Gas chromatography-mass spectrometry analysis, phytochemical screening and antiprotozoal effects of the methanolic Viola tricolor and acetonic Laurus nobilis extracts

Background The antiprotozoal and antioxidant activities of Viola tricolor and Laurus nobilis have been reported recently. Thus, the existing study pursued to assess the growth inhibition effect of methanolic extract of V. tricolor (MEVT) and acetonic extract of L. nobilis (AELN) against five Babesia parasites and Theileria equi in vitro and in vivo. Results MEVT and AELN suppressed Babesia bovis, B. bigemina, B. divergens, B. caballi, and T. equi growth at half-maximal inhibitory concentration (IC50) values of 75.7 ± 2.6, 43.3 ± 1.8, 67.6 ± 2.8, 48 ± 3.8, 54 ± 2.1 μg/mL, and 86.6 ± 8.2, 33.3 ± 5.1, 62.2 ± 3.3, 34.5 ± 7.5 and 82.2 ± 9.3 μg/mL, respectively. Qualitative phytochemical estimation revealed that both extracts containing multiple bioactive constituents and significant amounts of flavonoids and phenols. The toxicity assay revealed that MEVT and AELN affected the mouse embryonic fibroblast (NIH/3 T3) and Madin–Darby bovine kidney (MDBK) cell viability with half-maximum effective concentrations (EC50) of 930 ± 29.9, 1260 ± 18.9 μg/mL, and 573.7 ± 12.4, 831 ± 19.9 μg/mL, respectively, while human foreskin fibroblasts (HFF) cell viability was not influenced even at 1500 μg/mL. The in vivo experiment revealed that the oral administration of MEVT and AELN prohibited B. microti multiplication in mice by 35.1 and 56.1%, respectively. Conclusions These analyses indicate the prospects of MEVT and AELN as good candidates for isolating new anti-protozoal compounds which could assist in the development of new drug molecules with new drug targets.


Background
Piroplasms, the causative factors of piroplasmosis, are among the most prevalent blood parasites in the world and therefore show a significant economic, medical and veterinary impact all over the world [1,2]. The problems of parasite resistance, as well as the toxic residues to most of the commercially available antipiroplasmic drugs (diminazene aceturate (DMA), atovaquone (ATV), clindamycin, imidocarb dipropionate, azithromycin, and quinine) severely weaken their effective curative and protective approaches [3,4]. Therefore, it is clear that the development of treatment options for piroplasmosis is vital for improving disease treatment and control.
Up to date, medicinal plants have been documented as an important source for discovering new pharmaceutical molecules that have been used to treat serious diseases [3,5,6]. Strikingly, previous reports stated that natural products and their derived compounds exhibit lesser side effects and improved efficacy than other synthetic counterparts [7]. Many plant species have been reported to have pharmacological activities attributable to their phytoconstituents such as glycosides, saponins, flavonoids, steroids, tannins, alkaloids, terpenes and accordingly.
Although V. tricolor and L. nobilis have been reported for several medicinal values, there is no evidence on their antipiroplasmic activity. Thus, the current study examined the effectiveness of methanolic extract of V. tricolor (MEVT) and acetonic extract of L. nobilis (AELN) on the multiplication of T. equi, B. bigemina, B. bovis, B. caballi and B. divergens using the in vitro fluorescence assay and their chemotherapy prospects against B. microti-infected mice.

Ethical statement
The in vivo experiments were performed in conformity with the local guidelines for animal experiments, as approved by the Obihiro University of Agriculture and Veterinary Medicine, Japan (accession number of the animal experiment: 28-111-2/28-110). This ethical approval was developed through the basic guidelines for the proper conduct of animal experimentation and related activities in Academic Research Institutions, Ministry of Education, Culture, Sports and Technology (MEXT), Japan.

The chemical reagents
Stock solutions (100 mg (crude extract) / 1 mL (DMSO) and 10 mM) in dimethyl sulfoxide (DMSO) of crude extract and DMA (Ciba-Geigy Japan limited, Tokyo, Japan) and ATV (Sigma-Aldrich, Japan), respectively were stored at − 30°C and used for antibabesiosis evaluation. Reference drugs (DMA and ATV) were used either singly and/or in combination with the two extracts for both the in vivo and in vitro experiments. For the fluorescence assay, SYBR Green I (SGI) stain (10,000×, Lonza, USA) was mixed with the lysis buffer containing saponin (0.016% w/v), EDTA (10 mM), Triton X-100 (1.6% v/v), and Tris (130 mM at pH 7.5) which was filtered using a polyethersulfone (0.22 μm) and kept at 4°C.

Herbal plants
V. tricolor flower and L. nobilis leaves were gathered from Delta, North part of Egypt from June 2016 to August 2016 and identified by the members of the Pharmacology and Chemotherapeutics Department, Faculty of Veterinary Medicine, Damanhour University, Egypt. V. tricolor and L. nobilis voucher specimen numbers are A0177103 (DPV) and A0177104 (DPV), respectively. An electric dryer (Sanyo Electric Co., Ltd., Osaka, Japan) was used to dry the plants at a temperature of 30°C, then ground using a 60-80 mm mesh to a fine powder. Subsequently, fine plant powder (100 g) was extracted using methanol (99.8%) (Wako pure chemical Industrial, Ltd., Osaka, Japan) or acetone (99.5%) (Nacalai Tesque, Kyoto, Japan) (50 mL) and incubated for 72 h at a temperature of 30°C. The preparation of slurry extract was performed following the method as previously described [3,33,34] and the extracted stock (100 mg / 1 mL DMSO) was stored at − 30°C and used for antibabesial evaluation. The obtained extracts of the MEVT and AELN weight were 7.09 and 7.25 g, respectively, and the yield percentage was measured using the following formula [35] Phytochemical examination of plant extracts MEVT and AELN were examined for the existence of terpenoids, saponins, tannins, and alkaloids using several qualitative tests as previously reported elsewhere [36].

Determination of total phenolic material
The total phenolic material concentration present in MEVT and AELN was detected using Folin-Ciocalteu (FC) assay as described elsewhere [37]. A volume of 500 μL of both extracts (1 mg/mL) was added to 1.5 mL of 10% FC reagent and mixed for 5 min. After that, the reaction mixture was further incubated for an additional 2 h after the addition of aliquot (3 mL) of 7.5% Na 2 CO 3 solution. Finally, the absorbance was calculated at 760 nm and the total phenolic compounds were detected from a gallic acid standard curve and expressed as mg/g gallic acid equivalent (GAE) of the dry weight of the extract (mg GAE/g DW).

Determination of total flavonoid material
Aluminium chloride (AlCl 3 ) colorimetric assay was used for the examination of total flavonoid material in MEVT and AELN as previously determined [37]. Briefly, an aliquot (1 mL) of both extracts was added to 3 mL of solvent extracts, 3.8 mL of distilled water, 200 μL of 1 M potassium acetate and 200 μL of 10% AlCl 3 and incubated for 30 min. The flavonoid content was detected from a catechin standard curve after measuring the absorbance at 420 nm and expressed as mg/g catechin equivalents of the dry weight of individual extract (mg CAE/g DW).
Gas chromatography-mass spectrometry (GC-MS) analysis The chemical composition of MEVT and AELN was performed using Trace GC-ISQ mass spectrometer (Thermo Scientific, Austin, TX, USA) with a direct capillary column TG-5MS (30 m × 0.25 mm × 0.25 μm film thickness) as previously described [38,39]. The column oven temperature was initially held at 50°C and then increased by 5°C/min to 250°C withhold 1 min then increased to 300 at the rate of 30°C /min. The injector temperatures were kept at 260°C. Helium was used as a carrier gas at a constant flow rate of 1 mL/min. The solvent delay was 4 min and diluted samples of 1 μL were injected automatically using an AS3000 Autosampler coupled with GC in the split mode. EI mass spectra were collected at 70 eV ionization voltages over the range of m/z 50-650 in full scan mode. The ion source and transfer line were set at 250°C and 270°C, respectively. The components were identified by comparison of their retention times and mass spectra with those of WI-LEY 09 and NIST 11 mass spectral databases.

Parasites and mice
For the in vitro experiments, a German strain B. divergens, Argentine strain B. bigemina, Texas strain B. bovis, were cultivated in cattle RBCs, while USDA strains of equine piroplasm parasites (T. equi and B. caballi) were maintained in horse RBCs [34]. The parasite incubation occurred at 37°C in a humidifying chamber under 90% N 2 , 5% O 2 , and 5% CO 2 atmosphere using a microaerophilic stationary-phase culture. For conducting the in vivo study, female BALB/c mice, aged six-week, obtained from CLEA Japan were infected with B. microti Munich strain [1].

In vitro cultivation of parasites
Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma-Aldrich, Tokyo, Japan) replenished with 40% cattle serum and used as a growth medium for B. divergens parasite culture and culture medium 199 (M199) was used as a growth medium for B. bigemina and B. bovis replenished with 40% cattle serum. While T. equi was grown in M199 complemented with 40% horse serum containing hypoxanthine (MP Biomedicals, USA; final concentration 13.6 μg/mL). GIT medium replenished with 40% horse serum was used as a growth medium for B. caballi parasite culture. To ensure free-bacterial contamination, all medium was supplemented with amphotericin B (0.15 μg/ mL) (Sigma-Aldrich, USA), streptomycin (60 U/mL) and penicillin G (60 U/mL).

Assessment of the impacts of MEVT and AELN on RBCs of host
Prior to parasite subculture, 400 μg/mL of MEVT and AELN were mixed with fresh bovine and equine RBCs and incubated at a humidifying incubator for 3 h. Then B. bovis and T. equi-infected RBCs (iRBCs) were washed thrice with PBS and mixed with the pretreated-RBCs to achieve 1% parasitemia. Thereafter, using a 24-well plate, an aliquot of iRBCs (100 μL) was mixed with culture media (900 μL); the control RBCs were left untreated.
To monitor the parasitemia and any side effects due to the pretreatment, Giemsa-stained smears were prepared every 24 h for 4 days.

The inhibition assay of MEVT and AELN in vitro
The Babesia fluorescent assay was carried out on the in vitro culture as previously reported elsewhere [3]. Briefly, in three separate trials, using two-fold dilution, different concentrations MEVT, AELN, DMA, and ATV were prepared in the culture medium and added in 96well plates in triplicate with 1% parasitemia for equine piroplasm parasites (T. equi and B. caballi) and B. divergens at 5% hematocrit (HCT), while for B. bigemina and B. bovis using 2.5% HCT. The positive control had iRBCs with final concentration of 0.2% of DMSO, whereas uninfected RBCs with medium served as the negative control. Afterward, parasite cultures were incubated for 4 consecutive days without changing medium at 37°C humidifying incubator in 90% N 2 , 5% O 2 , and 5% CO 2 atmosphere. On day four of culture, an aliquot (100 μL) of lysis buffer was added to 0.2 μL/mL SG1 per well; subsequently, it was covered with aluminum foil to prevent exposure to light. After a 6-h incubation at 37°C, fluorescence readings were acquired on a spectrofluorimeter (Fluoroskan Ascent, Thermo Fisher Scientific, USA) with a 485 nm excitation wavelength and a 518 nm emission wavelength.

Parasite viability test in vitro
The viability studies were monitored via microscopy as described elsewhere [1,33]. Briefly, an aliquot (20 μL) of infected RBCs (1% parasitemia) was cultivated in 200 μL of media containing various concentrations of MEVT and AELN for 4 successive days, changing media daily. The concentrations used in this experiment were 0.25 ×, 0.5 ×, 1 ×, 2 ×, and 4 × the IC 50 . On the fifth day, a mixture of iRBCs (6 μL) from each well and fresh equine or bovine RBCs (14 μL) was transferred to another plate, cultured in a medium free from drug and then left for an additional 6 days. The total parasite clearance was recorded as negative, while the relapse of parasites was recorded as positive.

Cytotoxicity assay of MEVT and AELN on normal cells
The cell viability test was conducted in a 96-well plate as described elsewhere [2,39]. Briefly, an aliquot of (100 μL) cells was implanted at 5 × 10 4 cells/mL in DMEM or MEM with fetal bovine serum and incubated overnight under atmosphere 5% CO 2 at 37°C for attachment. Using two-fold dilutions, aliquots (10 μL) of herbal extracts and reference drugs were added in triplicate per well and further incubated for 24 h. The positive control wells containing cells mixed with the medium in 0.4% DMSO, whereas the negative control wells containing culture medium only. After a 24-h incubation, Cell Counting Kits-8 (CCK-8) (10 μL) was added per well and then plate incubation was conducted for an additional 3 h and a microplate reader was used to assess the absorbance at 450 nm.

In vitro combination treatment of MEVT and AELN with DMA and ATV
Combination therapies of MEVT and AELN with DMA and ATV were tested using the fluorescence inhibition method as reported elsewhere [40,41]. Five selected dilutions (0.25 ×, 0.5 ×, 1 ×, 2 × and 4 × the IC 50 ) of the two herbal extracts with DMA and ATV were set up in three sets of duplicate wells and the parasite cultures were incubated for four consecutive days at 37°C humidifying incubator in 90% N 2 , 5% O 2 , and 5% CO 2 atmosphere without changing medium. The drug cultivation and the fluorescence values were detected after the addition of 2 × SGI mixed with lysis buffer to each well of the 96-well plate as described above. Compu-Syn software was used for combination index (CI) values calculation and the synergetic degree was established as the average weighted CI values by using the following formulae; [(1 × IC 50 ) + (2 × IC 75 ) + (3 × IC 90 ) + (4 × IC 95 )]/10 and the resulted values were demonstrated using the recommended CI scale; lower than 0.90 was considered synergetic, between 0.90-1.10 was considered additive, while higher than 1.10 was considered antagonistic developed previously [40,41].

Chemotherapeutic effects of MEVT and AELN against B. microti
MEVT and AELN were examined for its in vivo chemotherapeutic effectiveness using B. microti-infected BALB/ c mice according to a procedure described elsewhere [1]. Twenty-five mice were placed in an environment free

Data analysis
The IC 50 values of the two extracts, DMA and ATV were established from the in vitro growth inhibition by nonlinear regression curve fit on a GraphPad Prism (GraphPad Software Inc., USA). While for in vivo, the significant variations (p < 0.05) among group mean values on parasitemia and one-way ANOVA Tukey's test in GraphPad Prism version 5.0 was used to analyze hematology profiles in mice infected with B. microti [3].

Plant extraction
The yield percentage of the MEVT and AELN were 7.09 and 7.25% w/w dry matter and dark in color.
The inhibition assay of MEVT and AELN in vitro MEVT ( Fig. 1) and AELN (Fig. 2) Table S1. The preparatory assessment of MEVT and AELN was carried out to detect their effect on the cattle and horse RBCs. The parasite proliferation did not show a significant difference between RBCs treated with MEVT or AELN and the untreated one for B. bovis and T. equi ( Fig. 3a and b).

Phytochemical evaluation of MEVT and AELN extracts
The primary examination of MEVT and AELN pointed to the existence of different phytoconstituents such as tannins, saponins, terpenoids and alkaloids that may be the main cause of their pharmaceutical properties ( Table 2).

Gas chromatography-mass spectrometry (GC-MS) analysis
The GC-MS analysis of MEVT and AELN revealed the existence of 27 and 20 phytochemical compounds, respectively. The identified chemical composition of MEVT is shown in Table 4 and represented 29 compounds. While the identified chemical composition of AELN is shown in Table 5 and represented 24 compounds. The phytochemical compounds' identification was established on the basis of the peak area, and retention time. The active principles with their retention time (RT) and percentage of peak area (%) are expressed in Fig. 4a and b.

Viability assay
The    Table 7). The combination effects of MEVT and AELN with ATV were shown in Table 5.

The in vivo chemotherapeutic potential of MEVT and AELN in mice
To investigate the chemotherapeutic potential of MEVT and AELN in vivo, six-female BALB/c mice were infected by B. microti and the two extracts were administered for 5 days after the infection reach 1% parasitemia.
On eighth day post-infection (p.i), the control group treated with double distillate water (DDW) exhibited rapid growth of parasitemia reached 58.2% and the parasitemia reduced slowly on the subsequent days. The level of parasitemia in all treated groups reached 37.8, 25.5 and 3.9% in MEVT (150 mg kg − 1 ), AELN (150 mg kg − 1 ), and DMA (25 mg kg − 1 ), respectively, at 8 days p.i (Fig. 5). Additionally, the hematology parameters; HCT percentage, RBCs count and HGB concentration (Fig. 6a-c) showed a significant difference in the MEVT-and AELN-treated groups when compared with the positive control group. Whereas, the comparison of the hematology parameters during in vivo studies showed no significant difference (p < 0.05) between MEVT-, AELN-treated groups as compared to the DMA-treated group.

Discussion
Plant extracts possess significant therapeutic effects with minimal side effects for the treatment of many infectious diseases [33], thus making medicinal plants an attractive choice for the source of new therapeutic compounds [34]. In spite of the antiparasitic effect of V. tricolor and L. nobilis, they have not been evaluated against piroplasmosis. Therefore, this study investigates the in vitro as well as in vivo antipiroplasmic efficacy of MEVT and AELN.
The existing study revealed that MEVT and AELN possess various biologically active compounds and the primary screening emphasized that both extracts contain terpenoids, alkaloids, flavonoids, and tannins ( Table 2). The qualitative examination revealed the presence of significant amounts of polyphenols and flavonoids (Table  3). Notably, this finding conforms to the report by Chandra et al. [42] and Alejo-Armijo et al. [43] who revealed the existence of these active constituents in both extracts. It has been shown that all these secondary metabolites have many therapeutic properties and are known to be pharmacologically active components.
The in vitro experiments indicated that MEVT and AELN suppressed the in vitro multiplication of piroplasm parasites. Previous reports documented that V.  [14] reported that L. nobilis  Results are calculated as the mean values from three separate trials ± SD, a positive (+) indicates parasites regrowth, and a negative (−) shows the parasites total clearance after drug pressure withdrawal using microscopy assay and V. tricolor has strong antioxidant, antimicrobial and antibacterial activity. Interestingly, recent studies documented the antiprotozoal activities of various bioactive molecules identified in our GC-MS analysis. For instance, Castaño Osorio and Giraldo García [45]. reported that different sesquiterpene lactones and costunolide, isolated from Laurus nobilis, showed antiprotozoal efficacy against Trypanosoma brucei rhodesiense. Colares et al. [46] and Le et al. [47] proved the antileishmanial activity of eugenol and methyleugenol against promastigotes of L. amazonensis. Moreover, Charma et al. [48] documented the antileishmanial effect of cinnamaldehyde and eugenol with IC 50 values of 0.681 and 1.426 g/ml, respectively. Eucalyptol and caryophyllene showed antitrypanosomal and antileishmanial activities, respectively [49]. Therefore, we hypothesized that eucalyptol, caryophyllene, cinnamaldehyde, eugenol, methyleugenol, and costunolide are the main active compounds responsible for the antipiroplasmic activity of MEVT and AELN. The CCK test used to examine the cytotoxicity of MEVT and AELN revealed their effect on NIH/3 T3, HFFs, and MDBK cell viability with a slightly high selective index value. Meaning that MEVT and AELN are more likely to affect the viability of piroplasm parasites rather than host cells. Notably, this finding conforms to the report by Dua et al. [5], who revealed that Viola extract exhibited antiplasmodial activity with some cytotoxic efficacy toward the L-6 cell line. Furthermore, Kivçak and Mert [28]. documented the safety of water and ethanol L. nobilis extracts on the Brine shrimp. Previous reports documented the cytotoxic activities of the  Their EC 50 values were higher than 400 μg/mL concentration, suggesting their safety on the normal cell lines [17,23]. Nowadays, combination chemotherapies are being reported to alleviate serious diseases, including pulmonary tuberculosis, malignancy, immune deficiency syndrome, and some protozoal diseases to promote higher therapeutic efficacy. The MEVT-DMA or ATV combined effects were additive toward all tested parasites. Likewise, AELN combined with DMA and ATV showed additive and synergistic effects toward T. equi, B. caballi, B. bigemina, B. divergens and B. bovis. These results are compatible with Batiha et al. [3] who previously investigated the in vitro combined efficacy of methanolic extracts of S. aromaticum and C. sinensis with DMA and ATV toward piroplasm parasites. They concluded that these combined effects are property for developing new chemotherapy techniques against piroplasm parasites. Together, these findings in our study emphasize that these combinations have prospects to be used as a treatment option of animal and human babesiosis.
In the in vivo experiment, the oral administration of MEVT and AELN resulted in 35.1 and 56.1% suppression in the parasitemia level on the eighth-day p.i., respectively when compared with 93.2% restriction showed by DMA. The effectiveness of MEVT and AELN was comparable to that shown by Batiha et al. [3], who reported that the methanolic extracts of S. aromaticum and C. sinensis on the eighth-day p.i. resulted in 69.2 and 42.4% inhibition in the parasitemia at day 8 p.i., respectively. Interestingly, no obvious toxic signs were observed in MEVT-and AELN-treated mice.
Nevertheless, MEVT and AELN, like DMA, prohibited anemia development in mice, although temporal reductions were observed in HCT, RBCs, and HGB. Furthermore, neither the MEVT nor the AELN treatments showed any apparent toxic symptoms or promoted anemia in uninfected mice. Interestingly, many reports have shown remarkable antioxidant and pro-oxidative effects of ethanol and water extract of the whole plant of Viola by prohibiting the reactive oxygen species (ROS) generation or stimulating the protein detoxification [11]. Additionally, Emam et al. [22] observed the significant antioxidant, antipyretic, and anti-inflammatory properties after L. nobilis extracts treatment. Moreover, Ozcan et al. [6] reported that essential oil, methanolic extract of seed oil and seed oil from L. nobilis possess antioxidant and antimicrobial activities. Such medicinal characteristics are important for piroplasmosis treatment because piroplasmosis infection is not only correlated with emaciation and poor growth performance in cattle but also with immunosuppression and overproduction of reactive oxygen and nitrogen species [3]. The limitation of this study is performing the cytotoxicity assay in vitro using cell lines, and it is recommended to evaluate the cytotoxic activity of our extracts in vivo. These findings emphasize the MEVT and AELN ability to eradicate B. microti in mice. Taken together, these findings support that MEVT and AELN could be a potential source of alternate chemotherapy against B. microti infection in humans.

Conclusions
To our knowledge, this is the first antipiroplasmic evaluation of methanolic V. tricolor and acetonic L. nobilis extracts against piroplasm parasites. MEVT and AELN exhibited an in vitro growth inhibitory effect against five piroplasm species as well as chemotherapeutic efficacy toward B. microti in vivo. Furthermore, the combination treatment of our herbal extracts with DMA and ATV demonstrated synergistic and additive effectiveness against all testes parasites. Our GC-MS analysis results documented the existence of several phytochemical molecules that may be responsible for the babesicidal activities of MEVT and AELN. Therefore, it is recommended to evaluate the antipiroplasmic efficacy of the GC-MS identified compounds for the future discovery of a novel potential drug against piroplasmosis. And evaluate the actual mode of action employed against the recovery of piroplasm parasites.
Additional file 1: Table S1. The IC 50 and selective indexes value of ATV and DMA.