1. bookVolume 68 (2021): Issue 1 (January 2021)
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25 Nov 2011
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access type Open Access

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

Published Online: 14 Apr 2021
Page range: 1 - 9
Received: 09 Sep 2020
Accepted: 01 Feb 2021
Journal Details
License
Format
Journal
First Published
25 Nov 2011
Publication timeframe
2 times per year
Languages
English
Abstract

Sedative phytomedications continue to play an important role in the management of a considerable amount of anxiety symptoms because of the various side effects of synthetic sedatives and tranquilizers. However, developing new herbal drugs needs their appropriate quality control according to the relevant requirements. The aim of the study was to determine the sedative properties of the tinctures obtained from the American basil (Ocimum americanum L., Lamiaceae Martinov family) herb and to develop the standardization parameters for the promising herbal medicinal product. The open field test was used to evaluate the sedative effect of the prepared tinctures: (1) with the added of O. americanum essential oil (OATEs) and (2) without adding O. americanum essential oil (OAT). The standardization parameters for the OATEs were developed using validated High-Performance Thin Layer Chromatography (HPTLC) and High-Performance Liquid Chromatography (HPLC) methods. The HPTLC analysis was used for the chromatographic fingerprints of polyphenols and for identifying linalool in the OATEs. The HPLC analysis found the significant content of rosmarinic acid (RA) (0.26%) in the OATEs. In conclusion, the developed OATEs can be considered as the new herbal medicinal product with significant sedative properties.

Keywords

INTRODUCTION

Rational phytotherapy is a modern concept of herbal medicines that uses the standardized herbal medicinal products (Choudhary et al., 2011). A significant number of components in the tinctures and extracts from the plant raw materials are accompanied by the problem of their standardization. Quality control of herbal drugs should be done according to the requirements of relevant regulations (American Herbal Pharmacopoeia, 2011; European Pharmacopoeia, 2016).

Anxiety and depression are the important health problems in a modern society leading to low quality of life (Gutiérrez et al., 2015). Sedatives continue to play an important role in managing many anxiety symptoms in the context of medical illness. However, the side effects of synthetic anxiolytic drugs such as various syndromes of withdrawal, dependencies, and impairment of cognitive function and memory are of concern to physicians and scientists. Searching of new sources of plant raw material with anxiolytic action among the Lamiaceae Martinov species is a promising direction of scientific investigations is the area of phytochemistry and ethnopharmacology (Rabbani et al., 2015; Caputo et al., 2018; Bittner Fialová et al., 2019). Pharmaceutical industries produce a lot of phytomedications of a sedative action using the aerial parts of the Lamiaceae representatives such as Leonurus cardiaca, Melissa officinalis, Mentha×piperita, Lavandula angustifolia, and so on (Kowalczyk et al., 2012; Derzhavnyi rejestr, 2020).

Tincture, as a water-ethanol extract of a plant material being prepared at room temperature, preserves the complex as non-volatile so as volatile compounds (Doughari, 2012). The quality of tinctures or any herbal medicinal products primarily depends on the presence and content of the certain ingredients with proven biological activity (Hudz et al., 2019). High-Performance Thin Layer Chromatography (HPTLC) and High-Performance Liquid Chromatography (HPLC) methods have become increasingly practiced for routine analysis of herbal medicinal products obtained from the representatives of Mint Family (Staszek et al., 2013; Shafqatullah et al., 2014; Asha et al., 2015) as well as synthetic drugs (Logoyda et al., 2018).

American basil (O. americanum L.) belongs to the genus Ocimum L., subfamily Nepetoideae Burnett, family Lamiaceae. It is an annual essential oil-bearing plant native to Africa and Southeast Asia used locally as a spice or ornamental plant as well as a sedative, digestive, and anti-inflammatory remedy (The Genus Ocimum, 2006; Shanaida et al., 2017). Nowadays, this species is successfully naturalized on most continents. The aerial part of O. americanum accumulates several valuable groups of primary and secondary metabolites such as terpenoids, polyphenols, and amino acids, which are perspective for the sedative purpose (Koteswar and Babu, 2011; Shanayda, 2012; Shanaida et al., 2017; Shanaida and Golembiovska, 2018; Shanaida et al., 2018). Linalool (49.84%) that possesses significant sedative properties (Linck et al., 2009; Aprotosoaie et al., 2014) was the major component of essential oil hydrodistillated from the O. americanum herb collected in Ukraine (Shanayda, 2012). Rosmarinic acid (19.59 mg/g) was revealed by HPTLC as a predominant polyphenolic compound in the methanol extract of this plant raw material (Shanaida et al., 2020). However, the raw material of this species is not included in any Pharmacopoeias (American Herbal Pharmacopoeia, 2011; European Pharmacopoeia, 2016). Thus, studying the sedative properties of tinctures obtained from the herb of O. americanum and developing their quality parameters can be considered as quite a perspective.

The aim of the study was to determine the sedative effects of the tinctures obtained from the Ocimum americanum herb and to develop the standardization parameters for the promising herbal medicinal product by the validated HPTLC and HPLC methods.

MATERIALS AND METHODS
Plant material and preparing of tinctures

The herb of O. americanum was collected from the experimental plots in Ternopil region (Ukraine). Plants were grown from the seeds obtained from the collection of M. Hryshko National Botanical Garden (Kyiv, Ukraine). A voucher specimen (OM: 10011) has been deposited in the Herbarium of I. Horbachevsky Ternopil National Medical University for further references. The herb was harvested during the flowering stage, then dried in the shadow at 25–35°C. Two tinctures were prepared from the O. americanum herb using 70% ethanol (V/V): (1) with added O. americanum essential oil (OATEs) and (2) without adding O. americanum essential oil (OAT).

The powdered herb of the plant (20.0 g) was macerated with 200 mL ethanol (70%, V/V) at room temperature for 24 hours using forced periodic stirring. The supernatant was separated and filtered with a Buchner funnel, then placed for 2 days in the refrigerator (at 8–10 °C) for the sedimentation of ballast substances. The total ratio of raw material and tincture obtained by the described method was 1:10 (if necessary, 70% ethanol was added to get 200 mL of OAT). The OATEs was obtained by the adding 0.5 mL of O. americanum essential oil hydrodistillated from its herb to 100 mL of OAT. The volume of O. americanum essential oil chosen for dissolving into basic ethanol extract was analogous to the dose of Mentha × piperita (Lamiaceae) essential oil, which is added to the ‘Menthae piperitae tincture’ to enhance its pharmacological effect (Derzhavnyi reiestr, 2020). The essential oil of O. americanum was first dissolved in ethanol (96%, V/V) in the ratio 1:4, and then this solution was added to 100 mL of OAT to obtain a homogeneous solution.

Drugs and chemicals

Ethanol, ethyl acetate, and formic acid were purchased from Merck (Kennborough, NJ, USA). Aluminium chloride, toluene, Tween-80, anisaldehyde were purchased from Sigma-Aldrich (Germany). HPLC-grade acetonitrile, methanol, and trifluoracetic acid were purchased from Sigma-Aldrich (USA). Double-distilled water prepared using a Milli-Q water purification system (Millipore system). Silica gel plates for HPTLC and reference standards such as linalool, rutin, caffeic acid, and rosmarinic acid (RA) were purchased from Merck (Germany). Diazepam (5 mg/mL) was obtained from the Elegant (India). All the chemicals used in this study were of analytical grade.

Open field test

The spontaneous locomotor activities of experimental animals were assessed according to Linck et al. (2009) and Sarker Apu et al. (2013). Open field device consisted of a wooden field of one square meter with a series of squares and holes at their intersection; the floor was divided into 25 marked small squares of equal dimensions (20 × 20 cm). This device was placed in a dimly lit room.

Animals: male albino rats (220–240 g) were obtained from the Vivarium of I. Horbachevsky Ternopil National Medical University (Ukraine). Animals were kept in the standard laboratory conditions: relative humidity 55±5%; room temperature 22±2°C under a 12 h light/dark cycle. The animals were provided with standard diet and clean water ad libitum during acclimatization period. They were fasted overnight, with free access to water before the experiments. All the experimental animals were conformed to internationally accepted standards, which were approved by the Bioethical Committee of I. Horbachevsky Ternopil National Medical University, in accordance with ‘Ethical Principles and Guidelines for Experiments on Animals’ (2005).

The tested animals were randomly divided into four groups (n = 6). The rats were fasted for 12 h before the experiment. The tinctures were dissolved in the vehicle (1% solution of Tween-80 in distilled water) to obtain the dose 100 mg/kg body weight, calculated in terms of dry residue of tincture. Each group of animals received a particular treatment. Group I: negative control (vehicle). Group II: treatment with OAT. Group III: treatment with OATEs. Group IV: positive control (reference drug Diazepam at a dose of 0.55 mg/kg body weight). After one hour of the treatment, each animal was placed individually at the centre of the device and observed for 3 min to count the number of actions. The open field arena was thoroughly cleaned between each test to avoid the influence of the urine odours and faeces of the previous animal.

HPTLC analysis of OATEs

The HPTLC method was performed as described by Shanaida et al. (2020) with some modifications. 5 μL of OATEs and 10 μL of standard solutions (0.25 mg/mL of phenolic compounds and 5 μL/mL of linalool) were applied to HPTLC plates using an automatic HPTLC application device (Linomat 5, CAMAG, Muttenz, Switzerland). The chromatographic separation was performed on 20 × 10 cm HPTLC plates. Two mobile phases were used for eluting process: the ethyl acetate – toluene (5:95, V/V) was used for method A, and the ethyl acetate – formic acid – water (15:1:1, V/V) was applied for method B. The chromatographic plates were evaluated after derivatization with anisaldehyde reagent (method A) or with 1% aluminium chloride solution in methanol (method B). The resulting bands were observed under the daylight (method A) and with UV at 254 and 366 nm. The obtained chromatograms were analysed using HPTLC software (vision CATS, CAMAG). The compounds were identified using their Rf values and colours of the fluorescence comparatively to those of the reference standards. The applied HPTLC method was validated using specificity and robustness parameters.

HPLC analysis of OATEs

The quantitative analysis of RA in the OATEs was performed with Shimadzu HPLC-DAD system (Shimadzu, Japan). Good separation was achieved by a Phenomenex Luna C18 column (Phenomenex Inc., 250 × 4.6 mm i.d., 5 μm particle size) at 35°C. The UV absorption spectra of the reference standards and test samples were recorded in the range from 190 to 400 nm. The mobile phase consisted of two solutions: A (0.1% trifluoroacetic acid in water) and B (0.1% trifluoroacetic acid in acetonitrile). The flow rate was 1.0 mL/min and injection volume was 5 μL. The developed HPLC method for analysis of RA content in the OATEs was validated according to the linearity, specificity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy, and robustness parameters.

Statistical analysis

The variables for parametric data were analysed by one-way analysis of variance (ANOVA). Statistical analyses were performed using Statistica 12 software (StatSoft Inc., Tulusa, OK, USA). The data were expressed as mean ± standard error of the mean (SEM). The statistical significance was set at p ≤ 0.05.

RESULTS AND DISCUSSION
Sedative activity of OAT and OATEs

The obtained OATEs demonstrated the noticeable sedative effect when compared to OAT for all the studied parameters in the open field test (Table 1). Anxiety level of animals after the single administration of OATEs decreased significantly. Sedative effect for OAT compared to Diazepam was observed only for the influence on defecation. Thus, adding essential oils of O. americanum to OAT led to increasing sedative activity. It was established that linalool as a predominant compound of O. americanum essential oil (Shanayda, 2012) added to tincture to obtain OATEs, possess significant sedative properties (Linck et al., 2009). As it can be seen from the Table 1, the number of squares crossed by the rats was the smallest after the treatment with OATEs. The frequency of defecation as an important indicator of anxiety after the OATEs influence was at the same level as a reference drug. The levels of tentative research activities such as vertical racks and examined holes were also the least numerous in the OATEs group compared to OAT and control groups.

The influence of tinctures prepared from Ocimum americanum herb on the spontaneous behavior of rats in the open field test (n = 6).

Group Number of squares crossed Tentative research activity Indicators of anxiety (emotional reactions)
Vertical racks Examined holes Defecations Urinations Grooming
Control 32.61 ± 2.29 9.52 ± 0.89 5.21 ± 0.47 1.03 ± 0.1 0.51 ± 0.05 2.32 ± 0.21
OAT 29.17 ± 1.09 8.83 ± 0.28 4.83 ± 0.35 0.67 ± 0.091 0.5 ± 0.05 2.33 ± 0.24
OATEs 26.0 ± 1.021 8.0 ± 0.421 4.5 ± 0.21 0.5 ± 0.051 0.33 ± 0.021,2 2.17 ± 0.19
Diazepam 18.5 ± 0.541 6.5 ± 0.181 3.17 ± 0.091 0.5 ± 0.021 01 1.17 ± 0.041

Notation.

– the differences are statistically significant for the values of the control group (p ≤ 0.05);

– the differences are statistically significant for the values of the Diazepam group (p ≤ 0.05)

Chromatographic analyses and standardization procedures for OATEs
HPTLC analysis

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 Rf = 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 Rf values 0.03, 0.11, 0.19, and 0.85 in the chromatogram of test solutions. The maximum difference in the values of the Rf zones of linalool corresponding the test and reference solutions within one plate was not exceed 0.01 units of Rf. The robustness studies for method A are presented in Table 3.

Figure 1

HPTLC chromatograms of the OATEs (1), Ocimum americanum essential oil (2), and linalool standard (A) after the derivatization with anisaldehyde solution at day light. Mobile phase: ethyl acetate – toluene (5:95).

The results of HPTLC analyses of biologically active compounds in OATEs.

Compound Rf value Colour Presence in OATEs Presence in essential oil of O. americanum
Method A
Unknown compound 0.03 Violet +
Unknown compound 0.11 Violet +
Unknown compound 0.19 Violet +
Linalool 0.29 Violet + +
Unknown compound 0.85 Violet + +
Method B
Rutin 0.09 Yellow + *
Unknown compound 0.14 Blue + *
Unknown compound 0.27 Yellow + *
Unknown compound 0.29 Blue + *
Unknown compound 0.43 Dark blue + *
Unknown compound 0.64 Blue + *
Rosmarinic acid 0.75 Blue + *
Caffeic acid 0.79 Blue + *

Notation. ‘+’ compound is presented; * – parameter was not studied

The robustness testing for the identification of linalool in OATEs by HPTLC.

Indicator Level of changes Rf variability (RSD, %)
Mobile phase composition (ethyl acetate – toluene, V/V) 5:95 0.6
5.2:95 1.3
4.8:95 0.8
Development distance, cm 8 1.3
18 1.8
Chamber 3 different containers 1.3
Analysts 2 different analysts 0.8

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 Rf = 0.75 (Fig. 2, Table 2). The less intense blue zone of caffeic acid was found at Rf = 0.79 and the yellow band of rutin was revealed at Rf = 0.09. The unknown compounds of blue shades were identified at 0.14, 0.29, 0.43, and 0.64 of Rf 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 Rf zones of RA as a predominant component corresponding the test and reference solutions within one plate did not exceed 0.01 units of Rf. The robustness studies of the RA identification in the OATEs are presented in Table 4.

Figure 2

HPTLC chromatograms of the OATEs (A) and polyphenols standards (1 – rutin; 2 – caffeic acid; 3 – RA) after the derivatization with 1% methanolic AlCl3 at λ=366 nm. Mobile phase: ethyl acetate – formic acid – water (15:1:1).

The robustness testing of RA identification in OATEs by HPTLC.

Indicator Level of changes Rf variability (RSD, %)
Mobile phase composition (ethyl acetate – formic acid – water, V/V) 15:1:1 0.8
15:1.05:1 1.3
15:0.95:1 1.7
15:1:1.05 1.3
15:1:0.95 1.3
Development distance, cm 8 0.8
18 1.3
Chamber 3 different containers 1.7
Analysts 2 different analysts 0.6

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.

HPLC analysis

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

UV absorption spectrum of RA in the HPLC method

Figure 4

HPLC chromatogram of the RA standard solution (A) and OATEs (B) (λ=330 nm)

The system suitability parameters for the determination of RA by HPLC.

Standard Retention time (Rt) Tailing factor (T) Theoretical plates (N)
Rosmarinic acid 38.0 1.2 2399586

The linearity, regression equation, limit of detection, limit of quantitation, precision parameters of RA standard in the HPLC analysis.

Standard Linearity range [μg/ml] Regression equation R2 LOD [μg/ml] LOQ [μg/ml] RSD [%]
Inter-day Intra-day
Rosmarinic acid 5–50 Y = 0.235x + 0.164 0.9998 0.005 0.015 0.16 0.04

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.

Standardization procedure

The standardization of the OATEs as a perspective herbal medicinal product has been carried out in accordance with the requirements of European Pharmacopoeia (2016) using such a scheme: definition (source of a plant material); production (method of extraction and solvent); characters (appearance, taste, odour), identification (by HPTLC); assay (content of the main biologically active compound by HPLC). Definition. Tincture is produced from the O. americanum herb and its essential oil (OATEs).

Production

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.

Characters

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

Identification. Method A
Test solution

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

Reference solution

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.

Results of TLC analysis

The chromatogram of the test solution in the lower third should be characterized by the weak violet zone of linalool (Rf = 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.

Identification. Method B
Test solution

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.

Results of TLC analysis

The chromatogram of the test solution should be characterized by intense blue zone of RA (Rf = 0.75) and less intense blue zone of caffeic acid (Rf = 0.79) just above it. The yellow zone of rutin should be evaluated at the bottom (Rf = 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.

HPLC analysis of RA content
Test solution

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.

Reference solution

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).

The gradient mode in the HPLC analysis.

Time (min) Mobile phase A (vol%) Mobile phase B (vol%)
0–5 95 5
5–35 95 → 75 5 → 25
35–40 75 25
40–60 75 → 50 25 → 50
60–65 50 → 20 50 → 80
65–70 20 80
70–85 95 5

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 GABAA-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 GABAA 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.

CONCLUSIONS

This study demonstrates the significant sedative effect of the OATEs developed from O. americanum herb using 70% ethanol and essential oil isolated from this plant. Such sedative property could be due to the common influence of RA and linalool as the predominant biologically active compounds in OATEs as well as some amino acids and triterpenoids. More tests and studies are needed to clarify the exact components with sedative activity in the developed herbal medicinal product. The necessary standardization parameters for the chromatographic analysis of the OATEs were established. The validated HPTLC method was successfully applied for identifying linalool and RA as the predominant compounds of the OATEs. The HPLC method was developed and validated to quantify the RA content in the OATEs. The current study provides a basis for the assessment of developed OATEs as the new perspective herbal medicinal product with sedative action.

Figure 1

HPTLC chromatograms of the OATEs (1), Ocimum americanum essential oil (2), and linalool standard (A) after the derivatization with anisaldehyde solution at day light. Mobile phase: ethyl acetate – toluene (5:95).
HPTLC chromatograms of the OATEs (1), Ocimum americanum essential oil (2), and linalool standard (A) after the derivatization with anisaldehyde solution at day light. Mobile phase: ethyl acetate – toluene (5:95).

Figure 2

HPTLC chromatograms of the OATEs (A) and polyphenols standards (1 – rutin; 2 – caffeic acid; 3 – RA) after the derivatization with 1% methanolic AlCl3 at λ=366 nm. Mobile phase: ethyl acetate – formic acid – water (15:1:1).
HPTLC chromatograms of the OATEs (A) and polyphenols standards (1 – rutin; 2 – caffeic acid; 3 – RA) after the derivatization with 1% methanolic AlCl3 at λ=366 nm. Mobile phase: ethyl acetate – formic acid – water (15:1:1).

Figure 3

UV absorption spectrum of RA in the HPLC method
UV absorption spectrum of RA in the HPLC method

Figure 4

HPLC chromatogram of the RA standard solution (A) and OATEs (B) (λ=330 nm)
HPLC chromatogram of the RA standard solution (A) and OATEs (B) (λ=330 nm)

The robustness testing of RA identification in OATEs by HPTLC.

Indicator Level of changes Rf variability (RSD, %)
Mobile phase composition (ethyl acetate – formic acid – water, V/V) 15:1:1 0.8
15:1.05:1 1.3
15:0.95:1 1.7
15:1:1.05 1.3
15:1:0.95 1.3
Development distance, cm 8 0.8
18 1.3
Chamber 3 different containers 1.7
Analysts 2 different analysts 0.6

The system suitability parameters for the determination of RA by HPLC.

Standard Retention time (Rt) Tailing factor (T) Theoretical plates (N)
Rosmarinic acid 38.0 1.2 2399586

The gradient mode in the HPLC analysis.

Time (min) Mobile phase A (vol%) Mobile phase B (vol%)
0–5 95 5
5–35 95 → 75 5 → 25
35–40 75 25
40–60 75 → 50 25 → 50
60–65 50 → 20 50 → 80
65–70 20 80
70–85 95 5

The linearity, regression equation, limit of detection, limit of quantitation, precision parameters of RA standard in the HPLC analysis.

Standard Linearity range [μg/ml] Regression equation R2 LOD [μg/ml] LOQ [μg/ml] RSD [%]
Inter-day Intra-day
Rosmarinic acid 5–50 Y = 0.235x + 0.164 0.9998 0.005 0.015 0.16 0.04

The results of HPTLC analyses of biologically active compounds in OATEs.

Compound Rf value Colour Presence in OATEs Presence in essential oil of O. americanum
Method A
Unknown compound 0.03 Violet +
Unknown compound 0.11 Violet +
Unknown compound 0.19 Violet +
Linalool 0.29 Violet + +
Unknown compound 0.85 Violet + +
Method B
Rutin 0.09 Yellow + *
Unknown compound 0.14 Blue + *
Unknown compound 0.27 Yellow + *
Unknown compound 0.29 Blue + *
Unknown compound 0.43 Dark blue + *
Unknown compound 0.64 Blue + *
Rosmarinic acid 0.75 Blue + *
Caffeic acid 0.79 Blue + *

The influence of tinctures prepared from Ocimum americanum herb on the spontaneous behavior of rats in the open field test (n = 6).

Group Number of squares crossed Tentative research activity Indicators of anxiety (emotional reactions)
Vertical racks Examined holes Defecations Urinations Grooming
Control 32.61 ± 2.29 9.52 ± 0.89 5.21 ± 0.47 1.03 ± 0.1 0.51 ± 0.05 2.32 ± 0.21
OAT 29.17 ± 1.09 8.83 ± 0.28 4.83 ± 0.35 0.67 ± 0.091 0.5 ± 0.05 2.33 ± 0.24
OATEs 26.0 ± 1.021 8.0 ± 0.421 4.5 ± 0.21 0.5 ± 0.051 0.33 ± 0.021,2 2.17 ± 0.19
Diazepam 18.5 ± 0.541 6.5 ± 0.181 3.17 ± 0.091 0.5 ± 0.021 01 1.17 ± 0.041

The robustness testing for the identification of linalool in OATEs by HPTLC.

Indicator Level of changes Rf variability (RSD, %)
Mobile phase composition (ethyl acetate – toluene, V/V) 5:95 0.6
5.2:95 1.3
4.8:95 0.8
Development distance, cm 8 1.3
18 1.8
Chamber 3 different containers 1.3
Analysts 2 different analysts 0.8

Agatonovic-Kustrin S, Kustrin E, Gegechkori V, Morton DW. Anxiolytic Terpenoids and Aromatherapy for Anxiety and Depression. Adv Exp Med Biol. 2020; 1260:283–296. doi: 10.1007/978-3-030-42667-5_11. Agatonovic-KustrinS KustrinE GegechkoriV MortonDW Anxiolytic Terpenoids and Aromatherapy for Anxiety and Depression Adv Exp Med Biol 2020 1260 283 296 10.1007/978-3-030-42667-5_11 Open DOISearch in Google Scholar

American Herbal Pharmacopoeia. Botanical pharmacognosy – microscopic characterization of botanical medicines. CRC Press, 2011. 735 p. American Herbal Pharmacopoeia Botanical pharmacognosy – microscopic characterization of botanical medicines CRC Press 2011 735 p. Search in Google Scholar

Aprotosoaie AC, Hancianu M, Costache Ii, Miron A. Linalool: a review on a key odorant molecule with valuable biological properties. Flavour Frag J. 2014; 29:193–219. AprotosoaieAC HancianuM CostacheIi MironA Linalool: a review on a key odorant molecule with valuable biological properties Flavour Frag J 2014 29 193 219 Search in Google Scholar

Asha D, Mathew L, Kalappurakkal R. Evaluation of HPTLC fingerprints of flavonoids and antioxidant activity of selected medicinal plants of Lamiaceae Family. Int Journal of Pharmacognosy and Phytochemical Research. 2015; 7(2):240–245. AshaD MathewL KalappurakkalR Evaluation of HPTLC fingerprints of flavonoids and antioxidant activity of selected medicinal plants of Lamiaceae Family Int Journal of Pharmacognosy and Phytochemical Research 2015 7 2 240 245 Search in Google Scholar

Benedec D, Hanganu D, Oniga I, Tiperciuc B, Olah NK et al. Assessment of rosmarinic acid content in six Lamiaceae species extracts and their antioxidant and antimicrobial potential. Pak J of Pharm Sci. 2015; 28(6):2297–2303. BenedecD HanganuD OnigaI TiperciucB OlahNK Assessment of rosmarinic acid content in six Lamiaceae species extracts and their antioxidant and antimicrobial potential Pak J of Pharm Sci 2015 28 6 2297 2303 Search in Google Scholar

Bittner Fialová S, Harris Ch, Ordsmith V, Nagy M, Jonáš F, Mučaji P. Polar phenolic compounds in peppermint rhizomes and leaves. Eur. Pharm. J. 2019; 66(1): 28–31. Bittner FialováS HarrisCh OrdsmithV NagyM JonášF MučajiP Polar phenolic compounds in peppermint rhizomes and leaves Eur. Pharm. J 2019 66 1 28 31 Search in Google Scholar

Caputo L, Reguilon MD, Mińarro J, De Feo V, Rodriguez-Arias M. Lavandula angustifolia essential oil and linalool counteract social aversion induced by social defeat. Molecules. 2018; 19; 23(10). pii: E2694. doi: 10.3390/molecules23102694. CaputoL ReguilonMD MińarroJ De FeoV Rodriguez-AriasM Lavandula angustifolia essential oil and linalool counteract social aversion induced by social defeat Molecules 2018 19 23 10 pii: E2694. 10.3390/molecules23102694 Open DOISearch in Google Scholar

Choudhary N, Sekhon BS. An overview of advances in the standardization of herbal drugs. J Pharm Edu Res. 2011; 2 (2):55–70. ChoudharyN SekhonBS An overview of advances in the standardization of herbal drugs J Pharm Edu Res 2011 2 2 55 70 Search in Google Scholar

Colla ARS, Rosa JM, Cunha MP, Rodrigues ALS. Anxiolytic-like effects of ursolic acid in mice. European Journal of Pharmacology. 2015; 758(5):171–176. CollaARS RosaJM CunhaMP RodriguesALS Anxiolytic-like effects of ursolic acid in mice European Journal of Pharmacology 2015 758 5 171 176 Search in Google Scholar

Cory H, Passarelli S, Szeto J, Tamez M, Mattei J. The role of polyphenols in human health and food systems: A mini-review. Front. Nutr. 2018; 5(87). CoryH PassarelliS SzetoJ TamezM MatteiJ The role of polyphenols in human health and food systems: A mini-review Front. Nutr. 2018 5 87 Search in Google Scholar

Derzhavnyi rejestr lіkarskyh zasobiv Ukrainy (2020). [ONLINE] Available at: http://www.drlz.com.ua (in Ukrainian) Derzhavnyi rejestr lіkarskyh zasobiv Ukrainy 2020 [ONLINE] Available at: http://www.drlz.com.ua (in Ukrainian) Search in Google Scholar

Doughari HJ. Phytochemicals: extraction methods, basic structures and mode of action as potential chemotherapeutic agents. In: Phytochemicals – a global perspective of their role in nutrition nd health. Ed. by V. Rao. 2012:1–32. DoughariHJ Phytochemicals: extraction methods, basic structures and mode of action as potential chemotherapeutic agents In: Phytochemicals – a global perspective of their role in nutrition nd health Ed.by RaoV. 2012 1 32 Search in Google Scholar

European Pharmacopoeia. 2016. 8th Ed. [ONLINE] Available at: https://www.edqm.eu/en/european-pharmacopoeia-ph-eur-8th-edition European Pharmacopoeia 2016 8th Ed. [ONLINE] Available at: https://www.edqm.eu/en/european-pharmacopoeia-ph-eur-8th-edition Search in Google Scholar

Ethical Principles and Guidelines for Experiments on Animals. 2005. 3rd ed. Available at: https://publisher.medfak.ni.ac.rs/ASN_1/Ethical%20Principles-S.pdf Ethical Principles and Guidelines for Experiments on Animals 2005 3rd ed. Available at: https://publisher.medfak.ni.ac.rs/ASN_1/Ethical%20Principles-S.pdf Search in Google Scholar

Gontova T, Ilyinska N, Golembiovska O, Mashtaler V. Study of the component composition of phenolic compounds obtained from Dahlia varieties Ken’s Flame herb. Der Pharma Chemica. 2016; 8(18):455–459. GontovaT IlyinskaN GolembiovskaO MashtalerV Study of the component composition of phenolic compounds obtained from Dahlia varieties Ken’s Flame herb Der Pharma Chemica 2016 8 18 455 459 Search in Google Scholar

Gutiérrez SL, Chilpa RR, Jaime HB. Medicinal plants for the treatment of “nervios”, anxiety, and depression in Mexican Traditional Medicine. Rev Bras Farmacogn. 2014; 24 (5):591–608. GutiérrezSL ChilpaRR JaimeHB Medicinal plants for the treatment of “nervios”, anxiety, and depression in Mexican Traditional Medicine Rev Bras Farmacogn 2014 24 5 591 608 Search in Google Scholar

Hirai M, Ito M. Sedative effects of the essential oil and headspace air of Ocimum basilicum by inhalation in mice. J Nat Med. 2019; 73(1):283–288. HiraiM ItoM Sedative effects of the essential oil and headspace air of Ocimum basilicum by inhalation in mice J Nat Med 2019 73 1 283 288 Search in Google Scholar

Hudz N, Yezerska O, Shanaida M, Horčinová Sedláčková V, Wieczorek PP. Application of the Folin-Ciocalteu method to the evaluation of Salvia sclarea extracts. Pharmacia. 2019; 66(4):209–215. HudzN YezerskaO ShanaidaM Horčinová SedláčkováV WieczorekPP Application of the Folin-Ciocalteu method to the evaluation of Salvia sclarea extracts Pharmacia 2019 66 4 209 215 Search in Google Scholar

Kessler A, Sahin-Nadeem H, Lummis SC, Weigel I, Pischetsrieder M et al. GABA(A) receptor modulation by terpenoids from Sideritis extracts. Mol. Nutr. Food Res. 2014; 58:851–862. doi: 10.1002/mnfr.201300420 KesslerA Sahin-NadeemH LummisSC WeigelI PischetsriederM GABA(A) receptor modulation by terpenoids from Sideritis extracts Mol. Nutr. Food Res 2014 58 851 862 10.1002/mnfr.201300420 Open DOISearch in Google Scholar

Koteswar SB, Babu AV. Pharmacognostic and phytochemical studies of Ocimum americanum. J Chem Pharm Res. 2011; 3(3):337–347. KoteswarSB BabuAV Pharmacognostic and phytochemical studies of Ocimum americanum J Chem Pharm Res 2011 3 3 337 347 Search in Google Scholar

Linck VM, da Silva AL, Figueiró M, Piato AL, Herrmann AP et al. Inhaled linalool-induced sedation in mice. Phytomedicine. 2009; 16:303–307. LinckVM da SilvaAL FigueiróM PiatoAL HerrmannAP Inhaled linalool-induced sedation in mice Phytomedicine 2009 16 303 307 Search in Google Scholar

Logoyda L, Abdel-Megied A, Kondratova Y, Trofimenko O, Korobko D, Dakhym I. Development and validation of HPLC method for the simultaneous determination of enalapril maleate in present of their impurities: application to tablet analysis. International Journal of Applied Pharmaceutics. 2018; 10(1):98–102. LogoydaL Abdel-MegiedA KondratovaY TrofimenkoO KorobkoD DakhymI Development and validation of HPLC method for the simultaneous determination of enalapril maleate in present of their impurities: application to tablet analysis International Journal of Applied Pharmaceutics 2018 10 1 98 102 Search in Google Scholar

Olayiwola G, Ukponmwan O, Olawode D. Sedative and anxiolytic effects of the extracts of the leaves of Stachytarpheta cayennensis in mice. Afr J Tradit Complement Altern Med. 2013; 10(6): 568–579. OlayiwolaG UkponmwanO OlawodeD Sedative and anxiolytic effects of the extracts of the leaves of Stachytarpheta cayennensis in mice Afr J Tradit Complement Altern Med 2013 10 6 568 579 Search in Google Scholar

Rabbani M, Sajjadi SE, Vaezi A. Evaluation of anxiolytic and sedative effect of essential oil and hydroalcoholic extract of Ocimum basilicum L. and chemical composition of its essential oil. Res Pharm Sci. 2015; 10 (6):535–543. RabbaniM SajjadiSE VaeziA Evaluation of anxiolytic and sedative effect of essential oil and hydroalcoholic extract of Ocimum basilicum L. and chemical composition of its essential oil Res Pharm Sci 2015 10 6 535 543 Search in Google Scholar

Richard EJ, Illuri R, Anandhakumar B, Anandhakumar S, Bhaskar A et al. Anti-stress activity of Ocimum sanctum: possible effects on hypothalamic–pituitary-adrenal axis. Phytotherapy Research. 2016; 30(5):805–814. RichardEJ IlluriR AnandhakumarB AnandhakumarS BhaskarA Anti-stress activity of Ocimum sanctum: possible effects on hypothalamic–pituitary-adrenal axis Phytotherapy Research 2016 30 5 805 814 Search in Google Scholar

Romanenko Ye, Koshovyi O, Kireyev I, Tryshchuk N, Ilyina T, Borodina N. Study of the relations between the main BAS groups content in Leonurus cardiaca tincture and its psychotropic activity. Ukraïns’kij bìofarmacevtičnij žurnal. 2018; 4 (57): 69–74 (in Ukrainian). RomanenkoYe KoshovyiO KireyevI TryshchukN IlyinaT BorodinaN Study of the relations between the main BAS groups content in Leonurus cardiaca tincture and its psychotropic activity Ukraïns’kij bìofarmacevtičnij žurnal 2018 4 57 69 74 (in Ukrainian). Search in Google Scholar

Rytsyk O, Soroka Y, Shepet I, Vivchar Z, Andriichuk I et al. Experimental evaluation of the effectiveness of resveratrol as an antioxidant in colon cancer prevention. Natural product communications. 2020; 15(6):1934578X2093274 RytsykO SorokaY ShepetI VivcharZ AndriichukI Experimental evaluation of the effectiveness of resveratrol as an antioxidant in colon cancer prevention Natural product communications 2020 15 6 1934578X2093274 Search in Google Scholar

Sarker Apu A, Hossan Bhuyan S, Matin M, Hossain F, Khatun F, Taiab A. Analgesic, neuropharmacological, anti-diarrheal, and cytotoxic activities of the extract of Solanum sisymbriifolium (Lam.) leaves. Avicenna J Phytomed. 2013; 3(4):302–312. Sarker ApuA Hossan BhuyanS MatinM HossainF KhatunF TaiabA Analgesic, neuropharmacological, anti-diarrheal, and cytotoxic activities of the extract of Solanum sisymbriifolium (Lam.) leaves Avicenna J Phytomed 2013 3 4 302 312 Search in Google Scholar

Shafqatullah, Khan R, Hassan W. Development of HPLC method by UV-VIS detection for the quantification of phenolic acids in different Ocimum sanctum Linn. extracts. Pak J Pharm Sci. 2014; 27(5):1271–1275. Shafqatullah KhanR HassanW Development of HPLC method by UV-VIS detection for the quantification of phenolic acids in different Ocimum sanctum Linn. extracts Pak J Pharm Sci 2014 27 5 1271 1275 Search in Google Scholar

Shafaei A, Ab Halim NH, Zakaria N, Ismail Z. Analysis of free amino acids in different extracts of Orthosiphon stamineus leaves by high-performance liquid chromatography combined with solid-phase extraction. Pharmacogn Mag. 2017; 13(Suppl 3):385–391. ShafaeiA Ab HalimNH ZakariaN IsmailZ Analysis of free amino acids in different extracts of Orthosiphon stamineus leaves by high-performance liquid chromatography combined with solid-phase extraction Pharmacogn Mag 2017 13 Suppl 3 385 391 Search in Google Scholar

Shanaida M, Kernychna I, Shanaida Y. Chromatographic analysis of organic acids, amino acids, and sugars in Ocimum americanum L. Acta Poloniae Pharmaceutica – Drug Research. 2017; 74(2):729–734. ShanaidaM KernychnaI ShanaidaY Chromatographic analysis of organic acids, amino acids, and sugars in Ocimum americanum L. Acta Poloniae Pharmaceutica – Drug Research 2017 74 2 729 734 Search in Google Scholar

Shanaida M, Golembiovska O. Identification and component analysis of triterpenoids in Monarda fistulosa L. and Ocimum americanum L. (Lamiaceae) aerial parts. ScienceRise: Pharmaceutical Science. 2018; 3(13):26–31. ShanaidaM GolembiovskaO Identification and component analysis of triterpenoids in Monarda fistulosa L. and Ocimum americanum L. (Lamiaceae) aerial parts ScienceRise: Pharmaceutical Science 2018 3 13 26 31 Search in Google Scholar

Shanaida M, Golembiovska O, Hudz N, Wieczorek PP. Phenolic compounds of herbal infusions obtained from some species of the Lamiaceae family. Current Issues in Pharmacy and Medical Sciences. 2018; 31(4):194–199. ShanaidaM GolembiovskaO HudzN WieczorekPP Phenolic compounds of herbal infusions obtained from some species of the Lamiaceae family Current Issues in Pharmacy and Medical Sciences 2018 31 4 194 199 Search in Google Scholar

Shanaida M, Jasicka-Misiak I, Makowicz E, Stanek N, Shanaida V, Wieczorek PP. Development of high-performance thin layer chromatography method for identification of phenolic compounds and quantification of rosmarinic acid content in some species of Lamiaceae family. J Pharm Bioall Sci. 2020; 12 (2):139–145. ShanaidaM Jasicka-MisiakI MakowiczE StanekN ShanaidaV WieczorekPP Development of high-performance thin layer chromatography method for identification of phenolic compounds and quantification of rosmarinic acid content in some species of Lamiaceae family J Pharm Bioall Sci 2020 12 2 139 145 Search in Google Scholar

Shanayda MI. Comparative analysis of essential oils components of Ocimum L. genus. Farmatz zhurnal. 2012; 4: 99–102. (in Ukrainian). ShanaydaMI Comparative analysis of essential oils components of Ocimum L. genus Farmatz zhurnal 2012 4 99 102 (in Ukrainian). Search in Google Scholar

Staszek D, Orłowska M, Waksmundzka-Hajnos M, Sajewicz M, Kowalska T. Marker fingerprints originating from TLС and HPLC for selected plants from the Lamiaceae family. J Chromatography. 2013; 36 (17):2463–2475. StaszekD OrłowskaM Waksmundzka-HajnosM SajewiczM KowalskaT Marker fingerprints originating from TLС and HPLC for selected plants from the Lamiaceae family J Chromatography 2013 36 17 2463 2475 Search in Google Scholar

Tankam JM, Ito M. Sedative, anxiolytic and antidepressant-like effects of inhalation of the essential oil of Ocimum gratissimum L. from Cameroon in mice. Journal of Pharmacognosy and Phytochemistry. 2014; 2(5):1–9. TankamJM ItoM Sedative, anxiolytic and antidepressant-like effects of inhalation of the essential oil of Ocimum gratissimum L. from Cameroon in mice Journal of Pharmacognosy and Phytochemistry 2014 2 5 1 9 Search in Google Scholar

The Genus Ocimum (Basil) / Ed. by R. Hiltunen and Y. Holm. Taylor & Francis e-Library. 2006. The Genus Ocimum (Basil) Ed. by HiltunenR. HolmY. Taylor & Francis e-Library 2006 Search in Google Scholar

Yamane H, Kurauchi I, Denbow DM, Furuse M. Central functions of amino acids for the stress response in chicks. Asian-Aust. J. Anim. Sci. 2009; 22(2): 296–304. YamaneH KurauchiI DenbowDM FuruseM Central functions of amino acids for the stress response in chicks Asian-Aust. J. Anim. Sci 2009 22 2 296 304 Search in Google Scholar

Yang Y, Zhang Z, Li S, Ye X, Li X, He K. Synergy effects of herb extracts: pharmacokinetics and pharmacodynamic basis. Fitoterapia. 2014; 92:133–147. YangY ZhangZ LiS YeX LiX HeK Synergy effects of herb extracts: pharmacokinetics and pharmacodynamic basis Fitoterapia 2014 92 133 147 Search in Google Scholar

Yeong Ok Kwon, Jin Tae Hong, Ki-Wan Oh. Rosmarinic acid potentiates pentobarbital-induced sleep behaviors and non-rapid eye movement (NREM) sleep through the activation of GABAA-ergic systems. Biomol Ther (Seoul). 2017; 25(2):105–111. Ok KwonYeong HongJin Tae OhKi-Wan Rosmarinic acid potentiates pentobarbital-induced sleep behaviors and non-rapid eye movement (NREM) sleep through the activation of GABAA-ergic systems Biomol Ther (Seoul) 2017 25 2 105 111 Search in Google Scholar

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