UPLC-ESI-MS/MS Profiling and Cytotoxic, Antioxidant, Anti-Inflammatory, Antidiabetic, and Antiobesity Activities of the Non-Polar Fractions of Salvia hispanica L. Aerial Parts

Salvia hispanica L. is an annual herbaceous plant commonly known as “Chia”. It has been recommended for therapeutic use because of its use as an excellent source of fatty acids, protein, dietary fibers, antioxidants, and omega-3 fatty acids. A literature survey concerning phytochemical and biological investigations of chia extracts revealed less attention towards the non-polar extracts of S. hispanica L. aerial parts, which motivates us to investigate their phytochemical constituents and biological potentials. The phytochemical investigation of the non-polar fractions of S. hispanica L. aerial parts resulted in the tentative identification of 42 compounds using UPLC-ESI-MS/MS analysis with the isolation of β-sitosterol (1), betulinic acid (2), oleanolic acid (3), and β-sitosterol-3-O-β-D-glucoside (4). GLC-MS analysis of the seeds’ oil showed a high concentration of omega-3 fatty acid, with a percentage of 35.64% of the total fatty acid content in the seed oil. The biological results revealed that the dichloromethane fraction showed promising DPPH radical-scavenging activity (IC50 = 14.73 µg/mL), antidiabetic activity with significant inhibition of the α-amylase enzyme (IC50 673.25 μg/mL), and anti-inflammatory activity using in vitro histamine release assay (IC50 61.8 μg/mL). Furthermore, the dichloromethane fraction revealed moderate cytotoxic activity against human lung cancer cell line (A-549), human prostate carcinoma (PC-3), and colon carcinoma (HCT-116) with IC50s 35.9 ± 2.1 μg/mL, 42.4 ± 2.3 μg/mL, and 47.5 ± 1.3 μg/mL, respectively, and antiobesity activity with IC50 59.3 μg/mL, using pancreatic lipase inhibitory assay. In conclusion, this study’s findings not only shed light on the phytochemical constituents and biological activities of the non-polar fractions of chia but also should be taken as a basis for the future in vivo and clinical studies on the safety and efficacy of chia and its extracts. Further study should be focused towards the isolation of the active principles of the dichloromethane fraction and studying their efficacy, exact mechanism(s), and safety, which could benefit the pharmaceutical industry and folk medicine practitioners who use this plant to cure diseases.


Introduction
The Lamiaceae (Labiatae, Mint) family comprises 245 genera and about 7886 species worldwide. Many genera belonging to this family have important uses in medicine, the culinary arts, and cosmetics [1]. The chemical components of the family members have biological roles with therapeutic value; these chemicals include essential oils, alkaloids, flavonoids, glycosides, steroids, coumarins, tannins, and terpenoids [2]. Salvia hispanica L. is an annual herb that is commonly known as "Chia", native to southern Mexico and northern Guatemala [3]. Salvia hispanica L. is mainly grown for its seeds, which are widely consumed because of their high nutritional and medicinal value [4][5][6][7][8][9]. Globally, research has been conducted investigating the benefits of chia seeds and oil and their applications in the food, cosmetic, medical, and pharmaceutical industries. A literature survey revealed more concern towards chia seeds' constituents and biological activities, with less attention to other parts of the plant. Previous phytochemical analyses of S. hispanica seeds' constituents indicated the presence of flavonoids and phenolic acids that are linked to their antioxidant, antiobesity, antidiabetic, and antimicrobial activities [4][5][6][7][8][9][10][11][12][13].
In contrast, only a few studies have reported on the phytochemical and biological activities of S. hispanica L. aerial parts, which exhibit the presence of neoclerodane-type diterpenoids with the tentative identification of different phenolic compounds [14][15][16].
To the best of our knowledge, there are no bibliographic data in the literature about the phytochemical composition and biological activities of the aerial parts of S. hispanica cultivated in Egypt except our previous work that focused on the investigation of the main bioactive constituents of the polar fraction of the aerial parts, which resulted in the tentative detection of 37 compounds, using UPLC-ESI-MS/MS analysis with the isolation of 1,2,4,5 tetrahydroxy benzene, leucantho flavone, and rhamnetin [17]. The current study focused on the identification of the active constituents of the non-polar fractions of the aerial parts of S. hispanica cultivated in Egypt with the investigation of their potential biological activities, including cytotoxic, antioxidant, anti-inflammatory, antidiabetic, and antiobesity activities, to attract attention and provide evidence for their therapeutic value.

Structural Identification of Constituents by UPLC-ESI-MS/MS
UPLC-ESI-MS/MS in positive ionization mode was used to analyze the light petroleum fraction of S. hispanica L. aerial parts ( Figure 1). The tentative detection of nine compounds was based on the fragmentation patterns that were compared with the available literature data, as shown in Table 1. culinary arts, and cosmetics [1]. The chemical components of the family members have biological roles with therapeutic value; these chemicals include essential oils, alkaloids, flavonoids, glycosides, steroids, coumarins, tannins, and terpenoids [2].
Salvia hispanica L. is an annual herb that is commonly known as "Chia", native to southern Mexico and northern Guatemala [3]. Salvia hispanica L. is mainly grown for its seeds, which are widely consumed because of their high nutritional and medicinal value [4][5][6][7][8][9]. Globally, research has been conducted investigating the benefits of chia seeds and oil and their applications in the food, cosmetic, medical, and pharmaceutical industries. A literature survey revealed more concern towards chia seeds' constituents and biological activities, with less attention to other parts of the plant. Previous phytochemical analyses of S. hispanica seeds' constituents indicated the presence of flavonoids and phenolic acids that are linked to their antioxidant, antiobesity, antidiabetic, and antimicrobial activities [4][5][6][7][8][9][10][11][12][13]. In contrast, only a few studies have reported on the phytochemical and biological activities of S. hispanica L. aerial parts, which exhibit the presence of neoclerodane-type diterpenoids with the tentative identification of different phenolic compounds [14][15][16].
To the best of our knowledge, there are no bibliographic data in the literature about the phytochemical composition and biological activities of the aerial parts of S. hispanica cultivated in Egypt except our previous work that focused on the investigation of the main bioactive constituents of the polar fraction of the aerial parts, which resulted in the tentative detection of 37 compounds, using UPLC-ESI-MS/MS analysis with the isolation of 1,2,4,5 tetrahydroxy benzene, leucantho flavone, and rhamnetin [17]. The current study focused on the identification of the active constituents of the non-polar fractions of the aerial parts of S. hispanica cultivated in Egypt with the investigation of their potential biological activities, including cytotoxic, antioxidant, anti-inflammatory, antidiabetic, and antiobesity activities, to attract attention and provide evidence for their therapeutic value.

Structural Identification of Constituents by UPLC-ESI-MS/MS
UPLC-ESI-MS/MS in positive ionization mode was used to analyze the light petroleum fraction of S. hispanica L. aerial parts ( Figure 1). The tentative detection of nine compounds was based on the fragmentation patterns that were compared with the available literature data, as shown in Table 1.   . This fragmentation pattern was in good agreement with the previous report of oleanolic acid [24].
For the dichloromethane fraction, the UPLC-ESI-MS/MS in negative and positive ion modes led to the identification of 33 compounds ( Figure 2). The compounds were arranged according to retention time (R t ) and classified accordingly into different classes including phenolic acids, flavonoids, diterpenoids, alkaloids, tannins, steroids, triterpenoids, fatty acids, and miscellaneous compounds ( Table 2).  The dichloromethane fraction is high in diterpenoids ( Figure 3A), most of which are abietane quinones. There were 13 diterpenoids compounds tentatively identified as follows.      Moreover, seven flavonoid aglycones were tentatively identified in the dichloromethane fraction ( Figure 3B), including compound 16 (Rt, 11.99 min), which showed a molecular ion peak at [M-H] − at m/z 359, as well as fragment ions at m/z 344, 329, and 314, due to successive losses of CH 3 , and a fragment ion at m/z 195 that formed after cleavage of the flavone skeleton. Based on this result, the compound was classified as 5,7,3 -trihydroxy-6,4 ,5 -trimethoxy flavone [34].  Moreover, seven flavonoid aglycones were tentatively identified in the dichloromethane fraction ( Figure 3B), including compound 16 (Rt, 11.99 min), which showed a molecular ion peak at [M-H] − at m/z 359, as well as fragment ions at m/z 344, 329, and 314, due to successive losses of CH3, and a fragment ion at m/z 195 that formed after cleavage of the flavone skeleton. Based on this result, the compound was classified as 5,7,3′-trihydroxy-6,4′,5′-trimethoxy flavone [34].  of diosmetin or chryseriol (flavone) [42], whilst compound 28 revealed fragment ions at m/z 284 and 255, characteristic of 3 -O-methylorobol or gliricidin (isoflavone) [43].
Three alkaloids were tentatively identified from the dichloromethane fraction of aerial parts ( Figure 3C
Other identified miscellaneous compounds ( Figure 5B)

Cytotoxic Activity
The cytotoxic activity of the dichloromethane fraction was tested using a viability assay with vinblastine as a standard against human lung cancer cell line (A-549), human prostate carcinoma (PC-3), and colon carcinoma (HCT-116). The presence of flavonoids, phenolic compounds, tannin, and glycosides is responsible for cytotoxic activities [50]. The results revealed that the fraction had a moderate cytotoxic activity against A-549, PC-3, and HCT-116 cell lines with IC 50 of 35.9 ± 2.1 µg/mL, 42.4 ± 2.3 µg/mL, and 47.5 ± 1.3 µg/mL, respectively, and when compared with vinblastine sulfate as a positive control, the IC 50 was 24.6 µg/mL, 42.4 µg/mL, and 3.5 µg/mL, respectively ( Figure 7A-C).

Antioxidant Activity
The promising antioxidant result of the dichloromethane fraction refers to the flavonoids and phenolic contents. The hydroxyl groups in phenolic compounds are responsible for antioxidant activity because of their radical-scavenging properties [51]. The DPPH scavenging percentage of the dichloromethane fraction (IC 50 = 14.73 µg/mL) was approximately comparable to that of ascorbic acid (IC 50 = 12.50 µg/mL, as shown in Figure 7D.

Anti-Inflammatory Activity
The dichloromethane fraction showed stronger anti-inflammatory activity than the light petroleum fraction, with IC 50s of 61.8 µg/mL and 458.6 µg/mL, respectively, compared to diclofenac sodium as a positive control, with IC 50 of 17.9 µg/mL ( Figure 7E). The contents of diterpenes and phenolics in the dichloromethane fraction play important roles in antiinflammatory activity [52]; sterols, such as β-sitosterol, betulinic acid, oleanolic acid, and β-sitosterol-3-O-β-D-glucoside, are also known to exhibit anti-inflammatory activity [53].

Antidiabetic Activity
The antidiabetic activity of the dichloromethane fraction was tested using the α amylase enzyme and acarbose as a positive standard. The results showed that the dichloromethane fraction inhibited the α-amylase enzyme, with IC 50 of 673.25 µg/mL compared to acarbose, which showed IC 50 of 34.71 µg/mL ( Figure 7F). S. hispanica contains a high concentration of omega-3 fatty acids (35.64% of total fatty acid content), which have been shown to reduce insulin resistance [54].

Antioxidant Activity
The promising antioxidant result of the dichloromethane fraction refers to the flavonoids and phenolic contents. The hydroxyl groups in phenolic compounds are responsible for antioxidant activity because of their radical-scavenging properties [51]. The DPPH scavenging percentage of the dichloromethane fraction (IC50 = 14.73 µg/mL) was approximately comparable to that of ascorbic acid (IC50 = 12.50 µg/mL, as shown in Figure 7D.

Anti-Inflammatory Activity
The dichloromethane fraction showed stronger anti-inflammatory activity than the light petroleum fraction, with IC50s of 61.8 µg/mL and 458.6 µg/mL, respectively, compared to diclofenac sodium as a positive control, with IC50 of 17.9 µg/mL ( Figure 7E). The contents of diterpenes and phenolics in the dichloromethane fraction play important roles in anti-inflammatory activity [52]; sterols, such as β-sitosterol, betulinic acid, oleanolic acid, and β-sitosterol-3-O-β-D-glucoside, are also known to exhibit anti-inflammatory activity [53].

Antiobesity Activity
There are numerous reports on the antiobesity activity of S. hispanica L. seeds but none on the activity of the aerial parts. The antiobesity activity was determined using a pancreatic lipase inhibitory assay, and the results showed that the dichloromethane fraction has moderate antiobesity activity, with IC 50 59.3 µg/mL, versus orlist, with IC 50 23.8 µg/mL ( Figure 7G). The antiobesity activity is due to the presence of poly phenolics, flavonoids, and terpenoids [55].

Instruments for Spectroscopic Analyses
Infrared spectral analysis was recorded using the potassium bromide disk technique on a PyeUnicam SP 3000 and IR spectrophotometer of Alpha (I-00523), Jasko, FT/IR-460 plus, Japan. Mass spectra were obtained on Shimadzu GC-MS-QP5050A mass spectrometer at 70 eV. 1 H and 13 C-NMR spectral analyses were carried out at the faculty of pharmacy, Ain Shams University, Egypt, using Bruker (Zurich, Switzerland) at 400 and at 100 MHz, respectively. Chemical shifts were given in ppm with the TMS as the internal standard.

Plant Material
Salvia hispanica L. aerial parts were collected at the flowering stage from Mushtohor farm (Tokh, Egypt) in March 2018. This plant was identified and verified by Dr. Hussein Abdelbaset (Professor of Plant Taxonomy, Faculty of Science, Zagazig University). A voucher specimen (Lam.S-10) was deposited in the herbarium of the pharmacognosy department, faculty of pharmacy, Zagazig University, Egypt.

Extract Preparation
The air-dried powdered aerial parts of Salvia hispanica L. (3 kg) were extracted by cold maceration (5 times × 7 L) using 70% aqueous ethanol. The total extract was evaporated under reduced pressure at 50 • C, yielding 540 gm of dark green viscous residue. The residue (400 gm) was dissolved in a methanol: water mixture (1:9) then subjected to fractionation using light petroleum and dichloromethane. The fractions were washed with distilled water and dried over anhydrous sodium sulfate, then the solvent of each fraction was distilled off under reduced pressure at 50 • C to yield a light petroleum fraction (68 gm) and a dichloromethane fraction (4 gm).

Chromatographic Investigations
The light petroleum fraction was investigated by normal phase TLC using dichloromethane and methanol 99:1. The TLC plates were visualized with anisaldehyde and sulfuric acid, and the promising fractions were subjected to chromatographic investigations.

LC/MS Instrument and Separation Technique
Each fraction (100 µg/mL) solution was prepared using HPLC analytical-grade solvent MeOH, filtered with a membrane disc filter, and then subjected to LC-ESI-MS analysis. Fractional injection volumes (10 µL) were injected into the UPLC instrument equipped with a reverse-phase C-18 column (ACQUITY UPLC-BEH C 18 1.7 µm particle size-2.1 × 50 mm column). The mobile phase was prepared by filtering solvents using a filter membrane disc and degassing by sonication before injection. The flow rate was 0.2 mL/min with a gradient mobile phase comprising two eluents: H 2 O acidified with 0.1% formic acid and MeOH acidified with 0.1% formic acid. The parameters for analysis were carried out using positive 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 and 1000. The peaks and spectra were processed using Maslynx 4.1 software and tentatively identified by comparing their retention time and mass spectrum with the reported data.

GLC-MS of Salvia Seeds' Oil
The seeds were pressed using the Ixtaina et al. method [56], and the oil was derivatized using the Metcalfe et al. method [57] and recorded using Shimadzu GCMS-QP2010 (Tokyo, Japan) equipped with Rtx-1MS fused bonded column and a split-splitless injector. The initial column temperature was kept at 45 • C for 2 min (isothermal), programmed to 300 • C at a rate of 5 • C/min, and kept constant at 300 • C for 5 min (isothermal). The injector temperature was 250 • C. The helium carrier gas flow rate was 1.41 mL/min. All the mass spectra were recorded under the following conditions: (equipment current) filament emission current, 60 mA; ionization voltage, 70 eV; ion source, 200 • C. A series of hydrocarbon samples (1% v/v) were injected in split mode (split ratio 1:15). The components were identified by matching the retention indices and mass spectra with those reported in NIST17-1 libraries and literature.

Cytotoxic Activity
The anti-cancer activity was carried out using a cell viability assay [58]. Briefly, the cell lines used were the human lung cancer cell line (A-549), human prostate carcinoma cells (PC-3), and colon carcinoma cells (HCT-116), and they were obtained from VACSERA company (Tissue Culture Unit), Cairo, Egypt) [59,60]. The dichloromethane fraction was used in various concentrations (500 to 0 µg/mL). The IC 50 values of the fractions and the standard (vinblastine sulfate) were calculated.

Antioxidant Activity
The antioxidant activity was determined using the DPPH method according to the Leaves et al. method [61]. Briefly, the dichloromethane fraction was used at different concentrations, 2.5, 5, 10, 20, 40, 80, 160, 320, 640, and 1280 µg/mL, which were each added to 3 mL of DPPH solution, and the decrease in absorbance at 515 nm was determined continuously, with data being recorded at 1min intervals until the absorbance stabilized (16 min). The 50% inhibitory concentrations (IC 50 ) of the dichloromethane fraction and the standard (ascorbic acid) were determined.

Anti-Inflammatory Activity
In vitro histamine release assay was performed on light petroleum and dichloromethane fractions according to Venkata et al.'s assay [62]. The results were expressed as inhibition percentage, which was calculated using the following formula: Inhibitory activity (%) = (1 − As/Ac) × 10 As is the absorbance in the presence of the test substance and Ac is the absorbance of the control substance. The IC 50 value in µg/mL was estimated.

Antidiabetic Activity
The α-amylase inhibition method was used to determine the antidiabetic activity [63]. Briefly, 1 mL of the dichloromethane fraction of various concentrations (1000 to 7.81 µg/mL) and 1 mL of the enzyme solution were mixed and incubated at 25 • C for 10 min. After incubation, 1 mL 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 2 mL of dinitro-salicylic acid, followed by heating the mixture in a boiling water bath for 5 min. After cooling, the absorbance was measured colorimetrically at 565 nm, and the IC 50 values of the dichloromethane fraction and the standard (acarbose) were estimated.

Antiobesity Activity
The antiobesity activity was determined by pancreatic lipase inhibitory assay [64]. Briefly, the dichloromethane fraction at different concentrations (1000 to 7.81 µg/mL) was pre-incubated with 100 µg/mL of lipase for 10 min at 37 • C. The reaction was then started by adding 0.1 mL of p-nitrophenyl butyrate substrate after incubation at 37 • C for 15 min. The amount of p-nitrophenol released in the reaction was measured using a multiplate reader (Sigma Aldrich, Burlington, Massachusetts, USA). The IC 50 values of the dichloromethane fraction and the standard (orlistat) were determined.

Conclusions
This study represents the first report on the phytochemical constituents of the nonpolar fraction of S. hispanica aerial parts cultivated in Egypt as well as their pharmacological potentials. The UPLC-ESI-MS/MS analyses of the non-polar fractions (light petroleum and dichloromethane fractions) resulted in the tentative identification of 42 compounds of different chemical classes, including fatty acids, steroids, di-and tri-terpenoids, flavonoids, phenolic acids, and alkaloids. The phytochemical investigation of the light petroleum fraction resulted in the isolation of four compounds, including β-sitosterol (1), betulinic acid (2), oleanolic acid (3), and β-sitosterol-3-O-β-D-glucoside (4). The GLC-MS analysis of the seeds' oil revealed that seeds contain a high concentration of omega-3 fatty acids, with a percentage of 35.64% of the total fatty acids content.
Biologically, the dichloromethane fraction showed moderate cytotoxic activity against the human lung cancer cell line (A-549), human prostate carcinoma (PC-3), and colon carcinoma (HCT-116). It also exhibited remarkable antioxidant results that can be attributed to its contents of polyphenolic compounds, in addition to antidiabetic, antiobesity, and anti-inflammatory activities, which are attributed to the fatty acids, steroids, terpenoids, flavonoids, and phenolic acid contents.
In conclusion, these data are considered an addition to the bibliographic data about chia and a contribution towards the exploration of its chemical diversity as well as nutritional and therapeutic value. Henceforth, further studies should be focused towards the isolation of the active principles of the dichloromethane fraction and studying their efficacy, the exact mechanism(s), and safety, which could aid in the development of a new therapeutic agent and/or using chia as a safe natural alternative therapy and nutritional strategy for the treatment of diabetes and obesity in addition to its use as an excellent source of omega-3 fatty acids.