9
1.1 : Natural products and plant defense.
Plants have to defend themselves from soil and air bacteria and fungi, so over millions of years they developed biochemical pathways to produce antimicrobial molecules. These form part of a huge repertoire of chemicals called secondary metabolites. They are chemically dissimilar (e. g. flavonoids, alkaloids, phenylpropanoids, coumarins, terpenoids…) and have different targets of activity.
Not all secondary metabolites are synthetized with the aim of protecting the plant from microorganisms : some of them have the function of pollinator attractors or act as anti-herbivore, some others are antioxidants, protect against UV-light and other abiotic stress, serve as allelopathic agents or help wound-healing after an insect or herbivores’ damage. Secondary metabolites influence interactions of the plant with the environment and other organisms (Anne Osbourn, New Phytologist, 2006). For example, many monoterpenoids have insecticidal properties, such as pyrethroids, α and β-pinene, limonene and myrcene (Mazid et al., 2011). Sesquiterpenoids play an important role as well in defense against herbivorous animals (e.g. costunolides), plant response to water-stress, oxidative and UV-light stress (e.g. abscisic acid) (Mazid et al., 2011).
Each plant species produces a different assortment of chemicals and there is often variation between two plants of the same species. However there are some similarities in classes of chemicals synthetized within the same Family. This chemical biodiversity is a precious resource to investigate, because it may be a source of useful chemicals for therapeutic use and it is still mostly unknown. (Dixon, 2001 ; Mazid et al., 2011)
10
1.2 : Natural products and drug discovery: botany and
ethno-pharmacology.
More than a half of existing drugs are derived from plants. To date, natural products-related molecules continue to be discovered and accepted as active ingredients of pharmaceuticals, some being the basis for new chemical classes of compounds (Harvey, 2008).
These natural products are effectively used in a wide range of therapeutic conditions, such as anti-infective, anti-diabetic, cardiovascular, gastrointestinal, anti-inflammation, anti-cancer. They have extremely variable chemical structures and they are usually absorbed more quickly than synthetic drugs which is a remarkable benefit for the drug discovery process (Harvey, 2008; Lamari and Cordopatis, 2008).
Despite the notable advantages related to natural products, like the well-known traditional use in folk medicines and the higher affordability especially for rural inhabitants in underdeveloped countries (80% of the whole world population depends on natural remedies), the lower environmental impact and health risks associated with them compared with synthetic chemicals, large pharmaceutical companies have stop research on them owing to certain difficulties, such as:
- Provision of the plant material for extraction (risk of impoverishment of the flora, agreements with local authorities, storage).
- Chemical complexity of biosynthesized molecules (stereogenic centers, high molecular weight) and difficulty to synthetize them in the laboratories.
- Chemical complexity of extracts, which are a miscellanea of natural molecules (lack of reproducibility of composition, interferences between compounds) - Problems to take out a patent on a natural product.
(Lamari and Cordopatis, 2008; Harvey, 2008).
Natural products are not only investigated as possible drugs, and have recently regained popularity among people and industries (especially food, agriculture, cosmetics and perfume), because of their numerous fields of applications. Natural chemicals could represent a good substitute of preservatives and pesticides used till now. A particular field of application of natural products is as pharmacological tools: they can help to discover new aspects of physiology by binding biological receptors. There are a lot of
11
examples of this possible application, like nicotine, muscarine, tubocurarine, morphine etc.
If we look at data for 2008, we can find that there were more than 100 natural products from plants at different stages of development (preclinical, phase 1, phase 2, phase 3, preregistration) waiting to come on the market. There were also products coming from microbes (bacteria and fungi) and animals: which underlines the fact that there is a very wide source of molecules present in nature, which is not only restricted to plants.
(Harvey, 2008)
Only a small amount of the total biodiversity that nature offers us has been screened, estimated at less than 152 of the world flowering plants. A lot of work has been done on plants, but even so in this field the greater bulk is still unexplored. (Vuorela et al., 2004)
An issue which is not to be undervalued while studying natural agents is that research undertaken to isolate structurally characterized compounds are both expensive and time consuming: that is one reason why studies are often initially focused on crude extracts. An important topic to discuss is the criterion (or criteria) adopted in the choice of the plant extract to study. How can we forecast which plant is more suitable for our research purposes?
There are various approaches that can be followed:
- Random screenings: those are possible only for big pharmaceutical companies which can rely on automated cutting-edge high-throughput screening assays and purification and isolation techniques.
- Rational screenings, like: o Taxonomic approach o Phytochemical approach o Chemical ecology approach o Ethno-pharmacology approach
The taxonomic approach consists in the screening among a particular Family or Genus which has one or more species acknowledged for a curative property. For example, since it is established that Taxus brevifolia extracts have anti-cancer properties, one can
12
screen other species of the Genus Taxus which probably have similar biochemical pathways and produce anti-cancer compounds as well. This method may lead to a better supply of extracts, but unfortunately decreases the possibility to find novel chemical structures.
The phytochemical approach consists in the screening among plants which contain the same classes of compounds. For example, if a plant containing mainly indole-alkaloids has anti-cancer properties, we look for this action in other plants containing indole-alkaloids. This method also leads to a better supply of extracts, but decreases the possibility to find novel chemical structures.
The chemical ecology approach studies the interaction among organisms and between organisms and their environment, for example poisonous plants for insects or herbivores or the plants chosen by animals to cure themselves. This method give the chance to discover new-structured compounds, but not all chemicals active on animals are active as well on humans (Harborne, 2001).
The ethno-pharmacology approach is the one which leads to the best results: plants collected after ethno-pharmacologic surveys often show higher in-vitro activity than randomly chosen plants (Khafagi and Deweder, 2000) and this approach allows the discovery of new-structured chemicals.
Ethno-pharmacology is a multidisciplinary subject which requires notions of pharmacology, ethnology, botany and medicine. It comprises both anthropological field studies and studies of ancient literature, because often the traditional medicine is oral-transmitted in underdeveloped countries or tribes. This is quite a big issue for researchers in this field, because they have to find a way to communicate with healers and shamans in their language; furthermore the risk of loss of these folk remedies is higher if they are not written down, so the investigations should be made as quickly as possible. The interviews between the ethno-pharmacologist and the traditional healer give the researcher a lot of cues about how to carry on his study. For example the pharmaceutical form (decoction, infusion in alcohol, powder) is often an indication of the proper extraction method to use. The part of the body which is supposed to heal is an indication of the therapeutic properties (for example, if a plant is used as anti-infective to heal skin wounds, it probably can be active against bacteria and fungal
13
pathogens of skin as well and so it has to be tested first of all on them). Likewise, the duration of the cure is an indication of the strength of the activity etc.
The fundamental steps of an ethno-pharmacologic investigation are the following: - Plants and medicinal uses by indigenous healers have to be recorded - Samples of the plants have to be taken back and identified by botanists
- The herbal formulas, the dosage schedules, the period of therapy and the side effects need to be observed and noted.
The mission of the ethno-pharmacologist is not only to discover traditional remedies, but also to offer improved alternatives taken by western medicine to the tribe healers. So it could be an exchange of information and a development of global health, since around 80% of world population still relies on folk medicine to treat its diseases. (Lamari and Cordopatis, 2008; “Manuale dell’erborista”, Morelli et al., 2006)
Ethno-pharmacologic surveys lay the foundations for further researches in order to validate scientifically the use of compounds previously used in popular medicine. The process of validation is hard, and passes through several steps:
- The selection of sources
- The screening and identification of active drugs or lead compounds - The in vitro and in vivo studies of activity and toxicity
- The production and synthesis (if possible) in high quantities - The preclinical and clinical evaluation
- The approval
- The development of analogues with better characteristics
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1.3 : Antimicrobial agents : natural, synthetic and natural scientifically
researched.
The discovering of antimicrobial agents followed the same trend of all other drugs: initially they came from natural sources according to folk medicines (to preserve food, to heal diseases, especially skin and gastrointestinal, to prevent contaminations etc.). With the progresses in synthetic chemistry, natural treatments were considered obsolete and researchers focused on the purpose to find new synthetic antibiotics, which for sure gave people a precious instrument to limit diffusion of a lot of microbial diseases. Unfortunately most of molecules already discovered are progressively losing their efficacy, due to careless use or abuse of taking them even when there is no need. Numerous bacteria and fungi are developing resistance to a wider range of antibiotics. Some of the best known examples are penicillin –resistant Streptococcus pneumonia (PRSP), methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), amphotericin-B and azole-resistant Candida spp. . There are also multidrug-resistant strains (Quave, 2003). Another problem relating to antibiotics are side effects on the body and include problems such as immune-suppression, allergic reactions or hypersensitivity. Therefore the fact that research has intensified to find new antibiotics is not surprising and one of the latest trend is to study novel natural agents scientifically researched.
Several reasons therefore support the effort to seek different antimicrobial agents in natural products including plants. These are:
• Bacteria and fungi develop resistance to essential oils or other extracts with more difficulty, because EOs are a mixture of chemicals in which often more than one component is active and they generally are synergic; it is harder for microbes to become resistant to all of them than if there is a single active compound.
• Antimicrobial products discovered after ethno-pharmacologic investigations usually rely on a very old tradition of use, so they were been tested on humans for a long time.
• The use of plant-derived products in agriculture (for example fungicides) should have lower environmental impact and health risks than synthetic products.
15
• The usage of natural preservatives in food and cosmetics should reduce the risk of allergies and should have fewer health consequences for a prolonged exposure to them.
1.4 : Mode of action of antibiotics.
The ideal antibacterial is a single molecule but more often a natural extract (which is a mixture of a lot of chemicals) that is able to inhibit the growth of bacteria but does not cause any damage to the mammalian cells.
How could it be possible? The answer can be found if we look at the differences between eukaryotes and prokaryotes organisms.
Bacteria are prokaryotic organisms, being always unicellular, they do not have a well-defined membrane-bound nucleus, they lack any other membrane-bound organelles and cytoskeleton. They only have a circular DNA without histone proteins and smaller ribosomes. Their reproduction is always asexual, their division is by binary fission. They also have a huge variety of metabolic pathways which are very dissimilar from eukaryotic organisms. Their cell wall is made of murein, a peptidoglycan interspersed with glycoproteins. They can be distinguished into two main group: Gram positive and Gram negative. The Gram positives are spore formers. They have a thick cell wall, they may have a capsule, a thick outer layer made of polysaccharides. They are penicillin susceptible. The Gram negatives have a thinner cell wall and an outer lipid layer, they have no capsule and they are not penicillin susceptible.
Conversely mammals and plants are eukaryotic organisms. Eukaryotic cells have a well-developed nucleus, they have membrane-bound organelles (as mitochondria) and cytoskeleton, DNA is linear and associated with proteins to form chromatin, they have larger ribosomes, their cell division is by mitosis or meiosis. Animal cells have not any cell wall.
Antibacterial agents can benefit from the differences between eukaryotic and prokaryotic, with several possible targets such as the cell wall, the cell membrane, the synthesis of nucleic acids, the synthesis of proteins and the energy metabolism.
16
1.5 : Essential oils.
Essential oils (EOs) are mixture of lipophilic and volatile chemicals extracted from plants.
The part of the plant from which essential oil is extracted is referred to as the drug (Herba medica). Different parts of the same plant can produce essential oils though not of the same composition, so it is important to specify from which drug the essential oil is derived. Frequently EOs are the most bioactive plant extracts, probably because they are highly concentrated.
Not all plants produce essential oils and also the yield varies among families.
Even between individuals of the same specie there are factors which may influence the yield and the composition of the essential oil. This will be discussed later.
There are different ways to extract EOs:
• Hydro-distillation with Clevenger apparatus
• Extraction by supercritical fluids (e.g. carbon dioxide)
• Enfleurage technique
• Pericarps maceration or cold pressing
These methods have their own advantages and disadvantages: for example hydro-distillation, which is the most wide-spread, is quite a cheap technique, but it takes a long time to be execute (usually about three hours) and the prolonged exposition of the plant material to the heat can lead to deterioration of some componenst of the EO. Extraction by supercritical fluids is quicker and does not require high temperatures, but it is expensive and needs a specific apparatus. Enfleurage is an ancient method which consists in putting petals of some flowers onto a thin layer of fat material like butter or lard and leaving the hydrophobic EO to diffuse in it for some days; the butter is then extracted with ethanol. This historical technique is really slow, but it is still used to produce special EOs from highly degradable plant materials. Pericarp maceration or cold pressing is used only with citrus fruits, which have a very high quantity of EO in oil glands situated in their external layer.
These oils are widely used in the cosmetic and perfume industries because they are often aromatic and good-smelling, in the food industry to enrich the aroma of products
17
and to preserve them, in the pharmaceutical industry both as excipients (to modify the smell or the flavour of a medicine) and as active ingredients, because some of them have particular medicinal properties. They have to be used with care, because some of them can be dangerous. For example there are some potential toxic effects of sage essential oils, especially on the liver, and because of the neurotoxicity of thujones some nations have imposed restrictions on their use. Due to all these implications, it is necessary to standardize EOs of the same species so they have the as uniform a composition as possible.
(“Manuale dell’erborista”, Morelli et al., 2006; “Manuale di Botanica Farmaceutica”, Maugini
et al., 2006; “Farmacognosia generale e applicata”, Bruni 1999; “Dizionario di fitoterapia e
piante medicinali”, Campanini 2008; “Chimica, biosintesi e bioattività delle sostanze naturali”, Fattorusso 2001; “Metabolismo e prodotti secondari delle piante”, Maffei 2003)
1.5.1 : Terpenes.
The main components of essential oils, especially in Lamiaceae family, are terpenoids, mostly monoterpenoids (C10) and sesquiterpenoids (C15). The terpenes which have a bigger number of carbons (C20, C30, C40) are usually not volatile and so they are not present in the essential oils.
Table 1.1 : Classification of Terpenes
Name Number of carbons Number of isoprene
units (C5) Monoterpenes 10 2 Sesquiterpenes 15 3 Diterpenes 20 4 Sesteterpenes 25 5 Triterpenes 30 6 Tetraterpenes 40 8
18
The five carbon isoprene unit (C5) can be formed through two different pathways: the mevalonate-dependent biosynthesis and the mevalonate-independent biosynthesis.
Piruvate + glyceraldehyde-3P
Figure 1.1: Terpenes biosynthesis. DXP = 1-deoxy-D-xylulose-5-phosphate; MEP= 2-methylerythrol-4-phosphate
(organicavirtuale.altervista.org)
Figure 1.2: Two pathways of the terpenes biosynthesis with the inter-conversion of isopentenyl pyrophosphate to dimethylallyl pyrophosphate
19
When the isopentenyl- pyrophosphate is formed, it can rearrange to dimethylallyl- pyrophosphate: the condensation reaction between IPP and DMAPP leads to Geranyl- pyrophosphate, which is the precursor of all monoterpenes.
Figure 1.3: Last step of the monoterpenes biosynthesis
Monoterpenes derived from the condensation of two isoprene monomers. They boil in a range of 140-180 ° C and can be classified as acyclic (like geraniol), monocyclic (like limonene) and byciclic (like α and β-pinene).
Figure 1.4: Image of cyclization of terpenes
They may be functionalized, generally by oxidation, to become alcohols (e.g. menthol) , aldehydes (e.g. citronellal) or ketones (e.g. menthone).
Isopentenyl-PP Dimethylallyl-PP Geranyl-PP Monoterpenes Linalyl cation Α-terpinyl cation Pinyl cation
20
Sesquiterpenes are derived from the condensation of geranyl pyrophosphate with isopentenyl pyrophosphate: the result is the 15-carbon farnesyl pyrophosphate, the precursor of all of them. Their boiling point is above 200 ° C and they can be classified as acyclic (like farnesol or neridol), monocyclic (like bisabolene or zingiberene), bicyclic (like cadinene, selinene, azulene ) and tricyclic (like cedrene).
farnesyl pyrophosphate
Figure 1.5: Image of the farnesyl pyrophosphate, the precursor of all sesquiterpenes.
(“Chimica, biosintesi e bioattività delle sostanze naturali”, Fattorusso 2001; “Metabolismo e prodotti secondari delle piante”, Maffei 2003)
21
1.6 : The Lamiaceae Family.
Lamiaceae, also called Labiatae or the Mint Family, is the largest family of the Order Lamiales. It consists of 236 Genera and about 7000 species worldwide but especially in Africa and temperate boreal regions. A lot are endemic in the Mediterranean zones. Most of Lamiaceae are perennial or annual herbs, sub-shrubs and shrubs. They usually have square stems and opposite, paired and simple leaves, which can be toothed and without stipules. Leaves are usually covered by glandular hairs which produce and contain essential oil. Their flowers have two-lipped, tubular corollas (five united petals), with five-lobed calyxes (united sepals) and they grow in long clusters, heads, or interrupted whorls on the stem; they are bisexual and zygomorphic. The fruit has 4 nutlets, each forming a single-seeded achene.
22
Figure 1.7: Typical features of the Mint Family flowers
23
Many members of the Lamiaceae family are of high economic interest due to their flavour, fragrance or medicinal properties. Therefore a lot of Lamiaceae are used in food, cosmetic, perfume and pharmaceutical industries. They are also commonly used as spices and in folk medicine. The most known and used Lamiaceae are: lemon balm (Melissa officinalis), basil (Ocimum basilicum), hyssop (Hyssopus spp.), lavender (Lavanda spp.), marjoram (Origanum majorana), mint (Mentha spp.), oregano (Origanum vulgare), rosemary (Rosmarinus officinalis), sage (Salvia spp.), savory (Satureja hortensis), thyme (Thymus vulgaris).
The smell, the flavour and the pharmaceutical properties associated with Lamiaceae are from essential oils, produced and stored in special glandular hairs or trichomes. EOs are typically rich in terpenoids, which are aromatic and volatile molecules (especially mono and sesquiterpenes).
Figure 1.9: Glandular hairs and trichomes
(“Manuale dell’erborista”, Morelli et al., 2006; “Manuale di Botanica Farmaceutica”, Maugini
24
1.7 : Salvia species.
1.7.1 : Botanic information and distribution.
Kingdom: Plantae (Plants)
Subkingdom: Tracheobionta (Vascular plants) Super-division: Spermatophyta (Seed plants) Division: Magnoliophyta (Flowering plants) Class: Magnoliopsida (Dicotyledons)
Subclass: Asteridae Order: Lamiales
Family: Lamiaceae (Mint family) Genus: Salvia L. (sage)
The genus Salvia belongs to the Lamiaceae and it is particularly rich in both annual and perennial species, used as herbs or spices, for therapeutic purposes or simply as ornamentals in gardens. It is the largest genus, with about 900 species organized in five subgenera (Audibertia, Jungia, Leonia, Salvia and Sclarea).
Sage is very wide-spread in all mild climate countries (mainly Central and South America, Central and Eastern Asia, the Pacific Islands, tropical Africa and the Mediterranean zone) and it usually forms shrubs. Branches are first green and they become woody when the plant matures, they are square in section and very ramified. The shrubs can reach one meter in height or more, depending on the species.
The genus name derives from Latin “salvus”, which means “healthy”, or “salus”, which means “health” and due to the virtues as a curative plant that Romans awarded to sage.
1.7.2 : Salvia officinalis.
The most common herb and spice of this genus is Salvia officinalis (or common sage), a perennial species native to European mild climate zones. It is an evergreen shrub which has green-grey and lightly hairy leaves and a particular smell. Leaves are oval with a crenate edge; flowers are grouped in purple vertical inflorescences and usually come into bloom in April- May in warm climates or June-July for more temperate areas.
25
(“Manuale dell’erborista”, Morelli et al., 2006; “Manuale di Botanica Farmaceutica”, Maugini
et al., 2006; “Farmacognosia generale e applicata”, Bruni 1999)
Figure 1.10 : Salvia officinalis scheme.
26 1.7.3 : Salvia somalensis.
Salvia somalensis (Somalia sage) is a perennial shrub growing endemically in Somalia.
It generally grows on mountains (1200-2100 m elevation) . Salvia somalensis can grow up 1.5 m, it has yellow-green oblong leaves and long terminal racemes with pale-blue flowers. The flowers and leaves are strongly aromatic.
Figure 1.13 : Salvia somalensis shrub.
Figure 1.14 : Salvia somalensis flower.
27 1.7.4 : Salvia dolomitica.
Salvia dolomitica (South African sage) is a perennial shrub endemic to South Africa (Transvaal). It usually grows at elevations of 900-1500 m. S. dolomitica can grow up 2 m, it has grey leaves and pale-lilac or pink flowers, which blooms during summer. It is called dolomitica because it grows on a type of rocky soil known as dolomite.
Figure 1.15 : Salvia dolomitica shrub
28 1.7.5 : Salvia patens.
Salvia patens (Gentian sage) is a perennial herbaceous native to the central Mexico. It
typically grows up 60-80 cm and it is very sensitive to frost. This sage is tuberous, it has bright-green elliptic-lanceolate leaves and electric-blue flowers which blooms from June to October.
Figure 1.18 : Salvia patens plant.
29
1.8 : Ethnobotany and traditional uses.
Sage has been used a lot since ancient times to heal several diseases and this genus has been treated like a sort of panacea. The Medical School of Salerno, one of the most famous during the Middle age, give it the appellation of “Salvia Salvatrix”, which means “Salvia the Savior”, due to its historical reputation for promoting health.
Many folk medicines all around the world uses their endemic Salvia species, for example:
• S. canariensis is used as anti-inflammatory , wound healing and antiseptic. Its
infusion is recommended for stomach complaints (Garcia Vallejo et al., 2005).
• S. bracteata and S. rubifolia are used to prevent sedation in patients affected by
tuberculosis, to cure bronchitis, to treat microbial infections, diabetes, cancer, malaria, anxiety, inflammation and to disinfect homes after sickness (Cardile et
al., 2009).
• Turkish Salvia species are used as antiseptic, stimulant, diuretic, wound-healing, diaphoretic, spasmolytic, stomachic and carminative agents (Nurhayat Tabanca et al 2006; Kotan et al., 2008).
• S. africana-lutea is used to treat coughs, colds, bronchitis and female ailments
(Watt & Breyer-Brandwijk 1962, Kamatou et al., 2009).
• Chinese Salvia is used to heal abscesses, calluses, hard swellings, warts and cancer (Kamatou et al., 2007).
• S. milthiorrhiza is used to treat hematological abnormalities, heart diseases,
hepatitis, hemorrhage, menstrual abnormalities and edema (Dong-Sun Lee et al., 1999).
• S. desoleana is used by Sardinians to treat menstrual, digestive and central nervous system diseases (Sokovic et al., 2008).
• S. fruticosa is used by Greeks as leaf infusion to treat inflammation of the mouth and the throat (Pitarokili et al., 2003).
• S. sclarea is used for coughs, colds, blood cleaning, on wounds and sore eyes, as
a diuretic, as a stomachic and anticonvulsive. It is also used in food and cosmetics (Pitarokili et al., 2002).
• S. officinalis is used as antidiabetic, antihydrotic, spasmolytic, antiseptic,
30
and for treating cardiovascular, mental and nervous conditions (Bouaziz et al., 2009).
• S. tomentosa is used in the treatment of flatulent dyspepsia, laryngitis, pharyngitis, stomatitis, gingivitis, glossitis, hyperhidrosis and galactorrhea (Zeki
et al., 2001).
• S. pomifera is used as an infusion for digestive, kidney and nervous disorders.
There is a lotion for ulcers and to heal abrasions of the skin (Glamoclija et
al.,2006).
1.9 : Bioactivity studies carried out to date on Salvia spp.
Various studies have been done on the bioactivity of Salvia species and the results usually give a scientific validation to their traditional uses. Most research has been done to test antimicrobial and antifungal activity, but there are also studies on cytotoxic, antioxidant, insecticidal, disinfectant, anti-inflammatory, antiplasmodial, antiviral activity, as shown in the following table.
Table 1.2: Summary of antimicrobial research using EO from Salvia species. Purple= study on bacteria. The EOs highlighted are the same tested in this research. For full details see the table 6.1 in the Appendix.
Article E.O. Microorganisms tested Method
1 Antibacterial and antimycobacterial activities of South African Salvia species and isolated compounds from S. chamelaeagnea (G.P.P. Kamatou et al., 2007) S. chamelaeagnea, S.dolomitica, … Staphylococcus aureus, E. coli, … Micro -dilution assay, radiometric tecnique 2 Trichomes, EO composition and biological activities of
Salvia albicaulis and S. dolomitica, two species from
Salvia albicaulis, S. dolomitica E. coli, Staphylococcus aureus,… Broth micro-dilution assay (micro-well dilution assay)
31 the Cape region of South
Africa (G.P.P. Kamatou et al., 2007) 3 Composition and antimicrobial activity of Salvia amplexicaulis EO
(Silvana Petrovic et al., 2009) Salvia amplexicaulis Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, E. coli, … Agar diffusion method Broth microdilution method 4 Antibacterial diterpenes from
the roots of Salvia blepharochlaena
(Ayhan Ulubelen et al., 2001) Diterpenes from Salvia blepharochlaena Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis… Disc-diffusion method
Tube dilution test
5 EOs of Salvia bracteata and
S. rubifolia from Lebanon:
chemical composition, antimicrobial activity and inhibitory effect on human melanoma cells
(Venera Cardile et al., 2009)
Salvia bracteata, S. rubifolia Staphylococcus aureus, Staphylococcus epidermitis, E. coli, Pseudomonas aeruginosa, … Broth micro-dilution assay
6 Chemical composition and biological activity of the EOs of S. canariensis
(M. C. García Vallejo et al., 2006) S. canariensis GRAM+: Staphylococcus aureus, S. epidermidis, Enterococcus faecalis GRAM-: Escherichia coli, Pseudomonas aeruginosa, … Two-fold dilution method
7 Antimicrobial activity of the ornamental species Salvia
corrugata, a potential new
crop for extractive purposes
Diterpene quinones from S.corrugata S. aureus (MSSA, MRSA) S. epidermidis (MSSE, MRSE), Enteroccocus faecalis Liquid spotted on agar plates Micro-dilution method
32
(Angela Bisio et al., 2008) (VAN-S, VAN-R), E. coli, Pseudomonas aeruginosa…
8 Antimicrobial and antioxidative activities of the EOs and methanol extracts of Salvia cryptantha and S.
multicaulis
(Bektas Tepea et al., 2004)
Salvia cryptantha, S. multicaulis Staphylococcus aureus, E. coli, Pseudomonas aeruginosa, … Disc diffusion assay Broth micro-dilution assay
9 Antimicrobial, cytotoxic and antiviral activities of Salvia
fructicosa EO
(Afroditi Sivropoulou et al. , 1997)
Salvia fruticosa E. coli, Pseudomonas aeruginosa, Staphylococcus aureus, … Disc diffusion assay 10 Antimicrobial and insecticidal activities of essential oil isolated from Turkish Salvia hydrangea (Recep Kotan et al., 2008)
Salvia hydrangea Pseudomonas sp., Enterococcus sp., Staphylococcus sp., E. coli, … Fungi: radial growth on PDA plates containing EO at a range of concentration Bacteria: disc diffusion assay 11 Stucture and antibacterial
activity of a new labdane diterpenoid from S. leriaefolia
(Zohreh Habibi et al., 2000)
Labdane diterpenoid from S. leriaefolia E. coli, Staphylococcus aureus, Enterococcus faecalis, Pseudomonas auroginosa Disc method of Kirby and Bauer
12 Antibacterial activity of different EOs obtained from spices widely used in Mediterranean diet
(Manuel Viuda-Martos et al., 2008) S. officinalis Lactobacillus curvatus, L. sakei, Staphylococcus carnosus … Agar disc diffusion
13 Antibacterial activity of EOs of S. officinalis and S. triloba
S. officinalis, S. triloba E. coli, Pseudomonas aeruginosa, Broth microdilution
33 cultivated in South Brazil
(Ana Paula Longaray Delamare et al., 2007) Staphylococcus aureus, S. epidermidis … susceptibility assay 14 Disinfectant properties of EOs from S. officinalis cultivated in Tunisia
(Mohamed Bouaziz et al., 2009)
S. officinalis E.coli, Pseudomonas Staphylococcus aureus, … Micro -dilution assay (resazurin as indicator) Agar vapor-inhibitory assay 15 Antimicrobial and antioxidant properties of rosemary and sage EOs (Biljana Bozin et al., 2007)
S. officinalis Pseudomonas aeruginosa, E. coli, Staphylococcus sp., … Micro-dilution technique Hole-plate agar diffusion method 16 Antibacterial activity studies
of flavonoids from S. palaestina
(Mahmutm Iski et al., 1983)
Flavonoids of S. palaestina E. coli, Staphylococcus aureus, S. epidermidis, Pseudomonas auroginosa… Disc diffusion method, tube-dilution test
17 Chemical composition and antimicrobial activity of EO of Salvia ringens baldacciana
(Katarina P. Šavikin et al., 2008) Salvia ringens baldacciana E.coli, Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, … Broth micro-dilution assay 18 Gas chromatographic-mass spectrometric analysis of volatiles obtained by four different techniques from
Salvia rosifolia and
evaluation for biological activity
(Gulmira Ozeka et al., 2010)
Salvia rosifolia Bacteria: E.coli, Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa … Broth micro-dilution assay Application of EO on TLC plates
19 Antibacterial screening and phytochemical study of nine
Crude extracts of Salvia schimperi, S. E.coli, Staphylococcus aureus, Pseudomonas Disc diffusion assay
34 medicinal plants from Eritrea
(Teklab Gebrehiwot et al., 2009)
merjamie, S. nilotica
aeruginosa…
20 Volatile constituents and antimicrobial activities of
Salvia suffruticosa from Iran
(Hassan Norouzi-Arasi et al., 2005)
Salvia suffruticosa Staphylococcus aureus, Staphylococcus epidermitis, E.coli, … Well method 21 Antibacterial activity of S. tomentosa EO (M. Zeki Haznedaroglu et al., 2001)
S. tomentosa E. coli, Staphylococcus aureus, S. epidermidis, Pseudomonas auroginosa, Enterococcus faecalis… Disc diffusion method 22 Antimicrobial and antioxidant activities of the EO and various extracts of S.
tomentosa
(Bektas Tepe et al., 2005)
S. tomentosa Staphylococcus aureus, E. coli, Pseudomonas aeruginosa, … Agar-well diffusion method Disc diffusion method Broth micro-dilution assay
Apart from essential oils decoctions, hexane extracts, dichloromethane extracts, methanol extracts, isolated compounds (flavonoids and terpenoids) have also been screened. It is hard to compare such a lot of results generated by diverse materials and methods, but as a general rule essential oils generally showed the highest in vitro activity.
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The composition of the essential oil strongly depends on various factors, like: • The species, subspecies and cultivar of the plant
• The part of the plant • The age of the plant
• Eventual elicitors
• The soil where the plant is growing
• The geographic area (latitude, longitude, elevation…)
• The collecting period of the year • The collecting period of the day • The storage of the plant
• The method of extraction
• Eventual infections or atmospheric conditions which may stress the plant.
(“Manuale dell’erborista”, Morelli et al., 2006; “Manuale di Botanica Farmaceutica”, Maugini et al., 2006; “Farmacognosia generale e applicata”, Bruni 1999; “Dizionario di fitoterapia e piante medicinali”, Campanini 2008; “Chimica, biosintesi e bioattività delle sostanze naturali”, Fattorusso 2001; “Metabolismo e prodotti secondari delle piante”, Maffei 2003)
Previous studies referring to sage essential oil composition, carried out by gas chromatography and mass spectrometry (GC-MS), showed a significant variation between species. Monoterpenoids and sesquiterpenoids are always the most abundant components in percentage. Some examples of EO composition are given in the following table.
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Table 1.3 : Main components of some different Salvia species. Legend: Pink= monoterpenes; Red= monoterpene oxides; Light blue= sesquiterpenes; Blue= sesquiterpene oxides. The minimum percentage considered is 7% (* for S. bracteata is 3.8% and for S. rubifolia is 5.1% because there was not any compound in a major amount)
S .c a n a ri en si s S .o ff ic in a li s S .p o m if er a S .f ru ti co sa S .t ri lo b a S .t o m en to sa S .h yd ra n g ea S .r in g en s S .s cl a re a S .r o si fo li a S .a eg yp ti ca S .d o lo m it ic a S .a lb ic a u li s S .c ry p ta n th a S .m u lt ic a u li s S .r u b if o li a * S .b ra ct ea ta * S .d es o le a n a S .s u ff ru ti co sa S .a m p le xi ca u li s α-pinene X X X X X X X camphe ne X X X β-pinene X X X myrcen e X X cyclofe nchene X limone ne X bornyl acetate X α-thujone X X X X β-thujone X X X campho r X X X X X X X X 1,8- cineole X X X X X X X X X X X borneol X X linalyl acetate X X X linalool X X X X geranil acetate X X α-terpine ol X geranio l X
37 trans-pinocar vyl acetate X pulego ne X α-terpinyl acetate X β-caryop hyllene X X X X α-caryop hyllene X gamma muurol ene X E-caryop hyllene X X germac rene D X δ-gurjune ne X viridifl orol X X X X caryop hyllene oxide X X
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1.10 : Purposes of this study.
The main scope of this study was to test the antimicrobial activity of the essential oils from five Salvia species using the disc diffusion assay method. The EOs of species which were tested were: S. officinalis (two different essential oils, one from branches and leaves and the other from flowers), S. dolomitica, S. patens and S. somalensis. The experiments were carried out on five ATCC certified bacteria strains, two gram-negative and three gram-positive:
• Escherichia coli (gram -) ATCC 28922
• Pseudomonas aeruginosa (gram -) ATCC 27853 • Enterococcus faecalis (gram +) ATCC 29212
• Staphylococcus aureus (gram +) ATCC 29213 • Staphylococcus epidermidis (gram +) ATCC n/a
There are some previous publications about antimicrobial activity of S. officinalis, but none of them was relating S. officinalis grown in Malta. The other species under investigation were not tested yet with disc diffusion assay on these bacteria strains. Every essential oil was also analyzed by gas chromatography and mass spectrometry at University of Pisa in order to find out the composition.
Salvia officinalis from Argotti Botanic Garden was hydro-distilled with a Clevenger apparatus at the scene while other oils were extracted with the same technique at University of Pisa, Department of Pharmaceutical Sciences.
