Phytochemical Investigation and Biological Screening of Ethyl Acetate Fraction of Salvia hispanica L. Aerial Parts

Family Lamiaceae consists of about 250 genera and 7000 species worldwide.1 The genus Salvia has about 1000 species.2 Mexican chia seeds protein content ranged from 18.5 to 22.3%, fat content ranged between 21.5 and 32.7%with their highquality fatty acids3. The survey involving chia seeds indicate the presence of phenolic acids and flavonoids have the most appropriate antioxidant activity4-9 and showed anti-obesity, anti-diabetic, anti-oxidant and anti-microbial activities.10-13 On the other hand, the study of S. hispanica L. aerial parts indicate the presence of neoclerodane diterpenoids,14, 15 also tentative identification of phenolics.16 There is no phytochemical investigation about S. hispanica cultivated in Egypt so this work focuses on it, resulted in the isolation of main bioactive phytochemical constituents that including 1,2,4,5 tetrahydroxy benzene (first report to be isolated from nature), leucantho flavone and rhamnetin (first report to be isolated from Salvia), UPLC-ESI-MS/MS analysis for the first time on S. hispanica L. aerial parts cultivated in Egypt and evaluation of biological activities of ethyl acetate fraction.


INTRODUCTION
Family Lamiaceae consists of about 250 genera and 7000 species worldwide. 1 The genus Salvia has about 1000 species. 2 Mexican chia seeds protein content ranged from 18.5 to 22.3%, fat content ranged between 21.5 and 32.7%with their highquality fatty acids 3 . The survey involving chia seeds indicate the presence of phenolic acids and flavonoids have the most appropriate antioxidant activity [4][5][6][7][8][9] and showed anti-obesity, anti-diabetic, anti-oxidant and anti-microbial activities. [10][11][12][13] On the other hand, the study of S. hispanica L. aerial parts indicate the presence of neoclerodane diterpenoids, 14,15 also tentative identification of phenolics. 16 There is no phytochemical investigation about S. hispanica cultivated in Egypt so this work focuses on it, resulted in the isolation of main bioactive phytochemical constituents that including 1,2,4,5 tetrahydroxy benzene (first report to be isolated from nature), leucantho flavone and rhamnetin (first report to be isolated from Salvia), UPLC-ESI-MS/MS analysis for the first time on S. hispanica L. aerial parts cultivated in Egypt and evaluation of biological activities of ethyl acetate fraction.
dichloromethane (7× 500 ml) and finally ethyl acetate (5× 500 ml). The fractions were washed with distilled water and dried over anhydrous sodium sulphate then the solvent of each fraction was distilled off under reduced pressure at 50 °C to yield 68 gm of light petroleum fraction, 4 gm of dichloromethane fraction and 14 gm of ethyl acetate fraction.

Isolation
About 11gm of ethyl acetate fraction was dissolved in a least amount of methanol and adsorbed on 250 gm silica gel for column and the solvent was evaporated completely. The dry zone was applied on the top of silica gel column (5 x 120 cm, 200 g) packed by wet method using dichloromethane, the development was started with dichloromethane and the polarity was increased gradually using methanol to yield 55 fractions. Fractions (15)(16)(17)(18)(19)(20) eluted by 4% MeOH/CH 2 Cl 2 were combined, concentrated then subjected to TLC examination , revealed the presence of one major orange spot using anisaldehyde-sulphuric acid and crystallized from dichloromethane-methanol mixture to afford pale yellow needle shaped crystals of compound 1. Fractions (21)(22)(23)(24)(25)(26) eluted by 6% MeOH/CH 2 Cl 2 were combined, concentrated then subjected to TLC examination, revealed the presence one major yellow spot using ammonia vapour and crystallized to afford yellow powder of compound 2. Fractions (27)(28)(29)(30)(31)(32)(33) eluted by 8% MeOH/CH 2 Cl 2 were combined, concentrated then subjected to TLC examination, revealed the presence of two major yellow spots using ammonia vapour, the fractions were crystallized by dichloromethane to afford yellow powder of compound 3, the mother liquor was concentrated and recrystallized from dichloromethane-methanol mixture to afford compound 4. Fractions (44)(45)(46)(47) eluted by 16% MeOH/CH 2 Cl 2 were combined, concentrated then subjected to TLC examination, revealed the presence of one major pink spot using anisaldehyde-sulphuric acid, the fractions were crystallized from dichloromethane-methanol mixture to afford yellowish brown crystals of compound 5. Fractions (51-53) eluted by 30% MeOH/CH 2 Cl 2 were combined, concentrated then subjected to TLC examination, revealed the presence of two yellow spots, fractions were pooled, concentrated and subjected to rechromatographic separation using Sephadex column LH-20 (2 × 50 cm, 3gm), eluted with 100% methanol. Compound 6 was isolated as a result of Sephadex column whose crystals was isolated after crystallized from dichloromethane-methanol mixture.

LC/MS instrument and separation technique
The sample (100μg/mL) solution was prepared using HPLC analytical grade solvent of MeOH, filtered using a membrane disc filter (0.2μm) then subjected to LC-ESI-MS analysis. Samples injection volumes (10μL) were injected into the UPLC instrument equipped with reverse phase C-18 column (ACQUITY UPLC -BEH C18 1.7 µm particle size -2.1 × 50 mm Column). Sample mobile phase was prepared by filtering using 0.2 μm filter membrane disc and degassed by sonication before injection. Mobile phase elution was made with the flow rate of 0.2 mL/min using gradient mobile phase comprising two eluents: eluent A is H 2 O acidified with 0.1% formic acid and eluent B is MeOH acidified with 0.1% formic acid. Elution was performed using the above gradient. The parameters for analysis were carried out using negative ion mode as follows: source temperature 150 °C, cone voltage 30 eV, capillary voltage 3 kV, desolvation temperature 440 °C, cone gas flow 50 L/h, and desolvation gas flow 900 L/h. Mass spectra were detected in the ESI between m/z 100-1000. The peaks and spectra were processed using the Maslynx 4.1 software and tentatively identified by comparing its retention time and mass spectrum with reported data.

Biological activities
The biological activities of ethyl acetate fraction of S. hispanica L. aerial parts were carried out at Regional Center for Mycology and Biotechnology (RCMB) at Al-Azhar University, Cairo, Egypt.

Antioxidant activity
The antioxidant activity using DPPH method ac. 17 Briefly, ethyl acetate fraction was determined at different concentration 2.5, 5, 10, 20, 40, 80, 160, 320, 640 and 1280ϻg/ml that were added respectively to 3 ml DPPH solution, the decrease in absorbance at 515 nm was determined continuously, with data being recorded at 1 min intervals until the absorbance stabilized (16 min). The 50%inhibitory concentration (IC 50 ) of ethyl acetate fraction and the standard (ascorbic acid) were estimated

Anti-obesity activity
The anti-obesity activity was determined by pancreatic lipase inhibitory assay. 18 Briefly, ethyl acetate fraction with different concentrations (1000 to 7.81 μg/mL) were pre-incubated with 100 µg/mL of lipase for 10 min at 37°C. The reaction was then started by adding 0.1 mL p-nitrophenyl butyrate substrate, after incubation at 37°C for 15. The amount of p-nitrophenol released in the reaction was measured using Multiplate Reader. IC 50 value of ethyl acetate fraction and the standard (orlistat) were estimated.

Anti-diabetic activity
The anti-diabetic activity was determined by α -amylase inhibition method 19 . Briefly, 1ml of the fraction of various concentrations (1000 to 7.81 μg/ml) and 1ml of enzyme solution were mixed together and incubated at 25°C for 10 min. After incubation, 1ml of starch (0.5%) solution was added to the mixture and incubated at 25°C for 10 min. The reaction was then stopped by adding 2ml of dinitro salicylic acid, heating the mixture in a boiling water bath (5min). After cooling, the absorbance was measured colorimetrically at 565 nm, the IC 50 value of ethyl acetate fraction and the standard (acarbose) were estimated.

Cytotoxic activity
The anti-cancer activity using cell viability assay. 20

Compound 1
IR spectrum showed broad absorption band at 3100 cm -1 for OH groups, 3030, 1511, 1460 and 1360 cm -1 indicated aromaticity. EI-MS exhibited a molecular ion peak at m/z 142(M + ) for molecular formula C 6 H 6 O 4 , the fragmentation pattern showed loss of hydroxyl groups by formation of peaks at m/z 110 (relative abundance 100%). 1 H˗NMR spectrum showed a singlet signal at δ 6.56 ppm for two aromatic protons and singlet signal at δ 4.90 ppm for four protons of OH groups. 13 C˗NMR (APT) spectrum showed only two types of carbons: at δ 115.42 ppm (C-H) and at δ 149.85ppm(C-O). These data confirmed the structure to be 1,2,4,5 tetrahydroxybenzene which was reported synthetically but it is the first time to be isolated from nature.

Structural identification of constituents by UPLC-ESI-MS/MS
UPLC-ESI-MS/MS in both negative and positive ion modes were used to analyze ethyl acetate fraction of S. hispanica L. aerial parts. The tentative detection of 37 compounds based on the fragmentation patterns that were compared with the available literature data as seen in Table 1. The compounds were arranged according to retention time (R t ) and divided according to different classes to phenolic derivatives (11), flavonoid aglycones (8), flavonoid-O-glycosides (7), flavonoid-Cglycosides (4), tannins (1), diterpenoids (2), lignin (2), coumarin (1), and triterpenoids(1).

Anti-oxidant activity
This promising result is due to presence of flavonoids and phenolic content as the presence of hydroxyl groups in the phenolic compounds are responsible of anti-oxidation effect as the hydroxyl group consider necessary component as a radical scavenger. 51 The DPPH scavenging percentage of ethyl acetate fraction of S. hispanica L aerial parts (IC 50 = 13.11 µg/ml) is shown in (Figure 4A) in comparison with ascorbic acid (IC 50 = 12.50 µg/ml).

Anti-obesity activity
There are many reports about S. hispanica L. seeds antiobesity action 10,[52][53][54][55][56] but no reports about aerial parts activity. The antiobesity activity is due to presence of poly phenolics, flavonoids and triterpenoides. 57 The results showed that ethyl acetate fraction has antiobesity activity with IC 50 114.9 compared to orlistat that showed IC 50 23.8 ( Figure 4B).

Anti-diabetic activity
The inhibition activity of ethyl acetate fraction of S. hispanica L. aerial parts was investigated on the α amylase enzyme using acarbose as standard then IC 50 value was calculated ( Figure 4C). The results showed that ethyl acetate farction significantly inhibited the α-amylase enzyme with IC 50 95.2 compared to acarbose with IC 50 34.71. S. hispanica is rich in omega-3 fatty acids which have positive effect on insulin resistance. 58

Cytotoxic activity
Cytotoxic activity of ethyl acetate fraction of S. hispanica L. aerial part was evaluated against human Lung cancer cell line (A-549), human prostate carcinoma (PC-3) and colon carcinoma (HCT-116) using viability assay with vinblastine as standard. The criteria used to categorize the activity against cancer cell lines based on IC 50 values as follows: IC50 ≤ 20 μg/ml = highly active, IC50 21 -200 μg/ml = moderately active, IC50: 201 -500 μg/ml = weakly active and IC50 > 501 μg/ml = inactive. 59 The presence of flavonoids, phenolics,tannin and glycosides are responsible for cytotoxic activities. 60 The results revealed  that the ethyl acetate fraction showed a highly cytotoxic activity against A-549 and HCT-116 cell lines with IC 50 of 15 ± 0.8 µg/ml and 19.5 ± 0.6 µg/ml µg/ml respectively but showed a moderately cytotoxic activity against PC-3 with IC 50 of 26.3 ± 1.1 µg/ml ( Figures 5A, 5B and 5C).

CONCLUSION
The biological study of S. hispanica L. aerial parts indicates that the ethyl acetate fraction has powerful anti-oxidant, cytotoxic, antiobesity and anti-diabetic activities. Phytochemical study indicated the presence of phenolic acids, flavonoids, tannins, diterpenoids, lignans and triterpenoids. Further studies are required to detect the exact mechanism of action and to characterize more chemical compounds responsible for the pharmacological activities of S. hispanica L.

DECLARATION OF COMPETING INTEREST
There are no conflicts to declare.