Issues with standardization and rationalization of the pharmacological activity of multicomponent phytoadaptogens considering their chemical composition are an active research area [1,2,3,4,5,6].

A multiphytoadaptogen (MPA) pharmaceutical composition for preventive oncology and gerontology has been developed at N. N. Blokhin National Medical Research Center of Oncology [7]. The MPA contains components of extracts from 40 official plants, including adaptogens from ginseng, aralia, Eleutherococcus, Rhodiola roseus, Oplopanax elatus, and Schizandra. The MPA was shown to be effective for preventive oncology, which is known to encompass primary (prevention of genesis or chemoprevention), secondary (prevention of recidivism and tumor metastasis), and tertiary prevention of oncological diseases (prevention of chemotherapy and beam side effects) [3]. Drugs for preventive oncology should absolutely possess antitumor and protective effects as the main properties. Antimutagenic (which is important for primary prevention of cancer), antitumor (important for secondary prevention), radioprotective, hormone-modulating, antioxidant, neuroprotective, and immunomodulating including adhesiogenic and interferonogenic effects of the MPA (important for tertiary prevention of cancer) have been found in experimental and clinical research [7,8,9,10,11,12,13,14,15,16,17,18]. The effectiveness of the MPA is undoubtedly due to the complex of biologically active compounds (BACs) included in it.

Research on the determination of the BACs in the MPA components is being conducted to assess the possibilities for quality control and standardization of the MPA. For example, polyphenolic compounds, essential oils, amino acids, and vitamins were found in the MPA using reversed-phase HPLC with UV detection, GC-MS, and NMR spectroscopy [19,20,21]. In addition, HPLC in combination with tandem mass spectrometry (HPLC-MS/MS) identified the main BACs among the MPA components, particularly ginseng and aralia, as triterpene saponins, i.e., ginsenosides Rb1, Rb2, Rc, Rd, Rg1, Rg2, Re, Rf, and Ro and aralosides A, B, and C [22, 23]. The phenylethanolglycoside salidroside, phenylpropanoid glycosides rosavin and rosarin, monoterpene glycoside rosiridin, and flavonoid rhodionin were determined as constituents of R. rosea BACs [24]; the phenylpropanoid eleutheroside B and the lignan eleutheroside E, of Eleutherococcus [25]; and the lignans schizandrin (schizandrol A) and schizantherin A (gomisin C), from Schizandra [26].

The next stage in the analysis of the constituent composition of the MPA was a determination of the BACs in the extract of rhizomes and roots of O. elatus, which is included in the MPA, using HPLC-MS/MS. This method has excellent specificity and high accuracy that enables the determination of trace amounts of compounds.

O. elatus Nakai (Araliaceae) is a deciduous thorny bush with long, creeping, woody rhizomes. Its lifespan is >300 years although it grows very slowly by 5 – 10 cm per year. The plant is like ginseng with respect to its medicinal activity although weaker. It contains a complex of BACs with useful properties. It is distributed in forests of the Far East and Ussuriysk Territory of Russia. It grows in fir and fir-birch forests and is shade tolerant. It is endemic to Primorsky Territory and is protected, i.e., included in the Red Book of Russia and Primorsky Territory. O. elatus is dying out because of overharvesting for medicinal purposes, forest fires, and low seed productivity. The biologically active complex of the plant includes triterpene saponins such as echinacosides, flavonoid glycosides, coumarins, lignans, essential oil, and resinous substances. However, the chemical composition of O. elatus is poorly studied. It possesses adaptogenic, immunomodulating, cardiotonic, neuroprotective, hormone-modulating, antidiabetic, and diuretic activity. The infusion of O. elatus is used for heart failure, physical and mental overexertion, and erectile dysfunction in men [27].

The aim of the present study was to determine the BACs of O. elatus in the MPA using HPLC-MS/MS and to assess the biological activity profile of the identified phytoconstituents using in silico analysis.

Experimental Part

Two samples were investigated in the work, i.e., the MPA and extract of rhizomes and roots of O. elatus included in the MPA composition. Samples of extracts were obtained using certified raw material and the same extraction technology, i.e., specific weight of raw material, extraction temperature and time regimes, extractant composition, and raw-material: extractant ratio.

An MPA sample was mixed with MeOH (1:2 ratio) and centrifuged for 5 min at 13,000 rpm. The supernatant liquid was passed through a 0.22-μm filter and centrifuged at 13,000 rpm for 1 min.

An aliquot (1 mL) of the O. elatus extract was evaporated to dryness in a Concentrator 5301 rotary evaporator (Eppendorf, Germany) at 30°C. The residue was dissolved in MeOH (100 μL) and centrifuged at 13,000 rpm for 1 min.

Chromatographic analysis of samples used a TSQ Vantage triple quadrupole mass spectrometer (Thermo Scientific TSQ series) connected to an Accela HPLC.

The analytical chromatography conditions were ACQUITY UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm; Waters); mobile phase composition: phase A (100% H2O) + formic acid (FA, 0.1%) and phase B (Me2CO 95%, H2O 5%, FA 0.1%).

Gradient elution was used to analyze the extracts (%B): 0 – 68 min (0 – 60%), 68 – 70 min (60 – 100%), 70 – 75 min (100%), and 75 – 80 min (0%). Samples (5 μL) were injected into a 25-μL loop (mobile phase, 20 μL). The flow rate was 450 μL/min.

Electrospray ionization was used. The conditions were negative polarity, spray capillary potential 4 kV, spray gas 60 psi, bypass gas 15 rel. units, capillary temperature 270°C. Total ion spectra were scanned with selected-ion monitoring in the range 150 – 1500 Da and scan time 0.1 sec.

Mass spectra were obtained by direct sample introduction through a syringe at 5 μL/min. The gas pressure in the impact chamber was 0.9 Torr. The potential in the impact chamber was selected separately for each compound.

Analysis in silico. Spectra of antitumor activity of O. elatus phytoconstituents were calculated using the PASS Refined 2022 software. The PASS software is based on a naive Bayesian algorithm and representation of structures as MNA descriptors [28]. The used version of the PASS software allowed the prediction of 1957 types of biological activity with an average accuracy of 97% [29]. The set of predicted activities was limited in the present work to 38 antitumor effects and 54 mechanisms of action associated with them.

The input data for PASS were structures of the compounds given in MOL or SDF format [30]. The result of the prediction was a list of biological activities with the calculated estimates Pa (probability of a compound being assigned to an active class) and Pi (probability of a compound being assigned to an inactive class) for each analyzed medicinal-like compound. All activities for which the calculated Pa exceed the Pi value were considered probable [28].

Additive/synergistic activity and possible drug(drug interactions of the studied chemical compounds were analyzed using the PharmaExpert software [31], which is based on a knowledge base containing information on >15,000 known interactions between pharmacological effects and mechanisms of action of pharmacological compounds.

Results and Discussion

A review of the literature showed that the chemical composition of O. elatus was poorly studied. Specific data on the main BACs included in it had not been published.

A chromatogram of the extract of O. elatus included in the MPA composition was taken in total ion scanning mode to study the main BACs (Fig. 1).

Fig. 1.
figure 1

Chromatogram of total ion current of O. elatus Extract.

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Table 1 lists the results of tandem mass spectrometric analysis of O. elatus extract (Rt, m/z of the pseudomolecular ion and its fragments) and the molecular mass of the compound.

Table 1. Experimental Data for Extract of Oplopanax elatus Nakai
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Table 1 shows that the studied compounds from O. elatus formed ion-adducts [M + FA (H]( with FA included in the mobile phase. One of the peaks with m/z 945.34 and retention time Rt = 30.12 min corresponded to a compound identical to ginsenoside Re from ginseng. A peak with m/z = 945.25 and Rt = 45.6 min corresponded to a compound identical to ginsenoside Rd, also from ginseng. Compounds giving peaks with m/z 1087.5 and Rt = 41.8 and 42.3 min had the same experimental characteristics (retention times and MS/MS spectra) as araloside C and stipuleanoside R2 from aralia extract. The extract of O. elatus probably contained the same compounds as in ginseng and aralia extracts or isomers very similar to them.

Thus, the main BACs of O. elatus were not specific compounds for this extract but compounds similar to those from other adaptogens, i.e., ginseng and aralia. In turn, these were ginsenosides Re and Rd (tetracyclic triterpene saponins) and araloside C and stipuleanoside R2 (pentacyclic triterpene saponins). They were also identified in O. elatus extract.

The chromatogram of the MPA was analyzed for content of O. elatus BACs knowing the retention time and m/z value of the main molecular ion. Figure 2 shows the chromatogram of the MPA in total ion scanning mode.

Fig. 2.
figure 2

Chromatogram of the MPA in total ion current mode.

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Chromatograms of the MPA obtained under the same conditions as that for O. elatus extract were analyzed by detecting separate ions corresponding to the main molecular ions of O. elatus ions (based on data given in Table 1). The sought ions were identified in the MPA chromatogram and the presence of ginsenosides Re and Rd, araloside C, and stipuleanoside R2 in the composition was confirmed by the analysis.

Results of analysis in silico of the antitumor effects of O. elatus phytoconstituents are given in Fig. 3.

Fig. 3.
figure 3

Thermal map of Pa – Pi assessments calculated for antitumor effects and Oplopanax elatus Nakai compounds.

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Figure 3 shows that 26 antitumor effects were predicted for O. elatus compounds with positive Pa – Pi values. Of these, 15 were predicted with Pa – Pi values >0.5 for at least one of the studied compounds. Table 2 lists the compounds for which antitumor effects were predicted with Pa – Pi values >0.7.

Table 2. Most Probable Antitumor Effects of Oplopanax elatus Phytocomponents
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Table 2 shows that an antitumor (Antineoplastic) effect (lung cancer) was predicted for four of the studied compounds with Pa – Pi values >0.7. Antineoplastic effects (ovary cancer, melanoma, and breast cancer) with an analogous threshold were predicted for araloside C and stipuleanoside R2.

The most probable mechanisms of action associated with the antitumor effects were established by us using the PharmaExpert software. Table 3 gives the corresponding results.

Table 3. Most Probable Mechanisms of Action Associated with Antitumor Effects
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Table 3 shows that two mechanisms of action, i.e., inhibition of transcription factor NF-κB and agonistic activity for apoptosis, were predicted for all studied compounds with threshold Pa – Pi > 0.9. Also, antioxidant activity was predicted for ginsenosides at this threshold. Four mechanisms of action were predicted for threshold Pa – Pi > 0.6. They included inhibition of tumor necrosis factor alpha release, stimulation of caspase 3, stimulation of AMP-activated protein kinase, and antioxidant activity predicted for araloside C and stipuleanoside R2. Inhibition of transcription factor NF-κB had the greatest estimates for all four compounds with Pa – Pi = 0.99.

An analysis of literature on the biological activities of the studied compounds gave the following results.

Ginsenosides Rd and Re, araloside C, and stipuleanoside R2 possessed neuroprotective properties [32,33,34,35,36] and antitumor activity against glioblastoma, hepatocarcinoma, colorectal cancer, and breast cancer, including activation of apoptosis and suppression of angiogenesis [37,38,39,40,41,42]. They were also shown to have geroprotective activity [43] with suppressed development of atherosclerosis [44, 45] and diabetes [46]. Also, these compounds exhibited antioxidant, immunomodulating, antiviral, and antiallergic activity [47,48,49,50,51,52,53] and had positive effects on the cardiovascular system [54,55,56].

Besides, it should be emphasized that the properties identified for ginsenosides Rd and Re, araloside C, and stipuleanoside R2 from the analysis in silico and from the scientific literature were analogous to those observed in studies of the MPA, i.e., immunomodulating, including antiviral, antioxidant, neuroprotective, antistress, chemopreventive (against skin squamous cell carcinoma and hepatocarcinoma), and antitumor against ovarian and cervical adenocarcinoma, hepatocarcinoma, and kidney, lung, and stomach cancer. The MPA also suppressed increased IL-6 levels and activated apoptosis of tumor cells and TNF-α production [8,9,10,11,12,13,14,15,16,17,18, 57,58,59,60,61,62,63,64,65,66,67,68].

The above activities characteristic of ginsenosides Rd and Re, araloside C, and stipuleanoside R2 that were analyzed in silico and using literature data agreed fully with the concept of drugs intended for preventive oncology.

However, the analysis in silico and literature data demonstrated additional properties for ginsenosides Rd and Re, araloside C, and stipuleanoside R2 that were not previously studied for the MPA pharmacological composition. In particular, these compounds could be effective against glioblastoma, colorectal cancer, breast cancer, prostate cancer, pancreatic cancer, liver cancer, bladder cancer, and others, including suppression of tumor angiogenesis. They possessed cardioprotective and antiallergic activity and prevented development of atherosclerosis and diabetes. All these properties could be subjects of future research on the MPA pharmacological composition and could be considered for development of new complex adaptogens, i.e., geroprotectors for preventive oncology.

Conclusion

Chromatograms and spectra obtained during the research could be used for standardization and quality control of the MPA.

Data obtained in silico and from the literature agreed with known characteristics of the biological activity of ginsenosides Rd and Re, araloside C, and stipuleanoside R2. The analysis in silico of the biological activity profiles identified the most probable mechanisms of antitumor activity of ginsenosides Rd and Re, araloside C, and stipuleanoside R2 and their possible additive/synergistic effects, including a broader spectrum of antitumor activity. Also, several pharmacological effects that were not previously known for the identified compounds were discovered. These data could become the basis for further research on ginsenosides Rd and Re, araloside C, and stipuleanoside R2 and extract from O. elatus rhizomes and roots.

Also, the research demonstrated the possibility for quality control and standardization of complex phytoadaptogens containing tetracyclic triterpene glycosides (ginsenosides Rd and Re) and pentacyclic triterpene glycosides derived from oleanolic acid (araloside C and stipuleanoside R2).