Chemical composition, antioxidant, cytotoxic, antiviral, and lung-protective activities of Salvia officinalis L. ethanol extract herb growing in Sinai, Egypt

Abstract

Background

Pulmonary fibrosis (PF) is a respiratory ailment that causes the substitution of healthy lung tissue with scar tissue due to collagen deposition and fibroblast proliferation. The incidence of PF increased with the successive waves of Coronaviruses and other viruses affecting the lungs. Therefore, the priority is moving toward treatment with medicinal plants for their active constituents. Salvia officinalis L. herbal drug (sage, family; Lamiaceae) is characterized by its strong antioxidative activity. This study aims to declare the effect of using sage ethanol extract (SOEE) as a protective agent from PF through an in vivo study on mercuric chloride (HgCl₂)-induced lung fibrosis and in vitro evaluation of its anticancer, antiviral, and antioxidant activities concerning its phytoconstituents.

Results

Twelve compounds were isolated and identified as apigenin, luteolin, genkwanin, quercetin, hispidulin, luteolin-7-O-β- glucopyranoside, rutin, rosmarinic acid, caffeic acid, ferulic acid chlorogenic acid, and ellagic acid. The results of antioxidants indicated that SOEE exhibited the greatest efficacy as an antioxidant agent in the 1,1-Diphenyl-2-picrylhydrazyl assay with inhibitory concentration (IC₅₀) 23.21 ± 1.17 µg/ mL followed by 1864.71 and 1793.80 µM Trolox equivalent/mg extract for ferric ion reducing antioxidant power and 3-ethylbenzothiazoline-6-sulfonic acid (ABTS), respectively. SOEE showed notable cytotoxic activity against A549. Oral administration of SOEE at 69.4 and 173.5 mg/kg.bw afforded a noteworthy protective effect against HgCl₂-induced pulmonary fibrosis. Also, lung superoxide dismutase (SOD), catalase (CAT), and reduced glutathione (GSH) were increased by SOEE. That was in parallel with working to reduce the malondialdehyde (MDA), necrosis factor kappa (NF-κB), interleukin-1β (IL-1β), and cyclooxygenase-2 (COX-2) levels. The lung P53, Bcl-2, Bcl-XL, AKT, and vanin-1 gene expression was upregulated in SOEE-treated rats compared to HgCl₂-treated rats.

Conclusion

SOEE reduces acute lung injury and can be used to inhibit the proliferation and migration of lung fibrotic cells. These activities may be related to its high antioxidant activity, which could result from the phenolic constituents. The authors recommended using sage in the treatment of lung diseases to protect from lung fibrosis. Additional animal and human experiments are needed for future research.

Graphical abstract

1 Background

Pulmonary fibrosis (PF) or lung fibrosis (LF) is a dangerous complication of many respiratory disorders; it is considered the end stage of heterogeneous interstitial lung diseases, injury, and inflammation [1]. LF causes an increase in the thickness of the alveolar wall and a decrease in lung functions, compliance, and capacity, which leads to failure in the respiration process [2, 3]. The occurrence of viral infections (VI) represents a risk factor for exposure to pulmonary fibrosis even after a long-term infection [4]. It can appear as a secondary lung viral infection. The relationship between LF and VI has become clearer and more widespread after the spread of severe acute respiratory syndrome (SARS) and coronavirus (COVID-19) pandemic diseases [2]. It was documented that most viruses like adenoviruses [5], influenza virus, avian influenza virus, SARS-CoV and SARS-CoV-2 [1], human T-cell leukemia virus (HTLV), hepatitis C virus (HCV) [5], human immunodeficiency virus (HIV), cytomegalovirus (CMV), Epstein–Barr virus (EBV), Middle East respiratory syndrome (MERS)-CoV, and murine γ-herpes virus 68 (MHV-68) cause long-term damage to the lung. Due to the enormous scope of COVID-19 with different waves, we expect an increase in PF that may be attributed to COVID-19 infection or other viruses that have the same symptoms and similar infections [3]. There is three-phase model in the treatment of PF which includes injury, inflammation, and repair [6].

Human adenoviruses (Ads) have numerous serotypes which cause various clinical syndromes. Notably, among the recognized serotypes, Ad type 7 (Ad7) is the most commonly associated with severe disease. This type most commonly causes mild upper respiratory tract illness and conjunctivitis, but it can also result in more severe lower respiratory tract illness, disseminated disease, and even mortality, particularly among infants and individuals with compromised immune or respiratory systems. Furthermore, Ad7 infections have been linked to central nervous system disorders and long-term respiratory complications like bronchiectasis and hyperlucent lung, also known as McLeod syndrome. WHO reported that Ad7 accounts for nearly 20% of all Ads, and there have been family clusters as well as institutional and community-wide outbreaks of Ad7 disease [7].

Human influenza subtype 1 (H1N1) influenza, also known as swine flu, is a variant of influenza A virus that causes a communicable viral disease resulting in upper and lower respiratory tract infections. The major symptoms include nasal secretions, fever, chills, reduced appetite, and, in severe cases, lower respiratory tract disease [8]. World Health Organization (WHO), 2009 [9], declared that H1N1 influenza and SARS-CoV-2 (Corona) were global pandemic diseases in 2009 and March 2020, respectively. They have the same symptoms, but Corona had the most severity [10].

The inflammation accomplished by VI resulted in a disturbance between antioxidant defense and oxidative stress [11]. SOD, CAT, and GSH enzymes are considered crucial enzymes in the enzymatic antioxidant defense system [12, 13]. SOD facilitates the conversion of superoxide radicals into H₂O₂ which is harmless through a process called dismutation [14]. Catalase is a heme protein that facilitates the reduction of hydrogen peroxide and safeguards the tissues against hazardous-harmful hydroxyl radicals [12].

GSH has a complex role in protecting against oxidative damage by acting as an antioxidant. It scavengers free radicals and also serves as a co-substrate for the detoxification of peroxide by glutathione peroxidases [15].

The Bcl-2 family proteins, which include both pro- and antiapoptotic members, play a role in the p53 apoptotic pathway [16]. The balance between these proteins, which have positive and negative regulatory functions, is crucial for determining the susceptibility to apoptosis [17]. All Bcl-2 family-associated proteins are functionally linked to mitochondria. Bcl-2, which is a representative of anti-apoptotic proteins, and Bcl-XL, which is commonly known as a pro-apoptotic factor, play roles in the later stage of programmed cell death and have contrasting activities. Cells are protected from apoptosis, which is induced by cytotoxic substances or cellular stress when there is a significant amount of Bcl-2 expression [18].

Vanin1 is an external enzyme that primarily carries out physiological tasks through the products of enzyme catalysis, which include pantothenic acid and cysteamine. The relation between Vanin1 metabolism and oxidative stress has gained significant attention in recent years [19]. Due to Vanin1's capability to impact several metabolic pathways and either worsen or alleviate pathological processes through oxidative stress, it has emerged as a crucial factor in the advancement of diseases [20].

Akt, a serine/threonine protein kinase, has been involved in suppressing cell death triggered by various stimuli such as growth factor withdrawal, cell cycle disruption, DNA damage, and loss of cell adhesion in diverse types of cells. Studies have demonstrated that organ toxicity stimulates Akt phosphorylation at Thr308 and Ser473 residues, leading to phosphorylation of the Bcl-2, which leads to the release of Bcl-XL [21].

Many rural people around the world still depend on the traditional use of herbal plants for the cure of some diseases [22,23,24]. In that respect, obtaining a scientific foundation for the potential application of herbal medicines in the treatment of illnesses, including viral infection, cancer, infectious disorders, and those linked to oxidative damage seems appropriate [25, 26].

Salvia officinalis L. (sage, family; Lamiaceae) is one of the most important herbal drugs widely used in folk medicine, cooking, and cosmetics [27]. The aerial parts of sage are characterized by its flavoring and seasoning properties that make them popular in the preparation of many foods [28]. The plant is common in the Middle East and Mediterranean areas [29], and nowadays, it is widely spread all over the world; Europe and North America [30]. In Egypt, it is a particular appendage in the South Sinai. Traditionally, sage has been used as an anti-inflammatory, antioxidant, antimicrobial, antidementia, and hypoglycemic drug around the world [28]. In Egypt, it is used in the treatment of upper respiratory tract disorders; asthma [31]. Several reports were traced concerning the biological activities of sage; antioxidant, anti-inflammatory, antimicrobial, and others that were attributed to its main phenolic constituents [32].

As a result of the widespread of successive waves of Coronavirus and other viruses that have similar and vigorous symptoms, causing LF and moving toward treatment with medicinal plants for their active constituents, this study aims to show the results of using SOEE as a protective agent from PF through in vivo study on HgCl₂-induced lung fibrosis and in vitro evaluation of its anticancer, antiviral, and antioxidant activities concerning the phytoconstituents, especially phenolics. This study was carried out on sage growing in South Sinai, Egypt. This plant was chosen based on ethnobotanical knowledge, evidence of continued widespread use, and local availability. The study proceeded from October 2020 to February 2023 in Egypt.

2 Methods

2.1 Plant material

Salvia officinalis L. herb was collected from South Sinai, Egypt, during Jun., 2020. The herb was kindly identified by Dr. Therese Labib, the chief consultant of Botanical Gardens in Egypt.

2.2 Biological material

Ad 7, hep-2 cells, H1N1 virus, and Vero E6 cells were provided from Nawah-Scientific, Egypt. The antibiotic & anti-mycotic solution, trypsin–EDTA, fetal bovine serum, and Dulbecco’s Modified Eagle Medium (DMEM) were provided from Gibco BRL (USA). Chemicals as ethanol (Fisher Scientific UK), phosphate buffer tablets (PBS, pH 7.4, Bio Shop Canada Inc.), Tween 80, and cholesterol (Sigma Aldrich, USA) were used.

Animals Ninety Swiss Albino rats (210 ± 10 g; 60 for lethal dose (LD₅₀) determination & 30 for S. officinalis ethanol extract 70% anticancer activity) were obtained from the National Cancer Institute's animal house, Cairo University, Egypt. Rats were kept in an environmentally friendly environment, observed, and served with water and standard diet ad libtum.

2.3 Plant extraction

2.3.1 Preparation of S. officinalis ethanol extract

The maceration method [33] was used for the preparation of the ethanol extract 70% (SOEE). Powdered herb (350 g) was extracted in boiled ethanol 70% (1 L/ 3days/ 3 times) at room temperature.

2.4 Phytochemical analysis

2.4.1 Qualitative phytochemical screening

Phytochemical tests were done for SOEE using reference procedures [34, 35] to estimate its constituents. The screening was conducted on various substances, including terpenoids/steroids, saponins, carbohydrates, flavonoids, tannins, proteins/amino acids, and alkaloids [36,37,38].

2.4.2 Quantitative estimation of total phenolic & flavonoid contents

All tests were carried out spectrophotometrically utilizing the FluoStar Omega microplate reader, owing to settled standard calibration curves. The folin–Ciocalteau method was used to evaluate the total phenolic (TP) content [39], while the total flavonoid (TF) content was estimated by quantifying the color intensity formed when the aluminum chloride reagent made a complex with flavonoids [40]. According to Attard [41], the results were expressed as gallic acid (GAE) equivalents and rutin (RE) equivalents, respectively.

2.4.3 Quantitative estimation of total soluble carbohydrate (TSC) contents

The assay was conducted in accordance with the Gerhardt et al. method [42]. For the method in detail, see the Additional file 1: S1.

2.4.4 High-performance liquid chromatography (HPLC) analysis

The major phenolic constituents in the SOEE were characterized and identified by HPLC (Agilant 1260 series, Germany) combined with a diode array detector, depending on UV spectra and retention time. For descriptions of the analysis, see the Additional file 1: S2.

2.5 Isolation and identification of the Phenolic constituents

SOEE extract (1.5 g) was exposed to preparative separations using a PuriFlash 4100 system (Interchim Software 5.0, France). After collecting similar fractions and sub-fractions using paper chromatography (PC, 1MM), they were purified using preparative paper chromatography (3MM) and columns (Sephadex LH-20) [43]. Twelve compounds were isolated and subjected to structural elucidation using various investigative techniques: physical, chromatographic, and spectral analyses (ultra violet (UV), nuclear magnetic resonance (NMR), and mass spectrophotometer (MS) [44,45,46]. Also, all data obtained were compared with the previously reported values of identified compounds [47,48,49]. For a detailed method, see the Additional file 1: S3.

2.6 Biological activities

2.6.1 Antioxidant activity

2.6.1.1 DPPH assay

The radical scavenging activity of SOEE was investigated by DPPH method developed by Boly et al. [50]. This method relies on reducing the color intensity of the DPPH reagent, measured at 540 ℷ nm [51]. Graph Pad Prism version 5 [52] was used for calculating IC₅₀ values. The greater antioxidant power of the extract was indicated by the lower IC₅₀ values. For more details, see the Additional file 1: S4.1.

2.6.1.2 FRAP assay

The FRAP assay for SOEE was conducted using the previously mentioned method [53] with the use of microplates as a minor modification. The rapid reduction of ferrictripyridyltriazine (FeIII-TPTZ) and forming ferrous-tripyridyltriazine (FeII-TPTZ) represent the power of the plant extract's antioxidant properties. The intensity of the blue color was measured by the microplate reader FluoStar Omega at ℷ593 [51]. The increase in the iron reduction of the sample was indicated by the increase in absorbance. The results were expressed as μM Trolox equivalent per milligram sample (TE/mg sample) by utilizing the linear regression equation derived from the Trolox calibration curve (linear dose–response curve of Trolox).

2.6.1.3 ABTS assay

The ABTS assay was established in accordance with the Arnao et al. method [54] using microplates as a minor modification. The results were expressed as μM TE/mg sample. For the concept and procedure in detail, see the Additional file 1: S4.2.

2.6.2 Antiviral activity

The potential antivirals against human Ad7 and H1N1 were assessed by the sulforhodamine B (SRB) assay and the crystal violet method, respectively. The cytotoxicity assays were demonstrated according to the recently reported cytopathic (CPE) inhibition effect [55, 56]. The efficacy of the antiviral was determined by dose–response assays; IC₅₀ and CC₅₀ (the range of cytotoxicity). Hep-2 cells and Vero E6 cells were cultured separately in a DMEM medium containing 10% fetal bovine serum and 0.1% antibiotic/antimycotic solution.

2.6.2.1 Human adenovirus type 7 (Ad7)

To assess the cytotoxicity, hep-2 cells were used in the steps described for the assay of antiviral activity [57]. Calculating the cell viability percentage was based on the determination of Ad7 infectivity using the SRB method, which monitored CPE [55, 56]. see the Additional file 1: S5.

2.6.2.2 H1N1 virus (swine flu)

The assessment of antiviral activity and cytotoxicity assays was conducted using the crystal violet technique [58]. Vero E6 cells were used for the cytotoxicity assay. The cell viability percentage was calculated by the determination of the influenza H1N1 infectivity through the monitoring of CPE using the crystal violet method. For detailed methods, see the Additional file 1: S6.

2.6.3 Cytotoxic activity

2.6.3.1 Determination of SOEE cytotoxicity on A549 lung carcinoma cell line

The viability of A549cell lines was assessed by using MTT assay [59] for measuring the impact of SOEE, see the Additional file 1: S7.

2.6.4 In-vivo lung-protective activity of SOEE against HgCl₂-induced lung fibrosis

2.6.4.1 LD₅₀ Determination for SOEE

The LD₅₀ was calculated by administering different doses of SOEE; 1200, 1800, 3000, 4000, 5000, and 6000 mg/kg to animal groups (n = 10) orally according to Maul et al. method [60]. The doses of the SOEE detected to be administrated to rats were 1/50 LD₅₀, 69.4 mg/kg.b.w. and 1/20 LD₅₀, 173.5 mg/kg.b.w. SOEE.

2.6.4.2 Experimental design

Thirty adult male Swiss Albino rats weighing 200–220 gm were divided into five groups (n = 6/each) containing normal and treated rats. Group 1 was the negative control group, and its rats were administered distilled water (3ml/21 days). SOEE was suspended through distilled water and administrated by intragastric intubation. Group 2 administered SOEE (173.5 mg/k.g.) only, 3 times per week for 21 days. HgCl₂ (5 mg/ kg.b.w.) was administrated orally to group 3 to induce lung fibrosis in rats 3 times per week for 21 days [61]. Groups 4&5 were treated with HgCl₂ (5 mg/kg) and different doses of SOEE (69.4 mg/k.g./mL and 173.5 mg/k.g./mL, respectively). Blood samples were collected in heparinized tubes at 22nd day and centrifuged (1000 xg, 20 min). Total triglycerides (TG), cholesterol (TC), and cholesterol-high-density lipoprotein (HDL-C) levels were evaluated in the Plasma. Commercial kits (Asan and Youngdong Pharmaceutical Co., Korea) were used.

2.6.4.3 Lung specimens

The lung tissue was homogenized in PBS (3 mL, pH 7.5) and then subjected to centrifugation (10 min, 3000 g). CAT, GSH, SOD, and TBARs were measured from the supernatant using a kit from Cayman Chemical Company (An Arbor, MI), while lung IL-1β, NF-κB, and COX-2 levels were assessed by an enzyme-linked immunosorbent assay (ELISA) kit from Shanghai YL Biotech Co. Ltd. (China).

2.6.4.4 Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

The rats' lung tissues were used for the extraction of RNA under the method [62]. The method and the sequence of primers Table (1) used are discussed in detail in the Additional file 1: S8.

2.7 Statistical analysis

Results were reported as the mean ± standard deviation (SD) for six distinct spectrophotometric and ELISA measurements, in addition to three separate assessments for in vitro cytotoxicity and gene expression PCR analysis. The data were subjected to statistical analysis using SPSS/20 software, employing ANOVA (one-way analysis of variance), followed by Bonferroni's multiple comparison test. P-value (P < 0.01) was considered to indicate statistical significance.

3 Results

3.1 Phytochemical analysis

3.1.1 Phytochemical screening tests

The results of the preliminary phytochemical screening tests listed in Table 1 showed the presence of a wide range of phytoconstituents such ascarbohydrates, phenolics, flavonoids, steroids and/or terpenoids, glycosides, tannins, and traces of alkaloids in the SOEE.

Table 1 Preliminary phytochemical screening tests of S. officinalis L. ethanol extract (70%) (SOEE)

3.1.2 Yield, total phenolic, total flavonoid, and total soluble carbohydrate contents in the SOEE of S. officinalis herb

The yield of SOEE was 19 g/100 g herb powder. The estimation of TP, TF, and TC contents was possessed utilizing gallic acid, rutin, and glucose as references, respectively, through the linear regression equation of the calibration curve. The TP, TF, and TSC contents were 140.29 ± 0.9 mg GAE/g extract, 60.85 ± 0.6 mg RE/g extract, and 187.93 µg glucose/mg extract, respectively. It seems to be the first record of the total flavonoid amount and total carbohydrate contents of the Egyptian species.

3.1.3 HPLC analysis of SOEE 70% of S. officinalis L. Herb

Fourteen compounds were identified from HPLC analysis of SOEE, including nine phenolic acids and five flavonoids (Table 2). Rosmarinic and ferulic acids were the major phenolic compounds (326.7496% and 6.9838%), respectively. The major flavonoid constituents were luteolin aglycon (24.2018%) and luteolin-7-O-glucoside (18.5305%).

Table 2 HPLC analysis of S. officinalis L. ethanol extract (70%) (SOEE)

3.1.4 Characterization of the isolated compounds from SOEE

Chromatographic and spectroscopic analysis of the SOEE of sage approved the isolation and identification of twelve compounds (Fig. 1).: apigenin (1); luteolin(2), genkwanin (3), quercetin (4), hispidulin (5), luteolin-7-O-glucoside (6), quercetin-3-O-α- rhamnosyl (1‴ → 6″)-β-glucoside (rutin) (7), rosmarinic acid (8), caffeic acid (9), ferulic acid (10), chlorogenic acid (11), and ellagic acid (12). Dimethyl sulfoxide (DMSO)-d₆ was the solvent used for compound solubility in the ¹H-NMR.

Fig. 1

Structures of isolated compounds (1–12) from S. officinalis L. (SOEE)

Apigenin yellowish-white powder, m.p. 345-347 °C, soluble in ethyl acetate and methanol. It appeared as a strong purple fluorescence (λ254) and turned yellow with ammonia vapor which sharpened after spraying with 5% aluminum chloride (AlCl₃) reagent. Mass spectrum (EI-Ms) showed the molecular ion peak (M⁺); m/z 270 corresponding to C₁₅H₁₀O₅ formula and fragments at 242, 213, 153, and 69 as major fragments. ¹H-NMR obtained results were similar to those published by Ateya et al. [63]. For data in detail, see the Additional file 1: S9.

Luteolin yellow powder, mp328–330 °C, soluble in ethyl acetate and methanol. It gives a strong purple fluorescence at λ254, 365 nm, which turned yellow with ammonia vapor, and the color was intensified by spraying with 5% AlCl₃ reagent. R_f value on PC; 0.49 (BAW, 4:1:5), 0.27, (CH₃COOH: H₂O; 1:1). EI-Ms spectrum: M⁺; m/z 286 related to a molecular formula C₁₅H₁₀O₆ and fragments at m/z 258,153, 134 and 69 as major fragments. The data of UV spectra in methanol (MeOH): λ253, 296 sh, 294 sh., 345. Sodium methoxide (NaOMe): λ272, 310 sh., 412. AlCl₃: 274, 300 sh., 328 sh.,420. AlCl₃/ HCl: 267, 297 sh., 353, 388. Sodium acetate (NaOAc): 267,310 sh., 75. NaOAc/H₃BO₃: 270, 297 sh., 362. Besides these, the¹H-NMR was similar to those published by Ateya et al. [63]. For data in detail, see the Additional file 1: S10.

Genkwanin yellow powder, mp278–280 °C, purple spot (UV; λ254, 365), yellow with ammonia and intensified with 5% aluminum chloride reagent, ¹H-NMR (DMSO -d₆, 500 MHz), δ 7.98 (¹H, d, 2'/6'-H, J = 8.5 Hz), 6.98 (¹H, d, 3'/5'-H J = 8.5 Hz), 6.81 (¹H, s,3-H), 6.85 (¹H, d,8-H), 6.44 (¹H, d,6-H), 3.85 (¹H, s,OCH₃-C7). EI- mass (m/z), 283(M⁺), 267, 253, 167,152, 127, 62 (major peaks). The UV spectral data in methanol: λ 262, 340. NaOMe: λ 260,382. AlCl₃: 270, 297, 350, 390. AlCl₃/ HCl: 270, 297, 350, 390. NaOAc: 260,340. NaOAc/H₃BO₃: 262,330. Genkwanin was reported to have anti-tumor activity [64].

Quercetin yellow amorphous powder, mp 345–347 °C, soluble in ethyl acetate and methanol. It had a dark purple color (UV; λ254) and turned yellow with ammonia vapor. After spraying with 5% aluminum chloride reagent, the color was intensified. EI-Ms spectrum exhibited the molecular ion peak (M⁺) at m/z 270, representing a molecular formula C₁₅H₁₀O₅, and fragments at 242, 213, and 153, with 69 as major fragments. The ¹H-NMR (DMSO-d₆, 500 MHz) was the same as published by Amer et al. [51]. For data in detail, see the Additional file 1: S11.

Hispidulin yellow powder dissolved in methanol. The UV spectrum at MeOH (λmax, nm: 278, 336. The ¹H-NMR (DMSO-d₆, 400 MHz): δ 13.05 (1H, s, 5-OH), 10.88 (1H, s, 7-OH), 10.44 (1H, s, 4'-OH), 8.2 (2H, d, J = 8.4 Hz, H-2', 6'), 6.98 (2H, d, J = 8.4 Hz, H-3', 5'), 6.98 (1H, s, H-3), 6.65 (1H, s, H-8), 3.70 (3H, s, O-CH₃). The data were in agreement with previously reported for Salvia plebeia [48, 65]. It was identified before by LCMS/MS, but it is the first time to be isolated from the S. officinalis herb. Co-chromatography with a standard also confirmed the compound.

Luteolin-7-O-β- glucopyranoside yellow powder, soluble in methanol, mp 260–261 °C. The ¹ H-NMR (DMSO -d₆, 500 MHz), aglycone: δ7.407 (¹H, m, 2'/6'-H), 6.88 (¹H, d, 5'-H, J = 8.5), 6.58 (¹H, s,3-H), 6.77, 6.47 (¹H, d, 6 /8-H), sugar moiety: 5.04 (¹H, d, H-1``, J = 7.5). The UV spectral data in methanol: λ 253, 268(sh.), 347. NaOMe: λ 264, 403 AlCl₃: 273, 298 (sh.),427. AlCl₃/ HCl: 273, 296 (sh.), 354, 389. NaOAc: 265, 408. NaOAc/H₃BO₃: 263, 374. Acid hydrolysis was done and revealed the presence of aglycone (luteolin), and the sugar moiety was characterized by paper chromatography as glucose.

Rutin yellow amorphous powder, soluble in methanol, mp 240–242 °C, appeared as a dark purple spot under UV light (λ254, 365), and their yellow color appeared and intensified after being subjected to ammonia solution and spraying with 5% aluminum chloride reagent, respectively. The data of UV spectrophotometry and ¹H-NMR at DMSO-d₆ (400 MHz) were similar to that published by Amer et al., [51] in the identification of quercetin-3-O-rutinoside (rutin). For data in detail, see the Additional file 1: S12.

Rosmarinic acid yellowish white powder; the UV spectrum data (λmax, nm) at MeOH: 232, 329. EI-Ms spectrum exhibited (M⁺) at m/z 361. ¹H-NMR (CD₃OD, 400 MHz): δ 7.56 (1H, d, J = 16 Hz, H7), 7.10 (1H, d, J = 2 Hz, H-2'), 6.95 (1H, dd, J = 8,2 Hz, H-6), 6.92 (1H, d, J = 8 Hz, H-5), 6.75 (1H, d, J = 8 Hz, H-5'), 6.75 (1H, d, J = 2 Hz, H-2), 6.69 (1H, dd, J = 8, 2 Hz, H-6'), 6.28 (1H, d, J = 16 Hz, H-8), 5.20 (1H, dd, J = 8.4, 4.4 Hz, H-8β), 3.11 (1H, dd, J = 14.2, 4.4 Hz, H-7β), 3.02 (1H, dd, J = 14.2, 8.4 Hz, H-7α); two doublets were found at 7.56 and 6.28 with large proton–proton coupling (J = 16 Hz) assigned to a pair of trans-olefinic protons, the same ABX system of three protons δ 3.02, 3.11 (J = 14) which represent H-7α& H-7β, and δ 5.20 (J = 8.4, 4.4 Hz, H-8β)). ¹³C-NMR (CD₃OD, 100 MHz): δ 48.99 ppm (deuterated methanol), δ 127.4 (C-1), 115.3 (C-2), 146 (C-3), 146.6 (C-4), 122.7 (C-5), 116.4 (C-6), 37.6 (C-7), 74.8 (C-8), 172.3 (C-9), 129.1 (C-1'), 114.6 (C-2'), 144.8 (C-3'), 147.6 (C-4'), 123.8 (C-5'), 116.0 (C-6'), 148.2 (C-7'), 117.4 (C-8'), 167 (C-9'). The¹H NMR spectrum showed all data were in agreement with previously reported for Salvia plebeia [48, 65]. It identified before by LCMS/MS, but it is the first time to be isolated from S. officinalis herb.

Caffeic acid yellowish-brown powder, soluble in methanol, melting point (mp) 220 °C. It was appeared as blue fluorescence at UV(λ254), blue at (λ 365), and dark blue after spraying with ferric chloride reagent (FeCl₃), the UV (λmax, nm); (MeOH) (242, 296, 325) confirmed a phenyl propene skeleton [66]. ¹H-NMR (CD₃OD, 400 MHz) (δ ppm): δ 7.45 (1H, d, J = 16 Hz, H-7), 7.01 (1H, d, J = 2.1 Hz, H-2), 6.90 (1H, dd, J = 8, 2.1 Hz, H-6), 6.75 (¹H, d, J = 8 Hz, H-5), 6.2 (1H, d, J = 16 Hz, H-8). ¹³C-NMR (CD₃OD, 100 MHz): δ 127(C-1), 115.8 (C-2), 146.3 (C-3), 148.6 (C-4), 121.8 (C-5), 114.3 (C-6), 145.8 (C-7), 114.6 (C-8), 170.3 (C-9). The data were similar to previously reported [67].

Ferulic acid off-white amorphous powder, it gave dark pot under short UV(λ254), and shiny violet under long UV (λ 365). ¹H-NMR (400 MHz) (δ ppm):, δ 7.49 (1H, d, J = 15.89, H-7), 7.08 (1H, dd,, J = 8.19, 1.93 Hz, H-6), 7.28 (1H, d, J = 1.9, H-2), 6.79 (1H, d, J = 8.06 Hz, H-5), 6.36 (¹H, d, J = 15.88 Hz, H-8), 3.7 (3H, s, O-CH₃).

Chlorogenic acid white powder, soluble in methanol, it′s melting point was 208–209 °C. It gave yellowish green fluorescence after the exposure of ammonia solution which turned to blue fluorescence after spraying with ferric chloride reagent. The results of ¹H-NMR (DMSO-d₆, 400 MHz), UV spectrum showed at methanol (MeOH), and co-chromatography was found to coincide with standard and with those previously published data for chlorogenic acid [68].

Ellagic acid white amorphous powder; the UV spectrum data at MeOH λ 255, 362. The ¹H NMR (DMSO, 400MHz); δ_ppm7.45 (2H, s, H-5, 5’). The compound was confirmed by co-chromatography with standard ellagic acid, and the obtained data were in agreement with that published by lil [69].

3.2 Biological activities

3.2.1 Antioxidant activity: DPPH, FRAP, and ABTS.

DPPH, FRAP, and ABTS methods were utilized to examine the antioxidant activity of SOEE (Table 3). The IC₅₀ for SOEE in the DPPH radical scavenging assay was 23.21 ± 1.17µg/mL compared with Trolox (IC₅₀ 42.42 ± 0.87) as a standard antioxidant agent. As a role, the lower the IC₅₀ value, the higher the antioxidant activity, while the values of the antioxidant activity quantified through FRAP and ABTS were 1864.71 and 1793.80 µM Trolox equivalent /mg extract, respectively. These data revealed that SOEE has highly antioxidant activity.

Table 3 Antioxidant activity of S. officinalis L. ethanol extract (70%) (SOEE)

3.2.2 Antiviral activity

The determination of CC₅₀ and IC₅₀ for both Ad7 and H1N1 viruses was fulfilled using GraphPad PRISM software (Graph-Pad Software, San Diego, USA). The results are shown in Figs. 2 and 3, Table 4. Regarding Ad7, the IC₅₀ of SOEE was 9.937µg/ml, and CC₅₀ was14 µg/ml. Thus, IC₅₀ of SOEE was lower than CC₅₀ against adenovirus 7, which indicates a promising reduction of the virus concentration and more activity.

Fig. 2

Graphs of the cytotoxic concentration and inhibitory concentration of SOEE against Adenovirus type 7

Fig. 3

Graphs of the cytotoxic concentration (CC₅₀) and inhibitory concentration (IC₅₀) of SOEE against Vero E6 cells and H1N1

Table 4 Antiviral activity of S. officinalis L. (SOEE) against H1N1 and adenovirus (Ad7)

IC₅₀ and CC₅₀ of SOEE against H1N1 were 9.641 µg/ml and 6.018 µg/ml, respectively. The lowest reduction in H1N1 virus concentration indicated SOEE has less activity against H1N1 than Ad7. There were no reported data about the effect of ethanol extract of sage against these types of viruses.

3.2.3 In vitro study

3.2.3.1 SOEE cytotoxicity

SOEE had low IC₅₀ value against the A549 lung cancer cell line. The results of MTT assay discussed that the SOEE incubation at different consternations (0, 7.8, 15.6, 31.25, 62.50, 125, 250, 500, and 1000 µg mL⁻¹) with A549 caused % of viability; 100, 100, 100, 99.26, 93.08, 79.54, 63.49, 39.02 and 28.75, respectively (Fig. 4), and toxicity % of 0, 0, 0, 0.74, 6.92, 20.46, 36.51, 60.98, and 71.25, respectively. The IC₅₀ value of SOEE against A549 cancer cells was 387.9 µg/mL.

Fig. 4

IC₅₀ value of SOEE against A549 lung cancer cell line

3.2.4 In vivo study

3.2.4.1 LD₅₀ dose of SOEE in rats

Oral administration of SOEE (10 rat/group) at doses 1200, 1800, 3000, 4000, 5000, and 6000 mg/kg b.w. resulted in mortalities 0, 1, 4, 7, 8, and 10, respectively. It was observed that the LD₅₀ dose of SOEE was 3470 mg/kg b.w. Regarding the criteria, when the LD₅₀ is greater than 2000 mg/kg, the plant extract is classified as relatively safe. The doses of SOEE administered to rats in the in vivo were determined with 1/50 LD₅₀, 69.4 mg/kg.b.w. and 1/20 LD₅₀, 173.5 mg/kg.b.w. SOEE.

3.2.4.2 Effect of SOEE on plasma TC, TG, and HDL-C levels

The levels of TC, TG, and HDL-C are shown in Table 5. Oral administration of mercuric chloride (HgCl₂) at a dose (5 mg/kg b.w.) resulted in a significant increase in TC and TG levels to 46.95 and 74.96%, with a significant decrease in HDL-C level, 33.58%, compared to the control, which is evidence of lipid peroxidation.

Table 5 Effect of SOEE on levels of lipid, oxidative stress, and inflammation markers

Treatment by SOEE (69.4 mg/kg.b.w.) caused significantly reduction in the TC and TG levels to 27.28 and 27.15%, respectively, and elevation in HDL-C level significantly to 34.72%, comparing to the HgCl₂—treated group, while administration of SOEE (173.5 mg/kg.b.w.) to HgCl₂-treated rats led to a significant decrease at the TC and TG levels to 31.56 and 37.17%, respectively, with a significant increase in HDL-C level to 44.23%, on the plasma comparing to the group treated by HgCl₂.

3.2.4.3 Effect of SOEE on lung tissues SOD, CAT, GSH, and MDA

Table 5 shows the levels of lung SOD, CAT, GSH, and MDA. The administration of HgCl₂ orally (5 mg/kg b.w.) led to a significant reduction in lung SOD, CAT, GSH, and MDA levels to 52.17%, 60.56%, 58.06%, and 146.15%, respectively, in comparison with the control group, indicating acute lung fibrosis. However, treatment with SOEE (69.4 mg/kg.b.w.) significantly elevated the lung SOD, CAT, and GSH levels to 55.06%, 66.75%, and 61.95%, with a significant decrease in lung MDA to 27.43%, respectively, comparing to the HgCl_2- treated group. Furthermore, the administration of SOEE (173.5 mg/kg.b.w.) to HgCl₂ -treated rats led to a significant increase in the level of lung SOD, CAT, and GSH to 114.02%, 185.43%, and 112.39%, as well as a significant decrease in the lung MDA level to 47.91%, compared to the HgCl₂- treated group. It can be concluded that the administration of SOEE orally provided remarkable protection against HgCl₂-induced lung damage [70].

3.2.4.4 Effect of SOEE on lung IL-1β, NF-κB, and COX-2 levels in treated rats

Lung levels of IL-1β, NF-κB, and COX-2 in HgCl2-treated rats were dramatically increased by 99.89, 124.97, and 225.05%, respectively, as compared to normal rats (Table 5) (p < 0.05). Also, administration of SOEE (64.9 mg/kg.b.w.) to HgCl₂-treated rats produces a significant decrease in lung IL-1β, NF-κB, and COX-2 levels by 27.39, 24.78, and 32.49%, respectively, compared to HgCl₂-treated group (p < 0.05). Additionally, the administration of SOEE (173.5 mg/kg.b.w.) significantly decreased lung IL-1β, NF-κB, and COX-2 levels by 42.42, 44.19, and 56.51%, respectively, as compared to HgCl₂-exposed rats (p < 0.05).

Figure 5a, b shows that severe lung damage occurred in the HgCl₂-treated compared with the normal rats group. They had significant in lung Bcl-XL, Akt, and vanin-1 genes expression to 183, 152.68%, and 248.15%, respectively, as well as a decrease in lung P53 and Bcl-2 to 53.60 and 62.14%, respectively, compared with the control. After administration of SOEE at 69.4 mg/kg, lung Bcl-XL, Akt, and vanin-1 gene expression was significantly decreased to 18.37, 57.78, and 21.28%, respectively, and lung P53 and Bcl-2 was increased to 60.0 and 84.62%, respectively, comparing with HgCl₂. The administration of SOEE to HgCl₂ -treated rats at 173.5 mg/kg.b.w. announced a notable reduction in Bcl-XL, Akt, and vanin-1 genes expression of the lung to 50.88, 61.47, and 69.68%, respectively, as well as an increase in lung P53 and Bcl-2 to 84.4 and 123.07% compared to rats administered HgCl₂.

Fig. 5

a. Effect of SOEE on lung P53 and Bcl-2 gene expression in control and treated rats. b. Effect of SOEE on lung Bcl-X_L, Akt and vanin-1 gene expression in treated rats

4 Discussion

The preliminary phytochemical screening examinations proved to be helpful in the estimation of the bioactive components, which provided the herb with its beneficial characteristics and then made it easier to estimate them quantitatively. It was reported that the biological activities of sage are attributed to its phytoconstituents, including essential oils, phenolics (phenolic acids, flavonoids, and glycosides), terpenes, tannins, etc. [71]. The results of the yield obtained from the current study were consistent with those reported by Pereira et al. [72]. The qualitative estimation of the extract revealed the presence of different categories of phytoconstituents, which are consistent with the previously reported data by Ghorbani and Esmaeilizadeh [28]. Meanwhile, the quantitative estimation of the TP content record was particularly near to those reported by Afonso [71]. Naturally, the differences in the levels of phenolic were attributed to the climatic changes. There was no previous record regarding the quantitative determination of the TF contents of this Egyptian species, Salvia officinalis, so our results seem to be the first record of the total flavonoid amount for this species. The Egyptian species contain TP and TF contents in more than ten folds of the species in Turkey [73]. Phenolics are responsible for a broad set of biological activities: antimicrobial, antioxidant, anti-inflammatory, and anticancer [28, 32, 74]. The data obtained from the quantitative estimation of the phenolic and flavonoid constituent of SOEE by HPLC were similar to those published by Herna´ndez [36], as rosmarinic and ellagic acids were the major phenolic acids but in different concentrations. The Egyptian herb contains 50-fold rosmarinic acid concentrations, and ellagic was nearly the same. Flavonoids and Phenolic acid concentrations might be influenced by some agricultural and climate factors [47]. Moreover, luteolin-7-O-glucoside and rosmarinic acid were the major phenolic acid and flavonoids in different countries [47, 49].

Herbal plants have a wide range of bioactive compounds with remarkable antioxidant properties, such as phenolic acids, flavonoids, catechins, and others. They are considered as a natural source of free-radical scavengers that protect the vital cells and organs from the dangerous effect of free radicals and remove reactive oxygen species (ROS) [75]. The antioxidant activity of major medicinal plants is strongly related to phenolic compounds (e.g., caffeic, rosmarinic, and carnosic acids). The twelve phenolic compounds isolated and identified from SOEE by chromatographic and spectroscopic techniques were previously identified in the plant by LC/MS/MS of S. officinalis L. herb and flowers [47,48,49]. It is the first record for the isolation of Hispidulin from this species.

Several studies reported that rosmarinic acid, caffeic acid, genkwanin, carnosol, carnosic acid, rosmanol, rosmadial, cirsimaritin, quercetin, and rutin are the most effective phenolic acids and flavonoids of S. officinalis [76, 77]. They protect cells from ROS-induced, lowering the risk of cancer, asthma, and fibrosis [78].

Oxidative stress has the main role in the development and progression of various diseases: diabetes, neurological diseases, cardiovascular disorders, bronchial asthma, and cancer [79, 80]. Normally, the body liberates free radicals through vital processes in a balance with naturally occurring scavengers such as glutathione. Excessive free radicals are liberated when this balance is out of control by age or injury, causing dangerous effects on cells and different diseases [81]. The authors used different methods with different mechanisms to determine the antioxidant capacity because only one method is insufficient and to obtain widely diverging results [82, 83].

In our study, the antioxidant activities of SOEE were powerful and relatively high. The low IC₅₀ value for SOEE in the DPPH assay (23.21 ± 1.17µg/mL) represents twofold activities than Trolox (42.42 ± 0.87), therefore a high antioxidant activity. The reducing power (FRAP) and antioxidant ability of SOEE to reduce the ABTS were 1864.71 and 1793.80 µM Trolox equivalent /mg extract, respectively, that means it has 1.8- and 1.7-fold activities than Trolox. The obtained data were similar to that reported for DPPH of the S. muirii and S. dolomitica extracts [84] and 10 folds for ABTS than the other species. Our results were in agreement with the reported potent anti-oxidant activities of sage [76, 77].

The high content of phenolic constituents, especially rosmarinic acid, ellagic acid, caffeic acid derivative, chlorogenic acid as phenolic acids, and luteolin-7-O-glucopyranoside, quercetin, rutin as flavonoids, may be responsible for this powerful activity as that reported about their activities [77, 85]. In addition, phenolic constituents were responsible for other activities, such as the anti-inflammatory process, healing of pulmonary injury, and preventing pulmonary fibrosis [77]. It is the first report concerning the effect of SOEE as an antiviral.

Polyphenols have a significant impact on the prevention of cancer initiation and progression by employing diverse mechanisms. These mechanisms encompass the inhibition of oncogenes and genes associated with oxidative stress and inflammation [48, 65], as well as the modulation of epigenetic aberrations, including histone modifications, DNA methylations, and microRNAs [86]. Our phytochemical studies proved the presence of rosmarinic acid and ellagic acids in high concentrations in the SOEE formula, which were reported to have cytotoxic effects on cancer cells [56,57,58,59,60,61].

In the current study for acute toxicity, the administration of SOEE in different doses 1200, 1800, 3000, 4000, 5000, and 6000 mg/kg to animal groups (n = 10) orally didn’t reveal any signs of mortality in all animals. Based on the documents provided by the Globally Harmonized System (GHS) of classification and labeling of chemicals, the substances having LD₅₀ values greater than 2000 mg/kg are classified as relatively safe [87]. The use of such high doses as in previous studies of Nikitakis and Kowcz used 1290, 2020, 3200, or 5000 mg/kg b.w. for LD₅₀ estimation of different plant extracts [88]. Regarding these criteria, SOEE was considered to be very safe. The doses of SOEE administered to rats in the in vivo were determined with 1/50 LD₅₀, 69.4 mg/kg.b.w. and 1/20 LD₅₀, 173.5 mg/kg.b.w. SOEE.

It is considered the first report to assess lung protection of SOEE on HgCl₂-induced lung damage in rats. HgCl₂ is a recognized substance that causes lung toxicity, principally through impairing the function of pulmonary surfactants [70]. A variety of signal transduction cascades are first activated in lung cells because of the host receptor first recognizing HgCl₂.

Several studies reported that HgCl₂ induced the production of ROS [63] and led to inflammation and a remarkable elevation in lung enzymes and lipid peroxidation [89, 90], as well as a significant rise in MDA and a decline in the activity of antioxidant enzymes in the lung [91]. This effect was compatible with Mohamed et al., who documented the increase in the inflammatory mediators’ levels in rats administered with LPS [92, 93].

Based on these reports, our study observed substantial changes in lipid peroxidation (MDA) and IL-1β, NF-κB, and COX-2 levels. Additionally, there was a significant depletion of the pulmonary antioxidant system (SOD, CAT and GSH). Our study suggested that HgCl₂ causes oxidative damage to the lungs by inhibiting the NADPH oxidase pathway and reducing the levels of pulmonary SOD, CAT, and GSH in rats treated with HgCl₂. In addition, the Nrf-2/HO-1 pathway, which plays a crucial role in regulating the antioxidant system, was found to be imbalanced in lung tissues treated with HgCl₂. This imbalance resulted in the suppression of GSH and SOD activities, as well as the formation of O²⁻, H₂O₂, NO, and iNos.

The administration of SOEE resulted in a hypolipidemic effect and inhibition of IL-1β, NF-κB, and COX-2, leading to a decrease in MDA levels and an increase in CAT, SOD, and GSH levels [94].

The improvements in SOD, CAT, and GSH levels, which play a critical biological function in eliminating ROS, are caused by SOEE's antioxidant and cell-protective activities [91, 95]. The current findings indicate that SOD, CAT, and GSH are activated by SOEE, which may aid in inhibiting the inflammatory reaction by lowering the oxidative stress brought on in rats treated with HgCl₂. Through several processes, SOEE may shield cellular components from HgCl₂ oxidative damage [96]. These include the up-regulation and activation of antioxidant enzymes, metal ion chelation, and ROS scavenging [97, 98].

The excessive production of MDA and inflammatory mediators (IL-1β, NF-κB, and COX-2) was associated with DNA damage. SOEE administration had a vital role in accelerating the repair of DNA. It is also considered a good protector of lung tissues. SOEE-treated rats had increased SOD and CAT activity, which resulted in decreased MDA production. GSH resynthesis also promotes DNA repair, which protects the lungs [99].

Our findings were consistent with previous studies that have demonstrated the efficacy of phenolic administration in reducing levels of IL-1β, NF-κB, and COX-2. This reduction in inflammatory markers leads to a decrease in lung inflammation and the generation of reactive oxygen species (ROS). Additionally, phenolics inhibit the expression of adhesion molecules and monocyte adhesion in lung tissue [100,101,102,103,104]. Antioxidant enzymes appear to be involved in this effect [105].

Our study predicted that the active phenolic compounds in SOEE, rosmarinic acid, caffeic acid, genkwanin, carnosol, carnosic acid, rosmanol, rosmadial, cirsimaritin, quercetin, and rutin, inhibit the production of IL-1β, NF-κB, and COX-2, prevent lung tissue degradation, protect cell membrane integrity, and delay inflammation. All these led to a reduction in lung fibrosis in HgCl2-treated rats' lungs.

The results of our study indicate that HgCl₂ decreased the expression of p53 and Bcl₂, as well as elevated the Bcl-XL, Akt, and vanin-1 gene expression in the lung of treated rats.

Furthermore, SOEE was found to be effective in significantly regulating biologically active lung P53, Bcl-X_L, Akt, and vanin-1 and induced Bcl-2 gene expression. Bcl-2 assumes a significant role in the development of fibrosis across various organs, such as lung fibrosis. It stimulates the production of collagen and fibronectin in fibroblasts [106]. Also, it has the ability to inhibit the protease production that degrades the extracellular matrix [107]. Bcl-2 has been found to be elevated in the alveolar inflammatory infiltrate of LPS-induced lung fibrosis [108].

In the current study, SOEE, as an antioxidant, plays its role in two levels. In the first level, eliminating ROS, preventing ROS synthesis, directly scavenging ROS, or controlling the activity of antioxidant enzymes, SOEE, eliminates ROS. SOEE stops the generation of ROS and inhibits ROS-producing enzymes at the second level [109].

SOEE could be demonstrating its anti-inflammatory activity through various mechanisms. Firstly, it can exert antioxidant and radical scavenging activities. Additionally, SOEE can modulate the metabolism of arachidonic acid by regulating enzymes such as cyclooxygenase, phospholipase A2, and lipoxygenase, thereby influencing the inflammatory response. Moreover, it can also affect the activity of nitric oxide synthase [110]. This protective effect is demonstrated by a decrease in the inflammatory response.

To the best of our knowledge, this is the first report highlighting the protective effect of SOEE against HgCl₂-induced pulmonary fibrosis. All the results fulfilled the objective and succeeded in proving the protective effect of S. officinalis from pulmonary fibrosis.

5 Conclusion

Salvia officinalis L. is considered one of the most powerful antioxidants, anti-inflammatory, and antiviral plants for their enrichment of phenolic acids and flavonoids, especially rosmarinic acid and luteolin-7-O-glucopyranisde. Also, we demonstrated that SOEE has a promising effect as an anti-inflammatory, healing the injury of the pulmonary after the viral infection and protecting from lung fibrosis. These effects organize us to expect the relation between SOEE-containing phenolics and flavonoid compounds and cytokines as well as oxidative stress biomarkers, which results in a modification in the inflammatory process associated with lung fibrosis. So, SOEE was suggested as an ideal herbal medicine in the treatment the lung injury and protects the lung from fibrosis.