Sedative Effect and Standardization Parameters of Herbal Medicinal Product Obtained from the Ocimum americanum L. Herb

The HPTLC method for the qualitative determination of phenolic compounds and linalool in the OATEs was developed for the first time. A number of solvent systems were tried for OATEs chromatographic analysis. The satisfactory resolutions were reached using such solvent systems as ethyl acetate – toluene (5:95, V/V) for linalool (method A) and ethyl acetate – formic acid – water (15:1:1, V/V) for phenolic compounds (method B).

The validation of HPTLC procedure for the identification of linalool in the OATEs (method A) included the specificity and robustness studies. During the specificity study, it was revealed that chromatograms of the reference solution had the violet zone of linalool at R_f = 0.29 at daylight (Fig. 1, Table 2). The chromatogram of the test solution showed the weak violet zone at the level of linalool spot. The additional violet bands of unknown compounds possessing the specific for terpenoids violet shades were also available at R_f values 0.03, 0.11, 0.19, and 0.85 in the chromatogram of test solutions. The maximum difference in the values of the R_f zones of linalool corresponding the test and reference solutions within one plate was not exceed 0.01 units of R_f. The robustness studies for method A are presented in Table 3.

Figure 1

The specificity study of the polyphenols identified in OATEs (method B) showed that the chromatograms of reference and test solutions had the prominent blue fluorescent zone of RA (at λ = 366 nm) in the upper part at R_f = 0.75 (Fig. 2, Table 2). The less intense blue zone of caffeic acid was found at R_f = 0.79 and the yellow band of rutin was revealed at R_f = 0.09. The unknown compounds of blue shades were identified at 0.14, 0.29, 0.43, and 0.64 of R_f values, and yellow one corresponding to flavonoids was found at 0.27 in the chromatogram of test solutions. The differences in the values of the R_f zones of RA as a predominant component corresponding the test and reference solutions within one plate did not exceed 0.01 units of R_f. The robustness studies of the RA identification in the OATEs are presented in Table 4.

Figure 2

Summarizing the above mentioned results, it was considered that the validated techniques for the identification of linalool and polyphenols in OATEs by the HPTLC method is a suitable procedure to control its quality in the ‘Identification’ test.

The content of RA as a predominant biologically active ingredient in the OATEs was assayed and validated by the HPLC for the first time. The mobile phase for HPLC consisted of two solutions: A (trifluoroacetic acid 0.1% in water), and B (trifluoroacetic acid 0.1% in acetonitrile) at 1.0 mL/min flow rate, volume of injection: 5 μL. All the solvents should be filtered through a 0.45 μm Millipore filter before use and degassed in an ultrasonic bath. Detection was made with spectrophotometer at 330 nm. HPLC was performed at 35°C column temperature. RA was determined using external standard method. The gradient elution was provided by mixing mobile phases A and B.

The validation of the HPLC test procedure for the analysis of RA content in the OATEs included specificity, linearity, accuracy, precision, and robustness studies. The specificity of the test was confirmed by the coincidence of retention time of the RA peak on the chromatogram of OATEs test solution with the retention of this peak on the chromatogram of RA standard solution (Fig. 3, 4). The chosen chromatographic conditions allowed to separate the peak of the RA from the peaks of other phenolic components. The values for the system suitability were an average of 6 replicates (Table 5). The linearity, recovery, accuracy, and range of HPLC method were defined on model mixtures with known content of active ingredients in the range of 80% to 120% of the RA nominal value (Table 6). The regression equation was calculated from the calibration curve for nine concentrations of the RA reference standard. The LOD and LOQ calculated for signal to noise ratio of 3 and 10, respectively. The aliquots of OATEs test solution was split and stored at 4ºC. The aliquots were compared against the fresh-prepared reference standards at various intervals to determine the standard stability. It was recommended to perform all the experiments during a maximum of 3 days and to store stock and standard solutions in a refrigerator.

Figure 3

Figure 4

According to the results of the robustness study, the column temperature and the mobile phase flow within ±5% did not affect the test results significantly.

Tincture is produced from one part of dried herb using ethanol (70%, V/V) by a maceration method to obtain 10 parts of the finished product. Essential oil was dissolved first in ethanol (96%, V/V) in ratio 1:4, and then added to the tincture until 0.5% solution was obtained.

Greenish-brown transparent liquid with a specific aromatic small and bitter-burning taste.

The tincture (OATEs) to be examined (10 μL).

Dissolve 25 μL of linalool in 5 mL of toluene.

Plate: TLC silica gel F₂₅₄ plate (10 × 20 cm).

Mobile phase: ethyl acetate – toluene (5:95, V/V); application: 5 μL, as bands; development: 8 cm from the line of start; drying: in air.

Detection: treat with anisaldehyde solution, heat at 100–105°C for 5 min and examine in daylight immediately.

The chromatogram of the test solution in the lower third should be characterized by the weak violet zone of linalool (R_f = 0.29). Furthermore, other faint violet zones may be present in the chromatogram obtained with the test solution. Sequence of the zones present in the chromatograms obtained with the reference and test solutions (comparatively with the essential oil of O. americanum) are shown in Fig. 1.

The tincture (OATEs) to be examined (10 μL). Reference solutions. Dissolve (separately) 1 mg of rutin, 1 mg of caffeic acid, and 1 mg of RA in 4 mL of methanol.

Plate: TLC silica gel F₂₅₄ plate (10 × 20 cm).

Mobile phase: ethyl acetate – formic acid – water (15:1:1, V/V); application: 5 μL, as bands; development: 8 cm from the line of start; drying: in air.

Detection: Visualization should be performed under UV light (254 nm, 366 nm) after spraying with 1% aluminium chloride solution and heating at 100–105°C for 5 min.

The chromatogram of the test solution should be characterized by intense blue zone of RA (R_f = 0.75) and less intense blue zone of caffeic acid (R_f = 0.79) just above it. The yellow zone of rutin should be evaluated at the bottom (R_f = 0.09). Furthermore, other faint blue and yellow zones may be present in the chromatogram obtained with the test solution. Red zones of chlorophyll may be present at the top. Sequence of the zones present in the chromatograms of the reference and test solutions are shown in Fig. 2.

Introduce 1 mL of OATEs into a volumetric flask (100 mL) and dilute it by adding 70% ethanol to the mark. Filter the obtained solution through 0.45 μm Millipore filter.

Dissolve 15 mg of RA standard in 70% ethanol and dilute to 100.0 mL with the same solvent.

Column: Phenomenex Luna C18 column (250 × 4.6 mm i.d., 5 μm particle size) at 35°C as appropriate.

Mobile phase: Trifluoroacetic acid 0.1% in water (solution A), and trifluoroacetic acid 0.1% in acetonitrile (solution B) at 1.0 mL/min flow rate in a gradient elution mode (Table 7).

Detection: DAD (330 nm).

Results of HPLC analysis: 0.26 ± 0.01% of RA. Identification of RA achieved by comparison of the retention time on the chromatogram and the absorption spectra obtained for its peak in tincture with those obtained for the standard. The quantification of RA made by measuring its peak area at λ = 330 nm. The content of RA in tincture should be at minimum 0.2%.

As it is known, the healing properties of the herbal medicinal products can be viewed as a result of the synergistic effect of a lot of biologically active compounds (Yang et al., 2014). As the complexes of active compounds in the obtained tinctures can interact synergistically, the dominating of both RA and linalool in the OATEs could be the reason for the manifestation of its noticeable sedative action. A number of different pharmacologically active compounds with the possible sedative action was established in the Ocimum americanum water-ethanol extracts and essential oil (Shanayda, 2012; Shanaida and Golembiovska, 2018). The noticeable reduction in locomotor activity of the OATEs could either be due to the effect of its biologically active compounds on the central nervous system or a direct influence on the periphery as it was established for the related species Ocimum sanctum (Richard et al., 2016). Researchers observed a marked anxiolytic dose-dependent effect, similar to Diazepam, after using the water-ethanol extracts and essential oil of Ocimum basilicum herb (Rabbani et al., 2015). The sedative influence of the essential oil released by Ocimum basilicum plants was found in mice using open field test (Hirai and Ito, 2019). The sedative, anxiolytic, and antidepressant-like effects were established after the inhalation of Ocimum gratissimum essential oil in mice (Tankam et al., 2014).

The obtained data about the prevalence of RA among phenolic compounds in the OATEs revealed by the chromatographic methods correlates with the results of phytochemical analyses of the O. americanum herb (Shanaida et al., 2018; Shanaida et al., 2020). RA was also the ‘marker component’ of the ethanol extracts obtained from a lot of plants belonging to the Lamiaceae family such as Origanum vulgare, Melissa officinalis, Rosmarinus officinalis, Ocimum basilicum, Salvia officinalis, and Hyssopus officinalis (Staszek et al., 2013; Benedec et al., 2015). RA can potentiate the pentobarbital-induced sleeping through the activation of GABA_A-ergic systems (Yeong et al., 2017). It can also demonstrate the antioxidant effect and other valuable biological activities similar to the other polyphenols containing more than 3 OH-groups in their molecules (Gontova et al., 2016; Rytsyk et al., 2020). Polyphenols possess free radicals scavenging activity, which can prevent neurodegenerative diseases (Cory et al., 2018). The methanol extract of Stachytarpheta cayennensis (order Lamiales) and its butanol, aqueous, and ethylacetate fractions demonstrated the sedative effect in a dose-dependent manner (Olayiwola et al., 2013).

Linalool as a key volatile molecule of many essential oils of the Ocimum species possess the sedative activity (Aprotosoaie et al., 2014). Linalool and other terpenoids can modify the function of GABA_A receptors (Kessler et al., 2014). It was found that linalool and linalool-containing essential oil obtained from Lavandula angustifolia (Lamiaceae) aerial part were useful alternative tools to the available traditional treatments for social stress-induced mental illnesses such as anxiety and depression (Caputo et al., 2018; Agatonovic-Kustrin et al., 2020). The relationships between the contents of certain bioactive compounds of terpenoid and polyphenol nature in the Leonurus cardiaca (Lamiaceae) tinctures and their sedative effect were revealed by Romanenko et al. (2018).

The sedative properties of the obtained tinctures can be enhanced by some amino acids, which are extracted from the plant raw material with water and water-ethanol solutions (Shafaei et al., 2017; Shanaida et al., 2017). Besides γ-aminobutyric acid and glycine as the famous inhibitory neurotransmitter in the central nervous system, it was also established that the sedative effect of a lysine, arginine, alanine, serine, and cysteine acting via γ-aminobutyric acid receptors (Yamane et al., 2009). All these amino acids were found in the Ocimum americanum herb (Shanaida et al., 2017). The triterpenic ursolic acid possessing sedative action (Colla et al., 2015) was revealed in the 70% ethanol extract of Ocimum americanum (0.12%) (Shanaida and Golembiovska, 2018). Thus, the revealed sedative properties of the OATEs can be due to synergistic effects of RA and linalool as well as some amino acids and triterpenoids extracted from the Ocimum americanum raw material with water-ethanol solutions.