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

Phytotherapy for the correction of excess body weight is widely used. However, a comprehensive study of herbal preparations on the organism of model animals has been carried out only for a few plant species. Supplementing the diet of rats with closely related sage species (Salvia officinalis L. and S. sclarea L.) against the background of high-fat hypercaloric diet triggered multidirectional changes in their metabolism. The addition of crushed dry shoots of S. officinalis to the diet of animals led to a sharp increase in their body weight (up to 130.8% of the initial one in 30 days of the experiment). The body weight of the rats treated with S. sclarea for 30 days increased only up to 103.8% of their initial weight and was lower than in the control group. Addition of S. officinalis caused an increase in daily weight gain up to 253.1% of the control group, and S. sclarea – its decrease to 27.8% of the daily weight gain in the control group. In the S. officinalis group, the relative weight of the brain, spleen, and thymus decreased, while in the S. sclarea group, the relative weight of the thymus decreased and that of the colon increased. Under the influence of S. officinalis, the concentration of urea, total bilirubin, and triglycerides in the blood plasma of male rats decreased and the concentration of total protein and the activity of alkaline phosphatase increased. While consuming S. sclarea shoots, there was an increase of alkaline phosphatase activity in the rats’ blood, but atherogenic index (23.1% of the level of the control group) sharply dropped due to an increase in the concentration of high-density lipoprotein cholesterol (286.9% of the control) and a decrease in the concentration of low-density lipoprotein cholesterol (67.7% of control). In rats feeding on S. sclarea shoots, we observed a decrease in the concentration of triglycerides in the blood (39.9% of the control), a decrease in the activity of gammaglutamyl transferase (62.8%), and an increase in the Ca/P ratio (132.5% of the control group). No significant changes were observed in CBC and WBC differential of male rats when eating S. officinalis and S. sclarea shoots. According to the results of the open field test, the physical and orientational activity of male rats under the influence of S. officinalis significantly decreased by the end of the experiment. Emotional status of rats, on the contrary, decreased when they ate dry crushed shoots of S. sclarea in the composition of the food. Thus, excess body weight of rats in the conditions of hypercaloric diet led to more pronounced deviations from the norm while consuming dry crushed shoots of S. officinalis. The addition of S. sclarea dry crushed shoots to the animals’ diet normalized the body weight in comparison with the control group, reduced the negative manifestations of obesity at the biochemical and organismal levels. In this regard, the substances that contains S. sclarea should be carefully studied for anti-atherosclerotic activity, and tea supplemented with S. sclarea shoots can be recommended as a corrective supplement in the diet of overweight people.


Introduction
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;Jakovljević 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).
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.
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-α-pinene, cineole (about 15%), α-and β-thujone, D-borneol and D-camphor. The leaves also contain alkaloids, flavonoids, tannins, oleanolic and ursolic acids, and the fruits contain 19-25% 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 field crop used for essential oil production (Cui et al., 2015;El-Gohary et al., 2020;Grigoriadou et al., 2020;Tuttolomondo et al., 2020). Essential oil of clary sage is isolated from the flowers 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 γ-tocopherol and α-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 (IC 50 17.45 μM), 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 caryophyllene 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).
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 Tcells (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 IFNgamma 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 immunostimulator by reducing regulatory T-cells (T regs ) frequency and size of tumour. Sclareol also demonstrated immuno-therapeutic efficacy after direct intra-tumoral injection.
The lectin isolated from seeds of S. sclarea (SSL) recognizes the Tnantigen (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 Tnantigen (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 .
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).
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 sativa L., Lepidium sativum L. and Portulaca 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
The choice of animals for the experiment, research protocols, withdrawal of animals from the experiment were approved by the local ethical committee of the Dnipro State Agrarian and Economic University. The maintenance, nutrition, care for animals and their withdrawal from the experiment were carried out in compliance with the principles described in the "European Convention for the Protection of Vertebrate Animals used for Experiments and for Other Scientific Purposes" (Strasbourg, March 18, 1986, ETS No. 123) and in the order No. 3447-IV from February 21, 2006 "About protection of animals from cruel treatment" (Ukraine).
A control group and two experimental groups of 8 animals in each were formed from 24 adult white outbred male laboratory rats weighing 200 ± 10 g. The rats were housed in polycarbonate cages with steel lattice lids and a stern recess, 4 rats per cage. Rats were kept in a room with a temperature of 20-22 °C and a relative humidity of 50-65%. The light regime was 12 hours of light and 12 hours of darkness. Ventilation was carried out according to the regime. The animals received water ad libitum. The duration of the experiment was 30 days.
The control group of animals was fed a high-fat diet (3600 kcal/kg), which was based on the standard diet (75% grain mixture (corn, sunflower seeds, wheat, barley, soybeans), 8% roots (potatoes, carrots), 2% meat and bone flour, 2% mineral-vitamin complex) with the addition of 15% sunflower oil. Animals in the experimental groups received semi-synthetic high-fat diets supplemented with 5% of medicinal plants. To the diet of the first experimental group,we added 5% of dry shredded shoots of S. officinalis, and the second experimental group was given 5% of dry shredded S. sclarea shoots. Herbal raw material was collected manually, dried without access to direct sunlight in the room temperature. The main ingredients of the diet were ground in a mill (grain, meat and bone meal, mineral-vitamin complex, dry shoots of medicinal plants) and mixed, then sunflower oil was added and granules were made at the rate of 4.200 g for each group for the entire experiment period (30 days). Fresh root crops were additionally given in an appropriate amount every day. The animals had free access to the food. During the experiment, we monitored the amount of food consumed by each group per day and the total amount for the entire period of the experiment.
Morphometric parameters (body weight, abdominal circumference) were determined on the first day and the 30th day of the experiment. The total increase in animal weight and the daily increase in live body weight were calculated Brygadyrenko et al., 2019;Lieshchova et al., 2018Lieshchova et al., , 2019Lieshchova et al., , 2020. The locomotor activity and the emotional status in the experimental rats were studied in the open field test. We used an installation consisting of a square area of 1 m 2 , divided into 16 squares and bounded by an opaque wall 20 cm high. The experiment was carried out in complete silence with intense illumination of the field itself. An experimental rat, was taken from a cage in a previously darkened room, and was placed in the center of the field. The exposure time was 2 minutes. The animals were tested for four days (1-4 days) at the beginning of the experiment and four days at the end (26-30 days). The number of squares that rats crossed was counted: peripheral and central ones -motor activity was assessed; peripheral (with support on the wall) and central (without support on the wall) standings upright squaresorientational activity; the number of acts of grooming, defecation and urination -emotional status (Lieshchova et al., 2021).
The animals were euthanized on the 30th day of the experiment under anaesthesia (80 mg/kg ketamine and 12 mg/kg xylazine, intraperitoneally) by blood withdrawal with cardiac puncture. After autopsy, the state of the internal organs was visually assessed for the presence of pathological changes. The sampling of organs and tissues (heart, liver, lungs, thymus, spleen, stomach, small and large intestines, kidneys, brain) was performed with surgical instruments and they were weighed. The mass of internal organs was determined with the accuracy of 0.01 g.
Blood samples that we collected during euthanasia were subjected to biochemical and morphological studies. Biochemical parameters were determined using an automatic analyzer Miura 200 (I.S.E. Srl, Italy) and with High Technology reagent kits (USA), PZ Cormay S.A. (Poland) and Spinreact S.A. (Spain). The erythrocytes and white blood cells (WBC) count in the rats' stabilized blood was determined using an automatic hematological analyzer BC-2800Vet and Mindray (Lieshchova et al., 2018(Lieshchova et al., , 2020Brygadyrenko et al., 2019). For the white blood cell differential, blood smears were prepared by Pappenheim method with their further staining according to Romanowsky-Giemsa. The total protein was deter-mined using the biuret method; globulins and protein coefficient -calculated; albumin -by reaction with bromcresol green; the activity of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) -kinetic, based on the optical Warburg test; alkaline phosphatase -enzymatic with n-nitrophenyl phosphate; glucose -by glucose oxidase method; ureaenzymatic, kinetic using urease and glutamate dehydrogenase; creatinineby the modified Jaffe method without deproteinization; total bilirubin -by a method based on oxidation in the presence of vanadate as an oxidizing agent; total calcium -by colorimetric method with Arsenazo III, inorganic phosphorus -by direct phosphomolybdate method without deproteinization, gamma-glutamyl transferase -kinetic method with L-gamma-glutamyl-3-carboxy-4-nitroanilide. The concentration of total cholesterol was determined using the colorimetric, enzymatic method with esterase and cholesterol oxidase; triglycerides -by colorimetric, enzymatic methods with glycerophosphoric oxidase; HDL and LDL -by direct enzymatic method, the atherogenic index were calculated. From biochemical markers of inflammation, C-reactive protein was determined by the method of highly sensitive latex-enhanced immunoturbidimetry.
All the data were analysed using Statistica 8.0 software (StatSoft Inc., USA). Results in the tables are demonstrated as x ± SD (mean ± standard deviation). Differences between the control and experimental groups values were determined using the Tukey test (with consideration of Boniferroni's correction), where the differences were considered significant at P < 0.05.

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.
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 (  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 of the experiment, taken as 100% for each of the experimental animals); small square -median, upper and lower borders of the rectangle -25% and 75% quartiles, vertical line -minimum and maximum values, circles -outliers; n = 8 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 of S. 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 of S. 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.

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)     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).
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.  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 1 * n 2 = 32: n 1 = 8 animals, n 2 = 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 or 26-30th), on the ordinate axis -the absolute number of markers of this behaviour type in 120 seconds of the experiment: for physical activitythe 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 of the 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 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 of AST 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) 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).
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 of the 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 , 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 IC 50 of 80.69 = 0.01 µg /mL. Staining with propidium iodide (PI) showed the presence of apoptosis in cells that were treated with oil (Durgha et al., 2016). Treatments with essential oil from S. sclarea in different concentrations reduced inflammation (Durgha et al., 2016). However, histopathological assay of the liver tissue treated with 1.5 µg 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 µg of essential oil of S. sclarea (Durgha et al., 2016).
The influence of sage and medicinal preparations made with it on the nervous system attracts considerable interest of researchers (Cavalcante et al., 2018;Dinel et al., 2020). Ther study revealed significant neuroprotection in the MTT assay by 39.5% and inhibition of intracellular ROS by 61.4% caused by eighty percent methanol extracts of S. sclarea in 100 µg/mL concentration (Tavakkoli et al., 2014). In the future, researchers may discover new natural therapeutic agents for treating disorders such as Alzheimer and Parkinson diseases (Tavakkoli et al., 2014). A group of female patients exposed to vapours of S. sclarea essential oil were observed to have a significant decreases in systolic blood pressure, diastolic blood pressure and respiratory rate compared with the control (Seol et al., 2013). Inhaling S. sclarea essential oil statistically significantly reduced the respiratory rate.
Salvia sclarea oil contains two times more omega-3 fatty acids than other Salvia species, which take part in eicosanoid synthesis and contain significant levels of monoterpane linalool, a psychoactive ingredient (Gross et al., 2013). In dominant mice, pregnant mothers of which received S. sclarea oil-enriched food from the date of conception, dominant and anxiety behaviour significantly decreased, compared with their counterparts treated with sunflower oil. A similar tendency and a prominent reduction in blood corticosterone levels were seen in submissive mice treated with S. sclarea oil (Gross et al., 2013). These data and findings confirm the hypothesis that S. sclarea oil has anxiolytic properties.
In the study, where rats were intraperitoneally injected or inhaled essential oils (Seol et al., 2010), 5% (v/v) S. sclarea oil had the strongest antistress effect in rats during the forced swim test. However, pre-treatment with buspirone (a 5-HT1A agonist), SCH-23390 (a D-1 receptor antagonist) and haloperidol (a D-2, D-3, and D-4 receptor antagonist) significantly blocked the anti-stress effect of S. sclarea oil (Seol et al., 2010). The antidepressant-like effect taken by oil from S. sclarea oil is related to the modulation of the dopaminergic pathway.
In our study, the behavioural responses of rats from the experimental groups which consumed dry crushed sage shoots with high-fat diet had changed by the end of the experiment compared with the control group. Thus, consuming S. officinalis caused only significant decrease in rats' physical activity. At the same time, S. sclarea caused decrease of physical activity at the level of tendency, significant decrease in orientational activity, and decrease in the emotional status of experimental animals. Therefore, further research will be aimed at studying the effects of different doses of extracts from these medicinal plants (S. officinalis and S. sclarea) on the organism of laboratory animals during various metabolic disorders at the tissue, cellular and subcellular levels.

Conclusions
The addition of closely related sage species from the same genus (Salvia officinalis L. and S. sclarea L.) to the food of rats caused multidirectional changes in their metabolism. The addition of S. officinalis to the diet led to sharp increase in body weight (up to 130.8% of the initial weight for 30 days of the experiment). The body weight of the rats that had been treated with S. sclarea increased only by 103.8% of their initial weight and was lower than in the control group. Oil from S. officinalis increased daily weight gain up to 1771 µg/day (253.1% of the control group), and S. sclarea -decreased it to 194 µg/day (27.8% of the control group). In the group that had received S. officinalis, the relative weight of the brain (77.7% of control), spleen (80.4%), and thymus (59.1%) decreased. In the group treated with S. sclarea, the relative weight of the thymus decreased (43.4%) and the relative weight of the colon increased (159.7% to the control group).
Under the influence of S. officinalis, the concentrations of urea (78.3%), total bilirubin (59.5%) and triglycerides (67.1%) decreased, and the concentration of total protein (109.8%) and the activity of alkaline phosphatase (435.1% of control) increased. Consumption of S. sclarea shoots increased the activity of alkaline phosphatase (276.5%), drastically decreases the atherogenic index (23.1% of the level of the control group) as a result of increasing the concentration of high density lipoprotein cholesterol (286.9%) and decreasing the concentration of low density lipoprotein cholesterol (67.7%). Also, in rats that had received S. sclarea, we observed increase in the concentration of cholesterol (128.5%) and decrease in the concentration of triglycerides (39.9%), decrease in the activity of gamma-glutamyl transferase (62.8%) and increase in the Ca/P ratio (up to 132.5% of the control group). No significant changes were observed in CBC and WBC differential of male rats that consumed S. officinalis and S. sclarea shoots.
According to the results of the open field test, the physical and orientational activity of male rats under the influence of S. officinalis significantly decreased by the end of the experiment. Emotional status of rats, on the contrary, decreased when they ate S. sclarea dry crushed shoots in the composition of their food.
Thus, excess body weight against the background of hypercaloric diet in rats led, in general, to more pronounced deviations from the norm when the animals ate S. officinalis dry crushed shoots. And vice versa, the addition of dry crushed shoots of S. sclarea to the diet of animals normalized body weight in comparison with the control group, and reduced the negative manifestations of obesity at the biochemical and organismal levels. According to these results, the substances contained in S. sclarea should be carefully studied for anti-atherosclerotic activity, and tea supplemented with S. sclarea shoots can be recommended as a corrective supplement in the diet of overweight people.