Effect of Salvia officinalis and S. sclarea on rats with a high-fat hypercaloric diet

Aromatic plant species of the genus Salvia are important medicinal herbs that are highly recommended because of their therapeutic properties. antiseptic, antispasmodic, antimicrobial, antirheumatic, antidiabetic and flatus-relieving (Pop et al., 2016; JakovljeviC et al., 2019; Ghowsi et al., 2020). Species of the Salvia genus have significant antioxidant and antiproliferative properties against tumour cells (Loizzo et al., 2014). Sage antioxidant features are explained by a high level of phenolic compounds (Hudz et al., 2019).

Abietane diterpenoids (ADs) - compounds synthesized from the roots of various species of Salvia - contain aethiopinone, 1-oxoaethiopinone, salvipisone, and ferruginol, which displayed promising cytotoxic activity toward several lines of human tumour cells, e.g., the breast adenocarcinoma MCF7, HeLa, epithelial carcinoma, prostate adenocarcinoma PC3, and human melanoma A375 (Vaccaro et al., 2020). Aethiopinone, an o- naphto-quinone diterpene, produced from the roots of various Salvia plants, exerted selective cytotoxicity against the A375 melanoma cell line (Vaccaro et al., 2019). Such compounds as ferruginol, salvipisone, and carnosic acid also showed antiproliferative activity (Vaccaro et al., 2019). The antioxidant activity of these extracts was evaluated in vitro using

DPPH (alpha, alpha-diphenyl-beta-picrylhydrazyl), FRAP (ferric reducing antioxidant power) and HAPX (haemoglobin / ascorbate peroxidase activity inhibition) methods (Hanganu et al., 2019). The polyphenolic profile of some Salvia species (S. aetiopsis, S. austriaca, S. sclarea, S. nutans, S. verticilatta and S. nemorosa) was found to have some common elements (caffeic acid, p-cumaric acid, isoquercitrin, hyperoside, luteolin, api- genin) and other rare compounds (chlorogenic acid, caftaric acid, ferulic acid, quercitrin, rutin, quercetin, kaempferol). All studied species of the Salvia genus were confirmed to contain significant amounts of polyphenolic compounds (22.25-118.75 mg GAE/g dry weight material plant) and could be used as a valuable resource of natural antioxidants together with S. officinalis, the most known one (Francik et al., 2020).

It was found that extracts from collected plants were effective antioxidants in three different in vitro assays (DPPH, ABTS, FRAP and superoxide anion scavenging activity) (Vergine et al., 2019). Therefore, they can be used as natural ingredients in herbal medicines, functional foods, or as bioactive molecules sources. phytotherapy herbal animals

Common sage (S. officinalis L.) is a well-known medicinal plant that is cultivated in several European countries. In sage, leaves or flowering tops are used as raw materials. The value of sage as a medicinal herb lies in its essential oil, which contains D-a-pinene, cineole (about 15%), a- and Я-thujone, D-bomeol and D-camphor. The leaves also contain alkaloids, flavonoids, tannins, oleanolic and ursolic acids, and the fruits contain 1925% fatty oil, represented mainly by linoleic acid glycerides. Having confirmed antioxidant properties, S. officinalis was used as a reference plant (Miliauskas et al., 2004). Free radical bounding capacities of extracts and their total content of phenolic compound were observed to correlate.

Clary sage (S. sclarea L.) is an equally important medicinal herb of this genus. It is a widespread feld crop used for essential oil production (Cui et al., 2015; El-Gohary et al., 2020; Grigoriadou et al., 2020; Tutto- lomondo et al., 2020). Essential oil of clary sage is isolated from the fo- wers and leaves. The plant is cultivated in Moldova, Russia, Bulgaria, France, Germany and other countries. Currently, there are micropropagation techniques to industrially produce clonally stable clary sage plants that would be used for commercial purposes (Erisen et al., 2020).

The essential oils of S. sclarea have been known since Ancient Greece (Pitarokili et al., 2002). Sixty six compounds have been identified, which is 93.3-98.2% of oils, their main compounds are linalyl acetate (19.8-31.1%), linalool (18.5-30.4%), geranyl acetate (4.5-12.1%), and a-terpineol (5.1-7.6%). There are two types of aromatic products - sage concrete and sage absolute. The essential oil of varies in composition depending on the sage's growing conditions and is determined by a wide variety of environmental factors (Dogan, 2020). It was found that the rate of essential oil in the plant grown without boron was 0.11%, while in the plant grown with pure boron it was 0.44% (Karayel 2020). Therefore, pure boron has a positive effect on the ratio of essential oil and components of clary sage.

The volatile oil compounds of S. sclarea and S. officinalis were identified to be relatively richer in terpenes but the amount of volatile oil depended on ecological factors (Karayel & Akcura, 2019).

While seed oil of S. officinalis could be categorized as oleic-linoleic oil, the fatty acid prevailing in S. sclarea was linolenic acid (around 54%). In both seeds and oils, main isomers among tocols were y-tocopherol and a-tocopherol. The concentration of carotenoids accounted for about 0.75 mg/100 g of seeds and 0.16 mg/100 g of oils, lutein being prevailent. Antioxidant potential exerted by oil from S. officinalis and its seeds was greater than such of investigated samples of S. sclarea, possibly because of larger amounts of overall vitamin E and carotenoids (Zivkovic et al, 2017).

Effects of essential oil from S. sclarea on pulse, blood pressure and mood were analyzed on healthy female and male humans (Mitic et al., 2020). The pulse rate decreased more significantly in women than in men. In general, these effects may depend on the method of application (inhalation or dermal application) and sex. Clinical studies confirmed that oil from S. sclarea is effective in reducing stress, depression and as analgesic agents in primary dysmenorrhea (Mahboubi, 2020). Studies have confirmed that clary sage has antioxidant, antimicrobial, cytotoxic and antiinflammatory effects.

By applying S. sclarea topically as part of aromatherapy, the severity of menstrual cramps was effectively reduced (Han et al., 2006). Therefore, women who experience menstrual cramps or dysmenorrhea may be offered aromatherapy as part of their nursing care (Han et al., 2006). In aromatherapy, oil from clary sage is used for women during their menstrual cycle, childbirth and menopause (Mahboubi, 2020).

Essential oil from S. sclarea damages the cell membrane and changes its permeability, thereby leading to the release of macromolecular substances of cytoplasm such as ATP and DNA. The antimicrobial effect of essential oil from S. sclarea is a result of a series of events occurring on the cell surface and within the cytoplasm (Cui et al., 2015).

Abietane diterpenoids isolated from culture of S. sclarea transformed roots inhibited pathogenic microorganisms as Acanthamoeba spp. (Kuzma et al., 2015). Microorganisms of the Acanthamoeba genus are well known pathogens of different diseases like granulomatous amoebic encephalitis (GAE), chronic progressive disease of the central nervous system, amoebic keratitis (AK), chronic eye infection, amoebic pneumonitis (AP), chronic lung and skin infections. The activity of Acanthamoeba was notably inhibited by ferruginol. This component produced 72% growth inhibition of Acanthamoeba in a 3-day exposure period (IC₅₀ 17.45 pM), whereas such activity of aethiopinone and 1-oxo-aethiopinone was only at the level of 55-56% (Kuzma et al., 2015). Essential oil S. sclarea was observed to have notable antifungal activity (Fraternale et al., 2005). 2,3-Dehyd- rosalvipisone, sclareol, manool, 7-oxoroyleanone, spathulenol and caryo- phyllene oxide were active against Staphylococcus aureus, the first and third components exerted significant action toward Candida albicans, while the last compound was active against Proteus mirabilis (Ulubelen et al, 1994). Lately, the antimicrobial and fungicidal effects of S. sclarea essential oil has found application in other areas of life. Results of the recent studies showed high fungicidal efficacy of S. sclarea for the packaging materials (Kostova et al., 2020). During the investigated shelf life period, inhibition of Candida albicans accounted for around 100%. Suppression of C. albicans during the investigated shelf life of the package accounted for 100%. Recycled paper showed a high efficiency against Aspergillus brasiliensis (81.9-99.2%). Bactericidal effect of the studied packaging materials processed with sage oils was higher against Escherichia coli and lower against Gram-negative bacterium Salmonella abony (Kostova et al., 2020). Therefore, clary sage essential oil could be used as an antimicrobial agent in the food industry because of its good antimicrobial features so as to improve products' quality and extend their shelf life.

After the essential oil is obtained from the raw material of clary sage, sclareol is isolated from the green parts of the plant - diterpene alcohol, a representative of terpenoids. Methanol was experimentally selected as the optimal solvent for ultrasound-assisted extraction of phenolic compounds from these plants (Jasicka-Misiak et al., 2018). In glandular trichomes of S. sclarea, bicyclic diterpene (-)-sclareol is accumulated (Schmiderer et al., 2008). In the flavours and fragrances industry, it is also used as the raw material for synthesis of Ambrox, a synthetic analogue of ambergris (Gunnewich et al., 2013).

Acetone extract of S. sclarea contains nine diterpenes: sclareol ma- nool, salvipisone, ferruginol, microstegiol, candidissiol and 7-oxoroylea- none, 2,3-dehydrosalvipisone and 7-oxoferruginol-18-al. Furthermore, the plant was found to contain two sesquiterpenes, caryophyllene oxide, spa- thulenol, alpha-amyrin, beta-sitosterol and the flavonoids apigenin, luteolin, 4'-methylapigenin, 6-hydroxyluteolin-6,7,3',4'-tetramethyl ether, 6-hyd- roxyapigenin-7,4'-dimethyl ether (Ulubelen et al., 1994).

The methanol extracts of S. sclarea inhibited the growth of all tested bacterial strains, also this plant had a prominent antioxidant activity (Firuzi et al., 2013). Sclareol caused inhibition of prostaglandin (PG) F-2 alpha-, oxytocin-, acetylcholine-, carbachol-, KCl-, and Bay K 8644-induced uterine contraction and led to an analgesic effect in the writhing test (Wong et al., 2020). Sclareol takes effect on Ca²⁺ level and mediates oxytocin receptor (OXTR), myosin light chain kinase (MLCK), extracellular signal-regulated kinase, p-p38, cyclooxygenase-2 (COX-2), and phospho- myosin light chain 20 (p-MLC20) protein expression. Summarizing these results, sclareol is a promising alternative supplement in treatment of dysmenorrhea (Wong et al., 2020).

The strongest antioxidant capacity among 5 studied species of Salvia was exhibited by extract from S. officinalis (Pop et al., 2016). Extracts from S. sclarea and S. officinalis displayed antibacterial activity toward Bacillus cereus, Staphylococcus aureus and Pseudomonas aeruginosa. Nonetheless, research has revealed resistance of Salmonella typhimurium to extract of S. sclarea and resistance of Escherichia coli to the extract of S. officinalis (Pop et al., 2016; Zazharskyi et al., 2019).

Intraperitoneally injected sclareol was studied for its effects that reduce the tumour volume and shift the cytokine profile. Also, there has been an investigation of whether injection of sclareol intraperitoneally can improve the outcome of cancer therapy by suppressing the regulatory T- cells (Noori et al., 2013). The results demonstrated significant decrease in tumour size. Moreover, in the group that was intraperitoneally injected with sclareol, the level of IL-4 significantly dropped and the level of IFN- gamma increased. An assay of cell proliferation also revealed that the splenocytes of the experimental animals significantly increased. Noori et al (2013) proposed that sclareol can enhance cancer therapy as an immu- nostimulator by reducing regulatory T-cells (Tregs) frequency and size of tumour. Sclareol also demonstrated immuno-therapeutic efficacy after direct intra-tumoral injection.

According to Fiore et al. (2006), there was potential antitumoral activity of S. sclarea extracts, determined by MTT test, on nine human cancer cell lines: glioblastoma (DBTRG-05MG, T98G, U-87MG), colorectal adenocarcinoma (WiDr and HT-29), prostate adenocarcinoma (MDA Pca2b), choriocarcinoma (JEG-3), endometrium adenocarcinoma (HEC-1A) and B lymphoblast (CIR). Extracts of S. sclarea also demonstrated cytotoxic activity to various degrees depending on the type of cell line (Fiore et al., 2006). The results confirmed that Salvia may be considered as a natural source of potential antitumor agents (AlMotwaa et al., 2020).

There were four diterpenoids isolated from transformed roots of S. sclarea, namely ferruginol, salvipisone, aethiopinone and 1-oxoaethi- opinone (Rozalski et al., 2006). Determining caspase-3 activity showed that salvipisone and aethiopinone could induce apoptosis in time- and concentration-dependent ways. Thus, aethiopinone and salvipisone diterpenoids from S. sclarea could be useful in treating human cancers, specifically in the drug-resistant cases (Rozalski et al., 2006).

The lectin isolated from seeds of S. sclarea (SSL) recognizes the Tn- antigen (GalNAc alpha 1 -> Ser/Thr), which is expressed in some human carcinomas (Wu, 2005). This was confirmed by Medeiros et al. (2000), whose studies found that SSL from S. sclarea seeds recognized the Tn- antigen (GalNAc alpha-O-Ser/Thr), a specific marker of many human carcinomas. SSL is an acidic (pi 5.5), 60-61 kDa dimeric glycoprotein that is composed of identical subunits, interlinked by a single disulfide bond.

Specificity of lectin from S. bogotensis seeds and its high affinity for the Tn-antigen, commonly found in tumour cells, is making this protein a useful element for immunohistochemical and cellular studies (Vega & Perez, 2006).

The antiviral effect of sclareol also has a significant potential. It is well known that filoviruses can trigger severe hemorrhagic fever in humans. So far, the most contagious filovirus is Ebola virus (EBOV). Sclareol and sclareolide exerted inhibitory activity toward all tested filoviruses, indicating their broad range of activities against filoviruses (Chen et al., 2020).

Inhibitory effect of sclareol was investigated on osteoclastogenesis and progression of postmenopausal osteoporosis (Jin et al., 2019). Sclareol was seen to have a protective effect against bone loss in the conditions of ovariectomy-induced mouse model. Jin et al. (2019) results suggested that sclareol is a potentially valueable therapeutic agent during postmenopausal osteoporosis.

Traditionally, S. sclarea is used as an agent against gingivitis, stomatitis and aphthae (Kosti et al., 2017). Treatment with S. sclarea extract significantly lowered the inflammation process by decreasing the levels of IL-1 beta, IL-6 and TNF-alpha, reducing lesions in the gingival tissue and preserving alveolar resorption of the bone, compared with the untreated group of the rats (Kosti et al., 2017). Extract from S. sclarea showed an anti-inflammatory effect in periodontitis induced by E. coli lipopolysaccharide, suggesting that it could be used as a therapeutic agent in treating periodontal diseases. There was increase in the inhibition depending on time of contact. Essential oils from S. sclarea can demonstrate better results when used in bioadhesive formulations that would take peripheral analgesic and anti-inflammatory actions on a local level (Peana et al., 1999).

Diffusion of essential oil of S. sclarea and its infiltration through porcine buccal mucosa was has been considered useful in stomatology. Compared to the other compounds, the ones having terpenic structure penetrated through the porcine buccal mucosa best (Ceschel et al., 1998).

Strongyloides papillosus (Wedl, 1856) is a common nematode of ruminants, and 24 hour-influence of S. sclarea at different doses had significant effects on the survival of invasive and noninvasive types of larvae (Boyko & Brygadyrenko, 2016). Aqueous extracts of S. sclarea inflorescences killed 50% of S. papillosus invasive larvae in 464 +/- 192 mg/L concentration and non-invasive larvae in 327 ± 186 mg/L concentration (Palchykov et al., 2019).

Saivia sclarea is a rich source of terpenoid compounds and may be used as a bio-based pesticide in nature (Bozok & Ulukanli, 2016). In the phytotoxicity assay, essential oil from S. sclarea showed significant dose dependent potential toxicity on Lactuca saliva L., Lepidium sativum L. andPortulaca oleracea L. (Bozok & Ulukanli, 2016).

Therefore, the objective of the study was the effect of the herbs on the organism of laboratory rats on a high-fat diet during a 30-day experiment.

Materials and methods

Results

The addition of S. officinalis dry shoots to the male rats' diet notably increased the body weight of the animals compared with the control group: the body weight in the control group increased to 111.5% of the initial, and in the S. officinalis group - up to 130.8% in 30 days of the experiment (Fig. 1a). The body weight of rats that had consumed diet supplemented with dry crushed shoots of S. sclarea was only 103.8% of their initial weight (Fig. 1b), that is, it was significantly lower than in the control group.

Fig. 1. Change in the rats' body weight when chopped shoots of Salvia officinalis L. (a) and S. sclarea L. (b) were added to their diet: the abscissa is the day of the experiment, the ordinate is the body weight of the animals (% relative to the initial body weight before the start ofthe experiment, taken as 100% for each of the experimental animals); small square - median, upper and lower borders ofthe rectangle - 25% and 75% quartiles, vertical line - minimum and maximum values, circles - outliers; n = 8

Rats that consumed dry crushed shoots of S. officinalis were observed to have insignificantly (at the tendency level, P > 0.05) reduced food intake to 85.3% of the control group level (Table 1). Animals that were fed on diet supplemented with S. sclarea shoots did not change feed intake, but increased water intake at the level of the trend to 116.9% of the level of the control group. With a change in the daily body weight gain in animals, there were multidirectional changes as compared with the control group (Table 1): S. officinalis caused an increase in weight gain up to 1771 pg/day (253.1% of the control group), and S. sclarea - a decrease in body weight gain to 194 pg/day (27.8% of the control group). Moreover, such abrupt changes in body weight in animals did not affect the abdominal circumference, which did not differ from the control group.

Table 1

Change in the body weight and food consumption ofyoung male rats under the influence of Salvia officinalis L. and S. sclarea L. addition to their diet (x ± SD, n = 8, duration of experiment 30 days)

Note: different letters indicate values that differed one from another reliably within one line of the table according to the results of comparison using the Tukey test with Bonferro- ni correction.

Table 2

Change in organs' relative mass (%) of male rats under the influence of Salvia officinalis L. and S. sclarea L. supplementation to their diet (x ± SD, n = 8, duration of experiment 30 days)

The overall increase in the body weight in the group consuming S. officinalis led (Table 2) to a significant decrease in the relative weight of the brain (to 77.7% compared with the control group), spleen (to 80.4%) and thymus (to 59.1%). After consumption ofS. sclarea, the relative weight of the thymus in male rats also reduced (to 43.4% of the control), but, at the same time, there was a significant increase in ther relative weight of the colon (up to 159.7% compared with the control group, Table 2). No other significant changes in the relative mass of the organs in animals from experimental groups were recorded.

Change in blood biochemical parameters of male rats fed on S. officinalis manifested itself in a decrease of the urea concentration (78.3% of control), total bilirubin (up to 59.5%), triglycerides (up to 67.1%), a moderate increase of the total protein in blood plasma (up to 109.8%) and a drastic increase in alkaline phosphatase activity (up to 435.1% of control, Table 3). There were no significant changes compared to the control group in other biochemical blood parameters that we studied in the S. officinalis group.

Consuming S. sclarea shoots in the composition of feed by rats also caused an increase in the alkaline phosphatase activity in blood plasma (however, weaker than in the previous group - only up to 276.5% of control, Table 3). More prominent changes in the group of animals eating S. sclarea were observed in fat metabolism: the index of atherogenicity decreased sharply - to 23.1% of the control group level (Table 3). This was due to a sharp increase in the concentration of high density lipoprotein cholesterol (up to 286.9% of the level of the control group) and an insigni ficant (at the tendency level, P > 0.05) decrease in the concentration of low density lipoprotein cholesterol (up to 67.7% of the level of the control group). Also, in the group of rats that were feeding on S. sclarea, an increase in the concentration of cholesterol (up to 128.5% of the control group) and a strong decrease in the concentration of triglycerides (up to 39.9% of the control, more pronounced than in the group of S. officinalis) were recorded. In addition, there was a decrease in gamma-glutamyl transferase activity in the blood of animals that consumed S. sclarea (up to 62.8% of the control group) and an increase in the Ca/P ratio (up to 132.5% of the control group). Other investigated blood biochemical parameters in the group of animals that consumed S. sclarea did not show significant differences (Table 3).

Under the influence of the S. officinalis and S. sclarea chopped shoots added to the diet of rats, there were no significant changes in complete blood count (CBC) and white blood cell (WBC) differential of male rats (Table 4).

Physical activity (Fig. 2a) in male rats fed S. officinalis significantly decreased by the end of the experiment; the addition of S. sclarea to the diet of animals caused an insignificant decrease (at the tendency level, P > 0.05) in the physical activity of rats. The effect ofS. officinalis also significantly decreased the orientational activity in animals (Fig. 2b). On the other hand, the emotional status (Fig. 2c) of rats on the diet with S. officinalis did not significantly change, while eating dry crushed shoots of S. sclarea as part of the compound feed significantly reduced the emotional status of experimental animals. Note: see Table 1.

Table 3

Change in blood biochemical parameters ofmale rats under the influence ofS. officinalis L. andS. sclarea L. (x ± SD, n = 8, duration of experiment 30 days)

Note: see Table 1.

Table 4

Change in CBC and WBC differential of male rats under effect of addition to S. officinalis L. and S. sclarea L. (x ± SD, n=8, duration of experiment 30 days)

Note: no statistically significant changes were found between samples.

There were no significant changes in the open field test between the groups and within groups at the beginning and the end of the experiment (Table 5), except for the group of rats that ate dry shoots of S. officinalis according to the number of peripheral squares that they visited (decrease from 24.9 ± 7.8 to 6.1 ± 6.6).

Discussion

In modern scientific medicine, sage herb is used very broadly. Scientific studies have revealed antitumour, anti-inflammatory, analgesic, antioxidant, antibacterial, antimutagenic, hypoglycemic, neuroprotective, hypolipidemic properties of sage extracts (Hamidpour et al., 2014; Monsefi et al., 2015, 2017). Sage contains essential oil, coumarins, alkaloids, flavonoids, saponins, sclareol, rosmarinic acid, dihydroquercetin, rutin, couma- rin, umbelliferone, gallic, chicoric and ferulic acids, salts of K, Ca, Fe, Mn, Zn, Li, Sn, and also identified polyphenols, phenolic and flavonoid glycosides. Of the phenolic substances, glycosides of luteolin (42%) and apige- nin (27%) were identified, the main volatile compounds of the extract weie a-thujone (32.3%), camphor (29.7%), 1,8-cineole (6.2%) and hu- mulene (5.1%).

Fig. 2. Changes in the motor (a) and orientational activity (b), as well as the emotional status (c) of male rats when Salvia officinalis L.

and S. sclarea L. chopped shoots were added to their diet: on the abscissa - groups of animals (n=n * n₂ = 32: n = 8 animals, n₂ = 4 experiments

with each animal) on a high fat diet with and the addition of shredded herbal shoots (the days after the start of the experiment are indicated in parentheses:

1--4th or26-30th), on the ordinate axis - the absolute number of markers of this behaviour type in 120 seconds ofthe experiment: for physical activity -

the number of visited squares of the open field, for orientational activity - the number of standings upright, for emotional status - the number

of grooming actions, defecation and urination; small square - median, upper and lower borders ofthe rectangle - 25% and 75% quartiles,

vertical line - minimum and maximum values, circles - outliers; different letters within each figure indicate significant differences

between groups (P < 0.05) according to the results of the Tukey test

Bioactive hydrophobic diterpene in the composition of the essential oil from S. sclarea is sclareol, it is broadly studied for its anti-inflammatory and antioxidant effects (Cerri et al., 2019). Cerri et al. (2019) did a very similar study to the one we did, where one of the randomly composed groups of Swiss male mice was fed standard diet (STD) and the other one received high-fat diet (HFD). After obesity in the mice had been induced, three treatment groups were made out of each of the two: free sclareol (Sc), sclareol-loaded lipid nanoparticle (L-Sc) and empty lipid nanoparticle (L). Treatments were conducted every day for the period of 30 days. The study revealed that L-Sc had positive effects on metabolic profiles in obese mice by decreasing adiposity, improving insulin sensitivity, tolerance to the glucose and increasing the HDL plasma levels. Furthermore, L-Sc caused decreases in the expressions of NF-KB, MCP-1 and SERBP-1. Combination treatment using sclareol and lipid nanocarriers, which can reduce adipose tissues, could be effective against metabolic disorders.

In our study, the two sage species showed the opposite effect on weight gain rats that received high-fat diet. Thus, the addition of S. officinalis dry crushed shoots led to sharp increase in the rats' body weight during the 30-day experiment, while the addition of S. sclarea, on the contrary, slowed down the weight gain both in comparison with the control and the first experimental group. This fact is of interest, since at the same time a decrease in food intake was observed in the group of rats that consumed S. officinalis, in comparison with the control group and animals that consumed S. sclarea.

An indicator that is often used as a sensitive index for assessing the toxic effects of various substances, including medicinal ones, is the mass of the organs (Balogun et al., 2014; Varcholyak & Gutyi, 2019). Increase in the total body weight against the background of high-fat diet is accompanied by increase in the absolute mass of the internal organs, while the relative mass may not always increase. In our experiment, the intake of sage led to significant decrease in the relative mass of the immune system organs (thymus and spleen), but no changes in the composition of the blood (WBC count, WBC differential) were found. In the studies by Tkachuk & Shapoval (1987), it was also indicated that S. sclarea essential oils affects the immune system, as well as the fermentative activity of the blood. In our experiment, we also observed change in the enzymatic activity of the rats' blood when chopped sage was added to the high-fat diet. Thus, the consumption of S. officinalis caused a strong increase in the activity of alkaline phosphatase, and consumption of S. sclarea caused moderate increase, while the activity ofAST and ALT did not significantly differ from those of the control animals. Also, in comparison with the control group, the rats of the first experimental group were observed to have insignificant decrease in the activity of gamma-glutamyl transferase, whereas in the second group this parameter significantly and reliably decreased; this indicator was within the reference values in all the groups.

Table 5

Change in behavioural characteristics of three rats groups during 2 minutes of the experiment, in which diet Salvia officinalis L. and S. sclarea L. was added (x ± SD, n = 32, duration of the experiment was 30 days)

Note: there were no significant diffйrences between the groups for most of the parameters studied; differences in the number of visited peripheral squares are indicated by different Latin letters (P < 0.05) according to the Tukey test results with Bonferroni correction.

In studies that were carried out on broiler chickens, sage powder of S. officinalis was added to the diet in the doses of 4 and 8 g/kg, its effect on the immune organs and blood biochemical parameters was indicated. Such a diet did not affect the ratio of albumin and globulins and the ratio of granulocytes and blood lymphocytes, as well as the concentration of cholesterol, HDL and LDL. At the same time, a high concentration of the sage powder (8 g/kg) significantly increased the relative mass of the cloacal bursa in poultry, and also led to a decrease in the concentration of total protein and triglycerides in the blood serum (Toghyani et al., 2012).

A study by Ekin et al. (2019) focused on antialzheimer, antidiabetic, antioxidant and antiobesity activities of ethanol extracts from S. sclarea. Leaf extract of S. sclarea significantly inhibited butyrylcholinesterase (51.76 ± 1.04%). The Salvia species demonstrated the highest total antioxidant activity. Extracts from flowers of S. sclarea (1.34 ± 0.08) and leaves (1.34 ± 0.08) exihibited high ferric-reducing antioxidant power. Extracts of S. sclarea displayed notable inhibitory activity and are considered possible sources ofalpha-glucosidase inhibitors (Ekin et al., 2019).

The most active chemotype of S. sclarea was determined by monitorring the acute and subchronic antidiabetic activities of essential oils along with linalool and linalyl acetate. Plants of chemotypes 1 (linalool content), which grow on low altitudes in Lebanon and Poland, was seen to have significantly higher acute and subchronic antidiabetic activities compared with chemotype 5 (high linalyl acetate content). Essential oils from S. sclarea demonstrated potential antidiabetic activities, and may be used as a complementary or alternative treatment of diabetes and complications that accompany it (Raafat & Habib, 2018; Pereira et al., 2019; Salehi et al., 2020; Abu-Odeh & Talib, 2021).

In studies by Agadzhanyan (2015) on human blood serum with a glucose concentration of 10.0 mmol/L and a total cholesterol level of 8.2 mmol/L in vitro, the hypoglycemic and hypolipidemic activity of S. officinalis leaf extracts was investigated. The results revealed both aqueous and alcoholic extracts of the studied plant being effective in reducing glucose concentration and serum cholesterol levels (Bassil et al., 2015). In studies on mice with diet-induced obesity, ethanolic sage extract caused rosiglitazone-like effect, improved insulin sensitivity, inhibited adipocyte lipogenesis, and reduced inflammation (Ben Khedher et al., 2018).

In our study, in rats on a high-fat diet, the glucose level was at the upper level ofthe reference range, and the addition of sage to the feed led to a slight and insignificant decrease in the blood glucose. At the same time, the indicators of fat metabolism in animals were significantly altered by the consumption of S. sclarea. Thus, in comparison with the control group, the rats of the second experimental group (that consumed S. sclarea) had an increase in cholesterol levels, a sharp increase in the concentration of high-density lipoproteins and a decrease in low-density lipoproteins, which led to a sharp significant decrease of the atherogenic index.

In our previous study (Zazharskyi et al., 2019), we observed a high activity of an alcoholic tincture of the S. sclarea against Escherichia coli and Proteus vulgaris, and, at the same time, a very low activity against Salmonella typhimurium, Klebsiella pneumoniae, Listeria monocytogenes, Corynebacterium xerosis and fungus Candida albicans.

A study (Yang et al., 2014) of efficiency of S. sclarea in treatment of endothelial dysfunction induced by chronic immobilization stress in rats demonstrated that treatment with 5%, 10%, and 20% S. sclarea significantly decreased systolic blood pressure. Treatment with 20% S. sclarea also significantly reduced the heart rate compared with the group that had chronic immobilization stress (Yang et al., 2014). Also, S. sclarea caused decreases in corticosterone (10% and 20%) and malondialdehyde (10%, 20%) in serum. Similar results were observed with nifedipine. Moreover, 20% S. sclarea provoked significant increases in nitric oxide production and eNOS expression level and relaxed aortic rings in rats suffering stress from chronic immobilization (Yang et al., 2014). By increasing production of NO and eNOS level, as well as decreasing oxidative stress, treatment of rats that were under immobilization stress with S. sclarea led to their recovery from endothelial dysfunction. Therefore, treatment of endothelial dysfunction with appropriate S. sclarea concentration could lead to recovery. These data indicate that oil from S. sclarea may be efficient in prophylaxis and treatment of stress-induced cardiovascular diseases (Yang et al., 2014).

In studies by El-Shafeial et al. (2013), against the background of pathological and toxic changes in the body of mice caused by the action of carbon tetrachloride, the use of S. officinalis oil before and after carbon tetrachloride had a levelling effect on the state of the liver and kidneys, and there was also a tendency towards restoration of their functions. At the same time, the preventive use of oils was less effective. Essential oil isolated from S. sclarea inhibited growth of HeLa cells with an IC50 of 80.69 = 0.01 pg /mL. Staining with propidium iodide (PI) showed the presence of apoptosis in cells that weie treated with oil (Durgha et al., 2016). Treatments with essential oil from S. sclarea in different concentrations reduced inflammation (Durgha et al., 2016). However, histopatho- logical assay of the liver tissue treated with 1.5 pg lipopolysaccharide (LPS)/30 g body weight of BALB/c mice revealed severe inflammation and necrosis. Hepatocytes' necrosis and infiltration were barely seen after using 100 pg of essential oil ofS. sclarea (Durgha et al., 2016).