Any damage of cells or tissues results in inflammation, often accompanied by pain [27,28,70,71]. Chronic diseases can develop as a result of the persistent exposure to these different stresses, which can include viruses, toxins, UV radiation, and pollution, among others. The components isolated from the Allochrusa species have never been studied for their anti-inflammatory effects. However, there is evidence that saponins have anti-inflammatory properties. In many studies, the mice paw model with histamine-induced acute inflammation was employed to determine edema development [57]. There were two methods to administer saponins: orally and intraperitoneally. In the first experiment, compound 5 (20 mg/kg and 50 mg/kg body weight), compound 6 (20 mg/kg and 50 mg/kg BW), indomethacin (20 mg/kg BW), and water were given orally to six groups of eight mice each (control). An hour after receiving the medications, each mouse received a subcutaneous injection of 0.05 mL 0.1% histamine in the right hind paw. Edema production was evaluated after the injection of histamine. The anti-inflammatory effect of compounds 5 and 6 was measured by the decrease in the index of edema compared to the control group, which is calculated as the percentage difference between the mass of the healthy and the inflamed paw, relative to the mass of the healthy paw. In contrast to the experiment with oral administration, saponins 5 and 6 had an anti-inflammatory effect that was generally dose dependent. Compound 6 failed to demonstrate any consistent anti-inflammatory activity in the trial based on oral dosing. The results of the experiments showed that compound 5 had stronger anti-inflammatory effects than compound 6 in the trial based on intraperitoneal dosing [58]. The ecdysteroid 11 isolated from A. gypsophiloides was studied for its anti-inflammatory activity by Tuleuov et al. [47]. In this screening, the anti-inflammatory effect of compound 11 was performed on male rats. Peritonitis (acute exudative response) was induced by injecting 1% acetic acid intraperitoneally (1 mL per 100 g of the rat body weight). Compound 2 decreased the exudation volume in rats by 24% at a dosage of 50 mg/kg body weight when compared to the control group. Saponins may exert corticomimetic properties or can inhibit phospholipase A2α (PLA2α); these properties could explain the anti-inflammatory activities [72].

Saponins from A. gypsophiloides (Algiox) can form immunostimulatory complexes (ISCOMs) that stimulate cellular immunity in mice as documented by Turmagambetova et al. [32]. As control preparations, ISCOMs were used containing the saponin Quil-A and micelles of H7N1 influenza virus glycoprotein antigens. The results demonstrated that a single-dose subcutaneous immunization with ISCOMs containing Algiox saponins was able to stimulate different phases of immunity (similar to ISCOMs containing the saponin Quil-A). A water-extractable fraction of saponins, Quil A, is derived from the plant Quillaja saponaria. When compared to immunization with micelles of H7N1 influenza virus glycoprotein antigens, the studied ISCOMs significantly increased the levels of IFN-, IL-2, IL-4, and IL-10 cytokines. The findings demonstrated that Algiox saponins could form immunostimulatory nanocomplexes with a similar immunostimulatory efficacy and structure to ISCOMs containing the saponin Quil-A. It has been proposed by the authors [32] that Algiox saponins obtained from A. gypsophiloides can be used to stimulate cellular and humoral immunity.

Saponins are known as effective adjuvant molecules. Extracts from A. gypsophiloides exhibited nonspecific activity compared to various antigenic ргераrаtions such as bovine serum albumin, the parainfluenza virus, and РG-3 [73]. Acanthophylloside (allochroside or a mixture of acanthophyllosides A-D) mediated the most potent adjuvant efficiency, which was similar or even superior to the other saponin preparations obtained from the ”Меrck”, ”Spofa”, and ”DVN” companies. The agent (allochroside) was also аn effective adjuvant when used as аn ingredient in the inactivated adsorbed vaccine for foot and mouth disease. It substantially enhanced its immunogenicity in cattle experiments. The data obtained in the course of the experiments showed that the administration of acanthophylloside to guinea pigs significantly activated the process of their antibody formation against immune bovine serum albumin. Based on the data they obtained, the authors concluded that saponins, the aglycones of which are represented by gypsogenin and quillaic acid, have a pronounced adjuvant activity [73]. Khatuntseva et al. [58] recently evaluated the adjuvant potential of triterpene glycosides 5 and 6 isolated from A. gypsophiloides. Test immunizations were carried out by using the keyhole limpet hemocyanin (KLH) protein carrier and synthetic vaccine neoglycoconjugate 3′SL-KLH (α-NeuAc-(2→3)-β-Galp-(1→4)-β-Glcp-KLH) on the basis of 3′SL (3′-sialyllactoside) ligands. The specific anti-3′SL IgM and IgG responses were measured after immunizing four groups of mice with the saponin of Quillaja bark, 40 µg of 3′SL-KLH along with 50 µg of compounds 5 and 6, or without an adjuvant. Comparing the test vaccine formulation with the antigen alone in terms of the control vaccine formulation, a substantial specific response was seen for saponins 5 and 6. The results demonstrated that high blood titers of IgM and IgG antibodies were recorded in the vaccination using compound 6 as an adjuvant, while the IgG level was less with the saponin from Quillaja bark. The levels of such antibodies in the saponin 5 experiment were rather modest. The authors concluded that antigen compound 6 demonstrated strong adjuvant qualities when combined with 3′SL-KLH and can thus be viewed as a potential component of vaccination formulations. The investigations of Turmagambetova et al. (2017) demonstrated that Algiox was capable of forming immunostimulatory nanocomplexes that were similar to ISCOMs containing the saponin Quil-A in terms of structure and immunostimulatory effectiveness. The adjuvant effect of the Algiox was studied in mice by subcutaneous injection with lipids and glycoproteins of the H7N1 influenza virus and ISCOMs containing saponins. The findings showed that H7N1 viral glycoprotein antigens, ISCOMs containing lipids, and saponins induced a high level of IgG and IgG2a serum antibodies. The level of the immune response and protective efficacy was greater than that induced by micelles containing glycoproteins combined with an aluminum hydroxide adjuvant, viral glycoproteins, or whole virions; the response was also similar to that elicited by ISCOMs containing Quil-A. As a result of the investigations, Algiox was recommended as an additional source of effective adjuvants for both human and veterinary vaccines [32].

Triterpene saponins are amphiphilic substances that can interfere with biological membranes. Saponins with lipophilic moiety can complex cholesterol and related lipids inside biomembranes; the hydrophilic side chains bind to proteins on the cell surface. This tension leads to a cell lysis. It can be best demonstrated with red blood cells, which disrupt when exposed to saponins [27,28,70]. The hemolytic activity of a bacterium or molecule is its ability to destroy red blood cells, leading to the release of hemoglobin.

The researchers Bazhenova and Aliev [74] investigated the hemolytic index of the saponin (a mixture of seven paniculatosides) isolated from A. paniculata. The hemolytic activity of paniculoside was determined and paniculatoside has a pronounced activity equal to 1 in 5000. For the purified saponin Algiox, the investigation of in vitro hemolysis was performed by Turmagambetova et al. [32]. The Quil-A concentration that caused 50% hemolysis (HD₅₀) was 225 µg/mL, whereas the HD₅₀ for Algiox was 700 µg/mL. The Algiox preparation that was tested contained derivatives of quillaic acid [75]. An in vitro hemolysis investigation was conducted by Khatuntseva et al. [58] using compounds 5, 6, and Quillaja bark saponin (as a control). According to the results, the saponin from Quillaja bark induced 100% of the hemolysis at the dose of 5.5 µg/mL. The hemolytic activity of saponins 5 and 6 was substantially lower; 50% hemolysis was seen at doses of 11 to 18 µg/mL, respectively. Compounds 5 and 6 caused hemolysis of 85–95% at a concentration of 62.5 µg/mL. Reference saponins QS-17, 18, and 21 obtained from Quillaja bark induced hemolysis at doses of 7–25 µg/mL. The findings are in good agreement with expectations that bidesmosidic saponins (as in the case of compounds 5 and 6) and the existence of glucuronic acid moiety at C-3 position of the aglycone have limited hemolytic activity [58].

Saponins exhibit cytotoxic activities that are promoted by their membrane-disrupting activities described for hemolysis. The in vitro and in vivo toxicity of the Algiox saponin from A. gypsophiloides was experimentally evaluated [32]. In comparison to the saponin Quil-A, which was employed as a reference, the saponins isolated from A. gypsophiloides displayed lower cytotoxicity in tests performed with model cell cultures. The cytotoxic concentrations (IC₅₀) of Quil-A were 21 μg/mL and 28 μg/mL in macrophages and MDCK (Madin–Darby canine kidney) cells, respectively. The IC₅₀ values of Algiox in macrophages and MDCK cells were 103.5 μg/mL and 93.75 μg/mL, respectively. The cytotoxicity of the essential oil isolated from the aerial parts (leaves, stems, flowers) of A. gypsophiloides was determined in human colon adenocarcinoma (HT-29) and prostate cancer (PC-3) cells by MTT assay [49]. The results showed a moderate cytotoxicity against PC-3 (IC₅₀ = 89.9 ± 2.01 µg/mL) and HT-29 cells (IC₅₀ = 43.6 ± 2.38 µg/mL). In a different study, biologically active compounds were extracted from the aerial parts of A. gypsophiloides using methanol, chloroform, and water as solvents [76]. The water extract had mild cytotoxic effects on leukaemia cell lines (CEM/ADR-5000: IC50 = 31.9 g/mL), leukemic lymphoblasts (CCRF-CEM), and breast cancer cells (MCF-7: IC50 = 145.9 g/mL).

Tuleuov et al. [47] studied the analgesic activities of 3-epi-2-deoxyecdysone (11) isolated from A. gypsophiloides. The peritoneal chemical irritation test (also known as the acetic acid writhing test) in white outbred mice was used to examine the analgesic effects of the compound 11. The mice were injected with 0.1 mL/10 g BW intraperitoneally with an acetic acid (0.75%) solution. The samples were administered intragastrically at a dose of 50 mg/kg BW before the acetic acid injection. The “writhes” were measured immediately following administration and for a period of 30 min. Tests were conducted using a concentration of 50 mg/kg BW of the reference painkiller diclofenac. The results of the test showed that compound 11 exerted an analgesic effect.

Triterpene glycosides also interfere with biomembranes of fungi and thus have often shown antifungal properties [27,28,70]. Compounds 5 and 6 were tested against four fungal cultures: basidiomycetous (Cryptococcus terreus, Filobasidiella neoformans) and ascomycetous (Saccharomyces cerevisiae, Саndida albicans) yeasts [58]. The results demonstrated that compounds 5 and 6 exhibited an antifungal effect against all the tested yeasts, especially the therapeutically significant C. albicans and F. neoformans. Growth-inhibiting tests revealed that saponins 5 and 6 had stronger antifungal effects at a pH of 4.0 than at a higher pH. At pH 7.0, the tested substances had no effect against C. albicans and F. neoformans. However, against these two strains both saponins were antifungal at pH 4.0. Notably, S. cerevisiae was resistant to compound 5 at either pH level.

The DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) assays are a simple, rapid, and sensitive method for evaluating free radical scavenging ability. The results demonstrated that the DPPH free radical scavenging ability of the essential oil of A. gypsophiloides was moderate (IC₅₀ = 456.4 ± 5.39 µg/mL) [49]. According to the IC₅₀ value (745.0 ± 6.87 µg/mL), a weak ABTS scavenging activity was observed for the essential oil.

The toxicity of the mixture of paniculatosides (paniculatoside) was investigated on white mice that had been treated with saponins (intravenously, subcutaneously, and orally) [74]. With an intravenous administration of paniculatoside in doses of 5–20 mg/kg BW, mice showed an increase in motor activity, followed by depression, and the mice huddled together. At high doses, mice died on the first day, and at low doses some mice died on the second and third days after treatment. Therefore, observations were carried out for 8 days. The following LD₅₀ values were obtained: the LD₅₀ value for the intravenous administration was 11.1 mg/kg, in the case of subcutaneous administration it was 77.5 mg/kg, and for oral administration 2550 mg/kg. Thus, paniculoside is a relatively toxic drug. Its toxic effect is reduced by subcutaneous and oral administration, which is typical for saponins [74]. The acute toxicity study of two triterpene glycosides (5 and 6) isolated from the roots of A. gysophiloides was carried out in vivo with male mice [58]. After a single oral or intraperitoneal dose, the corresponding LD₅₀ values were calculated: The LD₅₀ for oral administration ranged between 252 ± 57 mg/kg for compound 5 and 304 ± 55 mg/kg for compound 6. The LD₅₀ for the intraperitoneal administration of compounds 5 and 6 was 5.4 ± 2.8 mg/kg BW and 15.1± 5.6 mg/kg BW, respectively. The strong differences in LD₅₀ values between oral and intraperitoneal experiments indicated that the saponins were not being absorbed in the intestines. In in vivo tests, the toxicity of Algiox was evaluated [32]. Within the study, the toxicity was evaluated based on lethality, weight, and hair loss. For chickens and chicken embryos, the Quil-A LD₅₀ was 225 µg/animal, while for mice, it was 187.5 µg/animal. Algiox’s LD₅₀ in mice was found to be 650.4 µg/animal, whereas it was 850 µg/animal in chickens and chicken embryos. Lethality experiments performed on mice, chicken embryos, and chickens revealed that the assessed Algiox saponins were not toxic in the range of concentrations used. The standard threshold dose for vaccines is 15.0 µg per animal, which is at least 40 times less than the LD₅₀ calculated for the Algiox saponins.

Bazhenova and Aliev [74] investigated several pharmacological parameters (the effect on the central nervous system, blood pressure, heart arrhythmia, smooth muscle, diuresis) of the saponins isolated from A. paniculate (a mixture of seven paniculatosides) in rats. In doses of 1–10 mg/kg body weight, paniculoside briefly decreases blood pressure by 40–50%, followed by its restoration. There are no significant changes in respiration. In experiments with the in situ rat heart, paniculatoside at a concentration of 3 mg/kg body weight increased the amplitude (by 14%) and slightly slowed down the heart rate. With ECG research, the R-R interval increased by 31%. In doses of 3–5–10 mg/kg BW, the drug did not relieve or prevent the development of calcium chloride arrhythmia caused by the intravenous administration of calcium chloride (270 mg/kg). The drug at a concentration of 5 mg/kg body weight significantly, and at a concentration of 20 mg/kg almost completely, suppressed the components of the orientation reaction in white mice. At a concentration of 50 mg/kg, paniculatoside does not affect the sleep of white mice induced by chloral hydrate (300 mg/kg), hexanal (75 mg/kg), and nеmbutal (60 mg/kg). At the same time, at the concentration 50 mg/kg, it remarkably lengthened the latent period of corazole seizure onset. Paniculotoside did not have a ganglioblocking effect and did not change the blood pressure response caused by the intravenous administration of acetylcholine (0.5 μg/kg BW) and adrenaline (10 μg/kg BW). Paniculotoside enhanced the action of histamine (0.5–1 µg/kg BW) both in the acute experiment and in an isolated segment of the intestine of rats, which occurs 30–60 min after the administration of the drug and lasts 1.5–2 h. In experiments on rats, the drug in doses of 10–20 mg does not have a diuretic effect. It briefly lowers blood pressure and enhances the effect of histamine when administered exogenously [74]. Saponin isolated from the roots of A. gypsophiloides decreased the convulsive and toxic effects of corazole, potentiated the convulsive effect of strychnine, antagonized the narcotic effect of chloral hydrate, and increased diuresis in mice [77].