The Phytochemical and In Vitro Pharmacological Testing of Maltese Medicinal Plants

The Maltese archipelago is composed of a small number of islands with a total surface area of approximately 457 km2. Albeit this small size the Maltese islands host a vast number of plant and animal species. Plant biodiversity, with its 1264 vascular species, is mainly attributed to the strategic position of Malta within the Mediterranean, in which throughout the years several conquerors and civilisations sought to possess Malta particularly for military purposes. In part, the plant diversity of Malta is attributed to introductions brought about by various military forces, as an aid during injury and sickness. Naturally, the phytodiversity has an inclination towards the Mediterranean type of flora with an approximately 66% of the Maltese flora pertaining to this region (E. Attard, 2004). Typical Mediterranean medicinal plants include conifers (Pinus halepensis and Cupressus sempervirens), broad-leaved trees (Laurus nobilis, Morus nigra and Tamarix gallica), fruit trees (Ceratonia siliqua, Citrus trees, Nerium oleander, Olea europaea and Punica granatum), and others (Allium sativum, Aloe ferox, Capparis spinosa, Opuntia ficus-indica, Origanum vulgare, Papaver somniferum, Phytolacca decandra and Pistacia lentiscus). The other portion (34%) is attributed to plants originating from the warm North African (Cynomorium coccineum, Ficus carica and Myrtus communis) and the colder South Europaean regions (Crataegus monogyna, Populus alba and Salix species).


Introduction 1.1 General background
The Maltese archipelago is composed of a small number of islands with a total surface area of approximately 457 km 2 .Albeit this small size the Maltese islands host a vast number of plant and animal species.Plant biodiversity, with its 1264 vascular species, is mainly attributed to the strategic position of Malta within the Mediterranean, in which throughout the years several conquerors and civilisations sought to possess Malta particularly for military purposes.In part, the plant diversity of Malta is attributed to introductions brought about by various military forces, as an aid during injury and sickness.Naturally, the phytodiversity has an inclination towards the Mediterranean type of flora with an approximately 66% of the Maltese flora pertaining to this region (E.Attard, 2004).Typical Mediterranean medicinal plants include conifers (Pinus halepensis and Cupressus sempervirens), broad-leaved trees (Laurus nobilis, Morus nigra and Tamarix gallica), fruit trees (Ceratonia siliqua, Citrus trees, Nerium oleander, Olea europaea and Punica granatum), and others (Allium sativum, Aloe ferox, Capparis spinosa, Opuntia ficus-indica, Origanum vulgare, Papaver somniferum, Phytolacca decandra and Pistacia lentiscus).The other portion (34%) is attributed to plants originating from the warm North African (Cynomorium coccineum, Ficus carica and Myrtus communis) and the colder South Europaean regions (Crataegus monogyna, Populus alba and Salix species).
There are approximately 458 medicinal taxa, used in the past to treat one or more ailments (Lanfranco 1993;Lanfranco 1975).Most popular treatments were for the gastrointestinal system, nervous system, cardiovascular system and dermatological conditions.The most predominating plant family within this group is the Asteraceae family, followed by the Lamiaceae and Fabaceae families (Attard, 2004).In spite of their use, these medicinal plants were administered on a trial and error basis.Today, with the advent of modern scientific techniques, the ethnobotanical attributes of a medicinal plant can be challenged by phytochemical and pharmacological testing.

Technical approaches
The evaluation of plant species using different solvent systems has been widely exploited in previous studies (Rodriguez-Lopez et al., 2003;Kumarasamy et al., 2002;Calderon et al., 2003;Konning et al., 2004).A wide spectrum of solvents may be employed when a small number of plants (1-15) are investigated, but when investigating larger numbers or a new group of plants for the first time, the solvents used in ethno-medicine are preferentially selected (Punjani and Kumar, 2003;Guarrera, 2003).
Phytochemical analysis for major classes of metabolites is an important first step in pharmacological evaluation of plant extracts.Some journals require that pharmacological studies be accompanied by a comprehensive phytochemical analysis.Details of such analysis are found in several text books (Harborne, 1984;Evans, 2009).The main secondary metabolite classes include flavonoids, terpenoids and alkaloids, which have been widely tested by the acidified vanillin test, the Salkowski test and the Dragendorff's test, respectively.
Bench top bioassays have been devised to facilitate screening of a large number of samples (Meyer et al., 1982;Carballo et al., 2002).They are based on the principle that pharmacology is simply toxicology at low doses, while toxicology is pharmacology at high doses.Several researchers have used these bioassays for primary pharmacological screening of medicinal plants (Franssen et al., 1997;Kanegusuku et al., 2001;Javidnia et al., 2003).The brine shrimp lethality test (BST), which involves the exposure of brine shrimps to different extract concentrations, is considered as a useful tool for preliminary assessment of cytotoxicity (Jaki et al., 1999).It is a rapid (24 hours), inexpensive and simple technique.A positive correlation has been found between the brine shrimp test and cytotoxicity of the 9KB human nasopharyngeal carcinoma, and other cell lines (Meyer et al., 1982;Kim et al., 2000).
The DNA methyl green bioassay is a simple and comprehensive technique with a high throughput.Methyl green, binds quantitatively to DNA forming a DNA-methyl green complex, hence identifying agents with a high affinity for the DNA.This affinity determines the displacement of methyl green, hence leading to a colourless carbinol (N.Kurnick, 1950;B. Kurnick and Foster, 1950;Krey and Hahn, 1975).

Aims of study
We believe that Maltese medicinal and aromatic plants have a great pharmacological potential.This is based on the concept that, in the past, these plants had important medicinal uses.Therefore, we aimed our study at ethnobotanical research by: Herb/Emollient as skin softener (Borg, 1927)

IOA-AMP-453
Aster squamatus (Sprengel) Hieron, Asteraceae Settembrina selva a (Narrow leaved aster) A very abundant plant, said to be introduced to the Maltese Islands sometime around the 1930s

Brine Shrimp Test (BST)
In a set of 12-well plates, each well contained 10 nauplii, 1 ml sea water and 1 ml of extract diluted to final concentrations of 1%, 0.1%, 0.01%, 0.001% and 0.0001% respectively.The tests were set out in triplicate so that a total of fifteen wells per extract were used.numbers of living nauplii were counted after 24 hours.The LC 50 values and 95 % confidence intervals were determined in μg/ml, using the Finney probit analysis computer program.A median lethal concentration (LC 50 ) smaller than 1000 μg/ml (Alkofahi et al., 1997) indicates pharmacological activity.

DNA-methyl green (intercalation) tests
DNA intercalation assay for DNA activity.Samples were incubated with 200 µl of DNAmethyl green in the dark at 25 °C for 24 h.The decrease in absorbance at 650 nm was calculated as a percentage of the untreated DNA-methyl green absorbance value.The median inhibitory concentration (IC 50 ) was calculated (Desmarchelier et al., 1996) through regression analysis.Cucurbitacin E and Dexamethasone were used as potent and moderate positive controls, respectively.Data was analyzed using Student's t-test.

Phytochemical analysis
The results for the four phytochemical classes are illustrated in table 2 and a generalised picture of the number of extracts, containing phytochemicals for each solvent system used, is illustrated in figure 1  The predominating compound classes were terpenoids (56.07 %), followed by proteins (53.57%) and flavonoids (48.93 %).Alkaloids were limited to a smaller number of extracts (7.50 %).The majority of the polar solvents, aqueous, aqueous-ethanol and ethanol contained terpenoids and proteins (p<0.05, n=4).The chloroform extract contained mainly flavonoids (p<0.05,n=4), while the petroleum ether extracts contained predominantly flavonoids and terpenoids.
The highest terpenoid contents were found in the ethanol and aqueous-ethanol extracts.In fact, it was observed that 70.70 % of the positive extracts were polar extracts, i.e. using water, water-ethanol and ethanol as extracting solvents.This is due to the fact that most terpenoids are present in the glycosidic form rather than the non-polar or low polarity terpene aglycone form.Some plants exhibited the presence of terpenes and related compounds in all solvent systems.Typical examples included Acanthus mollis, which mainly contains -sitosterol as the triterpene-like compound (Loukis & Philianos, 1980), Antirrhinum tortuosum, with mono and sesquiterpene volatile derivatives (Nagegowda et al., 2008), Arum italicum, with the tetraterpene carotenoids (Bonora et al., 2000), Asparagus aphyllus with saponins and sapogenins (Shao et al., 1996), Olea europaea containing mainly triterpenoids (Caputo et al., 1974;Elamrani, 2011), Palaeocyanus crassifolius containing sesquiterpene lactones (Koukoulitsa et al., 2002) and Phlomis fruticosa, mainly containing mono-and sesquiterpenes (Amor et al., 2009).In the case of Fumaria capreolata the main constituents mentioned in previous studies were the alkaloids (Soušek et al., 1999;Maiza-Benabdesselam et al., 2007).In this present study, there was the strong presence of terpenoids.
The distribution of alkaloids in polar and non-polar solvents was almost equal (52.38 % and 47.62 %, respectively).Alkaloids may be present either as the non-polar organic form or as the polar ionised alkaloid salt.The highest content was recorded in the ethanol extract of Gladiolus italicus and in the chloroform extract of Aloe vera.For Gladiolus, this result goes in accordance with that obtained by Ameh and coworkers (2011) and for Aloe, a similar result was obtained by Waller and coworkers (1978).Other plants with an alkaloidal content include Asparagus aphyllus (Negi et al., 2010), Calendula arvensis (Shamsa et al., 2008), Glactites tomentosa, Glebionis coronaria, Oxalis pes-caprae, Parietaria judaica, Carlina gummifera, Hyoscyamus albus (Doerk-Schmitz et al., 1993), Laurus nobilis (Nayak et al., 2006), Pinus halepensis (Tawara et al., 1993) and Plantago lagopus (Hultin & Torssell, 1965).Fumaria species are known to contain alkaloids (Soušek et al., 1999;Maiza-Benabdesselam et al., 2007).However, no alkaloids were detected for Fumaria officinalis and Fumaria capreolata in this present study.Although Ceratonia siliqua is claimed to contain no alkaloids (El Hajaji et al., 2011), in this present study, alkaloids were detected in the aqueous, chloroform and petroleum ether extracts.It was also observed that for Papaver somniferum no alkaloids were detected in the leaves.This depends on several factors.Primarily, the wild variety might have a low potential for the production of morphinan alkaloids, and other plant parts such as the stem, roots and capsules, tend to accumulate more alkaloids than the leaves (Williams & Ellis, 1989).
The absence of flavonoids in these species for the current study may be due to several factors that include a different chemotype, different environmental conditions and the presence of these compounds below the detection limit, amongst others.Antirrhinum siculum is palely pigmented and this may contribute to the insignificant content of flavonoids (C.Martin et al., 2010).
Proteins prevail in many plants.Within the positive response group, 74.67 % were polar extracts while 25.33 % were non-polar extracts.This indicated that three-forths of the detected proteins were functional proteins including enzymes.Anagallis arvensis, Asparagus aphyllus, Palaeocyanus crassifolius and Prasium majus exhibited the presence of proteins in all their extracts.This goes in accordance with previous studies carried out on these plants (Alignier et al., 2008;King et al., 1990).Plants that were devoid of proteins in all their extracts include Aloe vera, Gladiolus italicus, Laurus nobilis and Sonchus oleraceus.In previous studies, Aloe vera revealed the presence of glutathione peroxidase (Sabeh et al., 1993), Gladiolus italicus contained arabinogalactan-protein (Gleeson & Clarke, 1979) and Laurus nobilis contained lipase (Isbilir et al., 2008).Although most plant material was collected at flowering time, the inclusion of seed protein in the extract would have been possible in cases where fruit were harvested alongside the flowers.

The Brine Shrimp Test
The results for the tested extracts are given in Table 3.Primary screening involves the use of bench-top bioassays.Extracts exhibiting LC 50 values above 1000 μg/ml are generally regarded as ineffective extracts.In this study, 42.26 % of the extracts were therefore inactive (Table 4).The most inactive were the petroleum ether extracts, while the most active were the ethanolic extracts.Correlating the BST lethal concentrations to phytochemical classes, it was observed that inactive extracts contained several phytochemicals.The reason may be due to the low concentration or possible antagonistic activity between the phytochemicals from the different classes.55.68 % of the extracts exhibited LC 50 values below 1000 μg/ml.The most active were the ethanolic extracts (72.97 %), while the least active were the petroleum ether extracts (35.14 %).Four plants exhibited activity for all their five extracts.These were Nerium oleander, Olea europaea, Opuntia ficus-indica and Pinus halepensis, all exhibiting LC 50 values below 0.01 μg/ml.These four plant species are amongst the most popular Maltese traditional medicinal plants.It was also observed that some extracts with non-detectable phytochemicals exhibited significant LC 50 values.Typical examples include the aqueous extract of Lactuca virosa, the aqueous-ethanol extract of Pinus halepensis, the ethanolic extracts of Hypericum aegypticum and Lactuca virosa, and the chloroform extracts of Ferula communis, Foeniculum vulgare and Pistacia lentiscus.On the other hand, there were extracts that exhibited significant LC 50 values as opposed to other studies.For example, for Fumaria officinalis aqueous-ethanol and ethanol extracts, in the present study, exhibited significant effects on brine shrimps as opposed to the ethanol extract reported in the study by Erdoğan (2009).

The DNA-methyl green assay
Table 5 shows the IC 50 values obtained for the DNA-methyl green assay.Although low IC 50 values have been reported for pure compounds (Burres et al., 1992) , such as rubiflavin and www.intechopen.com Bioactive Compounds in Phytomedicine 104 distamycin A (17 and 18 μg/ml, respectively), it is reasonable that in the case of extracts higher IC 50 values are acceptable as for pyrido [2,3-d]pyrimidin-4(1H)-one and pyrido [2,3d]triazolo [3,4-b]pyrimidine analogs (40 -53 μg/ml) (Goda & Badria, 2005).Since plant extracts are complex matrices with several phytochemicals, IC 50 values are expected to be higher than for pure compounds.Therefore, extracts with IC 50 values below 70 μg/ml were considered as active (Figure 2).Only 15 % of the extracts displaced methyl green from the methyl green DNA complex.It is likely that these compounds act as intercalating agents at the DNA level.86.67 % were active polar extracts with proteins predominating in these extracts.The other extracts either exhibited an IC 50 value higher than 70 μg/ml or else a 50 activity was never achieved.From the remaining 85 %, only one-third of the extracts exhibited values above 70 μg/ml.Alkaloids only featured in one active aqueous extract of Ceratonia siliqua.Terpenoids, flavonoids and proteins predominated mainly in aqueous and aqueous-ethanol extracts.For a few extracts, there was no correlation between the phytochemical class and DNA-methyl green activity.These include the aqueous-ethanol extract of Gladiolus italicus, the aqueous extract of Hedera helix and the ethanolic extract of Hypericum aegyptiacum.For example H. aegyptiacum contains hypericin that can inhibit DNA topoisomerase II (Peebles et al., 2001), but the naphthodianthrone was not detected by the phytochemical tests.

Conclusions
This study has confirmed the presence of useful phytochemicals and biological activities of several extracts from selected Mediterranean plants.It is expected that these results will serve as a stimulus for further investigations into the active phytochemicals.

Acknowledgment
This work was supported and funded by the Malta Council for Science and Technology, Malta (Project code: RTDI-2004-074).

The
Phytochemical and In Vitro Pharmacological Testing of Maltese Medicinal Plants 99

Fig. 1 .
Fig. 1.A generalised profile of the number of extracts containing terpenoids, alkaloids, flavonoids and proteins for each solvent system used (n=280).

Fig. 2 .
Fig. 2. The number of extracts classified as (a) below 70 μg/ml, (b) above 70 μg/ml range and (c) non-active extracts with the different solvent types for the DNA methyl green assay.

Plants 97 Voucher specimen number Botanical Name, family Maltese, (English) Names Part/s used, preparation and Maltese Traditional uses
The Phytochemical and In Vitro Pharmacological Testing of Maltese Medicinal
The phytochemical analysis of the extracts under investigation for the main phytochemical classes: Flavonoids (F), Terpenoids (T), Alkaloids (A) and Proteins (P)

Table 3 .
The result for the effect of extracts on the Brine Shrimp Test

Table 4 .
The percentage of results classified as (a) above 1000 μg/ml, (b) 0.01 -1000 μg/ml range, (c) less than 0.01 μg/ml and (d) not determined (ND) with the different solvent types for the brine shrimp test.

Table 5 .
The median inhibitory concentration (IC 50 in μg/ml) values obtained for the DNAmethyl green assay (NA no activity -50% effect was never reached).