Sagan Dalya Tea, a New “Old” Probable Adaptogenic Drug: Metabolic Characterization and Bioactivity Potentials of Rhododendron adamsii Leaves

Adams’ rhododendron (Rhododendron adamsii Rehder) or Sagan Dalya tea is a famous Siberian evergreen medical plant of the Ericaceae family used in traditional medicines of Buryats, Yakuts, and Mongols as a tonic, stimulant, and adaptogenic drug. The high popularity of R. adamsii coupled with poor scientific knowledge prompted the addressing of gaps related to metabolic and biomedical data of Sagan Dalya tea. The application of solid-phase extraction and liquid chromatography-mass spectrometric techniques for the metabolomic study of R. adamsii leaf extracts resulted in the identification of more than 170 compounds, including carbohydrates, organic acids, simple phenol glycosides, triterpene glycosides, flavonoids, prenylated phenols, benzoic acid derivatives, hydroxycinnamates, dihydrochalcones, catechins, and procyanidins, most of which were identified for the first time in the plant. Extended surveys of the seasonal content of all detected compounds prove that specific metabolite variations reflect the bioactivity of R. adamsii extracts. Regarding in vitro methods, the expressed antioxidant potential of R. adamsii extracts was investigated via radical-scavenging, nitric oxide scavenging, and ferrous (II) ion chelating assays. The animal-based swimming to exhaustion test demonstrates the stimulating influence of R. adamsii extract on physical performance and endurance, concluding that the drug could act as an adaptogen. Thus, Sagan Dalya tea (R. adamsii) has confirmed its “old” application as a tonic remedy and requires further precise study as a novel adaptogenic plant.


Introduction
Modern changes to human life, as well as the harmful effects of the environment observed in our time, often lead to a sharp decrease in the body's adaptive capacity and functional reserves. Therefore, the study of the mechanisms underlying the adaptation process and the search for new drugs and ways to increase the body's functional reserves are among the main aims of modern biomedical sciences. To increase the body's resistance to adverse factors, drugs of various groups are used, the most universal of which are natural adaptogens, which increase the body's performance and transfer it into a state of nonspecific increased resistance [1]. The relative safety and breadth of the therapeutic action of these natural remedies make them especially valuable for increasing the performance of people in unusual climatic conditions with static and dynamic industrial overloads, professional athletes, and the elderly, including those suffering from chronic diseases, alongside increasing the performance and mental activity of all people [2]. The effects of adaptogens on the human body are multi-faceted: adaptogens exhibit immunostimulating Rhododendron adamsii has numerous folk names, such as Sagan Dalya tea, White Wing, or Belgorod tea, owing to the story that the mountain spirit lives in it and helps to recover the health of warriors. The history of the Sagan Dalya name is enveloped in many poetic legends; according to one such legend, two lovers, Sagan and Dalya were separated by the evil shamaness, causing tears of the girl to fall to the ground and turn into the evergreen flowering shrubs also known as White Wing or Sagan Dalya [6]. In Buddhist mythology, Sagan Dalya is one of the seven plants surrounding the teacher of healing, the All-Enlightened Bhaishajyaguru [7].
Ethnopharmacological data indicates that the Buryat medicinal decoction of R. adamsii leaves (сaгaн дaли) is used as an elixir to strengthen the human organism [8]. Yakutian nomads used R. adamsii (хaскaрa) decoction as a stimulant, diaphoretic, antipyretic, antibacterial, and analgesic drug [9]. In Mongolia, Buryatia, and Altai, shamans traditionally drink R. adamsii decoction as a tonic beverage, to enter a trance, and as a panacea for any disease. In Tibet, R. adamsii decoction is used to treat nervous disorders while, throughout Siberia, it is known as a powerful energy drink [10]. procyanidin B 1 equivalents) [24] in R. adamsii extracts. All the analyses were carried out five times and the data were expressed as mean value ± standard deviation (S.D.).

One-Step Swimming to Exhaustion Test
The mice (n = 90) were randomly divided into ten groups received saline (0.9% NaCl; 0.5 mL), R. adamsii leave extract obtained by the various solvent (0-100% methanol) at dose 50 mg/kg, and Rodiola rosea rhizome extract (5% rosavins, Vitaforest Ltd., Saint Petersburg, Russia; 50 mg/kg). In 60 min, the mice were individually placed in a glass cylinder (height 40 cm, diameter 20 cm) filled with water (height 15 cm; 22 ± 1 • C) and exhaustive swimming of rodents continued until the first immersion in the water. After the swimming sessions, the mice were towel-dried and returned to their housing. Each animal was used only once. The swimming time was measured using a stopwatch and was expressed in min. The experiment was realized in two versions, the first was the one-day application of plant extracts and the second was the 10-day application of plant extracts.

Two-Step Swimming to Exhaustion Test
The mice (n = 54) were randomly divided into six groups received saline (0.9% NaCl; 0.5 mL), R. adamsii leave extract (January, May, July, October samples) at dose 50 mg/kg, and R. rosea extract (50 mg/kg) during 10 days and on the 10th day, the rodents were tested as described in Section 2.5.1. One hour later, the mice have been retested in the same conditions. The swimming times at each step were measured using a stopwatch and were expressed in min. At the end of the experiment, laboratory animals were decapitated and the homogenates of skeletal muscles (quadriceps femoris) and liver, and blood serum were assayed for the following biochemical parameters: skeletal muscles-adenosine triphosphate (fluorimetric ATP assay kit; Sigma-Aldrich, cat. No. MAK190), creatine phosphate (colorimetric phosphocreatine PCr ELISA kit; Abbexa Ltd., Cambridge, UK, cat. No. abx258965) and lactate (fluorimetric lactate assay kit; Sigma-Aldrich, cat. No. MAK064); blood serum-pyruvic acid (fluorimetric pyruvate assay kit; Sigma-Aldrich, cat. No. MAK071), glucose (colorimetric glucose assay kit; Sigma-Aldrich, cat. No. MAK264), malondialdehyde (colorimetric MDA assay kit; Abcam, Cambridge, UK, cat. No. ab238537), and catalase (catalase colorimetric activity kit; Invitrogen, Carlsbad, CA, USA, cat. No. EIACATC); liver-glycogen (colorimetric glycogen assay kit; Cayman Chemical, Ann Arbor, MI, USA, cat. No. 700480).

Polyamide Solid-Phase Extraction (SPE)
The pre-chromatographic solid-phase extraction (SPE) of R. adamsii extract was used before HPLC separation as described before [33,34] with a slight modification. Polyamide cartridges Chromabond (Polyamide 6; 6 mL, 1000 mg; Sorbent Technologies, Inc., Norcross, GA, USA) were eluted with methanol (70 mL) and water (90 mL). The extract of R. adamsii (100 mg) was dissolved in 90% methanol (1 mL) followed by dilution of 20% methanol (8 mL), then centrifuged (6000× g, 15 min), and the supernatant was transferred in the volumetric flask (10 mL). Aliquots of internal standards were added to volumetric flask including 20-hydroxyecdysone (100 µL; 500 µg/mL in 50% methanol; internal standard-I), apigenin-7-O-glucoside (150 µL; 400 µg/mL in 70% methanol; internal standard-II), apigenin-7-O-glucuronide (150 µL; 400 µg/mL in 70% methanol; internal standard-III), and epigallocatechin 3-O-gallate (100 µL; 1000 µg/mL in 30% methanol; internal standard-IV). The final volume reached 10 mL with 25% methanol in the volumetric flask (solution A). Solution A (2 mL) was passed through polyamide SPE-cartridge and eluted with water (40 mL; eluate I), methanol (50 mL; eluate II), 0.55% NH 3 in methanol (50 mL; eluate III), pure water (120 mL), and DMSO (40 mL; eluate IV). Eluates I-IV stored at 4 • C before chromatographic analysis (Section 2.7). Qualitative chromatographic analysis of metabolic profiles of G. bifida extracts was done by high-performance liquid chromatography with photodiode array detection and electrospray ionization triple quadrupole mass spectrometric detection (HPLC-PDA-ESI-tQ-MS) technique using a liquid chromatograph LC-20 Prominence coupled with photodiode array detector SPD-M30A (wavelength range 200-600 nm), and triple-quadrupole mass spectrometer LCMS 8050 (all Shimadzu, Columbia, MD, USA) and C18 columns. Twoeluent gradient elution was used for the successful separation of compounds in four chromatographic modes (HPLC conditions) described in Table 1. The injection volume was 1 µL in all modes. The UV-Vis spectra were registered in the spectral range of 200-600 nm. Mass spectrometric detection was performed in negative ESI mode and the temperature levels of ESI interface, desolvation line, and heat block were 300 • C, 250 • C, and 400 • C, respectively, and the flow of nebulizing gas (N 2 ), heating gas (air), and collision-induced dissociation gas (Ar) were 3 L/min, 10 L/min, and 0.3 mL/min, respectively. The mass spectra were registered as 3 kV source voltage and collision energy −10-35 eV by the scanning range of m/z 80-2000. The managing of the LC-MS system was realized by LabSolution's workstation software equipped with the inner LC-MS library. The final identification of metabolites performed after an integrated analysis of retention time, ultraviolet, and mass spectra in comparison with the reference standards and literature data.

Metabolite Quantification
The quantitative analysis of compounds found in R. adamsii was done using the above described HPLC-PDA-ESI-tQ-MS conditions (Section 2.7) and full scan MS peak area used for calculation. One hundred and one compounds were quantitatively analyzed using 55 reference standards (Table S1). Each compound was carefully weighed (10 mg), dissolved in the methanol-DMSO mixture (1:1) in volumetric flasks (10 mL), and the reference standard calibration curves were built using the stock solutions in methanol (1-100 µg/mL). Mass spectrometric peak area data were used to plot 'concentration-peak area' graphs and determination the validation criteria (correlation coefficients, r 2 ; standard deviation, S YX ; limits of detection, LOD; limits of quantification, LOQ; and linear ranges) calculated as described previously [35] (Table S2). All quantitative analyses were carried out five times, and the data were expressed as mean value ± standard deviation (S.D.).

Statistical and Multivariate Analysis
Statistical analyses were performed by one-way analysis of variance, and the significance of the mean difference was determined by Duncan's multiple range test. Differences at p < 0.05 were considered statistically significant. The results are presented as mean values ± standard deviations (S.D.) of some replicates. The linear regression analysis and generation of calibration graphs were conducted using Advanced Grapher 2.2 (Alentum Software Inc., Ramat-Gan, Israel). Principal component analysis based on a data matrix (171 markers × 215 samples) was performed using Graphs 2.0 utility for Microsoft Excel (Komi NTc URO RAN, Syktyvkar, Russia) to generate an overview for group clustering.

Chemical Composition and Bioactivity of Rhododendron Adamsii Leaves: Impact of Solvent Type
Prior to the in-depth chemical and biological study of R. adamsii leaves, we analyzed various extracts to identify the best solvent for providing the most active remedy. Various methanols (0-100%) were used and characterized chemically ( Table 2). The total yield of the extracts varied from 12.5% to 35.0% of dry plant weight. The basic nutrient content of R. adamsii leaves fluctuated depending on the methanol concentration: proteins and soluble carbohydrates showed the highest amounts in water extractions (23.69 mg/g and 326.03 mg/g, respectively), while lipids dominated in the pure methanol extract (254.12 mg/g). The main explanation for the distribution of nutrients in extracts lies in the rule 'like solves like', i.e., hydrophilic solvents extract hydrophilic compounds, and vice versa; the same rule applies to polysaccharides due to their high content in the water extractions (57.60 mg/g). The phenolics are a class of compounds with a medium polarity that demonstrate optimal extractability using 40% methanol, with yields of 293.35 mg/g for total phenolic content, 138.88 mg/g for total flavonols, 16.84 mg/g for flavanols, 89.27 mg/g for catechins, and 27.98 mg/g for procyanidins. This impacted the radical scavenging ability of R. adamsii extracts against 2,2-diphenyl-1-picrylhydrazyl radicals (DPPH • ), well-known artificial free radicals used in the detection of plant antioxidants [36]. The 40% methanol extract demonstrated the best DPPH scavenging activity with IC 50 value 4.82 µg/mL (half maximal inhibitory concentration). The use of solvents with lower or higher concentrations of methanol negatively affects the antioxidant potential of the extracts.
Unlike the antioxidant properties of Rhododendron species, the impact of rhododendron extracts on physical endurance has not been studied previously, therefore, the adaptogenic potential of R. adamsii is disputed and requires further investigation; in our current study, we use the one-step swimming to exhaustion test of mice to examine this, using swimming time as an indicator of effectiveness. The extract of R. rosea roots (total rosavins 5%) was used as a plant remedy known to enhance resistance activity to physical training [28]. The eight extracts of R. adamsii leaves created using 0-100% methanol as a solvent were studied using a single dose of 50 mg/kg, the same dose as used for R. rosea extract ( Figure 2). Preliminary experiments demonstrated lower (25 mg/kg) and higher doses (100 mg/kg) of the extracts to be less effective. The effect of R. adamsii leaf extracts (solvent: 0-100% methanol) and R. rosea extract (50 mg/kg) on swimming time of mice in swimming to exhaustion test on day 1 (empty bars) and day 10 (shaded bars). *-p < 0.05 vs. control group, day 1; **-p < 0.05 vs. control group, day 10. In the swimming test of mice experiments, one-day application of R. adamsii extract resulted in no statistically significant (p < 0.05) increase of swimming time of mice, in contrast to R. rosea extract, which resulted in a 36% increase in swimming time compared with the control group (19.4 min vs. 14.2 min, respectively). The ten-day experiments demonstrated increases in animal endurance in all R. adamsii extract groups, with various levels of intensity. Of the extract groups, the 40% methanol extract showed the best effectiveness (31.1 min) when compared to the R. rosea extract group (32.7 min). Based on these results, we can conclude that R. adamsii extract enhances physical endurance, however, the properties of this endowment differ from those provided by R. rosea, as the latter medicine is characterized by a rapid influence on the animal subject while the R. adamsii extract's impact develops over time (cumulative effect). It is easily noted that the 40% methanol extract of R. adamsii leaves demonstrated the maximal antioxidant power and adaptogenic potential, indicating a possible linkage of both activities. Interestingly, a strong correlation between the radical scavenging ability of R. adamsii extracts against DPPH radicals and the swimming time of mice in swimming to exhaustion test on day 10 was observed with correlation coefficient r = 0.8118 ( Figure 3). Some researchers have previously expressed their views on the role of antioxidants in the realization of adaptogenic properties of plant extracts; these researchers have a cautious view that antioxidants decrease the risk of complications induced by oxidative stress, thus contributing to the positive effect of adaptogens [42,43]. In light of known data concerning stress-induced increases in free radical processes in mammals [1], it makes sense that a close relationship between adaptogens and antioxidant properties exists.

Rhododendron Adamsii Leaves Metabolites: LC-MS Characterisation and Seasonal Variation
Rhododendron genus demonstrates the presence of numerous chemical classes with specific chromatographic behavior [9]. Critical to the success of metabolite separation are the preliminary procedures for purification and partitioning of the total plant extract on the specific fractions. Solid-phase extraction (SPE) is a helpful tool for the pre-treatment of plant samples prior to chromatographic analysis [28,33,34]. Application of polyamidebased SPE of methanol extract of R. adamsii leaves yielded four fractions enriched with highly hydrophilic compounds (SPE-1), neutral phenolics (SPE-2), acidic and acylated phenolics (SPE-3), and tannin-like compounds (SPE-4); this method avoided overlapping of the chromatographic zones and enabled better identification of the compounds.
Sakakin (21), the orcinol O-glucoside that was identified after comparison with the reference standard, was found in rhododendrons for the first time, although orcinol itself has previously been detected in R. dauricum twigs [47]. Additionally, orcinol di-O-hexoside (18) and its O-acetate (24) were components of R. adamsii leaves. The unusual combination of simple phenolic glycosides in R. adamsii probably relates to its specific metabolomic features, which are extraordinary for the Rhododendron genus but understandable for the Ericaceae family.

Triterpene Glycosides
Preliminary analysis of SPE-1 eluate using acidic hydrolysis followed by HPLC separation allowed the detection of ursolic acid and unquantifiable traces of oleanolic acid ( Figure S2 [59,60] demonstrated the presence of a low level of free ursolic acid (<1 mg/g), owing to the domination of the glycosylated form of triterpene acids in the plant.
Grifolic acid (137), or 2,4-dihydroxy-6-methyl-3-[(2E,6E)-3,7,11-trimethyl-2,6,10-dodecatrien-1-yl]-benzoic acid, was identified by comparison with a reference standard. Grifolic acid demonstrated a UV pattern close to 120 and mass spectra indicating the presence of a longer prenyl fragment in the side chain ( Figure 7); this compound has previously been detected in Rhododendron dauricum [64], although not in R. adamsii. Some esters of grifolic acid were also found in the plant as mono  Compound 130 has a lower retention time than 137 (t R 10.67 vs. 11.64 min) and a 16 a.m.u. higher mass of the deprotonated ion (m/z 387 vs. 371) while the general mass spectral pattern is similar, indicating an additional hydroxyl functional group in molecule 130 (Figure 7). The difference in the UV spectrum of hydroxy-grifolic acid was that it demonstrates a shoulder-like maxima at 270 nm, in contrast to the true extreme curve of grifolic acid. The hydroxy-grifolic acid derivatives were four unknown glycosides, di-O-hexoside 125, mono-O-hexosides 126 and 127, and mono-O-pentoside 128.
Compound 141 was identified as daurichromenic acid, or 2-[(3E)-4,8-dimethyl-3,7nonadien-1-yl]-5-hydroxy-2,7-dimethylchromene-6-carboxylic acid, due to its specific UV and mass spectral patterns [54], which were matched with a reference standard (Figure 8).  The m/z value of the deprotonated ion of compound 135 was 16 a.m.u. higher than that of daurichromenic acid, although the way of fragmentation was close to 141 ( Figure 8). It was obvious that 135 was a hydroxy-derivative of daurichromenic acid with the additional hydroxyl group in the chromene fragment of the molecule, as indicated by the extra shortwave band in the UV spectrum. Additionally, the O-methyl ester of hydroxydaurichromenic acid (136) was found in R. adamsii. All derivatives of daurichromenic acid and hydroxy-daurichromenic acid have not previously been described.
Finally, thirty-eight prenylated phenols were found in R. adamsii, with only three of these compounds (cannabigerorcinic acid, cannabigerorcinic acid methyl ester, and daurichromenic acid) being previously described in the plant. Existing data regarding the bioactivity of Rhododendron cannabinoids demonstrate anti-HIV, antiallergic, anti-inflammatory, antithrombotic, antipsychotic, anticancer, and anti-Alzheimer potential [62,65], indicating a need for further study of R. adamsii phytocannabinoids.  (150, 151). Previously, compounds 148 and 151 have been found in Rhododendron plants [16,49], however, benzoic acid O-hexosides are still unknown in the genus.
Only one dihydrochalcone phloretin (63) was found in the methanol eluates (SPE-2) of R. adamsii leaves. A previous case of the discovery of 63 in Rhododendron genus refers to R. molle [49].

Catechins and Procyanidins
Eight catechins and five procyanidins were detected in R. adamsii leaves, including the reference standard identified compounds catechin (155) [49], with catechin/epicatechin O-hexosides referred to in genus for the first time.

Seasonal Variation of R. adamsii Metabolites
Non-deciduous (or evergreen) plants, such as R. adamsii, can protect the integrity of their leaves, irrespective of environmental temperatures and season. Due to this feature, people collect the leaves of R. adamsii year-round, however, there is no information about the seasonal variation of metabolites. Quantification data of 171 compounds in 215 samples of R. adamsii leaves, collected across four seasons, demonstrated significant variability in the contents of all non-trace compounds (Table 3). Grouping the compounds into two clusters (phenolics and non-phenolics), it can be seen that the content of both clusters increased from January to July and reduced in December ( Table 4). The maximal phenolics/nonphenolics level was in July (186.28/131.47 mg/g) while the lowest level was in January and March (97.26/48.95 mg/g). This means that more extractable compounds accumulate in summer samples. The content levels of the smaller groups of compounds conform to the same rule, with the exception of prenylated phenols. Increased levels of grifolic acid derivatives were observed in October samples, with values up to 17.53 mg/g vs. 8.35 mg/g in May samples, while cannabigerorcinic acid derivatives in the December samples reached 47.14 mg/g vs. 25.18 mg/g in July samples and daurichromenic acid derivatives in December reached 8.04 mg/g vs. 1.29 mg/g in May samples. Close variations in content were found for the flavonoid aglycones that showed the highest amounts in December samples, such as flavonol aglycones (2.54 mg/g in December vs. 0.28 mg/g in July) and flavone aglycones (1.58 mg/g in December vs. trace content in the spring and summer). Lipophilic compounds differed from the core metabolites with medium and high polarity in a seasonal variation pattern.
The flavonoid aglycones are the usual components of leaf surface wax, which accumulates in the winter period and plays an ecophysiological function in plant development [75,76], unlike the little-known prenylated phenols of rhododendrons. In this regard, we assumed that the prenylated phenols of R. adamsii are the leaves' surface components, which was confirmed after the analysis of surface diethyl ether extract. Derivatives of cannabigerorcinic acid, grifolic acid, and daurichromenic acid were detected in ether extract at a high level, whilst trace content was detected in the extract of ether-treated leaves ( Figure S3). The total yield of ether extract was at a maximal in December samples (25.3% of dry leaf weight) and a minimal in July samples (11.24% of dry leaf weight) ( Figure S4); this enables us to suggest that lipophilic prenylated phenols of R. adamsii have a protective effect in the winter period on an equal basis with other lipids covered on the leaf surface of evergreen plants [77][78][79].
The results of principal component analysis (PCA) of the content of 171 compounds in 215 samples of R. adamsii leaves, collected in various months of the year, showed that the specific distribution of individual points on the diagram, all located on the sides of the round that means the metabolic changes occur gradually (Figure 9). The chemical study undertaken has demonstrated that the extractable metabolites of R. adamsii leaves are a very complex mixture of compounds of different nature and polarity, with significant variation depending on the season of collection. We expected to discover variable bioactivity in R. adamsii extracts prepared from plant materials collected during different seasons, an idea that was confirmed by our subsequent experiments.
The ability of R. adamsii extracts to scavenge nitric oxide molecules was poor (3.67 mg/mL for July sample) in comparison to Trolox (0.83 mg/mL), in contrast to the Fe 2+ -chelating ability of R. adamsii extract, which reached a maximum in the July sample (211.74 mg Fe 2+ /g) and minimum in the January sample (129.03 mg Fe 2+ /g), still exceeding the Trolox value (42.72 mg Fe 2+ /g). The compounds providing the most significant impact on artificial/oxygen/chlorine/nitric oxide radical scavenging ability and Fe 2+ -chelating ability were gallic acid, catechin, myricetin-3-O-glucoside, and quercetin-3-O-glucoside, which are also known as radical-scavengers [81], unlike prenylated phenols, phenol glycosides, triterpenes, and organic acids, which were inactive or at low activity levels. Thus, the high seasonal content of flavonoids and catechins is the reason why July samples of R. adamsii extract are the most active in all antioxidant assays studied.
Previously, some fungal prenylated phenols have been concluded to be medium effectiveness antioxidants, with IC 50 values in the DPPH assay ranging from 60-80 µg/mL [82]; therefore, our results are not that surprising. The HPLC-DAD assay coupled with precolumn incubation of R. adamsii extract with DPPH • solution showed an almost complete reduction of the flavonoid peaks area, indicating their primary activity in the free radical scavenging process ( Figure 10). Figure 10. HPLC-UV chromatograms of R. adamsii leave total extract (July sample) before (black) and after preincubation (red) with DPPH • radicals solution. The excess of DPPH • radicals signed as DPPH. The basic peaks are numbered as described in Table 3.
Derivatives of cannabigerorcinic, grifolic, and daurichromenic acid did not demonstrate visible changes of peak area due to weak activity. There is a clear demonstration of the important role of selected phenolic compounds (flavonoids, catechins) in the antioxidant properties of R. adamsii extracts.

Adaptogenic Activity
In further examination of the adaptogenic potential of R. adamsii, we analyzed the effectiveness of four leaf extracts with different seasonal origins-January, May, July, and October, in a two-step to exhaustion swimming test assessing the influence of the remedy on physical performance (first step) and endurance (second step). Animals in the control group, which received saline, showed 16.5 and 3.9 min swimming times in the 1st and 2nd steps of the test, compared to the R. rosea extract group used as a positive control, which showed 37.4 and 18.8 min swimming times in the two steps, respectively ( Figure 11). The observed differences in the results of the R. rosea group compared to the saline group demonstrate stimulation of physical performance and the endurance of the animals. Figure 11. Effects of four different R. adamsii leaf extracts (January, May, July, and October samples) and R. rosea extract (50 mg/kg) on swimming time of mice in a two-step swimming to exhaustion test. * -p < 0.05 vs. saline group.
Statistically significant (p < 0.05) increases in swimming time were observed in all experimental groups receiving R. adamsii leaf extracts when compared to the control group. The July group showed the best results, with values of 34.1 and 21.3 min in the first and second swimming steps, respectively, indicating that R. adamsii positively influences physical performance and endurance. The remaining R. adamsii groups demonstrated progressive decreases in swimming times when analyzing samples from the May to January groups, similar to the pattern observed for changes in the antioxidant potential of the extracts.
The two-step swimming to exhaustion test, as an exhaustive exercise, reflects on the biochemical parameters of the experimental animals. This exercise leads to fatigue and inability by the animals to perform any further active swimming movements, resulting from decreased macroergic compound content in the skeletal muscles, almost complete exhaustion of glucose concentrations in the blood and glycogen in the liver, and, accordingly, the accumulation of lactate and pyruvate [83]. Compared with intact mice, the saline group animals showed significant decreases in adenosine triphosphate (ATP; 359 vs. 71 pmol/g) and creatine phosphate (CP; 3215 vs. 937 pmol/g) in their skeletal muscles, as well as decreased glucose levels in the blood serum (9.5 vs. 1.1 mmol/L) and decreased glycogen content in the liver (23.3 vs. 5.7 mg/g), accompanied by increased levels of lactate in the skeletal muscle (3.7 vs. 8.8 µmol/kg) and pyruvic acid in the blood serum (210 vs. 1408 pg/mL) ( Table 6). The expressed consumption of macroergic compounds (ATP, CP) and accumulation of acidic products in the animals points to very intense physical stress resulting in oxidative misbalance due to the accumulation of malondialdehyde (MDA; 1.8 vs. 6.7 nmol/L) and decreases catalase activity in the blood serum (11.5 vs. 6.2 mcat/L).
A different picture emerged following similar physical activity after intake of either R. adamsii or R. rosea extracts by the animals. Both extracts, in doses of 50 mg/kg, resulted in increased working ability due to metabolic restructuring through an improvement in the energy supply of skeletal muscles, as evidenced by increases in ATP and CP contents equivalent to 1.7-2.4 times the values of the control group. Also, increased carbohydrate reserves were observed, as indicated by elevated levels of blood serum glucose and liver glycogen, while reduced levels of lactate, observed in the skeletal muscle, and pyruvate, in the blood serum, pointed to decreased levels of metabolic acidosis. The level of MDA in blood serum decreased, from 6.7 nmol/L in the control group to 2.8 nmol/L in the R. adamsii group and 2.3 nmol/L in the R. rosea group, while catalase activity levels were similar in these groups to the intact group value. Thus, under strong physical stress, R. adamsii demonstrated the most important properties of an adaptogenic drug, providing more economical use of the energy substrates and increasing the body's ability to function optimally with less energy consumption [84].
It is worth noting that the oral administration of R. adamsii extract to intact mice, in the absence of physical activity, was not accompanied by pronounced changes in the analyzed parameters of carbohydrate and energy metabolism; these results meet the basic requirement of an adaptogenic drug, which should mainly work during periods of stress and provide minimal activity under normal conditions [2]. All of the foregoing indicates that R. adamsii leaf extract provides a positive therapeutic effect on animals during situations of strong physical stress, similar to the way known herbal adaptogens (Rhodiola rosea, Eleuterococcus senticosus, Schizandra chinensis) affect these animals [4].

Conclusions
In the situation of the global catastrophe caused by the COVID-19 epidemic, scientific findings relating to drugs that mobilize the internal reserves of protective barriers of the human body are highly relevant [85]. Adaptogenic plant drugs are considered as possible prophylactic agents for alleviating the severity of COVID-19 symptoms [86]. The expansion of the range of known plant adaptogens in this regard is an extremely important scientific mission. Thus, we can give the first detailed opinion concerning the veracity of early ethnopharmacological views of R. adamsii leaf extracts providing positive effects on humans as an adaptogenic remedy. Additional experiments will be necessary to understand the mechanisms of activity of R. adamsii extracts, as well as to elucidate their safety data. Nonetheless, it is now possible to say that Sagan Dalya tea has good prospects for medical and therapeutic use.

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/10 .3390/antiox10060863/s1, Table S1: Reference standards used for the qualitative and quantitative analysis by HPLC-DAD-ESI-tQ-MS and HPLC-UV assays, Table S2: Regression equations, correlation coefficients, standard deviation, limits of detection, limits of quantification and linear ranges for 55 reference standards, Table S3: Retention times, UV-and ESI-MS spectral data of compounds 1-171 found in Rhododendron adamsii, Table S4: UV-spectral patterns of compounds found in R. adamsii, Figure S1: High-performance anion-exchange chromatography with photodiode detection chromatograms of free carbohydrates of Rhododendron adamsii leaves and reference standards, Figure S2: High-performance liquid chromatography with photodiode detection chromatograms of triterpenic acids of R. adamsii leaves and reference standards, Figure S3: HPLC-PDA chromatograms of ether and methanolic extracts of fresh R. adamsii leaves, Figure S4: Yield of ether extract from R. adamsii leaves collected in various months.