bioactive substances in plant extracts (Sam, 1993). Meyer et al. (1982) performed the brine shrimp bioassay on seed extracts of 41 species of Euphorbiaceae and compared the results with 9KB and 9PS cytotoxicities and concluded that the method was rapid, reliable, inexpensive, and convenient as an in-house general bioassay tool for natural product research. Brine shrimp bioassay has not been used on common mullein extracts. 25Radish seed bioassay has been used in various assessment of toxicity and was established by Einhellig and Ramussen (1978). They showed the effects of vanillic and p-hydroxybenzoic acids on radish and grain sorghum germination. Since then there have been many studies using allelochemics or plant extracts to examine the effects on germination of seeds of any weeds and cfops (Williams and Hoagland, 1982) Pardo et al. (1998) isolated iridoid glycosides lateroside 1, harpagoside 2, ajugol 3, and aucubin 4 from an ethanolic extract of the roots of mullein that exhibits antigermination activity on seeds of barley (Hordeum vulgare). Bioassays indicated that at 3 mM concentration, compounds 1, 2, and 4 showed moderate inhibition of seed germination. Aucubin 4 was the most active against root elongation and ajugol 3 showed no activity in the bioassays on barley seed germination and growth 26CHAPTER TWO In Vitro Culture of Common Mullein (Verbascum thapsus L.) 2.4. Introduction All normal living cells within the plant body possess the potential capacity to regenerate an entire plant. This potentiality has been exploited through the culture of protoplasts, cells, tissues and organs in vitro. Cells and tissues, which are mitotically quiescent or already committed to some function or pathway of development, can be (re) directed into organ or embryo formation. Organogenesis is the process by which cells and tissues are forced to undergo changes which lead to the production of a unipolar structure, namely a shoot or root primordium, whose vascular system is often connected to the parent tissues (Thorpe, 1994). The earliest report of controlled shoot formation in vitro was by White (1939). In the same year, the first observation of root formation from callus was reported by Nobecourt (1939), using carrot callus. White's observation was confirmed and extended by Skoog (1944), who showed that auxins could stimulate root formation and inhibit shoot formation. A similar conclusion on the role of auxin in rooting also was made by Gautheret (1945). In addition, Skoog (1944) found that the inhibitory effect of auxin on shoot formation could be partially overcome by increasing the concentration of sucrose and inorganic phosphate in the medium. Skoog and Tsui (1948) demonstrated that adenine sulfate was active in promoting shoot formation and 27counteracted the inhibitory action of auxin. They further reported (Skoog and Tsui, 1951) that the extent of organ formation was dependent on both the concentration and proportion of these additives. The discovery of kinetin (Miller et al., 1956) led to the classic finding of Skoog and Miller (1957) that a basic regulatory mechanism underlying organogenesis involved a balance between auxin and cytokinin. Both of these substances are required for cell division and enlargement in tobacco callus, but a relatively high level of auxin to cytokinin favored root formation and the reverse favored shoot formation. These observations led them to suggest that quantitative interactions between diverse growth factors rather than specific morphogenetic substances provided a common mechanism for the regulation of all types of morphogenetic phenomena in plants that is basic concept on the regulation of organogenesis in vitro (Thorpe, 1980) Christianson and Wamick (1988) determined that there are three phases of organogenesis: dedifferentiation, induction and —_ differentiation. Dedifferentiation involves callus production and ends when cells become competent. During the induction phase, cells become determined and in the differentiation phase, cells form roots or shoots. There has been a significant increase in popularity of common mullein as a medicinal plant and because of the market demand for this herb, in vitro culture protocol providing multiple regenerants per explant would be very useful for mass propagation of common mullein. Although this plant can easily be 28found in the wild, it is generally subjected to some herbicides and attacked by some insects and pathogens. In vitro micropropagation of common mullein provides pesticide or disease-free plants and produces large numbers of vegetative planting stock. In addition, with an in vitro propagation method, unlimited plant material can consistently be obtained throughout the whole year and uniform plant materials (less genetic diversity) can be produced that will be higher with seed germination Caruso (1971) showed that excised internodal segments of flowering specimens of V. thapsus L. with vascular tissues grew and formed numerous buds on a simple nutrient medium which lacked added growth regulators. However, a defined protocol for in vitro culture of common mullein has not been reported, Therefore, the objective of this study was to establish an in vitro culture protocol for Verbascum thapsus L. to provide a source of plant material for the extraction and analysis of biologically active ingredients. The results given in this chapter were already published (Turker et al., 2001). 2.2. Materials and Methods Seeds were collected from South Carolina Botanical Garden, Clemson, SC, in July and August of 1998. They were washed with anti-bacterial soap, rinsed with distiled water and surface disinfested by shaking for 20 min in 95% E1OH, for 20 min in 20% clorox® (1.05% sodium hypochloride) with tween 20 (three drops per 100 ml), and then rinsed with sterile water three times. Seeds 29were placed in sterile, disposable petri dishes (60 x 15 mm) containing 15 ml of Murashige and Skoog Minimal Organics medium (4.43 g/l, MSMO, Sigma Chemical Co., St. Louis, MO; Murashige and Skoog, 1962) with 30 g/l sucrose, 8.0 g/l Difco Bacto-agar (pH 5.7, autoclaved for 20 min at 121 °C and 105 kPa). Twenty-day-old seedlings were transferred to Magenta containers (GA-7 Vessel, Sigma Chemical Co., St. Louis, MO) containing liquid MSMO. Explants excised from 20- to 30-day-old plants (3 om in height) were leaf discs (7 mm in diameter), petioles (7-8 mm) and roots (2 om). Explants were placed in 96 x 25 mm shell vials containing 10 ml MSMO. Either BA (1,2,3,4 or 5 mgjl) or kinetin (1,2,3,4 or 5 mg/l) was added to the medium. Alll cultures were incubated at 25 °C under a 16-h photoperiod (cool-white fluorescent lights, 22-28 ymol m?s”). Shoot number and % explants producing shoots were recorded after 6 weeks for leaf discs and petioles, and after 8 weeks for root segments. Tests had 10 replications for each explant and the experiment was repeated three times Leaves (youngest three to four leaves of first-year rosette) were obtained from plants grown under greenhouse conditions in commercial potting soil. Leaves were washed with anti-bacterial soap, rinsed with distilled water, surface disinfested by agitating in 70% EtOH for 2 min, 20% clorox® with tween 20 (three drops per 100 mi) for 15 min, and subsequently rinsed with sterile distilled water three times. Leaf discs (9 mm, four per dish) were placed onto MSMO medium (15 mi per 60 x 15 mm sterile disposable petri dishes). After 1 week, single non-contaminated leaf discs were placed into 95 x 25 mm shell vials 30containing MSMO medium with combinations of auxin and cytokinin: BA (3 mg/l) and 2,4-D (0.1, 0.5 or 1 mg/l); BA (3 mg/l) and NAA (1, 3 or 5 mg/l). Shoot number and percent explants producing shoots were recorded after 6 weeks. There were 10 replications and the experiment was repeated twice. Well-developed shoots were excised from the various explants and subcultured for 15 days on the same initiation medium. Shoots were then separated individually and placed on rooting medium containing MSMO and various levels of either 2,4-D (0.1, 0.5 or 1 mg/l) or NAA (1, 3 or 5 mg/l). Shoots were subcultured onto medium containing the same auxin used to induce root formation. After 5 weeks, the number of roots and % of explants producing roots were recorded. There were 20 replications and the experiment was repeated three times. Rooted explants were transferred to vermiculate (Patterson Vermiculate Co., Enoree, SC) in Magenta containers and after 3 weeks transferred to 4.5 inch-plastic pots containing potting soil (Garden Magic® Potting Soil containing peat moss). Flow chart of the experiments is given in Figure 2.1. Data was subjected to ANOVA and Duncan's Multiple Range Test (SAS, 1996). 31PLANT SOURCE ‘Seedlings from sterilized seeds) (Plants grown under greenhouse condition) EXPLANTS (Leaf Discs, Petioles, Roots) (Leaf Discs) SHOOT FORMATION 1 ROOTING ! PLANTLETS Figure 2.1. Protocol for in vitro culture of Verbascum thapsus L. (common mullein). 322.3. Results and Discussion Verbascum thapsus L. is a widely-used medicinal herb, but there has been almost no interest in the in vitro propagation of this species. We, therefore, aimed at developing a reliable protocol for in vitro regeneration of V. thapsus L. and thus examined the effects of several combinations of plant growth regulators using different explants. Our initial attempts to sterilize explants taken from field-grown plants were unsuccessful due to significant contamination on the leaves, mostly located among dense and woolly hairs. We then had to germinate seeds under aseptic conditions to obtain a sterile source of explant. When leaf disc, petiole and root explants were cultured on MSMO medium containing 1-5 mg/l BA or kinetin, all explants tested formed shoots with either BA or kinetin (Table 2.1). The greatest number of shoots per explant and percentage of leaf and petiole explants forming shoots was observed on media with 3.0 mg/l BA (Table 2.1; Figure 2.2a,b). It was clear that the BA induced more shoot formation than kinetin at all concentrations regardless of the explant type. Root segment explants formed fewer shoots than leaf and petiole explants. In addition, root explants were considerably slower in shoot induction since the majority of the leaf and petiole explants had already formed shoots by 6 weeks while it took 8 weeks to see root explants formed shoots. Although we 33UIUYIM su9y9] Bes a4} YIM SUea\y “squawuedxe sad sayeoyjdei 0} YUM sjueuiadxe sary) Jo URAL ay) “GO'00.05. Sample Saponin-1 content (mg/g) Mean + SE Leaves from field-grown plant 0.198 + 0.024% Leaves from in vitro cultured plant 0.081 + 0.012" Commercially obtained leaves 0.215 + 0.0147 Capsules from field-grown plants 0.016 + 0.001° 55level detected (0.016 mg/g tissue). The higher levels in the commercial product may be related to the method of harvest and product preparation. In vitro cultured leaf material was obtained from micropropagated plants in our laboratory (Turker et al, 2001). For HPLC experiments, leaves of micropropagated plants developed from leaf disc with 3 mg/l BA + 1 mg/l NAA were used. Secondary product synthesis by disorganized callus or cell suspension is typically low in un-manipulated cultures. In contrast to disorganized cultures the behavior of differentiated cultures is much more predictable (Parr, 1989). Shoot cultures often produce higher product levels and more conventional product profiles than their corresponding callus or suspension cultures (Stafford, 1991). But, sometimes the level of the detected secondary products in shoot cultures is lower than donor plants. For example, in Catharanthus roseus shoot cultures, the dimeric alkaloids have been detected, although at lower levels than those found in the leaves of the plant, while cell suspension cultures contained no trace of these complex structures Although tropane alkoloids were obtained from multiple shoot cultures of shoot tip, lateral bud or leaf discs of Atropa belladonna, detected level was about 7% of donor plant (Benjamin et al., 1987). Also, production of steroids obtained from shoot tip cultures of Digitalis species was much lower than those found in the donor plant (Seidel and Reinhard, 1987). If the secondary product synthesis is low, there are some procedures for enhancing productivity. Optimization of hormone regime is often effective. The type and concentration of 56phytohormones available to cultured cells is probably the most important factor influencing their potential for secondary product synthesis. For example 2,4-D can stimulate both cell division and cell expansion in many systems. However, it can also bring about a dramatic suppression of secondary product synthesis in cell cultures. Alterations in other environmental factors such as nutrient levels, light regime and temperature may also be effective in increasing productivity and reduced phosphate levels often stimulate product accumulation (Parr, 14989). Future studies will focus on isolation of saponins from V. thapsus L. with subsequent elucidation of their structures. Perhaps this information can assist in standardization of commercial samples and elucidation of active ingredients and their mode of action. 57CHAPTER FOUR Biological Activity of Common Mullein 4.1. Introduction As an antimicrobial susceptibility testing, the most widely used procedure is the disk diffusion method. Bacterial susceptibility to antimicrobial agents may be measured in vitro by utilizing the principles of agar diffusion. At the end of the 1950's, antimicrobial susceptibility testing was marked by lack of an acceptable standardized procedure, which led to variable results depending on which laboratory was used (Atlas, 1988). The disc diffusion procedure (Kirby-Bauer method) (Barry and Thornsberry, 1985) has been accepted by the Food and Drug Administration (FDA) and as a standard by the National Committee for Clinical Laboratory Standards (NCCLS). Reasonably accurate and precise results can be obtained with Kirby-Bauer procedure, which all-procedural details are carefully standardized and controlled (Barry and Thornsberry, 1985). The qualitative susceptibility of microorganisms to antimicrobial agents can be determined on agar plates by using filter paper disks impregnated with antimicrobial agents. The Kirby-Bauer test system is a standardized antimicrobial susceptibility procedure in which a culture is inoculated onto the surface of Muller-Hinton agar, followed by the addition of antibiotic impregnated disks to the agar surface. In such agar diffusion methods the antibiotics diffuse into the agar, establishing a concentration gradient. A clear area (zone of inhibition) indicates inhibition of microbial growth around the antibiotic disk. The diameter of the zone of inhibition reflects the solubility properties of the 58particular antibiotic; that is, the concentration gradient established by diffusion of the antibiotic into the agar, and the sensitivity of the given microorganism to the specific antibiotic. Standardized zones for each antibiotic disk have been established to determine whether the microorganism is sensitive (S), intermediately sensitive (|), or resistant (R) to the particular antibiotic (Appendix A). Kirby-Bauer agar diffusion test procedure is designated for use with rapidly growing bacteria. It is not directly applicable to filamentous fungi, anaerobes, or slow-growing bacteria (Atlas, 1988). The disk diffusion method has been employed by using plant extracts in antibacterial susceptibility testing (Caceres et al., 1991; McCutcheon et al., 1992; Mousa, 1994; Mongelli, 1995; Essawi and Srour, 2000; Kelmanson, 2000; Samy and Ignacimuthu, 2000). For antibacterial assay in our study, bacteria were chosen according to medicinal properties of common mullein (Appendix B).. Crown gall is a neoplastic disease of plants induced by specific strains of Agrobacterium tumefaciens, a Gram-negative bacterium. The bacterium contains a large Ti (tumor-inducing) plasmid, which carties genetic information (T-DNA) that transforms normal, wounded, plant cells into autonomous tumor cells (McLaughlin, 1991). In 1980, Galsky et al. (1980; 1981) demonstrated that inhibition of crown gall tumors on discs of potato tubers showed an apparent correlation with compounds and plant extracts known to be active in the 3PS (in vivo, murine leukemia) antitumor assay (McLaughlin et al., 1998). Later, Ferrigini et al. (1982) modified the Galsky potato disc method (Galsky et al., 1980; 1981) and tested the effectiveness, with appropriate statistical evaluation, 59of the modified assay as a prescreen for 3PS activity in crude plant extracts (McLaughiin et al., 1991; McLaughlin et al., 1993). Finally, Ferrigini et al. (1982) concluded that crown gall tumors on potato discs could routinely be employed as a comparatively rapid, inexpensive, safe, animal-sparing, and statistically reliable prescreen for in vivo 3PS antitumor activity. Bioactive compounds are almost always toxic in high doses Pharmacology is simply toxicology at a lower dose, or toxicology is simply pharmacology at a higher dose. Thus, in vivo lethality in a simple zoologic organism can be used as a convenient monitor for screening and fractionation in the discovery of new bioactive natural products. The eggs of brine shrimp, Artemia salina (Leach) are readily available in pet shops at low cost and remain viable for years in the dry state. Upon being placed in seawater, the eggs hatch within 48 hours to provide large numbers of larvae (nauplii) for experimental use. A positive correlation was found between brine shrimp toxicity and 9KB (human nasopharyngeal carcinoma) cytotoxicity, and now the brine shrimp test has been used as a prescreen for a panel of six human solid tumor cell lines, at the Cell Culture Laboratory of Purdue Cancer Center (McLaughlin et al., 1998). It is possible to detect and then monitor the fractionation of cytotoxic, as well as 3PS (in vivo murine leukemia) active extracts using the brine shrimp lethality bioassay rather than more tedious and expensive in vitro and in vivo antitumor assays. The brine shrimp assay has the advantages of being rapid (24 hours), inexpensive, and simple. It easily utilizes a large number of organisms for statistical validation and requires no special equipment and a relatively small 60amount of sample (50 mg for crude extracts). Active compounds thus obtained could then be subjected to more elaborate bioassays for specific pharmacologic activities (Meyer et al., 1982). Furthermore, it does not require animal serum as is needed for cytotoxicities. Animal rights advocates have not yet objected to the use of these invertebrates in experimental work (McLaughlin et al., 1991). Allelopathy is an anti-competition mechanism in plants and described as inhibition of the germination, growth or reproduction of an organism affected by chemical substances released from another organism. Weed species are frequently considered to be competitive because they show vigorous growth in crops and reduce crop yield. An ideal weed should show strong interspecific competition via special mechanisms such as allelopathic processes Allelopathic chemicals may play an important role in determining the persistence and abundance of weed species in mixtures of plants (Pardo et al., 1998) Radish seed bioassay is suggested in order to demonstrate the effects of allelochemics on seed germination and to evaluate the allelopathic potential of V. thapsus L. (Patterson, 1986) In spite of the use of mullein in folk medicine, many of the medicinal activities of mullein are not supported by scientific studies. The objective of this study was to assess the biological activity of common mullein extracts using selected bioassays including antibacterial, antitumor and toxicity (brine shrimp and radish seed bioassays). The results of these bioassays were already submitted for publication (Tuirker and Camper, 2002). 614.2. Materials and Methods 4.2.1, Plant material and extraction Plant materials were extracted with different solvents (MeOH, EtOH and water) and their biological activities assessed. For extractions, three different sources of leaves (field-grown, in vitro cultured and commercially obtained) and capsules (from field-grown plants) were used. Leaves and capsules from field- grown plants were collected from native mullein plants between August and September of 2000. Leaves of in vitro cultured plants were collected from mullein plants that were previously micropropagated in our laboratory (Turker et al., 2001). In addition, mullein leaves were purchased from a health food store. Commercially obtained leaves were dried and pulverized by Frontier Herbal Company, but field-grown leaves, capsules and in vitro cultured leaves were dried in oven at 60 °C and then ground into a powder. In addition, commercial products of common mullein (tea bags, swallow capsules, an alcoholic extract and flower oil) were purchased from health food stores. Purified saponins were also included in the bioassays for comparison. Treatments, plant materials and extraction procedures are summarized in Table 4.1. 62“y uluodesisuem|| UISIINW :O4 eNKe plaly :O4 ‘sereaj paureygo AI | deg ‘uuodes oing :d deg Syeq elfen wioy uluodeg :D deg ‘pioe oz1yUhofIO :AID joyooiy “av ‘aInsdeo moljemg :9¢ ‘sjonpoud pauleyqo JOLIN :19 ‘seAea| painyno ay/A Uj °7] {UOYOONEG :9a ‘uOIsNyU| {Jul ‘seAee| Piel 14 « (SIOMOY, OIBUILUOD :WOD ‘Beg eal 'a ‘sainsdeo TUTE TT TOS OG) SIEM HORN UI UOHAIOS OOS WON D SUBHT EuEIMITONS WOH Y UVOEESSURNT Tees ‘(qeuibig) uNDdeS sing aes (juy6u |) 203m ur uoanios 01g nuodes odes 2 MOA, Aig__suuodes snouen 18 BAIS SINT PDEINS SOROS SPE RUSH", JOHVOIS) HO 765 Uk WOS-O4 WoRRIORT SATOH TTT) UCSTE TEAS-GP UI PEISEINS SANES| USAT BUT SqRH BRO) TOUODTE U EAN Ur wosa¥ TU G6 10) PUETS Pue JeIeM pajoq Jo W Og Uy SayNSdeS USSU Tq FeIE|OS) SINSTED MOTENS UF Wog-0S “TN 10} pUETS PUR TayeM aja jo TU OB UI paoeId seq ea) SOIL TaeaIED BAL GSH Hoyas) Beg Goh uk WOI-GL.sjonpord fe1eusw09 (weduiOg TequoIg) (B OF) Senee] jeRVAUILOD BeG-10 unnoea sepun ju! 08 0} paonpas JO}EMA "UW OF 40} pojog pue Jejem partisip 1 QOOL Ul paveid-uoH}I0O3q (6 oy) saneay paimyno oxy uy (6 oy) sonea) piel TUNROBA TOpUN TU OB OF PETNS! TIEN ue uly OF JO} 49}2m pa}OG jw DOL Ul PaDeId-UOISTYUL (6 op) soneat pies eI JOT HueaTOD FaWaI (OFT 56K -JaJeM |W 08 UI PEAIOSSIp enpiseu pue wNnoeA JepUN (6 op) saneay payelodena HOI (Xe) 0, 0S 1 HOIS %OS WM Pees (6 oy) soneay pias ea HOI “JVEM |W OB Ul PaAjOssip eNpises TE OF) seinsdes pisiz HOSW-O4 pue wnnoea Jepun payesodens HOS “S14 > (Auedwo9 senuois) (6 Oy) sanzay jenseuw0D HOeW-19 40} (Xe) HOBW Wim peyDeNK UO ‘papleOsIp pue Sy E (6 op) senza, paimro ona Uy Ho=W- 20} (XE) Wi0jo10140 YAP PEYDELND YOHOS (6 or) seneei piel HoAN-14 72243 HOON ‘unpadoig WORSEN qweunees, ui steuaIeW _.uoneUBISeG TEHOIeW 3891 ‘senpaooid uoqoenxe s18u) pue sKesseoig ul pasn suluodes jualayip pue s}oes}xe Jo uoneUbISEG Jy 81GeL 634.2.2. Antibacterial assay The disc diffusion assay (Kirby-Bauer method) was used to screen for antibiotic activity (Prescott et al., 1990). Six bacterial strains were used in the screening: Escherichia coli (ATCC®25922), Pseudomonas aeruginosa (ATCC®27853), and Klebsiella pneumoniae (ATCC®13883) which are Gram- Negative bacteria and Streptococcus pyogenes (ATCC°19615), Staphylococcus aureus (ATCC®25923), Staphylococcus epidermidis (ATCC®12228) which are Gram-Positive bacteria. Bactrol™ disks (Difco Laboratories) containing different bacterial strains were transferred to test tubes containing 2 mi of Tryptic Soy Broth (TSB) and incubated for 3 hours at 37 °C. After 3 hours, one bacteriological loop from each broth was streaked on Tryptic Soy Agar (TSA) plates and incubated for 2 days at 37 °C. After 2 days, a single colony was removed and streaked on a new TSA plate and incubated 37 °C for 2 additional days. After 2 days, 4-5 loops of pure culture were transferred to 20 ml of TSB in a test tube for each bacterial strain and incubated overnight at 37 °C; then stock bacterial suspensions (100-200 jl) were transferred to test tubes containing 2 ml of TSB and turbidity was measured at 625 nm. For Gram (-) bacteria, suspensions were adjusted to absorbance values of 0.08+0.02 and for Gram (+), 0.140.02. These absorbance values correspond to a 0.5 Mac Farland standard. This turbidity standard is used in hospitals for antimicrobial susceptibility testing and endorsed by the National Committee for Clinical Laboratory Standards (NCCLS) (Coker, 1999). 64A sterile cotton swab was dipped into the standardized bacterial suspension and used to evenly streak the entire surface of a (60 X 15 mm) Mueller-Hinton (M-H) agar plate (one test tube was used for each agar plate). Agar plates were streaked three times, each time tuming the plate at a 60° angle and finally rubbing the swab through the edge of the plate. All extracts were sterilized by filtering through a 0.22 um filter (Pal-Gelman Laboratory). Filter paper discs (Glass microfibre filters, Whatman®; 7 mm in diameter) were soaked in the extract and then blotted on a sterile paper towel and placed on the inoculated plates. There were four replicates in each plate and two plates for each extract tested for each bacterium. Positive controls consisted of five different BBL™ Sensi-Disc™ antimicrobial susceptibility test discs (Becton Dickinson Microbiology Systems): erythromycin (15 1g) (E-15), cephalothin (30 1g) (CF-30), carbenicillin (100 1g) (CB-100), sulfisoxazole (2.0 mg) (G-25) and chloramphenicol (30 1g) (C-30) (Appendix A). Four antibiotic discs were used for each plate and run in duplicate. Negative controls consisted of water, MeOH, 50% MeOH and 50% EtOH. Inoculated plates with disks were placed in a 37 °C incubator. After 16 to 18 hrs of incubation, inhibition zone diameter (mm) was measured. All experiments were repeated three times. 4.2.3. Anti-tumor assay Antitumor activity of mullein extracts was assessed with the potato disc method as modified by McLaughlin's group (Fertigini et al., 1982). Agrobacterium tumefaciens (strain B6) was cultured on Yeast Extract Media un nORod ge A yMANTASHON(YEM) for 2-3 days at 28 °C. Camptothecin (Sigma) (tumor suppressant) served as a positive control. All extracts were aqueous; therefore, water was used as a negative control. As a second negative control, 50% MeOH was also used for ilwensisaponin A and commercial products. Six to seven loops of A. tumefaciens were added to 10 ml phosphate buffered saline (PBS) (pH=7.2). Suspensions of A. tumefaciens in PBS were standardized to 1.0 X 10° colony forming units (CFU) as determined by an absorbance value of 0.96+0.02 at 600 nm. Inoculum: All extracts, controls and solutions were filter sterilized (sterile 0.22 ym filter, Pal-Gelman Laboratory). The following design was followed: Positive control: 600 yl camptothecin stock + 150 ul sterile distilled water + 750 ul A.tumefaciens in PBS; Solvent control |: 600 11! control (water, 50% MeOH or 50% EtOH) + 150 ul sterile distilled water + 750 pl A. tumefaciens in PBS; Solvent control Il: 600 ul control (water, 50% MeOH or 50% EtOH) + 150 yl sterile distilled water + 750 jl PBS; Test extracts: 600 pl test extract + 150 ul sterile distilled water + 750 jl A.tumefaciens in PBS. Red-skinned potatoes (Solanum tuberosum L. var. Red Russett) were scrubbed with a brush under running water and surface sterilized by immersion in 10% clorox for 20 min. Tubers were then placed on sterile petri dishes and cut along either side revealing the largest flat surface area available; tubers were immersed in 20% clorox for 15 min and then placed on sterile petri dishes. Cylinders (10 mm diameter) were cut from the center of potato tissue (skin portion was eliminated) and placed in sterile distilled water. Cylinders were 66rinsed twice more. Each cylinder was cut into 0.5 cm discs after excluding 1 cm end pieces. These discs were transferred to 24-well culture plates containing water-agar (15 g/l). One well plate (24 cell wells) was used for each experiment; each experiment had 24 replicates. Each disc was overlaid with 50 ul of appropriate inoculum. No more than 30 min elapsed between cutting the potato discs and inoculation (McLaughlin, 1991). Plates were incubated at room temperature (25 °C) in the dark for 2 weeks. After 2 weeks, disks were stained with Lugol's reagent (I2KlI; 5% lp plus 10% KI in distilled water) and tumors on each disc were counted. Lugol's reagent stains the starch in potato tissue to dark blue to dark brown color, but the tumors do not take up the stain and appear creamy to orange. Experiments were repeated three times. Percent inhibition of tumors was calculated (McLaughlin, 1991; McLaughlin et al., 1993; McLaughlin and Rogers, 1998) Significant activity was indicated when two or more independent assays gave consistently negative values of approximately 20% or greater inhibition. 4.2.4, Brine shrimp bioassay Brine shrimp (Artemia salina Leach) eggs (San Francisco Bay Brand, Inc.) were purchased from a pet store. Seawater was prepared by dissolving 36 g sea salt (Instant Ocean Salt Mix, Aquatic Eco-Systems, Inc. Florida) in 1 liter of distilled water and put in V-shaped, Plexiglas hatching container. Oxygen was supplied and 60-watt lamp was positioned near the container to provide direct light and heat (~ 27-28 °C). One-teaspoon brine shrimp eggs were 67placed in 1 liter of seawater. After 10 to 12 hr, cysts began hatching, Two days was allowed for the shrimp to hatch and mature as nauplii (Figure 4.1) (shrimp can be used 48-72 hrs after the initiation of hatching). After 72 hr they should be discarded (Mclaughlin et al., 1991), Nauplii were harvested by turning off the aeration and letting the culture settle for about 10 min, Hatched, empty shells floated on the surface and unhatched cysts sank to the bottom. Newly hatched nauplii concentrated just above the unhatched cysts on the bottom. Since the nauplii are positively phototropic (attracted to light), shining a light at the middle of the container and shading the container at the bottom helped direct them’ to an area where they can be easily harvested by siphoning or draining (Hoff and Snell, 1987). Stock solutions of extracts and other commercial products (Table 4.1) were prepared by suspending dried extract in saltwater to prepare a 10,000 mg/ml solution. The suspension was mixed for 5 min; then, 1000, 100 and 10 mg/l solutions were prepared by dilution. Twenty-four-well culture plates were used. A suspension of nauplii was removed and 10 nauplii were placed into each well and 2.5 ml of appropriate concentration of extract+salt mixture was added. Uncovered plates were incubated for 24 hr at room temperature under illumination. There were three replicates for each concentration (10,000, 1000, 100, 10 mg/l) and experiments were repeated three times. The same saline solution used to prepare the stock test sample solution was used as a negative control. As a positive control, MS-222 (tricaine methane sulfonate, Aquatic Eco- Systems, Inc. Florida), a common fish anesthesizer, was used at concentrations of 1, 10, 100, 1000 mg/l. 68