Bioactive Compounds from Boerhavia Erecta L
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2010 Bioactive compounds from Boerhavia erecta L. : an African medicinal plant Ari Nugraha University of Wollongong
Recommended Citation Nugraha, Ari, Bioactive compounds from Boerhavia erecta L. : an African medicinal plant, Master of Science - Research thesis, School of Chemistry, University of Wollongong, 2010. http://ro.uow.edu.au/theses/3674
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Bioactive compounds from Boerhavia erecta L.: an African medicinal plant
A thesis submitted in partial fulfilment of the requirements for the award of the degree
Master of Science - Research
from
University of Wollongong
by
Ari Satia Nugraha, SF (GMU), GDipSc (UoW), Apt (GMU)
School of Chemistry
December 2009
ii Certification
I, Ari Satia Nugraha, declare that this thesis, submitted in partial fulfilment of the requirements for the award of Master of Science-Research, in the School of Chemistry, University of Wollongong, is wholly my own work unless otherwise referenced or acknowledged. The document has not been submitted for qualifications at any other academic institution.
Ari Satia Nugraha 17 December 2009
iii Acknowledgement
I would like to give my sincere thanks to my supervisor, A/Prof. Paul A Keller, for guidance and support during the whole course of my study in the School of Chemistry
University of Wollongong. My thanks also go to Prof. William E Price for his generous support in my study. Thanks are extended to Australian Development Scholarships
(AusAID) for the financial support and the approval for MSc by research conversion.
I would like to thanks to our collaborator, Dr. Adama Hilou, for providing plant samples. My utmost thanks go to the following people who provided their help in completing this work: Dr. Wilford Lie for training me to operate the NMR and the never ending NMR discussion; Dr. John Korth for his helps dealing with electron impact mass spectrometry; Dr. Thitima Urathamakul for the assistance to operate ESMS and MS2; Prof. Stephen G Pyne for the idea of acetylated natural products; Dr. Kittiya
Somphol for her help on the HPLC and the friendship; Hayden Matthews for training using prep-HPLC; Sarah Murphy for her assistance in my early time in KRG lab and correcting my thesis; Melinda for correcting my written English; Other former and current KRG members (Rajee (in memoriam), Shane, Steve, Andrew, Mohammed,
Aaron, Nick, Akash, Qing, Ashraf, Phillip, Verena for help and friendship.
Finally, I would like to thanks to my beloved wife, Ira and my son, Abdee, for the support and their patience. The big family in Indonesia for the long distance support.
iv Table of Contents
List of Abbreviations ...... vii Glossary of Botanical Terms ...... viii Abstract ...... ix Chapter 1 Introduction ...... 1 1.1. African Traditional Medicine ...... 2 1.2. Antimicrobial Directed Screening on Tropical African Medicinal Plants ...... 4 1.3. Research on Boerhavia Genus Plants ...... 4 1.3.1. Boerhavia plumbaginea ...... 5 1.3.2. Boerhaavia chinensis ...... 7 1.3.3. Boerhavia repens ...... 7 1.3.4. Boerhaavia diffusa ...... 9 1.3.5. Boerhavia erecta ...... 17 1.4. Aims of The Research ...... 19 Chapter 2 Isolation and Characterization of Sterols and Glycosides from Boerhavia erecta .. 21 2.1. Research Scheme ...... 21 2.2. Isolation and Structure Elucidation of Less Polar Constituents from Boerhavia erecta ...... 23 2.2.1. LPF01 (β-Sitosterol) ...... 24 2.2.2. LPF02 ...... 27 2.2.3. LPF03 and LPF04 ...... 28 2.3. Isolation and Structure Elucidation of Polar Compounds from Boerhavia erecta ...... 29 2.4.1. Isolate PF3.1 ...... 32 2.4.2. Isolate PF3.2 ...... 39 2.4.3. Isolate PF3.2a ...... 42 2.4.4. Isolate PF3.3 ...... 44 Chapter 3 Acetylation of PF3.1 ...... 51 3.1. General ...... 51 3.2. Synthesis ...... 51
v 3.3. Molecular Structure Elucidation ...... 51 Chapter 4 Bioactivity Testing Results ...... 57 4.1. Introduction ...... 57 4.2. Anti-microbial Activity Testing ...... 57 4.3. Bio-testing Against HIV Infected Cells ...... 57 4.4. Reported Anti-microbial Activities ...... 58 4.4.1. Isolate LPF01 (β-sitosterol, steroid type compound) ...... 58 4.4.2. Isolate PF3.1 (~Rutin) and PF3.3 (~Isorhamnetin-3-O-rutinoside), Flavonoids ...... 59 Chapter 5 Conclusion and Future Directions ...... 63 5.1. Conclusion ...... 63 5.2. Future work ...... 64 Chapter 6 Experimental ...... 65 6.1. General ...... 65 6.2. Plant Sample, Extraction and Re-extraction ...... 67 6.2.1. Extraction of Stem Bark of Boerhavia erecta (SBBeL) ...... 67 6.2.2. Extraction of Stem of Boerhavia erecta (SBeL) ...... 67 6.2.3. Re-extraction ...... 67 6.3. Isolation and Purification ...... 68 6.3.1. Isolation and Purification of Sterols from Boerhavia erecta ...... 68 6.3.2. Isolation and Purification of Polar Constituents from Stem Bark of Boerhavia erecta (SBBeL)...... 68 6.3.3. Isolation and Purification Polar Constituents from Stem of Boerhavia erecta (SBeL)...... 69 6.4. Structure Elucidation...... 70 6.4.1. The Less Polar Constituents ...... 70 6.4.2. Polar Constituents from Sub-fraction PF3 of SBeL...... 71 6.5. Acylated PF3.1 ...... 74 6.5.1. Semi-synthetic Procedure ...... 74 References ...... 77 Appendixes ...... 85
vi List of Abbreviations
ACN Acetonitrile APT Attach Proton Test 13C-NMR Carbon Nuclear Magnetic Resonance COSY Correlation Spectroscopy DCM Dichloromethane DEPT Distortionless Enhancement by Polarisation Transfer gHSQC Heteronuclear Single Quantum Coherence HIV Human Immunodeficiency Virus gHMBC Heteronuclear Multiple Bond Correlation 1H-NMR Proton Nuclear Magnetic Resonance HPLC High Performance Liquid Chromatography HRESMS High Resolution Electrospray Mass Spectrometry IHD Index of Hydrogen Deficiency LPF Less Polar Fraction LREIMS Low Resolution Electronimpact Mass Spectrometry LRESMS Low Resolution Electrospray Mass Spectrometry MHz Megahertz MIC Minimum Inhibitory Concentration MRSA Methicillin Resistant Staphylococcus aureus 15N-gHMBC Nitrogen Heteronuclear Multiple Bond Correlation NMR Nuclear Magnetic Resonance NOE Nuclear Overhauser Effect NOESY Nuclear Overhauser Exchange Spectroscopy PF Polar Fraction PTLC Preparative Thin Layer Chromatography RP-HPLC Reverse Phase High Performance Liquid Chromatography SBBeL Stem Bark of Boerhavia erecta Linnaeus SBeL Stem of Boerhavia erecta Linnaeus TFA Trifluoric Acid TLC Thin Layer Chromatography TOCSY Total Correlation Spectroscopy UV Ultraviolet
vii Glossary of Botanical Terms
Achene : A small, dry, indehiscent one-seeded fruit with a thin wall Anthocarp : A false fruit consisting of the true fruit and the base of the perianth Bisexual flower : Flower which possesses both male (pollen-producing) and female (seed-producing) parts Club-shaped : Enlarged gradually at the end Elliptical leaves : Oval, with a short or no point (the blade’s shape) Erect plant : Plant/plant parts growing vertically or at right angles to the parts from which they arise Lanceolate leaves : Long, wider in the middle (the blade’s shape) Obconical fruit : Fruit or similar part shaped like a cone and attached at the pointed end Opposite leaves : Two per node, facing opposite sides of the stem (leaves arrangement) Ovate leaves : Oval, egg-shaped, with a tapering point (the blade’s shape) Pedicel : A small stalk or stalklike part bearing a single flower in an inflorescence (the arrangement of the flowers on the stalks) Perennial plant : A plant lasting for three seasons or more Perianth : The outer envelope of a flower, consisting of either the calyx or the corolla, or both Simple leaves : Show an undivided blade or, in case it has divisions, they do not reach the midrib Stipule : Outgrowths borne on either side of the base of a leafstalk Taproot : The large single root of plants such as the dandelion, which grows vertically downwards and bears smaller lateral roots
viii Abstract
A protocol was developed for the extraction of the traditional African medicinal plant
Boerhavia erecta L. and this resulted in eight compounds being isolated. From the less polar fraction, four constituents were isolated using thin layer chromatography techniques but only one was fully characterised and was elucidated as β-sitosterol. This sterol was previously reported from the same species. The remaining three constituents were only partially identified due to insufficient material but are likely to also be sterols.
From the polar fraction four glycosides were isolated using reverse phase high performance liquid chromatography. Only two of the four isolates were fully characterised and were shown to be 3′,4′,5,7-tetrahydroxyflavone-3-O-α-D- rhamnopyranosyl-(1Æ6)-O-β-D-glucopyranoside and 4′,5,7-tetrahydroxy-3′-methoxy flavone-3-O-α-D-rhamnopyranosyl-(1Æ6)-O-β-D-glucopyranoside. The remaining two constituents were partially elucidated due to limited yield but are likely to also be flavonoids. The one and two dimensional nuclear magnetic resonance spectral analysis were successful in assisting the molecular structure elucidation of these glycosides. The absolute configuration of the sugar was assigned using anomeric proton-carbon coupling constant analysis in which we found α-D-rhamnopyranoside and β-D- glucopyranoside. While the α-L-rhamnopyranoside derivative has been reported previously, the D-form isolated here is new and further, it is the first time these compounds (regardless of stereochemistry) have been found in this genus, Boerhavia.
The bioassay of the isolates against microbes (S. aureus, S. epidermidis, MRSA, Mu50,
E. faecium WT) did not possess significant activities. The anti-viral activity against
HIV-infected cells, revealed 3′,4′,5,7-tetrahydroxyflavone-3-O-α-D-rhamnopyranosyl-
(1Æ6)-O-β-D-glucopyranoside as the most active isolate.
ix
Chapter 1
Introduction
The war against disease has been an inevitability faced by human-kind throughout history. The methodologies by which societies treat diseases from generation to generation culminate into valuable information when searching for novel treatments.
Historically, medicinal plants have supported the health needs of human-kind by exploring and marketing the active constituents of traditional medicinal plants in the early modern pharmaceutical industries.1 Traditional medicinal plants continue to be important and significant leading to specific, potent and safe drug leads. Nearly 50% of new small-compound drugs introduced between 1981 and 2002 were natural products.2
Although in the period of 2001-2004, there was a slight decrease in funds allocated by large pharmaceutical companies to screen medicinal plants, this area is still considered to be a rich source of prospective leads. Evidence for this is based on the knowledge that more than 30% of total pharmaceutical market is based on natural products.2,3
Modern drug discovery can use traditional knowledge to discover new drug leads, often referred to as ethnobotanical and ethnopharmacologial studies. For example, in East
Kalimantan, Indonesia, the indigenous people of Kenyah have relied on Lansium domesticum Correia for malarial fever therapy, with later studies indicating the bioactive constituent to be methyl 15-acetoxylansiolate.4,5 Indeed, the application of modern concepts of drug research to indigenous medicinal plants has generated valuable discoveries with the isolation of active compounds. Moreover, the World Health
Organisation encourages the use of traditional knowledge to accompany the pharmacochemical screening of medicinal plants.6 A continent rich in medicinal plants in which its indigenous communities share their traditional knowledge is Africa.7 1 1.1. African Traditional Medicine
Africa is the second-largest continent and is covered by vegetation types ranging from desert to savanna to rainforest jungle. Together with Madagascar, Africa is reputed to be rich in biodiversity with of several tens of thousands of plant species including 5,000 medicinal plants used by up to 80% of the population.8-11
The long history of human civilization in Africa has recorded traditional medication including the use of medicinal specified plants and some edible plants. To the old indigenous African, a medication was thought as an understanding of the intricate relationship between humans and their environment.12 This traditional medication was then narrowed to a personalistic system in which religion strongly influenced the medication. In this system, healers utilize the human energy, the environment (including medicinal plants) and cosmic balance.13,14 This unique attribute of total African traditional medicine has resulted in some limitation in their exploration including inadequate information to separate the physical properties of the plants from their spiritual attributes. In the early investigation, this condition was exaggerated by investigators, mainly anthropologists, who were more concerned with bizarre habits of primitive tribes than the chore recording the remedies. Furthermore, modern techniques simplified the investigations in finding active compounds in a single plant and disregarded the concepts of mixed herbs medicine and the general relationship between communities and medicinal plants.1,13,15
Research results on African medicinal plants was initially disseminated in an inter-
African symposium in 1968 which reported numerous isolations of natural product constituents without considering the results of assay screening of biological activities.16
The symposium imposed to intensify the efforts to document the ethnomedical data of
2 medicinal plants, since most African herbalists kept no written records and passed the information from generation to generation verbally.17 This resulted in African traditional medicine being less 'developed' compared to Asian countries such as India, China and other South East Asia regions where old manuscripts were used as records.17,18,19
Newer research trends involving African traditional medicine have resulted in several notable works. In 1978, with the support from the World Health Organization, 20,000 plant species were identified and 2000 recipes recorded.7 The first African pharmacopoeia, which contained 100 monographs describing medicinal plants and their uses was published in 1986 and was being used to standardise traditional medicine.20
Further, medicinal plant research now involves several bioassays (see Table 1) with almost 16% of total African natural product publications reporting screening for anti- microbial activity.17 A current theme in exploration also concerns the conservation of medicinal plants due to a high degree of loss of biodiversity, climate change and population growth.17
Table 1. Publications of African medicinal plant research involving biological testing from 1987 to 1991.17
Types of biological Percent of total African activities papers Anti-microbial 16 Molluscicidal 11 Anti-malarial 7 Toxicology 7 Anti-tumour related 4 Others 54
In the 1980s, a sequel to the first African Pharmacopoeia was published providing tropical West African medicinal plants of which 1062 species were used as anti- infective agents, 106 species were used in nervous system treatments and 83 species were used in cardiovascular therapy.21-23
3 1.2. Antimicrobial Directed Screening on Tropical African Medicinal Plants
The humid climate of the tropical regions of Africa provides an excellent environment for microbial growth including pathogenic microbes. To the indigenous people of tropical Africa, many infectious diseases caused by these pathogens have been treated traditionally using particular plants.24 From 1987 to 2007, research on tropical West
African medicinal plants used a range of bioassays to test 109 plant species of which 48 species showed anti-plasmodial activity.25
Located in tropical Africa, Burkina Faso is a country rich with medicinal plants including prospective medicine sources of the Boerhavia erecta Linnaeus species which was investigated in this research. Past research on this plant revealed many activities including antimicrobial and anti-malarial activities (see section 1.3.1.5). Interestingly, the previous collaborative project between University of Wollongong, Australia and
Université de Ouagadougou, Burkina Faso (Africa), revealed significant activity against
HIV infected cells from a polar fraction of the crude methanol extract of the stem bark and the whole plants of B. erecta.26 Based on this, collaborative research continues in an intensive pharmacochemical screening to pursue the bioactive compound which is responsible for activity against HIV infected cells and/or other activities. Given this interesting range of possible bio-activities, it is therefore worthwhile to examine what is known about related species and their use in medicinal chemistry.
1.3. Research on Boerhavia Genus Plants
The Nyctaginacea, commonly called the four o’clock plant family, is occupied by at least 33 genus consisting of 290 species of flowering plants. Though many traditional medicines are prepared from these families, there are a only few chemical and
4 pharmacological studies on the constituents of these plants including the Bougainvillea,
Mirabilis and Boerhavia genus.27 The latter genus is endemic to tropical regions which are claimed to be the old world tropics.28 The taxonomy hierarchy of this genus is described in Figure 1.
Kingdom : Plantae (Plants) Subkingdom : Tracheobionta (vascular plants) Division : Magnoliophyta (angiosperms, flowering plants) Class : Magnoliopsida (dicotyledons) Subclass : Caryophyllidae Order : Caryophyllales Family : Nyctaginaceae (four o’clocks) Genus : Boerhavia Figure 1. Taxonomic classification of genus Boerhavia.27
Apart from the taxonomical ambiguity, this genus is occupied by many species including some commonly used as traditional medicine, including Boerhavia plumbaginea Cavanilles, Boerhavia chinensis Linnaeus, Boerhavia repens Linnaeus,
Boerhavia diffusa Linnaeus and B. erecta. Therefore, modern pharmacochemical research is needed to reveal the bioactivity claimed by traditional knowledge. The botanical, ethnomedical information and recent research results arising from investigations of these Boerhavia plants are described further in this section.
1.3.1. Boerhavia plumbaginea
This species is grown extensively from Southern Spain, Africa to Saudi Arabia and is also known as Commicarpus plumbagineus. It is a shrub with a branched, scandent stem up to 4-10 m tall. It has opposite, simple, stipules absent leaves with 1.5-12 cm × 0.5-8 cm blade ovate dimension. The flower is bisexual, regular with a one-celled ovary. The fruit is an achene enclosed by an anthocarp (see Figure 2 and Figure 3).29
5
1 2
3 1 2 3
Figure 2. Typical fruits of genus Boerhavia. 1: Achene, 2: Anthocarp, 3: Perianth.30
1
Figure 3. The B. plumbaginea showing the achene (1).29
In indigenous populations, different sections of the plant have been and are still used for different treatments.29 The decocted root is used to treat gonorrhoea and back pain
(Namibia) and Guinea worm sores (West Africa). The crushed roots are used to treat yaws (Gana) and leprosy (Nigeria). The boiled leaves are used to treat ulcers, Guinea worm (West Africa), jaundice (Ethiopia) and wounds (Kenya). The crushed leaves are used in swollen glands therapy (Kenya). The decocted leaves are applied to cattle as a laxative agent (DR Congo) and a decoction of the whole plant is used as a laxative agent
6 (Madagascar), as a veterinary medicine for skin disease (Ethiopia) and as an insecticide
(Kenya).
1.3.2. Boerhaavia chinensis
Boerhavia chinensis is distributed throughout the Old World tropics, Malaysia and some regions of Indonesia, eastern Java, Madura, Kangean, Lesser Sunda and the
Moluccas Islands which have a distinctly seasonal climate and altitude of up to 700 m.
The plant has similar characteristics to Boerhavia repanda Willdenov, Boerhavia helenae Schultes, and B. chinensis. It is an erect to climbing herb from 1-4 m in height, and is figured by lanceolate to ovate leaves with 2.5-4.5 cm x 1.5-4 cm dimension. In addition, the flower is bisexual.31
This plant is traditionally used by the Maduranese on Madura Island (Indonesia) to treat scabies and skin itching by applying the crushed leaves to the affected areas.
Phytochemical and pharmacological screening has not been performed on this species.31
1.3.3. Boerhavia repens
Vernacularly called creeping spiderling (see Figure 4), B. repens grows in regions with a distinct dry season including the Mediterranean countries and Africa. This annual creeping herb has a flushed red stem up to 60 cm long with a slender taproot. The leaves are opposite, simple and unequal with a 1-2.5 cm × 0.5-1.5 cm blade broadly ovate to elliptical dimension. The flowers are bisexual and possess a one-celled ovary. The fruit is an achene enclosed by obovoid anthocarp (see Figure 2). The seed is ovoid, pale brown with epigeal germination seedling.
7
Figure 4. The plant flower of B. repens (left) and showing the plant is creeping (right).32
In India, this plant is traditionally used as a diuretic, but also as a stomachic, cardiotonic, hepatoprotective, laxative, anti-helminthic, febrifuge and expectorant agent, while in higher doses it is used as an emetic and purgative agent. The roots are used to treat asthma, leprosy, syphilis, ulcers resulting from Guinea worm infection, chicken pox (West Africa), stomach-ache, and filarial infection (Central Africa). An infusion of the whole plant is used as anti-convulsive, amenorrhoic, laxative and febrifuge agent while the leaves are used to treat jaundice (Nigeria).33
1.3.3.1. Current Research on Boerhavia repens
Although exploration in this species has not been extensively conducted, this plant is considered to have a chemical and pharmacological similarity to the B. diffusa. In 1923, simple analytical studies reported that quinolzilidine alkaloidal constituents, punarnavines,* occurred in all parts of the plant although the molecular structure has still not yet been established (see section 1.3.4.1.).34,35 Myristic acid was also isolated from this species36 as were a number of rotenoids (Figure 5)37 and sterols (Figure 6).38
* Exact structure not reported in reference 8 OAc O HO O R=H, repenone (1) HO R=OH, repenol (2) OH O R
Figure 5. Rotenoids isolated from B. repens.38
R
R=e, 24-ethylcholest-3β-ol (3)
HO R=a, cholest-5-en-3β-ol (4) R=b, 24-methylcholest-5-en-3β-ol (campesterol) (5) R=c, 24-ethylcholest-5-22(E)-dien-3β-ol (stigmasterol) (6) R=d, 24-ethylcholesta-5-en-3β-ol (citosterol) (7) R=e, 24-(Z)-ethylidencholest-5-en-3β-ol (8) R=f, 24-ethylcholest-5-23(E)-dien-3β-ol (9) R=g, 24-ethylcholest-5,24-dien-3β-ol (10)
R=d, 24-ethylcholesta-7-en-3β-ol (11) R=e, 24-(Z)-ethylidencholest-7-en-3β-ol (12)
a b c d
e f g
Figure 6. Steroid compounds existed in Boerhavia species including B. repens, B. diffusa, B. erecta.37
1.3.4. Boerhaavia diffusa
This species grows throughout the tropical regions of the African and Indian continents.
Taxonomically, B. diffusa is similar to Boerhavia coccinea Miller, Boerhavia paniculata Richard, and Boerhavia adscendens Willdenov. It is an erect to creeping annual to perennial herb up to 1 m height with a thick taproot (see Figure 7) as distinguished from other species. Its green-red flushed stem is ascending to erect when 9 flowering. The leaves are opposite, simple, with unequal blades and broadly ovate to elliptical in shape, with 1.5-6 cm × 0.5-5 cm in dimension. It has bisexual, regular flowers with pedicel up to 1 mm long. The fruit is an achene (see Figure 2) enclosed by an obconical or club-shaped anthocarp. The seed is obovoid and pale brown.29,39,40
Figure 7. The whole plant of B. diffusa 1: flowering and fruiting stem; 2: taproot (left). The plant grows as creeping plant (right).29,41
Several medicinal uses have been reported in India, especially including its roots, which are recorded in the Indian Pharmacopeia, 'Punarnava'. The plants are traditionally used as cardiotonic, hepatoprotective, laxative, diuretic, and anti-helminthic† agents.
Moreover, it is also applied for febrifuge,‡ as an expectorant and emetic agent. A decoction of the roots is used to treat corneal ulcers and night blindness (South-East
Asia), hypertension via diuretics (Malaysia), abscesses and Guinea worm (tropical
Africa), anaemia, heart troubles, palpitations and jaundice (Angola and Ghana), spleen troubles, diarrhoea, dysentery, haematuria and gonorrhoea (Congo), gastroenteritic
† Anti-parasitic worms ‡ Anti-pyretic 10 problems and a prolapsed uterus (Bergdamara people, Namibia). The root sap is used as snakebite antidote, an aphrodisiac and used in mumps, laryngitis and burns treatment
(DR Congo). The powdered leaves are applied to the chest to relieve asthma, and to the forehead to relieve headache (Côte d’Ivoire). The decocted leaves are used in gonorrhoea and to calm pain treatment (DR Congo), and to induce sterility in women
(Papua New Guinea). People in Mauritania use the seeds in specific cakes for dysentery medication.29,39
1.3.4.1. Current Research on Boerhavia diffusa
The various uses of B. diffusa has led to further studies investigating these reported bioactivities. A non-toxic water extract of the leaves possessed anti-nicociceptive42,43 and anti-diabetic activity, which was more effective than glibenclamide 13 (Figure
8).44,45 The aqueous extract of the roots contained anti-viral proteins46,47, and therefore an ethanolic extract of the roots can enhance an immune response.48-51 A methanolic extract of the roots was reported to have protective activity against damage caused by gamma radiation.52 Chemical screening of this species resulted in the isolation of alkaloids, flavonoids, rotenoids, lignans, sterols (see Figure 6), glycosides and naturally occuring purine nucleosides.53
O O O S OMe O N N H H N H
Cl Glibenclamide (13)
Figure 8. Glibenclamide, a sulfonylurea anti-diabetic agent which is commonly used in oral 45 dosage form for non-insulin dependent diabetes mellitus (Type II diabetes) therapy.
11 Alkaloids from Boerhavia diffusa
The chemical studies of the alkaloidal constituents of the roots of B. diffusa involved firstly isolating and identifying the alkaloids punarnavine I and II with the molecular
54-57 formula C17H22N2O. Although the molecular structure of these alkaloids has not been established, punarnavine was proposed to have a quinolizidine group.35
Flavonoids from Boerhavia diffusa
Phytochemical screening on the leaves of B. diffusa isolated two flavonoids, eupalitin-
3-O-β-D-galactopyranoside 16, and eupalitin 17.58 Further studies on the roots of the plants resulted in isolation of six other flavonoids, eupalitin-3-O-β-D-galactopyranosyl-
(1Æ2)-β-D-glucopyranoside 14, eupalitin-3-O-β-D-galactopyranosyl-(1Æ2)-β-D- galactopyranoside 15, 6-meth oxykaempferol-3-O-β-D-(1Æ6)-robinoside 18, 3,3',5- trihydroxy-7-methoxyflavone 19, 4',7-dihydroxy-3'-methoxyflavone 20 and 2'-O- methylbronisoflavone 21.59-61
The bioactivity studies revealed that compound 16 could be developed as an immunosuppressive agent.58 The other flavonoids 14 and 18 exhibited a significant inhibitory effect against bone resorption induced by parathyroid hormone.60 Compound
21 was found to have anti-spasmolytic activity against contractions induced by acetylcholine.61
12
R1=β-D-gal-β-D-glc, R2=CH3 (eupalitin-3-O-β-D-galactopyranosyl-(1Æ2)-β-D-glucopyranoside) (14) R1=β-D-gal-β-D-gal, R2=CH3 (eupalitin-3-O-β-D-galactopyranosyl-(1Æ2)-β-D-galactopyranoside) (15) R1=β-D-gal, R2=CH3 (eupalitin-3-O-β-D-galactopyranoside) (16) R1=H, R2=CH3 (eupalitin) (17) R1=β-D-(1-6)-robinobiose, R2=H (6-methoxykaempferol 3-O-β-D-(1Æ6)-robinoside) (18)
OH CH3 OH
H3CO O H3CO O
OH OH OH O O 3,3',5-trihydroxy-7-methoxyflavone (19) 4′,7-dihydroxy-3'-methoxyflavone (20)
2'-O-methylbronisoflavone (21)
Figure 9. Flavonoid compounds isolated from the whole plant of B. diffusa.58-61
Isoflavonoids (Rotenoids) from Boerhavia diffusa
Rotenoids are commonly found in Fabaceae and Leguminase plants as isoflavonoid derivatives.62 Compared to the flavonoids found in this species, which are present mostly in the leaves, these isoflavonoids are isolated from the roots. The B. diffusa is similar to Boerhavia coccinea Miller, and therefore some of the isoflavonoids are named as coccineone instead of boeravinone (see Figure 10).
Studies were conducted on these rotenoids including cytotoxicity, breast cancer resistant protein inhibition62 and intestinal motility activities.61 Boeravinone A-C 22-24, E 27, G-
J 29-33, coccineone B 35 and E 39 showed no cytotoxicty against several cancer cell
13 lines. Boeravinone G 29 and H 30 strongly inhibited the breast cancer resistant protein
(BCRP) drug-efflux activity (Table 2).
R1=CH3, R2=H, boeravinone A (22) R=CH3, boeravinone C (24) R1=R2=H, boeravinone B (23) R=H, 10-demethylboeravinone C (25)
R=OCH3, boeravinone D (26) R=OH, boeravinone E (27) R=H, boeravinone G (29) R=O, boeravinone F (28) R=CH3, boeravinone H (30)
6-O-demethylboeravinone H (31) boeravinone I (32)
boeravinone J (33) coccineone A (34)
coccineone B (35) 9-O-methyl-10-hydroxycoccineone B (36)
O H H3CO O H CO 3 OH OH O
R=CH3, coccineone C (37) coccineone E (39) R=H, coccineone D (38)
Figure 10. Isoflavonoid compounds isolated from B. diffusa.62-70
14 Table 2. The inhibition activity of rotenoids against breast cancer resistant protein (BCRP).62
Concentration % maximal mitoxantrone Rotenoids (μM) accumulation Boeravinone A (22) 10 27 ± 5.1 Boeravinone B (23) 10 55 ± 5.8 Boeravinone C (24) 10 31 ± 4.2 Boeravinone E (27) 10 56 ± 5.0 Boeravinone G (29) 5 92 ± 6.5 Boeravinone H (30) 5 68 ± 6.1 Boeravinone I (32) 20 12 ± 5.4 Boeravinone J (33) 20 15 ± 3.1 Coccineone B (35) 10 29 ± 5.3 Coccineone E (39) 10 15 ± 5.2 Note: % maximal mitoxantrone accumulation indicates efficiencies of the rotenoids to inhibit BCRP-mediated mitoxantrone efflux leading to the drug accumulation.62
The roots of this species have been used traditionally to treat gastroenteric problems which was confirmed by pharmacological studies on the rotenoids constituents isolated from the roots.61,71 These studies showed compounds 25, 28, 29, 36 and 39 inhibit acetylcholine-induced contractions (Table 3).
Table 3. The inhibition activity of rotenoids against acetylcholine induced contractions.61,71
Rotenoids Emax Boeravinone C (24) inactive 10-Demethylboearavinone C (25) 19.4 ± 1.3 Boeravinone D (26) inactive Boeravinone E (27) 100 Boeravinone F (28) 50.0 ± 8.2 Boeravinone G (29) 60.0 ± 6.9 Boeravinone H (30) inactive 6-O-Demethylboeravinone H (31) 100 Coccineone B (35) inactive 9-O-Methyl-10-hydroxycoccineone B (36) 36.1 ± 3.3 Coccineone E (39) 25.2 ± 2.0 The study was conducted at 30 μg/mL. Emax indicates the percentage of maximum inhibition
Lignans from Boerhavia diffusa
A study on the constituents of the roots of B. diffusa resulted in the isolation of two known lignans, liriodendrin 40 and syringaresinol mono-β-D-glucoside 41 with the
15 former acting as an antagonist of the Ca2+ channel.72 This lignan is proposed to be the first water soluble lignan showing selective blocking of the Ca2+ channel.
OMe O H MeO OR H H 2 R O 1 OMe H O MeO R1=R2=β-D-glc, liriodendrin (40) R1= β-D-glc,R2=H, syringaresinol mono-β-D-glucoside (41)
Figure 11. Liriodendrin (40) and syringaresinol mono-β-D-glucoside (41) isolated from the roots of B. 72 diffusa.
Glycosides from Boerhavia diffusa
Three glycosides have been isolated from the roots of B. diffusa, 3,4-dimethoxy-1-O-β-
D-glucopyranoside 43, 3,4-dimethoxyphenyl-1-O-β-D-apiofuranosyl-(1′′Æ3′)-O-β-D- glucopyranoside 4359 and 2-glucopyrano-4-hydroxy-5-(p-hydroxyphenyl)propionyl diphenylmethane 44 although only the last compound was tested and found to be active as antifibrinolitic agent.73
3,4-dimethoxyphenyl-1-O-β-D-apiofuranosyl- 3,4-dimethoxy-1-O-β-D-glucopyranosde (42) (1′′Æ3′)- O-β-D-glucopyranoside (43)
OH O HO O O O HO OH HO
OH
2-glucopyrano-4-hydroxy-5-(p-hydroxyphenyl)propionyldiphenylmethane (44)
Figure 12. Phenol glycosides isolated from the roots of B. diffusa.59,73
16 A Naturally occurring purine nucleoside isolated from Boerhavia diffusa
Based on ethnobotanical studies, the roots of B. diffusa, have been traditionally used as a cardiotonic agent.74 Examination of the roots of the plant resulted in the isolation of a purine nucleoside hypoxanthine-9-L-arabinofuranoside 45 which possessed a direct vasodilatation effect on the isolated coronary arteries. The mechanism was not reported to have a correlation with the stimulation of coronary vascular adenosine receptors.
Figure 13. A purine nucleoside hypoxanthine-9-L-arabinofuranoside (45) from the roots of B. diffusa.74,75
1.3.5. Boerhavia erecta
Boerhavia erecta, commonly known as the erect spiderling, may originate from the New
World and grows throughout tropical Africa (from West Africa to Somalia) to Asia
(Singapore, Sumatra, Java, Lesser Sunda and New Guinea Islands). The plant is an erect annual to perennial herb up to 1 m in height. It is branched ascending from an erect stem with a thick taproot. The leaves are opposite, simple, about equal, with absent stipules and a 2.5-8.0 cm × 1.5-6.5 cm blade to ovate dimension (see Figure 14). The flowers are bisexual, regular with a one-celled superior ovary. The fruit is an achene (see Figure
2) enclosed by the obconical or club-shaped anthocarp. Around 25,000 obovoid, pale brown seeds are produced annually by a mature B. erecta. A distinct difference among the other species is the mucous coat of the anthocarp which swells when ripe.29,76
The roots of this plant have been traditionally used in India as a diuretic, but also as a stomachic, cardiotonic, hepatoprotective, laxative, anti-helminthic, febrifuge and
17 expectorant agent. The roots are also prepared to treat jaundice, enlarged spleen, gonorrhoea and other internal inflammations (India), asthma, and the stump of a newly severed umbilical cord (Sudan). The crushed leaves are used for treating diarrhoea
(Kenya), rheumatism and scabies (Tanzania) and conjunctivitis (Benin). The whole plant is commonly used to treat gastrointestinal, liver and infertility problems (Mali), fungal infection on skin (Nigeria) and convulsion in children (Benin).29,76
1
2
29,77 Figure 14. A typical flower (1) and leaves (2) of B. erecta which the plant stands above the ground.
1.3.5.1. Current research on Boerhavia erecta
Bioactivity screening on the relatively non-toxic extract of B. erecta78 revealed some activities, including anti-microbial79, anti-malarial effects80-82 and muscle contractility inhibition.83 A chemical screening narrowed on steroidal constituents on Boerhavia plants including B. erecta indicated the presence of some sterol compounds 4-12
(Figure 6).37 A further pharmacochemical screening isolated betanin 46, which was proposed to be responsible for the anti-malarial activity.80-82 Recently research in our laboratory on the extracts of the stem bark and whole plant of B. erecta revealed significant activity against HIV infected cells in which the polar fraction was
18 responsible for the activity.26 Based on these studies (see Table 4), an intensive pharmacochemical screening is worth conducting in order to find the bioactive constituents of B. erecta.
Table 4. The biological activities of what extracts of B. erecta.26,79,80,83
Biological activity Value Detail Anti-microbial 24 mm/40 mg Inhibition diameter/weight againts Bacillus cereus
Anti-malarial 564.95 ± 6.23 ED50 (mg/kg) against Plasmodium berghei Muscle contractility inhibition 10% - 35% % inhibition at dosage 71.5 μg/mL 26 Against HIV infected cells < 1.6 IC50 (μg/mL), stem bark extract
HOH C 2 O O H O HO OH HO N O HO
H O O N H OH OH
Figure 15. Molecular structure of betanin 46 isolated from B. erecta.82
1.4. Aims of The Research
As mentioned, the modern preliminary screenings on B. erecta have revealed several biological activities including anti-bacterial, anti-malarial and against HIV infected cells. The elucidation of structures of the active constituents forms the major component of this project. Specially, they are:
• To establish a protocol for separation of constituents in B. erecta.
• To investigate the chemical constituents of the plants by separation, isolation
and structure elucidation.
19 • To evaluate the anti-bacterial and anti-HIV activity of the extracts, fractions,
sub-fractions.
• To identify the constituents responsible for any observed bioactivity.
20 Chapter 2
Isolation and Characterization of Sterols and Glycosides from Boerhavia erecta
2.1. Research Scheme
The plant samples used in this research were stem bark and the stem of B. erecta which were obtained as dried and powdered plant material, respectively. These samples were supplied by our collaborator, Dr Adama Hilou from Université de Ouagadougou,
Burkina Faso, Africa. The extraction protocol applied to the samples is outlined in
Figure 16. The initial samples were extracted with MeOH followed a single extraction using EtOAc:DCM:CH3COOH (4.75:4.75:0.5) to produce the less polar fraction (LPF) and the more polar fraction (PF), respectively. A further fractionation was performed on the polar fraction to produce four sub-fractions (PF1, PF2, PF3 and PF4). This procedure was slightly different to a standard fractionation using sequential extraction using solvents from non-polar to polar (hexane, DCM, EtOAc, MeOH and water)84 as we were specifically targeting the polar fractions.
The separation and isolation was conducted using both the less polar fraction and polar fraction by employing chromatographic techniques in which the LPF mainly relied on preparative thin layer chromatography (PTLC) techniques and the polar fraction used preparative reverse phase high performance liquid chromatography (RP-HPLC). On the basis of TLC analysis, which is described further in section 2.3, the isolation of PF focussed only on the sub-fraction PF3 as this sub-fraction has the largest mass.
21
Figure 16. This research protocol was started from extraction, re-extraction, and chromatographic separation and isolation. Preparative TLC and preparative HPLC were the key step to obtain the pure isolates. Biological testing was performed at the 3 stages indicated.
By using PTLC with DCM:MeOH (9.5:0.5), four sterol type compounds (LPF01,
LPF02, LPF03 and LPF04) were collected from the LPF. The elucidation of the structures of these compounds is discussed in section 2.2. RP-HPLC was not able to isolate the different compounds from PF3 of stem bark of B. erecta (SBBeL), and therefore further exploration was redirected on PF3 of the stem of B. erecta (SBeL) which showed identical constituents based on TLC, HPLC and ESMS analysis. The detail of the separation method and its development is described on section 2.3. Overall,
22 preparative HPLC was able to isolate four compounds from sub-fraction PF3 labelled as PF3.1, PF3.2, PF3.2a and PF3.3.
To accompany the chemical screening, biological assays were performed at three different stages. These included anti-viral (initially against HIV-integrase) and anti- microbial activities (against Staphylococcus aureus, Staphylococcus epidermidis,
Methicilin resistant Staphylococcus aureus, Mu50, Escherichia faecium WT). The result of this testing process are discussed further in chapter 4.
2.2. Isolation and Structure Elucidation of Less Polar Constituents from Boerhavia erecta
The target compounds to separate from the LPF were defined by analytical TLC to give four compounds in reasonable purity (LPF01, LPF02, LPF03 and LPF04, see Figure
17).
LPF01
LPF02 LPF03 LPF04
Figure 17. TLC profile of extract of stem bark of B. erecta developed with DCM:MeOH (9.5:0.5) and stained with molybdate. The targets were classified LPF01, LPF02, LPF03 and LPF04. The other spots (marked in dotted boxes) were excluded from being targets due to impurities (shown as two overlapping spots).
Following the extraction protocol in Figure 16, 94.0 g of stem bark of B. erecta
(SBBeL) produced an 11.7 g extract which was then re-extracted to give 2.1 g of a
LPF. To separate and isolate the targets, routine silica gel column chromatography was
23 performed on the LPF and followed by PTLC using DCM:MeOH (9.5:0.5) to give isolates LPF01 (2.6 mg), LPF02 (1.3 mg), LPF03 (<1 mg) and LPF04 (< 1mg).
2.2.1. LPF01 (β-Sitosterol)
This sterol appeared as non-UV active creamy pale yellow material (2.6 mg, 0.17 mg/g dried plant weight). The PTLC was able to fully purify the β-sitosterol 48 (Figure 18).
Figure 18. Molecular structure of β-sitosterol (48)
The low resolution electron impact mass spectrometry (LREIMS) showed a peak at m/z
414 assigned to the molecular ion (M)+.. Additional ion fragments were observed at 396,
329, 315, 303, 273, 255, 231, 213, 57 and 43 (100 %). Mass spectral database searching
(Figure 19) suggested the structure of β-sitosterol.
Relative abundance
m/z
Figure 19. LREI MS library matching of β-sitosterol; experiment (above) and database (below).
24 The high resolution electron impact mass spectrometry (HREIMS) gave exact molecular weight and calculated molecular weight of 414.3846 and 414.3862, respectively. This corresponded to the molecular formula C29H50O and generated an index of hydrogen deficiency (IHD) of 5.
To confirm the structure of β-sitosterol, 1D and 2D NMR experiments were conducted and the results are summarized in Table 5.
Table 5. 1H-NMR and 13C-NMR spectral comparison of isolate LPF01 and β-sitosterol (aglycon of 3-O- glucosyl-β-sitosterol) from literature85
Carbon Carbon δC δH, J No. type Exp. Lit Exp. Lit. 1 CH2 37.2 37.2 1.01, 1.78 (m) 1.00, 1.86 (m) 2 CH2 29.7 29.5 1.86, 1.32 (m) 1.86, 1.32 (m) 3 CH 71.8 80.1* 3.46 (m) 3.46 (dd, 3J = 5.2, 10.9 Hz) 4 CH2 42.3 39.0 2.16, 2.24 (m) 2.20, 2.15 (m) 5 C 140.7 140.5 6 CH 121.7 122.2 5.28 (d, 3J = 7.0) 5.33 (d, 3J = 5.3) 7 CH2 31.9 32.0 1.44, 1.91 (m) 1.55, 1.94 (m) 8 CH 31.7 31.9 1.44 (m) 1.43 (m) 9 CH 50.1 50.2 0.86 (d, 3J = 6.5 Hz) 0.89 (dd, 3J = 3.0, 6.7 Hz) 10 C 36.5 36.8 11 CH2 21.1 21.1 1.45, 1.38 (m ) 1.45, 1.38 (m) 12 CH2 39.8 39.8 1.10, 1.94 (m) 1.19, 2.03 (m) 13 C 42.3 42.4 14 CH 56.8 56.8 0.93 (m) 0.97 (m) 15 CH2 24.3 24.3 1.01, 1.52 (m) 1.21, 1.59 (m) 16 CH2 28.2 28.2 1.20, 1.78 (m) 1.12, 1.12 (m) 17 CH 56.1 56.1 1.03 (m) 1.06 (m) 18 CH3 12.0 11.9 0.62 (s) 0.65 (s) 19 CH3 19.0 19.4 0.76 (s) 1.00 (s) 20 CH 36.1 36.1 1.30 (m) 1.36 (m) 3 3 21 CH3 18.8 18.8 0.85 (d, J = 8.5) 0.93 (d, J = 6.5) 22 CH2 33.9 34.0 0.96, 1.42 (m) 1.51, 1.38 (m) 23 CH2 26.1 26.2 1.10, 0.74 (m) 1.16, 0.74 (m) 24 CH 45.8 45.9 0.86 (m) 0.96 (m) 25 CH 29.2 29.2 1.60 (m) 1.65 (m) 3 3 26 CH3 19.0 19.1 0.74 (d, J = 6.5) 0.86 (d, J = 6.5) 3 3 27 CH3 19.8 19.8 0.77(d, J = 6.5) 0.81 (d, J = 6.5) 28 CH2 23.1 23.1 0.80, 1.21 (m) 1.23, 1.23 (m) 3 3 29 CH3 11.9 12.0 0.79 (t, J = 9.0) 0.85 (t, J = 7.0) 1 13 Note: H- and C -NMR experiment were performed in CDCl3 at 500 MHz and 125 MHz, respectively. 1H- and C13-NMR data for β-sitosterol (aglycon of 3-O-glucosyl-β-sitosterol) from literature were 85 obtained in CDCl3 at 300 Mhz and 75 MHz, respectively. *The downfield shift due to glycosidic residue effect on C3.
25 The 1H-NMR spectrum displayed peaks typical of sterols. For example, the peak at
3.46 ppm was ascribed as the oxymethine proton H3 (δ 3.46, m, 1H). Another key proton was the alkenic proton H6 which resonance at δ 5.28 (1H, d, 3J = 7.0 Hz). These key protons showed a COSY correlation (three bond length proton-proton correlation) which indicated coupling between H6 (δ 5.28, d, 1H, J = 7.0 Hz) and H7 (δ 1.44 and δ
1.91, m). Other correlations include H3 (δ 3.46, m, 1H) to H4 (δ 2.16, 2.24, m, 2H) (see
Figure 20, left).
s Figure 20. Examples of proton-proton correlation in gCOSY spectrum (left) and in TOCSY spectrum (right) of the molecular structure of isolate LPF01
From the TOCSY spectrum, the proton-proton connections within each of the sterol were clearly shown. For example, the proton H1 (δ 1.01, δ1.78, m, 2H) correlated with,
H2 (δ 1.20, δ1.60, m, 2H), H3 (δ 3.46, m, 1H) and H4 (δ 2.24, m, 1H) in ring A. In ring
B, H6 (δ 5.28, d, J = 7.0 Hz, 1H) had through bond connectivity with H7 (δ 1.44, δ
1.91, m, 2H) and H8 (δ 1.44, m, 1H) (see Figure 20, right).
The presence of two sp2 carbon was confirmed by peaks at δ 140.7 (C5) and δ 121.7
(C6). The C3 carbon attached to hetero atom was also detected at δ 71.8. From the gHMBC spectrum, the extensive proton-carbon correlations are illustrated in the Figure
21.
26 Figure 21. The proton-carbon correlations of the molecular structure of isolate LPF01
To finalize the structure elucidation, the chemical shift from the 1H- and 13C -NMR spectra were compared to published data (see Table 4). Although the stereochemistry could not be confirmed, the comparison showed only insignificant differences, and gives support for the assignment of the molecular structure of LPF01 as β-sitosterol.
The only discrepancy was shown at C3 where literature reports a slightly higher chemical shift of 8.3 ppm relative to experimental 71.8 ppm.85 This is likely due to the presence of a sugar unit present to form glycosidic linkage at C3 which changed the carbon’s chemical environment as the reported structure has a glycosidic present.
2.2.2. LPF02
The isolate LPF02 appeared as non-UV active creamy pale yellow material (1.3 mg,
0.02 mg/g dried plant weight). A routine PTLC procedure produced stigmasta-3,5-dien-
7-one 49 (Figure 22).
Figure 22. Molecular structure of stigmasta-3,5-dien-7-one (49)
27 Analysis of the LREIMS indicated a peak at m/z 410 assigned to the molecular ion (M)+. with ion fragments observed at 396, 395, 269, 187, 174, 161, 43 (100 %) and giving a clear match to stigmasta-3,5-dien-7-one by MS database searching (Figure 23).
Relative abundance abundance Relative
m/z Figure 23. LREI MS library matching of isolate LPF02 (top) with stigmasta-3,5-dien-7-one (below).
Due to the low yield, acquisition of spectral data required for a full characterisation was limited and hindered further molecular structure confirmation.
2.2.3. LPF03 and LPF04
The isolates LPF03 (<1 mg) and LPF04 (<1 mg) were not sufficiently pure (TLC analysis) and therefore further chemical and spectroscopic elucidation was unable to be conducted.
28 2.3. Isolation and Structure Elucidation of Polar Compounds from Boerhavia erecta
The PF of stem bark of B. erecta (SBBeL-PF, 8.2 g) was fractionated using normal silica gel column chromatography (solvent system (ACN, then ACN:MeOH:H2O
(9:0.5:0.5) and then ACN:MeOH:H2O (8.5:0.75:0.75)) to produce four fractions PF1
(158.9 mg), PF2 (162.3 mg), PF3 (789.4 mg), PF4 (500.4 mg) with the separation monitored by TLC analysis (see Figure 24).
PF3 PF1 PF2 PF4
Figure 24. A typical TLC fractionation profile of polar fraction from B. erecta extract. Mobile phase ACN:H2O:MeOH (9:0.5:0.5). Visualisation of components was by UV lamp.
To avoid excessive interaction between the polar compounds and silica, a reverse phase
HPLC was used to isolate our target compounds using a mobile phase of mixture of water, acetonitrile and small percentage of TFA (0.1%). Organic acids such as trifluoric acid and acetic acid are commonly employed in separation and purification of polyhydroxyl/polyphenolic compounds since mineral acid can lead to the lost of acyl groups.86 The fraction with the largest mass PF3, was further separated by HPLC and was found to contain the three polar constituents (see Figure 25) labelled as targets
PF3.1, PF3.2 and PF3.3.
29 PF3.3
PF3.1
PF3.2
PF3.3
PF3.1
PF3.2
Figure 25. Gradual development of HPLC method for polar constituents’ separation of PF3 from SBBeL. The methods employed solvent A (0.1% TFA in H2O) and solvent B (0.1% TFA in 90% ACN-H2O). First method (top) used gradient flow from 100% A to 0% A in 30 minutes. The last method (bottom) required 30 minutes to achieve 60% A. The targets were clearly defined through subsequent analytical HPLC as PF3. 1, PF3.2, PF3.3. Samples were dissolved in MeOH.
Although this protocol was able to isolate the PF3.1 (5.0 mg) and PF3.3 (8.0 mg), spectroscopic analysis was unable to characterize the compounds due to the presence of impurities. Since the PF from stem bark of B. erecta was limited, further exploration was redirected to the PF of stem of B. erecta (SBeL) which was based on analytical
HPLC and LRESMS analysis; both polar fractions appeared identical.
Using the standard protocol, a portion (3.1 g) of the PF of SBeL was further fractionated using silica gel column chromatography to obtain sub fractions PF1 (216.2 mg), PF2 (75.2 mg), PF3 (924.9 mg, the target) and PF4 (408.8 mg). The analytical
HPLC method was further developed for a better resolution which lead to produce targets PF3.1, PF3.2, PF3.2a and PF3.3 (see Figure 26, bottom).
30 PF3.1
PF3.3
PF3.2
PF3.1 shoulder
PF3.2
PF3.3
PF3.1
PF3.3
PF3.2 PF3.2a
Figure 26. Gradual development of HPLC method for the separation of the polar constituents of PF3 from SBeL. The methods employed solvent A (0.1% TFA in H2O) and solvent B (0.1% TFA in 90% ACN-H2O). First method (top) used gradient flow from 100% A to 0 % A in 30 minutes. Second method (middle) required 30 minutes to get 60% A. The best method (bottom) started with 90% A and finished with 75% A within 40 minutes. The last method resolved the shoulder peak to become two peaks of PF3.2 and PF3.2a. The purity of the individual fraction after separation was checked by analytical HPLC as PF3.1, PF3.2, PF3.2a, PF3.3. Samples were dissolved in MeOH.
The best method development successfully revealed the impurities (a shoulder peak) which separated as a single peak of PF3.2a. A reverse phase preparative HPLC was able to separate the four targets PF3.1 (39.6 mg), PF3.2 (3.8 mg), F3.2a (3.2 mg), and
PF3.3 (10.5 mg). The purity of each isolate was confirmed through analytical HPLC analysis (see Figure 27).
31
Figure 27. An example of analytical HPLC chromatogram showing purity of isolate PF3.3. Sample was directly collected from preparative HPLC (no MeOH present). The retention time was 2 minutes slower than in PF3 fraction’s chromatogram (see Figure 25) which used MeOH to dissolve the sample.
2.4.1. Isolate PF3.1
This isolate appeared as a yellow pale solid and showed yellow fluorescence under UV- light (λ 365 nm). In H2O:ACN (50:50) solvent, the compound crystallised as a unique floating yellow fibre which was composed of tiny crystal needles (see Figure 28).
200 μm
Figure 28. The fibre (a) development of PF3.1 in H2O:ACN:TFA (80:20:0.01) after 48 h (left). A cotton like PF3.1 appearance (right), taken from Leica Z16 APO lens controlled by Leica application suite V 3.0.1 (the magnification affected the compound’s color)
32 Spectroscopic and non-spectroscopic data analysis proposed a molecular structure of
PF3.1 as 3′,4′,5,7-tetrahydroxyflavone-3-O-α-D-rhamnopyranosyl-(1Æ6)-O-β-D-gluco pyranoside 50, a glycosidic compound (Figure 29).
Figure 29. Molecular structure of 3′,4′,5,7-tetrahydroxyflavone-3-O-α-D-rhamnopyranosyl-(1Æ6)-O-β- D-glucopyranoside (50)
The structure of the compound was found to contain no nitrogen atoms on the basis of negative results against Dragendorff, Mayer and Ninhydrin reagents, and no nitrogen resonance from a 15N-gHMBC experiment. Elemental analysis showed insignificant nitrogen present (< 0.1%).
Analysis of the LRESMS showed peaks at m/z 611 assigned to (M+H)+ and m/z 633 assigned to (M+Na)+. To examine the number of hydroxy units present, the isolate was acetylated and the resulting product was analysed by LRESMS revealing a peak at m/z
1053 (M+Na)+. This ion was 420 a.m.u. higher than the starting material which corresponded to the acetylation of 10 substituents, presumably hydroxyl groups, which
1 was confirmed analysis of the H-NMR spectra which showed 10 -OCOCH3 groups in the regions 1.80-2.50 ppm. Together with 13C-NMR (absolute mode conversion of APT spectrum), APT and gHSQC spectral analysis, an early molecular formula for 50 of
C27H30O10 was calculated. However, this was 97 a.m.u lower than what was expected
(611). Therefore, an additional 6 oxygens and 1 proton was proposed to a final
33 + molecular formula of C27H31O16 (M+H) . The sodium ion form was observed by
HRESMS showing m/z (M+Na)+ ion (633.1443, calculated mass 633.1432).
The total analysis of both 1D and 2D NMR spectra is summarised in Table 6.
Table 6. 1H- and 13C-NMR data of PF3.1 experiment and literature87
Carbon Carbon δC δH, J No. type Exp. Lit. Exp. Lit. 2 C 159.3 158.5 3 C 135.6 135.6 4 C 179.4 179.4 5 C 162.9 163.0 6 CH 99.9 100.0 6.21 (d, 4J = 2.0 Hz) 6.21 (d, 4J = 2.0 Hz) 7 C 166.0 166.0 8 CH 94.7 94.9 6.40 (d, 4J = 2.0 Hz) 6.40 (d, 4J = 2.0 Hz) 9 C 158.5 159.4 10 C 105.6 105.7 1′ C 123.1 123.2 2′ CH 117.7 117.7 7.67 (s) 7.66 (d, 4J=2.1 Hz) 3′ C 145.8 145.8 4′ C 149.8 149.8 5′ CH 116.0 116.1 6.88 (d, 3J = 8.5 Hz) 6.87 (d, 3J = 8.5 Hz) 6′ CH 123.6 123.6 7.62 (d, 3J = 8.5 Hz) 7.62 (dd, 3J = 8.5, 4J=2.1 Hz) 1′′ CH 104.7 104.7 5.10 (d, 3J = 7.5 Hz) 5.10 (d, 3J = 7.7 Hz) 2′′ CH 75.7 75.7 3.48 (m) 3.46 (dd, 3J = 7.7, 8.9 Hz) 3′′ CH 78.1 78.2 3.42 (m) 3.40 (t, 3J = 8.9 Hz) 4′′ CH 71.4 71.4 3.27 (m) 3.26 (t, 3J = 8.9 Hz) 5′′ CH 77.2 77.3 3.33 (m) 3.32 (ddd, 3J = 1.2, 6.1, 8.9 Hz) 6′′A HCH 68.5 68.6 3.39 (m) 3.38 (dd, 3J = 1.2, 11.0 Hz) 6′′B HCH 68.5 68.6 3.80 (dd, 3J = 8.5, 10.9 Hz) 3.80 (dd, 3J = 6.1, 11.0 Hz) 1″′ CH 102.4 102.4 4.52 (s, (3J≤1 Hz *)) 4.51 (d, 3J = 1.5 Hz) 2″′ CH 72.1 72.1 3.64 (s, 3J = 1.5 Hz ) 3.62 (dd, 3J = 1.5, 3.4 Hz) 3″′ CH 72.2 72.3 3.54 (d, 3J = 9.5, 3.0 Hz) 3.53 (dd, 3J = 3.4, 9.6 Hz) 4″′ CH 73.9 73.9 3.27 (m) 3.27 (t, 3J = 9.6 Hz) 5″′ CH 69.7 69.7 3.45 (m) 3.44 (dd, 3J = 6.2, 9.6 Hz) 3 3 6″′ CH3 17.9 17.9 1.12 (d, J = 6.0 Hz) 1.11 (d, J = 6.2 Hz) 1 13 Note: H-NMR spectrum of PF3.1 was collected at 500 MHz (in CD3OD) and C-NMR of PF3.1 was 1 13 obtained from absolute mode of APT spectrum which run at 125 MHz (in CD3OD); H- and C-NMR 87 literature were collected at 400 MHz and 100 MHz (in CD3OD), respectively. * Low vicinal coupling (3J≤1 Hz) normally shown as singlet.88 Few multiplicity disagreement due to resolution quality of the spectra which were then accomplished through analysis of acylated PF3.1.
The 1H-NMR (see appendix 7) spectrum comprised aromatic (aglycon) and glycosidic protons. From the TOCSY spectrum (see appendix 10), the aglycon has two separate aromatic proton systems and the sugar moiety also has two different proton systems.
34 The first aromatic system consisted of two protons which existed as doublet at δ 6.21 (d,
4J = 2.0 Hz, 1H, H6) and δ 6.40 (d, 4J = 2.0 Hz, 1H, H8), see Figure 26. The low coupling constant value clearly indicated a meta proton-proton correlation89 which was also shown as a weak correlation through the COSY spectrum (see appendix 9). Along with proton-carbon correlation analysis (gHMBC spectrum, see appendix 12) a tetra- substituted benzene ring fragment was suggested. The gHMBC spectrum also presented a four bond correlation between the protons H6 and H8 and a carbonyl carbon (C=O, δ
179.4, C4) with this carbon attached to C10 (δ 105.6). Again, the existence of the quaternary C5 (δ 162.9), C7 (δ 165.96) and C9 (δ 158.5) at a more downfield region suggested these were the position of -OH functionality. These analyses suggested the construct of the first aromatic ring labeled A (see Figure 30).
8 X X A 10 6 X O Figure 30. A proposed tetra-substituted aromatic ring fragment A of isolate PF3.1
The second aromatic system (Ring B) was identified by peaks resonanced at δ 7.67 (s,
1H, H2′), δ 6.88 (d, 3J = 8.5 Hz, 1H, H5′) and δ 7.62 (d, 3J = 8.5 Hz, 1H, H6′) which clearly indicate a 1,3,4 tri-substituted aromatic ring. From the COSY spectrum, the correlation of H5′ - H6′ was established and confirmed as one system in the TOCSY spectrum. From the gHMBC spectrum, a 3 bond correlation of a quaternary aromatic carbon (δ 159.3) with proton H5′ and H6′ suggested the upper field resonance of carbon
C1′ (δ 123.1). Finally the gHMBC spectrum was able to identify the more de-shielded aromatic carbons at δ 145.8 and δ 149.8 as C3′ and C4′, respectively. These downfield effects were caused by the presence of -OH substituents. The proposed tri-substituted benzene ring is illustrated in Figure 31 and is labelled B.
35 X 2' X B C 5' 6' Figure 31. A proposed tri-substituted aromatic ring fragment B of isolate PF3.1.
Although there are published methods for the identification of sugar moieties, including cellulose chromatographic90, micro-scale acid hydrolysis or gas chromatography analysis91 techniques, we relied on mass spectrometric and nuclear magnetic resonance spectral analysis to identify the molecular structure of the sugars present in 50.
The MS2 (MS/MS) experiment generated fragment ion at m/z 487 and 325 (see Figure
32) which were consistent with a typical one deoxyhexose fragment loss (146 a.m.u) and the loss of one hexose (162 a.m.u) fragment.89
633.0 2.25e5 100 (M+Na)+ 331.1 % 325.0 162 a.m.u 487.1 146 a.m.u 0 m/z 300 350 400 450 500 550 600 650 700 Relative abudance Relative abudance m/z Figure 32. MS2 (MS/MS) of isolates PF3.1 showed a deoxyhexose (146 a.m.u) and hexose (162 a.m.u) fragments loss.
From the 1H-NMR spectrum, the presence of sugar structures was clearly defined by the distinct peak at δ 5.10 (d, 3J = 7.5 Hz, 1H, H1′′) and δ 4.56 (s, 1H, H1′′′) assigned typically to two anomeric protons. The remaining peaks assigned to the sugars appear typically in the region δ 3.00-4.20.92 The carbons C1′′ and C1′′′ resonanced at δ 104.7 and δ 102.4 suggest a pyranose type sugar which has a lower chemical shift than a furanose configuration (Δδ 13C, 25.9 ppm).93 The anomeric protons and terminal protons were used as a toehold to begin tracing the proton assignment.94 In order to gain
36 the proton-proton correlation of the sugars, the COSY spectrum analysis, assisted with a
TOCSY spectrum obtained from 30 ms of mixing time, generated the same proton- proton correlation as COSY.95 In the COSY spectrum, H1′′′ coupled to H2′′′ with low vicinal coupling constant (3J ≤ 1 Hz), and therefore showed as a weak resonance and resulted in H1′′′ being presented as a singlet peak at 1H-NMR spectrum.88
Many published NMR studies on carbohydrates have established a correlation between the sugar molecular structure and their NMR properties.92 Every sugar has a unique configuration which specifies the proton-proton coupling constant patterns.96-98 In a pyranose structure, the axial-axial proton configuration gives large coupling constants
(7-8 Hz), whereas axial-equatorial proton configuration gives smaller coupling constants (~ 4 Hz) and even smaller coupling constants (< 2 Hz) are observed for the equatorial-equatorial configuration.97
In this case, 1H-NMR spectrum of PF3.1 gave poorly resolved coupling constants for
H2 to H6 of the sugar residues. However, a semi-synthetic trick of acylation of PF3.1 allowed NMR analysis, to elucidate the first sugar as glucopyranoside and the second sugar as rhamnopyranoside (please refer to chapter 3 for a detailed discussion of this analysis).
Further NMR studies could conclude the absolute configuration of the sugar by
1 measuring the JC1–H1, which is ~170 Hz for α-anomeric sugar configuration and ~160
Hz for β-anomeric sugar configuration in the D- sugar system.92,99 Therefore, the first
1 sugar ( JH1′′-C1′′ = 160.1 Hz) indicated a β-D-glucopyranoside and the second sugar
1 ( JH1′′-C1′′ = 167.5 Hz) indicated α-D-rhamnopyranoside.
37 Instead of using NOE to establish the glycosidic linkage, which might give inaccurate information100,101, the interglycosidic linkage was determined from the gHMBC data analysis (see Figure 33). The spectrum indicated a correlation between H6A′′, H6B′′ and C1′′′ suggesting a 6-O-linkage from glucopyranose to rhamnopyranose. The spectrum also indicated that H1′′ has a correlation with an aromatic carbon at δ 135.6
(C3). These data demonstrated that the sugars were O-linked at C3.
H OH O OH H 1''' OH H H H O H H 6'' 3 O O 1'' OH OH H HO H H Figure 33. A proposed glycosidic fragment of isolate PF3.1
By combining all the proposed structure fragments, a general picture of the structure of the isolate PF3.1 (see Figure 34) was visible to be constructed as flavonoid derived compounds 50.
Figure 34. General preview of proposed fragments of molecular structure of isolate PF3.1.
Total NMR proton-carbon correlations in support of this structure, are illustrated in
Figure 35.
The 13C-NMR comparative study with the closest molecular structure from literature87, quercetin-3-O-rutinoside (see Table 6), did not show any significant discrepancies, 38 which supported the proposed structure. However, the literature did not mention the absolute configuration of the sugar units87,102 in which the sugar units (rutinoside) commonly composed by α-L-rhamnopyranoside together with β-D-glucopyranoside.103
The paper assigns the structure to the NMR spectra, but do not explain how the structure is elucidated. What we found, again based on NMR spectral analysis92,99 was
α-D-rhamnopyranoside.
Figure 35. Proton-carbon correlation on the molecular structure of 3′,4′,5,7-tetrahydroxyflavone 3-O- α-D-rhamnopyranosyl-(1Æ6)-O-β-D-glucopyranoside.
Overall, the molecular structure of isolate PF3.1 is a flavonol derived compound, containing a 3′,4′,5,7-tetrahydroxyflavonol-3-O-α-D-rhamnopyranosyl-(1Æ6)-O-β-D- glucopyranoside.
2.4.2. Isolate PF3.2
This isolate appeared as a brown amorphous solid and showed a brown fluorescence under UV-light (λ 365 nm). The molecular structure (52, Figure 36) was established on the basis of spectroscopy and spectrometry analysis.
39
Figure 36. Molecular structure of 3′,4′,5,8-tetrahydroxyflavone-3-O-hexopyranoside (52).
Chemical testing for the presence of nitrogen using Dragendorff, Mayer and Ninhydrin reagents gave negative results. The LRESMS indicated ion at m/z 465 of (M+H)+, which was 146 a.m.u lower than (M+H)+ of PF3.1. Together with the TOCSY spectrum, the 1H-NMR spectral analysis clearly revealed typical of a glycosidic compound. The extensive NMR spectral analysis is summarized in the Table 7.
Table 7. 1H- and 13C-NMR data of PF3.1, PF3.2.
Carbon Carbon δC δH, J No. type PF3.1 PF3.2 PF3.1 PF3.2 2 C 159.3 159.2 3 C 135.6 135.7 4 C 179.4 179.5 5 C 162.9 162.3 6 CH 99.9 99.9 6.21 (d, 4J = 2.0 Hz) 6.21 (s) 7 C 166.0 166.1 8 CH 94.7 93.0 6.40 (d, 4J = 2.0 Hz) 6.40 (s) 9 C 158.5 158.5 10 C 105.6 104.5 1′ C 123.1 123.2 2′ CH 117.7 116.4 7.67 (s) 7.71 (s) 3′ C 145.8 145.9 4′ C 149.8 149.9 5′ CH 116.0 114.9 6.88 (d, 3J = 8.5 Hz) 6.87 (d, 3J = 7.5 Hz) 6′ CH 123.6 122.2 7.62 (d, 3J = 8.5 Hz) 7.60 (d, 3J = 7.5 Hz) 1′′ CH 104.7 103.3 5.10 (d, 3J = 7.5 Hz) 5.21 (m) 2′′ CH 75.7 75.2 3.48 (m) 3.48 (m) 3′′ CH 78.1 78.2 3.42 (m) 3.42 (m) 4′′ CH 71.4 70.9 3.27 (m) 3.21 (m) 5′′ CH 77.2 77.7 3.33 (m) 3.34 (m) 6′′A HCH 68.5 62.1 3.39 (m) 3.40 (m) 6′′B HCH 68.5 62.1 3.80 (dd, 3J = 8.5, 10.9 Hz) 3.82 (m) 1″′ CH 102.4 4.52 (s, (3J≤1 Hz *)) 2″′ CH 72.1 3.64 (s, 3J = 1.5 Hz ) 3″′ CH 72.2 3.54 (d, 3J = 9.5, 3.0 Hz) 4″′ CH 73.9 3.27 (m) 5″′ CH 69.7 3.45 (m) 3 6″′ CH3 17.9 1.12 (d, J = 6.0 Hz) Note: 1H- and 13C-NMR spectra were collected at 500 MHz and 125 MHz, respectively.
40 From the TOCSY spectrum analysis, two aromatic proton systems (aglycon) and two sugar type protons systems (sugar units) were clearly identified. The former system was established as tetra-substituted benzene ring A (Figure 37, left) with two protons at δ
6.21 (s, 1H, H6) and δ 6.40 (s, 1H, H8). The second aromatic systems had three protons resonant at δ 7.71 (s, 1H, H2′), δ 6.87 (d, 3J = 7.5 Hz, 1H, H5′) and δ 7.60 (d, 3J = 7.5
Hz, 1H, H6′) first from which the structure of a 1,3,4 tri-substituted benzene ring B was established (Figure 37, right).
X 2' X B C 5' 6'
Figure 37. A proposed tetra-substituted (left) and tri-substituted benzene ring (right) fragments of molecular structure of isolate PF3.2. X=hydroxyl group.
The 13C-NMR spectrum revealed the key carbonyl carbon of a flavonol type compound at δ 159.2. A comparative study on NMR spectral data between PF3.1 and PF3.2 enabled a complete elucidation of the PF3.2 skeleton.
The sugar unit was elucidated through MS2 experiment which gave peaks at m/z at 487
(M+Na)+ and 325 which indicated a hexose (162 a.m.u) fragment lost (Figure 38).
325.2 2.15e4 100 487.1
324.1 %
162 a.m.u
Relative abudance Relative abudance 377.1 361.1 439.1 469.3 0 m/z 280 300 320 340 360 380 400 420 440 460 480 500
m/z Figure 38. MS2 (MS/MS) of isolates PF3.2 gave ions at m/z 487 and 325 which represented the lost of hexose fragment.
41
The 13C-NMR spectrum revealed the chemical shift of the sugar’s carbons existed in the region of typical of a pyranose. A comparative study between the carbon chemical shifts in structures PF3.1 and PF3.2 revealed a good alignment which suggested a similar location of the 3-O linkage sugar. Due to insufficient yield, the spectrum analysis only established the sugar as 3-O-hexopyranoside. Reported NMR data was run in a different solvent104 which prohibits us from making a direct comparison to literature for structural confirmation. Time constraints also limited us from preparing more PF3.2 for more extensive spectroscopic experiments which is required for a full characterisation.
2.4.3. Isolate PF3.2a
Isolate PF3.2a appeared as a brown amorphous solid and showed a brown fluorescence under UV-light (λ 365 nm). The spectroscopic and spectrometric analysis enabled the elucidation of the isolate as 3′,5,7-trihydroxyflavone-3-O-hexopyranosyl-O- deoxyhexopyranoside 53 (Figure 39).
OH
2' 4' 8 B HO O 5' A C 6' 6 O OH O hexopyranoside-O-deoxyhexopyranoside
Figure 39. Molecular strcuture of 3′,5,7-trihydroxyflavone-3-O-hexopyranoside-O-deoxyhexo pyranoside (53)
The isolate did not contain nitrogen as it showed negative results in obtained from
Dragendorff, Mayer and Ninhydrin reagent tests.
42 The LRESMS experiment gave a peak at m/z 595, assigned as the molecular ion, which was 16 a.m.u lower than the (M+H)+ of PF3.1. This might be a deoxy-PF3.1 derivative with HRESMS experiment revealing an ion at m/z 617.1487 (calculated 617.1482)
+ (M+Na) suggesting a molecular formula of C27H30O15Na.
Selected NMR spectra analysis is summarized in Table 8.
Table 8. Selected 1H- NMR data of PF3.1, PF3.2a Carbon Proton PF3.1 PF3.2a 6 6.21 6.21 8 6.40 6.42 2′ 7.67 8.20 4′ - 7.98 5′ 6.88 8.07 6′ 7.62 6.91 1′′ 5.10 5.12 1″′ 4.52 4.52 6″′ 1.12 1.11 Note: Spectra were collected at 500 MHz (in CD3OD)
The 1H-NMR spectrum clearly suggested a typical glycoside in which the spectrum showed six aromatic protons and two anomeric protons.
The TOCSY spectrum analysis confirmed two aromatic proton systems with the former revealing a typical aromatic ring A of 1,2,4,6 tetrasubstitution (Figure 40, left) with H6
(δ 6.21) and H8 (δ 6.42). The second system consisted of a 4 aromatic proton construct with a 1,3 disubstituted benzene ring B (Figure 40, right) with protons resonant at δ
8.20 (H2′), δ 7.98 (H4′), δ 8.07 (H5′), δ 6.91 (H6′). From the spectrum, the meta proton correlation was clearly shown between H2’ and H4'.
X 2' 4' B C 5' 6'
Figure 40. A proposed tetra-substituted (left) and di-substituted benzene ring (right) fragments of isolate PF3.2a. X = hydroxyl group.
43
The sugar components were detected through MS2 (MS/MS) experiment (see Figure
41) which break the daughter ion of m/z 617 (M+Na)+ to give m/z at 471 and 309
(indicating the lost of deoxyhexose (146 a.m.u) and hexose (162 a.m.u)).
617.1 1.85e4 100
308.1 331.2 %
309.1 162 a.m.u 471.0 365.2 146 a.m.u 0 m/z Relative abudance Relative abudance 300 350 400 450 500 550 600 650 m/z Figure 41. MS2 (MS/MS) of isolates PF3.2a showed a typical deoxyhexose and hexose fragment lost.
From the TOCSY spectrum analysis the two sugar’s proton peak patterns were clearly defined with δ 5.12 (H1″), δ 4.52 (H1′′′) and the methyl group of a typical deoxyhexopyranoside at δ 1.11 (H6′′′). Due to limited yield, an extensive characterisation was not able to be conducted. For example, the sugars were only assigned as hexopyranoside and deoxy-hexopyranoside on the basis of comparative study with compound 50.
2.4.4. Isolate PF3.3
Isolate PF3.3 appeared as a brown amorphous solid and showed a brown fluorescence under UV-light (λ 365 nm).
44
200 μm
Figure 42. The solid state of isolate PF3.3 under magnification using a Leica Z16 APO lens controlled by Leica application suite V 3.0.1 (the magnification results in an improper compound color)
The spectrometric, spectroscopic and non spectroscopic data analysis suggest the molecular structure of PF3.3 as 4′,5,7-tetrahydroxy-3′-methoxyflavone-3-O-α-D- rhamnopyranosyl-(1Æ6)-O- β-D-glucopyranoside 54 (Figure 43).
Figure 43. Molecular structure of 4′,5,7-tetrahydroxy-3′-methoxyflavone 3-O-α-D-rhamnopyranosyl- (1Æ6)-O- β-D-glucopyranoside (54)
The compound is a non-nitrogen containing molecule based on results from
Dragendorff, Mayer and Ninhydrin tests.
Compared to the LRESMS of PF3.1, PF3.3 generated a 14 a.m.u greater molecular ion
+ 1 (M+H) at m/z 625. This suggested an extra -CH2- group present which from the H-
NMR spectra comparison (Figure 44), suggested the addition of a methoxy group (δ
56.8, s, 3H, OCH3).
45 The HRESMS indicated an ion at m/z 625.1743 (calculated 625.1769) of (M+H)+ which suggested a molecular formula of C28H33O16
.
-OMe
Figure 44. 1H-NMR spectra of PF3.3 (bottom) indicated a -OMe difference to that in PF3.1 (top) which affected the aromatic protons chemical shifts. The remaining peaks were analogous.
46 The total 1D and 2D-NMR spectral analysis is summarized in Table 9 together with spectral data of PF3.1 for comparison.
Table 9. The 1H- and 13C-NMR data of PF3.1 and PF3.3
Carbon Carbon δC δH, J No. type PF3.1 PF3.3 PF3.1 PF3.3 2 C 159.3 158.9 3 C 135.6 135.5 4 C 179.4 179.3 5 C 162.9 163.0 6 CH 99.9 100.0 6.21 (d, 4J = 2.0 Hz) 6.21 (s) 7 C 166.0 166.0 8 CH 94.7 94.9 6.40 (d, 4J = 2.0 Hz) 6.41 (s) 9 C 158.5 158.5 10 C 105.6 105.7 1′ C 123.1 123.0 2′ CH 117.7 114.6 7.67 (s) 7.94 (s) 3′ C 145.8 148.3 4′ C 149.8 150.8 5′ CH 116.0 116.1 6.88 (d, 3J = 8.5 Hz) 6.91 (d, 3J = 8.5 Hz) 6′ CH 123.6 124.0 7.62 (d, 3J = 8.5 Hz) 7.62 (d, 3J = 8.5 Hz) 1′′ CH 104.7 104.4 5.10 (d, 3J = 7.5 Hz) 5.23 (d, 3J = 7.0 Hz) 2′′ CH 75.7 75.9 3.48 (m) 3.49 (dd, 3J = 9.0, 6.5 Hz) 3′′ CH 78.1 78.2 3.42 (m) 3.46 (dd, 3J = 9.0, 9.5 Hz) 4′′ CH 71.4 71.6 3.27 (m) 3.26 (dd, 3J = 9.5, 7.0 Hz) 5′′ CH 77.2 77.3 3.33 (m) 3.38 (dd, 3J = 10.0, 6.0 Hz) 6′′A HCH 68.5 68.5 3.39 (m) 3.42 (Unresolved) 6′′B HCH 68.5 68.5 3.80 (dd, 3J = 8.5, 10.9 Hz) 3.82 (d, 2J = 11.0 Hz) 1″′ CH 102.4 102.5 4.52 (s, (3J≤1 Hz *)) 4.53 (s, 3J≤1 Hz *) 2″′ CH 72.1 72.1 3.64 (s, 3J = 1.5 Hz ) 3.62 (s, 3J≤1 Hz *) 3″′ CH 72.2 72.3 3.54 (d, 3J = 9.5, 3.0 Hz) 3.49 (dd, 3J = 9.0, 2.5 Hz) 4″′ CH 73.9 73.8 3.27 (m) 3.26 (dd, 3J = 9.5, 9.5Hz) 5″′ CH 69.7 69.8 3.45 (m) 3.42 (dd, 3J = 9.5, 6.0 Hz) 3 3 6″′ CH3 17.9 17.9 1.12 (d, J = 6.0 Hz) 1.10 (d, J = 6.0 Hz) -OCH3 56.8 3.95 (s) 13 1 Note: C-NMR spectrum was collected at 125 MHz (in CD3OD), H-NMR spectrum was collected at 3 88 500 MHz (in CD3OD). * Low vicinal coupling ( J≤1 Hz) normally exists as singlet.
Using the same characterization pathways of PF3.1, the aglycon moiety was elucidated to contain two aromatic proton systems. In the first aromatic ring labeled A, H6 (δ 6.21, s, 1H) and H8 (δ 6.41, s, 1H) existed at the same chemical shift to that in the isolate
PF3.1. The second aromatic ring labeled B, a typical 1,3,4-trisubstitued benzene ring, contains the proton H2′ resonance at δ 7.94 (s, 1H) which was slightly higher than H2′ of PF3.1. This is what we expected due to the less shielding effect of a -OMe substituent (compared to -OH) determined to be in the C3' position. The gHMBC
47 spectrum supported the -OMe position through correlation signals for the -OMe and
C3′. The proton H5′ and H6′ resonance at similar chemical shift, δ 6.91 (d, 3J = 8.5 Hz,
1H, H5′) and δ 7.62 (d, 3J = 8.5 Hz, 1H, H6′) to the same protons in isolate PF3.1.
However, the meta coupling of H2′ and H6′ was not observed.
The sugar moiety was analysed in a MS/MS experiment which showed ions at m/z 647
(M+Na)+, 501 and 339, indicating a typical deoxyhexose and hexose fragment loss (see
Figure 45).
331.1 4.49e4 100 647.0 (M+Na)+ % 338.0 649.0 162 a.m.u 501.0 146 a.m.u Relative abudance Relative abudance 0 m/z 300 350 400 450 500 550 600 650 700 m/z Figure 45. MS2 (MS/MS) of isolates PF3.3 showed a typical deoxyhexose and hexose fragment lost.
To fully characterize the sugar residues, proton-proton coupling constants were
3 3 3 analysed with the first sugar showing JH1-H2 = 7.0 Hz, JH2-H3 = 9.0 Hz, JH3-H4 = 9.5 Hz,
3 and JH4-H5 = 7.0 Hz, indicating a relative configuration of all protons as axial-axial (see
Figure 46, left). This agreed with the proton configuration of β-glucopyranoside.99 The
3 3 3 second sugar has coupling constant pattern of JH1-H2 ≤ 1 Hz, JH2-H3 ≤ 1 Hz, JH3-H4 = 9.5
3 Hz, and JH4-H5 = 9.5 Hz which indicates the relative configuration of the protons as equatorial-equatorial, axial-equatorial, axial-axial, axial-axial (see Figure 46, right).
This matched with a configuration of α-rhamnopyranoside.99
48
4 Figure 46. Relative configuration ( C1) of protons in β-glucose (all axial-axial) and α-rhamnose (equatorial-equatorial, equatorial-axial, axial-axial and axial-axial).
1 From the gHMBC spectrum, the anomeric proton-carbon ( JC-H) coupling constant of the first sugar was 162.0 Hz and second sugar was 169.0 Hz. This suggests the first sugar to be β-D-glucopyranoside and the second sugar to be α-D-rhamnopyranoside.99
The gHMBC spectral analysis enabled the identification of the glycosidic O-linkage by the H-1″ correlation with C3 whereas H6A″ and H6B″ correlated with C1′′′.
From the gHMBC spectrum analysis, the proton-carbon correlations were extensively determined (Figure 47).
OMe H OH H OH O H OH B H OH HO O H H H H A C H O H H O H H H O OH O OH H OH H HO H
OMe H OH H OH O H OH B H OH HO O H H H H A C H O H H O H H H O OH O OH OH H HO H H Figure 47. Proton-carbon correlation on 4′,5,7-trihydroxy-3′-methoxyflavone-3-O-α-D-rhamno pyranosyl-(1Æ6)-O-β-D-glucopyranoside
To confirm the proposed structure, comparative studies were carried out with published data105 (see Table 10) in which the proton and carbon chemical shifts did not show
49 significant differences other than systematic variations. However, the literature did not state the absolute configuration of the sugars in which rutinoside contains α-L- rhamnopyranoside103 instead of the D- form. Overall, our analysis strongly support
4′,5,7-tetrahydroxy-3′-methoxy-4-flavone-3-O-α-D-rhamnopyranosyl-(1Æ6)-O-β-D- glucopyranoside as the molecular structure of isolate PF3.3.
Table 10. The 1H- and 13C-NMR data of experiment and literature105
Carbon Carbon δC δH No. type Exp Lit. Exp Lit. 2 C 158.9 157.1 3 C 135.5 134.2 4 C 179.3 178.0 5 C 163.0 161.8 6 CH 100.0 99.2 6.21 (s) 6.18 (d, 4J = 2.2 Hz) 7 C 166.0 166.0 8 CH 94.9 94.0 6.41 (s) 6.37 (d, 4J = 2.2 Hz) 9 C 158.5 157.5 10 C 105.7 104.2 1′ C 123.0 121.8 2′ CH 114.6 113.6 7.94 (s) 7.92 (d, 4J = 1.8 Hz) 3′ C 148.3 147.2 4′ C 150.8 149.7 5′ CH 116.1 114.9 6.91 (d, 3J = 8.5 Hz) 6.90 (d, 3J = 8.4 Hz) 6′ CH 124.0 122.8 7.62 (d, 3J = 8.5 Hz) 7.62 (d, 3J = 8.6, 4J=2.4 Hz) 1′′ CH 104.4 103.3 5.23 (d, 3J = 7.0 Hz) 5.21 (d, 3J = 7.3 Hz) 2′′ CH 75.9 74.7 3.49 (dd, 3J = 9.0, 6.5 Hz) unresolved 3′′ CH 78.2 76.2 3.46 (dd, 3J = 9.0, 9.5 Hz) unresolved 4′′ CH 71.6 70.4 3.26 (dd, 3J = 9.5, 7.0 Hz) unresolved 5′′ CH 77.3 77.0 3.38 (dd, 3J = 10.0, 6.0 Hz) unresolved 6′′A HCH 68.5 67.4 3.42 (m) 3.45 (m) 6′′B HCH 68.5 67.4 3.82 (d, 2J = 11 Hz) 3.80 (d, 3J = 10.0, 4J =1.8 Hz) 1″′ CH 102.5 101.3 4.53 (s, 3J≤1 Hz *) 4.52 (d, 3J = 1.5 Hz) 2″′ CH 72.1 70.8 3.62 (s, 3J≤1 Hz *) unresolved 3″′ CH 72.3 71.1 3.49 (dd, 3J = 9.0, 2.5 Hz) unresolved 4″′ CH 73.8 72.6 3.26 (dd, 3J = 9.5, 9.5Hz) unresolved 5″′ CH 69.8 68.6 3.42 (dd, 3J = 9.5, 6.0 Hz) unresolved 3 3 6″′ CH3 17.9 16.7 1.10 (d, J = 6.0 Hz) 1.09 (d, J = 6.2 Hz) -OCH3 CH3 56.8 55.6 3.95 (s) 3.94 (s) 1 13 Note: H- and C-NMR spectra of experiment were collected at 500 MHz and 125 MHz (in CD3OD), 1 13 respectively. H- and C-NMR spectra of literature were obtained at 400 MHz and 100 MHz (in CD3OD), respectively.105* Low vicinal coupling (3J≤1 Hz) normally shown as singlet.88
50 Chapter 3
Acetylation of PF3.1
3.1. General
The structural elucidation of the sugar moiety of isolate PF3.1 was difficult due to unresolved peaks. A common technique to overcome this is acylation in which all -OH groups are converted into -OAc functionalities. As a result, the NMR spectra were more clearly defined and the number of -OH groups present was able to be calculated.
Overall, acetylation was able to assist in the structural elucidation of the sugars.
3.2. Synthesis
A standard acetylation method (see chapter 6, section 6.5) using acetic anhydride and pyridine (a base catalyst) successfully converted PF3.1 (7.6 mg) into 12.0 mg of brown solid which was identified as acetylated PF3.1. The acetylation started with the generation of the nucleophile from the hydroxyl groups by a Lewis base (pyridine) catalytic deprotonation. This species then attacked the polar carbonyl bond of the acetic anhydride to form a tetrahedral intermediate106 which then produced the ester (Figure
48).
Figure 48. Tetrahedral intermediate as the key step to produce the acetylated target compound.
3.3. Molecular Structure Elucidation
The compound was obtained as pale yellow solid. The spectroscopic and spectrometric data analysis established the molecular structure of 55 (Figure 49).
51 OAc H OAc O 2' OAc OAc 8 B H OAc AcO O H H 5' H A C 6' O 6 H O O H OAc O OAc OAc H AcO H H Figure 49. Molecular structure of 3′,4′,5,7-tetraacylflavone-3-O-α-D-2′′′,3′′′,4′′′-triacylrhamno pyranosyl-(1Æ6)-O-β-D-2′′,3′′,4′′-triacylglucopyranoside (55)
The structure was elucidated using 1H- and 13C-NMR (appendix 19 and 20) spectra and proton-proton connectivity was established through gCOSY (appendix 21) and TOCSY
(appendix 22) spectral analysis. The proton-carbon connectivity was assigned from the gHSQC (appendix 23) and gHMBC (appendix 24) spectra. The results are summarized in Table 11.
Table 11. 1H-NMR and 13C-NMR data of acetylated PF3.1 Carbon Carbon Chemical chift No. type δC δH, J 2 C 154.7 3 C 137.0 4 C=O 171.9 5 C 150.2 6 CH 113.4 6.82 (d, 4J = 2.0 Hz) 7 C 156.6 8 CH 109.0 7.30 (d, 4J = 2.0 Hz) 9 C 154.1 10 C 115.1 1′ C 128.6 2′ CH 124.7 7.89 (d, 4J = 2.0 Hz) 3′ C 141.8 4′ C 144.1 5′ CH 123.5 7.33 (dd, 3J = 9.0, 4J = 2.0 Hz) 6′ CH 127.2 7.94 (d, 3J = 9.0 Hz) -O-β-D-2′′,3′′,4′′-triacylglucopyranoside 1′′ CH 99.6 5.43 (d, 3J = 8.0 Hz) 2′′ CH 71.4 5.16 (dd, 3J = 10.0, 8.0 Hz) 3′′ CH 72.6 5.26 (dd, 3J = 10.0, 9.5 Hz) 4′′ CH 69.5 4.93 (t, 3J = 9.5 Hz) 5′′ CH 72.8 3.57 (ddd, 3J = 9.0, 6.0, 3.0 Hz) 6′′A HCH (HA) 67.0 3.52 (dd, 2J = 11.0 Hz, 3J = 3.0 )Hz 6′′B HCH (HB) 67.0 3.22 (dd, 2J = 11.0 Hz, 3J = 6 .0)Hz 1 Note: H-NMR spectrum was collected from acylated PF3.1 in CD3OD at 500 MHz while the 13C-NMR at 125 MHz. *Low vicinal coupling (3J≤1 Hz) normally exists as singlet.88
52
Table 11. (continue) 1H-NMR and 13C-NMR data of acetylated PF3.1
Carbon Carbon Chemical shift No. type δC δH, J -O-α-D-2′′′,3′′′,4′′′-triacylrhamnopyranoside 1′′′ CH 97.8 4.50 (s, 3J ≤ 1 Hz*) 2′′′ CH 69.4 5.07 (d, 3J = 3.0 Hz) 3′′′ CH 69.0 5.06 (dd, 3J = 9.0, 3.0 Hz) 4′′′ CH 70.9 4.93 (t, 3J = 9.0 Hz) 5′′′ CH 66.4 3.64 (m) 3 6′′′ CH3 17.2 1.05 (d, J = 6.5 Hz )
-OCOCH3 -OCOCH3 -OCOCH3 A C=O, CH3 169.2, 21.2 2.44 (s) B C=O, CH3 168.1, 21.1 2.34 (s ) C C=O, CH3 167.9, 21.1 2.34 (s ) D C=O, CH3 167.7, 20.9 2.29 (s) E C=O, CH3 169.7, 20.8 2.14 (s) F C=O, CH3 169.6, 20.7 2.08 (s) G C=O, CH3 170.2, 20.7 2.04 (s) H C=O, CH3 169.9, 20.6 2.02 (s) I C=O, CH3 170.1, 20.6 1.96 (s) J C=O, CH3 170.1, 20.6 1.94 (s) 1 Note: H-NMR spectra were collected from acetylated PF3. 1 in CD3OD at 500 MHz while the 13C-NMR at 125 MHz. *Low vicinal coupling (3J≤1 Hz) normally exists as singlet.88
The molecular ion of the acetylated compound was assigned to the peak at m/z 1053
(M+Na)+ on LRESMS which was 420 a.m.u higher than (M+Na)+ ion of starting material ion (m/z 633). This suggested 10 hydroxyl groups of PF3.1 being acetylated
1 which was confirmed with the existence of 10 peaks of -OCOCH3 in the H-NMR spectrum. The HRESMS gave ion at m/z 1053.2500 (calculated 1053.2488) for
+ (M+Na) with a suggested molecular formula of C47H50O26Na.
The structure of the aglycon fragment was previously determined for the starting material and in the spectrum for the acylated compound, the protons were shifted into a more downfield region, δ 7.89 (d, 4J = 2.0 Hz, 1H, H2'), δ 7.33 (dd, 3J = 9.0, 4J = 2.0
Hz, 1H, H5'), δ 7.94 (d, 3J = 9.0 Hz, 1H, H6'), δ 6.82 (d, 4J = 2.0 Hz, 1H, H6) and δ7.30
(d, 4J = 2.0 Hz, 1H, H8).
53
The gHSQC spectra comparison (see Figure 50) between acetylated and non acetylated compounds clearly shows 6 protons shifted downfield indicating the acylation of 6 hydroxyl groups of the two sugar units. The other four acyl group successfully coupled to the phenolic groups of the aglycon region 55. Overall, acetylation was able to
'magnify the chemical environment' within the region of the sugar which resulted in a well resolved spectrum.
Figure 50. gHSQC spectral comparison of PF3.1 (up) and the acetylated (bottom). The acetylation clearly gave a better resolution in the region of the sugar (dotted box). Proton H-2 to H-4 of the sugar were shifted due to chemical environment changes from acetylation.
54 The anomeric proton of the two sugar units was initially identified based on gHSQC
(see appendix 23) as peaks at δ 5.43 (d, 3J = 8.0 Hz, 1H, H1′′) and δ 4.50 (s, 3J≤1 Hz
1H, H1′′′). The assignment of these protons allowed the multibond correlations to be analysed. The C1′′ and C1′′′ carbons resonant at δ 99.6 and δ 97.8 suggest a pyranose type sugar.93
NMR studies on carbohydrates reveals that each sugar has a unique configuration that is specified by the proton-proton coupling constant patterns.92,97 Since acetylation retains the stereochemistry as well as the proton-proton coupling constant pattern. The well resolved 1H-NMR spectrum of the acetylated compound enabled complete coupling constant data to be obtained which eased the sugar structure elucidation. The first
3 3 sugar’s protons coupling constant pattern were composed of JH1-H2 = 8.0 Hz, JH2-H3 =
3 3 10.0 Hz, JH3-H4 = 9.5 Hz, and JH4-H5 = 9.5 Hz, indicating a relative configuration of all protons as axial-axial (see Figure 51, left) which had agreement with the configuration
3 of β-glucopyranoside. The second sugar has a coupling constant pattern of JH1-H2 ≤1
3 3 3 Hz, JH2-H3 = 3.0 Hz, JH3-H4 = 9.0 Hz, and JH4-H5 = 9.0 Hz which shows the relative configuration as equatorial-equatorial, axial-equatorial, axial-axial, axial-axial (see
Figure 51, right) and represented a configuration of α-rhamnopyranoside.
HOH H O H OH H H
4 Figure 51. Relative configuration ( C1) of the protons in β-glucose (all axial-axial) and α-rhamnose (equatorial-equatorial, equatorial-axial, axial-axial and axial-axial).
55 1 92,99 1 Based on NMR studies on the JC1–H1 of sugar compound , the first sugar ( JH1′′-C1′′ =
1 163.2 Hz) indicated a β-D-glucopyranoside and the second sugar ( JH1′′-C1′′ = 170.6 Hz) indicated α-D-rhamnopyranoside.
56 Chapter 4
Bioactivity Testing Results
4.1. Introduction
Traditionally used medicinal plants are enormous sources of drug leads from which pharmacochemical screening has identified many drugs.2 The B. erecta has been traditionally used to treat infectious diseases29,76 and previous work has successfully showed some anti-microbial activities of the species extract (see section 1.3.1.5.1). In our work the screening initially involved bio-testing against HIV infected cells as well as some bacteria.
4.2. Anti-microbial Activity Testing
Three sub-polar fractions and four pure isolates were tested against Staphylococcus aureus, Staphylococcus epidermidis, methicilin resistant Staphylococcus aureus, Mu50, and Escherichia faecium WT revealing insignificant activities (see Table 12).
Table 12. The anti-microbial activity of several fractions and pure isolates obtained from B. erecta
MIC (μg/mL) Entry S. aureus S. epidermidis MRSA Mu50 E. faecium WT PF1 >100 >100 >100 >100 >100 PF2 >100 >100 >100 >100 >100 PF4 >100 >100 >100 >100 >100 PF3.1 >100 >100 >100 >100 >100 PF3.3 >100 >100 >100 >100 >100 LPF01 >100 >100 >100 >100 >100 LPF02 >100 >100 >100 >100 >100 Vancomycin 1.56 3.125 1.56 6.25 >25
4.3. Bio-testing Against HIV Infected Cells
The bio-testing revealed the crude extract of stem bark of B. erecta to have significant activity in which the polar fraction was responsible for the activity. Further assay on the
57 sub-polar fraction and the pure isolates showed that both sub-fraction PF1 and isolate
PF3.1 possessed the highest activity (Table 13).
Table 13. The activities against HIV infected cells of several fractions and pure isolates of B. erecta
Entry IC50 (μg/mL) LPF 16 PF < 0.4 PF1 < 10 PF2 < 46 PF4 > 50 PF3.1 < 10 PF3.3 < 22 LPF01 > 50 LPF02 26
Although this research initially involved limited anti-bacterial and anti-HIV (whole cells) testing, some bioactivities tests have been reported previously on the pure compounds isolated from different species which warrants further discussion.
4.4. Reported Anti-microbial Activities
Although previous studies37 on the same plant identified the structure of LPF01, our research reported new isolates PF3.1 and PF3.3 from B. erecta. Regardless, the difference in the absolute configuration of the sugar units, in which we reported α-D- rhamnopyranosyl instead of α-L-rhamnopyranosyl, the L form has been isolated from different species and has been pharmacologically studied.
4.4.1. Isolate LPF01 (β-sitosterol, steroid type compound)
The β-sitosterol is commonly isolated from various species and has already been tested against some bacteria, viruses and yeast (see Table 14 and 15). The studies showed the compound appears to have a weak activity.
58 Table 14. Minimal inhibitory concentration (MIC) of β-sitosterol and standard drugs against some bacteria.108,109
MIC (μg/mL) Microbes β-Sitosterol Vancomycin* Fluconazole* Bacteria Staphylococcus aureus >100 2 Bacilus subtilis 50 0.75 Eschericia coli >100 NT Pseudomonas aeruginosa >100 NT Yeast Candida glabrata ≥64 2-4 Candida krusei 32-64 64 Candida neoformans 16-64 1 Note: *standard drug; NT=not tested
Table 15. In vitro antiviral activity of β-sitosterol and standard drugs against some virus.109
Activity (plaque reduction) Virus β-Sitosterol Foscarnet Acyclovir African swine fever virus 0 1.0 - Herpes simplex virus-1 - - 3.0 Herpes simplex virus-2 0.2 - 3.0 Note: The concentration used for β-Sitosterol, Foscarnet and Acyclovir were 15, 1000 and 100 μg/mL, respectively. The plaque reduction was showed as the log10 reduction of the viral plaques formed compared with control.
4.4.2. Isolate PF3.1 (~Rutin) and PF3.3 (~Isorhamnetin-3-O-rutinoside), Flavonoids
Flavonoids have been reported to possess various biological activities110, e.g. anti- bacterial111,112 and anti-viral.113,114 Flavonol is one of flavonoid class consisting of compounds that include quercetin and isorhamnetin which are commonly present in
asteraceae family.115
4.4.2.1. Rutin (Quercetin-3-O-rutinoside, Q3R)
Rutin (Quercetin-3-O-rhamnopyranosyl-(1Æ6)-glucopyranoside) mostly shows a weak activity against some bacteria, yeast and virus (see Table 16 and 17). Rutin expressed weaker microbicide activity against MRSA.116 Studies on a haematorporphyrin-rutin-
59 arginin complex (HpD-Rut2-Arg2) revealed an increase of the photosensitiser activity in its bactericidal photodynamic action compared to a haematorporphyrin-arginin complex
117 (HpD- Arg2). Rutin is inactive against the influenza virus neuromidase and Hepatitis
B virus, however the sulfated rutin provided a novel candidate for anti-HIV-1/HSV microbicide.118 The glycosylation might be responsible for the low anti-microbial activity.119,120
Table 16. Anti bacterial activity of rutin (Q3R), Isorhamnetin 3-O-rutinoside (I3R) and reference.116-118
MIC (μg/mL) Microbes Q3R Cefotaxim I3R Bacteria Enterococcus faecalis 8 R R Staphylococcus epidermidis 8 0.1 R Staphylococcus aureus 32 2 R Bacilus subtilis 128 32 R Enterobacter aerogenes 128 R R Eneterobacter cloacae 128 R R Eschericia coli 64 0.1 256 Kleibsiella pnemoniae 64 0.1 R Proteus mirabilis 16 0.03 R Proteus vulgaris 64 0.03 R Pseudomonas aeruginosa 64 1.6 256 Salmonela typhi 64 0.5 R MRSA S. aureus >256 (0.8 – 12.5c) 0.25 a NA Yeast Candida albicans 500 0.97 b NA Note: MIC= minimum inhibition concentration; MRSA: methicilin resistant Staphylococcus aureus; a= vancomycin; b=5-Flucitosine; c= MIC for HpD-Rut2-Arg2; R, absence of inhibition even in the highest concentration of 1000 μg/mL; NA= data not available
Table 17. Anti-viral activities of rutin, modified rutin and standard drug.119-123
Inhibition (IC (μM ± SD)) Antiviral activity 50 Q3R SQ3RS I3R Lamivudine Hepatitis B virus 0a NA NA 29.6b Human immunodeficiency virus-1IIIB >300 2.3 ± 0.2 inactive NA Human immunodeficiency virus -1Ba-L NA 1.9 ± 3.8 NA NA Human immunodeficiency virus -1Ada-M NA 4.5 ± 2.0 NA NA Neuromidase of influenza virus >100 NA NA NA Moloney murine leukimia virus reverse transcriptase 7.27c NA NA NA Note: a=reported as % inhibition with concentration 0.2 μmol/mL; a=reported as % inhibition with concentration 0.1 μmol/mL; NA=not available; c=reported as ID50 value.
60 4.4.2.2 Isorhamnetin 3-O-rutinoside (I3R)
The bioactivity data of this compound is quite limited (see Table 15 and 16). Limited anti-bacterial testing has been done, which indicated a very weak activity.121 The compound did not possess activity against HIV-1.123
61
62 Chapter 5
Conclusion and Future Directions
5.1. Conclusion
Biological assays were useful tools in a chemical screening of medicinal plant. A fractionation of crude extract helped in narrowing the screening. Our research was able to establish a chemical screening protocol which resulted in eight compounds being isolated from B. erecta. From the less polar fraction, four compounds were isolated but only one was fully elucidated as β-sitosterol. The remaining less polar constituents were partly characterised as sterols. From the polar fraction, other four compounds were isolated and only two were fully characterised and elucidated as 3′,4′,5,7- tetrahydroxyflavone-3-O-α-D-rhamnopyranosyl-(1Æ6)-O-β-D-glucopyranoside 50 and
4′,5,7-tetrahydroxy-3′-methoxy-4-flavone-3-O-α-D-rhamnopyranosyl(1Æ6)-O-β-D- glucopyranoside 54 (Figure 52). The α-L-rhamnopyranoside derivative has been reported previously87,102,103,105, however the D-form is novel. This is also the first time this molecule has been reported in in the Boerhavia genus.
Figure 52. Molecular strcuture of polar constituents. R = OH, 3′,4′,5,7-tetrahydroxyflavone-3-O-α-D- rhamnopyranosyl-(1Æ6)-O-β-D-glucopyranoside 50. R = OMe, 4′,5,7-tetrahydroxy-3′-methoxy-4- flavone-3-O-α-D-rhamnopyranosyl(1Æ6)-O-β-D-gluco pyranoside 54
The isolated compounds were inactive against Staphylococcus aureus, Staphylococcus epidermidis, methicilin resistant Staphylococcus aureus, Mu50, and Escherichia
63 faecium WT However, the initial biological assay against HIV infected cells revealed the extract of B. erecta to possess a significant activity. Further screening led to a polar fraction showing significant activity. The PF3.1 revealed the highest activity against
HIV infected cells than the other pure isolates.
Biological assay directed screening is a good tool to reveal chemical constituents for specific activity. Medicinal plants usually have various activities; therefore a pure chemical screening followed by several biological testing processes might give more benefit to provide a comprehensive description of constituents and their activities.
5.2. Future work
The extract of B. erecta displayed some potentially valuable activity, i.e. anti-malaria and anti-HIV (against whole infected cells). Although we have established the research protocol for the chemical screening of B. erecta, some sub-fractions (less polar and polar fraction) remain to be screened due to time constraint. Future work is needed to separate, isolate and characterize all remaining constituents. These will provide enough compounds for biological assay and will allow for the judgment of which compound/s is/are responsible for certain activities mentioned in both preliminary studies and folklore application.
64 Chapter 6
Experimental
6.1. General
Gravity chromatography was performed using Merck Silica Gel 60 (0.063-0.200 mm).
Analytical thin layer chromatography (TLC) was performed using Merck Kieselgel 60
F254 silica on aluminium sheets. UV light and molybdate staining agent was used to visualize the plates. Preparative thin layer chromatography plates (0.2 mm thickness, 20 x 20 cm) were supplied by Merck. Visualisation of the separated bands on preparative
TLC plates was performed on a single reference spot only using molybdate staining agent. Analytical high performance liquid chromatography (HPLC) was performed on a
4.9 x 150 mm, 5 μm, RP column (Symmetry ® C18) protected with a 3.9 x 20 mm, 0.5
μm Symmetry C18 guard column, with 0.1% TFA in water (A) and 0.1 % TFA in 90 % acetonitrile-water (B) as solvents, starting with 10 % B to obtain 25 % B at 40 minutes.
The flow rate was 1 mL/min and the injection volume 20 μL. The HPLC used a Waters
1525 binary HPLC pump and Waters 2487 dual λ absorbance detector, controlled by breeze software (version 3.30). The chromatograms were recorded at 254 nm. The preparative HPLC was employed on a 19 x 150 mm, 5 μm, RP column (OBD Sunfire)
TM protected with a 19 x 10 mm, 5 μm Sunfire C18 guard column, with the solvent systems mentioned above. The flow rate was 14.56 mL/min, and the injection volume
4.0 mL. The preparative LC system consisted of a Waters Preparative-LC controller, a
Waters prep degasser and a Waters 2489 UV/Visible detector, controlled by Empower software (series 2). The chromatograms were recorded at 254 nm and the fractions collected were managed manually. All analytical HPLC samples were filtered through
Whatman syringe filter PTFE 0.45 μm, 4 mm and preparative HPLC samples were filtered through Bonnet syringe filter 0.45 μm, 30 mm. A Büchi Rotary Evaporator (R-
65 114/200) with a high vacuum pump was used for evaporation of solvents under reduced pressure at 40 ºC. Nitrogen gas was used for evaporating smaller quantities of solvents.
A high vacuum system with a liquid nitrogen trap was used for the drying of compounds. Direct insertion electron impact mass spectra (DI-MS) were obtained on a
Shimadzu QP-5050 spectrometer with the DI probe heated from 40-200 ºC at 50
ºC/minute. HREIMS were run on a VG Autospec Mass Spectrometer. Electrospray mass spectra were obtained from Waters (micromass) platform LCZ mass spectrometer equipped with z-spray electrospray ionisation source and was controlled by MassLynx software (version 3.4). MS/MS experiment were carried out on Waters (micromass)
Quattro microTM equipped with z-spray electrospray ionisation source controlled by
MassLynx software (version 4.0). HRESIMS were run on Waters QTof Ultima mass spectrometer equipped with Lockspray electrospray ionisation source controlled by
MassLynx software (version 4.0). One and two dimensional NMR experiments including 1H-NMR, 13C-NMR, APT, DEPT, NOE, gCOSY, TOCSY, NOESY, gHSQC, gHMBC were obtained on a Varian Unity Inova-500 MHz NMR spectrometer, controlled by Varian VNMR software (version 6.1 revision C). Deuterated chloroform was used as NMR solvent (unless mentioned). 13C-NMR spectra were also obtained from APT spectrum through absolute value mode conversion. The proton with undefined multiplicity range (labelled as m), the chemical shift assignment was assisted by gHSQC spectral analysis.
All the solvents were AR (analytical reagent) grade or distilled except HPLC grade for the HPLC. Solvents mixtures are stated as volume to volume (v:v) proportion.
All the biological assays were peformed at Avexa Pty. Ltd in Melbourne, Victoria.
66 6.2. Plant Sample, Extraction and Re-extraction
The dried stem barks and stem of B. erecta samples were initially supplied by Dr.
Adama Hilou from Université de Ouagadougou, Burkina Faso (Africa).
6.2.1. Extraction of Stem Bark of Boerhavia erecta (SBBeL)
Dried extract of SBBeL (94 g) was blended with methanol (500 mL) and then evacuated into 2000 mL conical flask with 500 mL methanol. The mixture was stirred for 24 hours, then vacuum-filtered and concentrated yielding a 11.7 gram dried extract.
6.2.2. Extraction of Stem of Boerhavia erecta (SBeL)
To the powdered stem of Boerhavia erecta L. (350 g), was added methanol (2000 mL).
The slurry was poured into 2000 mL round bottom flask followed by stirring for 48 hours and then vacuum filtered and evaporated to dryness resulting in 45.0 gram of dried extract.
6.2.3. Re-extraction
The dried extracts of SBBeL (11.7 g) and SBeL (45.0 g) were separately extracted with
EtOAc:DCM:glacial acetic acid (4.75:4.75:0.5, 15 x 20 mL). The supernatants were separately collected and vacuum-dried to produce less polar fractions, SBBeL-LPF (2.1 g) SBeL-LPF (2.0 g). The insoluble solid was dried to give the polar fraction, SBBeL-
PF (8.2 g), and SBeL-PF (41.4 g).
67 6.3. Isolation and Purification
6.3.1. Isolation and Purification of Sterols from Boerhavia erecta
Analytical TLC analysis was performed on the less polar fraction of SBBeL with
DCM:MeOH (9.5:0.5) to define targets LPF01, LPF01, LPF02, LPF03 and LPF04.
6.3.1.1 Isolation of LPF01, LPF02, LPF03 and LPF04
The less polar fraction of SBBeL (2.1 g) was subjected to silica gel column chromatography and eluted with hexane:EtOAc (0.5:0.5, 600 mL), EtOAc (1.0 L),
DCM:MeOH (9.5:0.5, 500 mL), DCM:MeOH (9:1, 1.0 L). Twenty nine fractions were collected and every five were analysed by TLC (DCM:MeOH (9.5:0.5)). The fractions containing target compounds LPF01, LPF02, LPF03 and LPF04 were separately pooled and concentrated (100 mg). This was then subjected to silica gel column chromatography (30 cm) and was eluted with DCM:MeOH (92.5:7.5, 1.0 L) and
DCM:MeOH (9:1, 300 mL). Twenty five fractions (10 mL each) were obtained and were analysed by TLC (DCM:MeOH, 9.5:0.5). The fractions containing target LPF01 were pooled and was applied to PTLC (20 x 20 cm silica gel F254) with DCM:MeOH
(9.5:0.5) as eluent to separate and isolate the target LPF01 (2.6 mg, 0.17 mg/g dried plant). This was applied to the other fraction containing target compounds of LPF02
(1.3 mg, 0.02 mg/g dried plant weight), LPF03 (≤ 1 mg) and LPF04 (≤ 1 mg).
6.3.2. Isolation and Purification of Polar Constituents from Stem Bark of
Boerhavia erecta (SBBeL).
The polar fraction of extract stem bark of B. erecta (8.2 g) was redissolved in
ACN:H2O:MeOH (2:0.1:2) and subjected to silica gel column chromatography (2 cm diameter, 20 cm length). The column was eluted with the ACN (400 mL),
68 ACN:MeOH:H2O (9:0.5:0.5, 400 mL) and ACN:MeOH:H2O (8.5:0.75:0.75, 400 mL).
This fractionation was monitored using analytical TLC with the mobile phase
ACN:MeOH:H2O (9:0.5:0.5). After vacuum drying, this procedure produced sub- fractions PF1 (158.9 mg), PF2 (162.3 mg), PF3 (789.4 mg) and PF4 (500.4 mg) as brown, green, yellow-brown and brown solids, respectively.
6.3.2.1. Isolation of Compounds PF3.1 and PF3.3
To sub-fraction PF3, analytical HPLC was employed with a gradient elution from 100% of solvent A (0.1% TFA in H2O) to 60% of solvent A within 30 minutes (solvent B
(0.1% TFA in 90% of ACN-H2O)). Isolates PF3.1 and PF3.3 were subsequently collected from preparative HPLC process and vacuum dried to give pale yellow and yellow-brown solid with the purity of each confirmed by analytical HPLC.
6.3.3. Isolation and Purification Polar Constituents from Stem of Boerhavia erecta
(SBeL).
The polar fraction of the stem of B. erecta (3.1 g) was redissolved in ACN:H2O:MeOH
(2:0.1:2) and then subjected to silica gel column chromatography (2 cm diameter, 20 cm length). The column was eluted with ACN (400 mL), ACN:MeOH:H2O (9:0.5:0.5) (400 mL) and ACN:MeOH:H2O (8.5:0.75:0.75) (400 mL). After vacuum drying, this procedure produced sub-fractions PF1 (216.2 mg), PF2 (339.4 mg), PF3 (924.9 mg) and PF4 (408.8 mg) as brown, green, yellow-brown solids and brown solid, respectively.
6.3.3.1. Isolation of Compounds PF3.1, PF3.2, PF3.2a and PF3.3
To a small quantity of PF3 fraction, analytical HPLC was employed with a gradient elution from 90% of solvent A (0.1% TFA in H2O) to 75% of solvent A in 40 minutes
69 with solvent B (0.1% TFA in 90% of ACN-H2O). Isolates PF3.1, PF3.2, PF3.2a and
PF3.3 were collected from preparative HPLC and vacuum dried to give the solid isolates with the purity of each confirmed by analytical HPLC.
6.4. Structure Elucidation
6.4.1. The Less Polar Constituents
The isolates from the less polar fraction obtained from the extract of stem bark of
Boerhavia erecta L. were subjected into LREI mass spectrometry to give its fragmentation pattern which aligned to spectra database for the best match (proposed structure).
β-Sitosterol
Isolate LPF01: a non-UV active creamy yellow pale material (2.6 mg, 0.17 mg/g dried
1 3 plant sample); H-NMR (CD3OD, 500 MHz, nanoprobe), 5.28 (d, J = 7.0, 1H, H6),
3.46 (m, 1H, H3), 2.24 (m, 1H, H4B), 2.16 (m, 1H, H4A), 1.94 (m, 1H, H12B), 1.91
(m, 1H, H7B), 1.86 (m, 1H, H2A), 1.78 (m, 1H, H1B), 1.78 (m, 1H, H16B), 1.60 (m,
1H, H25), 1.52 (m, 1H, H15B), 1.45 (m, 1H, H11A), 1.44 (m, 1H, H8), 1.44 (m, 1H,
H7A), 1.42 (m, 1H, H22B), 1.38 (m, 1H, H11B), 1.32 (m, 1H, H2B), 1.30 (m, 1H,
H20), 1.21 (m, 1H, H28B), 1.20 (m, 1H, H16A), 1.10 (m, 1H, H23A), 1.10 (m, 1H,
H12A), 1.03 (m, 1H, H17), 1.01 (m, 1H, H1A), 1.01 (m, 1H, H15A), 0.96 (m, 1H,
H22A), 0.93 (m, 1H, H14), 0.86 (m, 1H, H24), 0.86 (d, 3J = 6.5 Hz, 1H, H9), 0.85 (d, 3J
= 8.5 Hz, 3H, H21), 0.80 (m, 1H, H28A), 0.79 (t, 3J = 9.0 Hz, 3H, H29), 0.77 (d, 3J =
70 6.5 Hz, 3H, H27), 0.76 (s, 3H, H19), 0.74 (m, 1H, H23B), 0.74 (d, 3J = 6.5 Hz, 3H,
13 H26), 0.62 (s, 3H, H18); C-NMR (CD3OD, 125 MHz, nanoprobe), 140.7 (C5), 121.7
(C6), 71.8 (C3), 56.8 (C14), 56.1 (C17), 50.1 (C9), 45.8 (C24), 42.3 (C13), 42.3 (C4),
39.8 (C12), 37.2 (C1), 36.5 (C10), 36.1 (C20), 34.0 (C22), 31.9 (C8), 31.6 (C7), 29.7
(C2), 29.2 (C25), 28.2 (C16), 26.1 (C23), 24.3 (C15), 23.1 (C28), 21.1 (C11), 19.8
(C27), 19.3 (C19), 19.0 (C26), 18.8 (C21), 12.0 (C18), 11.8 (C29); LRESMS, m/z 415
(M+H)+); LREIMS, m/z 414 (M+), 396, 329, 315, 303, 273, 255, 231, 213, 57, 43 (100
+ %); HREIMS: calculated for C29H50O (M ): 414.3862, found 414.3846.
stigmasta-3,5-dien-7-one
Isolate LPF02: a non-UV active creamy pale yellow material (1.3 mg, 0.02 mg/g dried plant sample); LRESMS, m/z 411 (M+H)+, LREIMS, m/z 410 (M)+, 396, 395, 269,
187, 174, 161, 43 (100%).
6.4.2. Polar Constituents from Sub-fraction PF3 of SBeL
13C-NMR was obtained from absolute value display mode conversion of APT spectrum.
3′,4′,5,7-tetrahydroxyflavone-3-O-α-D-rhamnopyranosyl-(1Æ6)-O β-D-glucopyranoside.
71 Isolate PF3.1: a pale yellow solid (1.64 mg/g dried plant sample); mp. 184-188 ºC; 1H-
3 3 NMR (CD3OD, 500 MHz), 7.67 (s, 1H, H2′), 7.62 (d, J = 8.5 Hz, 1H, H6′), 6.88 (d, J
= 8.5 Hz, 1H, H5′), 6.40 (d, 4J = 2.0 Hz, 1H, H8), 6.21 (d, 4J = 2.0 Hz, 1H, H6), 5.10 (d,
3J = 7.5 Hz, 1H, H1′′), 4.52 (s, 3J ≤ 1 Hz, 1H, H1′′′), 3.80 (dd, 2J = 10.9, 3J = 8.5 Hz,
1H, H6B′′), 3.64 (m, 1H, H2′′′), 3.54 (m, 1H, H3′′′), 3.48 (m, 1H, H2′′), 3.45 (m, 1H,
H5′′′), 3.42 (m, 1H, H3′′), 3.39 (dd, 2J = 10.9, 3J = 8.5 Hz, 1H, H6A′′), 3.33 (m, 1H,
H5′′), 3.27 (m, 1H, H4′′), 3.27 (m, 1H, H4′′′), 1.12 (d, 3J = 6.5 Hz, 3H, H6′′′); 13C-NMR
(CD3OD, 125 MHz), 179.4 (C4), 166.0 (C7), 162.9 (C5), 159.3 (C2), 158.5 (C9), 149.8
(C4′), 145.8 (C3′), 135.6 (C3), 123.6 (C6′), 123.1 (C1′), 117.7 (C2′), 116.0 (C5′), 105.6
(C10), 104.7 (C1′′), 102.6 (C1′′′), 99.9 (C-6), 94.7 (C8), 78.1 (C5′′), 77.2 (C3′′), 75.7
(C2′′), 73.9 (C4″′), 72.2 (C3′′′), 72.1 (C2′′′), 71.4 (C4′′), 69.7 (C5′′′), 68.5 (C6′′), 17.9
+ + (C6′′′); LRESMS, m/z 611 (M+H) ; HRESMS: calculated for C27H30O16Na (M+Na) :
633.1432, found 633.1443.
3′,4′,5,8-tetrahydroxyflavone-3-O-hexopyranoside
Isolate PF3.2: a yellow-brown solid (3.8 mg, 0.16 mg/g dried plant sample); 1H-NMR
3 3 (CD3OD, 500 MHz), 7.71 (s, 1H, H2′), 7.60 (d, J = 7.5 Hz, 1H, H6′), 6.87 (d, J = 7.5
Hz, 1H, H5′), 6.40 (s, 1H, H8), 6.21 (s, 1H, H6), 5.21 (m, 1H, H1″), 3.82 (m, 1H,
H6B″), 3.48 (m, 1H, H2″), 3.42 (m, 1H, H3″), 3.40 (m, 1H, H6A″), 3.34 (m, 1H, H5″),
13 3.21 (m, 1H, H4″); C-NMR (CD3OD, 125 MHz), 179.5 (C4), 166.1 (C7), 162.3 (C5),
159.2 (C2), 158.5 (C9), 149.9 (C4′), 145.9 (C3′), 135.7 (C3), 123.2 (C1′), 122.2 (C6′),
72 116.4 (C2′), 114.9 (C5′), 104.5 (C10), 103.3 (C1″), 99.9 (C6), 93.0 (C8), 78.2 (C3″),
77.7 (C5″), 75.2 (C2″), 70.9 (C4″), 62.1 (C6″); LRESMS, m/z 465 (M+H)+.
3′,5,7-trihydroxyflavone-3-O-hexopyranoside-O-deoxyhexopyranoside
Isolate PF3.2a: a yellow-brown solid (3.2 mg, 0.13 mg/g dried plant sample); 1H-NMR
(CD3OD, 500 MHz), 8.20 (m, 1H, H2′), 8.07 (m, 1H, H5′), 7.98 (m, 1H, H4′), 6.91 (m,
1H, H6′), 6.42 (s, 1H, H8), 6.21 (s, 1H, H6), 5.12 (m, 1H, H1″), 4.52 (m, 1H, H1′′′),
1.11 (m, 3H, H6′′′); LRESMS, m/z 595 (M+H)+, m/z 617 (M+Na)+; HRESMS:
+ calculated for C27H30O15Na (M+Na) : 617.1482, found 617.1487.
4′,5,7-tetrahydroxy-3′-methoxyflavone 3-O-α-D-rhamnopyranosyl-(1Æ6)-O-β-D-glucopyranoside.
Isolate PF3.3: a pale yellow solid (10.5 mg, 0.43 mg/g dried plant sample); mp. >250
1 3 ºC; H-NMR (CD3OD, 500 MHz), 7.94 (s, 1H, H2′), 7.62 (d, J = 8.5 Hz, 1H, H6′),
6.91 (d, 3J = 8.5 Hz, 1H, H5′), 6.41 (s, 1H, H8), 6.21 (s, 1H, H6), 5.23 (d, 3J = 7.0 Hz,
3 2 1H, H1′′), 4.53 (s, J≤1 Hz, 1H, H1′′′), 3.95 (s, 3H, OCH3), 3.82 (dd, J = 11.0, 1H,
H6B′′), 3.62 (s, 3J≤1 Hz, 1H, H2′′′), 3.49 (dd, 3J = 9.0, 6.5 Hz, 1H, H2′′), 3.49 (dd, 3J =
9.0, 2.5 Hz, 1H, H3′′′), 3.46 (dd, 3J = 9.0, 9.5 Hz, 1H, H3′′), 3.42 (m, 1H, H6A′′), 3.42
73 (dd, 3J = 9.5, 6.0 Hz, 1H, H5′′′), 3.38 (dd, 3J = 10.0, 6.0 Hz, 1H, H5′′), 3.26 (dd, 3J =
9.5, 9.5Hz, 1H, H4′′′), 3.26 (dd, 3J = 9.5, 7.0 Hz, 1H, H4′′), 1.10 (d, 3J = 6.0 Hz, 3H,
13 H6′′′); C-NMR (CD3OD, 125 MHz), 179.3 (C4), 166.0 (C7), 163.0 (C5), 158.9 (C2),
158.5 (C9), 150.8 (C4′), 148.3 (C3′), 135.5 (C3), 124.0 (C6′), 123.4 (C1′), 116.1 (C5′),
114.6 (C2′), 105.7 (C10), 104.4 (C1′′), 102.5 (C1″′), 100.0 (C6), 94.9 (C8), 78.2 (C3′′),
77.3 (C5′′), 75.9 (C2′′), 73.8 (C4″′), 72.3 (C3″′), 72.1 (C2″′), 71.6 (C4′′), 69.8 (C5″′),
+ 68.5 (C6′′), 56.8 (C (OCH3), 17.9 (C6″′); LRESMS m/z 625 (M+H) ; HRESMS:
+ calculated for C28H33O16 (M+H) : 625.1769, found 625.1743.
6.5. Acylated PF3.1
6.5.1. Semi-synthetic Procedure
OAc H O OAc 2' OAc OAc 8 B H OAc AcO O H H 5' H A C 6' O 6 H O O H OAc O OAc H OAc HAcO H 3′,4′,5,7-tetraacylflavone-3-O-α-D-2′′′,3′′′,4′′′-triacylrhamnopyranoside-(1Æ6)-O-β-D-2′′,3′′,4′′- triacylglucopyranoside.
To a solution of PF3.1 (7.6 mg) in acetic acid anhydride (0.5 mL) was added pyridine
(0.5 mL). The mixture was then stirred at RT for 48 h. The reaction was monitored by
LRESMS. Water (5 mL) was then added followed by extraction using EtOAc (3 x 20 mL). The combined organic layers were washed with NaHCO3 (2x10 mL) and brine solution (10 mL), dried (MgSO4) and then concentrated producing the acylated product
1 (12.0 mg, 94%) as a yellow solid; mp 130-134 ºC; H-NMR (CDCl3, 500 MHz), 7.94
(d, 3J = 9 Hz, 1H, H6′), 7.89 (s, 4J = 2.0 Hz, 1H, H2′), 7.33 (d, 3J = 8.5, 4J=2.0 Hz, 1H,
74 H5′), 7.30 (d, 4J = 2.0 Hz, 1H, H8), 6.82 (d, 4J = 2.0 Hz, 1H, H6), 5.43 (d, 3J = 8.0 Hz,
1H, H1′′), 5.26 (dd, 3J = 10.0, 9.5 Hz, 1H, H3′′), 5.16 (dd, 3J = 10.0, 8.0 Hz, 1H, H2′′),
5.07 (d, 3J = 3.0 Hz, 1H, H2′′′), 5.06 (dd, 3J = 9.0, 3.0 Hz, 1H, H3′′′), 4.93 (t, 3J = 9.5
Hz, 1H, H4′′), 4.93 (t, 3J = 9.0 Hz, 1H, H4′′′), 4.50 (s, 3J≤1 Hz, 1H, H1′′′), 3.64 (m, 1H,
H5′′′), 3.57 (ddd, 3J = 9.0, 6.0, 3.0 Hz, 1H, H5′′), 3.52 (dd, 2J = 11.0, 3J = 3.0 Hz, 1H,
2 3 H6A′′), 3.22 (dd, J = 11.0, J = 6.0 Hz, 1H, H6B′′), 2.44 (s, 3H, OCOCH3-A), 2.34 (s,
3H, OCOCH3-C), 2.34 (s, 3H, OCOCH3-B), 2.29 (s, 3H, OCOCH3-D), 2.14 (s, 3H,
OCOCH3-E), 2.08 (s, 3H, OCOCH3-F), 2.04 (s, 3H, OCOCH3-G), 2.02 (s, 3H,
3 OCOCH3-H), 1.96 (s, 3H, OCOCH3-I), 1.94 (s, 3H, OCOCH3-J), 1.05 (d, J = 6.5 Hz,
13 3H, H6′′′); C-NMR (CDCl3, 125 MHz), 171.9 (C4), 170.2 (OCOCH3-G), 170.1
(OCOCH3-I), 170.1 (OCOCH3-J), 169.9 (OCOCH3-H), 169.7 (OCOCH3-E), 169.6
(OCOCH3-F), 169.2 (OCOCH3-A), 168.1 (OCOCH3-B), 167.9 (OCOCH3-C), 167.7
(OCOCH3-D), 156.6 (C7), 154.7 (C2), 154.0 (C9), 150.2 (C5), 144.1 (C4′), 141.8 (C3′),
137.0 (C3), 128.6 (C1′), 127.2 (C6′), 124.7 (C2′), 123.5 (C5′), 115.1 (C10), 113.4 (C6),
109.0 (C8), 99.6 (C1′′), 97.8 (C1′′′), 72.8 (C5′′), 72.6 (C3′′), 71.4 (C2′′), 70.9 (C4′′′),
69.5 (C4′′), 69.4 (C2′′′), 69.0 (C3′′′), 67.0 (C6′′), 66.4 (C5′′′), 21.2 (OCOCH3-A),
21.1(OCOCH3-B), 21.1 (OCOCH3-C), 20.9 (OCOCH3-D), 20.8 (OCOCH3-E), 20.7
(OCOCH3-F), 20.7 (OCOCH3-G), 20.6 (OCOCH3-H), 20.6 (OCOCH3-I), 20.6
+ (OCOCH3-J), 17.2 (C6′′′); LRESMS, m/z 1053 (M+Na) ; HRESMS: calculated for
+ C47H50O26Na (M+Na) : 1053.2488, found 1053.2500.
75
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83
84 Appendixes
Appendix 1. Extraction protocol
This research protocol was started from extraction, re-extraction, and chromatographic separation and isolation. Preparative TLC and preparative HPLC were the key step to obtain the pure isolates.
85 Appendix 2. 1H- (top), 13C-NMR (middle), DEPT (bottom) spectra of LPF01
HO H-6 H-3
* Solvent * *
* = impurities C-7 C-7 -8 C -8 C-2 C-2 C-25 C-25 4 4 C-4 C-4 C-2 C-9 C-9 -13 C -13 C-12 C-1 -20 -20 C-10 C-10
C C-22 C-22 C-14 C-14 C-17 C-17 27 27 C-11 C-11 C-26 C-28 C-28 C-15 C-15 C-21 C-21 C-19 C-16 C-16 C-23 C-23 C-29 C-29 C- -18 C -18 C-3 C-3 C-6 C-6 C-5 C-5
86 Appendix 3. gCOSY spectra of LPF01
87 Appendix 4. gTOCSY spectra of LPF01
88 Appendix 5. gHSQC spectra of LPF01
89 Appendix 6. gHMBC spectra of LPF01
90 Appendix 7. 1H-NMR spectra of PF3.1
H-2' H-6' H-5' H-8 H-6 H-1" H-1'" H-6'"
H-4" H-6A" H-2'" H-3'" H-4'"
91 Appendix 8. 13C-NMR and APT (bottom) spectraof PF3.1
4''' 4''' C-3''' C-3''' C- C-2''' C-3'' C-3'' C-5''' C-5'' C-5'' C-4'' C-4'' C-6'' C-6'' C-2'' C-2'' C-1''' C-1''' 10 10 C-8 C-8 C-6 C-6 C- C-1'' C-1'' C-2' C-2' C-6' C-6' C-5' C-5' C-1' C-1' C-6''' C-6''' C-3 C-3 C-3' C-3' C-4' C-4' C-2 C-2 C-9 C-9 C-7 C-7 C-5 C-5 C-4 C-4
C-2''' C-2''' C-3''' C-3''' C-5''' C-5''' C-4''' C-4''' C-4'' C-4'' C-5'' C-5'' C-2'' C-2'' C-3'' C-3'' C-6'' C-6''
92 Appendix 9. gCOSY spectrum of PF3.1
93 Appendix 10. TOCSY spectra of PF3.1
94 Appendix 11. gHSQC spectra of PF3.1
95 Appendix 12. gHMBC spectra of PF3.1
96 Appendix 13. 1H-NMR spectra of PF3.3
H-5' H-6' H-2' H-8 H-6 H-1" H-1'" H-6'"
H-6B" H-4" H-3" H-4'" H-2" H-5'" H-3'" H-5"
-OCH3
H-6A" H-2'"
97 Appendix 14. 13C-NMR, APT (bottom) spectra of PF3.3
C-(OMe) C-(OMe) C-4''' C-4''' C-3''' C-3''' C-2''' C-2''' C-5'' C-5'' C-5''' C-5''' C-3'' C-3'' C-4'' C-4'' C-2'' C-2'' C-6'' C-6'' C-6''' C-6''' 10 10 C-1''' C-1''' C-8 C-8 C-6 C-6 C- C-1'' C-1'' C-2' C-2' C-6' C-6' C-5' C-5' C-1' C-1' C-3 C-3 C-3' C-3' C-4' C-4' C-2 C-2 C-9 C-9 C-7 C-7 C-5 C-4 C-4
C-2''' C-2''' C-3''' C-3''' C-5''' C-5''' C-4''' C-4''' C-5'' C-5'' C-6'' C-6'' C-4'' C-4'' C-2'' C-2'' C-3'' C-3''
98 Appendix 15. gCOSY spectra of PF3.3
99 Appendix 16. TOCSY spectra of PF3.3
100 Appendix 17. gHSQC spectra of PF3.3
101 Appendix 18. gHMBC spectra of PF3.3
102 Appendix 19. 1H-NMR spectra of acetylated PF3.1
H-1"
H-6'"
H-5' H-8 H-1" H-1'" H-6' H-2' H-6
H-4" H-1" H-3" H-4'" H-1'" H-6A" H-6B"
H-2" H-2'" H-5'" H-5" H-3'"
-OCH3-H
-OCH3-C -OCH3-J
-OCH3-F -OCH -A -OCH3-I 3 -OCH3-D -OCH3-G
-OCH3-B -OCH3-E
103 Appendix 20. 13C-NMR spectra of acetylated PF3.1
' C-5'' C-5'' C-5'' C-5'' C-2'' C-2' C-2'' C-3'' C-3'' C-3'' C-3'' -G -G 3 H C -OCO -F -F 3 H C - H - H 3 -I,J -D 3 3 H -E -E -OCO 3 H H -A C
3 -B,C -B,C C H C -D 3
3 H -C C H
3 C -B
-A 3 C
3 -F -OCO 3 -E -OCO -OCO OCH 3 -H
3 OCH -OCO C -I, J OCH -OCO 3 C OCH -G -G C-6''' C-6''' -OCO C 3 -O OCH C
C-5''' C-5''' C-5''' C-5''' OCH -O C-5 C-5 C C-9 C-9 C-3''' OCH C-6'' C-6'' C-6'' C-6'' C-2 C-2 -O C-7 C-7 C-3' C-3' C C-2''' C-2''' C-2''' C-2''' -O C-4' C-4' C-3 C-3 C-4'' C-4'' C OCH -O C-5' C-5' C-4''' C-4''' C-4''' C-4''' 10 10 C-6' C-1''' C-1''' C-2' C-2' OCH -O C C-1' C-1' -O C-6 C-6 C C-8 C-8 C- C-1'' C-1'' -O -O C-4 C-4
2''' 2''' C- C-4'' C-4'' C-4''' C-4''' C-3''' C-3''' C-5''' C-5''' C-5'' C-5'' C-2'' C-2'' C-3'' C-3'' C-6'' C-6''
104 Appendix 21. gCOSY spectra of acetylated PF3.1
105 Appendix 22. TOCSY spectrum of acetylated PF3.1
106 Appendix 23. gHSQC spectra of acetylated PF3.1
107
108 Appendix 24. gHMBC spectra of acetylated PF3.1
109 Appendix 25. HREIMS of LPF01 (up) and HRESMS of PF3.1 (bottom)
110 Appendix 26. HRESMS of PF3.2a (up) and PF3.3 (bottom)
111
Appendix 27. HRESMS of acetylated PF3.1
112