Medicinal Potential from Plant Biodiversity in a Mediterranean Scrubland, Thymo Piperellae-Cistetum Crispi Costa, Peris &Stubing 1986

Objective: The main aim of this study is establishing a research protocol for assessing the medicinal potential from the plant biodiversity in the plant association Thymo piperellae-Cistetum crispi Costa, Peris & Stübing 1986, an endemic scrubland. Methods: The proposed protocol was a multidisciplinary study that it included: a literature review on the traditional use from characteristic plants in the plant community, appropriate pharmacological assays and antimicrobial tests for confirming medicinal properties and phytochemical analysis for studying the active substances. Finally, the percentage of scientifically studied medicinal plants was compared before and after the study for assessing the effect of protocol implementation for establishing the real potential medicinal in plant community. Results: The review study of ethnobotanical literature showed that two of the characteristic species in plant community, E. scoparia and T. piperella, had traditional uses. The first species was used as medicinal plant for inflammations of the bladder and kidney and urinary tract disorders. The other one was used as preservative and flavoring of olives, and as an infusion for the tummy pain. After implementation the medicinal study protocol, medicinal properties of two species were demonstrated. E. scoparia showed analgesic and antiinflammatory activities and T. piperella presented spasmolytic and antimicrobial activities. In addition, the botanical characterization of the plant organs with medicinal activity was performed, and data, indumentum and other anatomical characteristics, allowed differentiating these specific plant drugs from another plant organ and other species, avoiding confusions and increasing the safety of medicinal herbal products. So the real percentage of scientifically studied medicinal plants increased in the studied vegetation after applied the proposed protocol for assessing its medicinal potential. Conclusion: The proposed protocol for assessing the real medicinal potential in a plant community is valid for this aim.


Introduction
The biodiversity conservation is one of the priorities for many Nations pursuing sustainable development. The plant resource potential use should be a criterion for protecting, controlling and conserving landscapes. The plant resource rational use can only be obtained through the knowledge of the plant properties. In this connection, the quest for medicinal properties in plants can go together with their control and conservation; this will hopefully turn out in a reduction of improper use of the plants, an event that could otherwise lead to hazards for both the environment and the population.
Given the above, we projected a research protocol designed for showing the real medicinal properties from characteristic species in the plant community, that they had not been scientifically studied, although references on their traditional use could exist. In order to obtain this objective, after an ethnobotanic bibliography review, we carried out different studies (botanical, toxicological, pharmacological (included microbiological tests) and phytochemical) from the characteristic species that were not considered as medicinal plants or their uses were not scientifically demonstrated, or not exists information on its use. This multidisciplinary research facilitates the rational use of medicinal plants because it provides a possibility for finding new medicinal species and new molecules that may drive, in the future, the design of new synthetic drugs.

Materials and Methods
The developed protocol is reported below: 1. Literature review and selection of plants for the study.
2. Selection of the drug (organ that was used for the assays).
3. Recollection of plant material. 4. Extraction from the leaves of the species and preparation of essential oils.

Literature Review and Selection of Plants for Study
A literature review on characteristic species traditional uses in the plant association was performed. Later, other literature review focused on the scientific evidence on the properties of traditionally used species. So, the not used and not scientifically studied medicinal plants came out.

Selection of the Plant Drug
The selection of the plant part for studying the medicinal properties was based on the bibliographic information about the plant organ of each species that has been traditionally used.

Plant Material Recollection
Plant material was collected from the locality Pla de Corrals in the Valencia region (Spain) and was authenticated at the Botanical Garden, University of Valencia.

Extraction from the Leaves of the Species and Preparation of Essential Oils
a. Preparation of the extracts Leaves (192 g) were air-dried, powdered and extracted at room temperature with 80% methanol (2 L, 2 days) and 50% methanol (2.5L, 2 days). Combined extracts were separated into two parts: 2L. were concentrated in vacuo for obtaining 1.78 g, while the remaining 3 L. were concentrated in vacuo with 0.5 L and extracted successively with hexane (3 x 0.5 L) and dichloromethane (3 x 0.5 L). Organic extracts were dried with a vacuum pump for yielding 4.4 g for hexane extraction, 5.20 g for dichloromethane extraction and 2.27 g for butanol extraction (w/w from dry plant). All extracts were stored at -20 º C.
b. Preparation of the essential oils Essential oil was obtained through hydrodistillation with a Clevenger's apparatus during 2 h 30 min. after with no further essential oil was collected. Qualitative analysis was performed with a GC-MS using a non-polar capillary column BPx5 (30 m x 0.22 mm i.d.) 5% phenylpolysiloxane (0.25 µm film thickness) and with the temperature programmed in two stages: T1=60 º C; isothermal time, 1.5 min; rate 1, 5 º C/min; isothermal temperature 2, 170 º C; isothermal time 2, 2 min; rate 2, 10 º C/min; isothermal temperature 3, 240 º C; isothermal time 3, 60 min; helium T3, 280 º C. The carrier gas was helium (flow: 50 kpa). The injector and detector temperatures were 250 º C and 200 º C respectively. The electron ionization voltage was 70 eV and the quantification of the different components of the essential oil was accomplished with a GC-FID using the same capillary column and temperature programmes as in the case of the GC-MS. Compounds were identified by comparing their retention indices (RI) on the capillary column with those of α-thujene and their mass spectra with the corresponding data of internal authentic samples. Quantification of the components was performed on the basis of their GC-FID peak areas.

a. Preparation solutions from extracts
Fresh solutions from extract and essential oils were prepared immediately before administration in Tween 20 (<5%) for in vivo studies and in DMSO (<10%) for in vitro assays. The solvents had previously been demonstrated to be pharmacologically inert in all experiments.

b. Experimental animals
Groups of albino mice weighing 25-30 g were employed for the writhing test and hot plate experiments for assessing analgesic activity (in vivo assays). Female and male Wistar rats were employed for assessing spasmolytic (in vitro assay) and anti-inflammatory (in vivo assay) activities. Animals were housed in standard cages at room temperature with food and water ad libitum. Each experimental group consisted of six animals. Treatments of animals during the experiments fulfilled international criteria for the care and use of animals in experimentation. In vivo essays, the solutions from extracts were administered intraperitoneally (i.p.) at a constant volume of 1 mL/kg. The tested doses were usually lower than 1/10 LD50. In vitro essay, essential oils were added to the organ bath.
c. Study of acute oral toxicity An acute oral toxicity study was performed in accordance with OECD guidelines [9]. The experiment was performed with male albino mice (25-30 g, n=5). Five mice were administered with a single dose of the extract via an intubation cannula. The initial dose was selected on the basis of an observational study, which showed it to produce some signs of toxicity without causing severe toxic effects or mortality. Based on the results of this study, the main study was carried out with a group of five mice administered with a dose of 2 g extract/Kg body weight. The dose volume administered was 0.2 mL in both the observational and the main study. Two control groups with which to compare the treated mice were included in each study and administered 0.2 mL saline (NaCl 0.9%) or 0.2 mL Tween 20 (<5%). Animals were kept under observation for a period of 14 days. This study was carried out with all the selected species and were discarded the toxic ones.

d. Pharmacological study
Analgesic activity Chemical stimulus: The Koster & Anderson method was employed in male albino mice, using i.p. acetic acid (3 % in a volume of 0.25 ml) as an algogenic substance. Control animals received a similar volume of saline. Thirty minutes after the administration of the different extracts, the acetic acid solution was injected and the number of writhings recorded over the following 20 minutes. Results were evaluated by calculating the average total number of writhings observed (n>6), and this number was represented as % of inhibition of writhing movements compared with the control vehicle group.
Thermal stimulus: the nociceptive threshold of male albino mice was determined by employing a hot plate and the method of Eddy and Leimbach. The device (Ugo Basile) consisted of a metal plate (25 x 25 cm) heated to a constant temperature (55º C), on which a plastic cylinder (20 cm diameter, 16 cm high) was placed. The maximum time allowed for an animal to respond was 90 sec. Both licking and jumping movements (determined in seconds) were recorded. The percentage of analgesic effect was evaluated by calculating the accumulated values (seconds) for each group (n>6 per group) and control.

Antiinflammatory activity
Carrageenan-induced paw edema [10]: carrageenan was injected into the plantar aponeurosis of the right hind paw of Wistar rats (0.1 mL, 2%). Animals (150-175 g, n=10) received extract (200 mg/Kg) 30 minutes prior to the injection of carrageenan, and the paw volume was measured with a plethysmometer (Ugo Basile, Italy) 1, 2, 3 and 24 h later. Results were expressed as a percentage of the inhibition of the edema in each group with respect to controls.

Spasmolitic activity
Assay: Male wistar rats (160-180 g) were used. The animals were killed by a blow on the head, and their tissues were then excised and suspended at 37 º C in a 10 mL organ bath with a Krebs Henseleit solution (composition in mmol/L: ClNa 118.4, ClK 4.7, Cl 2 Ca 2.5, SO 4 Mg 0.6, PO 4 H 2 K 1.2, CO 3 HNa 25 and glucose 11.1) and oxygenated with 95% O 2 and 5% CO 2 . 1 g was used as preload for all the preparations. Concentration-response curves to acetylcholine were recorded in the absence and presence of the essential oil (85, 42. 8.5 μg/mL) which was added to the bath 20 min. before the second curve. Modifications of the maximum effect (Emax) and efficient concentration 50 (EC50) were evaluated. A value of 100 was assigned to maximum effects obtained with acetylcholine in the absence of the essential oil. The mechanical activity was measured isometrically using a transducer (UF-1) connected to a system PowerLab/400 (Cibertec S.A).
Animal organ (rat ileum): strips of rat ileum were suspended in the same conditions as described above, and concentration-response curves to acetylcholine in the range 10 -9 to 10 -3 M were recorded. The essential oil (85, 42. 8.5 μg/mL) was assayed.

Characterization of the Plant Drug (plant leaf)
The morphology, epidermal micromorphology and anatomy of the plant leaf were studied. The morphological analysis was performed using a Wild Heerbrugg magnifying glass. The epidermal micromorphology was studied employing the Scanning Electron Microscope (HITACHI S-4100) at SCSIE-University of Valencia. Histologic techniques were applied for studying the foliar blade anatomy. A freezing microtome (LEICA CM 1325 coupled to CU MICRON COOLING SYSTEM K-400) was used for obtaining cross sections, and the safranine-fast green staining was employed for observing different plant tissues. The images were captured using an optical microscope with coupled digital camera (OLYMPUS PROVIS AX-70).

Evaluation of the Medicinal Potential in Plant Community
Medicinal potential was measured considering the characteristic species in the plant association, calculating the percentage of species with proven medicinal use, first taking into account data from literature on species that their medicinal properties were scientifically proven; subsequently, the same percentage was calculated taking into account the obtained results following the proposed protocol. Percentages were compared graphically.

Literature Review and Selection of Plants for Study
According scientific literature, essential oils from C. salvifolius and C. monspeliensis presented properties in prevention of neurodegenerative disorders [11]. Also, extracts from leaves of C. monspeliensis presented antimicrobial activity [12]. Another characteristic species in the plant community, L. stoechas, was a very used medicinal species and their properties had already been demonstrated [13,14]. The essential oils of the maritime pine or cluster pine, P. pinaster, presented a moderate antimicrobial activity against Staphylococcus aureus, Bacillus subtilis and Escherichia coli [15]. X. tuberaria was a species with ethnobotanical and ethnoveterinary uses, and the scientific evidence that explained their traditional medicinal uses was found, highlighting the interest of its decoctions and infusions as a source of bioactive compounds and functional beverages [16,17]. Tsumbu et al. [18] published a paper about the modulatory activities in extracts from tropical dietary plants with oxidant activity, one of the studied species was P. aquilinum, and the authors concluded that the role of those dietary and medicinal plants in the treatment of ROS-dependent inflammatory diseases could represent a new interesting starting point as a therapeutic approach.
After screening the literature, it resulted that only C. crispus, E. scoparia and T. piperella were not used for their medicinal properties, but our research group researched on T. piperella and preliminary tests showed pharmacological activity [19,20], which we proposed to confirm in this work. Thus the studied species were: C. crispus, E. scoparia and T. piperella According to popular usage, we deduced that E. scoparia might have anti-inflammatory and analgesic properties, because this specie and other ones of genus Erica were used for urinary problems that produce pain and inflammatory symptoms [4,21]. Regarding T. piperella, we thought that it had antimicrobial and spasmolytic properties, taking in account the preliminary studies performed by our research group [19,20]. Finally, we thought that C. crispus might present similar medicinal properties to other Cistus species.

Selection of the Plant Drug
Selection criteria of the plant organ for studying are, first, the information from literature [4,12,19,20]; subsequently, when information from literature was not existed, the characteristic of easiest collecting and most durable plant organ was taken in account. Also, the most used organ in other similar pharmacological studies was taken in account. So, the leaf was the selected organ.

Recollection of Plant Material
Plant material was collected in spring, when the plants were flowered and fructified and had all necessary plant organs for identifying the species using taxonomic botanical keys. Leaves from each species were collected and stored in a conventional refrigerator. Furthermore, the aerial part with flowers and fruits from each species was collected for the determination of species name.

Study on Medicinal Properties
Study of acute oral toxicity Oral Acute Toxicity was evaluated and no evidence of toxicity was detected at the assayed dose (2 g/Kg) for T. piperella and E. scoparia extracts. The animals died at the same doses of assayed C. crispus extracts, then lower doses were tested (2, 20, 200 mg/Kg) and toxic signs remained, therefore, pharmacological studies were not performed with this species.

Analgesic activity
Chemical stimulus: Pre-treatment with methanol, hexane and dichloromethane extracts (2, 20 and 200 mg/Kg) from E. scoparia leves showed an analgesic effect that reduced the percentage of writhing movements induced by the i.p. administration of 0.25 ml of a solution of 3% acetic acid by 11.0%, 21.4% and 85.0% respectively for the methanol extract, 59.6%, 78.0% and 100% respectively for the dichloromethane extract, and 22.4%, 49.6% and 77% respectively for the hexane extract ( Figure 1).

Spasmolitic activity
The in vitro contractile effect of acetylcholine on isolated rat ileum was reduced significantly in a concentration-dependent manner through preincubation with maximal concentrations of T. piperella essential oil. In this case, the E max was reduced by a 21.96% (p<0.001), by a 42.96% (p<0.001) and by a 27,03% (p<0.001) at the concentrations of 8.5, 43 and 85 µg/ml respectively, without a significant modification of the EC 50 (1.555 x 10-6 M, 1.420 x 10 -6 M, 1.641 x 10 -6 M and 1.406 x 10 -6 M for control, 85, 43 and 8.5 µg/ml of essential oil respectively, as shown in (Figure 8).

Antimicrobial tests
The antimicrobial test demonstrated that essential oil from T. piperella leaves showed activity against Staphylococcus aureus, Escherichia coli, Candida albicans, Streptococcus pyogenesand Mycobacterium phlei.

Phytochemical analysis
Phytochemical analysis from E. scoparia leaves showed the presence of lupeol and α-amyrin, that they were isolated. Column chromatographic fractionation of the hexane extract (4 g) on silica gel with dichloromethane: methanol (95:5) as eluent, provided lupeol and α amyrin, in a ratio of 90:10, as major constituents. The residue from the combined hexane extracts (4 g) was fractionated by column chromatography on silica gel 60 (230-400 mesh) with dichloromethane: methanol (95:5) and provided a crude mixture of lupeol and α -amyrin (figure 11). By TLC an approximate ratio of 9:1 was determined between the two triterpenes that represented the Essential oil yield from T. piperella dry leaves was 2.1v/w. The most abundant compounds obtained with GC-MS were ˠterpinene (11.3%), p-cymene (31.7%) and thymol (37.6%); indeed, they share the same biosynthetic route of which the precursor is ˠ-terpinene.

Characterization of the Plant Organ (drug) that was used for the Assays
Concerning the characterization of E scoparia leaves: Morphology: Leaves were small (4-7 x 0.5-0.9 mm) and arranged around the stem node in groups (3-4 leaves). They were linear with recurved margins. The young leaf had pilosity and the adult one was glabrous. There were two channels or furrows from the base to the apex of the leaf, next to the midrib, on the underside of the leaf ( Figure  9). Hispid and prickle trichomes, subglandular hairs and considerably ornamented indumenta with a small barrel form were in these channels ( Figure 10).
Anatomy (cross section of leaf blade): The leaf cross section ( Figure  11) had an elliptical-oval form. It showed a monostratified epidermis with a thick cuticle and stratified external wall, characteristic of mucilage presence. On the epidermis, the foliar indumenta were situated on the lower epidermis, but the ornamented indumentum was situated on the leaf blade margins. The palisade parenchyma showed one layer of thin-walled, long, and compactly arranged cylindrical cells. The spongy parenchyma showed isodiametric loose cells and was located underneath the palisade. Both parenchyma's showed druses in idioblasts. There were collateral vascular bundles in the spongy parenchyma. Primary vascular bundle or midrib was surrounded by sclerenchymatous cells in the oldest leaves.  Concerning the characterization of T. piperella leaves: Morphology: leaves are broad and oval with small glands located in deep fovea and small glandular trichomes, both essential oil reservoirs, and distributed on the upper and lower side of the leaf blade ( Figure  12).

Evaluation of the Medicinal Potential in Plant Community
It is observed that the medicinal potential from plant community, measured as the percentage of medicinal species, increased after this study, from 62% to 66%; it also increased the number of toxic species (Figure 14).

Discussion
The conservation of biodiversity is a fact of current interest. It is not a passing trend; it is a necessity connected to the industrial development of the world, by the migration of people and food, by the cultural interchanges and the effects of globalization. But do we ask ourselves, why actually we conserve the vegetation, what must we protect? And why do we do it? This work arose when we tried to answer these questions. We concluded that we must know the species and the ecosystems, but we must also see how useful they are for humans. The use of the natural resources must be an important subject within the plans of a sustainable development. It is not conserve just for the sake of conservation but also because of its possible medicinal use. We know that wild flora cannot always be used directly; in these cases, the plants will only be used to research, and it is possible to find some use for them to justify their growing and conservation ex situ. This work is an example about which we have discussed previously. We have observed that the medicinal potential in a plant community increases after doing the pharmacological test from characteristic species initially not considered as medicinal plants or no scientifically proven information that justifies their use.
The oral acute toxicity did not showed evidence of toxicity at the assayed dose for T. piperella and E. scoparia, but animals died at the same doses of assayed C. crispus extracts. So, the toxicity test showed that T. piperella and E. scoparia could be used as medicinal plants, as a tea that it is the most usual form of consumption, because they were not toxic plants. However, C. crispus was a toxic plant at low doses and should not be consumed; Mulet [7] already warned about the toxicity of this species.
With respect to the medicinal properties, on the one hand and in order to determine the type of analgesia induced by the E scoparia extract, the activity produced by two different pain stimuli was evaluated: heat and chemical agents, in this case acetic acid. Although both cerebral and peripheral analgesics respond by inhibiting pain stimuli of chemical and mechanical origin, only central analgesics increase the time of response in the hot plate test [22]. In this sense, methanol, hexane and dichloromethane extracts from E scoparia leaves showed an analgesic effect by significantly reducing the percentage of writhing movements induced by the acetic acid, and an analgesic effect arising from CNS action, observed in the increase in response time to licking and jumping in the hot plate test. Besides, anti-inflammatory activity was evaluated by the most commonly employed method according to the literature [23]. Oedema induction with carrageenan is preferable to that induced by other phlogistic agents because it is less influenced by non-specific factors such as vasodilatation and diuresis. The activity of anti-inflammatory drugs in this test exhibited a good correlation with their anti-inflammatory activity in humans [24,25]. In the present study, the evaluated E. scoparia extracts showed different profiles of anti-inflammatory activity in the rat when they were tested 1, 2, 3 and 24 h after the administration of the inflammatory agent. The extracts from the species showed a significant anti-inflammatory activity by inhibiting the oedema induced by carrageenin up to 24 h after the administration of this phlogistic agent.
On the other hand, the T. piperella spasmolytic activity was tested against acetylcholine induced contractions in isolated rat ileum. The different extracts inhibited significantly in a concentration-dependent manner. Maximum effect was obtained with the hexane extract. And the essential oil from leaves was active against Staphylococcus aureus, Escherichia coli, Candida albicans, Streptococcus pyogenes and Mycobacterium phlei The phytochemical study showed the presence of certain substances that could explain the medicinal properties of the studied species. In the T. piperella essential oil, p-cimeno, timol and terpineno monoterpenes were the main components; and several triterpenes, amirine and lupeol, in the hexane and diclorometane extracts from E. scoparia were isolated.
Finally, the study of plant organs used for evaluating the pharmacological activity (drugs) provided the needed differential characters for distinguishing these parts from other plant organ and other species, especially when the drug is adequately pulverized. The most interesting characteristics were related to the leaf indumentum. T. piperella leaf is well differentiated from other Thymus species more abundant, like Thymus vulgaris L., where the indumenta is formed by glands and glandular trichomes of different types such as star-shaped trichomes or branched ones but no glands in fovea [26]. E. scoparia presented simple eglandular trichomes located along the channel part of the abaxial surface of leaves. These trichomes are longer and thinner than the upper surface ones; this specie also presented characteristic trichomes on the margin of the leaf.
Given explained above, the medicinal and toxic properties of the Thymo piperellae-Cistetum crispi characteristic species are now disclosed and the results of previous works [27,28] are confirmed. It is deduced from this study that E. scoparia and T. piperella are medicinal plants and their popular uses are now scientifically proven. In addition, the drugs (leaves) are easily recognizable by their leaf morphology and anatomy, this is very important if the use of these species goes become popular. Finally, it is derived from this work that this multidisciplinary research type increases the percentage of medicinal plants from the studied community, thus increasing the value of this vegetation and offers reasons for protecting and preserving it for use.

Conclusion
The proposed protocol for assessing the medicinal potential from a plant community is valid and provides scientific data that justifies popular or new uses. This evaluation can increase the number of medicinal plant in a plant community.