A Comprehensive Science-Based Field Assessment of Bioactive Properties of the Native Plants of Palestine

Medicinal plants have been traditionally used in folk medicine for centuries as natural healing remedies with significant proven therapeutic effects in many areas including prevention of cardiovascular diseases, anti-inflammatory, antimicrobial, and anticancer activity. Several ethnobotanical surveys were conducted on Palestinian plants during the period 2000-2014 to investigate the traditional ecological knowledge of Palestinian plants used in Traditional Arabic Palestinian Herbal Medicine (TAPHM) as passed down through folk medicine over generations [1,3-12]. Many plant-derived medicines used in traditional medicinal systems have been recorded in pharmacopeias as agents used to treat infections and a number of these have been recently investigated for their efficacy against several diseases [13,14].

Efforts were made to collect plants with a history of known medicinal use and, when possible, with available scientific data for medicinal activity. Samples of plants known to belong to families of plants that were commonly used according to local folklore were also collected on the assumption that they might contain compounds of interest due to closeness in genus and species.
For each sampling site, the plant was photographed and its location and elevation were recorded using a portable GPS unit. Different parts from each plant species such as leaves, flowers, roots, etc. were collected. Each plant sample collected was identified vouchered, archived and deposited at the Herbarium of Biodiversity & Environmental Research Center (H-BERC), Til, Nablus, Palestine.

Plant extraction
An ethanol-based extract was prepared from each plant on the same day of sample collection. The extraction was performed as described in Dey [30], with some modifications. Two grams from each part were washed, mixed with 2 ml of 60% ethanol and crushed using mortar and pestle. The ethanol paste was left at room temperature for 10 minutes before it was filtrated and kept in dark at -20°C, extracts were used within 24 hours of extraction.

Bioassay methods
The assays used in this study targeted relevant health issues that mainly include infectious disease agents (bacteria, fungi, viruses, parasitic worms, and protozoan pathogens), metabolic disorders (diabetes), wound healing, and general health protection (potential or anti-inflammatory properties of antioxidant phytochemical constituents). A total of 13 assays were performed to estimate diverse biological activities of plant extracts, including general protozoa lethality, antibacterial and antifungal activity, roundworm lethality, flatworm lethality, planaria regeneration, oxidation inhibition, glucosidase and glucosidase inhibition, amylase inhibition, and protease and protease inhibition, as well as detect the presence of anthocyanin. Data generated from triplicate assays on the effectiveness of each plant extract was recorded in a computer-based database, and disclosed in the Bioxplore website (www.bio-xplore.org).

Antibacterial assay
The antibacterial assay was performed as described in Andrea-Marobela et al. [28] and Kellog et al. [27]. Briefly, nonpathogenic bacterial strains from human saliva were cultivated on LB agar, in 48well plates in the presence or absence of ethanol plant extracts. Agar was allowed to absorb liquids for 30 minutes before plates were inverted and incubated overnight at 37°C. The presence or absence of bacterial colonies was observed from duplicate assays classifying the complete absence of colonies as antibacterial activity. Penicillin (4 mg/ml) was used as positive control and 60% ethanol was used as negative control.

Antifungal assay
The antifungal assay was performed as described in Meletiadis et al. [31] and Andrea-Marobela et al. [28]. Yeast (Saccharomyces cerevisiae) suspension (50 mg/ml) containing 5% sugar was mixed with ethanolic plant extract and incubated at 30°C, the yeast viability was detected after adding (5 mg/ml) 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide (MTT) (Sigma Aldrich). Plant extracts activities were detected from duplicate assay by the lack of violet color formation from MTT (violet color indicates a metabolically intact organism). Cinnamon extract in ethanol was prepared as samples and used as a positive control and 60% ethanol was used as negative control.

General protozoa lethality assay
Bodo caudatus, a free living protozoa from the order of Kinetoplastida was used as a model organism for the general protozoa lethality assay. Hay medium solution (Ward's Science, Rochester, NY, USA) with Escherichia coli was used to culture the protozoa. The assay was performed in a round bottom 96-well plate using a technique similar to that described by Simpkin and Coles [32] and Andrea-Marobela et al. [28]. Plant extracts activity were detected by the lack of violet color formation after 12 hours incubation from 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma Aldrich) (violet color indicates a metabolically intact organism) with (5 mg/ml), CuSO 4 was used as a positive control and 60% ethanol as negative control.

Roundworm lethality assay
The assay was performed as described by Simpkin and Coles [32]. The free-living nematode, Panagrellus redivivus served as a model organism, with oat meal as culture medium and baker's yeast as a source of food. Plant extracts were added to roundworm suspension in a round bottom 96-well plate. Plates were covered and incubated at room temperature for 4 hours. 60% ethanol was used as negative control and a solution of CuSO 4 (Sigma Aldrich) was used as a positive control.

Flatworm lethality and regeneration assay
The assay was performed with a technique similar to that described by Simpkin and Coles [32] and Okumura and Kobayashi [33] with a few modifications as described below. The brown planaria (Turbellaria) organism was cultured in double distilled (dd) H 2 O, containing 5.7 mM NaCl, 600 μM CaCl 2 and 11.3 μM NaHCO 3 . Solution used for planaria culturing was added to 24-well plates and mixed with plant extracts. Healthy planaria were chosen and transferred under a dissecting microscope, planarian head was cut off using a scalpel, and a headless body was placed in each well. Viability of the organism was detected after 8 hours of incubation. Planaria regeneration was monitored daily, over a one week period. 60% ethanol was used as negative control and a solution of CuSO 4 was used as positive control for planaria lethality.

Glucosidase, glucosidase inhibition and amylase inhibition assays
The glucosidase, glucosidase inhibition, and amylase inhibition assays were performed as described by Harisha [34] and Andrea-Marobela et al. [28]. The assays were performed on Petri dishes containing agar starch. Glucosidase extracted from pea shoot was served as a glucosidase control, saliva sample was served as amylase control, and acarbose (Sigma) served as a glucosidase inhibitor control. The properties of plant extracts were qualitatively evaluated by exposure to solidified starch agar in the presence or absence of glucosidase and amylase. The intact starch surface was visualized with aqueous iodinesolution which results in a dark blue pigment formation.

Protease and protease inhibitor assays
Dot-Blot assay method for detection of Protease inhibitors on X-ray film developed by Pichare and Kachole [35] with minor modification was used to detect trypsin inhibitory activity of collected tissues. The principle of this technique is that the X-ray film is coated with gelatin, and as a drop of proteinase is placed on the film, it hydrolyzes gelatin and forms clear transparent spot against a dark background. Hence proteinases present in the sample are detected. Whereas in the presence of an inhibitor, the spot appears as un-hydrolyzed gelatin against a dark background. Trypsin and trypsin inhibitor (Sigma) served as controls.

Antioxidant assay
The antioxidant assay was performed using a technique similar to the ABTS radical cation decolorization assay [36]. In this assay, the preformed radical monocation of 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) is generated by oxidation of ABTS with potassium persulfate (blue color) and is reduced in the presence of such hydrogen-donating antioxidants. The ABTS radical cation is reactive towards antioxidants, which, when added, convert the blue ABTS radical cation to its colorless neutral form. In this assay, the antioxidant ascorbic acid was used as positive control.

Anthocyanin assay
The anthocyanin assay was performed as described by Giusti and Wrolstad [37]. Anthocyanins are pigment molecules common to all higher plants which change color from red to purple to blue depending on pH. The presence of anthocyanin was detected by the change in color, using water, hydrogen chloride and sodium hydroxide.

Bioactivity scoring
The results of each test were scored on a scale from 0 to 3 to evaluate qualitatively the activity of plant extracts, with score (0) representing no activity, (1) mild activity, (2) moderate activity, and (3) representing the highest bioactivity. In tests with positive or negative results only, a score of 0 represented the negative result while a score of 3 referred to the strongest possible positive result. The number and strength of bioactivities for each sample were registered and the ranges of activities per sample and per family were compared. For each assay, the number of plant samples exhibiting positive activity in each climate zone was compared. A comparison between plant geographical territories was performed by calculating the percent of plant samples from each territory that showed strong activity (score 3) in each of the carried assays.

Dose response tests
Plant samples scoring 3 out of 3 in antibacterial, antifungal, protozoa lethality, anthocyanin, round worm lethality protease and protease inhibitor assays were further analyzed using 4 more dilutions of the extracts. The assays were carried out in triplicates in 4 extract concentrations: (250,125, 62.5, 31.25 mg/ml). The extracts were diluted in the 60% ethanol. All experiments were repeated twice.

Distribution of plants by botanical families
A total of 1471 plant samples derived from 588 plant species belonging to 371 genera and 100 families were screened. Approximately 329 (56%) species, and 871 (59.2%) extracts belonged to only 12 families, notably the Papilionaceae, Asteraceae, Liliaceae, Lamiaceae, Brassicaceae, and Apiaceae families were represented by 254 plant species. Plants collected from the remaining 88 families each represented less than 2% of the total plant samples tested ( Table 1).

Distribution of extracts by plant parts
More than 35% and 29% of the plant samples tested were leaves and flowers, respectively, although roots, stems, seeds and other parts were also sampled ( Figure 1).
Thirteen assays were performed on each plant sample.

Screening results of plant extracts by STN screens
An analysis of the number of plant samples exhibiting positive activity in each of the different tests was performed. Figure 2 shows samples that exhibited scores of 3/3; lower scores were discarded. Of the 1471 plant samples tested, about 93.1% (1369/1471) of the extracts showed at least one high-potency bioactivity (3/3) on one of the assays performed; the remaining 7% did not show a high score on any assay. As seen in Figure

Screening results of plant extracts by phyto-geographical territories
Plants were collected from different areas of Palestine (West Bank) located in three phyto-geographical territories: the Mediterranean Sea Territory, a fertile agricultural area encompassing much of the hilly and semi-coastal central areas within the West Bank; and the Irano-Turanian, and Sudanese-Penetration territories, where annual rainfall ranges from 250 mm in northern and western areas down to barely 50 mm in the southern Valley running along the Syrian-African Rift Valley. Plants collected from the Irano-Turanian and the Sudanese-Penetration Territories with hot and dry areas, showed higher percentages of plants with high antioxidant (56%), glucosidase inhibitor (56%), amylase inhibitor (48%), planaria lethality (30.7%), glucosidase (23.4%), protozoa lethality (17.3%), planaria regeneration (10.7%), anthocyanin (8%), and protease (1.2%) activities, than those from the Mediterranean territory ( Figure 4).
On the other hand, plants collected in the zones with milder weather pattern, situated in the Mediterranean territory, showed higher percentages of plants with high antibacterial (15%), antifungal (11%), protease inhibitor (9%), and round worm lethality (0.46%) activities,    Table  2 presents the 30 plants that exhibited four to five high-potency antiinfectious activities.

Screening results of plant extracts with anti-infectious disease bioactivity
Plant samples exhibiting score 3 out of 3 in antibacterial, antifungal, protozoa lethality, anthocyanin, protease inhibitor, protease, and roundworm lethality assays were further analyzed using 4 extract concentrations: (250, 125, 62.5, 31.25 mg/ml). Eighteen plant samples have exhibited activity even at the lowest extract concentration 31.25 mg/ml and in a dose response manner ( Table 3).

Distribution of active STN screens by botanical families
A comparison between plant families (28), represented by 5 species or above from each family was performed to determine whether specific families have more medicinal activities than others (Table 4 and Figure  6). The remaining families (72), represented by ≤4 species each, were excluded from further discussion.       However, while there is significant knowledge regarding the traditional use of medicinal plants in the area, scientific and clinical data are scarce or lacking. In this study a broad range of Palestine flora was investigated using a methodology that enabled the discovery of plants
with potential medicinal characteristics. Some of the plants identified have a history of human use, while others have never been used before for medicinal purposes. A significant amount of data was accumulated to enable the selection of plants with potential to serve as source of biological material for therapeutic, and other purposes, and as a basis for further studies.
It was not surprising that the plant families most frequently collected in the field belong to the Papilionaceae, Asteraceae, Liliaceae, Lamiaceae, Brassicaceae, and Apiaceae families, which are the most common plant families in Palestine. All these families include many edible as well as medicinal plants that are sources of therapies used in the TAPHM [6,11].
Medicinal plants provide an enormous and highly varied chemical bank from which we can explore for potential therapeutic agents by bioactivity-targeted screenings [38]. As a country with rich plant biodiversity, Palestine has a lot of potential unexploited native plants to be developed as a source of therapeutics, nutritional or cosmetic products. However, less than 10% of the world's biodiversity has been tested for biological activity, many more useful natural lead compounds are awaiting discovery [39]. Therefore, in this study we have screened several plant species to explore the potential bioactive agents using the appropriate mechanism of action, e.g., α-glucosidase inhibition, to explore the potential antidiabetic agents. However, we found that the STN assays allowed the analysis of a large number of plant samples (1471 samples) taken from 588 plant species representing 100 families for 13 different bioactivities.

Antioxidant, α-glucosidase, and α-amylase activities
Our results demonstrate that plants with antioxidant, α-glucosidase, and α-amylase inhibitory activities are most common among the native Palestinian plants studied. It was not surprising that in an area with high levels of solar radiation such as Palestine, plants protect themselves by having higher levels of antioxidants. This indicates that compounds with such activities are abundant in Palestine plant biodiversity, and thus has an enormous potential to be developed as a source of antidiabetic agents [40].
Diabetes mellitus (DM) is a metabolic disorder characterized by a congenital (type I insulin-dependent DM) or acquired (type II noninsulin-dependent DM) inability to transport glucose from the bloodstream into cells [41]. One of the most beneficial therapies for type II diabetes is thought to be the control of postprandial hyperglycemia (PPHG) after a meal [42]. Stabilization of blood glucose is important for diabetic patients, because it prevents hyperglycemia and the complications associated with diabetes [43].
The best therapeutic approach to decrease PPHG is to retard absorption of glucose through inhibition of carbohydrate hydrolyzing enzymes in the digestive organs [42]. The enzymes (α-glucosidase, and α-amylase) are responsible for the breakdown of oligo-and disaccharides to monosaccharides (e.g., to glucose, which is the only sugar that can be utilized by the body) [44]. The inhibition of these enzymes leads to a decrease in blood glucose level, since monosaccharides are a form of carbohydrates which are absorbed through the small intestine [42,45]. α-Glucosidase inhibitors slow down the process of digestion and absorption of carbohydrates by competitively blocking the activity of glucosidase.
Compounds with α-glucosidase inhibitory activity seem to be abundant in medicinal plants. About 411 compounds (mainly flavonoids, phenylpropanoids, and terpenes) exhibiting α-glucosidase inhibitory activity isolated from medicinal plants were reported so far [40]. They have high α-glucosidase inhibitory potential, and can be clinically developed for treating diabetes mellitus.
In recent years, plants and their constitutions have received much attention in the treatment of diabetes for various reasons and many researchers have focused on hypoglycemic agents from medicinal plants [46,47]. Based on the existing studies, it is found that polyphenols, flavonoids, and terpenoids are among the natural active antidiabetic agents [48]. These compounds have been reported to exert various biological effects, including carbohydrate hydrolyzing enzyme inhibition and antioxidant activity. Polyphenolic compounds are able to inhibit the activities of digestive enzymes due to their ability to bind with proteins.
The inhibitory activities of plant phytochemicals, including polyphenols, against carbohydrate hydrolyzing enzymes contribute to the lowering of PPHG in the management of diabetes (i.e., delaying carbohydrate digestion and reducing the rate of glucose absorption, and consequently, decreasing postprandial rise in blood glucose) [43]. On the other hand, phenolic compounds and flavonoids are currently regarded natural antioxidants and considered to be important ingredients for human health [49,50]. Free radicals and reactive oxygen species (ROS) can react with biological molecules, leading to cell and tissue injuries and pathological events. The role of free radicals and ROS in the etiology of many chronic diseases has been well-known. Therefore, free radical scavengers and antioxidants, especially phenolic compounds, are important for human health and have been proposed as health-promoting natural products and in many cases high antioxidant activity is linked with anti-infectious activity [43,49,51]. It was therefore speculated that plants with abundant flavonoids and terpenoids (e.g., Rhus coriaria, [52]) could combat type 2 diabetes mellitus through targeting PPHG and OS [53].
Several α-glucosidase inhibitors, such as acarbose and voglibose obtained from natural sources, can effectively control blood glucose levels after food intake and have been used clinically in the treatment of diabetes mellitus [54]. Only a few α-glucosidase inhibitors are commercially available. All of them contain sugar moieties and their synthesis involves tedious multistep procedures. Moreover, clinically they have been associated with serious gastrointestinal side effects. Therefore, it is necessary to search for alternatives that can display α-glucosidase inhibitory activity but without side reactions. In recent years, research has been focused on the exploration of potent nonsugar based α-glucosidase inhibitors from natural sources because of the highly abundant compounds in nature and their promising biological activities [55].

Glucosidase activity
High bioactivity for glucose breakdown was found in many of the plants tested. The enzyme is essential to break down glucose, and it was hypothesized to be present in all plants. Enhanced glucose breakdown may be advantageous in humans especially for athletes, and plants with high glucosidase activity may prove valuable [29].

Anthocyanin activity
Anthocyanins are natural water-soluble pigments found in vascular plant vacuoles, they are responsible for different colors (orange, pink, red, violet and blue) found in many flowers and fruits [56]. Anthocyanins have different roles in plants including attraction of insects and animals to pollinate plants, and protection of the plant from UV damage to DNA [57]. They have also been shown to play a role in plant pest resistance and have anti-bacterial properties [58]. Anthocyanins may also protect humans against free radical damage and are of great interest due to their range of biological activities including the prevention of neuronal decline, prevention of cardiovascular disease and diabetes, anti-tumor activity, antiinflammatory activity and much more [56,58]. Their biological activity, as well as their use as natural dyes and food additives, makes anthocyanins an attractive area of investigation. In the present study however, only a small percentage (8%) of the studied plant samples exhibited high anthocyanin activity.
Anthocyanins are flavonoids, these are a class of plant secondary metabolite. The major classes of flavonoids are anthocyanins, flavonols, flavanols and proanthocyanidins [59]. There are differing concentrations of flavonoids depending on the plant species, developmental stage, tissue type and growth conditions, and this might explain the law percentage of plants possessing high activity of anthocyaniun in this study [59]. Also, the effect of pH is important to anthocyanin stability in solution. The color of anthocyanins can change from red to blue to colorless, with different pH. This color change is induced by reaction of the flavylium ion, which is stable in acidic conditions. When the pH is raised, the structures of the bonds around the flavylium ion change to form a colorless carbinol at around pH 4 and a purple/blue quinine compound at pH 6.5 and higher [60]. against these pathogens is therefore essential for survival. Our results are therefore in agreement with previous studies in Palestine which indicated that the majority of the plants tested are an important source of antimicrobial compounds that may provide renewable sources of useful antimicrobial drugs against infectious microbes in humans [15][16][17][18]21,24].

Protease activity
Plant extracts with high protease activity were considerably rare (5 extracts, 0.3%). Plant proteases are involved in many aspects of plant physiology and development [62]. They play an essential role in processes such as protein turnover, degradation of misfolded proteins, senescence and the ubiquitin/proteasome pathway [63]. In fact, proteases are involved in all aspects of the plant life cycle ranging from mobilization of storage proteins during seed germination to the initiation of cell death and senescence programs [64]. In this study high protease activity has been found in the extracts of a few plant families such as Asteraceae, Liliaceae, Meliaceae, and Solanaceae. Most plantderived proteases have been classified as cysteine proteases and more rarely as aspartic proteases [65]. These enzymes can be active over a wide range of temperature and pH, and thus can be used in a number of industrial processes that involve the breakdown of proteins by plantderived proteases [66].

Anti-parasitic (anthelmentic: anti-roundworm, antiflatworm; anti-protozoa) activities
About 31%, and 17%, of the plant extracts in this study showed anti-flatworm, and antiprotozoa activities, respectively. On the other hand, only 0.46% of the extracts showed anti-roundworm activities. This indicates that anti-flatworm, and antiprotozoa compounds are not uncommon in medicinal plants, whereas anti-roundworm compounds are much less frequent in plants.

Planaria regeneration
Planaria regeneration test was carried out as a model to screen plant extract for wound healing potential. In this study 122 plant extracts (8.3%) of the plant extracts in this study showed Planaria regeneration activity, of these 20, and 67 extracts exhibited antibacterial and antioxidant potential activities, respectively. Antioxidants play a determining role in the progression of wound healing, and antimicrobial agents are also useful in the management of microbial infection which may concomitantly occur in severe and chronic wounds [67][68][69]. However, it is worth mentioning that some of the plants species found in this study to possess high wound healing potential including Pistacia spp., Quercus spp., Punica granatum, Hypericum spp. and Polygonum spp. have also been reported by other researchers to possess wound healing activities [70].
TAPHM resources [1,[3][4][5][6][7][8][9][10][11][12] have introduced various medicinal plants including the above mentioned plants for wounds with confirmed effectiveness according to the present pharmacological study. Such herbs could be considered as potential sources for drugs for healing of wounds. However, further pharmacological and clinical investigations are needed for exploring safety, exact mechanisms, and efficacy of these herbal remedies [70].
The above-mentioned families and the later plants deserve further investigations to understand their potential use as a source of medicinal bioactives.

Dose response tests
To further investigate plant extracts which show high activity, we analyzed the activity of these extracts using the dose response with 4 more dilutions (250,125, 62.5, 31.25 mg/ml). 255 plant extracts which show 3 on a scale of 3 in one or more of the following STN screens were tested: antibacterial, antifungal, protozoa lethality, anthocyanin, protease and protease inhibitor, and roundworm lethality assays. However, the other STN screens were excluded from the dose response analyses either because the number of active extracts with high activity were very high (antioxidants, glucosidase inhibitors, amylase inhibitors, and glucosidase), or because the animal model was not available in the case of planaria lethality, and planaria regeneration assays.
Of the tested plant extracts 18 samples exhibited activity even at the lowest extract concentration 31.25 mg/ml and in a dose response manner (Table 3). It is worth noting that some of these plants are known as medicinal plants in TAPHM, including Pistacia lentiscus, Pistacia vera, Rhus coriaria, Ceratonia siliqua, Quercus calliprinos, Punica granatum, and Rubus sanctus [6][7][8][9][10]. Others, such as Erodium gruinum, Smilax aspera, Cistus creticus, and Terminalia chebula are not known or have limited use in TAPHM. These plants should be further investigated to understand their potential use as a source of medicinal bioactives.

Effect of environment on bioactivity
In this study, plants growing under harsh environmental conditions (Irano-Turanian and Sudanian Penetration Territories) showed higher percentages of plant samples showing high-potency bioactivities than plants found in phytogeograpical territories with moderate growing conditions (Mediterranean area), in congruence with the hypothesis that environment may be more important than plant characteristics in determining bioactivity [71] (Figure 4).