HPLC profiling of selected phenolic acids and flavonoids in Salvia eigii, Salvia hierosolymitana and Salvia viridis growing wild in Jordan and their in vitro antioxidant activity

Salvia eigii Zohry.

S. eigii Zohary. is a beautiful showy perennial herb native to the Mediterranean woodlands and shrub lands of Jordan, Palestine, Syria and Lebanon. It grows up to 30–50 cm high and 60–100 cm wide. The flowers have a white lip and the hood is a light mauve to purple. Flowering occurs in spring season during the period extending from March to June. Little literature describes S. eigii. The aqueous extract obtained from the aerial parts of this plant has been investigated for Pancreatic triacylglycerol lipase (PL) inhibition and was found to have moderate activity (Kasabri et al., 2014). The extract had also a dual inhibition activity for α-amylase and α-glucosidase and showed moderate cytotoxic activity against colon cancer cell lines HT29, HCT116 and SW620 (Kasabri et al., 2014). Prior to our study, the plant has never been evaluated for its phytochemical composition and antioxidant activity.

Salvia hierosolymitana Bioss.

S. hierosolymitanta Bioss. (known also as the Jerusalem sage) acquired its name from the Greek word “hieros” meaning holy and the Latin name for Jerusalem:“herosolyma”. This plant is native to the East Mediterranean region, growing wild in Jordan, Palestine, Syria, Lebanon, Cyprus and Turkey. It is a perennial herb, 30–60 cm tall, branched from the base with square purple stems. The plant is characterized by purple or red-wine color flowers that are 2–2.5 cm long. Leaves, stems, and floral parts are covered with small hairs. Flowering occurs during the period extending from March to June. The plant is known to grow wild in forest grounds of Irbid, Jerash, Ajloun, Tafila and Amman (Al-Eisawi, 1998). In Levine countries, especially in Jordan and Palestine, S. hierosolymitana leaves are edible. Green fresh leaves are boiled, stuffed with rice, minced meat and condiments and then made into rolls, cooked and eaten with yogurt (Al-Eisawi, 1998; Ali-Shtayeh et al., 2008). In Palestinian traditional medicine, grinded S. hierosolymitana seeds mixed with lanolin is prescribed for the treatment of skin cancer (Jaradat et al., 2016). Previous phytochemical investigation on the plant led to the isolation of dammarane type triterpenoids (Pedreros et al., 1990), polyhydroxylated triterpenes and rosmarinic acid (De Felice et al., 2006). The methanolic extract obtained from the roots and aerial parts was investigated for its anti-angeogenic activity (Abdallah et al., 2018). The crude ethanolic extract of the plant was evaluated for its cytotoxic activity against MCF-7, T47D, ZR-75-1 and BT 474 cancer cell lines (Abu-Dahab et al., 2012).

Salvia viridis L.

S. viridis L. (synonym S. horminum L.), commonly known as ‘Red topped sage’, naturally occurs in the Mediterranean region. It is a perennial, annual or biennial herb, having the erect stem of 50 cm and 4–8 axillary flowers (Rungsimakan & Rowan, 2014). S. viridis has been used in traditional medicine as gargle against sore gum (Grzegorczyk-Karolak & Kiss, 2018). In Turkey, an infusion of the shoots, flowers, and leaves of S. viridis have been used against a sore throat, throat inflammation, antitussive, ulcer, intestinal spasm and gynecological complications (Sharifi-Rad et al., 2018; Zengina et al., 2019).

In continuation of our concern in evaluating the chemical constituents of medicinal plants from the flora of Jordan, we report here the total phenolics and flavonoids content and the antioxidant activity of the methanolic extract obtained from the aerial parts of these three Salvia species growing wild in Jordan. Additionally, we also report the quantitative determination of six flavonoids including naringenin, hesperidin, apigenin, luteolin-7-O-glucoside, rutin, quercetin in addition to five other phenolic acids including gallic acid, chlorogenic acid, caffeic acid, rosmarinic acid and salvianolic acid B in the methanolic extract of these three species using Liquid Chromatography-Electron Spray Ionization- Tandom Mass Spectrometry (LC-ESI-MS/MS). Prior to our investigation, no phytochemical studies were carried out on these three species from Jordanian origin.

Plant material

Aerial parts of each of S. eigii Zohary. (Umm Qais–Irbid, 32.6544°N; 35.6881°E), S. hierosolymitana Benth. (Ajloun, 32.3327°N; 35.7518°E) and S. viridis L. (As Subayhi, Al-Salt, 32.1500°N; 35.7000°E) were collected during their full flowering stage. The plants were identified by Prof. Dr. Hala Al-Jaber, Department of Chemistry, Faculty of Science, Al-Balqa Applied University. A voucher specimen of each species (S. eigii: L/SE/2019; S. hierosolymitana: L/SHie/2019 and S. viridis: L/SH/2019) was deposited at the herbarium of Department of Chemistry, Al-Balqa Applied University, Al-Salt, Jordan.

Chemicals and standards

All solvents (HPLC grade) used in this investigation, rutin hydrate (≥94% HPLC), nariginin, hesperidin, gallic acid, chlorogenic acid and caffeic acid (HPLC, ≥98%) were obtained from Sigma-Aldrich (Buchs, Switzerland). Luteolin-7-O-glucoside (HPLC, ≥98%), apigenin (HPLC, ≥99%) were products of EXTRASYNTHESE (France). Apigenin-7-O-glucoside, rosmarinic acid and salvianolic acid B were purchased from EDQM (Strasbourg, France).

Extraction and sample preparation

Extraction of the selected Salvia species was performed according to the procedure described in the literature but with slight modification (Fotovvat, Radjabian & Saboora, 2019). Briefly, 10.0 g sample of the air dried and pulverized aerial parts of each selected species was soaked in HPLC-grade methanol (3 × 100 ml, 24 h each) at room temperature. After filtration, the solvents were evaporated under vacuum at 40 °C. The obtained crude extracts were then stored in dark at 4 °C until analysis. For LC-ESI-MS/MS analysis, the dried extracts were dissolved in 70% aqueous HPLC-grade ethanol, filtered and then prepared for analysis. Stock solutions of each extract (4,000 ppm) were prepared and then assayed immediately for their flavonoids and phenolic acid compounds using LC-MS/MS.

Total flavonoids content (TFC) and Total phenolics content (TPC)

The TFC of the methanolic extracts was determined colorimetrically according to the method described in the literature with slight modifications (Olajire & Azeez, 2011; Al-Jaber, 2017). Briefly, 1.0 mL sample of each extract (1 mg/ml) diluted with 4.0 ml distilled water were introduced into 10 ml volumetric flask and then 0.30 ml of NaNO₂ solution was added. After 5 min, 0.30 ml of AlCl₃ solution (10% w/v) was added to the mixture. The solution was incubated 6 min, and then 2.0 ml of 1.0 M NaOH solution was introduced and the final volume of the solution was adjusted to 10.0 ml with distilled water. After another 15 min, the absorbance of the resulting solution was measured at 510 nm using methanol as a blank. The TFC content in the plant extracts was determined and expressed in mg quercetin/g dry extract.

The TPC was determined using the Folin–Ciocalteu method described in the literature (Al-Jaber, 2017). Briefly, to 0.5 ml of extract, 2.5 ml of Folin–Ciocalteu reagent (2N diluted ten folds) and 2 ml of Na₂CO₃ solution (75 g/l) were added. After allowing 15 min period of incubation at room temperature, the absorbance of the resulting solution was measured at 765 nm. Methanol was used as a blank reference. The TPC of the different extracts is reported as mg/g gallic acid equivalent. All measurements were performed in triplicates.

Antioxidant activity

DPPH^• free radical scavenging activity

The antioxidant activity of the methanolic extracts obtained from the three Salvia species was evaluated using the DPPH^• radical scavenging method according to the procedure mentioned in the literature using ascorbic acid and α-tocopherol as positive controls (Olajire & Azeez, 2011; Al-Jaber, 2016; Al-Jaber, 2017). Briefly, 1 ml of 0.1 mM DPPH^• solution (dissolved in MeOH) was added to 2.0 ml of each methanolic extract solution at various concentration levels (0.005–0.5 mg/ml). The solutions were incubated in dark for 30 min at room temperature and then the absorbance of the solutions was measured at 517 nm against methanol blank sample. The ability to scavenge the DPPH^• radical was calculated using the following equation: ${\displaystyle {\text{DPPH}}^{•}\text{scavenging effect}\left(\text{\%}\right)=\frac{Ac-As}{Ac}\times 100\text{\%}.}$

Where A_C is the absorbance of the blank and A_S is the absorbance in the presence of the extract/compound. The scavenging activity assay of all extracts, positive controls and IC₅₀ value determinations were conducted in triplicates using the non-linear regression analysis of the GraphPad Prism 6 (GraphPad Software, San Diego, CA, USA). The UV- spectra were recorded on Wavelight II UV- visible spectrophotometer (Version 7120V1.8.0, USA).

ABTS radical scavenging assay

The total antioxidant activity determined by radical cation (ABTS^• +) decolonization assay was performed according to the procedure described in the literature with slight modifications (Zhao et al., 2011; Olajire & Azeez, 2011). The radical cation reagent (ABTS^• +) solution was prepared by mixing similar quantities of 7 mM of ABTS^• + and 2.4 mM of potassium persulfate (K₂S₂O₈) solutions and allowing them to react in dark for 16 h and at room temperature. Before use; this solution was diluted with methanol to get an absorbance of 0.75 ± 0.02 at 734 nm. To measure the antioxidant power of the plants’ extracts, 3 ml of ABTS^• + reagent solution and 1 mL of each extract at various concentration levels (ranging from 0.005–0.500 mg/ml) were mixed and the absorbance was then measured at 734 nm on Wavelight II UV- visible spectrophotometer (Version 7120V1.8.0, USA). Blank sample was run in each assay and all measurements were done after at least 5 min. Ascorbic acid and α-tocopherol were used as positive controls. All measurements and IC₅₀ value determinations were conducted in triplicates.

Ferrous Ion Chelating (FIC) effect

The ability of the tested extracts and EDTA used as a positive control to chelate ferrous ion from the formation of ferrozine-Fe²⁺ complex was determined according to the procedure listed in the literature (Al-Qudah et al., 2019). Briefly, a 3 ml of methanol solution containing different concentrations of the extracts (0.005–1.0 mg/ml) was added to a 0.25 ml of 2 mM ferrous chloride (FeCl₂) reagent. Then, 0.2 ml of 5 mM ferrozine solution was added to the mixture and after vigorous shaking, the mixture was allowed to stand at room temperature for 10 min. The reduction in the absorbance of the red colored complex was measured at 562 nm. All measurements and IC₅₀ values determination for the tested extracts and the positive control were conducted in triplicates.

LC-MS/MS analysis parameters

Analysis of flavonoids and phenolics compounds was performed on HPLC (Agilent 1200 series, USA) equipped with LC-ESI-MS/MS API 5000 system (AB Sciex Instrument, Framingham, USA) utilizing Analyst 1.6.3 software for data analysis. The chromatographic separation was conducted at 40 ± 1 °C using Inertsil ODS-3 column (4.6 × 150 mm, 5 µm, GL Sciences-Japan) at ambient temperature. The elution gradient consisted of mobile phase A (5 nM ammonium formate in water: Methanol (90:10; v/v)) and solvent B (5 mM ammonium formate in methanol). The gradient program with the following proportions of solvent B was applied (%B, min): 40–90% B (0.00–6.00 min), isocratic 90% (6.00–10.00 min), isocratic 40% (10.01–15.00 min). The solvent flow rate was 0.5 ml/min and the injection volume was 10 µl. MS/MS analysis was performed in negative ion mode with an ion spray voltage of −4,500 V. Nitrogen gas at a pressure of 60 psi was used as the nebulizing and drying gas. The mass spectra were obtained over the m/z range of 100–1,000 amu.

Preparation of standard solution

A stock solution containing eleven standard compounds (0.5 mg/ml) was prepared in HPLC-grade methanol and diluted to eight different concentrations to construct the calibration curve.

Extraction yield, TPC, TFC and antioxidant activity

The results of extraction yields, TPC and TFC and the antioxidant activities of the methanolic extracts of the three Salvia species are shown in Table 2.

The results in the table showed that S. heirosolymitana had the highest TPC (27.31 ± 0.92 mg gallic acid /g dry extract), TFC (770.85 ± 5.26 mg quercetin/g dry extract) followed by S. eigii (24.99 ± 3.12 mg gallic acid/g dry extract; 520.60 ± 6.24 mg quercetin/g dry extract). The obtained results clearly indicated that S. hierosolymitana had the highest radical DPPH^• scavenging activity.

LC-MS/MS profiling of phenolic acids and flavonoids

The results of the LC-MS/MS analysis of the three species are summarized in Table 3. LC-MS/MS analysis of the methanolic extract of the three Salvia species revealed that the three Salvia species were rich in caffeic acid derivatives, mainly rosmarinic acid. Among the different compounds studied, the plants also contained high concentration levels of luteolin-7-O-glucoside (Table 3).

Table 3:

LC-MS/MS data for standard phenolic and flavonoids compounds detected in S. eigii, S. hierosolymitana and S. viridis from Jordan and their concentration (mg/kg dry plant material).

Compound	Structure	Formula	Rt (min)	MS/MS	Concentration (mg/kg dry plant material)
					S. eigii	S. hierosolymitana	S. viridis
Gallic acid		C7H6O5	3.46	169, 125	100.09	84.15	27.36
Salvianolic acid B		C36H30O16	3.51	717, 519	13.79	896.11	890.90
Chlorogenic acid		C16H18O9	3.80	353, 191	173.75	201.19	6619.93
Caffeic acid		C9H8O4	4.15	179, 135	32.97	260.79	453.03
Rosmarinic acid		C18H16O8	5.00	359, 161	15783.33	27124.93	329.91
luteolin-7-O-glucoside		C21H20O11	7.45	447, 285	1756.73	21651.36	26125.14
Hesperidin		C28H34O15	7.58	609, 300	142.74	95.69	298.38
Rutin		C27H30O16	7.59	609, 300	54.49	3.71	100.89
Quercetin		C15H10O7	9.56	301, 179	6.10	6.91	7.10
Apigenin		C15H10O5	10.53	269, 117	111.31	26.08	39.37
Naringenin		C15H12O5	10.55	271, 150	154.02	418.59	67.77
				Total	18329.32	50769.51	34629.87

DOI: 10.7717/peerj.9769/table-3

As could be deduced from the LC-MS/MS results (Table 3), all eleven compounds were detected in the three species, but with variable concentration levels. Careful inspection of the results indicated that the most abundant component detected in S. hierosolymitana and S. eigii was rosmarinic acid (27124.93, 15783.33 mg/kgDW, respectively). S. heirosolymitana also contained high concentration levels of luteolin-7-O-glucoside (21651.36 mg/kgDW), naringenin (418.59 mg/kgDW), caffeic acid (260.79 mg/kgDW) and had the highest content of salvianolic acid B (896.11 mg/kgDW) when compared to the other two species. S. eigii was richer in each of gallic acid (100.09 mg/kgDW) and apigenin (111.31 mg/kgDW) and contained luteolin-7-O-glucoside detected at lower concentration level compared to S. hierosolymitana (1756.73 mg/kgDW). On the other hand, S. viridis had the highest content of luteolin-7-O-glucoside (26125.14 mg/kgDW), chlorogenic acid (6619.93 mg/kgDW), caffeic acid (453.03 mg/kgDW), hesperidin (298.38 mg/kgDW) and rutin (100.89 mg/kgDW).