Determination of Sesquiterpenic Acids with Sedative Properties in Extracts of Medicinal Lavender (Lavandula angustifolia Mill.)

Abstract

Plant raw materials with a calming effect on the nervous system are increasingly used in modern phytotherapy. Lavender belongs to this group of plants, due to the content of essential oil with known therapeutic properties and other phytoconstituents that can be responsible for the sedative effect. Our studies confirmed the presence of sesquiterpenic acids characterized by sedative activity in lavender extracts. The contents of valerenic acid and acetoxyvalerenic acids in flowers and leafy stalks of two various Lavandula angustifolia cultivars—‘Blue River’ and ‘Ellagance Purple’—were determined. Analyses of methanolic extracts performed using the HPLC method showed that content of these sesquiterpenic acids varied with the cultivars and the morphological parts of the plant. The amount of acetoxyvalerenic acid was significantly higher than the amount of valerenic acid. In the ‘Blue River’ cultivar, higher levels of both compounds characterized by sedative properties were found. The content of valerenic acid in flowers ranged from 0.50 mg/100 g d.m. in the ‘Ellagance Purple’ cultivar to 1.75 mg/100 g d.m. in the ‘Blue River’ cultivar. In turn, leafy stalks contained 0.81 mg/100 g d.m. of valerenic acid in the ‘Ellagance Purple’ cultivar and 1.16 mg/100 g d.m. in the ‘Blue River’ cultivar. Interestingly, the ‘Blue River’ cultivar contained about 10 times more acetoxyvalerenic acid (65.80 mg/100 g d.m.) in flowers and four times more acetoxyvalerenic acid in leafy stalks (50.1 mg/100 g d.m.), in comparison with the ‘Ellagance Purple’ cultivar. The higher content of valerenic and acetoxyvalerenic acids in the flowers and leafy stalks of the ‘Blue River’ lavender cultivar can be important for its possible medical applications.

1. Introduction

The dynamic development of the chemical industry at the beginning of the 20th century resulted in a retreat from traditional phytotherapy and natural methods of treatment. However, the development of advanced research and analytical methods also allowed for intensified studies on medicinal plant raw materials. The possibility of isolation of biologically active compounds from plants and their characterization contributed to the development of the pharmaceutical industry based on synthetic drugs, but also resulted in the intensive development of research on plant raw materials and plant-based medicines. The primary factor influencing the development of modern knowledge about various plant raw materials for medical applications is ability to determine their exact chemical composition, which allows for precise dosing, determining the mechanism of action, and confirming safety and effectiveness in randomized clinical trials. Nowadays, plant medicines are becoming more and more popular, and the therapeutic effectiveness of their constituents is recognized at the pharmacological, biochemical, and molecular levels. Compared to a synthetic drug as a single chemical compound, a herbal drug is a mixture of chemical substances with various directions and mechanisms of action. Their beneficial effects on health often result from synergism between compounds with different chemical structures.

Due to the fact that stress and nervous tension are an increasingly common cause of lifestyle diseases, special attention is paid to herbal medicines that can affect the central nervous system through a multi-directional calming effect. Medicines can influence the activity of the central nervous system by inhibiting or stimulating it. Currently, antianxiotic, antidepressants, and antineurotics drugs enable effective treatment of various mental disorders [1]. In this group of drugs, the basic mode of action is related to their impact on emotional processes: they inhibit the symptoms of fear, excitement, aggressiveness, or anxiety. The majority of the drugs that affect the central nervous system are of synthetic origin; however, plant-origin substances are very popular among psychodysleptic drugs. Plant medicines are usually characterized by a weaker effect and require a longer period of use than synthetic ones. However, when dosed correctly, they are less likely to cause side effects, excessive sleepiness, or addiction. A large group of plant raw materials are known for their calming and sleep-inducing effect. Among them, valerian (Valeriana officinalis), lemon balm (Melissa officinalis), purple passionflower (Passiflora incarata), common hop (Humulus lupulus), and medicinal lavender (Lavandula angustifolia) are successfully used to inhibit mental hyperactivity and to facilitate falling asleep [1].

Medicinal lavender, also known as common lavender, is a plant found in many European countries, but also cultivated in North America [2]. The medicinal raw materials of this plant, which belongs to the Lamiaceae family, are flowers (Flos Lavanduale), which should be collected before they bloom (in the bud stage). Lavender flowers contain a minimum 1.4% of essential oil, as well as tannins, triterpenes, coumarins, phenolic acids, flavonoids, phytosterols, anthocyanins, and minerals [1,2]. Lavender flowers, extracts, and essential oils are used traditionally for the reduction of stress and anxiety. In these applications, they are approved as plant medicines by the European Medicines Agency. Some studies performed on humans and animals show positive results in models of anxiety and stress, but there are still little available literature data related to the mechanisms of their action [1,2,3,4,5,6,7]. Medicinal lavender belongs to the plants with potential anticonvulsant, anti-depressant, anti-anxiety, hypnotic, and sedative properties [7,8,9,10,11].

Most scientific research on lavender has focused on the essential oil, which is credited with its main therapeutic activity. The calming effect of this essential oil is likely due to the effect of its components on GABA_A receptors, resulting in the inhibition of neuronal activity. This was confirmed by Linck et al., who, in a study on mice, assessed the calming properties of one of the main components of lavender oil—linalool [3]. In the experiment, mice were placed for an hour in inhalation chambers saturated with linalool in a concentration of 1% and 3%. It was shown that the inhaling of linalool in studied concentrations prolonged sleeping time and reduced body temperature. Additionally, at 3% concentration, it reduced locomotion of animals without disturbing their motor coordination [3]. Another study showed that the smell of lavender essential oil reduces the level of anxiety in dental patients [4]. Lavender flowers also have antidepressant properties, as demonstrated in a study that examined the effect of aqueous lavender extracts on rats. It has been shown that rosmarinic acid and apigenin glycosides found in the aqueous extract may be responsible for the antidepressant effect [1,2]. Despite the long tradition of application, research on specific aspects of lavender’s biological activity is continuing in both in vitro and in vivo experiments [7,12,13,14,15,16,17].

Data related to the composition of essential oils obtained from the various varieties of L. angustifolia are available in the literature, whereas only few data present the results of studies on the composition and application of extracts [9,18,19]. Moreover, there are scarce data on the content of sesquiterpenic acids in lavender extracts in the literature. In our previous studies, the presence of valerenic and acetoxyvalerenic acids in leafy stalks of L. angustifolia was found, but in those studies their contents in lavender flowers were not determined [20]. Interesting reports of biological activity of these compounds encouraged us to carry out broader research in relation to different morphological parts and cultivars of lavender. The aim of the present study was to determine and compare the contents of valerenic and acetoxyvalerenic acids in flowers and leafy stalks of two various cultivars of Lavandula angustifolia—‘Ellagance Purple’ and ‘Blue River’. The analyses of sesquiterpenic acids in methanolic extracts from the various samples of lavender were performed using the high-performance liquid chromatography (HPLC) method.

2. Results

Based on the performed experiments, it was found that all tested extracts obtained from various morphological parts of the two examined cultivars of L. angustifolia contained acetoxyvalerenic and valerenic acids. Their structures are shown in Figure 1, while an example of the HPLC chromatogram is shown in Figure 2. The comparative analysis of the lavender extracts and the chromatographic standards performed in the same conditions enabled the identification of both acids. The method showed high linearity of the calibration curve, with a coefficient of correlation (R²) of 0.9999. The results of the chromatographic analyses are presented in

Table 1. Sesquiterpenic acids contents in various cultivars and morphological parts of medicinal lavender (Lavandula angustofolia Mill.).

Sesquiterpenic Acid	Content of Sesquiterpenic Acids (mg/100 g d.m. ± SD 1)
	Flowers		Leafy Stalks
	‘Blue River’	‘Ellagance Purple’	‘Blue River’	‘Ellagance Purple’
Valerenic acid	1.75 a ± 0.02	0.50 b ± 0.06	1.16 a ± 0.02	0.81 b ± 0.02
Acetoxyvalerenic acid	65.80 a ± 1.51	6.50 b ± 0.18	50.10 a ± 1.14	12.50 b ± 0.43

¹ SD—standard deviation, n = 3; ^a,b—values with different letters differ significantly in flowers or leafy stalks between two different cultivars (p < 0.05).

. It can be noticed that the amounts of acetoxyvalerenic acid were significantly higher than the amounts of valerenic acid. Moreover, the content of analyzed sesquiterpenic acids in the flowers and the leafy stalks of various lavender cultivars were significantly different (p < 0.05). The content of valerenic acid in flowers ranged from 0.50 mg/100 g d.m. in the ‘Ellagance Purple’ cultivar to 1.75 mg/100 g d.m. in the ‘Blue River’ cultivar. In turn, leafy stalks contained 0.81 mg/100 g d.m. of valerenic acid in the ‘Ellagance Purple’ cultivar and 1.16 mg/100 g d.m. in the ‘Blue River’ cultivar. It should be noticed that the ‘Blue River’ cultivar contained about 10 times more acetoxyvalerenic acid (65.80 mg/100 g d.m.) in flowers and four times more acetoxyvalerenic acid in leafy stalks (50.1 mg/100 g d.m.) when compared to the ‘Ellagance Purple’ cultivar.

3. Discussion

Nowadays, there is a noticeable increasing need to use sedative drugs, including herbal medicines, which may be used in states of anxiety, fear, or difficulty sleeping. Valerian root (Valerianae radix) is widely considered to be one of the most powerful plant raw materials with calming properties. The sedative and hypnotic effects, according to some researchers, are attributed to rosmarinic and chlorogenic acid, flavonoids, and sesquiterpenic acids, mainly found in valerian (Valeriana officinalis) [21]. The sedative properties of valerian extracts are the result of the combined effect of many chemical compounds, but clinical studies have shown that they are conditioned mainly by the content of valeranone and sesquiterpenic acids: valerenic and acetoxyvalerenic [22]. Valerenic and acetoxyvalerenic acids are sesquiterpenoic compounds found in various species of valerian (V. officinalis). These compounds are known from their various pharmacological activities including sedative, anxiolytic, and antidepressant activities, as well as from their roles in brain-derived neurotrophic factor and gastrointestinal motility [23]. Valerenic acid and its derivatives are recognized as the main sedative factor of V. officinalis [24]. It was found that valerenic acid allosterically modulated GABA_A receptors in the central nervous system and induced an anxiolytic activity [24,25]. The variable chemical composition is a characteristic feature of plant raw materials, depending on many factors, including soil, climate, development stage, method and time of extraction, or genetic factors, which determine the existence of various chemotypes within one plant species [26,27,28]. For comparison, the results of the research carried out by Hassan et al. [29] showed that the content of valerenic acid in different species and morphological parts of Valeriana L. was in the range of 40–70 (mg/100 g d.m.) and acetoxyvalerenic acid was in the range of 20–210 (mg/100 g d.m.).

Recently, not only valerian root but also lavender flowers were recognized as possible alternative and milder substitutes for synthetic sedative drugs, such as benzodiazepines. This is important, because the demand for natural remedies is significantly growing due to the observed increase in stress-derived lifestyle diseases, resulted in the problem of benzodiazepine drug addictions [28]. In addition to numerous studies on the antimicrobial activity of lavender, it was reported that this plant is also characterized by sedative and somnifacient activity [5,11,20]. It was believed that lavender’s sedative properties are associated with the content of linalool, which determines this activity by inhibiting the release of acetylcholine and its effect on ionic conductivity in neurons [30]. Pharmacological studies confirmed the sedative properties of the essential oil from lavender flowers as well, as two of its most important components: linalool and linalool acetate [5]. However, it was found that the sedative effect of lavender on the central nervous system is not only related to linalool and its derivatives that are present in essential oil. It was noticed that content of sesquiterpenic acids in lavender extracts, which are characterized by a sedative effect, may also play a significant role [31]. There are not much data in the literature confirming the sedative, calming, and somnifacient activity of lavender extracts. Nevertheless, it was found that alcoholic and aqueous lavender extracts exert a strong sedative effect, comparable to that of diazepam [9].

Our studies confirmed the presence of sesquiterpenic acids characterized by sedative activity in L. angustifolia. Chromatographic analyses of extracts from lavender have shown that two studied cultivars and their various morphological parts differ in the contents of valerenic and acetoxyvalerenic acids. The ‘Blue River’ cultivar was characterized by significantly higher contents of both valerenic and acetoxyvalerenic acid in comparison to the ‘Ellagance Purple’ cultivar, which suggests that it can be more useful in some medical applications. Moreover, the obtained results indicate that various morphological parts of lavender, not only flowers but also leafy stalks, may be valuable sources of acetoxyvalerenic and valerenic acids and have potential therapeutic uses in the treatment of some central nervous system disorders. This contributes to the explanation and confirms the pertinence of traditional therapeutic applications of lavender in herbal medicine—e.g., for the cure of anxiety or insomnia.

4. Materials and Methods

4.1. Plant Material

The plants were collected from the Experimental Station of the Department of Horticulture at West Pomeranian University of Technology in Szczecin (Poland). The plant samples were identified at the Department of Horticulture, Faculty of Environment Management and Agriculture of West Pomeranian University of Technology (Szczecin, Poland), based on voucher specimen number 195 from the Institute of Natural Fibres and Medicinal Plants (Poznań, Poland). Two various cultivars of medicinal lavender (L. angustifolia) were used in the experiments: ‘Blue River’ and ‘Ellagance Purple’. Plant materials were collected in July by cutting the plants before they fully bloomed. The collected raw materials were cleaned, dried at room temperature, packed in paper bags, and kept in a dry place away from light until the research began. Before starting the research, the plant raw materials were separated into flowers and leafy stalks. The flowers were crushed in a mortar, while the leafy stalks were ground in a laboratory grinder (A10, IKA, Staufen, Germany).

4.2. Preparation of Extracts for Sesquiterpenic Acids Analyses

The dried and crushed flowers and the grounded leafy stalks of ‘Blue River’ and ‘Ellagance Purple’ lavender cultivars were effectively extracted using anhydrous methanol (99.8%, Sigma-Aldrich, Saint-Louis, MO, USA) under reflux. The samples of plant material (1.5 g of dried flowers or leafy stalks) were extracted with 20 mL of anhydrous methanol by heating for 30 min in a boiling water bath under reflux. After cooling and filtration using a glass wool, the precipitate with the filter was placed in a round-bottomed flask; 20.0 mL of anhydrous methanol was added and heated again under reflux for 15 min. After cooling, the solution was filtered and then both filtered solutions were combined and filled up to 50.0 mL with anhydrous methanol.

4.3. Determination of Sesquiterpenic Acids by High Performance Liquid Chromatography Method (HPLC)

Analyses were carried out using a Shimadzu HPLC chromatograph (Shimadzu Corporation, Kyoto, Japan) equipped with a binary pump, a column thermostat, an autosampler, and an SPD-M20A UV-VIS Photodiode Array Detector. Chromatographic analysis conditions were as follows: a stainless steel STR-ODS II column, 250 × 4 mm, filled with 5 μm octadecyl silica gel in stationary phase (Shinwa Chemical Industries Ltd., Kyoto, Japan). Mobile phase A: acetonitrile/phosphoric acid solution 5 g/L (20/80); mobile phase B: acetonitrile/phosphoric acid solution 5 g/L (80/20). The mobile phase components were of analytical grade and purchased from Merck Co. (Darmstadt, Germany). The fow rate of the mobile phase was 1.5 mL/min; elution was programmed by combining isocratic and linear gradient elution. The detector analytic wavelength was 220 nm; the injection volume was 20 μL. The quantification was carried out using an external standard method. Valerenic acid and acetoxyvalerenic acid standards were purchased from Supelco (Supelco Inc., Bellefonte, PA, USA). All lavender extracts samples were analyzed three times and the results obtained were averaged. The data about valerenic acid and acetoxyvalerenic acid from ChemBioDraw Ultra 13.0 programm described in Supplementary Materials.

4.4. Statistical Analysis

The results obtained from the chromatographic analyses were expressed as a mean value ± SD (standard deviation, n = 3). To analyze and compare the data obtained and to identify the values with significant differences (p < 0.05), one-way analysis of variance (ANOVA) coupled with the Tukey post hoc test was applied. Statistical analysis was performed using the PQStat v. 1.6.2 package (PQStat Software, Poznan, Poland).

5. Conclusions

The aspect of variability of chemical composition within a given plant species is interesting, and searching of new valuable plant varieties can be one of the innovative approaches in the development of plant-based medicines. Our results indicate that the ‘Blue River’ lavender cultivar was characterized by significantly higher contents of both valerenic and acetoxyvalerenic acid and, for this reason, it may be more useful for the production of herbal remedies characterized by a sedative effect. The obtained results encourage further research on a larger scale, which would allow a comparison of the composition of extracts from other different cultivars of L. angustifolia and correlate them with their sedative activity, by in vivo experiments. Furthermore, in-depth studies related to mechanism of action, safety in medicinal applications, and possible interactions with synthetic drugs should be conducted.