Anti-trypanosomal activity and DNA fingerprinting of fifteen Euphorbia species using ISSR and SCoT markers

Euphorbia is the largest genus in family Euphorbiaceae with a great biological and genetic diversity. The anti-trypanosomal activity of the crude extract of 15 Euphorbia species against Trypanosoma brucei brucei was carried out. Furthermore, DNA fingerprinting of the tested species using ISSR and SCoT markers was also investigated. The anti-trypanosomal activity of the 15 Euphorbia species revealed the highest activity of E. officinarum L. and E. milli Des Moul. against Trypanosoma brucei brucei with IC50 values < 10 μg/mL after 48- and 72-h incubation. Moreover, the assessment of the genetic diversity among the 15 tested species showed similar correlation coefficients of 0.76–0.98 which classified Euphorbia species into two main groups, one contained two species and the other contained 13 species. Some Euphorbia species exhibited significant growth inhibitory activity toward Trypanosoma brucei strain TC221. Results also indicated the suitability of both markers for genetic fingerprinting of the tested Euphorbia species. To our knowledge, this is the first detailed comparison of the performance of two targeted DNA molecular markers (SCoT and ISSR) on the tested 15 Euphorbia species. The results guide future efficient use of these molecular markers in the genetic analysis of Euphorbia.


Background
Human African trypanosomiasis, sleeping sickness, is a mortal vector-borne parasitic disease caused by Trypanosoma brucei transmitted by the tsetse fly (Glossina spp.), especially in sub-Saharan Africa [26]. Up till now, only a few drugs have been approved to treat human African trypanosomiasis as suramin, pentamidine, melarsoprol, eflornithine, and the combination of nifurtimox/ eflornithine. Most of the drugs discovered in the 1940s and 1950s had adverse effects such as nausea, vomiting, diarrhea, abdominal cramping, fever, hypoglycemia, hypertension, heart damage, seizures, peripheral neuropathy, and neutropenia [18].
Natural sources as plants, microorganisms, animals, and marines provide a high number of natural products with various chemical structures and novel pharmacological mechanism of action [16]. Therefore, discovering anti-trypanosomal drugs from natural sources is an urgent requirement. Euphorbia was reported for its antiarthritis, anticancer, anticonvulsant, anti-diabetic, antieczema, anti-inflammatory, antimicrobial, antioxidant, antispasmodic, anti-tumor, and myelopoiesis properties [5,12].
Euphorbia genus contained a wide variety of terpenoids, ranging from mono-, sesqui-, and diterpenes to triterpenoids and steroids [24]. Metabolic profiling of 15 Euphorbia species, using LC-HRMS, annotated of 44 natural compounds [11]. Despite the taxonomic effort discussing the variations and relationships among Euphorbia species, morphologically based relationship between different Euphorbia species is confusing in many cases [9]. They can be morphologically differentiated by their flower color, but it is difficult to distinguish them at the vegetative stage [7]. [6] identified 600 species of the genus Euphorbia and then branded into 26 sections. [27] included only the shrubby, fleshy, and cactus-like species in the genus Euphorbia. The final analysis as presented by [29] was to treat Chamaesyce as a separate genus, whereas all other taxa were downgraded to sub-generic status within Euphorbia. Thus Euphorbia is still a large polymorphic genus with seven subgenera, viz. Esula, Poinsettia, Euphorbia, Agaloma, Eremophyton, Lyciopsis, and Rhizanthium [25].
Plant identification is the first step to assure the efficacy, quality, and safety of a drug or an extract [23]. Molecular genetic markers have been widely used in the last decades in the search for valuable plant phenotypes [13]. They are mostly used for fingerprinting, diversity analyses, gene mapping, variability, tracking individuals or lines carrying particular genes, and linkage maps [17]. Using more than one marker is continuously recommended for a better analysis of the genetic homogeneity of plants [20]. DNA-based markers illustrate the germplasm and count the degree of genetic diversity more specifically than other markers [19].
Herein, the anti-trypanosomal activity of the methanolic extract of the 15 tested species is evaluated. In addition, two genetic markers, SCoT and ISSR, are also used for identification, authentication, and determining the degree of diversity and similarity between 15 Euphorbia species.

Plant materials
The aerial non-flowering parts of 15 Euphorbia species were collected during October 2018 from Helal cactus farm, Abdel Samad village, El-Mansouria, Giza, Egypt. All the collected species consisted mainly of stems and leaves. The species were identified and authenticated by Prof. Dr. Abdel-Halim Mohammed, Professor of Agriculture, Flora Department, Agricultural Museum, Dokki, Giza, Egypt. Fresh plants were stored at − 5 °C for the DNA study and coded as follows: E. abyssinica J.

Methanolic extracts for anti-trypanosomal activity
Methanolic extracts were prepared by maceration of 50 g fresh samples in (3 × 200 mL) 80% methanol (Sigma-Aldrich, Germany). The extracts were separately filtered and evaporated to dryness using a rotary evaporator (Buchi, G., Switzerland). Dried extracts were stored at 4 °C.

Anti-trypanosomal activity
The anti-trypanosomal activity had been investigated following the method reported by [1,14]. Complete Baltz medium was used for cultivation of Trypanosoma brucei strain TC 221 (Missionsärztliche Klinik, University of Wurzburg), Germany. Trypanosomes were seeded in 96-well plate chambers with a pore size of 0.4 μm at an initial cell density of 3 × 10 4 /mL in 140 μL of the medium. They were tested versus various concentrations of the tested 15 Euphorbia methanolic extracts at 0.25-40 μg/ mL in 1% DMSO to a final volume of 200 μL. 1% DMSO was used as the negative control, and suramin (Calbiochem-Novabiochem Co., purity > 98% by HPLC) was selected as the positive control (concentration of 1 to 0.008 μg/mL). Suramin gave an anti-trypanosomal activity against T. brucei at an MIC of 0.23 μg/mL. The plates were incubated for 48 h at 37 °C, 5% CO 2 with a humidified atmosphere, after which 20 μL of Alamar blue was added. Then, plates were incubated again for another 24 h under the same conditions. The activity was measured by light absorption at ʎ 550 nm with a reference wavelength of 650 nm using an MR 700 microplate reader (Dynatech Engineering Ltd., Willenhall, UK) after 48 and 72 h. The quantification of the IC 50 values of the investigated extracts (the dose of the extract which reduces survival to 50%) was processed by linear interpolation.

DNA analysis 2.4.1 DNA isolation procedure
Sample from each of the 15 Euphorbia species (fresh stem) was ground by liquid nitrogen to a fine powder and transferred to an appropriately sized tube. The bulked DNA extraction was performed via DNeasy Plant Mini Kit (QIAGEN) on Agriculture Research Center in Giza, according to [10,30] methods.

PCR (Polymerase Chain Reaction) condition for SCoT and ISSR
DNA amplifications were achieved in an automated thermal cycle (model Techno 512) which was programmed for one cycle at 94 °C for 4 min, followed by 45 cycles of 1

Gel preparation procedure
Agarose (1.5 g) was mixed with (100 mL) l × TBE buffer and boiled in a microwave. Ethidium bromide (5 µL) was added to the melted gel after the temperature became 55 °C. Then , the melted gel was poured into the tray of the mini-gel apparatus and a comb was inserted immediately after that comb was removed when the gel becomes hardened. The gel was covered by the electrophoretic buffer (1 × TBE) , and DNA-amplified product (15 µL) was loaded in each well. DNA ladder (100 bp) mix was used as standard DNA using molecular weights ranging from 100 to 3000 bp, and the run was done for about 30 min at 80 V in mini-submarine gel BioRad.

Data analysis for genetic study
The similarity matrices were done using Gel works ID advanced software UVP-England Program. The relationships among genotypes as revealed by dendrograms were done using the SPSS windows (version 10) program. DICE computer package was used to calculate the pairwise difference matrix and plot the phenogram among cultivars [8,31].

Anti-trypanosomal activity
The anti-trypanosomal assay was used here as one way to discriminate between the active and inactive extracts (

Genetic diversity revealed by start codon targeted (SCoT) markers
Ten SCoT polymorphic markers were screened to access the level of genetic diversity and similarity between 15 Euphorbia species, only five produced scorable bands and selected for the analysis (Additional file 1: Table 2 and Fig. 1). They produced a total of 25 reproducible and scorable bands ( Table 2). Each primer produced an amplification product in the range of 135-2465 bp, and the number of the scorable bands varied from 2 to 8 for each primer. The total polymorphism was estimated as 48%. The distribution of monomorphic (common) and polymorphic bands generated by the five SCoT markers showed a unique band at 1735 bp for SCoT 10 with E. trigone Mill. (3), while for SCoT 12 a unique band at 1500 bp with E. lactea Haw. was detected (13) ( Table 3). The dendrogram of the tested Euphorbia species revealed classification of the 15 species, according to the data obtained by the SCoT into two main clusters. The first cluster included species 1 and 2 corresponding to E. abyssinica J.F. Gmel. and E. caput-medusae L., indicating close similarity between these two species. The other cluster segregated the remaining species into three more sub-clusters (Additional file 1: Figure 1).

Genetic diversity revealed by inter-simple sequence repeat (ISSR) markers
In the case of ISSR markers, five markers yielded reproducible bands out of 15 screened markers (Additional file 1: Table 3 and Fig. 2). They yielded 26 scorable bands ( Table 4). The bands ranged from 130 to 1665 bp, while the band numbers ranged from 3 to 8 in each primer. The distribution of monomorphic and polymorphic bands produced by the five ISSR markers revealed that 14A showed unique bands at 1430 and 740 bp with E. milli Des Moul. (15), 44B provided unique band with E. grandialata R.A. Dyer (11) at 315 bp, and HB-12 had unique band with the same species at 360 bp. The total polymorphism was estimated as 57.7% (Table 5). The dendrogram of 15 Euphorbia species was represented in Additional file 1: Figure 2. The 15 examined species were segregated into two main clusters. The first cluster contained seven species, and the second contained the remaining eight.

Combination of ISSR and SCoT data obtained from the 15 Euphorbia species
The markers produced a total of 51 scorable bands ( Table 6). The number of bands for each primer varied from 2 to 8 and ranged from 130 to 2465 bp. The maximum number of bands was obtained in SCoT 10 and 44B, and the lowest number of bands was recorded in SCoT 8. A monomorphic banding pattern was observed for all the amplified bands from SCoT and ISSR in all Euphorbia species. Dendrograms were generated for the two markers (Additional file 1: Figure 3) and revealed two major groups, one contained E. abyssinica J.F. Gmel.
(2) and the other contained the remaining species.

Similarity index combination of SCoT and ISSR analysis
Results illustrated that the examined species were segregated according to the data obtained into two main clusters. The first cluster included only two species E. abyssinica J.F. Gmel. and E. caput-medusae L. with similarity 98.1% to each other and with the lowest similarities with the others. On the other hand, the second cluster was divided into two subclusters: Subcluster 1 contained six species, which showed some similarities with the remained species as it was between the first cluster and subcluster 2 and referred to E. officinarum L., E. obesa

Anti-trypanosomal activity
The anti-trypanosomal activity of three extracts of E. hirta Linn. (hexane, ethyl acetate, and methanol) was Table 3 The distribution of monomorphic and polymorphic bands generated by the SCoT markers

DNA analysis
Appropriate identification of plant materials is essential for the conservation of medicinal plant resources and to ensure the proposed biological efficacy. Molecular techniques have recently been used for plant diversity studies as well as for confirmation of the identity of plant species. [9] used multiple regions of DNA sequence data, cpDNA coding region matK including the partial trnK intron, the cpDNA coding region ndhF, and the internal transcribed spacer region of the nuclear ribosomal DNA (ITS), for taxonomic classification of Euphorbia species. Furthermore, RAPD and ISSR markers were reported to be used for genetics profiling of three Euphorbia species (E. prostrata Aiton, E. peplus L., and E. terracina L.) [22]. The current study uses two markers, SCoT and ISSR. Both markers were previously   Total SCoT  17  17  23  22  23  19  19  19  18  21  24  20  18  22  18   Total ISSR  18  17  22  20  17  17  17  21  20  19  20  20  20  21  15   Total  35  34  45  42  40  36  36  40  38  40  44  40  38 43 33 used for the diagnostic fingerprinting of Pistacia vera cultivars [4]. Moreover, the ISSR marker was previously used to distinguish between three varieties of E. milli at the vegetative stage, and it was observed to be a useful molecular marker as it produced 98.7% polymorphism, which proved high genetic diversity [7]. Results of the present study revealed that both markers generated high numbers of polymorphic bands that could be used in the diagnostic fingerprinting of Euphorbia species. ISSR was found to be more efficient in clarifying the genetic diversity and homogeneity between the 15 tested Euphorbia species. This was indicated by the percentage of polymorphism (57.7%) recorded for ISSR compared to 48% for SCoT. Considering unique bands, that allowed easier and faster genetic identification, ISSR markers produced more unique bands (four bands) than SCoT marker (two bands). Phylogenetic relationships among six Euphorbia species were previously, analyzed by RAPD-PCR markers, and the resulted dendrogram indicated similarity between E. milli and E. tirucalli [3]. However, the current study recorded diversity between these two species according to both SCoT and ISSR markers. Accordingly, these results confirmed that using more than one marker is mostly recommended for a better analysis of genetic homogeneity of plants, as previously stated by [20].

Conclusions
The present study highlighted the anti-trypanosomal activity of 15 Euphorbia species where four species exhibited high activity against T. brucei brucei with E. officinarum L. being the most active. Genetic fingerprinting of the 15 tested species, using ISSR and SCoT markers, indicated six unique bands, one for E. trigone Mill., one for E. lactea Haw., two for E. milli Des Moul., and two for E. grandialata R.A. Dyer. Moreover, the dendrogram, generated for the two markers, revealed genetic similarity between E. abyssinica J.F. Gmel. and E. caputmedusae L. picked them in one cluster, while the other remained species were further subdivided under the second cluster. It concluded successful employment of ISSR and SCoT molecular markers for detecting levels of DNA polymorphism and genetic relationship in genus Euphorbia. ISSR is recommended for better analysis of homogeneity among Euphorbia species.
Additional file 1. The nucleotide sequence of each marker, The result of each marker & the dendrograms of SCoT, ISSR and their combination.