Phytochemical analysis of leaves and stems of Physalis alkekengi L. (Solanaceae)

2.1 Plant material

According to the objectives of the study, two individual samples of the aerial parts of P. alkekengi plants – the leaves and the stems – were analyzed (Figure 1). To follow the phenotype-related differences in the phytochemical composition, two phenotypes of the species were compared, representing distant regions of plant occurrence in Bulgaria. The leaves and stems of the first phenotype (PA-NB) were collected from a natural habitat in North-Eastern Bulgaria (Ivanski village, Shumen region), with geographical coordinates 43°07′24″N 27°04′35″E. The plants of the second phenotype (PA-SB) were collected from a population in Central Southern Bulgaria (the city of Plovdiv, Plovdiv region), 42°14′26″N 24°70′24″E. The plants were identified at the Department of Botany, Plovdiv University ‘Paisii Hilendarski’. Both aerial parts were air-dried and kept in cloth bags at 5–8°C until analysis.

Figure 1

Fresh leaves and stems of P. alkekengi (photo by authors).

2.2 Methods of analysis

The moisture content of the raw materials was determined by drying to constant weight, and the results from the chemical analyses were given further on a dry weight (DW) basis.

The ash content was determined according to AOAC [19], by mineralization of the samples at 550°C for 5 h.

The cellulose content was determined applying a slight modification of the method described previously [20], in which hydrolysis was completed with 16.5 mL of 80% CH₃COOH and 1.5 mL of concentrated HNO₃ at boiling for 1.5 h, and the dried solid residue (105°C for 24 h) was quantified.

The protein content was determined according to the standard method [19], on a UDK 152 system (Velp Scientifica Srl, Usmate Velate, Italy). The HPLC analysis of amino acids followed a previously described procedure [21], using an Elite LaChrome (Hitachi, Tokyo, Japan) unit (DAD; reverse phase C18 AccQ-Tag column, 3.9 mm × 150 mm).

Mineral elements were determined by atomic absorption spectrophotometry using a Perkin Elmer/HGA 500 (Norwalk, CT, USA) instrument. The identification and quantification of metal ions were performed according to the analytical procedure described in ref. [21].

The dried leaves of P. alkekengi were extracted with a non-polar solvent, n-hexane, to obtain the respective crude extracts (leaf concretes) [22]. Two-fold extraction was carried out, at 40°C temperature and hydromodule 1:30 (raw material:solvent, w/v) for 1 and 0.5 h, respectively. The solvent was completely evaporated from the combined extracts using a rotary vacuum evaporator, at temperature not exceeding 40°C.

The GC-MS analysis of the leaf concretes was carried out on an Agilent 7890A gas chromatograph coupled to an Agilent 5975C inert XL EI/CI mass selective detector (Agilent Technologies Inc., Santa Clara, CA, USA). The column was HP-5 ms (30 m × 250 mm × 0.25 μm, i.d.); the carrier gas was helium, run at a constant rate of 1 mL/min. Oven temperature increase was at a rate of 5°C/min, from 35°C (held for 3 min) to 250°C (held for 3 min); the total run time was 49 min, and the split mode was 30:1. The MS conditions were as follows: ionization voltage 70 eV, electron impact mode, scan range 50–500 m/z, MS source temperature 230°C, injector temperature 150°C, and detector temperature 250°C. MS data were processed using the tools of 2.64 AMDIS software (NIST, Gaithersburg, MD, USA). The identification of volatiles was according to their retention times, the calculated retention (Kovat’s) indices (RI), and the obtained MS fragmentation data, compared with mass spectra library data [23,24, own library]. A standard mixture of n-alkanes (C₈–C₄₀) in n-hexane was used to calculate the relative RIs and analyzed under the same conditions as described above. Volatile contents were given as percentage of the total ion current (TIC) after normalization of peak areas in the TIC chromatograms.

2.3 Statistics

All analyses were repeated three times and the data were presented as the mean values ± the standard deviation (SD).

3.1 Basic chemical indices of P. alkekengi leaves and stems

The basic chemical indices of the analyzed plant materials are presented in Table 1. The moisture content of the air-dried leaves and stems was relatively low (9.37–12.11%), thus facilitating the safe storage of the plant materials [25]. Data revealed that there were no significant differences between the phenotypes with regard to the analyzed macro components – protein, cellulose, and ash. The two aerial parts differed by chemical composition; the leaves reasonably were with higher moisture content and contained more protein and ash and less cellulose than the stems of the respective phenotype.

The results from the identification of the amino acid composition of leaf and stem protein, after protein hydrolysis, are presented in Table 2. Seventeen amino acids were identified in each of the plant samples. As seen from the data, there was variation in the individual amino acid distribution, both on a phenotype and plant part basis. The most abundant amino acids in the leaves of the two phenotypes were arginine (22.3 mg/g), aspartic acid (8.8 mg/g), serine (8.5 mg/g), and alanine (8.2 mg/g) (in PA-SB) and arginine (21.3 mg/g), aspartic acid (18.4 mg/g), glutamine (11.2 mg/g), and alanine (11.0 mg/g) (in PA-NB), respectively. As seen from the table, the leaves of PA-NB phenotype contained correspondingly high levels of other essential amino acids, as well, such as lysine (10.6 mg/g), phenylalanine (10.0 mg/g), and isoleucine (7.5 mg/g). The dominant amino acids in the stems of PA-SB were proline (8.8 mg/g), aspartic acid (7.7 mg/g), and histidine (6.4 mg/g) and those in the stems of PA-NB were isoleucine (12.8 mg/g) and tyrosine (6.6 mg/g). As a general observation, the amino acid concentrations were higher in the leaves than in the stems, especially for amino acids, such as arginine, phenylalanine, lysine, alanine, valine, and others; some exceptions with higher stem contents were histidine and proline (in PA-SB) and tyrosine and isoleucine (in PA-NB). Therefore, the amino acid composition of P. alkekengi foliage protein was influenced by the phenotype factor, a finding that might be regarded in future use considerations. Data in Table 2 revealed substantial protein quality in P. alkekengi leaves and stems, due to the high proportion of essential amino acids. The ratio between essential and non-essential amino acids in the studied aerial parts of PA-SB and PA-NB phenotypes varied in the range from 0.8:1 to 1.1:1.

The results about the individual mineral composition of the leaves and the stems of P. alkekengi are presented in Table 3. Data revealed that both aerial parts contained considerable amounts of micro and micro minerals, as well as some differentiation between the two phenotypes in the study. The dominant macro mineral in all samples was K; the leaves of PA-NB contained comparable amounts of Ca, as well. The distribution of K and Mg was predominantly in the leaves, and that of Na was predominantly in the stems. In general, PA-NB phenotype contained higher levels of Ca, Mg, and Na, compared to PA-SB phenotype (both leaves and stems). The same trend was observed for the accumulation of micro minerals, as well, and the leaves and stems of PA-NB were richer in Cu, Fe, and Zn than the respective plant parts of PA-SB. The heavy metals had different presence in the two phenotypes, too, obviously reflecting the influence of soil and environmental conditions of the respective plant habitats [26]. Pb was identified only in the aerial parts of PA-NB, while Cr was practically found only in PA-SB leaves and stems. Cd was not detected in either of the plant samples. It is hard to parallel those results to previous data; as stated earlier, the phytochemical research on the species has regarded almost exclusively fruit and calyx constituents [2].

3.2 Volatiles (by GC-MS) in P. alkekengi leaf concretes

The diversification of plant species available for obtaining natural bioactive extracts, as a better alternative of synthesized components, has become essentially important for the fragrance industry and phytopharmacy [27], and rigorous research has been conducted in the last decades [28]. Therefore, the leaves of P. alkekengi alone were further processed by extraction with n-hexane to obtain the respective leaf concretes, and the general descriptions of the obtained aromatic products are presented in Table 4.

The results from the identification of GC-MS volatiles in the two concretes from P. alkekengi leaves revealed significant variations between the phenotypes, both in the presence of individual components (Figure 2 and Table 5) and in their distribution by chemical classes (Figure 3).

Figure 2

TIC chromatograms (GC-MS) of concretes from P. alkekengi leaves: (a) phenotype from Central Southern Bulgaria, PA-SB, and (b) phenotype from North-Eastern Bulgaria, PA-NB.

Figure 3

Distribution of compounds (GC-MS) in concretes from P. alkekengi leaves: PA-SB – phenotype from Central Southern Bulgaria; PA-NB – phenotype from North-Eastern Bulgaria.

In the concrete from PA-SB, leaves were identified 28 components (97.99% of the total composition); 12 of them were with over 1% content. The major components (over 3% content) were seven, as follows: phytol acetate (48.60%), (2E,6E)-methyl farnesoate (8.38%), (2E,6E)-farnesoic acid (5.88%), (2E,6E)-farnesyl acetate (5.79%), (5E,9E)-farnesyl acetone (4.89%), 1-octadecene (4.67%), and myristic acid (3.48%). The volatile profile of PA-SB leaves was dominated by diterpenes (49.96% of the identified composition) and oxygenated sesquiterpenes (35.61%).

In the concrete from the leaves of the second phenotype, PA-NB, 32 components (97.50% of the total composition) were identified; 19 were over 1%. The major compounds (with over 3% content) were as follows: phytol (17.90%), n-hexadecanol (10.72%), methyl hexadecanoate (9.25%), (2E,6E)-farnesoic acid (8.14%), 3α-acetoxy-manool (7.35%), 3α-14,15-dihydro-manool oxide (5.56%), oleic acid (4.69%), methyl octadecanoate (3.41%), 3α-hydroxy-manool (3.23%), and 4,8,12,16-tetramethylheptadecan-4-olide (3.16%). The dominant groups of volatiles in the PA-NB leaf concrete were oxygenated aliphatics (40.01%) and diterpenes (35.19%). As seen from Table 5, only four compounds were identified in both concretes, (2E,6E)-farnesoic acid, (2Z,6E)-farnesyl acetate, phytol, and phytol acetate, but at greatly different concentrations. The rest of the major and minor components in the two concretes were phenotype specific. Correspondingly, there were significant differences in the distribution of most of the chemical classes on a phenotype basis, as well. As seen from Figure 3, the major differences were with regard to the presence of oxygenated aliphatics (5.48% in PA-SB vs 40.01% in PA-NB) and oxygenated sesquiterpenes (35.61% in PA-SB vs 9.54% in PA-NB); significant variation was found in the share of oxygenated monoterpenes and aromatic compounds, as well. Therefore, the results suggested that the phenotype factor (environment, harvest period, probably genetics, and other details) should be considered important in the assessment of P. alkekengi leaves as a resource for obtaining aromatic products for cosmetic use, as it strongly affects the volatile composition of the products. Of course, additional research devoted to that subject is needed, to make conclusions that are more definite [10,11].

To the best of our knowledge, there were no previous data about the identification of P. alkekengi leaf volatiles, as well as previous attempts to obtain certain aromatic products for cosmetic use (leaf concretes), moreover, in a comparative mode regarding different phenotypes. As already stated, the identification of biologically active constituents in P. alkekengi tissues has been focused on fruit and calyces, as they are used in folk medicine, while phytochemical investigations of other plant parts (leaves, stems, roots) are rather scarce [29]. For those reasons, it is hard to make direct comparison between our results and published data, although some studies have identified similar chemical classes and individual volatile components in different P. alkekengi tissues, such as terpenoids, phenylpropanoids, aliphatic derivatives, organic acids, sucrose esters, and others [2,13,14,30,31,32].

The results characterized the leaves of P. alkekengi as good sources for obtaining aromatic products for cosmetic purposes, despite the significant difference in the concrete-yielding potential of the studied phenotypes. The registered concrete yields were sufficiently high (1.02%, PA-SB and 4.81%, PA-NB), approximating those from other Solanaceae species currently being used as plant materials for obtaining similar concentrated aromatic products [22,33]. The different concrete yields could be reasonably related to the impact of environmental factors, as the soil and climatic conditions of the regions of plant populations are considerably different [34]; some genetic differences might also be involved, although genotype characterization was not regarded in this study. Both leaf concretes were undistinguishable in terms of visual and olfactory assessment: semi-solid waxy masses with yellow-orange color and specific odor. The color and the appearance of the concretes correlated well with the chemical composition of the extracts (as presented below); the color was due to the extraction of natural pigments, and the semi-solid waxy texture was due to the presence of aliphatic hydrocarbons, with equal share in the two extracts.

The presence of various aroma active volatile classes identified in the study spoke in favor of the potential of P. alkekengi leaf extracts for cosmetic and perfumery purposes [28,33]. As seen from the data (Table 5 and Figure 3), the aroma-active terpenoid derivatives (in particular, diterpenes and oxygenated sesquiterpenes) and oxygenated aliphatic derivatives (alcohols, acids, and esters) were the major chemical classes in both concretes. Several individual constituents with established aroma-contributing properties were found in significant concentration in those classes; for example, farnesyl acetate (with floral type odor), farnesyl acetone (with fruity, winey, creamy odor), phytol acetate (with waxy, fruity, balsamic odor), methyl farnesoate, 3α-hydroxy-manool, 3α-14,15-dihydro-manool oxide, 3α-acetoxy-manool, farnesoic acid, myristic acid, hexadecanol, methyl hexadecanoate, methyl octadecanoate, and others; a number of minor components with known aroma-active potential were also present [35]. In addition, the leaf concretes did not contain fragrance substances referenced as human allergens [28,36], with the single exception of farnesol identified as a minor component (0.67%) in PA-SB concrete. Considering the safe future cosmetic use of P. alkekengi leaf concretes, attention should be paid to the presence of phthalates in the extract from PA-NB leaves (isobutyl phthalate, 2.32% and monoethylhexyl phthalate, 0.61%), as they are specified in the list of banned ingredients of cosmetic products [36]. The identified phthalate esters in the concretes might be contaminants but also natural plant constituents, as previously reported [37]. Phthalates have been found as naturally occurring constituents in different edible and essential oils, and a number of edible plants; phthalate contamination in plants has been reported to occur through different transfer mechanisms, most probably through migration and accumulation from the surrounding environment into the roots and the aerial parts [38].

Finally, it should be outlined that the ecological impact of medicinal plants collection and processing is an important aspect in the general discussion about the prospective use of medicinal plants as sources of bioactive compounds. World Health Organization reports that various plant fractions and their dynamic constituents are utilized as traditional medicines by 80% of the world population. Medicinal plants are used for the treatment of different infections and received worldwide attention. As a medicinal plant within the scope of the national legislation [8,39,40,41,42], P. alkekengi is not under a special regime of protection and use, and there are reasonable grounds to suggest that the possible utilization of its aerial parts (e.g. for pharmaceutical and perfumery products) would not be problematic in terms of disturbances of the populations and species conservation [8,43,44]. Moreover, the plant is vigorous, highly adaptive, rapidly multiplying, invasive in some environments, disease resistant, and with a good cultivation potential [2,5,6,31,45]. Thus, in a broader sense, the natural occurrence and the targeted cultivation of P. alkekengi could provide sufficient availability of plant biomass; moreover, such considerations are in line with the contemporary requirement for the sustainable utilization of the natural Bulgarian medicinal flora [44].

Therefore, the results from the non-targeted GC-MS analysis, as well as those from the rest of the phytochemical assays in this study, could be considered confirmative for the potential of P. alkekengi leaves and stems for obtaining functional derivatives [22,27,46,47]. Similar practices have already been established in the utilization of various plant organs and wastes, including a number of medicinal and cultivated plants from the Solanaceae family, such as Solanum nigrum L., Solanum dulcamara L., Datura stramonium L., and Nicotiana tabacum L. [15,48,49,50,51].