Biological Activities and Cytotoxicity of Leaf Extracts from Plants of the Genus Rhododendron

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From Ethnomedicine to Application: Biological Activities and Cytotoxicity of Leaf Extracts from Plants of the Genus Rhododendron

Ahmed Rezk

a Thesis submitted in partial fulfillment of the requirements for the degree of

Doctor of Philosophy in Biochemistry

Approved Dissertation Committee

Prof. Dr. Matthias Ullrich, Prof. of Microbiology Prof. Dr. Klaudia Brix, Prof. of Cell Biology Jacobs University Bremen

Prof. Dr. Nikolai Kuhnert Prof. of Chemistry Jacobs University Bremen

Prof. Dr. Dirk Albach, Prof. of Plant Biodiversity University of Oldenburg

Date of Defense: 15.06.2015

This PhD thesis project was financed by Stiftung Rhododendronpark Bremen

Dedicated to: My Wife Rasha

Acknowledgment

First, I thank Allah for giving me the ability and strength to accomplish this study. I would like to express my gratitude to the following people for support during my work:

I would like to express my sincere appreciation and gratitude to my PhD supervisors, Prof. Dr. Matthias Ullrich, and Prof. Dr. Klaudia Brix, who gave me the opportunity to compose my doctoral thesis in their workgroups. I would like to thank them for their support, guidance and all the time they gave to discuss and help in designing experiments to achieve this work.

I would also like to thank my dissertation committee members, Prof. Dr. Nikolai Kuhnert and Prof. Dr. Dirk Albach for their time and for their valuable comments during our meetings and reviewing my thesis.

I would specifically like to thank AG Ullrich and AG Brix lab members, Amna Mehmood, Antje Stahl, Gabriela Alfaro-Espinoza, Khaled Abdallah, Neha Kumari, Maria Qatato, Joanna Szumska, and Jonas Weber for maintaining a friendly and family working environment. I want to thank my lab rotation students Alaa Al-Hashimi and Warren John for their contributions in my project.

Sincere thanks for Maren Rehders, who guided me in all cell biology methods and her great help for discussing results and all technical issues in the lab. I cannot forget our great team assistants Maike Last and Nina Böttcher for their technical helps.

I am thankful to the Stiftung Bremer Rhododendronpark and Jacobs University Bremen for providing the financial support throughout the period of my studies. I am particularly grateful to Mr. Wolfgang Klunker for his enthusiastic support. I cannot forget to thank Dr. Hartwig Schepker for providing plant materials and discuss the original habitat of Rhododendron.

Last but not least, I have great pleasure to express my deep sense of gratitude towards the woman who supported and stood by me in all circumstances, my wife Rasha El-Abassy. A very warm and affectionate thanks to my lovely daughters Judy and Lena.

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Table of contents

Table of Contents

Acknowledgment ...... 3

Abstract ...... 6

1. Introduction ...... 8

1.1. Historical Background ...... 8

1.2. Rhododendron Classification and Description ...... 8

1.3. Habitat and distribution of genus Rhododendron ...... 10

1.4. Use of Rhododendron in Traditional Medicine ...... 11

1.5. Plant metabolites ...... 14

1.6. Plant Secondary Metabolites as Drugs ...... 16

1.7. Antibiotics and Bacterial Defense Mechanisms ...... 19

1.8. Drug development ...... 21

1.9. Cell death ...... 22

1.9.1 Necrosis ...... 23

1.9.2 Apoptosis ...... 23

2. Aims of the study ...... 28

3. Results...... 29

3.1 ...... Phylogenetic spectrum and analysis of antibacterial activities of leaf extracts from plants of the genus Rhododendron ...... 31

3.2 ...... Assessment of Cytotoxicity Exerted by Leaf Extracts from Plants of the Genus Rhododendron towards Epidermal Keratinocytes and Intestine Epithelial Cells ...... 56

3.3 ...... Distinguishing the polyphenolic and antibacterial profile of the leaves, fruits and flowers of Rhododendron ambiguum and Rhododendron cinnabarinum using high performance liquid chromatography coupled with ion trap and time of flight mass spectrometry ...... 102

4. Discussion ...... 119

5. References ...... 125

6. Appendix ...... 139

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Abstract

The evolution of bacterial resistance to current antibiotics is one of the biggest threats to human health. There is an increasing interest to identify novel antimicrobial compounds from various natural sources. Plants-derived compounds are used as ingredients in traditional treatment of numerous human disorders including infectious diseases caused by pathogenic microorganisms. In planta, secondary metabolites like the polyphenols are known to act as potent antimicrobial agents against several plant pathogens. Plants of the genus Rhododendron are typically used in a range of ethno-medical applications. There are more than 1,000 Rhododendron species growing/spreading/distributed particularly in the Northern hemisphere. The City of Bremen harbors the Rhododendron-Park in which approximately 600 different species of Rhododendron and hybrids are grown. This enables research with about two thirds of all known Rhododendron species.

The aim of this study is to identify novel compounds from the secondary metabolite pool synthesized by Rhododendron that can be used as antimicrobial treatments of human diseases/maladies in form of ectopic application or as orally administered drugs. Therefore, leaf extracts of a total of 120 different Rhododendron species were tested using the agar diffusion assay towards twenty-six bacterial species representing different taxonomic clades of non- pathogenic strains Gram-positive and Gram-negative bacteria. The possible cytotoxic effects of the most promising, antimicrobial bioactive extracts from Rhododendron species were assessed in a concentration-dependent manner using epidermal keratinocytes of the skin and epithelial cells of the intestinal mucosa, respectively, as target cell systems.

The leaf extracts of 17 Rhododendron species exhibited significant growth-inhibiting activities against Gram-positive bacteria. In contrast, only very few of the leaf extracts affected the growth of Gram-negative bacteria. All leaf extracts with antimicrobial bioactivity were extracted from representatives of the subgenus Rhododendron, with 15 from the sub-section Rhododendron and two belonging to the section Pogonanthum. Six Rhododendron species out of the 17 species showed the lowest minimum inhibitory concentration with 50 µg leaf extract powder per mL solvent. Equally low and moderate concentrations (50 µg/ml) of leaf extracts from three of these plant species were non-toxic towards both mammalian cell lines, i.e. HaCaT keratinocytes and IEC6 enterocytes. However, higher doses such as 500 µg/mL of Rhododendron leaf extracts were potent in negatively affecting both, keratinocytes and, particularly, the intestine epithelial cells.

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We conclude that bioactive compounds with high antimicrobial activities can be extracted from the leaves of Rhododendron species mainly belonging to the subgenus Rhododendron, and that they acted mostly against Gram-positive organisms. The leaf extracts from R. minus, R. ferrugineum, and R. racemosum applied at a concentration of 50 µg/ml proved safe to be used in 24-h-incubations of monolayer cultures of both, HaCaT keratinocytes and IEC6 intestine epithelial cells. In contrast, high doses of most leaf extracts induced apoptosis evidenced by a significant increase in the levels of active caspase-3 in IEC6 intestine epithelial cells. Finally, keratinocytes proved more resistant than intestine epithelial cells against cytotoxicity exerted by leaf extracts of a large variety of Rhododendron species.

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1. Introduction

1.1. Historical Background

The name “Rhododendron” derives from the ancient Greek words “rhodo” that means rose and “dendron” meaning tree. The Rhododendron genus belongs to the Ericaceae which are woody plants. The first record of Rhododendron can be found in an artifact dated 401 B.C. on the Turkish coast of the Black Sea, when the army of Xenophon was retreating from Babylon. The soldiers were poisoned from eating a large quantity of honey made from toxic nectar of a yellow flowered, pontic Azalea, Rhododendron luteum [1]. The second known disaster took place with the troops of Alexander the Great of Macedonia on their way to India in 327 B.C. That time the perniciousness probably came from consuming honey made from R. afghanicum. Later, in 66 B.C., the armies of Pompey were poisoned during the war against King Mithridates of Pontus on the Black Sea coast of Turkey [2].

The earliest phylogenetic record for Rhododendron goes back roughly 50 million years ago at the Early Tertiary in Alaska, where the first fossils of Rhododendron leaves were discovered [3]. The history of Rhododendron cultivation goes back to ancient times with first records mentioned around AD 750. The first known cultivation of Rhododendron was in 1650 for R. hirsutum after seed material had been introduced from India and Nepal to the United Kingdom [4]. The evergreen azaleas have the longest history of hybridization, particularly those from Japan [5]. Most of the currently known Rhododendron species have been identified, and hybrids thereof have been generated within the last two centuries. Most of the species later used for hybrid generation and to develop ornamental garden plants were introduced to Europe and North America during the time period from 1790 to 1935 [6]. In the year 1792, R. luteum Sweet, a member of deciduous azaleas, was introduced to England and Western Europe. This was the basis of the first Azalea pontica hybrids. The first evergreen azaleas were introduced to England from China in 1833. On the other hand, in Germany, Jacobs Rinz started his hybridizing work for deciduous azaleas in 1834 [6].

1.2. Rhododendron Classification and Description

The genus Rhododendron belongs to the division Angiospermae, the sub-division Dicotyledoneae, the class Metachlamydeae, the order Ericales, the family Ericaceae, and the subfamily Rhododendroideae. The first description of Rhododendron has been achieved by the

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Swedish scientist Carl Linnaeus in his work termed Genera Plantarum in 1737 [7]. Initially when Linnaeus coined/classified the botanical group called genus Rhododendron, he recorded a separate genus for Azalea containing six species. However, in 1796 Salisbury proposed that Azalea and Rhododendron belong to the same genus, since the differences between them are not considered to be large enough to justify their separation into distinct genera [8]. Today, the most accepted phylogenetic system used for the taxonomic classification of Rhododendron is that of Chamberlain published in 1996 [9]. Rhododendron species are systematized in subgenera, sections, and subsections (Fig. 1). This systematization was based on the morphological synapomorphies i.e. sharing the same traits, which led to four large and four small subgenera as outlined here:

• Subgenus Rhododendron L.: several hundred species

• Subgenus Hymenanthes K.Koch: about 140 species

• Subgenus Pentanthera G. Don: 25 species

• Subgenus Tsutsusi: 15 species

• Subgenus Azaleastrum Planch: five species

• Subgenus Candidastrum Philipson & Philipson: one species

• Subgenus Mumeazalea: one species

• Subgenus Therorhodion: one species

There are a number of differences between azaleas and other members of the Rhododendron genus. The leaf structures differ, i.e. azalea leaves harbor long straight hairs which are parallel to the underside of the leaf surface. Nearly all Rhododendron species are characterized by so- called scales, which are tiny round structures on the underside of the leaf. These scales are characteristic features for the usually small leaves of the subgenus Rhododendron, which are also known as “lepidotes”. In contrast, these scales are absent in the rather large leaves of the subgenus Hymenanthes also termed “elepidotes”. Another distinctive feature is the number of stamens per flower differs with Rhododendron having ten stamens while most azaleas have flowers with only five or six stamens.

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Figure 1: The taxonomic organization of the genus Rhododendron at the levels of subgenera, sections, subsections, and species. Only representative examples are given.

1.3. Habitat and distribution of genus Rhododendron

Rhododendron plants are original to mountainous areas characterized by well-drained and acidic soils, mild summer temperatures, and regular precipitation. Rhododendron plants are not common at low altitudes in hot equatorial forests, and rare or absent in desserts, grasslands, or boreal forests, except for R. lapponicum, which has adapted to the extreme climate of the arctic regions. Rhododendron plants are abundant in the tropical mountains of the Indonesian archipelago. In comparison to many other woody plants, Rhododendron plants have low nutritional requirements. However, deficiency of nitrogen and iron as well as toxic levels of nitrate were noticed to cause chlorosis, and may result in the loss of young leaves [10]. The genus Rhododendron encompasses approximately one thousand species, 90% of which are indigenous to Asia. They are mainly distributed from the Northwestern Himalayas through

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Nepal, Northeastern India, Western and Central China, as well as in Vietnam, Thailand, Malaysia, Indonesia, and the Philippines [11] (Fig. 2). The latter tropical areas are populated by species belonging to the subgenus Rhododendron section Vireya. A small number of Rhododendron species is native to Europe and Northern America. The majority of the genus exists at moderate temperatures including the subgenus Hymenanthes [12]. Nowadays, many Rhododendron hybrids have become native to Europe and the USA as a consequence of crossbreeding between various Rhododendron species originally derived from Asia.

Figure 2. The worldwide distribution map of plants of the genus Rhododendron. The color code indicates the abundance of the genus Rhododendron from “high” to “moderate”. (Figure modified from Ref. [3] )

1.4. Use of Rhododendron in Traditional Medicine

In general, plants had been used as traditional treatments for numerous human diseases ever since prehistoric times. They are still used for these purposes by the vast majority of the human population all over the world. Several studies estimated the use of plants for health care dating back to at least the Middle of Paleolithic age [13]. According to the World Health Organization (WHO), almost 65% of the World’s population integrates medicinal plants into their primary health care needs [14, 15]. In developing countries about 80% of the population use traditional

11 | Page Introduction medicinal plants as the initial source of medicine [16]. Furthermore, at least 25% of modern pharmaceuticals contain compounds extracted from plant material or synthetic compounds that are produced by chemical industries based on the original compounds isolated from plants [17].

There are many purposes of using plants as medicinal agents. For instance, the pharmaceutically active compounds are extracted from plants for direct use as medicine. Alternatively, the whole plant or parts of it are used as a herbal remedy provided that there are no side effects or toxicity affecting patients upon long term therapeutic application [18]. Despite the huge number of higher plant species occurring worldwide (roughly 500,000) [19, 20], only 6% of them have been screened for biological activities and only about 1% has been evaluated by phytochemical means [18, 20].

Several species of Rhododendron are used as medicine in their native habitats. In Nepal, the leaves of R. anthopogon are used to alleviate liver disorders, or to treat common cold, lung problems, and sore throat [21]. Although young leaves of R. arboreum are known to be poisonous, they are used as medication by applying them on the forehead to relieve headache [22, 23]. The flowers of R. arboreum are crushed and snuffed to stop nasal bleeding [24]. The leaves of R. edgeworthii are used after distillation as a cure for skin diseases in West Kameng, India [25]. Several other Rhododendron species were recorded as having anti-inflammatory effects. R. ponticum is used to alleviate rheumatic pain and for treatment of inflammatory disease [26]. In addition, many other species are widely used for the same purpose, such as R. ferrugineum. R. chrysanthum and several other species listed in Table 1 were also reported to be used as therapy against rheumatoid disorders [27]. A few Rhododendron species are already introduced to the market as homeopathic medicine to treat symptoms of common diseases. For example, R. molle is used as an anti-hypertensive treatment and can be found with the trade name “Rhomitoxin” [28]. R. ferrugineum exists in German pharmacies in a combination with compounds isolated from other plant species as a bone pain relief agent called “Rhododendron cp” paste.

Several Rhododendron species were shown to exhibit antimicrobial effects against different microbes. The ethanolic and aqueous extracts of R. arborerum flowers revealed antibacterial activity against different bacteria such as Staphylococcus aureus and Escherichia coli [29]. The ethanolic leaf extract of R. setosum showed antibacterial activity against Gram-negative bacteria, i.e. E. coli, which was attributed to the presence of high concentrations of terpenoids [30]. Moreover, the essential oil from the flowers and leaves of R. anthopogen indicated a

12 | P a g e Introduction significant antibacterial activity against S. aureus, Bacillus subtilis and Enterococcus fecalis [21]. Using antifungal activity tests with a group of clinical Candida ssp. strains, it was demonstrated that Rhododendron oil was active against Candida pseudotropicalis [21] The authors claimed that the antimicrobial activity of the essential oil was due to high concentrations of hydrocarbons belonging to the monoterpenes, mainly α-pinene, β-pinene and limonene. Similarly, the hexane fraction of the aerial parts of R. campanulatum showed high antibacterial activity against S. aureus, B. subtilis, E. coli, and Salmonella typhi. The corresponding active fraction contained oleanane-type triterpene [31]. According to most recent phytochemical analyses, there are more than 200 different compounds isolated from different Rhododendron species with their majority being represented by flavonoids and diterpenoids [32]. The pharmacological properties of several of these compounds have been investigated including anti-inflammatory, antibacterial, antihyperglycemic, antioxidant and analgesic effects [33, 34].

Likewise, Rhododendron is also used for non-medicinal purposes. In Nepal, people use the wooden parts of this plant as house building material and fuel. The most common species that are used for such purposes are R. arboreum, R. barbatum, R. campanulatum, and R. cinnabarum. Furthermore, R. anthopogon is widely used in the Buddhist monasteries mixed with Juniper as incense [35].

Table 1. Traditional applications of selected Rhododendron species (Table modified from [32])

Species Folk medicine Plant part Region Reference

R. aureum rheumatoid arthritis, diarrhea, Leaves, flowers Korea, Russia, [36, 37] emesis, diuretic, gout Turkey, India

R. calendulaceum Rheumatism Twigs North America [38]

R. campanulatum sore throat, digestion, skin disease, whole plant India, Nepal [38, 39] rheumatism, cold, fever

R. ferrugineum Rheumatism, hypertension, muscle Leaves, flowers Europe [40] pain, migraine, diuretic, diarrhea, sore throat, cough, bronchitis, common cold, prostatic diseases

R. hirsutum Sore throat, bronchitis, rheumatism, Leaves, flowers Austria [32] common cold, lung disorders, cardio-vascular disease, gastrointestinal disorders, prostatic diseases

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R. lepidotum Fever, cough, cold, tonsillitis, Leaves, flowers Nepal, Bhutan, Tibet, [41] headache, stomachic, bile and lung India disease, back pain, blood disorders, bone disease

R. luteum Diuretic, analgesic in rheumatic Leaves Turkey [42] pains, fungal foot infections

R. micranthum Cough, chronic tracheitis, regulates Unspecified China [43] menstruation and relieves pain

R. molle Analgesic, anesthetic, rheumatism, Flowers, fruits, China [44] arthritis, hypertension, migraine, roots itch, toothache, narcotic

R. mucronulatum Fever, asthma, cough, dysuria, Leaves, bark China, Korea, Russia [45] chronic tracheitis, tonic, tuberculosis, rheumatism, neuralgia

R. ponticum Edema, common cold, toothache, Leaves, stems Turkey [26] rheumatic pain, diuretic, fungal foot infections, narcotic

R. schlippenbachii Hypertension, cholinesterase Leaves Korea [46] inhibitor, discharge of cardiotonic phlegm

R. simsii Bronchitis, cough, amenorrhea, Unspecified China [47] antiallergic, diarrhea

R. spinuliferum Phlegm, cough, asthma Leaves, flowers, China [48] stems

R. tomentosum inflammation, abortifacient, Leaves China, Korea, Japan, [49, 50] diuretic, lactagogue, narcotic, Russia, Poland, arthrosis, rheumatism, bronchitis, North America cough, fever, sore throat, dysentery, gout, itch, tuberculosis, tonic, eye problems, dry skin, diarrhea

1.5. Plant metabolites

Plant metabolites are organic compounds synthesized by plants for different purposes. Those compounds can be divided into two major classes, primary metabolites (PM) and secondary metabolites (SM). PM are basic compounds for many essential functions in plant metabolism, for instance, in cell division, growth, sexual reproduction, anabolism and catabolism, as well

14 | P a g e Introduction as in energy acquisition and respiration. PM are often found as substrates, intermediates, or products of biosynthesis pathways for essential components of living organisms, such as amino acids or nucleotides. In contrast, SM have initially been defined as “waste products” that are not necessary for the living cells because they are not found in every species, and are often linked with their occurrence in distinct taxonomic groups [51]. The overall number of distinct SM from plants is not known but is estimated to exceed 200,000 [52].

SM have important functions in the plant’s life cycle because they might serve as attractants for pollinators (odor, color, and taste), protect plants against being infected by microbes (i.e. bacteria, fungi, and viruses), or act against herbivores [53]. In general, plants produce SM in response to environmental biotic or abiotic stimuli or stresses. Many of these compounds may be assigned to the groups of phytoalexins or phytoanticipins, respectively [54]. Phytoalexins are defense metabolites synthesized de novo when a plant gets attacked by a microbial pathogen or herbivore. They are the basis of disease resistance mechanisms and often lead to an active response during plant-microbe interactions. In contrast, phytoanticipins are usually constitutively synthesized and thus serve as the basis of resistance mechanisms prior to an attack [55]. However, their production can increase in response to different types of stress.

SM can be furthermore sub-divided into distinct groups based on their chemical lead structure(s) or biosynthesis pathway(s) [56]. The three main structural classes of SM are terpenoids, alkaloids, and phenolics. Additionally, there are many other specific classes of SM chemicals less abundant and potentially occurring only in specific plant families or species. Terpenoids constitute the largest class of SM and belong to the large family of isoprenoid- based/derived compounds, which are made of variable number of the C5 isoprene units. Interestingly, all terpenoids are derived from the same type of building block and are synthesized originally from two main pathways, the mevalonate and the deoxyxylulose phosphate pathway (Fig. 3). Terpenoids are sub-divided according to the number of C5 isoprene units with monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), triterpenes (C30), tetraterpenes (C40), and polyterpenes, respectively [57]. The second largest SM class is chemically represented by alkaloids, which typically contain one or more heterocyclic nitrogen atoms and which are usually synthesized from one of the amino acids lysine, tyrosine or tryptophan [58]. Depending on their ring structures, alkaloids can be sub-divided into various groups such as quinolizidine, quinolone, acridone, piperidine, pyrrolidine, and many others. Alkaloids are synthesized and derived from numerous pathways such as shikimate or nicotinate pathway [59]. The third large group of SM is composed of phenolic compounds, which are

15 | Page Introduction classified according to the number of their phenolic ring structures and carbon chains, such as, i.e., flavonoids, coumarins, or tannins. The majority of phenolic compounds are synthesized through the phenylpropananoid pathway fueled by the products of the shikimic acid pathway (Fig. 4) [60].

1.6. Plant Secondary Metabolites as Drugs

Due to the high diversity and the unique structural differences, SM are prone to be considered for different medical or physiological purposes. Within this thesis, plants shall be investigated as a source of drug agents irrespective of the acknowledgement of a significantly high number of drugs previously developed from different organismal sources, e.g., bacteria, fungi or various marine resources such as algae, sponges, or ascidians [61]. Plant SM are a significant source of new drugs, especially as anticancer, anti-inflammatory, antimicrobial, antihypertensive, or anti- neurodegenerative compounds [62]. Historically, an impressive example for such a plant SM discovery is the synthesis of the anti-inflammatory agent, acetylsalicylic acid (aspirin), which is naturally derived from the bark of the willow tree Salix alba [63]. More than 60 and up to 75% of the anticancer and antimicrobial drugs, respectively, are of natural origin [61]. For example, Paclitaxel (Taxol®) is a secondary metabolite isolated from the bark of Pacific Yew tree Taxus brevifolia. This compound is a terpenoid, which was first approved by the food and drug administration (FDA) for treatment of ovarian cancer in 1992 and for breast cancer treatment in 1994 [64]. Ingenol-3-angelate is another example for an antitumor compound that was approved as a topical treatment of actinic keratosis in 2012 [65]. This compound represents a derivative of the diterpenoid, ingenol, which is one of the active compounds from Euphorbia peplus that has been used traditionally for the same purpose.

As a result of many additional studies, several promising plant SM from all three major chemical groups were reported to be active against both, Gram-positive and Gram-negative bacteria. Unfortunately, none of these have been developed further successfully or approved for clinical use as antibiotic. In vitro, several of multidrug-resistant (MDR) bacteria were susceptible to different SMs driven from natural sources [66, 67]. Berberine is a widely distributed antimicrobial plant alkaloid with a broad range of additional bio-activities such as being an anticancer [68], anti-inflammatory [69], and antidiabetic compound [70]. Moreover, other SMs exhibited in vivo a promising antimicrobial activity against MDR Mycobacterium tuberculosum and other microbes [71].

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Fig. 3. Biosynthesis of secondary metabolites compound in plant

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Fig. 4. Biosynthesis of phenolic compounds derived from phenylpropananoid pathway

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1.7. Antibiotics and Bacterial Defense Mechanisms

Antimicrobial substances are essential for treatment of various infectious diseases. In 1910, Paul Ehrlich introduced the first antimicrobial agent to the world, it was suitable to treat the symptoms of syphilis, which is a sexually transmitted infection caused by Treponema pallidum [72]. Afterwards, Alexander Flemming discovered penicillin in 1928 which was isolated from a fungus-contaminated agar culture and which was able to inhibit the growth of Staphylococcus aureus. In the 1940s, penicillin was approved as an official antibiotic and introduced for clinical applications [73]. From then on, the golden era of antibiotics discovery had started to last for the following 25 years [74]. During this period, several antibiotic products were discovered mainly from soil bacteria such as streptomycin, chloramphenicol, vancomycin, or tetracycline [75]. In 1960, the first generation of cephems (sub-group of β-lactam antibiotics) were developed and extensively used against Gram-positive bacteria and Escherichia coli. Thereafter, the second and third generations of cephems were developed to be used not only against Gram-positive organisms but also against Gram-negative bacteria. Some of the third- generation drugs were also effective against Pseudomonas aeruginosa [76]. However, from the early 1970s until 1999 the development of new generations of antibiotics came to a stop, and all launched antibiotics of this period were analogues of the existing ones. From 2000 to 2012, only five new classes of antibiotics have been approved for human application, out of which four are for systemic use (linezolid, daptomycin, bedaquiline and fidaxomicin) while one was developed for topical applications (retapamulin). It is worth mentioning that all these five classes were found to be limited in use to treat Gram-positive bacteria, only [77]. Penicillin and cephalosporin belong to the β-lactam antibiotics family and target bacterial cell wall synthesis. Tetracyclines and streptogramins inhibit protein synthesis by preventing the bacterial ribosome to associate with aminoacyl-tRNA species [78]. At the level of nucleic acids, fluoroquinolones and rifampicin inhibit the synthesis of DNA and RNA, respectively [79].

Several studies reported on pathogens showing resistance to different types of antibiotics [80- 82]. Such resistant bacteria have been known from the early period of antibiotics discovery with the first S. aureus strain producing penicillinase detected in the 1950s. In consequence, new antibiotics were developed in 1959 which were penicillinase-insensitive and termed methicillin. Unfortunately, within a year the first methicillin-resistant S. aureus (MRSA) was isolated in the United Kingdom [83]. According to estimations by the European Union in 2007, approximately 25,000 patients per annum die due to infection with major types of drug-resistant bacteria, which are responsible for bloodstream infections, such as S. aureus, Klebsiella

19 | Page Introduction pneumoniae or P. aeruginosa [82]. In many countries, antibiotics are offered without prescription, which in consequence leads to an overuse of the antibiotics resulting in the undesired increased occurrence of antibiotics-resistant pathogens. Furthermore, usage of antibiotics in the feed of livestock contributes to massive increases of drug resistance of many non-pathogenic bacteria since the thereby disseminated antibiotics reach natural habitats such as soil and water [84, 85].

Bacteria express resistance to antibiotics through a variety of mechanisms. Various bacterial species exhibit resistance against one or several antimicrobial agents as a result of inherited resistance e.g. low membrane permeability, and particularly composed outer membrane structure of Gram-negative bacteria, or they acquired resistance by various mechanisms. One of the well-known resistance strategies is vertical transmission, which means mutation- mediated genetic modification of cellular drug targets transferred from generation to generation. An example for this is given by mutations in the gene encoding for DNA gyrase or topoisomerase IV in Neisseria gonorrhoeae and S. aureus, respectively [86, 87]. Other types of resistance emerge from the production of specific enzymes, which degrade or modify antimicrobial agents. For example, bacterial penicillinases are hydrolytic enzymes which destroy the beta-lactam ring of penicillin to inactivate it [88]. A highly diverse and large group of antibiotics resistance-mediating enzymes is that of transferases. For instance, acyltransferases modify the vulnerable amine group of the antibiotics leading to inactive forms, which lose their ability to bind the target [89]. Furthermore, different types of multidrug efflux pump system are employed by resistant bacteria to exclude antibiotics from the cell [90]. The widespread resistance-nodulcation-cell division (RND)-type multidrug efflux pump system consists of three individual proteins, which are functionally and structurally connected. The three components include an energy pump (transporter protein) located in the inner membrane, an outer membrane porin, and a periplasmic membrane fusion protein connecting the other two proteins with each other [91]. This system allows drug transport across both the inner and outer membranes.

Since the occurrence of novel antibiotics and development of resistance mechanisms is an evolutionary process, there are different classes of antibiotics directed to counteract each of the possible resistance mechanisms.

Bacteria may acquire the mentioned target mutations-mediated or gene-associated resistance mechanisms through horizontal gene transfer, which is the transmission of genetic material

20 | P a g e Introduction from resistant organisms to susceptible ones by several mechanisms such as plasmid conjunction, phage-mediated transduction, or transformation (uptake of free DNA from the environment) [92]. Therefore, searching for and developing novel antimicrobial agents are critically required in modern medicine.

1.8. Drug development

Drug safety and effectiveness are the two most important issues to address when launching a new drug to the market. Due to occurrence of lethal incidences which happened due to the inaccuracy of new drugs launched in the 19th century, institutions such as the Food and Drug Administration (FDA) of the United States had been established with the main objective to ensure that drugs are both, safe and effective.

Drug discovery is the first step in a sequence of steps leading to the development of a new drug and includes an interdisciplinary research approach to identify molecules suited as lead compounds, and study their potential effects toward specific illnesses. The three major approaches for drug discovery are rational design of novel drugs, broad-scale and high- throughput search for innovative medicinal compounds, or taking advantage of the knowledge of the ethno-pharmacological past. For sure, a combination of all three approaches is most fruitful. Drug development is a time-consuming and elaborative process and divided into two main phases (Fig. 5). The first phase is the so-called preclinical phase, during which candidate compounds are tested on living cells and tissues as well as on two or more laboratory test animal species. Usually, this phase includes several thousands of candidate molecules and takes 3 to 4 years to complete. Subsequently, there are three essential clinical phases, for which pharmaceutical companies apply to the FDA in order to investigate the compounds in human volunteers. The clinical trials have different evaluation criteria and differ in the number of tested subjects. In phase I, the new drug is applied on relatively small numbers of healthy volunteers (20-100) and the main target is to identify the safe dose of the drug. This trial phase takes between 2 to 3 years to complete and roughly 70% of tested drugs move to the next trial phase II. The number of volunteers increases to 100-300 subjects, i.e. patients suffering from a specific medical condition of interest. In this phase, safety is still the main issue but effectiveness becomes important and the study takes between 4 to 5 years. The number of effective drugs in this phase is dramatically decreased to approximately 30 %. In phase III, the effectiveness and the long-term safety of the new drug is tested in a large number of volunteer patients (1,000-3,000 subjects) and may take 6 to 8 years to complete. By the end of these trials,

21 | Page Introduction the FDA approves the new drug to allow launching it for clinical application [93, 94]. During post-marketing trials, FDA continues to monitor the new drug since some rare side effects might appear after the launching. The overall time to develop a new drug estimates between 8 to 12 years [94].

Fig. 5. Schematic presentation of the drug development process

1.9. Cell death

Cell death is a natural phenomenon in all kinds of multicellular organisms, where it is often part of developmental processes [95]. Millions of cells die every minute in the human body in balance with cell division giving rise to new cells. In addition, there are further roles of cell death processes, such as elimination of lymphocytes, which lost functionality due to ageing. There are several external factors prompting cells to undergo cell death, such as exposure to radiation, heat, infection, or toxic substances.

Studies of cell death pathways demonstrated various types of cell death, including autophagy, oncosis, necrosis, or apoptosis [96, 97]. Apoptosis and necrosis are considered fundamental types of cell death. In some cases, necrosis can follow apoptosis, and is then termed late necrotic cell death, or secondary necrosis. More detailed descriptions of the characteristics of both types

22 | P a g e Introduction of cell death pathways are listed below. Apoptosis plays an important role in several biological processes such as embryogenesis and tissue morphogenesis, and the removal of damaged or infected cells. Disbalance of apoptosis and cell proliferation can lead to the development of tumors and is also thought to contribute to the onset and progression of neurodegenerative diseases such as Alzheimer disease and Parkinson disease [98].

1.9.1 Necrosis

Necrosis is the ancient term describing a so-called degenerative cell death and derives from the Greek word “necros". It refers to the accidental type of cell death, is considered a passive process requiring only minimal energy, and does not depend on de-novo protein synthesis. However, it is now understood that necrosis just like apoptosis follows a specific molecular pathway leading to cell death [99]. Several external stimuli such as lethal chemicals, metabolic poisons, and physical stress can induce necrosis.

A number of different biochemical and morphological features characterizes necrosis. Early changes in cell morphology include swelling of the organelles such as mitochondria and the nucleus before clumping of nuclear DNA and loss of plasma membrane integrity is observed [100] (Fig. 6). Once the plasma membrane ruptures, proteins are released from the cytosol such as heat shock proteins, lactate dehydrogenase, or high mobility group box proteins, which collectively with other cellular components have the capacity to induce inflammation [101].

1.9.2 Apoptosis

The Greek term “Apoptosis” means “falling off” as used to illustrate the falling of leaves from a tree. In 1972, Kerr et al. [102] were the first to explain the features of this physiological type of cell death and introduced the term apoptosis. Apoptosis is also often referred to programmed cell death (PCD), cellular suicide, or cellular self-destruction. Apoptotic cells are characterized by a number of different biochemical and morphological features. Morphologically, apoptotic cells lose junctional contacts to the neighboring cells. Furthermore, cell shrinkage, condensed chromatin, and formation of apoptotic bodies including organelles are observed. The most obvious feature of apoptosis is blebbing of the plasma membrane (Fig. 6). All of the morphological changes are outcomes of molecular and biochemical dysfunctions of the affected cells. One of the most remarkable biochemical processes is the cleavage of a multitude of specific protein substrate, which are responsible for maintaining the integrity and the shape of organelles and the entire cell. For instance, the activation of proteolytic enzymes from latent

23 | Page Introduction zymogenic forms, i.e. pro-caspase-9 and -3 ultimately leads to cleavage of chromatin DNA into inter-nucleosomal fragments of multiples of about 180 base pairs (bp) [103].

The molecular mechanism of apoptosis is complex. There are at least two pathways leading to apoptosis – the so-called extrinsic and intrinsic pathways. Common to both pathways is the action of cysteine proteases – the caspases - that are responsible to initiate (Caspase-9) and to execute (Caspase-3) the proteolytic cascade that finally results in apoptotic cell death. In vivo, the remnants, apoptotic bodies, are then removed from the tissue by professional phagocytes, thereby largely preventing inflammation [104]. In contrast, in vitro and in absence of macrophages this process may develop to another type of cell death called secondary necrosis [105].

Figure 6: The morphological features of cell death via apoptosis and necrosis. (Figure modified from [106])

24 | P a g e Introduction

1.9.2.1 Extrinsic pathway inducing apoptosis

The extrinsic apoptosis pathway begins with the binding of pro-apoptotic ligands that activate pro-apoptotic receptors on the cell surface. Death receptors (DR) belong to the family of tumor necrosis factor (TNF) receptors which transmit the apoptotic signals to the cell’s interior. The TNF receptor is characterized by a cysteine-rich extracellular domain and a 60-80 amino acid residues long cytoplasmic tail known as the death domain (DD), which is mainly responsible to enable the DR to initiate the death signal [107]. In the cytoplasm, the DD of DR binds to adaptor molecules with Fas-associated death domains which have their own death effector domain (DED). This association subsequently mediates the recruitment of caspases through the association with a corresponding DED. The complex of Fas Ligand, Fas receptors, and procaspase-8 is called the death-inducing signaling complex (DISC). Subsequently, active caspase-8 is released from the DISC into the cytosol, which results in the activation of the down-stream procaspases-3 and -7. Ultimately, once procaspases-3 and -7 are activated in cells, the latter are destined to apoptosis death (Fig. 7). Caspases cleave proteins after aspartic acid residues in a promiscuous manner, which consequently leads to the cleavage of numerous substrates in the cytosol of the dying cell.

1.9.2.2 Intrinsic pathway inducing apoptosis

The intrinsic or mitochondria-based apoptosis pathway is initiated in the cytoplasm by one or several different factors. Such factors include an increase in the intracellular concentration and availability of free calcium ions [108], DNA damage, oxidative stress, response to signals from extrinsic apoptosis pathways [109], or other types of severe cellular stress. These factors lead to the disruption of the membrane potential (Δψ) across the inner mitochondrial membrane leading to an increase of its permeability and the subsequent release of several mitochondrial constituent including cytochrome C into the cytosol [110]. Moreover, such stress leads to activation of B-cell lymphoma-2 (Bcl2) homology 3-only proteins. Consequently, Bcl-2- associated X protein and Bcl2-killer cells are activated. Next, the mitochondrial outer membrane permeabilization takes place making the outer mitochondrial membrane leaky. Cytochrome C is released and binds to both, the apoptosis protease activating factor 1 and procaspase-9, in such a way that the so-called apoptosome complex is formed. This complex is the initial point within the cytoplasm for activation of procaspase-9 which serves as an initiator caspase. It cleaves procaspase-3 and -7 to generate activated caspase-3 and -7 [111]. Finally, the activation of these so-called executioner caspases results in cell death by apoptosis (Fig. 7).

25 | Page Introduction

Moreover, the intrinsic pathway can also be induced by a secondary signal derived from the extrinsic pathway. Alternative to caspases-3 and -7, BID (BH3 interacting-domain death agonist can be cleaved by the initiator caspase 8 in order to generate truncated BID which translocates to the mitochondria, and may thus induce apoptotic pathways [112] (Fig. 7).

26 | P a g e Introduction

a) Extrinsic pathway

b) Intrinsic pathway

Apoptosis

Figure 7: Extrinsic and Intrinsic pathways of apoptosis (Figure modified from [113])

27 | Page Aims of the study

2. Aims of the study

Medicinal plants have been used for a long time as traditional treatments for numerous human diseases. Therefore, in many parts of the world different types of plant material are still used as a main source of medicinal drugs. In this study, we focused on one plant genus which is Rhododendron, belonging to the Family of Ericaceae. Several ethno-pharmacological studies hinted at the importance of bioactive compounds from Rhododendron species as a source of traditional medicine. Herein, the first comprehensive respective study on the genus Rhododendron had to be designed taking advantage of an interdisciplinary cooperation between microbiology, cell biology, genetics, and phytochemistry laboratories and using the World’s second largest collection of Rhododendron represented by the Rhododendron-Park Bremen.

The overall goal of this study was to identify, investigate, and isolate novel secondary metabolic compounds from the leaves of 120 different Rhododendron species. The respective compounds had to exhibit a reproducible antimicrobial activity against bacteria to be further tested for cytotoxic effects onto mammalian cells. In addition, if considered for exploitation as potential drugs to treat skin, metabolic, or internal organ disorders, these compounds had to be safe for use when applied topically or orally. Thus, the major aims of this study can be summarized as follows:

I. Investigation of the distribution of antimicrobial activities among Rhododendron species against a large set of Gram-positive and Gram-negative bacterial species.

II. Testing the effects of the most promising antimicrobial bioactive Rhododendron crude extracts on viability and proliferation rates of two different mammalian cell lines, intestinal epithelial and keratinocyte cell lines.

III. Study of the phytochemical profile of different plant parts (leaves, flowers, and fruits) in order to learn more about the distribution of bioactive or toxic compounds in Rhododendron.

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Results

3. Results

The results of this PhD thesis work are presented in form of three manuscripts, for which experimental work was conducted during the time of this PhD thesis and which are presented by the following three articles.

Ahmed Rezk, Jennifer Nolzen, Hartwig Schepker, Dirk C Albach, Klaudia Brix and Matthias S Ullrich, “Phylogenetic spectrum and analysis of antibacterial activities of leaf extracts from plants of the genus Rhododendron”, BMC Complementary and Alternative Medicine, 2015. 15(1): p. 1-10.

The results of this article proved the antibacterial activity of Rhododendron leaf extracts against a cluster of Gram-positive bacteria. Interestingly, this the first study correlated between the antibacterial effect and Rhododendron taxonomy and approved the most active extracts are mainly belong to a particular subgenus (Rhododendron)

Contributions: Ahmed Rezk contributed to the design of the study, carried out the experiments described in all figures and participated in manuscript writing

Ahmed Rezk, Alaa Al-Hashimi, Warren John, Hartwig Schepker, Matthias S. Ullrich, and Klaudia Brix, “Assessment of cytotoxicity exerted by leaf extracts from plants of the genus Rhododendron towards epidermal keratinocytes and intestine epithelial cells”, 2015 . Submitted.

This article asks a follow up question from the previous study, whether the most active antibacterial Rhododendron leaf extracts are cytotoxic. Therefore in this study we conducted several cytotoxicity assays towards two cell lines i.e. epidermal keratinocytes and intestine epithelial cells. The results proved that two Rhododendron leaf exacts are non-toxic towards both cell types at their bacterial inhibitory concentration. While high dose of Rhododendron extracts induced apoptosis for the intestine epithelial cells. It worth to mention that, the keratinocytes cells were most resistant against the treatment with Rhododendron leaf extracts.

Contributions: Ahmed Rezk contributed to the design of the study, carried out the experiments described in all figures and participated in manuscript writing

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Abhinandan Shrestha, Ahmed Rezk, Inamullah Hakeem Said, Jennifer Nolzen, Victoria von Glasenapp, Rachelle Smith, Matthias S. Ullrich, and Nikolai Kuhnert, “Distinguishing the polyphenolic and antibacterial profile of the leaves, fruits and flowers of Rhododendron ambiguum and Rhododendron cinnabarinum using high performance liquid chromatography coupled with ion trap and time of flight mass spectrometry”, 2015, In preparation.

It has become more interesting to identify and determine the phytochemical profile of the bioactive Rhododendron extracts. In this study the phytochemical profile was assessed for different leaves ages in addition to flowers and fruits of two most active Rhododendron. The results proved the random and uniform of polyphenols in the aerial parts of both plant species. Moreover, the antibacterial active compound/s are distributed equally in all plant parts.

Contributions: Ahmed Rezk was involved in manuscript writing, carried out the experiments described in figure 3 and discussion and interpretation of the results.

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3.1 Phylogenetic spectrum and analysis of antibacterial activities of leaf extracts from plants of the genus Rhododendron

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Abstract

Plants are traditionally used for medicinal treatment of numerous human disorders including infectious diseases caused by microorganisms. Due to the increasing resistance of many pathogens to commonly used antimicrobial agents, there is an urgent need for novel antimicrobial compounds. Plants of the genus Rhododendron belong to the woody representatives of the family Ericaceae, which are typically used in a range of ethno-medical applications. There are more than one thousand Rhododendron species worldwide. The Rhododendron-Park Bremen grows plants representing approximately 600 of the known Rhododendron species, and thus enables research involving almost two thirds of all known Rhododendron species.

Twenty-six bacterial species representing different taxonomic clades have been used to study the antimicrobial potential of Rhododendron leaf extracts. Agar diffusion assay were conducted using 80% methanol crude extracts derived from 120 Rhododendron species. Data were analyzed using principal component analysis and the plant-borne antibacterial activities grouped according the first and second principal components.

For the first time, our comprehensive study demonstrated that compounds with antimicrobial activities accumulate in the leaves of certain Rhododendron species, which mainly belong to a particular subgenus. The results suggested that common genetic traits are responsible for the production of bioactive secondary metabolite(s) which act primarily on Gram-positive organisms, and which may affect Gram-negative bacteria in dependence of the activity of multidrug efflux pumps in their cell envelope.

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Background

For a long time, medicinal plants have been used as traditional treatments of a variety of human diseases. In many parts of the World, different kinds of plant material are still in use as a major source of traditional medicine formulations [1-3]. According to the World Health Organization, approximately 65% of the World’s populations integrate medicinal plants and products generated thereof into their primary health care strategies [4,5]. Importantly, in developing countries about 80% of the population is used to prepare traditional medicine formulation from plant sources [6].

Bacterial pathogens have developed different types of resistance to antimicrobial agents, thereby causing a significant increase in the costs of diagnostics and pharmaceutical treatments. Moreover, resistant microorganisms contribute to the currently observed dramatic increments in mortality and morbidity of patients affected by infectious diseases [7]. Since patients remain inflicted for a longer time period due to persisting microbial infection, the person-to-person transmission rates are prolonged and thus, enhanced. Increase in morbidity caused by antibiotics-resistant bacteria was recorded in several recent out-breaks such as pneumococcal infections, typhoid fever, and shigellosis in different regions world-wide [8]. The increasing resistance of human pathogens to commonly used antimicrobial agents motivated a renewed interest in the discovery of novel antimicrobial compounds. Several secondary metabolites of plants proved effective as biologically active agents against pathogens [9-11]. This is explained by the notion that plants acquired most of their secondary metabolite repertoire during evolution as metabolic byproducts, which then serve as defense compounds against predators such as insects and other herbivores, or against pathogens such as bacteria, fungi, or viruses [12,13].

Rhododendron L. (Ericaceae) is one of the largest genera of vascular plants and comprises eight subgenera with more than 1,000 species that populate habitats mostly in the Northern hemisphere [14]. Extracts of several species of Rhododendron are used in traditional medicine in the countries of their indigenous habitats. Among them are R. ferrugineum L., R. anthopogon Don, or R. tomentosum (Stokes) Harmaja that are used for the treatment of inflammation, skin or gastrointestinal tract disorders, respectively [3,15-18]. These studies showed that the antibacterial activities of certain species of Rhododendron could be due to the presence of specific mono-, di-, or sesquiterpenoids, which had already been extracted from other plant

33 | P a g e Results families [19,20]. However, there is neither a comprehensive survey of the impressive number of chemical compounds in this genus nor have the later been tested systematically for their pharmacological potential [21]. The Bremen Rhododendron-Park possesses an unrivaled diversity of reliably identified Rhododendron species. Hence, the purpose of the current study was to comprehensively assess the spectrum of antibacterial activities of extracts prepared from 120 different Rhododendron species against a broad array of Gram-positive and Gram-negative bacteria. This study therefore aims to initiate the further identification and exploitation of novel plant-borne antibiotics derived from Rhododendron leaf extracts.

Methods

Plant material and extraction procedure

Fresh leaf material of 120 reliably identified Rhododendron species was collected from plants grown in the Rhododendron-Park Bremen (www.rhododendronparkbremen.de) from January 2012 to December 2013. Each plant species was sampled once without considering seasonal variations. The identities of all plant species have been verified according to the German Genebank Rhododendron Database provided by the Bundessortenamt (www.bundessortenamt.de/rhodo) (Additional file 1: Table S1). Material from all used plant species is publicly and freely available from the Rhododendron-Park Bremen upon request. The herein used Rhododendron species were chosen from five main subgenera: Rhododendron, Hymenanthes, Tsutsusi, Pentanthera and Azaleastrum, 10 sections, and 34 sub-sections in order to cover a broad phylogenetic spectrum of plants. Leaf material was immersed in liquid nitrogen and grinded to powder. Crude extracts were prepared by re-suspending 2 g (fresh weight) of leaf powder in 10 mL of the following solvents: 80% methanol (MeOH), ethyl acetate (EtOAc), or distilled water, respectively, each for 24 hours at 4°C. Non-dissolved leaf residues were removed by centrifugation (3,220 x g, 30 min, 4°C). The resulting supernatants were stored at-20°C, and the remaining, non-extracted powder was kept at-80°C for long-term storage to ensure reproducibilty of measurements from a standard reservoir of plant-derived material.

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Bacterial strains

Twenty-six bacterial species were randomly selected from different taxonomic clades to test for the susceptibility spectrum of crude extracts against a range of Gram-positive and Gram- negative bacteria (Figure 1). The phylogenetic tree was constructed by the Neighbor joining method in the Molecular Evolutionary Genetics Analysis (MEGA) software version 6 (Tamura, Stecher, Peterson, Filipski, and Kumar 2013). In order to investigate the role of multidrug efflux systems as bacterial defense mechanism, wild type and gene-specific mutants of the following bacterial species were analyzed: Erwinia amylovora 1189 (wild type), Escherichia coli TG1 (wild type) [22], Pseudomonas syringae DC3000 (wild type) [23] as well as the respective mutants with deletions in acrAB, tolC, or mexAB [24-28]. Additionally, knockout mutants of E. amylovora and E. coli with deletions in both, acrAB and tolC [25,27] were subjected to the antimicrobial analysis.

Antimicrobial susceptibility test

Antimicrobial activity screening was conducted by the agar diffusion method [29]. Briefly, Lysogeny Broth (LB) agar plates were inoculated with 200 μL of the inoculum of the tester organism (1 x 107 colony forming units per mL) by evenly spreading the cell suspensions over the agar surface. Holes with diameters of 5 mm were punched into the agar plates. Subsequently, 50 μL of the plant crude extracts were filled into each well. The plates were incubated overnight at 28°C or 37°C, according to the optimal growth temperature of each bacterial strain. Inhibition of microbial growth was determined by measuring the radius of the inhibition zone. For each bacterial strain, 80% methanol and 100% EtOAc solutions were used as negative solvent controls. All experiments were performed in triplicates and the results are presented as mean values.

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Figure 1 Phylogenetic tree based on bacterial 16S rRNA gene sequences. The phylogenetic tree was constructed using the neighbor-joining method showing the bacterial organisms (numbers in brackets) used in this study. Bootstrap values (1,000 replicates) lower than 50% are not shown. Filled triangles indicate the phylogenetic position of human pathogens which have not been used in this study. The scale bar 0.02 indicates 2% of nucleotide sequence substitution. The strain numbers are indicated in brackets.

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Principal component analysis and data analysis

Principal component analysis (PCA) as multivariate analysis of data was used to handle the multi-dimensionality of the data sets and to transform them into new, uncorrelated variables called principal components [30]. One hundred and twenty Rhododendron samples were tested against 26 bacterial strains (variables) using a random cross validation method. The matrix was designed by using the inhibition zone (mm). PCA was performed with The Unscrambler® 9.6 (CAMO AS, Oslo, Norway) software. All graphs were plotted using Origin (OriginLab, Northampton, MA).

Results

Bioactive metabolites and efficiency of solvent extraction

Initially, Bacillus subtilis 168 and Escherichia coli TG1 were used as model organisms for Gram-positive and Gram-negative bacteria, respectively. Crude leaf extracts from 120 Rhododendron species were obtained using different solvents in order to test for their efficacy to extract biologically active compounds from the plant material. The susceptibility of both bacterial model organisms was tested towards each of the crude leaf extracts. The antibacterial activity of Rhododendron leaf extracts obtained with MeOH or EtOAc as solvents was determined and categorized into three classes according to the radius of the inhibition zone: a) extracts of 13 Rhododendron species caused no inhibition, b) 71 extracts caused low inhibition (radius of less than 4 mm), and c) leaf extracts of 36 Rhododendron species caused inhibition (radius ranging from 4 to 12 mm) of growth of either E. coli or B. subtilis (Figure 2). None of the Rhododendron crude extracts obtained with distilled water showed any antibacterial activity (data not shown) indicating that bioactive compounds from Rhododendron are not readily water-soluble. The used solvent concentrations did not impact bacterial growth since treatment with MeOH and EtOAc alone did not induce inhibition zones (data not shown). In contrast, MeOH- and EtOAc-derived leaf extracts exhibited differential effects in that they were partially significantly active on both model organisms, suggesting that the contents or the concentration of potentially bioactive substances in the crude extracts might vary from one species of Rhododendron to the other. Interestingly, both tester organisms were generally more susceptible to treatment with the MeOH extracts while the EtOAc extracts showed less antimicrobial effects (Figure 2), indicating that methanol was more suitable to extracting

37 | P a g e Results bioactive compound (s) from powdered Rhododendron leaves. The extent by which the growth of the tester organisms was inhibited suggested a broad range of activities, which is possibly explained either by different compounds in various Rhododendron species or by diverse mechanisms of inhibition of bacterial growth mediated by the produced compounds.

Spectrum of microbial susceptibility towards Rhododendron crude extracts

In order to test a wider spectrum of bacterial organisms, the antimicrobial activity test with all 120 Rhododendron crude extracts was extended to a set of 24 additional bacterial tester strains representing both, a broad phylogenetic spectrum as well as intra-genus diversity (Figure 1). As observed above, MeOH and EtOAc as solvents alone had no impact on growth of any of the tested bacterial organisms (data not shown).

Figure 2 Antimicrobial activities of methanol- and ethyl acetate-obtained crude leaf extracts of different Rhododendron species against B. subtilis and E. coli. The radius of the inhibition zones was measured in triplicates and the values are given as means ± standard deviations. Treatment with solvents were used as negative controls and did not yield in inhibition zones (data not shown).

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PCA was used to classify the spectrum of bioactivities of all Rhododendron extracts against all bacterial tester organisms according to their phylogenetic relatedness (Figure 3).The first and second principal components PC1 and PC2 explained 82% of the data indicating a high robustness of analysis. The data revealed that the microbial susceptibility towards Rhododendron extracts can be grouped into one group representing all Gram-positive tester organisms, while another group contained 17 out of 18 Gram-negative species (Figure 3) irrespective of the nature of the Rhododendron extract applied. The only Gram-negative species showing a similar susceptibility pattern as Gram-positive organisms was the alpha- protobacterium Sinorhizobium meliloti.

Five out of the 120 Rhododendron leaf extracts showed no growth inhibitory activity against any of the Gram-positive tester organisms: R. elliottii Watt ex Brandis, R. hylaeum Balfour & Farrer, R. ponticum L., R. keiskei Miquel, and R. eriocarpum (Hayata) Nakai (data not shown). These Rhododendron species belong to the subgenera Hymenanthes (three species), Rhododendron, and Tsutsusi, respectively. However, crude extracts obtained from 38 other Rhododendron species exhibited a moderate antimicrobial activity against 12 out of the 26 tested bacterial species. These plant species belonged to the subgenera Azaleastrum, Hymenanthes, Rhododendron, or Tsutsusi. The crude extracts derived from the remaining 77 Rhododendron species showed significant bioactivities against at least one of the 26 tester organisms. It is important to note that the spectrum of microbial susceptibilities towards Rhododendron extracts varied widely and showed dramatic differences, i.e. some of the tester organisms were susceptible towards leaf extracts from most of the Rhododendron species while other bacterial tester organisms were susceptible towards very few Rhododendron leaf extracts. Bacillus thioparus was the most sensitive tester species susceptible to all 77 potentially bioactive Rhododendron leaf extracts. The other tested Gram-positive bacteria showed similar susceptibility only towards leaf extracts derived from the 17 most bioactive Rhododendron species (Figure 4, Table 1).

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Figure 3 PCA score plot. Principal component analysis for score plot (random cross validation method) of the entire dataset (only PC1 vs. PC2 shown). Every dot represents one bacterial species and each color represents a particular group of bacterial organisms.

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Figure 4 PCA loading plot. Principal component analysis for loading plot for 120 Rhododendron species (only PC1 vs. PC2 shown). Every dot represents one Rhododendron species and each color represents a subgenus of Rhododendron.

In contrast, susceptibilities of the tested Gram-negative bacterial strains were classified into either low or moderate extent. Only one Gram-negative species, Sinorhizobium meliloti, belonging to the order of alpha-proteobacteria exhibited susceptibility to most of the bioactive Rhododendron extracts and was therefore similar in its susceptibility to the majority of Gram- positive bacteria (Figure 3, sample no. 19).

On one side, the PCA allowed classification of the bacterial tester organisms according to their degree of susceptibility after incubation with Rhododendron extracts. On the other hand, the analysis performed in this study allowed to successfully categorized the representatives of the genus Rhododendron into bioactive and non-bioactive species (Figure 4), where bioactivity is understood as antimicrobial activity as assayed by growth inhibition on LB-agar plates. The results illustrated that 41% of all analyzed species of the subgenus Rhododendron were highly bioactive against the majority of Gram-positive bacteria tested herein. In contrast, only two of the 14 species (14%) of the subgenus Pentanthera showed similarly high antimicrobial

41 | P a g e Results bioactivity. The majority of the remaining Rhododendron species representing other subgenera exhibited only moderate or low antimicrobial activities.

Table 1 List of Rhododendron species with the highest antimicrobial activities against Gram-positive bacteria

Genebank-no.* Species name Section Sub-section

100.345 R. ferrugineum L. Rhododendron Rhododendron 100.007 R. ambiguum Hemsley Rhododendron Triflora 2006/232 R. anthopogon Don ssp. anthopogon Betty Graham Pogonanthum - NA R. hirsutum L. Rhododendron Rhododendron 100.906 R. anthopogon ssp. hypenanthum Bale. F. & Cullen Pogonanthum - 100.326 R. concinnum Hemsley Rhododendron Triflora 100.881 R. sichotense Pojarkova Rhododendron Rhodorastra 100.322 R. cinnabarinum Hooker Rhododendron Cinnabarina NA R. racemosum Franchet Rhododendron Scabrifolia 100.882 R. ledebourii Pojarkova Rhododendron Rhodorastra 100.404 R. rubiginosum Franchet Rhododendron Heliolepida 100.474 R. xanthostephanum Merrill Rhododendron Tephropepla 101.048 R. myrtifolium Schott & Kotschy Rhododendron Rhododendron 100.370 R. minus Michaux Rhododendron Caroliniana 100.392 R. polycladum Franchet Rhododendron Lapponica 100.464 R. spinuliferum Franchet Rhododendron Scabrifolia 100.353 R. hippophaeoides var. hippophaeoides Hutchinson Rhododendron Lapponica * Gene bank numbers used in the collection of the Rhododendron-Park Bremen.

NA: Not a plant of the German Genebank Rhododendron but a verified plant of the Rhododendron-Park Bremen.

Role of bacterial multidrug efflux pumps as potential resistance mechanisms

One important observation herein was the finding of higher susceptibility of Gram-positive over Gram-negative tester organisms towards Rhododendron leaf extracts, suggesting an easier passage of potential bioactive compounds into Gram-positive cells. In order to find out, whether one of the best studied antibiotics resistance mechanism, the RND-type multidrug efflux pump system of Gram-negative bacteria, is responsible for the lower susceptibility of the latter organisms, previously generated gene-specific knock-out mutants of E. coli, Erwinia amylovora, and Pseudomonas syringae with gene deletions in the multidrug efflux pump

42 | P a g e Results components acrAB, tolC, or mexAB were analyzed by treating them with crude extracts of R. ambiguum Hemsley, R. ferrugineum L., or R. fastigiatum Franchet (Figure 5). The E. coli wild type as well as all of its multidrug efflux pump mutants exhibited full resistance to all of the tested leaf extracts. However, data for the fire blight pathogen, E. amylovora, differed. While the wild type strain of this bacterium exhibited full resistance to each one of the tested Rhododendron extracts, single as well as double mutant strains with deletions of tolC and acrAB exhibited high sensitivity towards all three tested Rhododendron leaf extracts thereby rendering their phenotype to that of the Gram-positive bacteria (Figure 5). The mexAB multidrug efflux pump mutant of P. syringae did not show a significant reduction in growth when exposed to any of the Rhododendron leaf extracts.

In summary, these data suggested that some but not all of the multidrug efflux pump systems are likely to be directly involved in export of Rhododendron-derived bioactive antimicrobial compounds.

Figure 5 Role of the multidrug efflux pump in Gram-negative bacteria. Susceptibility tests for E. coli, E. amylovora, P. syringae and their respective RND-type multidrug efflux pump mutants to leaf extracts of three Rhododendron species effective against Gram-positive bacteria. WT, wild type. The radius of the inhibition zones was measured in triplicates and values are given as means ± standard deviations.

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Discussion

The purpose of this study was to conduct a comprehensive analysis of antibacterial activities of plants from the genus Rhododendron, which has the highest species richness among all woody plant genera [31]. Three different solvents were compared to optimize extraction efficiency for bioactive compounds from powdered Rhododendron leaf material. Our results demonstrated that methanol and ethyl acetate but not water extraction were suitable for this purpose. This finding is in agreement with those of previous studies, which reported that most of the bioactive secondary metabolites extracted from plant material are obtained by an aqueous methanol extraction [32].

The herein observed susceptibility of Gram-positive bacteria toward Rhododendron leaf extracts contrasted the results obtained with the majority of Gram-negative tester bacteria. This finding might be explained as follows: The cellular envelopes of either type of bacteria differ dramatically and the outer membrane of the Gram-negative cells with its lipopolysaccharide leaflet might pose an impermeable barrier for the methanol-extractable Rhododendron-derived bioactive compounds. Alternatively, various types of multidrug efflux pumps found in Gram- negative bacteria might extrude those bioactive compound (s). The latter hypothesis is strongly supported by findings of this study in that E. amylovora mutants with defects in the multidrug efflux pump system AcrAB/TolC were no longer resistant against treatment with Rhododendron leaf extracts. Similar conclusions have been drawn previously by our group with respect to treatment of E. amylovora with several chemical compounds originating from other plants [25,33,34]. Interestingly, multidrug efflux pump knock-out mutants of E. amylovora but not those of E. coli or P. syringae exhibted a significant growth inhibition when compared to the wild type strain upon exposure to Rhododendron extracts. These results indicated that presence of distinct multidrug efflux systems might explain the resistance towards some of the bioactive Rhododendron extracts. Since neither E. coli nor P. syringae mutants with the same defects exhibited growth inhibition upon exposure to these leaf extracts, additional factors must contribute to the resulting resistance in those organisms. This notion points to an interesting diversity of potential molecular mechanisms realized in different Gram- negative bacteria, thus providing some species with resistance towards bioactive compounds extracted from Rhododendron leaves while others will remain unaffected. In line with this conclusion, it has been described previously that the permeability of the outer membrane of E. coli differs from that of other Gram-negative microorganisms [25]. The diversity of potential

44 | P a g e Results resistance mechanisms among Gram-negative bacteria could also help explaining the unusual high susceptibility of Sinorhizobium meliloti. A future comparative multi-species genomic analysis is planned to shed light on the relationship of resistance and expression of individual genes among different Gram-negative bacterial species.

The herein studied bioactivities of Rhododendron leaf extracts showed a range of different effects towards the bacterial tester organisms possibly reflecting the phylogentic diversity of the analyzed Rhododendron species. The finding agrees with previously published results for R. anthopogon Don that exhibited antimicrobial activity against a group of Gram-positive bacteria (e.g. Staphylococcus aureus, Enterococcus faecalis, and Bacillus subtilis) [15]. We hypothesize that different Rhododendron genotypes may result in the formation of a broad range of compounds due to the diversity in structure and concentration of secondary metabolites resulting from different metabolic pathways. Previous authors had suggested that this might explain why certain plants become more or less susceptible to herbivores [35-37].

In contrast to the studies of others, which demonstrated that extracts from R. setosum Don, R. ponticum, R. luteum Sweet, R. arboreum Smith, R. smirnowii Trautvetter and R. campanulatum Don, inhibited the growth of E. coli, B. subtilis and S. aureus [18,38-41], corresponding extracts used in the current study did not show any antibacterial effects. This discrepancy might be attributed to a number of factors such as differences in compound extractability or in concentration of the secondary metabolites contained in the plants depending on the plants’ growing conditions with respect to variable ranges of biotic (e.g. herbivory, infection or allelopathy) or abiotic (e.g. nutrient, light, temperature and drought) stress factors. Some of the latter had been proven to trigger alterations of the secondary metabolite composition produced in one and the same plant species [42-46]. Taken together, we suggest that only a comprehensive and well-controlled analysis of the antimicrobial compound composition of plant-derived extracts will allow deciphering of the underpinning molecular and metabolic pathways, and only a delineation of genetically related phytomedical profiling will enable reproducible production of extracts which might then be useful as precursors for medicinal treatment options.

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Conclusions

The results obtained in this work revealed that 17 species of the genus Rhododendron exhibited antibacterial effects against Gram-positive bacteria. For the first time, a comprehensive study demonstrated that there is an accumulation of antimicrobial bioactivities among Rhododendron species of the subgenus Rhododendron. Consequently, a detailed phylogenetic assessment of the differences of various Rhododendron subgenera accompanied by in-depth phytochemical analysis of the extracts might shed light on the actual nature of the bioactive compound (s). Additionally, a further analysis of the bacterial susceptibility spectrum might indicate how the bioactive compound (s) act on certain microbes. Herein, potential resistance mechanisms of bacteria were shown to differ in a species-specific manner indicating the necessity to obtain an array of Rhododendron-derived bioactive compounds in order to inhibit a broad spectrum of potentially harmful bacteria.

Abbreviations

MeOH, Methanol; EtOAc, Ethyl acetate; PCA, Principal component analysis

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

AR designed experiments, conducted the experimental work, and prepared the manuscript; JN, DA, and HS collected, identified, prepared plant materials, and helped in the design of the study; KBr and MU designed the study, supervised the work, discussed the results, and contributed to manuscript writing. All authors read and approved the final manuscript.

Acknowledgements

This study was financially supported by the Stiftung Bremer Rhododendronpark. The authors are particularly grateful to Wolfgang Klunker for his enthusiastic support and would like to thank Rasha El-Abassy for help with The Unscrambler software.

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Additional file

Table S1: List and origin of Rhododendron species tested in the study

Genebank Species Name Subgenus Section Subsection -No 100.470 R. hongkongense Hutchinson Azaleastrum Azaleastrum - 100.390 R. ovatum (Lindley) Maximowicz Azaleastrum Azaleastrum 100.886 R. moulmainense Hooker Azaleastrum Choniastrum - 100.467 R. macabeanum Watt Balfour Hymenanthes Ponticum Grandia 100.463 R. arboreum ssp. arboreum Smith Hymenanthes Ponticum Arborea 100.799 R. wasonii Hemsley & Wilson Hymenanthes Ponticum Taliensia 100.891 R. decorum ssp. diaprepes (Balfour & Hymenanthes Ponticum Fortunea Smith) Ming 100.812 R. argyrophyllum ssp. nankingense Hymenanthes Ponticum Argyrophylla (Cowan) Chamberlain 100.393 R. ponticum L. Hymenanthes Ponticum Pontica 100.016 R. auriculatum Hemsley Hymenanthes Ponticum Auriculata 100.784 R. campanulatum ssp. aeruginosum Hymenanthes Ponticum Campanulata (Hooker) Chamberlain 100.420 R. strigillosum Franchet Hymenanthes Ponticum Maculifera 100.491 R. thomsonii ssp. lopsangianum Hooker Hymenanthes Ponticum Thomsonia 100.349 R. galactinum Tagg Hymenanthes Ponticum Falconera 100.794 R. annae Franchet Hymenanthes Ponticum Irrorata 100.496 R. elliottii Watt ex Brandis Hymenanthes Ponticum Parishia 100.483 R. griersonianum Balfour & Forrest Hymenanthes Ponticum Griersoniana 100.338 R. degronianum ssp. yakushimanum Hymenanthes Ponticum Pontica (Nakai) Hara 100.417 R. smirnowii Trautvetter Hymenanthes Ponticum Pontica 100.441 R. williamsianum Rehder & Wilson Hymenanthes Ponticum Williamsiana 100.412 R. selense ssp. jucundum (Balfour & Hymenanthes Ponticum Selensia Smith) Chamberlain 100.439 R. wardii var. puralbum (Balfour & Hymenanthes Ponticum - Smith) Chamberlain 100.880 R. protistum Balfour & Forrest Hymenanthes Ponticum Grandia 100.874 R. coriaceum Franchet Hymenanthes Ponticum Falconera 100.003 R. adenogynum Diels Hymenanthes Ponticum Taliensia 100.887 R. adenopodum Franchet Hymenanthes Ponticum Argyrophylla

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100.821 R. alutaceum var. iodes (Balfour & Hymenanthes Ponticum Taliensia Forrest) Chamberlain 100.830 R. aganniphum var. flavorufum Hymenanthes Ponticum Taliensia ((Balfour & Forrest) Chamberlain 100.824 R. beesianum Diels Hymenanthes Ponticum Taliensia 100.791 R. bureavii Franchet Hymenanthes Ponticum Taliensia 100.457 R. longesquamatum Schneider Hymenanthes Ponticum Maculifera 100.015 R. aureum Georgi Hymenanthes Ponticum Pontica 100.796 R. purdomii Rehder & Wilson Hymenanthes Ponticum - 100.889 R. ririei Hemsley & Wilson Hymenanthes Ponticum Argyrophylla 101.066 R. faberi Hemsley Hymenanthes Ponticum Taliensia 100.018 R. balangense Fang Hymenanthes Ponticum Grandia 100.466 R. arboreum ssp. delavayi (Franchet) Hymenanthes Ponticum Arborea Chamberlain 100.856 R. thayerianum Rehder & Wilson Hymenanthes Ponticum Argyrophylla 101.118 R. campanulatum Don Hymenanthes Ponticum Campanulata 100.795 R. sherriffii Cowan Hymenanthes Ponticum Thomsonia 1999/259 R. neriiflorum Franchet Hymenanthes Ponticum Neriiflora 100.315 R. campylocarpum ssp. caloxanthum Hymenanthes Ponticum Campylocarpa (Balfour & Farrer) Chamberlain 100.249 R. brachycarpum Don ex Don ssp. Hymenanthes Ponticum Pontica fauriei (Franchet) Chamberlain 100.248 R. brachycarpum ssp. brachycarpum Hymenanthes Ponticum Pontica Don ex Don 100.860 R. wasonii Hemsley & Wilson Hymenanthes Ponticum Taliensia 100.473 R. falconeri ssp. eximium (Nuttall) Hymenanthes Ponticum Falconera Chamberlain 100.394 R. praevernum Hutchinson Hymenanthes Ponticum Fortunea 100.489 R. hylaeum Balfour & Farrer Hymenanthes Ponticum Thomsonia 100.884 R. montroseanum Davidian Hymenanthes Ponticum Grandia 100.837 R. macrophyllum Don ex Don Hymenanthes Ponticum Pontica 100.355 R. insigne Hemsley & Wilson Hymenanthes Ponticum Argyrophylla 100.456 R. austrinum (Small) Rehder Pentanthera Pentanthera Pentanthera 100.363 R. luteum Sweet Pentanthera Pentanthera Pentanthera 100.431 R. vaseyi Gray Pentanthera Pentanthera - 100.408 R. schlippenbachii Maximowicz Pentanthera Pentanthera - 100.005 R. albrechtii Maximowicz Pentanthera Pentanthera Pentanthera 101.044 R. arborescens (Pursh) Torrey Pentanthera Pentanthera Pentanthera 100.012 R. atlanticum (Ashe) Rehder Pentanthera Pentanthera Pentanthera

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101.046 R. prunifolium (Small) Millais Pentanthera Pentanthera Pentanthera 100.391 R. periclymenoides (Michaux) Shinners Pentanthera Pentanthera Pentanthera 101.045 R. cumberlandense Braun Pentanthera Pentanthera Pentanthera 100.250 R. calendulaceum (Michaux) Torrey Pentanthera Pentanthera Pentanthera 100.380 R. occidentale (Torrey & Gray) Gray Pentanthera Rhodora Pentanthera X/1266 R. pilosum (MICHX. ex LAM.) Pentanthera Rhodora - CRAVEN NA R. multiflorum var. purpureum Pentanthera Sciadorhodion - (MAKINO) CRAVEN 100.397 R. prinophyllum (Small) Millais Pentanthera Sciadorhodion Pentanthera 101.115 R. canadense (Linneaus) Torrey Pentanthera Sciadorhodion 100.007 R. ambiguum Hemsley Rhododendron Pogonanthum Triflora 100.750 R. maddenii ssp. maddenii Hooker Rhododendron Pogonanthum Maddenia 100.426 R. tomentosum (Stokes) Harmaja Rhododendron Rhododendron Ledum 100.368 R. micranthum Turczaninow Rhododendron Rhododendron Micrantha 100.353 R. hippophaeoides var. hippophaeoides Rhododendron Rhododendron Lapponica Hutchinson 100.370 R. minus Michaux Rhododendron Rhododendron Caroliniana 100.404 R. rubiginosum Franchet Rhododendron Rhododendron Heliolepida 100.322 R. cinnabarinum Hooker Rhododendron Rhododendron Cinnabarina 100.468 R. leucaspis Tagg Rhododendron Rhododendron Boothia 100.345 R. ferrugineum L. Rhododendron Rhododendron Rhododendron 100.374 R. moupinense Franchet Rhododendron Rhododendron Moupinensia 100.329 R. davidsonianum Rehder & Wilson Rhododendron Rhododendron Triflora 101.054 R. tapetiforme Balfour & Kingdon- Rhododendron Rhododendron Lapponica Ward 100.384 R. russatum Balfour & Forrest Rhododendron Rhododendron Lapponica 100.803 R. trichanthum Rehder Rhododendron Rhododendron Triflora 100.882 R. ledebourii Pojarkova Rhododendron Rhododendron Rhodorastra 100.377 R. nitidulum var. omeiense Philipson & Rhododendron Rhododendron Lapponica Philipson 100.392 R. polycladum Franchet Rhododendron Rhododendron Lapponica 100.449 R. yunnanense Franchet Rhododendron Rhododendron Triflora 100.343 R. fastigiatum Franchet Rhododendron Rhododendron Lapponica 100.362 R. lutescens Franchet Rhododendron Rhododendron Triflora 100.464 R. spinuliferum Franchet Rhododendron Rhododendron Scabrifolia 100.326 R. concinnum Hemsley Rhododendron Rhododendron Triflora 100.484 R. genestierianum Forrest Rhododendron Rhododendron Genestieriana 100.498 R. rigidum Franchet Rhododendron Rhododendron Triflora

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100.477 R. scabrifolium var. spiciferum Rhododendron Rhododendron Scabrifolia (Franchet) Cullen 100.495 R. polylepis Franchet Rhododendron Rhododendron Triflora 100.906 R. anthopogon ssp. hypenanthum Bale. Rhododendron Rhododendron - F. & Cullen 100.474 R. xanthostephanum Merrill Rhododendron Rhododendron Tephropepla 100.748 R. keysii Nuttall Rhododendron Rhododendron Cinnabarina 100.848 R. bracteatum Rehder & Wilson Rhododendron Rhododendron Heliolepida 2006/232 R. anthopogon Don ssp. anthopogon Rhododendron Rhododendron - Betty Graham 100.009 R. amesiae Rehder & Wilson Rhododendron Rhododendron Triflora 100.654 R. augustinii ssp. chasmanthum Cullen Rhododendron Rhododendron Triflora NA R. hirsutum L. Rhododendron Rhododendron Rhododendron 101.358 R. keiskei Miquel Rhododendron Rhododendron Triflora 100.471 R. pleistanthum Balfour ex Wilding Rhododendron Rhododendron Triflora 100.424 R. tatsienense Franchet Rhododendron Rhododendron Triflora 1470.000 R. triflorum Hooker Rhododendron Rhododendron Triflora 100.376 R. mucronulatum Turczaninow Rhododendron Rhododendron Rhodorastra 2010/384 R. setosum Don Rhododendron Rhododendron Lapponica 2002/1084 R. scopulorum Hutchinson Rhododendron Rhododendron Maddenia 101.047 R. neoglandulosum Harmaja Rhododendron Rhododendron Ledum 100.881 R. sichotense Pojarkova Rhododendron Rhododendron Rhodorastra NA R. racemosum Franchet Rhododendron Rhododendron Scabrifolia 101.052 R. auritum Tagg Rhododendron Rhododendron Tephropepla 101.048 R. myrtifolium Schott & Kotschy Rhododendron Rhododendron Rhododendron 100.403 R. mucronatum (Blume) G. Don var. Tsutsusi Brachycalyx - ripense (Makino) Wilson 100.422 R. tashiroi Maximowicz Tsutsusi Tsutsusi - 100.904 R. dilatatum Miquel Tsutsusi Tsutsusi 100.357 R. kaempferi Planchon Tsutsusi Tsutsusi 100.872 R. oldhamii Maximowicz Tsutsusi Tsutsusi 100.650 R. eriocarpum (Hayata) Nakai Tsutsusi Tsutsusi NA : not a plant of the German Genebank Rhododendron but nevertheless a verified plant of the Rhododendron-Park Bremen

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3.2 Assessment of Cytotoxicity Exerted by Leaf Extracts from Plants of the Genus Rhododendron towards Epidermal Keratinocytes and Intestine Epithelial Cells

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Abstract

Rhododendron leaf extracts previously found to exert antimicrobial activities against a range of Gram-positive bacteria were tested for their cytotoxic potential with the aim to determine whether the bio-active compounds extracted from certain Rhododendron species can be used in medical treatments such as ectopic application or as orally administered drugs.

To test for cytotoxic effects exerted by Rhododendron leaf extracts towards epidermal keratinocytes and epithelial cells of the intestinal mucosa, cultures of human HaCaT and rat IEC6 cell lines were incubated with different concentrations of DMSO-dissolved remnants of methanolic leaf extracts for 24 h. Experimental tests were performed that quantified (i) plasma membrane integrity, (ii) cell viability and proliferation rates, (iii) intact cellular metabolism, (iv) compromised cellular architecture, and (v) morphological and biochemical assessments of cell death pathways induced by specific Rhododendron leaf extracts.

Concentrated extracts (500 µg/mL) from almost all Rhododendron species were potent in negatively affecting both, keratinocytes and intestine epithelial cells, except material from R. hippophaeoides var. hippophaeoides. Extracts of R. minus and R. racemosum were non-toxic towards both mammalian cell types at their minimal inhibitory concentration against bacteria (50 µg/mL). Leaf extracts from three other highly potent antimicrobial Rhododendron species proved non-cytotoxic at this concentration against one or the other mammalian cell type, with extracts of R. ferrugineum being non-toxic towards IEC6 cells, and extracts of R. rubiginosum as well as R. concinnum not affecting HaCaT cells. In general, keratinocytes proved more resistant than epithelial cells against the treatment with cytotoxic compounds contained in Rhododendron leaf extracts.

We conclude that 50 µg/mL leaf extracts from highly potent antimicrobial R. minus and R. racemosum are safe to use in 24-hours incubations of monolayer cultures of both, HaCaT keratinocytes and IEC6 intestine epithelial cells, while others, such as extracts from R. rubiginosum as well as R. concinnum or R. ferrugineum shall be applicable to either skin or intestinal cell layers, respectively. Further studies are required to evaluate safe application onto human tissue other than skin or gastrointestinal tract mucosa, and thus, for an assessment of using

57 | P a g e Results formulations based on leaf extracts from these Rhododendron species as antimicrobial phytomedical treatments.

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Background

Plant extracts are commonly used in formulations of alternative and traditional medicine such as for preparation of lotions applied to skin, or when used as ingredients in dietary treatments or teas [1]. Plant-based medications are typically well-accepted by the patients and often preferred over chemically produced therapeutics because many plants have been known since long for their health-beneficial, bio-active ingredients [2-6]. Moreover, plant-extractable compounds have gained a lot of attention in conventional medicine, for instance plant-based drugs are used for therapeutic treatment of several diseases including cancer and inflammatory disorders [7, 8]. Thus, the potential of plant-derived bio-active compounds is nowadays regarded substantial if not invaluable for drug development. This has generated increasing interest of the pharmaceutical industry in gaining the rights to identify and exploit plant-borne compounds from species-rich rain forests in countries of tropical and subtropical regions [9-11]. While the potential of identifying plant-derived medication is considered promising, it must be noted that the protection of biodiversity, acceptance of intellectual property rights, as well as biosafety of application are challenging issues which entered current discussions on exploitation of nature-borne bio-active compounds only recently [12, 13].

So far, roughly 6% of all worldwide occurring higher plant species have been or are being assessed for their phyto-medicinal potential. Only a minor portion of these plant species have been subjected to in-depth phytochemical profiling [14-16]. It turned out to be critical to consider different plant parts for extraction of bio-active compounds which in turn might have either trophic, anti-cancer, or antimicrobial effects. Bio-active compounds must be purified before further assessment and testing prior to eventually entering clinical trials, thereby ensuring the efficacy of a certain bio- molecule in therapeutic approaches for treatment of specific disorders. Simultaneously, drug safety and absence of undesired side-effects are of highest concern [17, 18]. These considerations are important, no matter whether pure compounds or crude extracts of the entire plant or parts thereof are used for the production of a pharmaceutically applicable plant ingredient.

The genus Rhododendron comprises the species-richest group of wooden plants, belongs to the family Ericaceae, and encompasses about one thousand species, the majority of which being indigenous to Asia [19]. In ethno-medicine, extracts of Rhododendron have been used in

59 | P a g e Results traditional treatments of various disorders such as inflammatory conditions, common symptoms of cold, gastrointestinal disorders, skin diseases, or as pain killers beyond others [20]. Recent research highlighted that Rhododendron leaf extracts might be highly potent and potentially health beneficial because they contain e.g. anti-bacterial [21, 22], anti-allergic, anti-inflammatory [23, 24] agents. The reported use of crude extracts of R. ferrugineum and R. anthopogon [20, 25-27] are most likely due to the presence of terpenoids in high concentrations [25].

Previously, we investigated leaf extracts of 120 different Rhododendron species for their efficacy as antimicrobials in killing a variety of Gram-positive and Gram-negative bacteria [25]. In the current study, extracts of 12 of the most anti-bacterial Rhododendron species were applied in different concentrations to monolayer cultures of human HaCaT epidermal keratinocytes and rat intestine epithelial cell line IEC6, respectively. We consider intestinal epithelial cells and keratinocytes among the first points of contact when drugs are administered orally or applied ectopically, respectively. In general, bio-active compounds are considered cytotoxic when they alter cellular morphology or metabolism, interfere with the cytoskeleton or cell adhesion, affect cell proliferation rates or cell differentiation processes, or initiate programmed cell death [28]. Different cell lines might exhibit differential responses towards a specific compound or plant extract. Consequently, it is neither sufficient to use only one cell line nor to apply just a single cytotoxicity assay.

Effects on cell survival and growth as well as on different cellular processes were monitored by an array of cell biological assays. The aim of this study was to assess possible cytotoxic effects of antimicrobial Rhododendron leaf extracts toward mammalian cells in order to identify potential candidate species for further analysis of safe use and thus to contribute to on-going investigations on the bioactivity potential of plant species such as Rhododendron.

Methods

Collection of plant material and leaf extract preparation

Leaves of the Rhododendron plants used in this study (Table 1) were collected from the Rhododendron-Park Bremen (www.rhododendronpark-bremen.de), and the species were

60 | P a g e Results identified and verified by references to the German Gene Bank Rhododendron Database provided by the Bundessortenamt (www.bundessortenamt.de/rhodo) [25].

Leaf material was frozen in liquid nitrogen and powdered using a Ksw 3307 mill (Clatronic, Kempen, Germany). Crude extracts were prepared by soaking two grams of Rhododendron leaf powder in 10 mL of 80% methanol for 24 hour at 4°C with constant shaking. Insoluble material was removed by centrifugation at 3,220 g for 30 min at 4°C, and supernatants were stored at -20°C until further use. Methanol was evaporated from the extracts using a Micro Modulyo lyophilizer (Edwards, Crawley, UK), and stock solutions were prepared by dissolving the residues in 100% dimethyl sulfoxide (DMSO) (Carl Roth, Karlsruhe, Germany) with 5, 50, or 500 µg of lyophilized extract per mL solvent. Prior to assays, the samples were mixed with the respective cell culture medium such that the final concentration of DMSO did not exceed 0.5% (v/v).

Table 1. List of Rhododendron species from which leaves were collected and used to prepare extracts that were screened for exhibiting cytotoxicity toward intestine epithelial cell cultures and monolayer of keratinocytes

Genebank-No.* Species name Section Sub-section 100.345 R. ferrugineum L. Rhododendron Rhododendron 100.007 R. ambiguum Hemsley Rhododendron Triflora NA R. anthopogon Don ssp. anthopogon Betty Grahamgon Pogonanthum - NA R. hirsutum L. Rhododendron Rhododendron 100.326 R. concinnum Hemsley Rhododendron Triflora 100.322 R. cinnabarinum Hooker Rhododendron Cinnabarina NA R. racemosum Franchet Rhododendron Scabrifolia 100.404 R. rubiginosum Franchet Rhododendron Heliolepida 100.474 R. xanthostephanum Merrill Rhododendron Tephropepla 100.370 R. minus Michaux Rhododendron Caroliniana 100.392 R. polycladum Franchet Rhododendron Lapponica 100.353 R. hippophaeoides var. hippophaeoides Hutchinson Rhododendron Lapponica *Gene bank numbers used in the collection of the Rhododendron-Park Bremen.

NA: Not a plant of the German Gene Bank Rhododendron but a verified plant of the Rhododendron-Park Bremen.

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Cell cultures

The normal rat small intestinal epithelial cell line IEC6 [29, 30] and the human keratinocyte cell line HaCaT [31, 32] purchased from the European Collection of Cell Cultures (ECACC, Salisbury, UK) were used throughout this study. IEC6 cells were grown in Dulbecco’s modified Eagle’s Medium (DMEM High Glucose) (Lonza Group, Basel, Switzerland) supplemented with 10% fetal calf serum (FCS) (Perbio Science, Bonn, Germany) and 10 μg/mL insulin (Sigma-Aldrich,

Steinheim, Germany). IEC6 cells were incubated at 37°C in a 5% CO2 atmosphere in an incubator (Heraeus, Osterode, Germany). HaCaT cells were cultured in DMEM containing 10% FCS and incubated at 37°C in an 8.4% CO2 atmosphere. Cell cultures were routinely passaged once per week. All experiments were performed with cultures for which approx. 70% and 95% confluence was reached for IEC6 and HaCaT cells, respectively.

Determination of the cell viability and proliferative activity by MTT assays

Effects of Rhododendron leaf extracts on the viability and proliferative activity of cultured IEC6 and HaCaT cells were quantitated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Carl Roth) indicative for mitochondrial NADH-dependent dehydrogenase activity, which is proporational to both, cell viability and proliferation rates of treated cultures [33- 35]. A total of 1 × 104 cells/well were seeded in single wells of 96-well plates (Greiner, Essen, Germany), and upon reaching the desired confluence, the cells were incubated with three different concentrations of Rhododendron leaf extracts (5, 50 and 500 µg per mL culture medium containing up to 0.5% DMSO) for 24 h in complete medium and at standard incubation conditions. Incubation of cells with culture medium containing DMSO at a final concentration of 0.5% (v/v) was used as a negative control. Culture supernatants eventually containing free-floating dead cells were removed at the end of incubation, replaced with fresh culture medium containing MTT at a final concentration of 0.5% (w/v), and the cell layers further incubated for another four hours. Subsequently, culture supernatants were removed, the cells adherent to the plate surface were collected in 100% DMSO and incubated for 15 min at 37°C to terminate the reaction and to dissolve formazan crystals. The absorbance of formazan formed by Rhododendron leaf extract- treated and non-treated control cell cultures was quantified at 595 nm in a microplate reader against

62 | P a g e Results the solvent (Tecan Group, Männedorf, Switerland). Percentages of cell viability were calculated from triplicates using Eq.(1):

푎푏푠표푟푏푎푛푐푒 표푓 푡푟푒푎푡푒푑 푐푒푙푙푠 % 표푓 푐푒푙푙 푣푖푎푏푖푙푖푡푦 = ( ) × 100 … … … … . . (1) 푎푏푠표푟푏푎푛푐푒 표푓 푐표푛푡푟표푙 푐푒푙푙푠

Propidium iodide staining of cells with ruptured plasma membranes

The two cell lines were grown on cover glasses in 24-well Bio-One Cellstar plates (Greiner) to reach the desired degree of confluence. Next, cells were incubated with three different concentrations of Rhododendron leaf extracts (i.e. 5, 50, and 500μg/mL) for 24 h as described above. Subsequently, cells were washed three times with phosphate-buffered saline (PBS) before incubating them for 45 min in 2 mg/mL propidium iodide (PI) (Carl Roth) and 5 μM Draq5™ (Biostatus, Leicester, UK) in culture medium at 37°C. After washing three times in PBS, cells were fixed in 4% paraformaldehyde (PFA) (Carl Roth) in 200 mM HEPES (pH 7.4) at room temperature for 20 min. Cells on cover glasses were washed again in PBS and distilled water before mounting them in mowiol for subsequent laser scanning microscopy as described before [36]. PI is not capable of penetrating cells with intact plasma membranes. However, if plasma membrane integrity is lost, PI gains access to the nucleus and forms complexes with the DNA. In contrast, Draq5™ serves as a nuclear counter-stain that penetrates intact plasma membranes and can therefore be used to determine the total cell number. Total cell numbers may differ depending on the treatment since some plant leaf extracts might exhibit anti-adhesive properties such that cell numbers are significantly diminished after incubation and washing steps. Therefore, the total cell numbers are reported herein as well.

Phalloidin staining of the filamentous actin cytoskeleton

IEC6 and HaCaT cells were grown on cover glasses in 24-well plates to reach 70% and 95% confluence, respectively, and exposed to Rhododendron leaf extracts for 24 h as described above, while 0.5% DMSO was used as a negative control. Cells were washed three times with PBS before fixation in 4% PFA in 200 mM HEPES (pH 7.4) at room temperature for 20 min. After fixation, cells were washed extensively with PBS before applying 0.2 % Triton X-100 in PBS for 5 min at room temperature followed by several washing steps in PBS. Finally, cells were stained with a

63 | P a g e Results mixture of 3 μM FITC-labeled phalloidin (Sigma Aldrich) and 5 μM Draq5™ as a counter stain of the nuclear DNA in PBS at room temperature for 30 min. Cover glasses were mounted in mowiol for subsequent laser scanning microscopy.

MitoTracker XMSRos staining of the mitochondrial matrix

Cells were incubated and treated as described above, before washing twice in phenol red-free HEPES-buffered culture medium (20 mM HEPES) for 5 min. Subsequently, the cells were incubated with phenol red-free culture medium containing 20 mM HEPES and 500 nM MitoTracker Red CMXRos (Molecular Probes, Oregon, USA) for 45 min at 37°C followed by several washes. The fluorescent dye reaches the mitochondrial matrix for accumulation therein if an intact membrane potential due to active cellular metabolism applies across the inner mitochondrial membrane. Cells were fixed with 4% PFA in 200 mM HEPES (pH 7.4) for 20 min at room temperature, rinsed, and mounted on microscope slides as described above for subsequent microscopic inspection.

Microscopy techniques

Stained cells were visualized with an LSM 510 confocal laser scanning microscope (Carl Zeiss, Jena, Germany) at excitation wavelengths of 488 nm, 543 nm, and 633 nm, for fluorophore excitation to visualize FITC-phalloidin, PI or MitoTracker XMSRos, and Draq5™, respectively. Scans at a resolution of 1024 x1024 pixels were taken in the line averaging mode and at a pinhole setting of one airy unit. Color coding and image analysis was performed by using the LSM 510 software, release 3.2 (Carl Zeiss).

Caspase-3 activity assay

For IEC6 cells, induction of apoptosis after incubation with R. ferrugineum and R. cinnabarinum leaf extracts at the highest concentration used (500 µg/mL) was evaluated at different time intervals ranging from 1 to 24 h. The apoptosis assay was performed using the EnzChek Caspase- 3 assay kit (Invitrogen, Karlsruhe, Germany) detecting activation of procaspase-3 and other Asp- Glu-Val-Asp (DEVD)-specific proteases. Lysates of treated IEC6 cells and non-treated controls were prepared according to the manufacturer’s protocol. Following clearance by centrifugation,

64 | P a g e Results the samples were incubated with 5 mM Z-DEVD-R110 substrate for 30 min at 4°C. Lysates of IEC6 cells treated for 4 h at 37°C with apoptosis-inducing staurosporin (10 mM) (Sigma-Aldrich) were used as positive controls whereas no treatment or incubation with the solvent served as negative controls. Additionally, staurosporin-treated cells incubated with 1 mM of Ac-DEVD- CHO for 10 min served as negative control since caspase-3 activity is blocked under these conditions. The extent of procaspase-3 activation was determined by excitation at 496 nm and reading the emission at 520 nm using a microplate reader (Tecan Group, Männedorf, Switzerland). The values were normalized to equal amounts of DNA in the pellets after lysis as determined by the Burton assay [37].

Determination of minimum inhibitory concentrations

The minimum inhibitory concentration (MIC) was defined as the lowest concentration of antimicrobial that will inhibit the visible growth of a micro-organism after overnight incubation. The MIC was determined by a two-fold dilution assay in Mueller-Hinton broth (MHB) (Becton Dickinson, Heidelberg, Germany). Bacillus subtilis S168 strain was tested with 12 Rhododendron crude extracts (Table 1) [1]. All tests were done in triplicate following the National Center for Clinical Laboratory Standards recommendations [38].

Statistical evaluation

All assays were performed in triplicates and repeated at least three times in independent experiments unless stated otherwise. All data were expressed as means ± standard derivation (SD) as determined by using Origin software (MicroCal Software, Northampton, USA). The heat map shown in Figure 9 was created using R (RStudio, Boston, USA). Levels of significance were calculated by One-Way ANOVA, and p < 0.05 was considered statistically significant. CellProfiler software was used to determine total cell numbers (Draq5™-positive cells) versus numbers of dead cells (PI-positive cells), and this software was also employed to quantify the MitoTracker XMSRos and FITC-phalloidin fluorescence signal intensities as described by us previously [39].

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Results

Classification of Rhododendron species based on antibacterial activities

In order to group the 12 selected Rhododendron species [25] according to their antibacterial activities, minimum inhibitory concentration (MIC) tests were conducted with B. subtilis as the tester organism. Accordingly, the plant species could be classified into four major groups. Six Rhododendron species formed the group with the highest antibacterial activity with an MIC of 50 µg/mL: R. minus, R. racemosum, R. ferrugineum, R. rubiginosum, R. anthopogon ssp. anthopogon, and R. cocinnum. Another three species formed the group with moderately active extracts: R. cinnabarinum, R. hirsutum, and R. ambiguum with an MIC of 100 µg/mL. The remaining Rhododendron species exhibited lower antibacterial activities with R. xanthostephanum and R. polycladum having a MIC of 150 µg/mL and R. hippophaeoides var. hippophaeoides requiring 300 µg/mL to efficiently kill the bacterial tester organism.

Cell viability and proliferation rates as quantified by the MTT assay

The effects of leaf extracts prepared from 12 different Rhododendron species on cell viability and proliferation rates were initially estimated with the help of the MTT assay since this test allows for a rapid screening of many samples. IEC6 and HaCaT cells were exposed to Rhododendron leaf extracts applied at three different concentrations (5, 50, and 500 µg/mL) for 24 h and results are shown in Figure 1. Incubations with extracts applied at the two lower concentrations revealed no detectable change in MTT conversion rates of HaCaT cells while the leaf extracts from R. polycladum Franchet, R. concinnum Hemsley, and R. xanthostephanum Merrill, affected IEC6 cellular metabolism negatively (arrows) in comparison to cells that were not treated or treated with 0.5% DMSO suggesting that even 5 µg/mL of these extracts caused cytotoxic effects on intestinal cells. Rhododendron leaf extracts used at higher concentrations (500 µg/mL) were more effective in reducing the MTT conversion abilities of the treated cells (Figure 1). Extracts of R. rubiginosum Franchet, R. cinnabarinum Hooker, and R. ferrugineum L., exerted cytotoxic effects as deduced from the significant decrease in the ability of both, IEC6 and HaCaT cells, to reduce MTT. In addition, samples from R. minus Michaux, R. polycladum, R. concinnum, R. ambiguum Hemsley, and R. hirsutum L., induced statistically significant reduction in the MTT conversion ability of

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IEC6 cells although not negatively effecting HaCaT cells. Interestingly, leaf extracts of R. hippophaeoides var. hippophaeoides Hutchinson, R. anthopogon Don ssp. anthopogon Betty Graham, and R. racemosum Franchet did not exhibit any significant alterations on metabolic activities and cell viability of both cell lines which highlights their potential non-cytotoxicity (Figure 1).

Figure 1: Effects of Rhododendron bioactive compounds on IEC 6 (a) and HaCaT (b) cells incubated with three different concentrations (5, 50, and 500 μg/mL) of leaf extracts as indicated. Cell viability and proliferation was analyzed by the MTT assay upon incubation at 37°C for 24h. The percentage of MTT reduction for each extract concentration was normalized to that of the 0.5% DMSO solvent. Values are given as mean ± standard deviations from three independent experiments, each performed in triplicates. Statistical evaluation was performed by one way Anova analysis; levels of significance are indicated as * for p < 0.05.

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Analysis of plasma membrane integrity

In order to verify the initially observed cytotoxic effects of Rhododendron leaf extracts on IEC6 and HaCaT cell cultures, changes in the integrity of the plasma membrane upon incubation of the cells with leaf extracts were tested by PI acquisition, which occurs in cells with ruptured plasma membranes. Cell staining with Draq5TM allowed an estimation of the total number of cells. The results summarized in Table 2, Figure 2, and Supplementary Figure 1 demonstrated that incubation of IEC6 cells with 5 and 50 μg/mL of most Rhododendron leaf extracts did neither significantly affect the total cell numbers nor membrane integrity. However, incubations of IEC6 cells with 50 µg/mL of leaf extracts from R. polycladum, R. concinnum, R. anthopogon ssp. anthopogon, and R. hirsutum resulted in a significant reduction of total cell numbers as compared to the control treatments (Figure 2a).

Table 2. Total cell number and percentage of dead cells of IEC6 and HaCaT cell cultures which were treated with three different concentrations of Rhododendron leaf extracts (5, 50 and 500 µg/mL). Data are expressed as mean ± standard deviations.

IEC 6 HaCaT Total cell Dead cells Total cell Dead cells Treatments Conc. numbers (%) numbers (%) μg/mL (Draq5) (Draq5) R. hippophaeoides var. hippophaeoides 500 99 ± 46 81 ±16 212 ± 57 0 ± 0 50 494 ± 188 2 ± 0.6 283 ± 123 0.2 ± 0.2 5 297 ± 139 1 ± 1 328 ± 138 2 ± 2 R. minus 500 74 ± 48 79 ± 19 221 ± 86 83 ± 12 50 497 ± 106 0.4 ± 0.4 348 ± 113 0.4 ± 0.5 5 683 ± 60 0.8 ± 0.2 372 ± 155 0.8 ± 0.8 R. rubiginosum 500 288 ± 120 97 ± 3 253 ± 122 4 ± 0.7 50 551 ± 147 0.9 ± 1 413 ± 197 0.9 ± 0.9 5 672 ± 101 0.5 ± 0.4 390 ± 41 0 ± 0 R. cinnabarinum 500 204 ± 40 100 ± 0 112 ± 23 94 ± 7 50 475 ± 183 0.8 ± 0.8 448 ± 77 2 ± 1 5 481 ± 128 1 ± 1 552 ± 68 2 ± 0.5

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R. ferrugineum 500 33 ± 11 100 ± 0 490 ± 122 85 ± 15 50 522 ±62 1 ± 0.6 287 ± 109 1 ± 1 5 439 ± 85 2 ± 2 481 ± 59 1 ± 1 R. polycladum 500 99 ± 25 100 ± 0 204 ± 62 13 ± 22 50 274 ± 106 1 ± 1 204 ±72 1 ± 1 5 384 ± 104 0.9 ± 0.5 274 ± 26 0 ± 0 R. concinnum 500 304 ± 109 100 ± 0 143 ± 58 93 ± 6 50 252 ± 58 45 ± 44 320 ± 66 0.5 ± 0.1 5 586 ± 160 0.9 ± 0.8 260 ± 108 0 ± 0 R. xanthostephanum 500 211 ± 58 12 ± 4 127 ± 56 0 ± 0 50 408 ± 68 0 ± 0 181 ± 70 0 ± 0 5 460 ± 115 0.7 ± 0.6 319 ± 89 0.3 ± 0.3 R. anthopogon ssp. anthopogon 500 164 ± 41 100 ± 0 196 ± 50 90 ± 4 50 235 ± 72 23 ± 9 370 ± 153 1 ± 1 5 578 ± 164 2 ± 1 395 ± 157 4 ± 5 R. ambiguum 500 666 ± 220 99 ± 2 179 ± 60 0 ± 0 50 439 ± 154 1 ± 1 327 ± 82 0.5 ± 0.6 5 535 ± 119 0.7 ± 0.7 357 ± 150 0 ± 0 R. hirsutum 500 189 ± 65 99 ± 0.6 270 ± 113 98 ± 0.7 50 267 ± 64 11 ± 15 258 ± 109 0.5 ± 0.1 5 271 ± 147 1 ± 0.5 298 ± 67 0 ± 0 R. racemosum 500 224 ± 54 81 ± 23 123 ± 45 94 ± 3 50 400 ± 262 0.4 ± 0.6 211 ± 33 0 ± 0 5 471 ± 183 1 ± 0.7 225 ± 79 0 ± 0

Application of almost all leaf extracts at the highest concentration resulted in significant and dramatic decreases in the total cell numbers and a dramatic decrease of membrane integrity (Table 2, Supplementary Figure 1) suggesting that the observed effects on IEC6 cell cultures were likely due to both, a massive cell de-adhesion and cell death by membrane rupture of the majority of remaining cells irrespective of the used leaf extract. Interestingly, the leaf extract of R. ambiguum had a remarkable divergent effect on IEC6 cells as opposed to all other extracts at 500 µg/mL: Although almost all cells had lost their membrane integrity, they remained adherent to the well

69 | P a g e Results surface (Supplementary Figure 1, Table 2) indicating that the extract from this Rhododendron species might induced different reaction than those of the other species.

Figure 2. Effects of Rhododendron leaf extracts on the cell numbers of IEC6 (a) and HaCaT (b) cells after 24h incubation at 37°C with three different concentrations (5, 50, and 500 μg/mL) of leaf extracts as indicated. The total number of cells as determined by Draq5 staining reflects the effects leaf extracts on cell viability and adhesion since only cell monolayer-associated cells were stained and counted in this assay. Values are given as mean ± standard deviations from three independent experiments, each performed in triplicates. Statistical evaluation was performed by one way Anova analysis; levels of significance are indicated as * for p < 0.05.

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HaCaT keratinocytes exposed to Rhododendron leaf extracts at any of the concentrations tested tolerated the incubation much better than IEC6 cells since the total cell numbers were only significantly diminished when HaCaT cells were incubated with 500 µg/mL leaf extracts from four Rhododendron species, i.e. R. cinnabarinum, R.concinnum, R. xanthostephanum, and R. racemosum (Figure 2b, Supplemental Figure 2). Three out of those treatments followed the previously observed major trend for a combination of cell de-adhesion and membrane disruption of HaCaT cells when a high concentration of plant extract was applied (Table 2). Interestingly, the extract of R. xanthostephanum led to de-adhesion but not to a disruption of membrane integrity. Irrespective of the level of reduction in total numbers caused by 500 µg/mL of extract (Figure 2b), five out of the 12 Rhododendron leaf materials did not induce membrane rupture in HaCaT cells (Table 2) indicating significant differences in susceptibility of different cell types towards Rhododendron leaf extracts.

Effects of Rhododendron leaf extracts on mitochondrial membrane potential

Changes of the mitochondrial membrane potential of IEC6 and HaCaT cells, respectively, induced by Rhododendron leaf extracts were determined by MitoTracker® staining. For this, fluorescence intensity of stained mitochondria was quantified by measuring the average intensity over arbitrarily chosen inspection areas (Figure 3). This intensity is directly proportional to the mitochondrial membrane potential. In addition, Rhododendron leaf extract-treated and MitoTracker-stained cells were inspected by fluorescence microscopy which allowed a determination of the shape of mitochondria (Figure 4, Supplementary Figure 3). IEC6 cells incubated with leaf extracts from all Rhododendron species at the highest concentration were dramatically affected with regard to the mitochondrial membrane potential as deduced from the drastically reduced MitoTracker staining although effects were somewhat milder for leaf extracts from R. hippophaeoides var. hippophaeoides, R. xanthostephanum, R. hirsutum, and R. racemosum (Figure 3a). Alterations in mitochondrial structure of IEC6 cells that were treated with leaf extracts at any concentration were frequently observed (Supplementary Figure 3). However, IEC6 cells treated with 5 or 50 μg/mL of extracts from R. hippophaeoides var. hippophaeoides, R. xanthostephanum, R. hirsutum, and R. racemosum did not show a significant difference in the mitochondrial structure when compared to controls (Figures 3a and 4a).

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Figure 3. Effects of Rhododendron leaf extracts on the mitochondrial membrane potential of IEC 6 (a) and HaCaT (b) after 24h incubation at 37°C with three different concentrations (5, 50, and 500 μg/mL) of leaf extract as indicated h. The intensity of MitoTracker red signal reflects the accumulation of the dye within the mitochondrial matrix which depends on an intact inner mitochondrial membrane potential, thus on the metabolic activity of the cells. Values are given as mean ± standard deviations from three independent experiments, each performed in triplicates. Statistical evaluation was performed by one way Anova analysis; levels of significance are indicated as * for p < 0.05.

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Effects of Rhododendron leaf extracts on mitochondrial structure and staining intensities were much less pronounced in HaCaT keratinocytes (Figures 3b and 4b, Supplementary Figure 3b). Exceptions were observed when HaCaT cell cultures were treated with high concentrations of leaf extracts prepared from R. minus, R. cinnabarium, R. ferrugineum, R. concinnum, R. anthoppgon ssp. anthopogon, and R. ambiguum (Figure 3b) with mitochondria appearing no longer elongated but round (Figure 4b, Supplementary Figure 3b).

Figure 4. Morphological changes of mitochondria in IEC6 (a) and HaCaT (b) after 24h exposure to three different concentrations (5, 50 and 500 μg/mL) of Rhododendron leaf extracts. Confocal fluorescence images of IEC6 cells labeled with MitoTracker Red CMXRos. Cells treated with 0.5% DMSO were used as controls, while also cells are depicted upon incubation with extracts from R. hippophaeoides var. hippophaeoides (A), R. Xanthostephanum (B), R. hirsutum (C) and R. racemosum (D). Bars represent 20 μm.

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Actin cytoskeleton analysis of Rhododendron extract-treated cells

Next, we inspected the filamentous actin cytoskeleton system of Rhododendron leaf extract-treated IEC6 and HaCaT cells as a measure of preservation of the overall cellular architecture. With regard to the intensity of FITC-phalloidin staining of the cortical F-actin system of both, ICE6 and HaCaT cells, the analyses revealed mostly mild effects of the Rhododendron leaf extracts when applied at concentrations of 5 or 50 µg/mL (Figure 5). Likewise, when inspecting the cytoskeleton of either cell type morphologically, lower concentrations of almost none of the leaf extracts caused visible changes to the cortical F-actin system detectable as fluorescent structures underneath the plasma membranes of most cells (Figure 6). However, IEC6 cells exposed to any of the concentrations of R. ambiguum leaf extracts showed a significant decrease in the intensity of phalloidin-staining (Figure 5a), and five Rhododendron leaf extracts applied at the highest concentration, i.e. R. cinnabarinum, R. ferrugineum, R. concinnum, R. xanthostephanum, and R. anthopogon ssp. anthopogon, resulted in a significantly reduced staining intensity of actin filamentous system in both cell lines (Figure 6), while the extracts of R. minus, R. rubiginosum, and R. polycladum exerted negative effects on the staining of F-actin in IEC 6 cells only (Figure 6a) suggesting a rather heterogenous spectrum of effects on actin cytoskeleton morphology by various Rhododendron extracts. No particular microscopic phenotype could be observed with Rhododendron extract-treated HaCaT keratinocytes (Figure 6b).

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Figure 5: Effects of Rhododendron leaf extracts on the F-actin cytoskeleton of IEC6 (a) and HaCaT (b) upon incubation with three different concentrations (5, 50, and 500 μg/mL) of leaf extract for 24 h. The intensity of the phalloidin signal reflects F-actin presence, which maintains the cellular architecture. Values are given as mean ± standard deviations from three independent experiments, each performed in triplicates. Statistical evaluation was performed by one way Anova analysis; levels of significance are indicated as * for p < 0.05.

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Figure 6. Morphological changes of F-actin structures in IEC6 (a) and HaCaT (b) after 24h exposure to three different concentrations (5, 50 and 500 μg/mL) of Rhododendron leaf extracts. Confocal fluorescence images of IEC6 cells labeled with phalloidin (green) and Draq5 (blue). Cells treated with 0.5% DMSO served as controls for cells treated with leaf extracts of R. hippophaeoides var. hippophaeoides (A), R. Xanthostephanum (B), R. hirsutum (C) and R. racemosum (D). Bars represent 20 μm.

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Inspection of sub-cellular architecture of floating cells that detached from monolayers

Partially contradictory findings detailed above suggested that IEC6 and HaCaT cells remained either adherent within or to the monolayers, or they detached upon incubation with the Rhododendron leaf extracts. Such observation could be false-interpreted such that both cell types are able to tolerate exposure to cytotoxic agents only to some extent. Because the above assays were technically restricted to cell monolayers, we next analyzed the fraction of free-floating cells which detached during treatment with Rhododendron leaf extracts using the same staining methods as described above. Here, Draq5 staining additionally served to examine the status of nuclear DNA and to identify nuclear fragmentation typical for apoptotic cells.

Treatment of IEC6 cells with 500 µg/mL leaf extracts from R. cinnabarinum and subsequent analysis of the detached cells revealed nuclear condensation, cell shrinkage, rounding-up, loss of contacts with adjacent cells, formation of typical membrane blebs and occurrence of apoptotic bodies (Figure 7a), thus suggesting that leaf extracts of this plant species might have induced apoptosis.

However, IEC6 cells exposed to 500 μg/mL R. ferrugineum leaf extracts became pycnotic and the actin filaments formed a ring surrounding the nucleus. In addition, IEC6 cells treated with leaf extracts from R. minus, R. rubiginosum and R. ambiguum showed different stages of chromatin condensation and shrinkage of the nuclei (Figure 7). Thus, five Rhododendron leaf extracts, namely R. hippophaeoides var. hippophaeoides, R. cinnabarium, R. ferrugineum, R. xanthostephanum, and R. racemosum induced signs closely related to the classical symptoms of programmed cell death –apoptosis– and they also exhibited typical phenotypes like formation of plasma membrane blebs.

HaCaT cells showed also cellular changes indicative of cell death upon exposure to 500 μg/mL of Rhododendron leaf extracts, however, these were different from the phenotypes observed in treated IEC6 cell cultures. HaCaT cells treated with both, leaf extracts from R. cinnabarinum and R. ferrugineum represented signs of final stages of cell death because they exhibited intense PI staining throughout the nuclei and the entire cytoplasm, and some cells had even lost their nuclei altogether (Figure 7G and H).

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Moreover, exposure of HaCaT cells to R. minus, R. concinnum, and R. anthopogon ssp. anthopogon induced changes that are exemplified by shrinkage of nuclei and chromatin condensation (Figure 7I).

Figure 7. Plasma membrane integrity and cell death by apoptosis as induced by a 24h- exposure of IEC6 (a) and (b) HaCaT keratinocytes to 500 µg/mL of Rhododendron leaf extracts. Merged micrographs taken with a confocal laser scanning microscope depict IEC6 (panels A-E) and HaCaT cells (panels F-J). Violet signals in merged images are due to overlapping red, PI-derived signals, in cells with ruptured plasma membranes, and blue, Draq5™ staining of nuclei in all cells. Pictures A and F are control cells treated with 0.5 % DMSO, R. cinnabarinum (B and G), R. ferrugineum (C and H), R. minus (D and I) and R. hippophaeoides var. hippophaeoides (E and J). Bars represent 20 μm.

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Determination of apoptotic cell death pathways through determination of procaspase-3 activation

In order to substantiate the interpretation of some of the observed morphological changes induced by specific Rhododendron leaf extracts, cell layers were subjected to an additional apoptosis- proving assay. Besides some of the above noted symptoms and among others, apoptosis is characterized by the activation of procaspase-3. Therefore, a caspase-3 activity assay was applied for all treatments of IEC6 cells. Incubation of the cells with 500 µg/mL of the leaf extracts from any of the 12 Rhododendron species induced apoptosis as evidenced by a significant increase in the levels of caspase-3 activity (data not shown). There was no significant activity of caspase-3 when IEC6 cells were treated with 5 or 50 µg/mL of all leaf extracts prepared from Rhododendron species. Interestingly, R. cinnabarinum and R. ferrugineum, which were treated with concentrations of 500 µg/mL clearly exhibited phenotypic changes characteristic for apoptosis (Figure 7). Thus, we selected the leaf extracts of these two Rhododendron species to analyze procaspase-3 activation in free-floating IEC6 cells in a time-dependent manner (Figure 8). The results revealed a steady increase in caspase-3 activity levels until 12 h of treatment with extracts of R. cinnabarinum, and to a five-fold lesser extent upon treatment with R. ferrugineum extracts. However, caspase-3 activity decreased during the next 12 h. These results argue that components contained in Rhododendron leaf extracts induce apoptosis in IEC6 cells when applied in high enough concentrations.

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Figure 8. Detection of caspase-3 activity in intestinal epithelial cell (IEC 6). Cells were treated with 500 μg/mL of leaf extracts from R. cinnabarinum (a) or R. ferrugineum (b) for the indicated time intervals. Reactions were carried out at room temperature and fluorescence was measured in a fluorescence microplate reader using 496 nm for excitation and emission detection at 520 nm .Non-treated cells and cells treated with DMSO (0.5%) were used a negative controls, while staurosporine (10 μg/mL) treatment was used a positive control (apoptosis inducer). Values are given as mean ± standard deviations from three independent experiments, each performed in triplicates. Statistical evaluation was performed by one way Anova analysis; levels of significance are indicated as * for p < 0.05.

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Summarizing integration of the results achieved with a variety of cell toxicity assays

The partially complex data acquired with different cell toxicity analysis assays as reported above were summarized by grouping the 12 Rhododendron species according to their antibacterial effectiveness with respective MICs of 50, 100, 150, or 300 µg/ml, and qualitatively comparing their effects against both types of cell cultures (Figure 9). In general, most Rhododendron extracts applied in high concentrations exerted stronger cytotoxic effect on IEC6 intestinal epithelial cells as compared to HaCaT keratinocytes. R. hippophaeoides var. hippophaeoides which exhibited the lowest antibacterial effect also proved to be least aggressive against both mammalian cell types. A total of five Rhododendron extracts with high antibacterial potential (MIC of 50 µg/mL) did not reveal cytotoxicity against the mammalian cell lines in any of the assays, at least, when applied at 50 µg/mL, indicating that these extracts are unlikely to harm mammalian cells while killing bacterial cells. This is to say they are likely to be further assessed as safe antimicrobials applied to skin or used for oral administration. The corresponding plant species were R. minus, R. racemosum, R. ferrugineum, R. rubiginosum, and R. concinnum. Interestingly, only the extracts of R. minus and R. racemosum proved to be non-cytotoxic to both, intestinal epithelial cells and keratinocytes (Figure 9) suggesting them as the most promising candidates for future investigations on the search for optimized antibiotic in bio-active plant extract, or to be used when purified compounds are identified in this plant species which exert no cytotoxicity.

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Figure9. Heatmap summarizing the results of cell biological assessment assays combined with the effects of Rhododendron crude extracts against B. subtilis depicted by minimum inhibitory concentration (MIC). The twelve Rhododendron species were classified into four groups (i.e. 50, 100, 150, and 300 µg/mL) according the MIC results (black vertical line). Three concentrations (5, 50, and 500 µg/mL) of Rhododendron crude extracts were applied to the two different cell lines for 24h. The colors are representative for the toxicity that Rhododendron crude extracts exerted on mammalian cells, i.e. non-toxic extract are depicted in gray, and cytotoxic extracts are shown by black boxes. Panel a represents the results of IEC6 cells for the assays on cell viability and proliferation rates (A), plasma membrane integrity (B), cellular architecture (C), total cell numbers (D), and cellular metabolism (E). Panel b represents the results of HaCaT cells.

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Discussion

Up to date, there are only few medicinal formulations on the market which contain compounds derived from Rhododendron. These comprise the anti-hypertensive agent ‘Rhomitoxin’ and ‘Rhododendron cp paste’ used to relieve arthritis pain [40]. In addition, few in vitro and in vivo studies for selected Rhododendron extracts and their isolated compounds have been reported for their validation as being useful in traditional remedies [20]. Importantly, plants of this genus are preferably used as alternative medicine in geographic regions of their native habitats such as Nepal, Northeastern India, Western and Central China, or Indonesia [20]. On the one side, the compositions of such medicinal formulations are not at all, or at least, not well defined [41, 42]. On the other side, Rhododendron plants are known to synthesize a large number of chemical compounds some of which exhibit pharmacological activity [43-46]. Several of those chemical compounds have been identified to belong to pro-anthocyanidins, polyphenols, or terpenoids which are typically synthesized by plants in defense reactions against pathogens and herbivores [47, 48].

Not surprisingly, various plant-derived compounds exert severe cytotoxic or mutagenic effects when applied to animal cells and tissues [49, 50]. Intoxications of domesticated or wild animals which fed on Rhododendron plants have been repeatedly reported, and were linked to the presence of grayanotoxins [51-53]. Therefore, a comprehensive number of cytotoxicity studies involving mammalian cells or tissue cultures must be conducted before a given extract or compound can eventually be considered for testing on animal models, or may even enter clinical trials [54, 55].

To our best knowledge, none of the previous studies had comprehensively analyzed the cytotoxicity of a group of pharmaceutically interesting Rhododendron species. Consequently, the current study introduces a multi-facetted approach by investigating the effects of leaf extracts on viability, morphology, and metabolic activity of two different types of mammalian cells with five different cytotoxicity assays.

The results obtained herein are consistent in as much as treatment of IEC6 and HaCaT cells with low concentrations of leaf extracts prepared from any of the 12 Rhododendron species exhibited no or rather mild cytotoxic effects, whereas the use of very high concentrations (500 µg/mL)

83 | P a g e Results resulted in an expectable dramatic induction of cytotoxicity. In total, five Rhododendron species which exhibited high antibacterial activities with MICs of 50 µg/mL proved to be non-cytotoxic at this concentration. Interestingly, extracts of R. minus and R. racemosum were non-toxic against any of the tow cell lines making them promising candidates for future studies. In contrast, incubation of either of the two cell lines with 500 µg/mL of most Rhododendron leaf extracts resulted in severe structural and functional alterations often associated with signs of apoptosis. The study thus confirms that simultaneous analysis of several, partially unlinked or only indirectly linked cellular parameters is a powerful tool to separate potentially cytotoxic extracts from potentially ‘safe-use’ Rhododendon extracts thus overcoming technical restrictions of previous studies.

Overall, keratinocytes were more resistant to cytotoxicity exerted upon incubation with Rhododendron leaf extracts than IEC6 cells. Resistance of HaCaT cells against cytotoxic agents was observed by us previously when studying dust exposure [32]. This remarkable feature of keratinocytes might be due to the specific lipid composition of their membranes and their ability to build a stratified epithelium when exposed to air during cornification [32, 56, 57].

Our results demonstrated that the inclubation of cells with high concentrations of Rhododendron leaf extracts induces apoptosis in intestinal epithelial cells. Interestingly, only two extracts, namely those from R. cinnabarinum and R. ferrugineum shared a similar pattern of cytotoxicity in all assays tested in this study. Leaf extracts of these two Rhododendron species were capable of inducing procaspase-3 activation prominently in IEC6 cells. The results of this study concur with other studies which have shown that several secondary metabolic compounds from Rhododendron species induce apoptosis in cultures of different mammalian cell lines [58, 59].

Conclusion

Using a comprehensive approach, the cytotoxicity of those Rhododendron species was determined that had previously been shown the exhibit highest antibacterial activities thus continuing our ongoing approach to identify a pharmaceutically feasible antibiotics or lead structure. Use of two tester cell lines as relevant models for ectopic or oral treatment and several different assays proved to be a suitable combination of screening tools. Two out of 12 Rhododendron species with

84 | P a g e Results antibacterial activities turned out to exhibit the desired traits: the extracts of R. minus and R. racemosum were non-cytotoxic at a concentration at which they efficiently killed the bacterial tester organism. We could furthermore conclude that Rhododendron leaf extracts induced apoptosis as evidenced by alteration of the cellular phenotypes (chromatin condensation and formation of plasma membrane blebs) as well as by increasing the levels of active caspase-3 when cells were exposed to higher doses. In future, we will extend our current study in order to determine whether the specific apoptosis-inducing effects of R. cinnabarinum and R. ferrugineum can be used to selectively target cancer cells, for instance, colorectal carcinoma cells. In our future research, we also intend to focus on phyto-chemically identifying the actual active compounds present in different Rhododendron species’ leaf extracts to study their individual cytotoxicity effects or lack of thereafter by a similar repertoire of different methods as laid out in the current study.

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Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

AR designed experiments, conducted the experimental work and the analysis, and prepared the manuscript; AH and WJ contributed to the experimental work presented in Figures 4, 6 and 7, HS collected, identified, prepared plant materials, MU and KBr designed the study, supervised the work, discussed the results, and contributed to manuscript writing. All authors read and approved the final manuscript.

Acknowledgements

This study was financially supported by the Stiftung Bremer Rhododendronpark. The authors are particularly grateful to Wolfgang Klunker for his enthusiastic support and would like to thank Maren Rehders for expert help with cell culture experiments.

Author details

1Molecular Life Science Research Center, Jacobs University Bremen, Campus Ring 1, Bremen 28759, Germany. 2Stiftung Bremer Rhododendronpark, Deliusweg 40, Bremen 28359, Germany.

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Supplemental Figure 1

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Overview of plasma membrane integrity and apoptotic cell death induced by 24h exposure of IEC6 cells to three different concentrations (5, 50, and 500 µg/mL) of Rhododendron leaf extracts. Single channel fluorescence, phase contrast and merged micrographs taken with a confocal laser scanning microscope. Violet signals in merged pictures are due to overlapping red, PI-derived signals with blue Draq5™ staining. Cells treated with 0.5 % DMSO served as controls, A) R. hippophaeoides var. hippophaeoides, B) R. minus, C) R. rubiginosum, D) R. cinnabarinum, E) R. ferrugineum, F) R. polycladum, G) R. concinnum, H) R. xanthostephanum, I) R. anthopogon ssp. anthopogon, J) R. ambiguum, K) R. hirsutum, and L) R. racemosum. Bars represent 20 μm.

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Supplemental Figure 2

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Overview of plasma membrane integrity and apoptotic cell death induced by 24h exposure of HaCaT keratinocytes to three different concentrations (5, 50, and 500 µg/mL) of Rhododendron leaf extracts. Single channel fluorescence, phase contrast and merged micrographs taken with a confocal laser scanning microscope. Violet signals in merged pictures are due to overlapping red, PI-derived signals with blue Draq5™ staining. Cells treated with 0.5 % DMSO served as controls, A) R. hippophaeoides var. hippophaeoides, B) R. minus, C) R. rubiginosum, D) R. cinnabarinum, E) R. ferrugineum, F) R. polycladum, G) R. concinnum, H) R. xanthostephanum, I) R. anthopogon ssp. anthopogon, J) R. ambiguum, K) R. hirsutum, and L) R. racemosum. Bars represent 20 μm.

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Supplemental Figure 3

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Overview of mitochondrial morphology in IEC6 (a) and HaCaT cells (b) after a 24h-exposure to three different concentrations (5, 50 and 500 μg/mL) of Rhododendron leaf extracts. Confocal fluorescence images of IEC6 (left) and HaCaT (right) labeled with MitoTracker Red CMXRos. Cells treated with 0.5 % DMSO served as controls, A) R. hippophaeoides var. hippophaeoides, B) R. minus, C) R. rubiginosum, D) R. cinnabarinum, E) R. ferrugineum, F) R. polycladum, G) R. concinnum, H) R. xanthostephanum, I) R. anthopogon ssp. anthopogon, J) R. ambiguum, K) R. hirsutum, and L) R. racemosum. Bars represent 20 μm.

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Supplemental Figure 4

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Overview of the structure of the F-actin system in IEC6 (a) and HaCaT cells (b) after a 24h- exposure to three different concentrations (5, 50 and 500 μg/mL) of Rhododendron leaf extracts. Confocal fluorescence images of IEC6 (left) and HaCaT (right) labeled with phalloidin (green) and Draq5 (blue). Cells treated with 0.5 % DMSO served as controls, A) R. hippophaeoides var. hippophaeoides, B) R. minus, C) R. rubiginosum, D) R. cinnabarinum, E) R. ferrugineum, F) R. polycladum, G) R. concinnum, H) R. xanthostephanum, I) R. anthopogon ssp. anthopogon, J) R. ambiguum, K) R. hirsutum, and L) R. racemosum. Bars represent 20 μm.

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Results

3.3 Distinguishing the polyphenolic and antibacterial profile of the leaves, fruits and flowers of Rhododendron ambiguum and Rhododendron cinnabarinum using high performance liquid chromatography coupled with ion trap and time of flight mass spectrometry

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Abstract

Polyphenols are secondary plant metabolites that serve different functions in plants. They are known to have beneficial health properties for humans and have been used to treat diseases like asthma, skin diseases. Rhododendron species are known to be a good source of polyphenols. These species are found throughout the world with the exception of some parts in America and Africa. It is a diverse genus of woody plants with different size, shape, texture and color of flowers. They have spirally arranged leaves which may be evergreen or deciduous. Rhododendron species have been traditionally used in countries like China, Nepal, Russia and North America for treating human diseases.

In this study, the phytochemical profile of fruits, flowers and leaves of different ages of Rhododendron ambiguum and Rhododendron cinnabarinum were studied by using HPLC-MS. 62 different polyphenols including isomers were identified in these species by their fragmentation pattern and high resolution data. Parallelly, the antibacterial activity of these parts was assessed against Gram-positive bacteria.

A total of 59 polyphenols were identified for both Rhododendron species. All plant parts for both species were determined to have a significant antibacterial effect against four Gram-positive bacteria. We conclude that the distribution of polyphenol in all crude extracts of aerial parts for both Rhododendrons species are randomly and age independent. Furthermore, the bioactive compounds are mostly distributed equally in leaves, flowers, and fruits which indicated by the similarity in the antibacterial activity assay.

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Introduction

Polyphenols are secondary plant metabolites that are known to have different physical, chemical and biological properties. Proanthocyanidins (PAs) are a class of polyphenols which are formed from the oligomerization and polymerization of flavan-3-ol units such as catechin and afzelechin (Figure 1). PAs are commonly found in fruits, vegetables and grains [1]. They are known to have antioxidant [2], anti-inflammatory [3] and antimutagenic effects and have been used in the treatment of asthma [4], skin diseases, and UV radiations [5].

Rhododendron is a genus of woody plants that belong to Ericaceae family. Most of the species have attractive flowers and more than 1000 species of the genus have been described. They differ in their range of size, shape, texture, growth habit and color of blossoms [6]. The genus is ranging from shrubs and small to large trees, having decorative flowers which are mainly used for ornamental purposes. In fact, Rhododendron was selected as a favorite shrub by planters in an informal survey [7]. There is variation in the height of the plant, starting from 10 cm to 1 m (smallest species), while the example of largest specie is R. giganteum which is almost 3 m tall. The leaves of most species are spirally arranged; the nature of leaves may be evergreen or deciduous.

The genus Rhododendron is found in almost all parts of the world except some parts in America and Africa. Species of the genus Rhododendron occur throughout the Northern Hemisphere and the Southern Hemisphere in South Eastern Asia and Northern Australasia. The plant is originally present in mountainous areas characterized by acidic well-drained soil, regular rainfall and cool summer temperatures [8]. On the other hand, majority of the genus prefers cooler temperature including the subgenus Hymenanthes. One of the subsections (Pontica) is indigenous to the areas outside the center of distribution and present in the areas of Japan, eastern China, Europe, North America and Russia [9].

Rhododendron have traditionally been used in China, Nepal, North America, Russia, Korea, Austria and Romania for treating arthritis, intestinal disorders, rheumatism, skin diseases, cough and other ailments [10]. They are known to be rich in polyphenolic compounds namely flavonoids and their glycosides, terpenoids and essential oils [1, 11]

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The new and exciting aspect of this study is the analysis of different plant organs which has limited data available in literature. The phytochemical profile of the leaves of different age, flowers and fruits were analyzed for R. ambiguum and R. cinnabarinum. Both species belong to the subgenus Rhododendron, section Rhododendron. R. ambiguum and R. cinnabarinum belong to the subsection Triflora and Cinnabarina respectively. The flowers of R. cinnabarinum are known to be toxic. It is known from our previous studies that these two species out of 17 Rhododendron species showed higher activity against several Gram-positive bacteria [11]. Moreover, the high dose of both Rhododendron species exhibited a toxic effect in two mammalian cell and induced phenotypic changes that are characteristic for apoptosis [12]. Thus, the aim of the study was to analyze the chemical profile of Rhododendron crude extracts in order to contribute to our on-going investigations on the bioactivity potential of Rhododendron.

OH OH OH HO O HO O OH

OH OH OH OH Catechin Epicatechin

OH OH OH OH HO O HO O OH OH OH OH OH OH Gallocatechin Epigallocatechin OH OH OH OH HO O

HO O O OH OH OH OH HO OH OH O OH O

OH HO HO OH Procyanidin B1 HO Procyanidin A1

Figure 1. Examples of flavan-3-ols and proanthocyanidins

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Materials and Methods

Plant material and chemicals

Fresh leaf material of R. ambiguum Hemsley and R. cinnabarinum Hooker (first, second, and third leaf) were collected from plants grown in the Rhododendron-Park Bremen (www.rhododendronparkbremen.de) from spring 2013. First and second year leaves were collected for R. ambiguum, and first, second and third year leaves were collected for R. cinnabarinum. The leaves were distinguished on the basis of their morphological features. Moreover, the flowers and fruits for both Rhododendron species were also sampled. Each sample species was collected from three different individual plants with the help of Dr. Hartwig Schepker. The identities of all plant species have been verified according to the German Genebank Rhododendron Database provided by the Bundessortenamt (www.bundessortenamt.de/rhodo). All chemicals (analytical grade) were purchased from Carl Roth (Karlsruhe, Germany).

Plant Extraction

The Rhododendron leaves, fruits and flowers were freeze dried using liquid nitrogen. A mortar and pestle was used to crush the dried brittle leaves. 2 g of the powdered material were dissolved in 10 mL of 80% aqueous methanol and for 24 h at 4°C. The mixture was then sonicated for 15 min and centrifuged at 3,220 x g for 10 min. The aliquot was then separated and stored at -20°C until further analyses.

LC-ESI-TOF (High resolution mass spectrometry)

The LC equipment (Agilent 1200 series, Bremen, Germany) consisted of a binary pump, an auto- sampler with 100 µL loop and a UV-Vis detector with a light-pipe flow cell. The UV detector was used at 280 nm to measure the polyphenols. The 5 µm diphenyl column having 250 x 3 mm inner diameter (Varian, Darmstadt, Germany) was used for separation. This was connected to the microTOF mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with an electrospray ionization source. The internal calibration was achieved by using 0.1 M sodium formate solution at 0.10 mL/min, which was injected through the six-port valve. The calibration was achieved by using the enhanced quadratic mode. Water/formic acid (1000:0.05 v/v) and

106 | P a g e Results methanol were used as solvent A and B respectively. The flow rate of the solvents was adjusted to 500 µL/min. A linear gradient was used from 10% B to 80 % B in 70 min and a further 10 min was assigned for the gradient to equilibrate from 80% B to 10% B for the next run.

LC-ESI-MSn (Tandem mass spectrometry)

The Liquid chromatography equipment (Agilent 1100 series) comprised of a binary pump, an auto sampler having a 100 μL capacity loop and a Diode Array Detector with a range from 200 to 600 nm. The detector recorded at 254, 280 and 320 nm, which is the best absorption wavelength for polyphenolic compounds. Chromatographic separation was performed using the same gradient method used in the LC-TOF analyses. A 5 μm diphenyl column of 250 x 3 mm i.d. (Varian, Darmstadt, Germany) with 500 μL/min flow rate of solvent was used. The LC equipment was connected with Ion-trap mass spectrometer which was fitted with an ESI source (Bruker Daltonics HCT Ultra, Bremen, Germany) operating in full scan auto MSn mode to obtain fragment ion m/z. Tandem mass spectra were acquired in Auto-MSn mode (smart fragmentation) using a ramping of the collision energy. Maximum fragmentation amplitude was fixed to 1 Volt. MS operating conditions (negative mode) had been optimized with a capillary temperature of 365 °C, a dry gas flow rate was of 10 L/min, and a nebulizer pressure of 10 psi.

Bacterial strains and antimicrobial susceptibility test

Four Gram-Positive bacterial species i.e. Bacillus subtilis S168, Bacillus aquimaris MB-2011, Bacillus thioparus, and Clavibacter michiganensis were selected to compare the susceptibility of crude extracts of leaves, flower and fruit of two Rhododendron species i.e. R. ambiguum and R. cinnabarinum GSPB 390. Antimicrobial activity screening was conducted by the agar diffusion method [13]. Briefly, Lysogeny Broth (LB) agar plates were inoculated with 200 μL of the inoculum of the tester organism (1 x 107 colony forming units per mL) by evenly spreading the cell suspensions over the agar surface. Holes with diameters of 5 mm were punched into the agar plates. Subsequently, 50 μL of the plant crude extracts were filled into each well. The plates were incubated overnight at 28 ºC. Inhibition of microbial growth was determined by measuring the radius of the inhibition zone. For each bacterial strain, 80% aqueous methanol solutions were used as negative solvent controls. All experiments were performed in triplicates and the results are presented as mean values.

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Results and Discussion

The hypotheses of this study are that fruits, flowers and leaves have different phytochemical profile and that the leaves of different ages which are exposed to numerous environmental conditions might lead to the production of new compounds. The crude extracts of all parts of the two selected species R. ambiguum and R. cinnabarinum (i.e. first, second, third leaves, flowers and fruits) were extracted with 80% aqueous methanol in order to extract polyphenols. The two species were chosen as their crude extracts were bioactive and their plants consisted of leaves of different age. These extracts were analyzed by reversed phase HPLC using a diphenyl column with a gradient using methanol and water/formic acid (1000:0.05 v/v). Negative ion mode was used to study the polyphenols using tandem mass spectrometry and high resolution mass spectrometry. The compounds were identified in the high resolution mass data by observing an absolute mass error below 5 ppm for their elemental composition. The UV spectrum at 280 nm was used to identify the PAs present in the samples. Also, the fragmentation pathway of PAs by heterocyclic ring fission (HRF) and retro-Diels-Alder (RDA) reaction described by Gu et al. were considered [14].

Total Ion Chromatograms from LC-MS

The chromatograms of Rhododendron cinnabarinum first year leaves, second year leaves, third year leaves, flowers and fruits were directly compared are shown in Figure 2. The chromatograms of the first, second and third year leaves consisted of similar peaks. The chromatogram of flowers and fruits were different compared to the leaves. The chromatogram of flowers consisted of peaks with lower intensity in the region 0 – 30 min, which suggest that the hydrophilic compounds are present in lower intensity compared to the leaves. However after 30 min, the chromatogram of the flowers was similar to the leaves. On the other hand, the chromatogram of the fruits was exhibited different pecks compare to other plant parts (leaves and flowers).

In this study, 59 polyphenolic compounds were identified from both Rhododendron species for the first, second third year leaves, flower and fruit (Table 1). It worth to mention that, Taxifolin-O- pentoside was present in a high concentration in the leaves, while low concentration in flowers and fruits of both Rhododendron species. Additionally, the fruits consisted of the highest variety and concentration of PAs among the four parts of R. ambiguum and R. cinnabarinum.

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Intens. R. cinnabarinum first year leaves x10 7 55 18 23 22 1.5 19 25 8 21 34 28 1.0 39 40 30 3 9 20 0.5 12 14 5 2 56 15 10 Intens.0.0 x10 7 R. cinnabarinum second year leaves

Intens.0 x10 7 R. cinnabarinum third year leaves

Intens.0 x10 7 R. cinnabarinum flowers 3

Intens.0 x10 7 R. cinnabarinum fruits 2.0 1.5 1.0 0.5 0.0 0 10 20 30 40 50 60 Time [min]

Figure 2. Total Ion Chromatogram of R. cinnabarinum first year leaves, second year leaves, third year leaves, flowers and fruits generated by LC-MSn in negative mode.

Table 1. Polyphenols present in different parts of R. ambiguum and R.cinnabarinum

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Species R. ambiguum R. cinnbarinum

Part 1st 2nd Fl. Fr. 1st 2nd 3rd Fl. Fr. m/z No. Compound RT [M-H]-

1 Methyl gallate hexoside 345.0827 11.8 Y

2 Vanillic acid hexoside 329.0864 10.2 Y Y Y Y Y Y Y

3 Vanillic acid hexoside 329.0867 11.7 Y Y Y Y Y Y Y

4 Vanillic acid hexoside 329.0864 13.9 Y Y Y

5 Salicylic acid-O-hexoside 299.0761 6.3 Y Y Y Y Y

6 Salicylic acid-O-hexoside 299.0762 8.5 Y

7 3-O-Caffeoylquinic acid 353.0877 13.0 Y Y Y Y Y

8 5-O-Caffeoylquinic acid 353.0873 18.2 Y Y Y Y Y Y Y Y

9 4-O-Caffeoylquinic acid 353.864 23.0 Y Y Y Y

10 Naringenin 271.0602 47.9 Y Y Y Y Y

11 Myricetin 317.0296 38.9 Y Y Y Y Y

12 Myricetin-O-hexoside 479.0820 30.8 Y Y Y Y Y Y

13 Myricetin-O-hexoside 479.0853 35.3 Y Y Y Y

14 Myricetin-O-rhamnoside 463.0899 33.0 Y Y Y Y

15 Myricetin-O-pentoside 449.0728 32.1 Y Y Y Y Y

16 Myricetin-O-pentoside 449.0725 37.8 Y Y Y

17 Myricetin-O-pentoside 449.0732 39.1 Y Y Y Y

18 Quercetin-O-hexoside 463.0898 34.2 Y Y Y Y Y Y Y Y

19 Quercetin-O-hexoside 463.0897 35.5 Y Y Y Y Y Y

20 Quercetin-O-pentoside 433.0771 36.6 Y Y Y Y Y

21 Quercetin-O-pentoside 433.0795 37.4 Y Y Y Y Y Y Y Y

22 Quercetin-O-pentoside 433.0798 41.2 Y Y Y Y Y Y Y Y

23 Quercetin-O-rhamnoside 447.0921 38.7 Y Y Y Y Y Y Y Y Y

24 Quercetin-O-rhamnoside-O-hexoside 609.1442 34.3 Y Y Y Y

25 Quercetin-O-glucoronide 477.0676 40.8 Y Y Y

26 Quercetin 301.0342 44.4 Y Y Y Y Y Y

27 Kamepferol 285.0414 49.5 Y Y Y Y

28 Kaempferol-3-O-rhamnoside 431.0982 43.6 Y Y Y Y Y Y Y Y

29 Kaempferol-3-O-pentoside 417.0827 41.0 Y Y Y

30 Kaempferol-3-O-pentoside 417.0833 43.9 Y Y Y Y Y Y Y

31 Kaempferol-3-O-glucuronide 461.0719 43.6 Y

32 Taxifolin 303.0507 29.1 Y Y

33 Taxifolin-O-pentoside 435.0936 27.8 Y Y Y Y

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34 Taxifolin-O-pentoside 435.0936 31.1 Y Y Y Y Y Y Y Y Y

35 Taxifolin-O-pentoside 435.0930 33.6 Y

36 (Epi)gallocatechin-(epi)gallocatechin 609.1259 6.5 Y Y

37 (Epi)gallocatechin-(epi)gallocatechin 609.1246 7.5 Y

38 (Epi)gallocatechin-(epi)gallocatechin 609.1251 10.2 Y (Epi)catechin-(epi)catechin (Procyanidin 39 577.1372 13.4 Y Y Y Y Y Y Y Y dimer B1) (Epi)catechin-(epi)catechin (Procyanidin 40 577.1377 14.3 Y Y Y Y Y Y Y dimer B) (Epi)catechin-(epi)catechin (Procyanidin 41 577.1373 18.6 Y Y Y dimer B) (Epi)catechin-(epi)catechin (Procyanidin 42 577.1367 20.3 Y Y Y Y Y Y Y dimer B2) (Epi)catechin-(epi)catechin (Procyanidin 43 577.1358 23.1 Y Y Y Y Y Y dimer B)

44 Procyanidin Trimer C 865.1994 5.6 Y Y Y Y Y Y

45 Procyanidin Trimer C 865.1953 25.6 Y

46 A type Procyanidin Trimer C 863.1805 22.2 Y Y Y Y

47 (Epi)gallocatechin-(epi)catechin 593.1310 7.7 Y Y Y Y

48 (Epi)gallocatechin-(epi)catechin 593.1309 9.7 Y Y Y Y Y Y

49 (Epi)gallocatechin-(epi)catechin 593.1314 10.5 Y Y

50 (Epi)gallocatechin-(epi)catechin 593.1307 12.6 Y Y

51 (Epi)gallocatechin-(epi)catechin 593.1323 13.7 Y Y Y

52 (Epi)gallocatechin-(epi)catechin 593.1311 18.2 Y

53 (Epi)catechin-(4,8/2,6)-(epi)catechin 575.1209 27.2 Y Y Y Y Y

54 (Epi)catechin-(4,8/2,6)-(epi)catechin 575.1201 32.4 Y Y Y Y Y

55 Catechin 289.0721 16.0 Y Y Y Y Y Y Y Y

56 Epicatechin 289.0713 23.0 Y Y Y Y Y

57 Gallocatechin 305.0656 8.5 Y Y Y Y

58 Epigallocatechin 305.0660 15.4 Y Y Y

59 (Epi)catechin-O-D-glucopyranoside 451.1258 10.5 Y Y Y Y

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Characterization of (epi)catechin-(4,8′/2,6′)-(epi)catechin [53, 54] (Mr 576)

Two peaks were detected at m/z 575 and were assigned as A-type dimer of (epi)catechin unit. The first peak produced an MS2 base peak of m/z 449 and secondary peak of m/z 287. The second peak produced an MS2 base peak of m/z 423 and secondary peak of m/z 285.

Characterization of catechin [55] and epicatechin [56] (Mr 290)

Two peaks were detected at m/z 289. The first peak was assigned as catechin and the second peak was assigned as epicatechin based on their polarity. Both peaks produced similar fragmentation, consisting of an MS2 base peak of m/z 245 and secondary peak of m/z 203.

Characterization of gallocatechin [57] and epigallocatechin [58] (Mr 306)

Two peaks were detected at m/z 305. The first peak was assigned as gallocatechin and the second peak was assigned as epigallocatechin based on their polarity. Both peaks produced similar fragmentation, consisting of an MS2 base peak of m/z 179 and secondary peak of m/z 164.

Characterization of (epi)catechin-(4,8′)-(epi)catechin [39, 40, 41, 42, 43] (Mr 578)

Five peaks were detected at m/z 577. They were assigned as the PA dimer. All the peaks produced similar fragmentation with the base peak of m/z 407 and secondary peaks of m/z 425 and 285. The standards of the dimers B1 and B2 were used to differentiate the isomers by their retention time.

Characterization of (epi)gallocatechin-(4,8′)-(epi)catechin and (epi)catechin-(4,8′)- (epi)gallocatechin [47, 48, 49, 50, 51, 52] (Mr 594)

Six peaks were detected at m/z 593. They were speculated to be dimeric B-type PA consisting of (epi)catechin and (epi)gallocatechin monomeric units. All the peaks produced similar fragmentation having the base peak of m/z 425 and secondary peak of m/z 407.

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Characterization of (epi)gallocatechin-(4,8′)-(epi)gallocatechin [36, 37, 38] (Mr 610)

Three peaks were detected at m/z 609 and were speculated to be dimeric B-type PAs with (epi)gallocatechin monomeric units. The three peaks produced the fragmentation with base peak at m/z 423 and secondary peaks of m/z 441and 283.

Characterization of Taxifolin [32] (Mr 304)

A peak was detected at m/z 303 and was assigned to be taxifolin. The peak produced the fragmentation with base peak at m/z 285 and secondary peaks of m/z 177 and 125.

Identification of other polyphenols

The polyphenols were identified by their specific fragmentation patterns, retention time and high resolution mass values. The other polyphenols that were identified in the leaf extracts are methyl gallate hexoside, three vanillic acid hexosides, two salicylic acid hexosides, three caffeoylquinic acids, naringenin, myricetin, two myricetin-O-hexosides, myricetin-O-rhamnoside, three myricetin-O-pentosides, four quercetin-O-hexosides, three quercetin-O-pentosides, quercetin-O- rhamnoside, quercetin-O-rhamnoside-O-hexoside, quercetin-O-glucoronide, quercetin, kaempferol, kaempferol-O-rhamnoside, two kaempferol-O-pentosides, kaempferol-O- glucuronide, three taxifolin-O-pentosides, two procyanidin trimmers C, one A-type procyandin trimer C and (epi)catechin-O-D-glycopyranoside.

Antibacterial activity

Four Gram-positive organisms were used to compare the antibacterial activity of different plant parts of two species from genus Rhododendron. Crude extract of first, second, third year leaves in addition to flower and fruits of R. ambiguum and R. cinnabarinum were obtained using 80% methanol. The bioactivity of R. ambiguum ranged between 0.5-0.7 cm, while for R. cinnabarinum between 0.5-0.8 cm (Figure 3). The results did not show a significant different between the leaves and fruits of both Rhododendron species. However, there was no antibacterial activity observed for the flower of R. cinnabarinum and a reduced effect for the flower of R. ambiguum. This could be

113 | P a g e Results due to the evolutionary aspect as flowers have a short blooming period in a year compared to the leaves and fruits. In general, B. thioparus was the most sensitive bacteria species towards the plant parts for both Rhododendron species. These results are in agreement with our previous study which showed a higher antibacterial effect of Rhododendron species against Gram-positive and higher effect for R. cinnabarinum [11]. The phytochemical analysis indicated that Taxifolin derivatives were present in high concentration in the leaves of both plant species which could be the reason of apoptosis like phenotype observed before in other studies [12]. Moreover, this finding is also supported by other studies which reported the effect of Taxifolin in different cancer cell lines by inducing apoptosis cell death [15, 16].

R. ambiguum

R. cinnabarinum

Figure 3 Antimicrobial activities of methanol crude extract of different plant parts. a) R. ambiguum and b) R. cinnabarinum. The radius of the inhibition zones was measured in triplicates and the values are given as means ± standard deviations. The 80% aqueous methanol used as negative controls did not yield inhibition zones (data not shown).

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12, 13 Myricetin-O-hexoside 33, 34, 35 Taxifolin-O-pentoside

1 1 [%] MS [%] 434.8 MS 478.8 100 80 100 336.9 60 50 40 20 290.8 392.9424.9 530.8 0 0 2 2 316.7 MS 284.7 MS 100 100 80 80 302.8 60 60 40 40 150.7 20 20 178.7 124.8 176.7 416.8 0 0 178.6 MS3 240.7 MS3 100 100 80 80 60 150.7 316.7 60 174.7 40 40 284.7 20 20 198.7 254.6 288.7 0 106.8 226.6 0 160.7 50 100 150 200 250 300 350 400 450 500 m/z 50 100 150 200 250 300 350 400 m/z

23 Quercetin-O-rhamnoside 39, 40, 41, 42, 43 Procyanidin dimer B

1 1 [%] 446.9 MS [%] MS 100 576.8 80 100 60 40 50 20 238.8 284.8 96.9 395.0424.9 478.8 0 0 2 2 300.7 MS 424.8 MS 100 100 80 80 60 60 40 40 288.8 20 20 450.8 150.7 244.7 558.8 0 178.6 0 380.8 3 3 178.6 MS 406.8 MS 100 100 80 150.7 299.7 80 60 60 40 40 20 272.7 20 106.9 228.7 272.7 0 0 338.8 380.8 50 100 150 200 250 300 350 400 450 m/z 100 200 300 400 500 m/z

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Conclusions

The different parts of the R. ambiguum and R. cinnabarinum are a rich source of polyphenols. 59 different types of polyphenols including isomers were identified in these parts based on their fragmentation pattern and high resolution mass spectra in negative ion mode. The phytochemical profile of different year leaves was found to be similar. Among all the parts, the fruits were found to contain the highest variety and concentration of polyphenols. PAs were mainly found in the fruits. However, there are many unidentified compounds present in leaves, flowers and fruits which need to be analyzed in future. We can conclude that both Rhododendron species have antibacterial effect towards Gram-positive bacteria, while there was no significant difference between different seasonal leaves and fruits, but low effect for flowers.

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Reference

1. Jaiswal, R., L. Jayasinghe, and N. Kuhnert, Identification and characterization of proanthocyanidins of 16 members of the Rhododendron genus (Ericaceae) by tandem LC- MS. J Mass Spectrom, 2012. 47(4): p. 502-15.

2. Luo, S., et al., Extraction, identification and antioxidant activity of proanthocyanidins from Larix gmelinii Bark. Nat Prod Res, 2014. 28(14): p. 1116-20.

3. Kruger, M.J., et al., Proanthocyanidins, anthocyanins and cardiovascular diseases. Food Research International, 2014. 59: p. 41-52.

4. Zhou, D.Y., et al., Grape seed proanthocyanidin extract attenuates airway inflammation and hyperresponsiveness in a murine model of asthma by downregulating inducible nitric oxide synthase. Planta Med, 2011. 77(14): p. 1575-81.

5. Deters, A., et al., High molecular compounds (polysaccharides and proanthocyanidins) from Hamamelis virginiana bark: influence on human skin keratinocyte proliferation and differentiation and influence on irritated skin. Phytochemistry, 2001. 58(6): p. 949-958.

6. Barceloux, D.G., Medical Toxicology of Natural Substances: Foods, Fungi, Medicinal Herbs, Plants, and Venomous Animals. 2008, New Jersey, USA: John Wiley and sons, Inc. .

7. Dirr, M.A., Manual of woody landscape plants: Their identification, ornamental characteristics, culture, propagation and uses, ed. 6th. 2009, Champaign, IL: Stipes Pub Llc.

8. Horn, C.N., Distribution and Ecological Preference of Rhododendron eastmanii Kron & Creel (May-White Azalea) in South Carolina. Castanea, 2005. 70(1): p. 1-12.

9. D.F., C. and T.-L. M., The distribution of Rhododendron subgenus Hymenanthes with special reference to Yunnan (W. China). Vol. 43. 1985, Edinburgh: Notes Roy. Bot. Gard.

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10. Popescu, R. and B. Kopp, The genus Rhododendron: an ethnopharmacological and toxicological review. J Ethnopharmacol, 2013. 147(1): p. 42-62.

11. Rezk, A., et al., Phylogenetic spectrum and analysis of antibacterial activities of leaf extracts from plants of the genus Rhododendron. BMC Complementary and Alternative Medicine, 2015. 15(1): p. 1-10.

12. Rezk, A., et al., Assessment of Cytotoxicity Exerted by Leaf Extracts from Plants of the Genus Rhododendron towards Epidermal Keratinocytes and Intestine Epithelial Cells. submitted, 2015.

13. Nathan, P., et al., A laboratory method for selection of topical antimicrobial agents to treat infected burn wounds. Burns, 1978. 4(3): p. 177-187.

14. Gu, L., et al., Liquid chromatographic/electrospray ionization mass spectrometric studies of proanthocyanidins in foods. J Mass Spectrom, 2003. 38(12): p. 1272-80.

15. Zhang, Z.R., et al., Taxifolin enhances andrographolide-induced mitotic arrest and apoptosis in human prostate cancer cells via spindle assembly checkpoint activation. PLoS One, 2013. 8(1): p. e54577.

16. Wätjen, W., et al., Low Concentrations of Flavonoids Are Protective in Rat H4IIE Cells Whereas High Concentrations Cause DNA Damage and Apoptosis. The Journal of Nutrition, 2005. 135(3): p. 525-531.

118 | P a g e Discussion

4. Discussion

Medicinal plants have been used as traditional treatments to counteract numerous human diseases for thousands of years. Therefore, it is not surprising that in many parts of the world different types of plant materials are still used as the main source of medicine. The increasing resistance of many pathogens towards the commonly used antimicrobial agents led to a renewed interest in the discovery of novel antimicrobial compounds. Several secondary metabolites of plants such as tannins, terpenoids, alkaloids, and flavonoids, have been proven as biologically active against pathogens to defend themselves [114, 115]. For instance, fabatin belongs to the plant defense compounds found in beans and is active against Gram-positive and Gram- negative bacteria [116]. Moreover, some mixtures of plant compounds act in a synergetic manner against bacteria [117, 118]. By way of example, the alkaloid berberine is a common compound in several plant species and particularly in the family Berberidaceae. The alkaloid berberine exhibits weak antimicrobial activity against Staphylococcus aureus. However, the antimicrobial active substances of berberine are dramatically increased in the presence of 5′- methoxyhydnocarpin; a phenomenon that is referred to as a synergetic effect [119].

Very few previous studies dealt with the biological activity of compounds derived from Rhododendron. Most of the published articles investigated only a few species, which existed regionally, e.g., in Nepal or in Turkey [30, 120, 121]. Although these studies focused on potential antimicrobial activities of the extracted compounds, the researcher of those studies did not consider investigating any cytotoxicity effects on mammalian cells. Consequently, none of the previous studies had systematically investigated a large number of Rhododendron species and none of the published studies had simultaneously analyzed antibiotics and cell toxic effects of Rhododendron extracts alike. Therefore, in the current thesis, the bioactivity of a large number of Rhododendron samples with respect to their antimicrobial and cell toxic effects was investigated and correlated to the biochemical and phylogenetic profile of such samples taken from different species of the genus Rhododendron.

In this study, agar diffusion assays were used to investigate the antibacterial effect of extracts from 120 phylogenetically distinct species of the genus Rhododendron against a group of Gram- positive and Gram-negative tester bacterial organisms. The plant species originated from different subgenera which were Rhododendron, Hymenanthes, Pentanthera, Tsutsusi, and Azaleastrum. The condensed outcome of this study was that, leaf extracts of 17 out of 120 Rhododendron species showed significant antibacterial effects (radius of growth inhibition ≥ 6

119 | P a g e Discussion mm) towards Gram-positive bacteria. The remaining 103 plant species were either only moderately active or did not show any antibacterial effects. Interestingly, all bioactive extracts were derived from Rhododendron species belonging to the subgenus Rhododendron. Thus, our study is the first to limit antimicrobial activity of Rhododendron leaf extract to a distinct subgenus. This result is in agreement with other study that reported some of Rhododendron species showed activity against Gram-positive and Gram-negative bacteria i.e R. lepidotum belongs to subgenus Rhododendron [122]. Consequently, we hypothesize that this group of Rhododendron species are producing specific secondary metabolite (SM) compounds which are responsible for the antibacterial effect. However, in previous studies there was no clear correlation between the bioactivity and taxonomical classification of genus Rhododendron.

Two Rhododendron species belonging to the subgenera Pentanthera and Hymenanthes, respectively, R. cumberlandense and R. wardii var. puralbum exhibited a moderate antibacterial effect (radius of growth inhibition of 5 mm) against E. coli (Gram-negative bacteria). These bioactivities may be attributed to specific compounds, which are particularly produced in these species but not in other Rhododendron species and which must differ in chemical structure from the above postulated compounds. However, these variances in bacterial sensitivity might also reflect the presence or absence of different multidrug efflux systems, or might be attributed to individual composition of cell surface structures of Gram-negative or Gram-positive bacteria [123-125].

In previous studies, the biological activity of the crude extracts of R. ponticum ssp. ponticum, and R. smirnovii were shown to inhibit the growth of different bacterial species [121, 126]. Moreover, the methanolic extract of R. campanulatum were shown to bear antibacterial activity against different Gram-positive bacteria (S. aureus and S. faecalis) [120]. In contrast, extracts from these Rhododendron species did not show antibacterial effects against either Gram- positive or Gram-negative bacteria in the current study. These conflicting results might be explained by the excessive amount of dried plant material used for extraction in the previous studies as compared to the current study (2 g). Alternatively, the composition of the secondary metabolites of plants might vary between different genotypes. This hypothesis could be confirmed by comparing the results from Ertürk et al. [127] with this study. In their study, three Rhododendron species were highly active against B. subtillis while in this study they did not show inhibition activity toward this bacterial species. Moreover, variation of biotic or abiotic ecological factors under which the plants were grown might contribute to further diversity of results. Some of such yet-to-be identified environmental factors might be essential or

120 | P a g e Discussion instrumental for the composition of antimicrobial metabolites in one and the same plant species [128-130]. Since in the current study, all plants from which leaf extracts were obtained were grown under the same macroclimatic conditions, the observed differences in results within the current study shall rather not be attributed to differences in environmental parameters.

In order to study potential resistance mechanisms of Gram-negative bacteria towards Rhododendron leaf extracts, two bacterial species were chosen. E. coli and Erwinia amylovora with gene deletions in multidrug efflux pumps (Δ tolC and Δ acrAB) were used and compared with the respective wild types. The E. coli wild type exhibited full resistance to Rhododendron leaf extracts. Interestingly, all E. coli mutants, irrespective of whether they were single or double mutants also showed full resistance to the tested plant extracts. The E. amylovora wild type also exhibited resistance to Rhododendron extracts. However, and in contrast to this, the single and the double mutants (Δ tolC and Δ acrAB) of E. amylovora exhibited a high sensitivity towards the leaf extracts. In order to test whether the increased susceptibility towards Rhododendron extracts was due to the lack of AcrAB or TolC, respectively, the corresponding single mutants of E. amylovora were transformed with recombinant plasmids, which carried the genes, acrAB or tolC, respectively, derived from E. coli. As expected and in line with previously published data [131], a resistant phenotype of transformants could be fully restored to wild type levels. These results indicated that the multidrug efflux system of E. amylovora – but not that of E. coli – is responsible for the resistance towards compounds in leaf extracts of some Rhododendron species. In contrast, other factors might contribute to the resistance of E. coli towards the same leaf extracts suggesting an interesting diversity of potential resistance mechanisms in different Gram-negative bacteria [132].

Additionally to the antibacterial activity assays within this study, Rhododendron leaf crude extracts were subjected to in vitro cytotoxicity assays with mammalian cell cultures. These tests were conducted to confirm the potential safety of the use of bioactive leaf extracts. Such assays are widely used in drug discovery research at pre-clinical stages. Epidermal keratinocytes of the skin and epithelial cells of the intestinal mucosa in form of cultures of the human HaCaT and the rat IEC6 cell lines, respectively, were used as tester systems modeling initial contact points when drugs are applied ectopically or administered orally, respectively. All assays were performed with 12 Rhododendron leaf extracts administered at three different concentrations (5, 50, and 500 µg/mL). According to the minimum inhibitory concentration of these extracts against B. subtilis, they could be classified into four groups with variable antimicrobial potential.

121 | P a g e Discussion

In general, high doses of the bioactive extracts from all 12 Rhododendron leaf extracts negatively affected the growth of both cell lines. Our results furthermore showed that leaf extract of R. hippophaeoides var. hippophaeoides was least toxic to both, bacteria and keratinocytes. In general, keratinocyte cells appeared to be more resistant to cell toxic effects of Rhododendron extracts as compared to intestinal epithelial cells when exposed to high doses of Rhododendron leaf extracts. This finding is in agreement with other studies in which it was argued that differences in chemical composition of the respective plasma membrane enable keratinocytes to build stratified epithelial layers in a process termed cornification to provide maximum protection against mechanical and chemical stressors [133-136].

In contrast, lower doses of 5 and 50 µg/mL of the tested Rhododendron leaf extracts did not cause as severe structural and functional alterations in mammalian cells as higher doses of the same extracts did. However, intestinal epithelial cells were once again more sensitive towards the Rhododendron extracts as compared to HaCaT cells. Interestingly, extracts of five Rhododendron species were demonstrated to be both, antimicrobial and not toxic to mammalian cell cultures at the tested concentrations: R. minus, R. ferrugineum, and R. racemosum were safe in use for both cell lines while R. rubiginosum and R. concinnum turned out to be safe in use for keratinocytes. All of the five species exhibited high antibacterial effect with MICs reached at 50 µg/mL. To our best knowledge, this is the first study in which a combination of antimicrobial and cell toxicity assays was used for Rhododendron crude extracts.

Interestingly, use of high concentrations of Rhododendron leaf extracts caused phenotypical alterations in mammalian cells which led to apoptosis-mediated cell death. This observation was more obvious for intestinal epithelial cells than for keratinocytes. When the intestinal cell cultures were exposed to extracts of R. minus, R. rubiginosum and R. ambiguum the typical early stages of apoptosis such as chromatin condensation and shrinkage of the nuclei were induced. Extracts of other Rhododendron species such as R. hippophaeoides var. hippophaeoides, R. cinnabarium, R. ferrugineum, R. xanthostephanum, and R. racemosum induced phenotypes in intestinal cells typical for late stages of apoptosis such as formation of plasma membrane typical blebs. In consequence, caspase activity assays were used to further define the cell death pathway and identify it as apoptosis, necrosis, or secondary apoptosis. Extracts of R. ferrugineum and R. cinnabarium induced higher levels of caspase-3 in IEC6 cells, suggesting that the high dose of these leaf extracts could be used as a potential anti-proliferative agent. Our results are in line with previous studies, in which the effects of Rhododendron crude extract or compounds extracted from Rhododendron were shown to increase the level of

122 | P a g e Discussion caspase-3 activity in different mammalian cell lines [137, 138]. In other studies, it have been suggested that different SM compounds such as ursolic acid, hyperin, oleanolic acid, and betulinic acid, isolated from Rhododendron are triggering apoptosis cell death in human endometrial cancer and human leukaemia [138-140]

Finally, the phytochemical profiles of two Rhododendron species that showed high antibacterial effects against Gram-positive bacteria (R. ambiguum and R. cinnabarinum) were analyzed in depth. For this, extracts from different seasonal samples such as first-, second- and third-year leaves as well as flowers and fruits were obtained and tested against bacterial tester organisms in order to study the distribution of potential bioactive compounds in different and differently aged plant parts. Furthermore, this attempt was meant to separate active from non-active chemical fractions. The corresponding results demonstrated that there were no differences in the quantity or quality of bioactive effects caused by extracts from either the first-, second-, or third-year leaves. These results might indicate presence of bioactive compounds in the leaves of Rhododendron independent of season and not linked to any potential stress factor such as herbivore or pathogen attacks. Moreover, the bioactive compounds were likely to be present in flowers and fruits, which supports the idea that potential bioactive compounds might be produced by most if not all aerial plant parts of Rhododendron. This results in agreement with pervious study which showed similar antibacterial effect of the leaves and flowers from three different Rhododendron species [127]. In the course of the phytochemical analysis, more than 60 polyphenolic compound and isomers could be identified. However, there were no clear patterns for the presence or absence of specific compounds in particular parts of the plant identifiable. Interestingly, the poly-phenolic compound, taxifolin, was present in high concentrations in the leaves of the tested plants. It is tempting to speculate that this compound might have contributed to the observed apoptosis phenotypes cited above. However, our study requires further experimental work with the aim to compare the cytotoxicity of extracts from the different plants in order to substantiate such a hypothesis. In previous studies, taxifolin had been demonstrated to cause similar apoptotic effects in different mammalian cell lines [141, 142].

Due to insufficient technical solutions and due to time constraints, we could not determine the precise nature of the bioactive compounds acting against bacteria and causing cytotoxicity onto keratinocytes or intestine epithelial cells. Consequently, future studies should focus on an intensified phytochemical mapping and an in-depth structural analysis of pure-fractioned samples of bioactive Rhododendron extracts. However, such newly obtained fractions must all

123 | P a g e Discussion be carefully tested again against bacterial tester organisms as well as in terms of their potential cytotoxicity towards mammalian cell cultures. This way, a separation of differently acting compounds and their structural determination shall be the next important steps in the analysis of bioactivity in the genus Rhododendron.

124 | P a g e References

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6. Appendix

Phenolic Profile and In Vitro Assessment of Cytotoxicity and Antibacterial Activity of Ziziphus spina-christi Leaf Extracts

139 | P a g e Appendix

Abstract

The phenolic profiles of Ziziphus spina-christi leaf extracts as well as their antibacterial activity and potential cytotoxicity towards keratinocytes and small intestine epithelial cells were investigated in vitro. We unambiguously assigned fifty-seven phenolic compounds to their regioisomeric level in the methanol extract of Z. spina-christi leaves using tandem mass spectrometry (MSn). To our knowledge 45 of them were not reported previously in Z. spina-christi and five for the first time in nature. Highly glycosylated flavonoids, proanthocyanidins, and chlorogenic acids were identified as the major components.

In vitro studies to determine antibacterial activities of aqueous and methanolic leaf extracts were carried out with six bacterial strains (Bacillus subtilis, Bacillus aquimaris, Clavibacter michiganensis, Escherichia coli, Erwinia amylovora, and Pseudomonas syringae) using an agar diffusion assay and revealing that the extracts were only active against B. aquimaris and P. syringae. Methanol extracts were found to show higher activity than the aqueous extracts indicating hydrophobic substances of Ziziphus leaves exerting antibacterial activity.

In vitro application of Z. spina-christi leaf extracts over 24 hours revealed no cytotoxic effects towards the human epidermal keratinocyte cell line HaCaT or rat intestine epithelial IEC-6 cells as assess by cytoskeletal and plasma membrane integrity or analyses of mitochondrial and proliferative activities.

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Introduction

Ziziphus spina-christi (L.) Desf. (Rhamnaceae) is a tropical evergreen tree of Sudanese origin. The plant has very interesting historical and religious aspects. It is repeatedly mentioned in Muslim as well as Christian traditions and was recorded by pilgrims visiting the Holy Land on numerous occasions. The boiled water extracts of the leaves of Z. spina-christi are used by Muslims in the cleaning of a dead body before burial suggesting antibacterial properties. In addition, the plant has been used in mummification by the ancient Egyptians [1]. It has been suggested that the plant material referred to in the Bible as the "thorns" or "bramble" (Judges 9; 14–15), "thorns" (Matthew 27:27–29) and the "crown of thorn" (John 19:5) might have been derived from Z. spina-christi [2]. The Holy Quran mentions the Lote tree (Cedar) three times (XXXIV: 16; LIII: 13-18; LVI: 28- 32), which was frequently identified as Z. spina-christi. Accordingly, this species is highly respected throughout the Middle East, has been widely used as a food and as medicinal as well as a environmental protection plant since antiquity, and is still in use at present [2, 3].

Z. spina-christi is commonly used in ethno-medicine for the treatment of many illnesses such as digestive disorders, weakness, hepatic disorders, obesity, urinary problems, diabetes, skin infections, fever, diarrhea, or insomnia [4, 5]. In Sudanese ethno-medicine, the leaves of Z. spina- christi are used for the treatment of malaria [6]. In addition, Michel et al. (2011) reported an antidiabetic activity of the leaves of Z. spina-christi due to their saponin and polyphenol constituents [7], which was supported in pharmacological studies by Glombitza et al. (1994) indicating that extracts of Z. spina-christi leaves or its main saponin glycoside, christinin-A, enhanced glucose consumption in diabetic rats [8]. Furthermore, Z. spina-christi leaves and fruits are reported to possess antibacterial activity [9], as well as antifungal activity on plant pathogens [10]. In addition, Adzu et al. (2001) found that root bark extracts showed significant antinociceptive activity in mice and rats [11].

The widespread and incorrect use of antibacterial agents has caused emergence of bacterial strains that are resistant to several antibiotics. Bacteria have developed different defence strategies to protect themselves from antimicrobial drugs such as alteration of the drug target, enzymatic inactivation, reduction of intracellular drug concentration by modifications in membrane permeability, or by efflux pumps [12, 13]. Accordingly, new antimicrobials with greater effectiveness and better tolerability than existing drugs are urgently required for treatment of bacterial infections. Plant extracts are well-known to combine a diversity of phytoconstituents that have a broad spectrum of antimicrobial activities, consequently plant-derived bioactive compounds counted as the main source for lead compound identification and development in pharmaceutical production [14].

Plant polyphenols comprise a large group of secondary metabolites that can extend from simple molecules, such as phenolic acids, to highly polymerized compounds such as tannins. They are constituents with a rich number of derivatives in the plant kingdom, from mosses and ferns to higher angiosperms including food plants. Many of these plant-derived polyphenols are essential components in our diets [15]. They are derivatives of the pentose phosphate, mevalonic acid, shikimate, and phenylpropanoid pathways in plants [16].

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Flavonoids have been reported to have numerous biological effects such as antioxidant activity [17], antimicrobial activity [18], anti-inflammatory activities [19], inhibition of platelet aggregation [20], and inhibition of mast cell histamine release [21]. Moreover, antioxidant phenolics have been suggested to possess preventive functions in the progression of heart diseases and different cancers including prostate, breast, lung, colon and rectal cancers [22]. In addition, epidemiological studies have demonstrated that there is an inverse relationship between the intake of flavonoids such as myricetin, apigenin, quercetin, kaempferol and luteolin, which are naturally present in fruits, vegetables and beverages, and the lowering of the risk of suffering from cardiovascular diseases [23].

From different species of Ziziphus, peptide and cyclopeptide alkaloids, flavonoids, sterols, tannins, betulinic acid and triterpenoidal saponin glycosides have been isolated and chemically identified [24, 25]. From the methanol extract of the fruits of Z. jujube and Z. spina-christi twelve compounds have been reported as representing quercetin, kaempferol, and phloretin derivatives [15]. In a qualitative as well as quantitative study, flavonoids, saponins, and triterpenic acids were isolated from the leaves of two Ziziphus species [26].

Herein, the aim of the present work was to significantly improve and deepen our knowledge about the phenolic composition of Z. spina-christi leaves using liquid chromatography coupled to electron spray ionization multi-stage mass spectrometry (HPLC-ESI-MSn) as a powerful tool for the analysis of natural products. The obtained results may contribute to a better understanding of the role of Z. spina-christi phenolics on biological, nutritional and medicinal properties.

Material and Methods

Chemicals

Procyanidin B1 (purity 95%), Procyanidin B2 (97%), 5-O-caffeoylquinic acid (> 98%), 3-O- caffeoylquinic acid (> 98%), and 4,5 di-O-caffeoylquinic acid (99%) were purchased from PhytoLab. Gallocatechin (98%), epigallocatechin (95%), catechin (97%), epicatechin (≥ 98%), kaempferol 3-O-glucoside (97%), kaempferol 3-O-(6-O-rhamnosyl-glucoside) (≥ 98%), quercetin 3-O-(6-O-rhamnosyl-glucoside) (rutin) (≥ 94%), quercetin 3-O-arabinoside (95%), quercetin 3-O- rhamnoside (≥ 97%), kaempferol-3-O-galactoside-rhamnoside-7-O-rhamnoside (robinin) (≥ 90%), quercetin 3-O-glucoside (90%), methanol (≥ 99.9%) and DMSO (≥ 95%) were purchased from Sigma–Aldrich. Ultrapure water with a resistance of 18.2 M was deionized in a Milli-Q system (Sartorius Stedim Biotech).

Plant material

The Z. spina-christi plant samples were freshly collected in June 2010 from its natural habitats in Omdurman (15° 38' 10" North, 32° 26'F 14" East), Khartoum, Sudan. The voucher specimen was identified by Dr. Hayder Abdel Gadir of Herbarium of Medicinal and Aromatic Plants Research Institute (MAPRI), Khartoum, Sudan, where the specimens (Voucher No. Zi-sp-ch-04) are maintained in a repository.

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Sample preparation

Three grams of the shade-dried green leaves of the plants were ground in a hammer mill to fine powder and extracted with methanol/water (7:3 v/v) using a Soxhlet apparatus (Buchi B-811 extraction system) for 5 h. These extracts were filtered through a Whatman no. 1 filter paper. The solvent was removed by evaporation in vacuo, and the extracts were stored at -20°C until required, then thawed at room temperature, dissolved in methanol/water (7:3 v/v; 60mg/10 mL), filtered through a membrane filter with a pore size of 0.45 μm (Carl Roth) and used directly for LC–MS.

UV Irradiation

UV irradiation experiments were performed as previously reported [35].

Liquid chromatography–mass spectrometry (LC/ESI/MSn)

The Agilent 1100 LC equipment (Agilent) comprised a binary pump, an auto-sampler with a 100- µL loop, and a diode array detector with a light-pipe flow cell (recording at 254, 280, and 320 nm). This was interfaced with an ion-trap mass spectrometer fitted with an electrospray ionization source (Bruker Daltonics HCT Ultra) operating in full-scan, auto-MSn mode to obtain fragment ion m/z. Tandem mass spectra were acquired in auto-MSn mode (smart fragmentation) using a ramping of the collision energy. Maximum fragmentation amplitude was set to 1 V, starting at 30% and ending at 200%. MS operating conditions (negative mode) had been optimized using B1- type and B2-type PAs with a capillary temperature of 365 °C, a dry gas flow rate of 10l/min, and a nebulizer pressure of 10 psi. The spectra full scan mass were performed within the range from m/z 50 up to 1500 in negative ion mode.

High-resolution LC–MS was performed using the same high performance LC equipped with a micrOTOF mass spectrometer (Bruker Daltonics) fitted with an electrospray ionization source, and internal calibration was achieved with 10 mL of 0.1M sodium formate solution injected through a six-port valve prior to each chromatographic run. Calibration was performed using the enhanced quadratic mode.

High-performance liquid chromatography

The HPLC separation was were performed as previously reported [58].

Antibacterial activity assay

Three Gram-positive bacterial strains (Bacillus subtilis S168, Bacillus aquimaris MB-2011, and Clavibacter michiganensis GSPB 390 as well as three Gram-negative bacterial strains (Escherichia coli DH5α, Erwinia amylovora 1189, and Pseudomonas syringae pv tomato DC300) were selected as model organisms to evaluate the antibacterial activity of the Z. spina-christi crude plant extract. The antimicrobial activity assay was performed using the agar diffusion method

143 | P a g e Appendix according to Nathan et al (1978) with slight modification as follows: Lysogeny broth (LB) agar plates were inoculated with 200 µL of the tester organism (1 x 107cfu/mL) by spreading the bacterial cell suspensions over the agar with the help of sterile glass beads. Holes (5 mm diameter) were punched into the agar with a sterile Pasteur pipette, and 50 µL of the crude extracts was pipetted into the wells [59]. The plates were incubated overnight at 28 ºC except for E. coli, for which incubation was done at 37 ºC. Inhibition of microbial growth was determined by measuring the radius of the zone of inhibition. For each bacterial strain, an equal volume of water and methanol was used as negative controls. As positive controls, the following antibiotics were tested effective against the following bacterial organism causing inhibition zones of 10.3 to 23.3 mm: 25 mg/mL kanamycin (grad ≥ 750 I.U./mg; Carl Roth) for B. subtilis and B. aquimaris; 50 mg/mL ampicillin (purity 99 %; Carl Roth) for E. coli and E. amylovora; 25 mg/mL streptomycin (grade 730 I.U./mg; Serva) for P. syringae and C. michiganensis. The experiments were conducted in triplicates, and the data are given as mean values ± standard deviation (SD).

Cytotoxicity Analysis

Cell Culture: HaCaT cells at passages 40 – 55 were used as a model for epidermal keratinocytes [60, 61]. They were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Lonza Biowhittaker) containing phenol red and supplemented with 10 % fetal bovine serum (FBS; Lonza Biowhittaker). The IEC-6 cell line at passage 16 – 40 was used as cellular model of small intestine epithelial cells (European Collection of Cell Cultures, ECACC), and cultured in DMEM containing 0.1µg/mL insulin (purity ≥ 99%; Sigma-Aldrich) and 5 % FBS. Cells were grown at 37 °C in a humified atmosphere at 8 % CO₂ for keratinocytes and at 5 % CO₂ for IEC-6 cells. Cell culture medium was exchanged every three to four days, confluent cell cultures were used for experimentation.

Cellular integrity analyses by acquisition of propidium iodide (PI) and counter-staining with Draq5™: PI (purity > 95% p. a.; Carl Roth) acquisition through ruptured plasma membranes was used as a measure of necrotic cell death, while Draq5™ staining of cellular DNA served to determine total cell numbers [55]. Cells were grown on coverslips for 24 hours and then treated with 50 µL of the respective extracts at final concentrations of 100 µg/mL, 10 µg/mL, and 1 µg/mL (weight/volume, final concentrations). Controls were treated with 0.5 % DMSO, since all extracts were diluted with DMSO to this final concentration. After incubation of the cells with extract preparations for 24 hours at 37°C, cells were washed with prewarmed PBS. Staining with 2 µg /µL PI (Carl Roth GmbH) diluted in the respective culture medium was for 30 minutes at 37°C. Afterwards cells were rinsed with PBS twice before fixation with 4 % paraformaldehyde (purity min. 95% assay titr.; Applichem) in 200 mM Hepes, pH 7.4 for 20 minutes at RT. Then, cells were rinsed in PBS and stained with 5 µM Draq5™ (Biostatus) in PBS for 45 minutes at 37 °C. Finally, cells were washed three times with PBS and then mounted in mowiol (33 % glycerol and 14 % Mowiol (Carl Roth) in 200 nM Tris-HCL in ultrapure H₂O at pH 8.5).

Images were taken at a Zeiss LSM 510 Meta confocal laser scanning microscope (Carl Zeiss GmbH) and analyzed using Zeiss LSM Image Browser software version 3.2.0.70.

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Staining of the actin cytoskeleton with FITC-Phalloidin and counter-staining with Draq5™: Cells were fixed as described above and stained with 3 µM FITC-Phalloidin (purity 90%; Sigma- Aldrich) and 5 µM Draq5™ in PBS for 45 minutes at 37 °C [56]. Cells were washed three times before mounting in mowiol.

Determination of cell counts using the Image Analysis Software CellProfiler™: Images were analyzed using Cell Profiler 2.0 [62] as previously described [55, 56, 60].

MTT Assay: Cells were grown for 24 hours at 37 °C and the respective CO₂ concentration in 24- well plates before treatment with 50 µL extracts per 1 mL culture medium to achieve final concentrations of the extracts to reach 100, 10 and 1 µg/mL (wt/vol), respectively. As controls, cells were treated with 0.5 % DMSO and mock-treated cells were used, to which no extracts were applied. MTT ([3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; purity ≥ 98%; Carl Roth GmbH) was applied in PBS to the respective cell culture medium at a final concentration of 0.5 mg/mL MTT. Cells were incubated in the dark for 1.5 hours at 37°C and the respective CO₂ concentration, before formazan crystals were dissolved in 500 µL of 100 % DMSO and quantified at 570 nm using a Thermo Spectronic Genesys 10 UV–vis Spectrophotometer (Thermo Scientific™).

Result and Discussion

Methanolic extract of the leaves of Z. spina-christi was separated by reversed-phase chromatography. Identification of compounds was obtained using high resolution-electrospray ionization-mass spectrometry (HR-ESI-MS) for determination of molecular formula and ESI with quadruple ion trap analyzer MS (ESI-QIT-MS) to obtain tandem MS data for further structure elucidation. Additionally, retention times and UV (320 nm) spectra were used for reversed phase compounds identification (Fig. 1). Phenolic compounds, for which commercial standards were available was carried out by the comparison of their retention times, UV–vis spectra and mass spectral data recorded in negative ion mode, while, the identity of other compounds was elucidated using the UV–vis spectrum to assign the phenolic class [27]. For all compounds the high resolution mass data were in good agreement with the theoretical molecular formulas (Table 1S, Supporting Information), all displaying a mass error of below 5 ppm, thus confirming their elemental composition. Moreover, the chromatographic elution order helped in some structural assignments as it was described previously [28]. The phytoconstituents characterized in Z. spina-christi are presented Table 1 and their chemical structures are shown in Fig. 2.

In the following section structure assignment of selected compounds is illustrated. Full assignment arguments are provided in the Supporting Information.

One peak (m/z 593) was detected at retention time 11.1 min in the extracted ion chromatogram (EIC) and was tentatively assigned as dimeric B-type proanthocyanidins with (epi)catechin and (epi)gallocatechin monomeric units (epi)catechin-(4, 8′)-(epi)gallocatechin 1 (Table 1 and Fig. 1S and 2S, Supporting Information) as evident from tandem MS data previously reported [29].

Six isomers (tR 6.0, 7.7, 9.6, 11.1, 11.9, 14.8 min) were detected at m/z 609 in the methanolic extract of leaves of Z. spina-christi (Fig. 3S and 4S, Supporting Information). Isomer 2-5 were

145 | P a g e Appendix assigned as (Epi)gallocatechin-(4, 8′)-(epi)gallocatechin as previously reported [30]. While, isomers 6-7 were tentatively assigned as (epi)gallocatechin-(4, 6′)-(epi)gallocatechin assuming that the latter are less polar and hence eluted later in the RP C18-Amid column (Table 1, Fig. 1 and 2). (See also Fig. 5S, Supporting Information for the fragmentation pathway).

At retention times 10.5 and 17.3 min, two peaks with m/z 305 were detected in the EIC and base peak chromatogram (BPC) and were assigned as gallocatechin 8 and epigallcatechin 9 (Table 1) after comparison with the retention time and fragmentation pattern of gallocatechin and epigallcatechin commercial standards.

Two peaks (tR 17.8 and 24.0 min) were detected at m/z 289 in the EIC and were identified as catechin 10 and epicatechin 11 (Table 1) by comparison of their UV and mass spectra with authentic standards.

[mAU] I 10.0

5.0

0.0 10 15 20 25 30 35 40 45 50 Time [min]

[%] 39 27 7 24 23 II x10 50 26 25 20 51 54 38 1.0 45 21 17 12 13 18 22 1 16 52 40 0.5 9 10 19 11 15 8 14 53 0.0 10 15 20 25 30 35 40 45 50 Time [min]

[%] 57 III 7 33 32 x10 30 31 37 1.5 49 34 56 29 48 42 1.0 36 47 55 46 28 43 44 41 0.5 0.0 40 42 44 46 48 50 52 54 56 58 Time [min]

Fig. 1. UV Chromatogram (320nm) (I) base peak chromatogram (II) and expansion (III) for Z. spina-christi leaves extract. Peaks assignment listed in Table 1.

146 | P a g e Appendix trans-5-O-caffeoylquinic acid 12, trans-5-O-p-coumaroylquinic acid 15 and cis-5-O-p- coumaroylquinic acid 16 were easily detected in the EIC and total ion chromatogram TIC (Table 1), while 3-O-caffeoylquinic acid 14 at m/z 353, and 4, 5 di-O-caffeoylquinic acid 18 at m/z 515 were observed in low intensities and were not determined in the BPC or total ion chromatogram (TIC). Subsequently, targeted tandem LC-MS experiments at m/z 353 and m/z 515, respectively, have been performed to confirm there presence in the plant extract. The chlorogenic acids were assigned after the comparison with the retention time and fragmentation pattern of authentic standard supported with the hierarchical keys previously reported in the literature [31-34]. At retention time 23.2 min, a further small peak (m/z 353) with low intensity (16%) compared to compound 12 (100%) was observed. Targeted tandem-LC-MS experiments at m/z 353 have been performed and it displayed fragmentation patterns identical to the 5-O-caffeoylquinic acid and we suspected that it might be a cis isomer of the 5-O-caffeoylquinic. For confirmation of this isomer, the extract of Z. spina-christi was irradiated with UV light at 245 nm for 60 min. After irradiation, we found that the cis isomer in the chromatogram as peak with considerably increased intensities if compared to trans isomer from the original plant extract (Fig. 7S and 8S, Supporting Information), which confirmed the presence of the cis-5-O-caffeoylquinic acids 13 [35].

One peak was readily detected at m/z 341 in the EIC and was tentatively assigned as caffeic acid 4-O-glucoside 17. The neutral loss of a glucosyl moiety (162 u) resulted in a dominant fragment ion at m/z 179.0 corresponding to deprotonated caffeic acid and secondary peak at m/z 161 ([M-  3 H-162-H2O] ) from the neutral loss of a water molecule and an MS base peak at m/z 135 by the neutral loss of CO2 (44 u) (Fig. 9S, Supporting Information). For further evidence the UV spectrum, MSn fragmentation and the retention time of this compound was compared with the compound (m/z 341) reported in the flowers of Chrysanthemum morifolium Ramat [36] and found that they are identical. Based on the above arguments compound 17 was identified as caffeic acid 4-O-glucoside. We have recently identified 6-O-caffeoyl-α-glucose and 6-O-caffeoyl-β-glucose in the bottle gourd (Lagenaria siceraria Stand) fruits [37]. Here we are unable to assign the anomeric structure for the glucose moiety.

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A OH OH 8 1 OH HO O OH 7 OH 8 1 2 HO O OH OH 1 6 7 2 OH 8 3 5 4 OH OH HO O OH 6 OH 3 7 2 OH 1' 5 4 OH 8' OH HO 7' O 6 3 OH 1' 5 4 OH OH 8' 2' HO 7' O OH 2' OH 6' 6' 3' 7' OH 5' OH HO 4' 5 OH 6' 3' OH 1 2-5 5' 4' 6 & 7 8' OH 4' O1' HO 3' 2' OH OH OH OH HO OH HO O HO O OH OH OH

OH OH OH 8 OH 9 OH OH

OH OH HO OH OH HO HO O HO O O O O O 5 4 OH 5 4 OH HO OH OH HO 1 3 1 3 OH OH OH OH O OH 10 11 12 O OH 13

OH HO O OH HO 5 1 4 OH O 3 1 OH O O HO 6 2 O O 5 3 5 4 OH OH 4 OH O HO 1 3 O O HO O 5 OH 1 4 OH O OH O 3 HO OH HO OH OH 15 16 OH 14 OH 17

OH OH 3' O O 2' 4' OH 8 1 O O O 5' 1' HO 7 2 6' HO OH O OH 6 O 3 OH O HO OH 5 4 5 4 O HO OH OH OH O 1 3 OH 19 O OH 18

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O OH B OH OH 1 O 1 8 8 HO O 2 HO O 2 7 7 OH O OH 3 HO OH 3 5 4 O 5 4 O OH OH O OH OH O OH O O OH O HO 8 1 OH OH O O 2 OH OH 7 HO O OH HO O OH 3 5 4 OH O O HO OH O HO 20 & 21 22 & 23 24

OH OH OH OH 1 8 OH HO O 2 OH 1 7 8 1 2 8 HO O HO O 2 3 7 5 4 O 7 OH O OH 3 5 4 O OH 3 O 5 4 O OH O O OH OH O OH OH OH O O HO O OH O OH OH OH O OH OH HO 25 & 26 27 & 28 29

OH OH OH OH OH OH 1 1 8 8 8 1 HO O 2 HO O 2 HO O 2 7 7 7

3 3 3 5 4 O 5 4 O 5 4 O OH O OH OH O OH OH O OH O O O

OH OH OH OH OH OH O OH 30 31 32 O OH OH OH OH 1 1 8 8 1 8 HO O 2 HO O 2 HO O 2 7 7 7

3 O 3 3 O 5 4 5 4 O 5 4 OH O OH OH O OH OH O OH O O O

OH OH OH OH OH OH OH HO O OH

33 34 O O 35

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C OH OH OH OH 1 3' O OH HO 8 2' OH HO O 2 1 4' 8 3 7 HO O 2 5' O O OH 1' 2 7 OH HO 4 3 O 6' 5 4 HO 5' 6' OH 5 3 HO 1 OH O OH 5 4 O 4' 6 O OH 1' OH O O O O HO 3' 2' OH OH O OH OH O HO O OH OH HO OH 36 37 38-40 OH OH O O OH OH HO OH OH

8 1 HO HO O O O O O O 7 2 3' 3 2' O 6 HO OH 4' HO O OH O 8 1 5 4 OH HO O O 1' OH OH OH O O 5' O 7 2 6' O 6 3 O OH HO OH OH 5 4 OH O HO O OH O 41 & 42 OH 43 & 44 O O HO OH OH OH OH OH HO HO 8 1 OH OH HO O 2 O OH O OH 7 HO HO 6 3 8 1 1 O HO O O 8 5 4 OH O O 7 2 7 2 OH O O 3 O 6 3 O HO 6 O OH 5 4 OH 5 4 O OH OH OH O O OH O OH O O HO O OH OH O O O OH OH OH O HO HO O OH OH HO OH 45 O 46 & 47 48 HO OH OH OH HO OH HO OH OH O OH O OH HO 8 1 HO 8 1 HO O 8 1 O O 7 2 O O 7 2 6 3 7 2 3 HO 3 6 5 4 O 6 O HO OH O 5 4 OH OH O O 5 4 OH O O O OH O OH O O OH O O OH OH OH OH OH HO O OH OH OH HO O OH HO O OH O O HO O HO HO 49 50 51 & 52

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OH OH D OH HO OH OH 8 1 HO O O OH 7 2 HO 8 1 6 3 O O 5 4 O 7 2 OH O OH 6 3 O 5 4 O OH O OH HO OH OH O HO O OH HO O OH OH O HO O OH O HO OH O 53 HO 54

OH OH O HO OH HO OH OH O OH HO O OH 8 1 HO O O 8 1 O O 7 2 7 2 6 3 HO 6 3 5 4 O HO OH 4 O OH O O 5 OH O OH O O O O OH OH OH O OH OH OH HO O OH HO O OH O HO 55 & 56 O HO 57

Fig. 2. Representative structures Z. spina-christi phytoconstituents.

One peak was detected at retention time 21.4 min with m/z 593 in the Z. spina-christi methanolic extract. It showed fragmentation patterns representing apigenin 7, 4′-di-O-glucoside 19. Compound 19 produced an MS2 fragment ion at m/z 431 ([(M-H)-162]) by the neutral loss of a glucosyl unit from C-4′ (Fig. 10S, Supporting Information). It produced the MS3 base peak at m/z 269 ([apigenin-H]) by the neutral loss of the second glucosyl unit from C-7 (Table 1). Based on this fragmentation behaviour compound 19 was assigned as apigenin7, 4′-di-O-glucoside.

Three C-glycosides derivatives of phloretin (compounds 38-40) were detected in the EIC of Z. spina-christi leaves extract (Table 1). They produced characteristic C-glycosides MS2 fragment ions ([M-H-120], [M-H-90] and [M-H-30]) (Fig. 11S, Supporting Information). Compound 40 was identified as phloretin 3', 5'-di-C-glucoside. This compound has been previously reported in Ziziphus species [15]. We speculate that compounds 38 and 39 were isomers of compound 40 alternative C-glycosylated hexoses. From the literature and our experiments we have found that the glycosides containing galactoside units are more polar than the glycosides containing glucoside units [38]. Based on the above arguments we have tentatively assigned isomer 38 as phloretin 3', 5'-di-C-galactoside (tR 32.6 min) and isomer 39 as phloretin 3'-C-glucoside 5'-C-galactoside or

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phloretin 3'-C-galactoside 5'-C-glucoside (tR 36.8 min) (Table 1). To our knowledge, compound 38 was not previously reported in the plant kingdom.

Two peaks, 43 and 44, were detected at m/z 769 in the EIC and showed different retention times 3 (tR 54.4 and 56.6 min). They showed in their MS fragment ions at m/z 299 (base peak), 255 and 284 corresponding to diosmetin aglycone [39], which suggested that, these compounds were diosmetin glycosides (Table 1). Based on the elution order and the data previously reported [40- 42] compound 43 was assigned as diosmetin 3'-O-galactoside 7-O-rutinoside and the later eluting isomer 44 as diosmetin 3'-O-glucoside 7-O-rutinoside (Table 1). To the best of our knowledge, compounds 43 and 44 were not reported previously in nature. (Further arguments are provided in the Supporting Information).

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Table 1. Retention times and MS4 fragmentation of Z. spina-christi phenolics

 No. Compound name tR (min) [M-H] Characteristic m/z of ions in negative ion mode 1 (Epi)catechin-(4, 11.1 593 MS2 → 423(100), 305 (46), 441(45), 467(25), 575(27); MS3 → 297 8′)- (100), 283(99), 285 (49), 405 (33) (epi)gallocatechin 2 (Epi)gallocatechin 6.0 609 MS2 → 423(100), 305 (39), 441(75), 483 (15), 591 (14); MS3 → -(4, 8′)- 297(100), 283 (85), 285 (31), 405(15), 269 (60), 255 (33), 243 (34); (epi)gallocatechin MS4 → 297 (100), 269 (30) 3 (Epi)gallocatechin 7.7 609 MS2 → 423(100), 305 (30), 441(81), 483 (11), 591 (13); MS3 → -(4, 8′)- 297(100), 283 (91), 405(21), 255 (28), 243 (23); MS4 → 297 (100), (epi)gallocatechin 269 (60) 4 (Epi)gallocatechin 9.6 609 MS2 → 441 (100), 423(99), 305 (27), 483 (18), 539 (10); MS3 → -(4, 6′)- 283(100), 255(29), 297 (81), 405 (16), 243 (21); MS4 → 297 (100), (epi)gallocatechin 269 (68) 5 (Epi)gallocatechin 11.1 609 MS2 → 423(100), 305 (40), 441(70), 483 (19), 591 (07); MS3 → -(4, 8′)- 297(100), 283 (83), 405(07), 255 (27), 269 (23); MS4 → 297 (100), (epi)gallocatechin 269 (42), 253 (16) 6 (Epi)gallocatechin 11.9 609 MS2 → 423(100), 305 (36), 441(76), 483 (14), 593 (08), 591 (13); -(4, 6′)- MS3 → 283 (100), 297(67), 255 (24), 405(22), 255 (24), 269 (14), (epi)gallocatechin 243 (34); MS4 → 283 (100), 255 (30) 7 (Epi)gallocatechin 14.8 609 MS2 → 423(100), 305 (29), 441(66), 483 (14), 539 (10), 591 (10); -(4, 8′)- MS3 → 283 (100), 297(89), 405(12), 243 (34); MS4 → 297 (100), (epi)gallocatechin 269 (99), 255 (34) 8 Gallocatechin 10.5 305 MS2 → 179(100), 219 (69), 261(10), 165 (26), 137 (22), 125(36); MS3 → 164 (100), 151(27), 135(36), 243 (34); MS4 → 120 (100) 9 Epigallocatechin 17.3 305 MS2 → 179(100), 219 (92), 261(28), 165 (24), 137 (26), 125 (36); MS3 → 164 (100), 151(14), 135(23), 247 (16) 10 Catechin 17.8 289 MS2 → 244(100), 205 (28); MS3 → 203 (100, 227(18), 186(16), 161 (20); MS4 → 174 (100), 187 (70) 11 Epicatechin 24.0 289 MS2 → 244(100), 205 (40); MS3 → 03 (100), 227(25), 186(16), 161 (21); MS4 → 174 (100), 187 (1) 12 trans-5-O- 20.2 353 MS2 → 191 (100); MS3 → 127 (10), 173(75), 85 (73); MS4 → 109 Caffeoylquinic (100) acid 13 cis-5-O- 23.2 353 MS2 → 191 (100), 179 (40), 135 (11); MS3 → 127 (100), 111 (55), Caffeoylquinic 85 (41), 59 (18) acid 14 3-O- 13.5 353 MS2 → 191 (100); MS3 → 111 (100), 173(85), 127 (45), 85 (33) Caffeoylquinic acid 15 trans-5-O-p- 27.2 337 MS2 → 191 (100); MS3 → 127 (100), 173(47), 85 (92) Coumaroylquinic acid 16 cis-5-O-p- 28 337 MS2 → 191 (100); MS3 → 127 (100), 173(51), 85 (71) Coumaroylquinic acid 17 Caffeic acid 4-O- 14.9 341 MS2 → 179 (100), 161 (26), 135 (15); MS3 → 135 (100) glucoside 18 4, 5-di-O- 41.7 515 MS2 → 353 (100), 203 (17), 173 (6), 179 (7); MS3 → 123 (100), Caffeoylquinic 179(23), 191 (19), 135 (7) acid

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19 Apigenin 7, 4'-di- 21.4 593 MS2 → 431 (100); MS3 → 269 (100), 161(45) O-glucoside 20 Isorhamnetin 3-O- 45.1 623 MS2 → 315 (100), 300 (27), 271 (14); MS3 → 300 (100); MS4 → (6-O-rhamnosyl- 271 (100), 255 (53) galactoside) 21 Isorhamnetin 3-O- 46.0 623 MS2 → 315 (100), 300 (15), 271 (11); MS3 → 300 (100); MS4 → (6-O-rhamnosyl- 271 (100), 255 (8) glucoside) 22 Kaempferol 3-O- 44.0 593 MS2 → 285 (100) 284 (55); MS3 → 255 (100), 257 (33), 229 (12), (6-O-rhamnosyl- 241 (13), 151 (10), MS4 → 255 (100), 229 (34), 163 (46) galactoside) 23 Kaempferol 3-O- 45.5 593 MS2 → 285 (100); MS3 → 257 (100), 229 (38), 241 (29), 255 (18), (6-O-rhamnosyl- 163 (26), MS4 → 255 (100), 229 (58), 163 (66) glucoside) 24 Quercetin 7-O-(6- 36.4 609 MS2 → 300 (100), 301(32), 271 (20), 255 (10), 445 (10), 489 (11); O-rhamnosyl- MS3 → 271 (100), 179 (14), 255 (59); MS4 → 271 (100), 255 (34), glucoside) 243 (49) 25 Quercetin 3-O-(6- 37.3 609 MS2 → 300 (100), 301(40), 271 (24), 255 (12); MS3 → 300 (100), O-rhamnosyl- 271 (77), 179 (11), 255 (41); MS4 → 271 (100) galactoside) 26 Quercetin 3-O-(6- 41.0 609 MS2 → 301 (100), 300(50), 271 (12); MS3 → 179 (100), 151 (72), O-rhamnosyl- 271 (63), 255 (38); MS4 → 151 (100) glucoside) (Rutin) 27 Quercetin 3-O-(2- 45.8 579 MS2 → 301 (32), 300 (100), 271 (22), MS3 → 271 (100), 255 (57); O-rhamnosyl- MS4 → 271 (100), 255 (78), 243 (79) arabinoside) 28 Quercetin 3-O-(2- 46.1 579 MS2 → 300 (100), 301 (28), 271 (18), MS3 → 271 (100), 255 (57), O-rhamnosyl- 179 (11), 151 (10); MS4 → 255 (100), 271 (41), 244 (18), 227 (44), xyloside) 215 (32), 199 (38), 151 (16) 29 Quercetin 3-O- 43.4 433 MS2 → 301 (100), 300 (63), 377 (17); MS3 → 271 (100), 255 (49), arabinoside 179 (68), 151 (53) 30 Quercetin 3-O- 45.2 447 MS2 → 301 (100); MS3 → 179 (100), 271 (42), 255 (24), 151 (56), rhamnoside MS4 → 151 (100) 31 Quercetin 3-O- 41.7 463 MS2 → 301 (100), 343 (10); MS3 → 179 (100), 151 (59), 255 (18), glucoside 271 (30); MS4 → 151 (100) 32 Quercetin -O- 45.2 505 MS2 → 463 (100), 301 (34), 447 (64); MS3 → 327 (100), 301 (9), acetyl hexoside 271 (9), 257 (6); MS4 → 223(100), 271 (53), 241 (21) 33 Kaempferol 3-O- 44.6 447 MS2 → 284 (100), 285 (58), 255 (22), 327 (13); MS3 → 255 (100); glucoside MS4 → 227 (100), 211 (39), 163 (19) 34 Kaempferol 3-O- 50.2 431 MS2 → 284 (100), 285 (88), 255 (12); MS3 → 255 (100), 267 (16), rhamnoside 229 (13) 35 Quercetin 3-O- 43.1 607 MS2 → 463 (100), 301 (11), 505 (41), 545 (18); MS3 → 301 (100); [6″-(3-hydroxyl- MS4 → 179 (100), 271 (24), 255 (15), 151 (56), 151 (71) 3-methylglutaryl)- β-D-galactoside] 36 kaempferol 3-O- 47.8 591 MS2 → 447 (100), 285 (25), 489 (50), 523 (31), 529 (45); MS3 → [6″-(3-hydroxyl- 285 (100); MS4 → 257 (100), 267 (87), 239 (37), 163 (37) 3-methylglutaryl)- β-D-galactoside] 37 Quercetin 3-O-ß- 46.9 725 MS2 → 579 (100); MS3 → 300(100), 301 (27), 447(12), 271 (22), D-xylosyl-(1→2)- 255 (12), MS4 → 271 (100), 255 (46) α-L-rhamnoside-

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4'-O-α-L- rhamnoside 38 Phloretin 3', 5'-di- 32.6 597 MS2 → 357 (100), 387(66), 417 (18), 477 (18); MS3 → 209 (100), C-galactoside 251 (11), 167 (7), 123 (7); MS4 → 123 (100), 165 (82), 191 (26), 93 (29) 39 Phloretin 3'-C- 36.8 597 MS2 → 357 (100), 387(65), 417 (17), 477 (53); MS3 → 209 (100), glucoside 5'-C- 251 (12), 167 (9), 123 (5); MS4 → 123 (100), 165 (45), 191 (18) galactoside or phloretin 3'-C- galactoside 5'-C- glucoside 40 Phloretin 3', 5'-di- 39.4 597 MS2 → 357 (100), 387(66), 417 (22), 477 (42); MS3 → 209 (100), C-glucoside 251 (12), 167 (8), 123 (7); MS4 → 123 (100), 165 (34), 191 (14) 41 Quercetin 3-O-(2, 57.5 1047 MS2 → 901 (100), 755 (60); MS3 → 755 (100), 781 (14); MS4 → 6-di-O- 300 (100), 301 (56), 271 (26), 255 (14), 609 (15), 489 (17), 591 (16) rhamnosyl- galactoside) 7-di- O-rhamnoside 42 Quercetin 3-O-(2, 58.4 1047 MS2 → 901 (100), 755 (72), 781 (22); MS3 → 755 (100), 781 (10); 6-di-O- MS4 → 300 (100), 301 (47), 271 (19), 255 (15), 343 (15), 609 (28), rhamnosyl- 489 (30), 591 (21) glucoside) 7-di-O- rhamnoside 43 Diosmetin 3'-O- 54.4 769 MS2 → 607 (100), 469 (20), 733 (10), 714 (73), 469 (22), 299(31), galactoside 7-O- 285 (20); MS3 → 299 (100), 255 (29), 284 (43) rutinoside 44 Diosmetin 3'-O- 56.6 769 MS2 → 607 (100), 469 (23), 733 (81), 714 (73), 453 (55), 315 (76), glucoside 7-O- 299 (60), 285 (37); MS3 → 299 (100), 255 (56) rutinoside 45 Kaempferol 3-O- 39.2 739 MS2 → 285 (100), 284 (60), 255 (42), 575 (76), 593 (29), 473 (13), (2, 6-di-O- 327 (23), 393 (45); MS3 → 284 (100), 285 (14), 255 (29), 151 (15); rhamnosyl- MS4 → 255 (100) glucoside) 46 Quercetin 3-O-(2, 42.2 917 MS2 → 755 (100); MS3 → 300 (100), 301 (50), 271 (29), 255 (12), 6-di-O- 343 (22), 609 (11), 591 (29); MS4 → 271 (100), 256 (50), 179 (20), rhamnosyl- 151 (17) glucoside) 7-O- galactoside 47 Quercetin 3-O-(2, 48.9 917 MS2 → 755 (100); MS3 → 300 (100), 301 (42), 271 (12), 255 (12), 6-di-O- 591 (34), 489 (18), 325 (21); MS4 → 284 (100), 271 (95), 255 (26), rhamnosyl- 243 (20), 169 (12) glucoside) 7-O- glucoside 48 Kaempferol 3 -O- 56.6 739 MS2 → 593 (100); MS3 → 285 (100), 284 (89), 429 (39); MS4 → rutinoside 7-O- 257 (100), 255 (32) rhamnoside 49 Kaempferol 3-O- 55.8 885 MS2 → 739 (100), 285 (25); MS3 → 285 (100), 284 (69), 255 (33), (2, 6-di-O- 575 (75), 473 (18), 393 (45), 327 (33); MS4 → 255 (100), 241 (17), rhamnosyl- 151 (42) glucoside) 7-O- ramnoside 50 Quercetin 3-O-(2, 35.6 755 MS2 → 300 (100), 301 (17), 271 (19), 255 (9), 591 (18), 609(13), 6-di-O- 489 (13), 343(19); MS3 → 271 (100), 255 (60), 179 (24), 151 (17); rhamnosyl- MS4 → 255 (100), 271 (83), 227 (37), 243 (99) glucoside)

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51 Quercetin 3-O- 49.2 755 MS2 → 609(100), 301 (33); MS3 → 301 (100), 300 (51), 271 (12), rhamnosyl- 343 (15); MS4 → 179 (100), 271 (90), 255 (85), 151 (83) galactoside 7-O- rhamnoside 52 Quercetin 3-O- 55.2 755 MS2 → 609 (100), 301 (47); MS3 → 301 (100), 300 (51), 343 (12); rutinoside 7-O- MS4 → 179 (100), 271 (71), 255 (62), 151 (63) rhamnoside 53 Quercetin 3-O- 38.8 771 MS2 → 301 (100), 609(7); MS3 → 179 (100), 151 (84), 273 (9); MS4 (rhamnosyl- → 151 (100), 169 (10) (1α→6)-O- glucoside) hexoside 54 Quercetin 3 -O- 24.6 771 MS2 → 609 (100), 301 (5); MS3 → 301 (100), 300 (38), 271 (9), 255 rutinoside 7-O- (4); MS4 → 300 (100), 273 (10), 255 (6), 179 (29), 151 (12) glucoside

55 Quercetin 3-O-(2, 52.9 901 MS2 → 755 (100); MS3 → 300 (100), 301 (44), 271 (30), 255 (15), 6-di-O- 609 (17), 343 (21); MS4 → 271 (100), 255 (48), 179 (14), 151 (18) rhamnosyl- galactoside) 7-O- rhamnoside 56 Quercetin 3-O-(2, 54.9 901 MS2 → 755 (100); MS3 → 300 (100), 301 (60), 271 (29), 255 (13), 6-di-O- 343 (25), 609 (14), 489 (21); MS4 → 271 (100), 255 (60), 179 (33), rhamnosyl- 151 (25) glucoside) 7-O- rhamnoside 57 Quercetin 3-O-(2, 44.9 931 MS2 → 755 (100), 769 (65), 781 (17), 795 (10); MS3 → 300 (100), 6-di-O- 301 (43), 271 (27), 255 (12); MS4 → 255 (100), 271 (74), 179 (11), rhamnosyl- 151 (17) glucoside) 7-O- glucuronide

Di, tri, tetra and penta glycosylated flavonoids were also detected in Z. spina-christi methanolic extract. The presence of the aglycone fragment ions at m/z 315, 285 and 301 in their MS2 and/or MS3, and characteristic fragment ions (m/z 151 and 179 for quercetin, m/z 151 for kaempferol and m/z 300 for isorhamnetin) [43], in addition to their UV spectra suggested that these compounds were isorhamnetin, kaempferol and quercetin derivatives [44, 45].

Compounds 23, 26, 29, 30, 31 and 33 were identified as kaempferol 3-O-(6-O-rhamnosyl- glucoside), quercetin 3-O-(6-O-rhamnosyl-glucoside) (rutin), quercetin 3-O-arabinoside, quercetin 3-O-rhamnoside, quercetin 3-O-glucoside, and kaempferol 3-O-glucoside, respectively, by the comparison of their UV spectra and retention times with commercial standards.

Compounds 20-22, 24, 25, 27, 28, 32, 34, 37 and 45-57 were tentatively identified as isorhamnetin 3-O-(6-O-rhamnosyl-galactoside), isorhamnetin 3-O-(6-O-rhamnosyl-glucoside), kaempferol 3- O-(6-O-rhamnosyl-galactoside), quercetin 7-O-(6-O-rhamnosyl-glucoside), quercetin 3-O-(6-O- rhamnosyl-glucoside), quercetin 3-O-(2-O-rhamnosyl-arabinoside), quercetin 3-O-(2-O- rhamnosyl-xyloside), quercetin -O-acetyl hexoside, kaempferol 3-O-rhamnoside, quercetin 3-O- [6″-(3-hydroxyl-3-methylglutaryl)-β-D-galactoside], kaempferol 3-O-[6″-(3-hydroxyl-3-

156 | P a g e Appendix methylglutaryl)-β-D-galactoside], quercetin 3-O-ß-D-xylosyl-(1→2)-α-L-rhamnoside-4'-O-α-L- rhamnoside, kaempferol 3-O-(2, 6-di-O-rhamnosyl-glucoside), quercetin 3-O-(2, 6-di-O- rhamnosyl-glucoside) 7-O-galactoside, quercetin 3-O-(2, 6-di-O-rhamnosyl-glucoside) 7-O- glucoside, kaempferol 3 -O-rutinoside 7-O-rhamnoside, kaempferol 3-O-(2, 6-di-O-rhamnosyl- glucoside) 7-O-ramnoside, quercetin 3-O-(2, 6-di-O-rhamnosyl-glucoside), quercetin 3-O- rhamnosyl-galactoside 7-O-rhamnoside, quercetin 3-O-rutinoside 7-O-rhamnoside, quercetin 3- O-(rhamnosyl-(1α→6)-O-glucoside) hexoside, quercetin 3 -O-rutinoside 7-O-glucoside, quercetin 3-O-(2, 6-di-O-rhamnosyl-galactoside) 7-O-rhamnoside, quercetin 3-O-(2, 6-di-O-rhamnosyl- glucoside) 7-O-rhamnoside, and quercetin 3-O-(2, 6-di-O-rhamnosyl-glucoside) 7-O-glucuronide, respectively, as previously reported [15, 26, 40-42, 46] (See also the Supporting Information for characterization of these compounds).

Two isomers of flavonoid pentaglycosides (41 and 42) were also detected in the Z. spina-christi methanolic extract. Both isomers showed a pseudomolecular ion at m/z 1047 ([M-H]), and the MS2 spectra showed a base peak at m/z 901 ([M-H-146]) due to the neutral loss of rhamnoside residue from position 7 of the flavonoid moiety [47] and a high abundance ions (≥ 60) at 755 ([M- H-146-146]) indicating a subsequent neutral loss of another rhamnoside residues from position 7 (Table 1 and Fig. 3), this was supported by observing the ion m/z 755 as a base peak in MS3 spectra. Their MS4 (755 → 300/3001) [neutral loss of triglyosides (454 u) moieties] were reminiscent to that of quercetin 3-O-(2, 6-di-O-rhamnosyl-glucoside) (50) (Table 1). These data suggested that these isomers were derivatives of compound 50 with an additional two O- rhamnoside substituent at position 7. Accordingly, isomer 42 was tentatively identified as quercetin 3-O-(2, 6-di-O-rhamnosyl-glucoside) 7-di-O-rhamnoside, and the earlier-eluted isomer (41) as quercetin 3-O-(2, 6-di-O-rhamnosyl-galactoside) 7-di-O-rhamnoside. To the best of our knowledge compounds 41 and 42 were not reported previously in nature.

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OH - [(M-H-146) -146] OH

OH 8 1 [M-H-146]- O O O OH 7 2 3 8 1 6 HO O O O O O 2 OH 5 4 O 7 2 MS HO 3 HO OH O OH 6 O HO O OH O HO 5 4 OH O OH OH OH O O O OH O OH OH O OH OH OH HO O OH 41, 42 [M-H] HO O OH O HO m/z 1047 O 41, 42 [(M-H-146)] HO m/z 901 MS3 OH OH OH OH 8 1 8 1 O O O O 7 2 [(M-H-146)-146 -(454)] 7 2 6 3 6 3 4 5 4 O O MS HO 5 4 OH O OH OH O O O - [Aglycone -H] O OH OH OH 41, 42 [(M-H-146) -146-(454)] HO O OH m/z 301 O 41, 42 [(M-H-146) -146] HO m/z 755

[%] MS2 41&42 901.3 100 755.3

0 MS3 755.2 100

0 MS4 299.9 100

489.1 609.1 343.0409.0 737.2 0 200 300 400 500 600 700 800 900 m/z

Fig. 3. Proposed fragmentation pathway and MS4 spectra of quercetin 3-O-(2, 6-di-O-rhamnosyl- galactoside) 7-di-O-rhamnoside 41 and quercetin 3-O-(2, 6-di-O-rhamnosyl-glucoside) 7-di-O- rhamnoside 42 of precursor ion at m/z 1047 in negative ion mode.

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The antibacterial activities of the plant extracts analysed in this study were evaluated for their efficacies against Gram-positive and Gram-negative bacteria using the agar diffusion method. As suitable model organisms B. subtilis S168, B. aquimaris MB-2011, and C. michiganensis GSPB 390 were chosen for Gram-positive and E. coli DH5α, E. amylovora 1189, and P. syringae pv tomato DC300 for Gram-negative. The results of the antibacterial activity tests of aqueous and methanolic extracts of the leaves of Z. spina-christi are given in Table 2 and Fig. 4. Interestingly, the plant extracts tested herein showed antibacterial activity only against the Gram-positive bacterium B. aquimaris MB2011 and the Gram-negative representative P. syringae pv tomato DC300 while the other bacterial strains were not affected at all. These results are in contrast to those of others [48] who found that ethanolic and methanolic extracts of Z. spina-christi leaves inhibited the growth of a variety of Gram-negative bacterial strains Salmonella typhi, Proteus mirabilis, Shigella dysenteriae, E. coli, Klebsiella pneumoniae, Brucella melitensis, Bordetella bronshiseptica and Pseudomonas aeruginosa. Previously, Moghadam et al. (2010) reported antibacterial activity of ethanolic extracts of the leaves of Z. spina-christi against a Gram-positive, Methicillin-resistance Staphylococcus aureus strain [9].

Fig. 4. Inhibition zones as means ± SD generated by extracts prepared from Z. spina-christi leaves and the standard antibiotics against bacterial species tested.

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Table 2. Antibacterial activities of Z. spina-christi leaf extracts and the standard antibiotics as assessed by the agar diffusion method.

Bacterial strains Inhibition zone diameters (mm) Extracts prepared in Antibiotic Water MeOH Kanamycin Ampicillin Streptomycin Gram-positive B. subtilis (S168) 0.0 ± 0.0 0.0 ± 0.0 20.0±0.0 NA NA B. aquimaris 16.0 ± 1.0 26.0 ± 2.0 19.7 ±0.6 NA NA (MB2011) C. michiganensis 0.0 ± 0.0 0.0 ± 0.0 NA NA 10.3± 0.6 (GSPB 390) Gram-negative E. coli (DH5α) 0.0 ± 0.0 0.0 ± 0.0 NA 16.3±0.6 NA E. amylovora (1189) 0.0 ± 0.0 0.0 ± 0.0 NA 13.3 ±0.6 NA P. syringae pv tomato 0.0 ± 0.0 18.3 ± 0.5 NA NA 23.3± 1.5 (DC300) NA = Not Applicable

In line with our findings, it has been shown before that many plant metabolites including flavonoids, phenols, tannins, and alkaloids exhibit moderate antimicrobial activity [49, 50]. Our data propose that antibacterial activities might be associated with the phenolic compounds from Z. spina-christi leaves, which were identified in the aqueous and methanolic extracts. Our data furthermore indicated that methanolic extracts appeared to be more effective in antibacterial activity against Gram-negative bacteria as compared to the aqueous extracts of the leaves from Z. spina-christi suggesting that the bio-active metabolites of Z. spina-christi were rather hydrophobic than hydrophilic. However, B. aquimaris MB-2011 was similarly affected by the aqueous and the methanolic extracts suggesting that it is either affected by at least two differently extractable compounds or that it is susceptible to a compound different from that effective against the Gram- negative strain P. syringae. Thus, if traditional use of the leaves of Z. spina-christi involves the preparation of a pre-extract in alcoholic solvents, their effectiveness may be more pronounced than simply brewing the leaves in water [3].

In order to use Z. spina-christi extract as a potential antibiotic and to enable safe use as topical treatment of skin or as systemic drug taken up by oral administration routes, cytotoxicity against the exposed cells and tissues must be analyzed in order to exclude potential negative, off-target effects during treatment. Here, we have used human keratinocyte (HaCaT) and rat intestine epithelial (IEC-6) cell lines in culture to assess potential cytotoxicity exerted by leaf extracts from Z. spina-christi in vitro. For all experiments, the cells were first grown to confluence with the aim to simulate the effect of the extracts on cell monolayers which are considered representative of intact epithelia of the respective tissue, i.e. epidermis of the skin and mucosa of the gastro-intestinal tract lining.

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Cytotoxicity was estimated by using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, which is a routine test evaluating cell viability and proliferative activity by means of cellular enzyme activity. The MTT is converted by mitochondrial NADH-dependent dehydrogenase into an insoluble dye made of dark blue formazan crystals [51]. MTT assays are suited to determine the activity of mitochondrial enzymes reducing the yellow tetrazole MTT to purple formazan in living cells, only [52]. Therefore MTT conversion is proportional to both, cell viability and proliferation rates of treated cell cultures.

The MTT assay has been applied by us and others before. In comparison with other cytotoxicity assays such as the neutral red assay or quantification of LDH latency, the MTT assay was suitable but less sensitive than the former when CdCl2 effects onto hepatoma cell lines were determined [53]. In another study, the above mentioned three different assays were compared and additionally ATP contents of the cultured cells were determined [54]. While both, the MTT and the LDH assay were somewhat non-suitable because of their sensitivity to inhibitory agents contained in the compound mixtures tested, it was also pointed out that the choice of assay depends on the pathway of death that is potentially initiated by the agents applied.

Here, we used the MTT assay to recapitulate the well-known cytotoxicity of DMSO by incubating cultures of HaCaT keratinocytes and IEC6 intestine epithelial cells with different concentrations thereof (Fig. 18S, Supporting Information). The results demonstrate that indeed DMSO is cytotoxic if applied at concentrations above 1%, because MTT conversion declines in a concentration dependent manner. However, the data also support the notion that concentrations of up to 0.5% DMSO are non-cytotoxic, and can be used to as solvent of lipophilic compounds.

We have used this assay before to determine cell viability upon exposure of fibroblasts, keratinocytes, and intestine epithelial cells to other stressors in addition to testing for the safe use of e.g. potential drug delivery tools [55-57]. The conversion of MTT by HaCaT keratinocytes (Fig. 5A) is not influenced upon exposure to 100 µg or 10 µg/mL Z. spina-christi methanolic extract. However, upon treatment with 1 µg/mL Z. spina-christi conversion of MTT was decreased in HaCaT cell cultures. On the other hand the MTT conversion by small intestine epithelial IEC-6 cells (Fig. 5A) increased with decreasing concentration although this was not statistically significantly different from controls. For the lower concentrations of 10 µg and 1 µg/mL the values of conversion were approximately even. We conclude that cytotoxicity towards IEC-6 cell cultures increased with increasing concentration of Z. spina-christi, and it was safe to apply extracts at a final concentration of 1 µg/mL to intestine epithelial cell monolayers. In contrast, for HaCaT cells the exposure to 1µg/mL Z. spina-christi extracts, only exerted cytotoxic effects that were detectable by a significant reduction of the MTT-conversion to approx. 75% in comparison to DMSO-treated controls.

In addition, MTT- and LDH-assays are usually backed up by additional tests that involve determination of cellular integrity as we have shown in a study in which the MTT assay was employed to test for cytotoxicity of dust samples applied to keratinocytes and fibroblasts [56]. Results achieved by the MTT assay were comparable with culture impedance measurements. In addition, phalloidin staining of the actin cytoskeleton and propidium iodide staining of the nuclei of cells with ruptured plasma membrane were performed in our previous work, again, supporting the results of the MTT-assay with regard to structural and metabolic integrity of the cells,

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respectively. In another study, the MTT assay was used by us to determine IC50 values of cucurbit[7]uril which were backed up by imaging studies and supported by in vivo toxicity studies revealing highly congruent data [57].Therefore, we believe that the MTT assay, especially when used in combination with additional measures of cytotoxicity, is a suitable method to conclude on cytotoxic effects of diverse samples such as dust simulants, drug delivery agents, or plant extracts as applied in this study.

Fig. 5. MTT conversion by HaCaT keratinocytes (A) and IEC-6 small intestine epithelial cells (B) upon incubation with 1, 10 or 100 µg/mL Z. spina-christi extracts for 24 h (n=3). Extent of MTT conversion was quantified spectrophotometrically and values are given as means ± standard deviations. Cells treated with 0.5% DMSO, only, were used as controls and set to 100%.

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Consequently, here we performed propidium iodide (PI) staining for both, HaCaT keratinocytes and IEC-6 intestine epithelial cells to determine the extent of potential plasma membrane rupture and the number of necrotic cells per viable cells when the cultures were treated with extracts of Z. spina-christi. This assay is based on the fact that PI intercalates with DNA, thereby visualizing nuclear DNA when PI passes ruptured membrane lipid bilayers. Therefore PI may stain for necrotic cells in which the lipid bilayer integrity of the plasma membrane is compromised [56]. With increasing concentration of Z. spina-christi extracts, the number of necrotic or ruptured cells increased for the exposed HaCaT cultures (Fig. 6A). For the IEC-6 cell cultures (Fig. 6B), the number of ruptured cells decreased upon exposure to leaf extract of Z. spina-christi. However, the numbers of ruptured cells in HaCaT or IEC6 cell cultures treated with Z. spina-christi leaf extracts remained moderate as they were determined to not even reach one percent upon 24 h of exposure.

Fig. 6. Plasma membrane integrity and extent of necrotic cell death exerted after 24 hours of exposure of HaCaT keratinocytes (A) or IEC-6 small intestine epithelial cells (B) to leaf extracts from Z. spina christi. The acquisition of PI indicates ruptured plasma membranes due to necrotic cell death. Fluorescence intensities of PI in three arbitrary chosen regions were determined by CellProfiler™ software to quantify the fluorescence of dead cells over that of all cells, which was determined by Draq5™ counter-staining of nuclear DNA. Values are given as permille and expressed as means ± standard deviations.

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As an additional and sensitive measure of cellular integrity, the actin cytoskeleton was analyzed using a Phalloidin Draq5™ staining. Upon treatment with 100 µg/mL leaf extracts of Z. spina- christi, HaCaT cells (Fig. 7) showed a slightly altered actin filamentous system, and some cells detached from their neighbors indicating loosening of cell-cell contacts. For the IEC-6 cell cultures (Fig. 7), the cortical F-actin system remained intact upon treatment with different concentrations of the extracts prepared from Z. spina-christi.

Fig. 7. Phalloidin staining of the filamentous actin system of HaCaT keratinocytes and IEC-6 small intestine epithelial cells after exposure to 100 µg/mL leaf extracts of Z. spina-christi for 24h. Single channel fluorescence, phase contrast and merged micrographs taken with a confocal laser scanning microscope of formaldehyde-fixed cells after staining of actin filaments with FITC-phalloidin, and Draq5™ counter-staining of nuclear DNA. Bars represent 20 µm and 50 µm, respectively.

In summary, mild cytotoxic effects can be observed on both, HaCaT and IEC-6 cell cultures upon a 24-h treatment with leaf extracts of Z. spina-christi. IEC-6 cells were less sensitive compared to HaCaT cells, suggesting Z. spina-christi extracts might be potentially suitable for topical applications.

Acknowledgements

This work was supported by Jacobs University Bremen and Deutscher Akademischer Austausch Dienst (DAAD). The authors would like to thank Anja Müller for excellent technical support. We are grateful to Dr. Hayder Abdelgader, Medicinal and Aromatic Plants Research Institute (MAPRI), Khartoum, Sudan, for providing the plant materials.

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Antimicrobial, Antiparasitic and Antioxidant Activities of Medicinal Plants from Sudan

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Abstract

The rural population of Sudan has traditionally used medicinal plants for treatment of several ailments and microbial infections. In the present study, we have investigated the antibacterial, antitrypanosomal, antiplasmodial, and antioxidant properties of selected Sudanese medicinal plants using various in vitro assays. Methanolic extracts of various parts of the plants were tested against six bacterial strains (Bacillus subtilis, Bacillus aquimaris, Clavibacter michiganensis, Escherichia coli, Erwinia amylovora, and Pseudomonas syringae) using agar diffusion and minimum inhibitory concentration (MIC) methods. The antiplasmodial activity was tested against a chloroquine sensitive strain of Plasmodium falciparum NF54, whereas the antitrypanosomal activity was evaluated against Trypanosoma brucei rhodesiense STI900 (African strain). The antioxidant activity of the plant extracts was assessed by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging.

Various extracts showed antibacterial, antiparasitic and antioxidant activities. Acacia nilotica, Ocimum basilicam, Ziziphus spina-christi, Balanites aegyptiaca, Sonchus oleraceus, Punica granatum, Mimosa pigra and Ixora coccinea were the most interesting ones with antioxidative, antiplasmodial, antitrypanosomal and antibacterial activities. By means of preparative HPLC, HPLC-ESI-TOF, HPLC-ESI-MSn, 1H-NMR and 13C-NMR, thirteen phytoconstituents were isolated and identified in the methanolic extracts of Z. spina-christi, S. oleraceus, and H. sabdariffa including chlorogenic acids, flavonoid glycosides, coumarins and derivatives. Sudanese plants represent an important alternative source of natural antioxidants, antiparasitic and antimicrobials. To our knowledge, this is the first report about the antibacterial, antiparasitic and antioxidant activities of some of these plants.

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Introduction

Medicinal plants continue to play a vital role as therapeutic agents in primary health care in developing countries (Tshikalange et al., 2005). Sudan is located in tropical Africa and has high plant diversity and a multinational population. In Sudan and other developing countries, traditional medicine plays a major role particularly in rural regions due to both economic and cultural reasons (Ali et al., 2002). Comprehensive ethnobotanical investigations on Sudanese folk medicine was reported previously (El Ghazali et al., 1994; El Ghazali, G. E. B. et al., 1997; El-Kamali and El- Khalifa, 1999; Khalid et al., 2012). (The ethnomedicinal data of selected plant materials are provided in Table S1 in the Supporting information).

The frequent use of medicinal plants for treatment of different diseases has encouraged a number of researchers to study their biological activities (Rubio et al., 2013; Silva and Fernandes Júnior, 2010). Additionally, natural products can contribute to the discovery of novel antimicrobial (Maroyi, 2013) and antioxidant components (Carocho and Ferreira, 2013). A number of pharmaceutical studies have demonstrated the antibacterial, antimalarial, antitrypanosomal, and antioxidant activities of Sudanese medicinal plants (Abd alfatah Abd alla, 2013; Abdelrahman, 2010; Ali et al., 2002; Almagboul et al., 1988; El-Kamali and El-Khalifa, 1997; El-Tahir et al., 1999; Elegami et al., 2001; Hilmi et al., 2014; Taha et al., 2010). It is well-known that plants, which are rich in a diversity of secondary metabolites such as polyphenols, tannins, terpenoids and alkaloids are usually interesting for their antiparasitic, antimicrobial, and antioxidant activities (Albagouri et al., 2014; Barreira et al., 2013; Cheung et al., 2003; Cowan, 1999; Gyawali and Ibrahim, 2014; Machumi et al., 2013). The antioxidant hypothesis is however under discussion, and recent evidence suggested that its role has heavily been overestimated (Mikutis et al., 2013). Therefore, the aim of the present study was to evaluate the antibacterial, antiplasmodial, antitrypanosomal, and antioxidant activities of some Sudanese medicinal plants. The antimicrobial effect of some phenolic compounds extracted from edible and medicinal plants from Europe was also assessed. We have also isolated thirteen bioactive phenolic constituents from Z. spina-christi, S. oleraceus and H. sabdariffa by preparative-HPLC (PHPLC).

Materials and methods

Chemicals and standards

All chemicals, solvents and authentic standards used in this study were analytical grade. 5-O- caffeoylquinic acid (> 98%), 3-O-caffeoylquinic acid (> 98%) and 4-O-caffeoylquinic acid (> 96.45%) were purchased from Phytolab (Vestenbergsgreuth, Germany). Quercetin 3-O-(6-O- rhamnosyl-glucoside) (rutin) (≥ 94%), quercetin 3-O-glucoside (90%), esculin (≥ 98%), luteolin 7-O-glucoside (≥ 98%), gallic acid (≥ 99%), methanol (≥ 99.9%), methanol-d4 (99.96%) and DMSO (≥ 95%) were purchased from Sigma-Aldrich (Steinheim, Germany). Ultrapure water with a resistance of 18.2 M was deionized in a Milli-Q system (Sartorius Stedim Biotech GmbH, Germany).

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Plant materials and preparation

All Sudanese plant samples were freshly collected June and July 2010 from their natural habitats in Omdurman (15° 38' North, 32° 26' East) and Khartoum (15° 33' North, 32° 31' East), Sudan. The voucher specimens were identified by Dr. Hayder Abdel Gadir of Herbarium of Medicinal and Aromatic Plants Research Institute (MAPRI), Khartoum, Sudan, where the specimens were also deposited. All plant species, part used and voucher specimen numbers are presented in Table 1. The plants were selected based on previous studies (Abdelgaleil et al., 2011; AbouZid and Orihara, 2005; El Ghazali et al., 1994; El Ghazali, G. E. B. et al., 1997; El-Kamali and El-Khalifa, 1999; Ghazanfar, 1994; Grosvenor et al., 1995; Han and Park, 1986; Hilmi et al., 2014; Hussein et al., 2000; Khalid et al., 2012; Kirtikar and Basu, 1984; Rosado-Vallado et al., 2000; Satayavati et al., 1976; Teugwa et al., 2013). (See also Table S1 in the Supporting information).

The flowers of Catharanthus roseus were purchased from Wassenaar Living Garden (Bremen, Germany). Quince fruits (Cydonia oblonga), green coffee (Coffea arabica) beans and black tea (Camellia sinensis) leaves were purchased from local markets in Bremen, Germany, while the cocoa (Theobroma cacao) beans and Bobgunnia madagascariensis pods were donated by Barry Callebaut, Wieze, Belgium and Prof. Dr. Philip Stevenson, Royal Botanic Gardens, Kew, Richmond, Surrey, UK, respectively. The sample preparation was achieved as described in our previous work (Jaiswal et al., 2014).

Table 1. List of the plant investigated in this study.

Family Plant species Part used Voucher

Malvaceae Hibiscus sabdariffa L. Petals Hi-sa-09 Fabaceae Acacia nilotica (L.) Willd. ex Del. Fruits Ac-ni-10 Bombacaceae Adansonia digitata L. Fruits Ad-di-11 Rhamnaceae Ziziphus spina-christi (L.) Desf. Leaves Zi-sp-ch-04 Rhamnaceae Ziziphus spina-christi (L.) Desf. Fruits Zi-sp-ch-04 Balanitaceae Balanites aegyptiaca (L.) Del Bark Ba-ae-03 Balanitaceae Balanites aegyptiaca (L.) Del stem Ba-ae-03 Balanitaceae Balanites aegyptiaca (L.) Del Leaves Ba-ae-03 Asteraceae Sonchus oleraceus L. Whole plant So-ol-01 Asteraceae Ambrosia maritima L. Whole plant Am-ma-02 Rubiaceae Ixora coccinea L. Stem Ix-co-07 Rubiaceae Ixora coccinea L. Leaves Ix-co-07 Lamiaceae Ocimum basilicam L. Aerial part Oc-ba-05 Fabaceae Mimosa pigra L. Stem Mi-pi-06 Fabaceae Mimosa pigra L. Leaves Mi-pi-06 Annonaceae Annona senegalensis Pers. Stem An-se-08 Annonaceae Annona senegalensis Pers. Leaves An-se-08 Tiliaceae Grewia tenax (Forssk.) Fiori. Fruits Gr-te-12 Cucubitaceae Cucurbita moschata Duch. Seeds Cu-mo-13 Apocynaceae Catharanthus roseus L. Leaves - Leguminosae Bobgunnia madagascariensis (Desv.) J. H. Pods STVP-1017 Kirkbr. & Wiersema Rosaceae Cydonia oblonga Miller. (quince) Fruits - Lythraceae Punica granatum L. (pomegranate) Peels Lythraceae Punica granatum L.(pomegranate) Juice - Rubiaceae Coffea arabica L. (green coffee) Beans - Malvaceae Theobroma cacao L. (cocoa) Beans - Theaceae Camellia sinensis L. (black tea) Leaves -

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Evaluation of antibacterial activities

Bacterial susceptibility determinations

The minimal inhibitory concentration (MIC) was defined as the lowest concentration of antimicrobial that will inhibit the visible growth of a micro-organism after overnight incubation. The MIC was determined by a twofold dilution assay in Mueller-Hinton broth (MHB) (Becton Dickinson, Heidelberg, Germany). Three Gram-positive bacterial strains (Bacillus subtilis S168, Bacillus aquimaris MB-2011, and Clavibacter michiganensis GSPB 390 and three Gram-negative bacterial strains (Escherichia coli DH5α, Erwinia amylovora 1189, and Pseudomonas syringae pv tomato DC300) were selected as model organisms to evaluate the antibacterial activity of the crude plant extracts and phenolic compounds. The plates were incubated overnight at 28 ºC except for E. coli, for which incubation was done at 37 ºC. All tests were done in triplicate following the National Center for Clinical Laboratory Standards recommendations (National Committee for Clinical Laboratory Standards (NCCLS), 2000).

Agar diffusion assays

Agar diffusion assays were performed as in our previous study (Karar et al., 2014).

Evaluation of antiplasmodial and antitrypanosomal activities

The antiplasmodial and antitrypanosomal activities were evaluated in vitro against a chloroquine sensitive strain of Plasmodium falciparum NF54 and Trypanosoma brucei rhodesiense STI900 (African strain), respectively. Both assays were carried out at two different concentrations (2 and 10 µg/mL). These experiments were conducted in collaboration with Swiss Tropical and Public Health Institute (Swiss TPH), Basel, Switzerland as previously described (Hata et al., 2011).

Evaluation of antioxidant activities

The scavenging activity of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals of the crude plant extracts was measured according to a method previously described (Chang et al., 2001). Assays were carried out in 3 mL reaction mixtures containing 2 mL of 0.1 mM DPPH-ethanol solution, 0.9 mL of 50 mM Tris-HCl buffer (pH 7.4) and 0.1 mL of plant extracts at two different concentrations (10 and 100 µg/mL). The reaction mixture was vortexed and left in the dark at room temperature (27 °C) for 30 min. The absorbance was measured spectrophotometrically at 517 nm. Gallic acid was used as a positive control, while ethanol was used as a blank sample. The inhibitory effect of DPPH was calculated using the standard equation (Liyana-Pathirana and Shahidi, 2005). The experiments were conducted in triplicates, and the data are given as mean values ± standard deviation (SD).

HPLC & HPLC-MSn

The HPLC separation and HPLC-MSn analysis was achieved as previously described (Jaiswal and Kuhnert, 2011; Kuhnert et al., 2010).

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Preparative-HPLC isolation

Preparative-HPLC isolation of compound 1-13 was carried out as in our previous study (Karar et al., 2014).

NMR

1H NMR and 13C NMR spectra were acquired on a JEOL ECX-400 spectrometer operating at 400 1 13 MHz for H NMR and 100 MHz for C NMR in CD3OD using a 5 mm probe. The chemical shifts (δ) are reported in parts per million (ppm) and were referenced to the residual solvent peak. The coupling constants (J) are quoted in hertz (Hz).

1 Esculin (5): H-NMR (400 MHz, CD3OD) δH 6.28 (d, J 9.62 Hz, H-3), 7.82 (d, J 9.16 Hz, H-4), 7.18 (s, H-5), 7.0 (s, H-8), 4.96 (d, J 7.33 Hz, H1'), 3.54 (dd, J 9.27 and 7.3 Hz, H2'), 3.50 (dd, J 9.2 and 7.3 Hz, H-3'), 3.40 (dd, J 9.62 and 8.7 Hz, H-4'), 3.51 (m, H-5'), 3.92 (dd, J 12.36 and 2.29 13 Hz, H6'a), 3.70 (dd, J 11.91 and 5.5 Hz, H6'b); C-NMR (100 MHz, CD3OD) δC 61.02 (C-6'), 69.93 (C-4'), 73.36 (C-2'), 76.03 (C-3'), 77.16 (C-5'), 101.55 (C-1'), 104.18 (C-8), 112.49 (C-3), 113.25 (C-10), 114.04 (C-5), 144.40 (C-6), 145.10 (C-4), 147.71 (C-7), 149.52 (C-9), 162.29 (C- 2). These data were in agreement with those reported in the literature (Kisiel and Michalska, 2002; Zhou Hai-Yan et al., 2009).

1 Luteolin 7-O-glucoside (6): H-NMR (400 MHz, CD3OD) δH 6.28 (1H, d, J 7.0 Hz, H-1''), 3.35 – 3.55 (4H, m, H-2'',3'',4'',5''), 3.91 (1H, dd, J 11.91 and 1.83 Hz, H-6''a), 3.70 (1H, dd, J 11.91 and 5.5 Hz, H-6''b), 6.59 (1H, s, H-3), 6.78 (1H, d, J 2.29 Hz, H-6), 6.48 (1H, d, J 2.29 Hz, H-8), 7.38 (1H, d, J 1.83 Hz, H-2'), 6.87 (1H, d, J 8.70 Hz, H-5'), 7.40 (1H, dd, J 8.24 and 2.29 Hz, H-6'); 13 C-NMR (100 MHz, CD3OD) δC 61.11 (C-6''), 69.93 (C-4''), 73.39 (C-2''), 76.52 (C-3''), 77.05 (C-5'), 102.61 (C-1''), 165.55 (C-2), 102.61 (C-3), 182.69 (C-4), 161.55 (C-5), 99.79 (C-6), 163.66 (C-7), 94.70 (C-8), 157.50 (C-9), 105.73 (C-10), 122.20 (C-1'), 112.70 (C-2'), 146.02(C-3'), 145.21 (C-4'), 115.53 (C-5'), 119.14 (C-6') (Gohari et al., 2011; Yin et al., 2008; Zhou Hai-Yan et al., 2009).

Results and discussion

Phytochemical profiling and characterization of the isolated constituents

A detailed phytochemical analysis using HPLC-MS was carried out on Z. spina-christi, S. oleraceus and H. sabdariffa in previous work (Da-Costa-Rocha et al., 2014; Ou et al., 2013; Pawlowska et al., 2008; Rodriguez-Medina et al., 2009). From these plants in our present study, preparative HPLC was used to isolate four compounds (1-4) from the methanolic extracts of Z. spina-christi, two compounds (5 and 6) from S. oleraceus and seven compounds (7-13) from H. sabdariffa. The structures of the isolated compounds (Fig. 1) were elucidated by HRMS, tandem MS, UV chromatograms, retention times (RT), authentic standards and data obtained from literature (Fig. 2 and Table 2). Chromatographic resolution and MS data were considered for isolated compounds showing a poor NMR spectral resolution. Tandem MS data and preparative HPLC chromatograms are provided in the supporting information. With these agreements

175 | P a g e Appendix compounds (1-13) were identified as quercetin 3-O-(2,6-di-O-rhamnosyl-glucoside), quercetin 3- O-(6-O-rhamnosyl-glucoside) (rutin), phloretin 3',5' di-C-glucoside, quercetin 3-O-(2-O- rhamnosyl-pentoside), esculin, luteolin 7-O-glucoside, hibiscus acid, quercetin 3-O-glucoside, 3- O-caffeoylquinic acid, 4-O-caffeoylquinic acid, 5-O-caffeoylquinic acid, 5-O-caffeoylshikimic acid and N-feruloyltyramine, respectively. The purity of these compounds (Table 1) was determined as in our previous study (Karar et al., 2014) by total ion chromatograms in negative ion mode and UV chromatograms at 280nm.

OH OH OH OH OH OH 1 1 1 8 8 8 2 2 HO O 2 HO O HO O 7 7 7 3 3 HO 3 O O 5 4 O 5 4 O 5 4 O O OH OH O O OH OH O OH OH O O O O HO O O OH HO OH OH OH OH OH HO HO O O HO OH 1 2 4 HO OH HO OH 1 HO 8 OH OH HO O 2 O 7 OH O 6 OH 3 1 HO O 8 5 4 O O O 2 3' OH HO 7 HO 2' OH O OH 4' 3 1' HO OH 5 4 HO 5' OH 6' 3 5 OH OH O OH OH 6 O OH O HO

HO OH OH O O OH 8 1 HO HO O 2 HO OH 1 1 7 O 5 3 CO2H O 5 3 OH O 3 HO OH 5 4 O OH 4 H HO 4 O CO H OH O OH O 2 O OH OH HO OH O 7 8 OH 9 10 OH OH

O O OH OH HO HO 1 O 1 O O 3 5 N HO 5 3 OH H O OH HO 4 O 4 OH 11 OH OH 12 13 HO OH O

Fig. 1. Chemical structures of compounds 1-13 isolated from Z. spina-christi, S. oleraceus and H. sabdariffa.

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[%] [%] MS 1 755.0 MS 2 608.9 100 100

0 0 299.7 MS2 MS2 100 100 299.7 488.8 0 0 270.5 MS3 150.5 MS3 100 100 178.5 270.6

0 0 200 400 600 800 m/z 200 400 600 m/z

[%] 596.9 [%] 578.9 MS MS 4 100 3 100

0 0 356.8 MS2 299.7 MS2 100 100 476.9 446.8 0 3 0 208.5 MS 270.6 MS3 100 100

150.5 0 0 200 400 600 m/z 200 300 400 500 600 m/z

[%] 339.0 MS [%] 446.8 MS 5 100 176.8 100 6

260.7 0 0 176.8 MS2 284.6 MS2 100 100

0 0 132.9 MS3 174.5 MS3 100 100 240.6 105.1 0 0 100 200 300 m/z 200 300 400 500 m/z

[%] [%] 7 188.5 MS 8 462.9 MS 100 220.5 100 126.6 158.6 0 83.0 0 126.5 MS2 300.6 MS2 100 100

82.9 0 0 82.9 MS3 178.5 MS3 100 100 299.7

0 0 100 150 200 250 m/z 200 300 400 500 m/z

177 | P a g e Appendix

[%] 9 352.8 MS [%] 10 352.8 MS 100 374.8 100 374.7

302.5 0 0 234.6 190.5 MS2 172.5 MS2 100 100

0 0 172.5 MS3 92.8 MS3 100 126.6 100

0 0 100 200 300 400 m/z 100 200 300 400 m/z

[%] [%] 334.7 MS 11 352.8 MS 12 100 100 248.5

238.4 0 0 190.5 MS2 160.5 MS2 134.6 100 100

0 0 126.6 MS3 132.6 MS3 100 172.5 100 84.9 0 0 100 200 300 m/z 100 200 300 m/z

[%] 13 311.8 MS 100

347.8 0 177.5 MS2 100 134.6 296.7 244.4 0 134.6 MS3 100 175.5

0 100 150 200 250 300 350 m/z

Fig. 2. Tandem MS spectra of compounds 1-13 in negative ion mode.

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Table 2. Retention times, high resolution MS data and amounts of the isolated constituents.

No. Compound identity RT Mol. Ther. m/z Exp. m/z Err. Amount Purity Ref./Std. (min) formula [M−H] [M−H] [ppm] [mg/5g] [%] 1. Quercetin 3-O-(2,6-di-O- 15.6 C33H40O20 755.2040 755.2055 -2.0 2.5 100.00 (Ferreres et al., rhamnosyl-glucoside) 2008) 2. Quercetin 3-O-(6-O- 41.0 C27H30O16 609.1461 609.1478 -2.8 25.0 85.62 Std. rhamnosyl-glucoside) (rutin) 3. Phloretin 3',5' di-C-glucoside 36.8 C27H34O15 597.1825 597.1850 -4.2 9.0 55.47 (Pawlowska et al., 2008) 4. Quercetin 3-O-(2-O- 46.2 C26H28O15 579.1355 579.1351 0.8 2.6 74.95 (Pawlowska et al., rhamnosyl-pentoside) 2008) 5. Esculin 15.0 C15H16O9 339.0722 339.0726 -1.3 1.1 100.00 Std.

6. Luteolin 7-O-glucoside 36.6 C21H20O11 447.0933 447.0929 0.8 0.9 100.00 Std.

7. Hibiscus acid 4.0 C6H6O7 189.0041 189.0039 0.9 54.1 100.00 (Fernandez-Arroyo et al., 2011) 8. Quercetin 3-O-glucoside 41.7 C21H20O12 463.0882 463.0877 1.1 2.8 100.00 Std.

9. 3-O-caffeoylquinic acid 13.6 C16H18O9 353.0878 353.0878 0.1 11.1 90.61 Std.

10. 4-O-caffeoylquinic acid 21.2 C16H18O9 353.0878 353.0877 0.3 12.1 100.00 Std.

11. 5-O-caffeoylquinic acid 20.9 C16H18O9 353.0878 353.0881 0.8 6.0 84.72 Std.

12. 5-O-caffeoylshikimic acid 27.0 C16H16O8 335.0772 335.0770 0.8 0.6 79.62 (Jaiswal et al., 2011) 13. N-feruloyltyramine 42.7 C18H19O4 312.1241 312.1241 0.2 0.7 78.42 (Fernandez-Arroyo et al., 2011) Std.: Compounds identified after comparison with their commercial standards

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Evaluation of antibacterial activities

The antibacterial activities of the crude plant extracts and some selected phenolic compounds isolated by preparative HPLC from the plant material analysed in this study were evaluated for their efficacies against Gram-positive and Gram-negative bacteria. As suitable model organisms B. subtilis S168, B. aquimaris MB-2011, and C. michiganensis GSPB 390 were chosen for Gram- positive and E. coli DH5α, E. amylovora 1189, and P. syringae pv tomato DC300 for Gram- negative. The organisms chosen can be viewed as suitable model organisms for both pathogenic Gram-positive and Gram-negative bacteria. Ampicillin and DMSO were used as positive and negative controls, respectively. The MIC values of the plant extracts obtained using the micro- dilution method are presented in Table 3. Interestingly, the plant extracts tested herein showed antibacterial activity only against Gram-positive bacteria, with MIC values varying from 195 to 1562 μg/mL, while Gram-negative strains were not affected at all. The higher sensitivity of the Gram-positive bacteria compared to Gram-negative bacteria could be attributed to their differences in cell envelope components. Gram-positive bacteria have an external peptidoglycan layer, which only is a permeable and thus ineffective barrier against toxic compounds (Baba and MaliK, 2014). Extracts of Acacia nilotica and Punica granatum (pomegranate) peals showed the highest activities against B. aquimaris MB 2011 with a MIC value of 195 μg/mL. The antibacterial activities of these two plants against Streptococcus viridans, S. aureus, E. coli, B. subtilis, Shigella sonnei and Salmonella typhimurium were reported in previous studies (Banso, 2009; Nuamsetti et al., 2012). However, the reference antibiotic as a positive control showed variable inhibitory activity on the all strains of bacteria with MIC values ranging from 3.9 to 250μg/mL (Table 3). No inhibition zone was detected for the negative control (DMSO).

Using agar diffusion assay, we have also investigated the antimicrobial activities of some of the crude extracts and the positive control ampicillin against the Gram-negative bacterium E. coli DH5α and Gram-positive bacterium B. subtilis S168. Surprisingly, some of the plant extracts showed growth inhibitory effects on both tested strains (Fig. 3). Regarding the Gram-positive bacterium, B. aegyptiaca bark extract exhibited the highest activity (4.65 ± 0.07 mm). These findings were in agreement with previous reports (Abdallah et al., 2012; Eldeen and Van Staden, 2007). While, for Gram-negative bacterium, the arial part extract of O. basilicum showed the strongest inhibitory effect (4.23 ± 0.03 mm). The positive control (ampicillin, 50mg/mL) showed zone of inhibition 18.5 ± 0.7 mm against B. subtilis and 16.0 ± 0.6 mm against E. coli. No inhibition zone was detected for the negative control (DMSO).

Phenolic compounds found in medicinal plants have been extensively studied against a wide range of microorganisms, and among them chlorogenic acids, flavanols and tannins received more interest due to their broad spectrum and the fact that most of them process antimicrobial properties (Daglia, 2012; Fiamegos et al., 2011; Su et al., 2014). Consequently, we have evaluated the in vitro antibacterial activities of some phenolic compounds against the selected bacterial strains (Table 4). Generally, the antibacterial activities the pure compounds were found to be comparatively higher than that of crude extracts. Additionally, Gram-positive bacteria were found to be more susceptible to the phenolic compounds than Gram-negative bacteria. Among the tested bioactive compounds, phloretin and resveratrol showed the strongest inhibitory activities against the all Gram-positive bacteria with MICs ranged from 9 to 125 μg/mL (Table 4), followed by luteolin 7- O-glucoside and then epigallocatechin gallate (EGCG) (MICs 62 to 625 μg/mL), whereas the MIC

180 | P a g e Appendix values of chlorogenic acids ranged from 260 to 540 μg/mL, and therefore showed comparably low inhibitory activity. Nevertheless, the antibacterial activity of chlorogenic acids was already documented in previous studies (Almeida et al., 2006; Fiamegos et al., 2011). The hydroxyl groups in the polyphenols are believed to be play an important role in the antimicrobial activity (Lai and Roy, 2004) because these groups can inactivate the microbial enzymes and interact with the cell membrane of bacteria to disrupt membrane structures and causing leakage of cellular components (Di Pasqua et al., 2007; Xue et al., 2013).

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Table 3. MIC of the plant extracts on the studied bacterial species.

Plant extract/ control Part MIC (µg/mL) B. subtilis B. quimaris C. michiganensis E. coli E. amylovora P. syringae pv tomato DC 3000 S168 MB 2011 GSPB 390 DH 5α 1189 H. sabdariffa T > 1000 > 1000 390 > 1000 125000 > 1000 A. nilotica F 1562 195 781 1562 > 1000 781 A. bombacaceae F > 1000 3125 781 > 1000 > 1000 > 1000 Z. spina-christi L > 1000 1562 781 > 1000 > 1000 > 1000 Z. spina-christi F > 1000 781 6250 > 1000 > 1000 > 1000 B. aegyptiaca B > 1000 781 1562 > 1000 > 1000 > 1000 B. aegyptiaca S > 1000 > 1000 > 1000 > 1000 > 1000 > 1000 B. aegyptiaca L > 1000 > 1000 > 1000 > 1000 > 1000 > 1000 S. oleraceus Wp > 1000 > 1000 1562 > 1000 25000 > 1000 A. maritima Wp > 1000 781 781 > 1000 > 1000 > 1000 I. coccinea S 1562 391 781 > 1000 > 1000 > 1000 I. coccinea L 1562 > 1000 781 > 1000 > 1000 > 1000 O. basilicum Ap > 1000 1562 > 1000 > 1000 > 1000 > 1000 M. pigra S > 1000 781 > 1000 > 1000 > 1000 > 1000 M. pigra L > 1000 781 > 1000 > 1000 > 1000 1562 A.senegalensis S 781 1562 390 > 1000 > 1000 > 1000 A. senegalensis L > 1000 > 1000 1562 > 1000 > 1000 > 1000 G.tenax F > 1000 > 1000 > 1000 > 1000 > 1000 > 1000 C. moschata D > 1000 > 1000 > 1000 > 1000 > 1000 > 1000 C. roseus L 781 > 1000 781 > 1000 > 1000 > 1000 B.madagascariensis O > 1000 390 3125 > 1000 > 1000 25000 C. oblonga) F > 1000 1562 > 1000 > 1000 > 1000 > 1000 P. granatum P 1562 195 390 > 1000 > 1000 3125 P. granatum J > 1000 > 1000 390 > 1000 > 1000 > 1000 C. arabica N > 1000 > 1000 390 > 1000 > 1000 > 1000 T. cacao N > 1000 781 390 > 1000 > 1000 > 1000 C. sinensis L > 1000 781 390 3125 > 1000 > 1000 Ampicillin - < 195 < 3.9 < 3.9 15.6 250 62.5 DMSO ------(-): Inactive; L: Leaves; S: Stem; B: Bark; Wp: Whole plant; F: Fruits; P: Peels; D: Seeds; T: Petals; O: Pods; Ap: Aerial part; J: Juice; N: Beans.

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Table 4. MIC of the pure phenolic on the studied bacterial species.

Compound/ control MIC (µg/mL) B. subtilis B. quimaris C. michiganensis E. coli E. amylovora P. syringae pv tomato DC 3000 S168 MB 2011 GSPB 390 DH 5α 1189 3,4-di-O-caffeoylquinic 540 540 540 > 1000 > 1000 > 1000 4,5-di-O-caffeoylquinic 540 540 > 1000 > 1000 > 1000 > 1000 3,5-di-O-caffeoylquinic 540 540 > 1000 > 1000 > 1000 > 1000 3,4,5-tri-O-caffeoylquinic acid 540 540 > 1000 > 1000 > 1000 > 1000 1,3-di-O-caffeoylquinic acid 260 > 1000 > 1000 > 1000 > 1000 > 1000 (cynarin) 5-O-caffeoylquinic acid 5000 > 1000 > 1000 > 1000 > 1000 > 1000 Phloretin 62 62 62 > 1000 > 1000 1000 Ellagic acid > 1000 > 1000 > 1000 > 1000 > 1000 10000 Epigallocatechin gallate (EGCG) 78 78 625 > 1000 > 1000 78 Epicatechin (EC) 2500 1250 5000 10000 > 1000 > 1000 Tannic acid > 1000 > 1000 > 1000 > 1000 > 1000 1250 Caffeic acid 10000 156 1250 > 1000 > 1000 5000 Quinic acid 10000 5000 10000 > 1000 > 1000 10000 Shikimic acid 10000 5000 10000 > 1000 > 1000 10000 Esculetin 6-O-glucoside (esculin) > 1000 > 1000 > 1000 > 1000 > 1000 > 1000 Quercetin > 1000 > 1000 > 1000 > 1000 > 1000 > 1000 Quercetin 3-O-(6-O-rhmanosyl- > 1000 > 1000 > 1000 > 1000 > 1000 > 1000 glucoside) (rutin) Quercetin 3-O-arabinoside 300 > 1000 > 1000 > 1000 > 1000 > 1000 Luteolin 7-O-glucoside 62 > 1000 > 1000 > 1000 > 1000 > 1000 Resveratrol 125 15 9 250 500 > 1000 Apigenin > 1000 > 1000 > 1000 > 1000 > 1000 > 1000 Ampicillin <4 <4 <4 <4 250 31 DMSO ------(-): Inactive

183 | P a g e Appendix

Fig. 3. Inhibition zones as means ± SD generated by different plant extracts against bacterial species tested.

Evaluation of antiplasmodial and antitrypanosomal activities

Table 5 shows the antiplasmodial and antitrypanosomal activities of selected Sudanese medicinal plants against a chloroquine sensitive strain P. falciparum NF54 and T. brucei rhodesiense STI900 (African strain), respectively. For most of these plants, no specific studies of antitrypanosomal and antitrypanosomal activities exist in the literature. Most of the plant extracts exhibited dose dependent antiparasitic activities. These plants (Table 4) are traditionally used for the treatment of many ailments, parasitic and microbial infections including malaria, virus infections, digestive disorders, weakness, hepatic diseases, obesity, diabetes, skin infections, fever, diarrhea, insomnia, heart problems, colds, toothaches, hypertension, bronchial asthma, spasms, frequent urination, urinary tract infections and elimination of kidney stones (Abdelgaleil et al., 2011; AbouZid and Orihara, 2005; El-Kamali and El-Khalifa, 1999; Ghazanfar, 1994; Grosvenor et al., 1995; Han and Park, 1986; Hilmi et al., 2014; Hussein et al., 2000; Kirtikar and Basu, 1984; Rosado-Vallado et al., 2000; Satayavati et al., 1976; Teugwa et al., 2013). This could explain the good observed inhibitory activities of the most of these extracts against the tested parasites. The extracts S. oleraceus and B. aegyptiaca were found to be the most promising ones. The plant extracts showed however weaker antiparasitic activity than that reported for Chrysanthemum cinerariifolium flower extract (86% inhibition against P. falciparum and 99% inhibition against T. brucei

184 | P a g e Appendix rhodesiense at test concentrations of 4.8 μg/mL) (Hata et al., 2011). Nevertheless, our findings were close to that reported by Karou et al.(Karou et al., 2011), which showed significant antimalarial activity for methanolic extract of B. aegyptiaca against P. falciparum (IC50 24.56 μg/mL). I. coccinea, A. senegalensis and Z. spina-christi extracts showed very low or no activities against P. falciparum NF54. On the other hand, T. brucei rhodesiense was sensitive towards the methanolic extracts of S. oleraceus (whole plant) and I. coccinea (stem) (38.4 and 25.5% inhibition activity at 10 µg/mL, respectively), when relatively compared to the reported antitrypanosomal drug suramin (IC50 0.03 ± 0.02 μg/mL) (Al-Musayeib et al., 2012).

Table 5. Antitrypanosomal and antiplasmodial activity of selected crude plant extracts.

Plant extract Part T. brucei rhodesiense STI900 P. falciparum NF54 % Inhibition % Inhibition % Inhibition % Inhibition at 10µg/mL at 2µg/mL at 10µg/mL at 2µg/mL S. oleraceus Wp 38.4 24.4 20.1 7.2 A. maritima wp 9.7 13.6 25.7 11.4 I. coccinea S 25.5 7.7 0.0 4.2 I. coccinea L 18.2 9.6 8.1 6.1 B. aegyptiaca B 25.3 10.4 25.9 3.8 B. aegyptiaca S 26.9 8.1 0.6 0.0 B. aegyptiaca L 18.3 25.0 2.6 0.7 Z. spina-christi L 24.7 15.3 6.6 0.0 O. basilicum Ap 29.1 21.8 7.5 12.4 M. pigra L 12.3 8.4 14.8 10.6 M. pigra S 5.3 1.9 5.2 3.6 A. senegalensis S 9.0 0.2 3.7 3.3 A. senegalensis L 7.5 3.4 0.0 3.0

Evaluation of antioxidant activities

It is believed that the antioxidant activity of plant extracts rich phenolic phytoconstituents is due to their ability to be donors of hydrogen atoms or electrons and to capture the free radicals. Scavenging activity for free radicals of 1.1-diphenyl-2-picrylhydrazyl (DPPH) has been commonly used to assess the antioxidant activity of medicinal plants and natural products. Plant extracts from Sudan were prepared for investigation of their antioxidant activities. They showed significant free radical scavenging activity at the high concentration (100 µg/mL) on DPPH (Fig. 4). The extracts of M. pigra stem, M. pigra leaves, O. basilicam arial part and I. coccinea leaves were the most effective radical scavengers with the inhibition of 33.4 ± 3.3%, 29.1 ± 2.4%, 26.3 ± 1.2% and 26.7 ± 1.4%, respectively, as compared to 83.5± 1.5% for gallic acid standard. Nevertheless, these crude plant extracts showed better DPPH scavenging activity than that reported for Crataegus monogyna fruits extract (15 ± 1% scavenging activity at a test concentration of 100 µg/mL) a flavonoid drug included in most European pharmacopeia (Barreira et al., 2013). At lower concentration (10 µg/mL) the extracts were not effective as the positive standard (Fig. 4), indicating that the activity was concentration dependent. Many studies have demonstrated that the antioxidant activity is significantly affected by the phenolic constituents of the sample (Barreira et

185 | P a g e Appendix al., 2013; Cheung et al., 2003). Thus, the radical-scavenging activity of the plant extracts may be attributed to their phenolic and flavonoid contents. Furthermore, the antioxidant property of M. pigra was in agreement with those mentioned in the literature (Perez-Vizcaino and Duarte, 2010; Rakotomalala et al., 2013).

Fig. 4. Percentage inhibition as means ± SD of free radical scavenging by the plant extracts.

Conclusions

In conclusion, our findings strongly support the traditional use of the studied Sudanese plants in the treatment of bacterial and parasitic infections. The results revealed that some of these plants, such as A. nilotica, O. basilicam, Z. spina-christi, B. aegyptiaca, S. oleraceus, P. granatum, M. pigra and I. coccinea have the potential to be investigated further to identify the antioxidative, antiplasmodial, antitrypanosomal, and antibacterial metabolites in these plants. The study also reports on the antibacterial activities of some naturally occurring phenolic compounds. Among the tested phytochemicals, phloretin, resveratrol, luteolin 7-O-glucoside and epigallocatechin gallate showed the highest antimicrobial activities. By means of preparative HPLC, HPLC-ESI-TOF, HPLC-ESI-MSn, 1H-NMR and 13C-NMR, thirteen phytoconstituents were isolated and identified in the methanolic extracts of Z. spina-christi, S. oleraceus and H. sabdariffa including chlorogenic acids, flavonoid glycosides, coumarins and derivatives. The results of this study highlight the importance of the Sudanese medicinal plants as potential source of plant derived antimicrobial and antiparasitic drugs. However, pharmacological and toxicological studies will be necessary to confirm this hypothesis.

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zizphus_inhibationStatement of contribution

I, Ahmed Rezk, certify that all co-authors have consented to their work being included in the thesis and they have accepted the candidate’s contribution as indicated below. The results of this PhD thesis work are presented in form of three manuscripts in chapter three in addition to two other contributions presented in the appendix.

Contributions: Ahmed Rezk contributed to the design of the study, carried out the experiments described in all figures and participated in manuscript writing. The percentage of the contribution: (90%)

Contributions: Ahmed Rezk contributed to the design of the study, carried out the experiments described in all figures and participated in manuscript writing The percentage of the contribution: (90%)

Contributions: Ahmed Rezk was involved in manuscript writing, carried out the experiments described in figure 3 and discussion and interpretation of the results. The percentage of the contribution: (40%) Mohamed Gamaleldin Elsadig Karar, Laura Quiet, Ahmed Rezk, Rakesh Jaiswal, Maren Rehders, Matthias S. Ullrich, Klaudia Brix, Nikolai Kuhnert, “Phenolic Profile and In Vitro Assessment of Cytotoxicity and Antibacterial Activity of Ziziphus spina-christi Leaf Extracts”, 2015 . Submitted.

Contributions: Ahmed Rezk was involved in manuscript writing, carried out the experiments described in figure 4 and 5. The percentage of the contribution: (30%)

Mohamed Gamaleldin Elsadig Karar, Ahmed Rezk, Omnia Tag Alkhatim Abdalla, Amna Ahmed Yousif Ebrahim, Matthias S. Ullrich, Nikolai Kuhnert, “Antimicrobial, Antiparasitic and Antioxidant Activities of Medicinal Plants from Sudan”, 2015 . Submitted.

Contributions: Ahmed Rezk was involved in interpretation of the results, carried out the experiments described in figure 3 and table 3 and 4. The percentage of the contribution: (40%)

Candidate’s Signature Date

Statutory Declaration (on Authorship of a Dissertation)

I, Ahmed Rezk hereby declare that I have written this PhD thesis independently, unless where clearly stated otherwise. I have used only the sources, the data and the support that I have clearly mentioned. This PhD thesis has not been submitted for conferral of degree elsewhere.

I confirm that no rights of third parties will be infringed by the publication of this thesis.

Bremen, May 25, 2015

Signature ______