1 Introduction

Since time immemorial, Iran has been reputed as the treasure-house of precious medicinal plants of the world on account of the vast diversity in climatic condition. Up to 8200 plant species are recognized throughout the country, of which 1900 are endemic [1]. The genus Alcea, widely scattered throughout different areas of the country, is a main component of this botanical richness. Alcea is a large genus recognized in the family Malvaceae, including about 70 species, mostly perennial, sometimes annual and biennial ones, indigenous to Asia and Europe [2]. The genus has undergone marked species radiation in Iran including 34 endemic species, and numerous taxa are naturally scattered across west and southwest Iran [3].

Given its striking appearance and geometry, Alcea could be easily distinguished from other genera; the flowers are either solitary or arranged in racemes or fascicles on an unbranched upright stem rising to over 2 m at a fast rate. This genus is also characterized by having long notched petals ranging in color from white and yellow to pink and purple. The leaves usually appear on long petioles and are often lobed or toothed, and adorn the whole parts of the herbage. From an industrial and medicinal point of view, Alcea is among the most important genera recognized in the family Malvaceae.

During the past decade, traditional systems of medicine have become a topic of global importance. In many developing countries, a large proportion of the population relies heavily on traditional systems of medicine to meet primary health care needs for historical and cultural reasons. The genus has long been traditionally used around the world as e.g. an antitussive, sedative, antibacterial and anti-inflammatory agent for thousands of years. Besides being well appreciated as ornamentals, members of the genus have also found a wide range of applications as either functional foods or nutraceuticals. One of the main claims of the food, cosmetic and dying industries nowadays is for natural colorants to replace synthetic dyes, and anthocyanins are the principal candidates [4]; with high levels of anthocyanins, purple-colored flowers of the genus Alcea have been utilized as a healthy food commodity and source of natural food colorants in Asia.

Today we clearly know that the biosynthesis of secondary metabolites in medicinally active plants is tightly regulated and is not by no means a random process. The expression of the genes involved in the biosynthesis of secondary metabolites shows tissue- and organ-specific distribution patterns, and hence the capacity for biosynthesis and accumulation can vary greatly within different tissues or organs of a plant [5, 6]. Alcea is a botanical source for various pharmaceutically active components. Even though different parts of the plant including flowers and herbage contain a variety of active substances e.g. starch, mucilage, pectin, sucrose, phenolics etc. [7], little is known about the distribution of these phytochemicals in different plant organs. This paper presents data on the qualitative and quantitative distribution of phenolic and mucilaginous compounds in the flowers and herbage of Alcea plants.

Moreover, few ethnobotanically important taxa have been scientifically evaluated for their possible medical application. Safety and efficacy data are available for even fewer plants, active ingredients and their extracts, and the preparations containing them. Furthermore, in most countries the herbal medicines market is poorly regulated, and herbal products are often neither registered nor controlled. Despite the potential importance of the Alcea-derived chemicals in several industries, little attention has been devoted to exploring this genus worldwide. Likewise, the industrial importance of the genus has always been regretfully neglected in Iran, and curative application of this national wealth has only been considered from the ethnobotanical point of view among indigenous people of the country. Assurance of the safety, quality, and efficacy of medicinal plants and herbal products has now become a key issue in industrialized and in developing countries. Therefore, modern assessments of the genus in Iran e.g. characterization of its active substances and biological activities, is still missing. To fulfill this information gap, here we reported the phytochemical properties and antioxidant activities of three species of the genus Alcea growing in Iran aimed at taking the first steps towards introducing the genus for further future investigations.

2 Materials and methods

2.1 Sample collection

The distribution range of three species of the genus i.e. Alcea koelzii Riedl, Alcea arbelensis Boiss. and Hausskn. and Alcea aucheri (Boiss.) Alef. (var. aucheri and var. lobata) in Iran was determined according to Flora Iranica and other reliable sources. The taxa were collected at the flowering stage from their natural habitats in the west and southwest Iran over the growing season in spring and summer 2016 (Table 1). Herbarium specimens (Alcea koelzii, 2420; A. arbelensis, 1833; A. aucheri var. aucheri, 3513; A. aucheri var. lobata, 1949) are kept at Agricultural and Natural Research Center of Chaharmahal and Bakhtiari (C and B) province. Taxonomical identification was made by an expert botanist (Asghar Shahrokhi, Education Organization, C and B Province, Shahrekord, Iran); samples were verified based on some material available at Agricultural and Natural Research Center of C and B province.

Table 1 Geographical distribution of three Alcea populations throughout southwest Iran
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2.2 Preparation of the ethanolic extracts

Samples were shade-dried at room temperature, and different plant organs, including petals and herbage (leaves and stems), were powdered to homogenous particle size. Plant materials were macerated in 70% ethanol at room temperature and were regularly shaken for 12 h. After filtration, the crude extract was evaporated under vacuum to dryness and kept for 24 h at 4 °C until assayed.

2.3 Phytochemical characterization

Total phenolic content (TPC), total flavonoid content (TFC), total anthocyanin content (TAC), total mucilage content (TMC) were measured. The TPC of extracts were determined according to the method published by Liang et al. [8] and results were expressed as mg gallic acid equivalents per g dry weight of the sample (mg/g GAE). TFC, expressed in mg quercetin per g dry weight of plant material (mg Q/g DW) was determined according to Nazif [9]. The methods referenced by Lee et al. [10] were used to measure the TMC and TAC, respectively.

2.4 Free radical scavenging activity

The antioxidant activity of the ethanol extracts was measured by the DPPH free radical scavenging assay method as previously described [11]. In the DPPH assay, the free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) was used to find the antioxidant activity of flower extracts. In a modified assay, 2 ml of 0.1 mM solution of DPPH radical in methanol was mixed with 2 ml of extract. After mixing, it was left for 15 min at room temperature. The DPPH radical inhibition was measured at 517 nm by using a spectrophotometer (Unico 2100). The antioxidant activity was calculated using the following equation:

$$100 - \left[ {\frac{\left( A \right)sample - \left( A \right)blank}{\left( A \right)control } \times 100} \right]$$

where A stands for the absorbance of the color formed in the spectrophotometer cell, using DPPH as control (no extract), and blank consisted of only methanol. The IC50 (µg/ml) of each sample (concentration in µg/ml required to inhibit DPPH radical formation by 50%) was calculated. This was obtained by interpolation and using linear regression analysis.

2.5 Statistical analysis

The data were statistically analyzed using nested ANOVA with organ, population and species effects (SPSS 16.0, SPSS Inc., USA) computer software. In order to understand the relationships among different Alcea samples and to determine the main constituents influencing the chemical variability, a covariance data matrix composed of 13 samples and 4 variables was prepared and subjected to Principal Component Analysis using Minitab 16.0 (Minitab Inc., USA).

3 Results and discussion

3.1 Phenolic composition

It has been widely proposed to prepare herbal medicines as mixtures (extracts) rather than isolates, as their activity is potentiated when delivered in mixtures [12]. Phenolic compounds are a vital part of the human diet and are of considerable interest due to their biological and physiological activities (e.g. antioxidant, anticarcinogenic, antimutagenic and anti-inflammatory effects) [13]. They can basically be categorized into several classes, of which flavonoids are bioactive substances occurring widely in food and industrial plants. Anthocyanins are among the most prevalent flavonoids and occur in a vast variety of plants [14].

3.1.1 Total phenolic content

TPC of Alcea extracts are shown in Tables 2, 3 and 4. TPC was quantified within and between species in both flowers and herbage. In general, findings showed that TPC in the flowers was higher than that of the herbage of the taxa examined (Table 2). The only exception was the values measured in A10 from A. aucheri var. aucheri, in which a reverse pattern was observed (Table 4). The highest TPC in flowers was found in A. aucheri var. aucheri, followed by A. koelzii, A. arbelensis and A. aucheri var. lobata (Table 3).

Table 2 Phytochemical components and antioxidant activity according to organ
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Table 3 Phytochemical components and antioxidant activity of flower and herbage according to species
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Table 4 Phytochemical components and antioxidant activity according to ecotype and organ for each species
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Regarding TPC in the floral organs, with the exception of the specimen A7 from A. aucheri var. lobata (27.26 mg GAE/g DW), in which TPC was markedly lower than other specimens, no significant difference was observed within the taxa in each species. However, this parameter varied significantly in the herbage of the taxa within each species. TPC in A1 (53.51) and A2 (52.69) from A. koelzii, A5 (53.69) from A. arbelensis and A10 (58.15) from A. aucheri var. lobatawas significantly higher, respectively, than that of other taxa within the species.

Moreover, the variation in TPC between four Alcea taxa was quantitatively significant. Generally, flowers showed high TPC, and yielded values ranging from 27.26 (A. aucheri var. lobata) to 82.59 (A. aucheri var. aucheri) mg GAE/g, showing a difference in TPC up to three-fold. The highest TPC levels were measured in A13 (A. aucheri var. aucheri) (82.59 mg GAE/g DW), and the lowest in A7 (A. aucheri var. lobata) (27.26 mg GAE/g DW). The highest (58.15 mg GAE/g DW) and lowest (26.83 mg GAE/g DW) values of TPC were recorded in the herbage of A10 and A7 samples, both belonging to A. aucheri var. lobata (Table 4).

3.1.2 Total flavonoid content

TFC for Alcea extracts is shown in Tables 3 and 4. TFC was mostly higher in the crude extracts obtained from the flowers (Table 2), ranging from 8.06 to 16.15 mg Q/g DW. TFC was higher in A1, A2, A3 (16.15, 16.12, 16.06 mg Q/g DW, respectively) all of which belonging to A. koelzii, while the lower values were achieved in A9 from A. aucheri var. lobata (8.06 mg Q/g DW), A6 from A. arbelensis (8.07 mg Q/g DW) and A4 from A. koelzii (8.08 mg Q/g DW). The highest and the lowest TFC in the herbage was detected in A5 from A. arbelensis (15.90 mg Q/g DW), and A4 from A. koelzii and A11 A. aucheri var. lobata (7.89 mg Q/g DW), respectively (Tables 3, 4).

The flavonoids are probably the most important single group of phenolic compounds in plants, comprising a group of thousands of aromatic compounds, including anthocyanins, flavonols, flavones, flavanones, flavan-3-ols, and isoflavones [12]. Epidemiological data associate some important biological properties e.g. antioxidant, anti-inflammatory, and antimicrobial activities, as well as cardioprotective and anticancer effects, to flavonoids. They are sensitive to degradation because of their hydroxyl and ketone groups and unsaturated double bonds [15].

3.1.3 Total anthocyanin content

Results showed that the content of anthocyanins markedly varied, as influenced by genetic structure of the examined taxa, environmental condition and the organ of choice [16,17,18,19]. As shown in Table 2, the flowers contained markedly higher TAC in comparison to the herbage of the examined taxa. The variation of total anthocyanin content (TAC) observed in the crude extracts of four Alcea species are summarized in Tables 3 and 4. TAC was highest (934.97, 871.83 and 261.42 mg/ml) in the flowers of A12 (A. auchrei var. aucheri), A9 (A. auchrei var. lobata) and A13 (A. auchrei var. aucheri), respectively. The minimum levels were found in A1 (6.67 mg/ml), followed by A5 (8.07 mg/ml) and A3 (8.53 mg/ml).

TAC in the flowers of A12 was over 30-fold higher that of the herbage. TAC in the herbage of the taxa yielded values between 1.34 mg/ml in A2 (A. koelzii) and 48.43 mg/ml in A6 (A. arbelensis). Thus, flowers contained a higher level of anthocyanin than that of the herbage of the same sample. Exceptions include the white-colored flowers taxa A1, A3 (A. koelzii) and A5 (A. arbelensis).

Anthocyanins, an important class of flavonoids, are water-soluble vacuolar pigments, plentiful in plant foods, many fruits, and vegetables. Anthocyanin-rich plants have long been used in folk medicine throughout the world for the treatment of vision disorders, microbial infections, diarrhea, and many others. It is only in recent years, however, that epidemiological data verified some of the specific, measurable pharmacological properties of isolated anthocyanins.

3.2 Total mucilage content

Total mucilage content (TMC) was measured both in the flowers and herbage of the examined taxa. Results showed that this parameter was under the influence of the genetic structure [20], geographical condition [21] as well as the organ of choice [20]. TMC was predominantly higher in the flowers as compared to the herbage of the taxa (Table 2). TMC in the flowers ranged between 8.6 and 32%. The highest TMC in the flowers was recorded in A6 from A. arbelensis (32%) followed by A9 from A. var. lobata (27.3%), whereas the lowest values were obtained in A12 (8.6%) (A. aucheri var. aucheri), followed by A7 (11.6%) and A8 (11.8%) from A. aucheri var. aucheri, respectively. The maximum (11.9%) TMC in the herbage of the taxa was found in A4 (A. koelzii), while A11 from A. aucheri var. lobata contained minimal content (Tables 3, 4).

Mucilages are large, highly branched polymeric structures built from many different kinds of sugar and uronic acid units [22] that contribute to several important ecological adaptations, particularly tolerance to water stress. Therapeutically, mucilage can reduce bowel irritation, toxin absorption, cough, bronchial and urinary spasm, and it is useful to increase expectoration.

3.3 Principal component analysis (PCA)

Principal component analysis (PCA) scatter plot, derived from TPC, TFC, TAC and TMC of flowers in 13 Alcea specimens was designed (Fig. 1). PCA gives a fairly well-resolved picture of the distribution pattern of the specimens in terms of the phytochemical properties. Two factors were extracted with PCA, accounting for 72.76% of the total variance. Results indicate that Alcea specimens are distributed across the plot in four main groups (A, B, C, D) based on their phytochemical properties. Three specimens A4 (A. koelzii), A6 (A. arbelensis) and A9 (A. aucheri var. lobata) grouped together based on TMC and formed group A. A13 (A. aucheri var. aucheri) was distinct in terms of TPC and alone took place within group B, while group C was formed by a set of 6 specimens (A1, A2, A3, A5, A11, A12), the representatives of all the taxa examined. As it can be seen in Fig. 1, the specimens representing A. aucheri var. lobata, (A7, A8, A10) were separated from other taxa by their lower TPC, TFC, TAC and TMC, and formed group D. They revealed a different quantitative phytochemical profile supporting their chemical distinctiveness from other taxa.

Fig. 1
figure 1

PCA performed on TPC, TFC, TAC and TMC in the flowers of 13 Alcea specimens

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In a general view, specimens placed in four groups with no evident relationships to genotype and bioclimatic zone.

3.4 Free radical scavenging activity

Free radical scavenging activity (DPPH assay) was used to determine the antioxidant capacity of the extracts obtained from both flowers and herbage of the Alcea taxa and results were reported as IC50 values. DPPH radical is a stable organic free radical with an absorption band at 517 nm [23]. The addition of Alcea extracts to the DPPH solution induced a rapid decrease in the optical density at 517 nm. Results of DPPH radical scavenging activity of the examined taxa are summarized in Tables 3 and 4. The samples obtained from flowers gave stronger radical scavenging capacity than those obtained from herbage (Table 2). The extracts obtained from the flowers yielded DPPH radical scavenging activity with IC50 values ranging from 34.06 to 236.09 μg/ml. The strongest antioxidant activity of the flowers belonged to A12 from A. aucheri var. aucheri (34.06 μg/ml), followed by A8 (41.88 μg/ml) and A9 (51.19 μg/ml) from A. aucheri var. lobata. A2 from A. koelzii (236.09 μg/ml), A10 from A. aucheri var. lobata (168.04 μg/ml) and A6 from A. arbelensis (157.81 μg/ml) showed weaker activities. Scavenging activity of the herbage yielded values between 62.96 μg/ml (A6 from A. arbelensis) and 358.39 μg/ml (A7 from A. aucheri var. lobata).

Except for the ecotypes within A. aucheri var. lobata (A7–A11) and A. aucheri var. aucheri (A12 and A13), the extracts obtained from the flowers showed more or less a weaker scavenging activity compared to the herbage of the same plants. This comes to mean that anthocyanins play an important role. The correlation matrix (Table 5) revealed a positive correlation between the antioxidant activity of the species studied with TAC (r = 0.387, α = ≤ 0.01), TMC (r = 0.321, α = ≤ 0.01) and TPC (r = 0.295, α = ≤ 0.01). It may be concluded that anthocyanins are likely to play an especially more important role in the overall scavenging activity of the genus Alcea. It should be noted that antioxidant activity of the tested ecotypes also correlates strongly (r = 0.418, α = ≤ 0.01) with the attitude of the collection site.

Table 5 Correlation analysis between altitude and phytochemical properties of the genus Alcea
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Overall, our findings indicated that the extracts from several specimens of Alcea could vary in quantity and quality according to genotype, climate, and the plant part used [24]. It is widely accepted that the production of secondary metabolites in medicinal plants is a complex interplay of intrinsic and extrinsic (e.g. environmental factors) elements. This is particularly modulated at the DNA level, where specific enzymes in the biosynthetic pathway of desired products are encoded [25]. Therefore, the genetic structure can create substantial variability in the distribution and composition of active substances in plants, which is to a large extent genotype-specific [26, 27]. For example, idioblastic mucilage cells typically occur within members of Malvaceae, Liliaceae, Cactaceae, and Orchidaceae [28]. Members of the genus Alcea are among those plants known to contain far greater concentrations of mucilage than is typically found in most plants. The accumulation of the mucilage in the genus Alcea constitutes an excellent strategy of the plants to adapt to harsh environmental conditions, particularly heat, drought and cold stresses [29,30,31].

On the other hand, metabolism of SMs is under the influence of extrinsic elements like environmental factors as well. Influenced by environmental elements, each class of secondary metabolites functions as a chemical interface between the environment and the plant. These phytochemicals exert their biological roles, as plastic adaptive drivers in response to the surrounding environment. Such chemical interactions often include variations in the production of plant metabolites, which could possibly be one of the main explanations behind plants metabolic diversity [32]. Therefore, the study of these variations is very useful in the chemical characterization of plants of the same species which are collected from different geographical areas, especially when they have genetic homogeneity [33]. In relation to our study, synthesis of phenolic derivatives (e.g. flavonoids)is reported to be upregulated when plants are overexposed to light [26]. It has also been reported that the color and stability of anthocyanin pigments are influenced by pH [27], light and temperature [34]; they may appear red, purple, or blue depending on the pH of their environment [14].

Moreover, today we clearly know that biosynthesis of secondary metabolites is not a random process, but rather is restricted to specific plant tissues or developmental stages [30]. Anthocyanins are found predominantly in flowers and fruits, but also in leaves, stems, and roots [31]. These are primarily recognized by their strong red to blue coloring [14]. Thus, it is not surprising that we recorded the maximum contents of anthocyanins in the flowers, particularly in those specimens with purple- and pink-colored petals. Interestingly, in the white-flowered taxa, the content of anthocyanins was higher in the herbage rather than floral organs (Tables 1, 4). Moreover, TPC, TFC, and TMC in all of the examined taxa were also abundant in the flowers compared with the herbage (Tables 2, 4).

A large number of medicinal plants contain chemicals with antioxidant activity, among which phenolics and their derivatives are receiving particular attention [29]. Flavonoids possess strong antioxidant activity which may be responsible for some vital biological activities e.g. age-related neurodegenerative disorders [12], prevention or lowering the risk of cancer, diabetes, arthritis and cardiovascular diseases [35]. The phenolic moiety in flavonoids can accept an electron to form relatively stable phenoxyl radicals, thereby disrupting chain oxidation reactions in cellular components [33].

Today we know that additive and synergistic effects of phytochemicals in plants are responsible for their potent antioxidant activity and that the benefit of a diet rich in fruit and vegetables is attributed to the complex mixture of phytochemicals present in the body of the plants, not a single compound [36]. It has been shown that the antioxidant capacity of the plant extracts largely depends on the composition of the extracts and conditions of the test system. Therefore, all the aforementioned factors (e.g. genotype, growth factors, laboratory conditions etc.) influencing the recovery of plant extracts would accordingly affect their antioxidant capacity [33, 37, 38]. Our findings suggest that antioxidant activity has a relatively strong correlation with the intensity of the plant color. A12, A8, A9 that showed the strongest activity also owned purple-, pink- and purple-colored flowers, respectively, while in the other taxa having white-colored flowers the antioxidant activity of the herbage was stronger than that of the flowers. Likewise, Sadighara et al. (2012) examined the antioxidant activity of the taxa belonging to Althaea officinalis L. and reported that the plants with more pigmentation have more biological effects in association with antioxidant potentials [39]. However, the stronger free radical scavenging activity of the vegetative organs in white-colored flowers may indicate that other compounds different from phenolics may contribute to the antioxidant activity observed. Accordingly, the strong antioxidant activity of A. officinalis to a large extent (approximately 70%) is attributed to the reference compound α-tocopherol [40].

4 Conclusion

In conclusion, flowers and herbage of several taxa belonging to the genus Alcea were evaluated for their total phenolics, flavonoids, anthocyanins, mucilage content as well as antioxidant activity, and results showed that the content and biological activity of such phytochemicals were markedly varied, as influenced by genetic structure, environmental condition and the organ of choice. Our findings indicated that in the majority of the taxa the content of the phytochemicals in the flowers was to some extent higher than that of the herbage. We also reasoned that there is a significant relationship between the flower color and its anthocyanin content. Except for the taxa within A. aucheri var. aucheri and A. aucheri var. lobata, extracts obtained from flowers showed, more or less, weaker antioxidant activity compared with the herbage of the same plants. Our findings suggest that the antioxidant activity has a relatively strong correlation with the intensity of the plant color, as the strongest activity was observed in the purple- and pink-colored flowers, while in the white-colored taxa, the antioxidant activity of the herbage was stronger than that of the flowers.