Two New Bioactive Biphenylpropanoids from the Roots of Salsola imbricata ( Chenopodiaceae ) Growing in Saudi Arabia

Phytochemical investigation of the roots of Salsola imbricata allowed to two new bioactive biphenylpropanoids (1) and (2) named, biphenylsalsonoid A and B, respectively. Their structures were established through spectroscopic methods (1D and 2D NMR, (ES)-HRMS, and IR). The isolates were tested for their antioxidant activity using DPPH• and ABTS•+ assays. 1 and 2 showed a moderate activity towards DPPH (IC50 = 86.5 ± 1.3 and 122.3 ± 0.63 mg/mL, respectively) and ABTS (IC50 = 95 ± 1.5, 137.7 ± 1.2 mg/mL, respectively). The antibacterial effect of the ethyl acetate extract and the isolates were assessed. Results obtained revealed that compounds showed important antibacterial activities against S. aureus, S. epidermidis, M. luteus, and E. coli with MIC values ranging from 16 to 32 mg/mL.


INTROdUCTION
The genus Salsola includes halophyte species and belongs to the family of Chenopodiaceae [1][2][3] .The genus is widespread in the dry regions of Middle East, Africa, and Europe.Many species among the genus are used in traditional medicine.In the Middle East, Salsola baryosma is used as a diuretic agent and against some inflammations 4 .This plant also exhibits antioxidant activities 5 , alkaloids (salsolin and salsolidin) have been isolated from Salsola tragus (synonym: Salsola kali) used in the treatment of hypertension by stimulating the activity of sleep 6,7 .At present, only few species of the genus have been studied chemically and biologically and were found to be a good source of phenolic compounds identified in S. kali, S. soda, S. oppositifolia and S. collina 8,9 .Furthermore, antioxidant triterpenes were isolated from S. baryosma and S. somalensis 10 and new antioxidant bibenzyl derivative and isoflavonoid were isolated from S. tetrandra 11 .Our previous work on the genus led to salsolanol and biphenylsalsinol isolation from S. villosa 12 and cleomiscosin D, norisoprenoid, long-chain hydroxyl fatty acids, taxiphyllin, trans-Nferuloyltyramine S-(-)-trans-N-feruloyloctopamine and coumarinolignan from S. tetrandra 13,14 .Recent research on Salsola imbricata showed the presence of triterpene saponins from the methanolic extract of roots 14 and new isorhamnetin derivatives from the leaves 15 .Thus, in order to continue our research on the genus of salsola growing in Saudi Arabia.We focused our study on the ethyl acetate extract from the roots of S. imbricata because it has not been studied previously.Indeed, the present study suggests isolating new compounds with important biological activities.We try to the isolation of new bioactive compounds from the roots of S. imbricate and the evaluation of its antibacterial activity against Gram-positive and Gram-negative bacteria.Furthermore, the in vitro antioxidant activity was tested by using DPPH• and ABTS• + assays of isolated compounds.

General Procedures
The optical rotations were recorded on a Perkin-Elmer 241-MC polarimeter.UV spectra were measured by using a Shimadzu UV-2401A spectrophotometer.Infra-red spectra were measured on a Perkin-Elmer 157G. 1 H, 13 C and 2D NMR spectra of isolated 1 and 2 were obtained in CD 3 OD on Bruker 300 MHz, 75 MHz( spectrometer using internal reference the residual solvent resonance.Coupling constants were measured in Hertz and chemical shifts were reported in ppm.ESI-HRMS was measured on a Shimadzu LC-MS Spectrometer.

Plant material
Roots of Salsola imbricata Delile.ex Schul.Were collected from Arar, Saudi Arabia, on November 2015.The plant was identified by Dr. Ahmed K. Osman, College of Sciences, Department of Biology, Kingdom of Saudi Arabia and deposited in the herbarium (Sv-26) of the above department.

Antibacterial Activities
The antibacterial activities were tested against Gram-positive (Staphylococcus aureus, S. epidermidis and Micrococcus luteus) and Gramnegative strains (Pseudomonas aeruginosa,

HO
Escherichia coli, and Salmonella typhimurium).The tested extracts were respectively: Methanol crude of the roots, the isolated compound 1 and the isolated compound 2. Mueller-Hinton agar (5ml) was used for the culture of bacteria (stored at -70 °C stock) and the media were incubated for 24 h at 37 °C.
The antibacterial activity was evaluated by minimum inhibitory concentration (MIC) 16,17 .Serial tube dilution was used to determine the values of MIC for the methanol crude extract and for the two isolated compounds.To obtain stock solution, 0.5 mg of plant extracts (methanol crude, compound 1 and 2) was suspended in 2 mL of distilled water and 2 drops of between-80 for the homogenization.The suspensions of micro-organisms consist of a medium with the concentration fixed at 10 7 organisms/mL and one drop of suspension (0.02 mL) was added to the broth dilution.The temperature of the incubation was fixed at 37 °C for 18 h and the tubes were examined for the growth.The MIC of the tested extract/products was fixed for the lowest concentration that showed the totally absence of the growth for the microorganisms.The negative and the positive control consist, respectively, of distilled water with 2 drops of tween-80 and kanamycin.

Antioxidant activities Free radical scavenging ability using dPPH • radical
The protocol was used as described, previously, by Tepe, B. et al. 2005 19 .Briefly, 2 mL of DPPH solution (100 ìg/mL, EtOH) was added to 0.5 mL of compounds (0.01-1 mg/mL).After 30 min, the absorbance was read at 517 nm.The blank consists of 2 mL of DPPH solution and 0.5 mL of methanol.The IC 50 was determined by the following equation: A blank and A sample were, respectively, the absorbance values of the control and the test sample.Vitamin C was used as reference and tests were measured in triplicate.

Free radical scavenging ability using ABTS •+ radical cation
ABTS was dissolved in distilled water and the concentration was fixed at 7 mmol/L 20 .For the completion of radical generation, ABTS• %+ radical cation was generated by adding the potassium persulfate (2.45 mmol/L) to the ABTS solution.The mixture was conserved in the darkness for 12-16 h at room temperature.After a dilution of the mixture with ethanol, the wavelength was fixed at 734 nm until to obtain an absorbance value of 0.70 ± 0.02.ABTS solution (50 mL) was added to 950 mL of compounds (0.01-1 mg/mL) and after 6 min.the absorbance was measured at 734 nm.The blank consists of 50 mL of ABTS solution and 950 mL of ethanol.The IC 50 was determined by the formula mentioned before in DPPH assays.
In the aromatic region of the spectrum 1 H NMR of 1 (Table 1) displayed proton signals at d H 6.98 (H-2', d, J = 1.9Hz) and 6.84 (H-6', d, J = 1.9Hz), attributable to the metacoupled protons, of the terasubstituted aromatic ring A and two aromatic proton signals at d H 7.01 (H-5, d, J =8.1Hz) and 6.78 (H-6, d, J = 8.1Hz) attributable to the two ortho-coupled protons of the tetrasubstituted aromatic ring B. The same spectrum displayed the presence of two trans-olefinic protons resonating at d H 5.55 (1H, d, J =15.9Hz) and at d H 6.25 (1H, dt, J =15.9Hz, J = 6.0Hz), attributable to H-7 and H-8, respectively, as well as two methoxy groups at d H 3.76 (3H, s) and 3.84 (3H, s) assignable to H-10 and H-10', respectively.
The 13 C-NMR and DEPT spectra of 1 showed signals for 14 sp 2 carbons (seven methines and seven quaternary carbons) and 6 sp 3 carbons (two methyl, two methylene and two methine groups) (Table 1).
The aromatic region of the 1 H-NMR 2 (table 1) spectrum showed characteristic singlets at d H 6.58 (2H, s) which were attributed to the equivalent protons H-2' and H-6' of the tetrasubstituted aromatic ring A. The same region exhibited two aromatic signals at d H 6.87 (d, J = 1.8Hz) and at d H 7.01 (d, J = 1.8Hz) attributed to H-2 and H-6.In addition, the spectrum showed a singletat d H 3.78 (6H, s) attributed to the two equivalent methoxy goups H-10'and H-11' (-OCH 3 ) attached to the aromatic ring A and another singlet at d H 3.76 (3H, s) corresponding to the methoxy group H-10 (-OCH 3 ) attached to the second aromatic ring B. The 1 H-NMR, 13 C-NMR and HMQC spectra exhibited characteristic resonances of two disubstituted epoxides 12,21,22  The 13 C spectrum of 2 showed resonance of 12 sp 2 carbons attributable to eight quaternary carbons which four are oxygenated and four tertiary carbons (Table 1).The same spectrum also showed three methoxy carbons at d C 56.8 and six  1).
The 1 H-1 H COSY experiment (Fig. 3) showed correlations H-8 with H-7and H-9on the one hand and /H-8' with H-7'and H-9'on the other hand provided evidence for the two epoxy propanoid moieties.The above spectral data indicate that 2 and 1 are two analogous compounds.

Antioxidant activities
For the antioxidant activities of the isolated compounds 1 and 2, two assays have been assessed: DPPH free radical scavenging and ABTS system (Table 2).
The isolate biphenylpropanoids 1 and 2 showed a moderate antioxidant activity towards DPPH with IC 50 values of 86.5 ± 1.3 and 122.3 ± 0.63 mg/mL, respectively, but less potent when compared to Vitamin C. On the other hand, the isolated biphenylpropanoids showed antioxidant activity against ABTS in the similarly order as against DPPH (IC 50 = 95 ± 1.5, 137.7 ± 1.2 mg/mL, respectively).Compound 1 has a relatively high activity due to the presence of two phenol groups by comparison with 2 bearing one phenol group.

Antibacterial Activities
The in vitro antibacterial activity of the EtOAc extract of the roots of S. villosa, compounds 1 and compound 2 was assessed using the MIC method against three Gram-positive bacteria; S. aureus, S. epidermidis and Micrococcus luteus and three Gramnegative bacteria; Escherichia coli, Pseudomonas aeruginosa and Salmonella typhimurium.
According to the results given in Table 3.The compounds 1 and 2 display the same activity against the three Gram-negative used and the Gram-positive strains S. aureus and S. epidermidis.This finding could be due to the common biphenyl skeleton bearing in the same position (C-4') present in both 1 and 2. On the other hand, Compound 2 (MIC = 16 mg/mL) was found to be two times more active than compound 1 against M. luteus.This dual sensitivity against the compound 2 might be explained by the difference in structure between the two compounds, mainly the apparition of a new epoxy moiety at C-4 and another methoxy group at C-5'.In addition to the modification in the position of the hydroxyl group by comparison to compound 1.

CONCLUSION
Two new natural biphenylpropanoid analogous 1 and 2 (named biphenylsalsonoids A and B) were isolated from the roots of Salsola villosa.Their structures were elucidated by spectroscopic methods including 1D, 2D-NMR experiments.Compounds 1 and 2 showed a moderate activity of radicalscavenging towards DPPH and ABTS.The ABTS scavenging activity of compounds was similar to the DPPH free radical scavenging.Their antibacterial activity was evaluated by the MIC method against Staphylococcus aureus, S. epidermidis, Micrococcus luteus, Escherichia coli, Salmonella typhimurium and Pseudomonas aeruginosa.The two compounds have shown the same activity towards the tested bacteria, except M. luteus which exhibited more sensitivity against compound 2.

Fig. 2 : 3
Fig. 2: Relevant HMBC ( ), COSY ( ) and NOEs ( ) correlations for compound 1 . The presence of the first epoxy propanoid at C-4 was established by the correlations of the proton H-7 (d H 5.51) with C-4 (d C 126.8), C-3 (150.1) and C-5 (152.4) observed in HMBC spectrum.The location of the methoxy group at C-5 was confirmed by the 3 J correlations H-10/C-5 deduced from the HMBC spectrum (Fig. and by the appearance of the noe between H-7 and H-10(-OMe) observed in the NOESY spectrum.The second epoxy propanoid at C-4' was clearly indicated by the correlations of the proton H-7' (d H 5.46) with (C-3'and C-5', d C 152.2) and C-4' (d C 126.2) observed in HMBC spectrum (fig 3).The position of the two equivalent methoxy groups at C-3' and C-5' was established by the 3 J C-H correlations of the protons resonating at d H 3.78 (H-10',11') and the aromatic quaternary carbons C-3', C-5' (d C 152.