Screening of plant materials and selection of the most prospective plants
Five plant species:
Sanguisorba officinalis,
Rumex obtusifolius,
Hypericum alpestre,
Lilium armenum and
Agrimonia eupatoria (Additional file
1: Table S1)
, were chosen based on initial
in vitro evaluation of antimicrobial activities of different parts of 28 wild plant species against five bacterial and two yeast strains. All plant materials are widely used in Armenian traditional medicine.
In order to select the most active parts of five plant species, agar well diffusion assay was used. We used 500 μg ml
−1 concentration of crude extracts (50 μg dry material for each well) for agar well diffusion assay according to recommendations [
12,
19]. Rios and Recio (2005) in their review stated that using crude extracts of plant materials with concentrations above 1000 μg ml
−1 in antimicrobial screening protocols should be avoided, because using high concentrations of plant crude extracts can bring to false positive results [
19].
According to the obtained data all plant parts possessed antimicrobial activity against at least three tested strains (see Table
2). In order to select prospective plant parts for further comprehensive studies we used two main criteria. At first, we paid attention to plant extracts with the highest growth inhibition zones. We also gave a priority toward the samples which had broad spectrum of action against various microbial groups (Gram-positive, Gram-negative, endospore forming bacteria and yeast). Thus, according to data obtained (see Table
2) following plant parts were chosen for further more profound investigation:
Sanguisorba officinalis (aerial part),
Rumex obtusifolius (seed),
Hypericum alpestre (aerial part),
Lilium armenum (bulb), and
Agrimonia eupatoria (whole plant).
Table 2
Antibacterial and anti-yeast activity of the tested plant extracts determined by agar well diffusion assay
Agrimonia eupatoria
| Whole plant | Water | – | – | – | 9 ± 0.6 | – | – | – |
Methanol | 10 ± 0.6 | 11 ± 0.6 | 11 ± 0.6 | 11 ± 0.6 | – | 11 ± 0.6 | – |
Chloroform | 9 | 11 ± 0.6 | 10 ± 0.6 | 10 ± 0.6 | 11 ± 0.6 | 12 ± 0.6 | 9 ± 0.6 |
Acetone | 12 ± 0.6 | 10 ± 0.6 | 11 ± 0.6 | 10 ± 0.6 | 10 ± 0.6 | 12 ± 1 | 9 ± 0.6 |
Hexane | 13 ± 0.6 | 10 ± 0.6 | 10 ± 0.6 | – | 9 ± 0.6 | 9 ± 0.6 | – |
Hypericum alpestre
| Aerial part | Water | – | – | 11 ± 0.6 | – | – | – | – |
Methanol | 10 ± 0.6 | 11 ± 0.6 | 18 ± 0.6 | 10 ± 0.6 | – | – | – |
Chloroform | 13 ± 0.6 | 12 ± 0.6 | 23 ± 1 | – | – | – | – |
Acetone | 15 ± 0.6 | 13 ± 0.6 | 21 ± 0.6 | – | 10 ± 0.6 | 9 ± 0.6 | – |
Hexane | 17 ± 0.6 | 16 ± 0.6 | 21 ± 0.6 | – | – | – | – |
Lilium armenum
| Stalk with leaf | Water | – | – | 10 ± 0.6 | – | – | – | – |
Methanol | 9 ± 0.6 | – | 13 ± 0.6 | – | – | – | – |
Chloroform | 9 ± 0.6 | – | 10 ± 0.6 | – | – | – | – |
Acetone | 10 ± 0.6 | 9 | 11 ± 0.6 | – | – | – | – |
Hexane | 9 ± 0.6 | – | 12 ± 0.6 | – | – | – | – |
Lilium armenum
| Bulb | Water | – | – | 9 ± 0.6 | – | – | – | – |
Methanol | 9 ± 0.6 | 9 ± 0.6 | 11 ± 0.6 | 9 ± 0.6 | – | – | – |
Chloroform | 9 ± 0.6 | – | 13 ± 0.6 | – | 9 ± 0.6 | – | – |
Acetone | 10 ± 0.6 | 9 ± 0.6 | 11 ± 0.6 | 9 ± 0.6 | 9 ± 0.6 | – | – |
Hexane | – | – | 11 ± 0.6 | – | 9 ± 0.6 | – | – |
Rumex obtusifolius
| Leaf | Water | – | – | – | – | – | – | – |
Methanol | 9 ± 0.6 | – | – | – | 10 ± 0.6 | 14 ± 0.6 | – |
Chloroform | 11 ± 0.6 | – | 10 ± 0.6 | – | 10 ± 0.6 | 9 ± 0.6 | – |
Acetone | 11 ± 0.6 | – | 12 ± 0.6 | – | 10 ± 0.6 | 12 ± 0.6 | – |
Hexane | 9 ± 0.6 | – | 11 ± 0.6 | 9 ± 0.6 | – | – | – |
Rumex obtusifolius
| Root | Water | – | – | 11 ± 0.6 | – | – | – | – |
Methanol | 10 ± 0.6 | – | 10 ± 0.6 | – | – | – | 9 ± 0.6 |
Chloroform | 11 ± 0.6 | – | 10 ± 0.6 | – | 9 ± 0.6 | 12 ± 0.6 | 9 ± 0.6 |
Acetone | 10 ± 0.6 | 9 ± 0.6 | 12 ± 0.6 | – | – | 13 ± 0.6 | 9 ± 0.6 |
Hexane | 9 ± 0.6 | – | – | – | – | 12 ± 0.6 | – |
Rumex obtusifolius
| Inflorescence | Water | – | – | – | – | – | – | – |
Methanol | – | 9 ± 0.6 | 9 ± 0.6 | 10 ± 0.6 | – | – | – |
Chloroform | – | 9 ± 0.6 | 9 ± 0.6 | 9 ± 0.6 | – | – | – |
Acetone | 11 ± 0.6 | 11 ± 0.6 | 9 ± 0.6 | 10 ± 0.6 | 9 ± 0.6 | 9 ± 0.6 | – |
Hexane | 9 ± 0.6 | 9 ± 0.6 | 10 ± 0.6 | – | – | – | – |
Rumex obtusifolius
| Seed | Water | 9 ± 0.6 | – | 10 ± 0.6 | – | – | – | – |
Methanol | 12 ± 0.6 | 11 ± 0.6 | 10 ± 0.6 | 11 ± 0.6 | 12 ± 0.6 | 10 ± 0.6 | – |
Chloroform | – | 9 ± 0.6 | 9 ± 0.6 | – | – | – | – |
Acetone | 12 ± 0.6 | 12 ± 0.6 | 10 ± 0.6 | 10 ± 0.6 | 12 ± 0.6 | 10 ± 0.6 | – |
Hexane | 9 ± 0.6 | 9 ± 0.6 | 10 ± 0.6 | – | – | – | – |
Sanguisorba obtusifolius
| Aerial part | Water | – | – | 10 ± 0.6 | – | 9 ± 0.6 | – | – |
Methanol | 12 ± 0.6 | 11 ± 0.6 | 10 ± 0.6 | 10 ± 0.6 | 12 ± 0.6 | 9 ± 0.6 | 10 ± 0.6 |
Chloroform | 10 ± 0.6 | 9 ± 0.6 | 10 ± 0.6 | – | – | 12 ± 0.6 | 11 ± 0.6 |
Acetone | 13 ± 0.6 | 13 ± 0.6 | 12 ± 1 | 12 ± 0.6 | 11 ± 0.6 | 10 ± 0.6 | 10 ± 0.6 |
Hexane | 11 ± 0.6 | 10 ± 0.6 | 10 ± 0.6 | 9 ± 0.6 | – | 10 ± 0.6 | 9 ± 0.6 |
Sanguisorba officinalis
| Root | Water | – | – | – | – | – | – | – |
Methanol | 10 ± 0.6 | – | 10 ± 0.6 | 10 ± 0.6 | – | 10 ± 0.6 | 10 ± 0.6 |
Chloroform | – | – | – | – | – | 10 ± 0.6 | 11 ± 0.6 |
Acetone | 10 ± 0.6 | 10 ± 0.6 | 10 ± 0.6 | 10 ± 0.6 | – | 10 ± 0.6 | 10 ± 0.6 |
Hexane | – | – | – | – | – | – | 10 ± 0.6 |
| PCb
| | 20 ± 0.6 | 30 ± 1 | 28 ± 1 | 19 ± 0.6 | 23 ± 0.6 | 24 ± 0.6 | 23 ± 0.6 |
All tested parts of
Rumex obtusifolius demonstrated substantial antimicrobial activity with some differences (see Table
2). For example, only root extracts exhibited antimicrobial activity against
C. albicans. In turn they did not inhibit the growth of
E. coli, whereas its other parts did.
B. subtilis was more sensitive to crude extracts of inflorescence and seed of
Rumex obtusifolius. In contrast, tested yeast strains were more sensitive to leaf and root extracts of this plant. Acetone and methanol extract of
Rumex obtusifolius seeds showed the highest antimicrobial activity against all tested strains except
C. albicans. Consequently, we have chosen this part for further study. Other parts of
Rumex obtusifolius had less efficiency compared to the seeds. Although root crude extracts had lower activity than seed extracts, however, they were also interesting due to their activity against
C. albicans.
Aerial part of
Lilium armenum inhibited the growth of only three bacterial test strains. Meanwhile, its bulb extracts exhibited activity against all tested bacterial strains. Moreover, growth inhibition zones of the bulb extracts exceed zones, induced by the aerial part. Therefore, the bulb part of
Lilium armenum was chosen for further studies. This plant is also considered interesting, as it is native to Armenia, where it is widely used in folk medicine. However, to this date there have been no studies about its antimicrobial properties [
20,
21].
Among all the tested plant materials the largest zones of inhibition were induced by
Hypericum alpestre extracts (see Table
2). They exhibited activity against almost all test strains with the exception of
C. albicans and
S. typhimurium. Even though other species of the genera
Hypericaceae were widely investigated and their high antimicrobial activity has already been shown in the literature [
22,
23], there was no information about
Hypericum alpestre’s antimicrobial properties. However, it was the most commonly used species in traditional medicine within the genera in the region from where it was collected.
Even though the use of the roots of
Sanguisorba officinalis is more common in traditional medicine [
20,
21], the obtained data demonstrated higher antimicrobial activity of the aerial part compared to the root. Acetone and methanol extracts of
Sanguisorba officinalis’s aerial part inhibited the growth of the tested strains whereas crude extracts of the root did not show activity against
S. typhimurium at tested concentrations. Therefore, we selected the aerial part for further research. Crude extracts of
Agrimonia eupatoria also inhibited the growth of all tested bacterial and yeast strains (see Table
2). Although
Sanguisorba officinalis and
Agrimonia eupatoria were already investigated in many research works and their high antimicrobial activity was shown [
24‐
26], it was interesting to study these plants in order to find out any possible differences between the activity of same plant species from various geographical areas.
We compared the activities of the tested plant materials against Gram-negative and Gram-positive bacteria based on agar well diffusion assay (Additional file
1: Table S2). Crude extracts of
Hypericum alpestre were more active against Gram-positive bacteria. In case of
Rumex obtusifolius, Lilium armenum, Agrimonia eupatoria and
Sanguisorba officinalis in general similar activity was observed against both Gram-negative and Gram-positive bacteria.
Thus, screening of different parts of plant species used in Armenian folk medicine allowed us to evaluate their antimicrobial properties at 500 μg ml−1 concentration and choose the most active plant parts for further studies. On the other hand, the obtained results enabled the evaluation of comparative effectiveness of five different solvents for their ability to solubilize antimicrobial compounds from plant materials.
Determination of MIC values of selected plant materials
MIC values of the selected five plant parts:
Lilium armenum (bulb),
Rumex obtusifolius (seed),
Hypericum alpestre (aerial part),
Agrimonia eupatoria (whole plant) and
Sanguisorba officinalis (aerial part) extracted with five solvents were determined (see Table
3). According to collected data MIC values of the tested extracts generally varied within the range from 64 μg ml
−1 to 1024 μg ml
−1. The MIC values of aqueous, chloroform and hexane extracts for almost all tested plants were relatively high. Therefore, they cannot be considered for any further practical use based on their weak activity. The exceptions were chloroform and hexane extracts of
Hypericum alpestre which had low (128 μg ml
−1) MIC values against
S. aureus. Moreover, hexane extract of this plant continued to be active till 64 μg ml
−1 concentration against
P. aeruginosa. Another exception was hexane extract of
Agrimonia eupatoria with 128 μg ml
−1 MIC value against
P. aeruginosa. In contrast, MICs of acetone and methanol extracts of the tested plant materials had fairly low values against some test strains. For instance, methanol and acetone extracts of
Hypericum alpestre had 128 μg ml
−1 and 64 μg ml
−1 MIC values respectively against
P. aeruginosa. The MIC of acetone extract of the same plant against
S. aureus and
B. subtilis was 128 μg ml
−1. The MIC value of acetone extract of
Sanguisorba officinalis against
P. aeruginosa was 64 μg ml
−1. Acetone and methanol extracts
of Rumex obtusifolius had 128 μg ml
−1 MIC value
against B. subtilis and
P. aeruginosa. On the other hand, selected plants’ crude extracts possessed broad range of activity inhibiting the growth of Gram-negative, Gram-positive and endospore forming bacteria. They also showed considerable activity against yeasts. Among the tested five plants
Agrimonia eupatoria,
Rumex obtusifolius and
Sanguisorba officinalis had lower MIC values against yeast strains, compared to
Hypericum alpestre and
Lilium armenum, which in contrast, inhibited the growth of yeast strains only in high concentrations.
Table 3
Determination of minimum inhibitory concentration (MIC) and Minimum bactericidal/fungicidal concentrations (MBC/MFC) of crude extracts of five selected plant materials. (Place in the text: page 10 after line 13)
Hypericum alpestre (aerial part) | Wat | 1024 | – | – | – | 256 | – | – | – | 1024 | | – | – | – | – |
Met | 256 | 512 | 256 | 512 | 128 | 1024 | 512 | – | – | – | – | – | – | – |
CF | 128 | – | 256 | 1024 | 256 | 1024 | 1024 | – | 1024 | – | 1024 | – | – | – |
Ace | 128 | – | 128 | 512 | 64 | 512 | 1024 | – | 512 | – | 1024 | – | 1024 | – |
Hex | 128 | 512 | 256 | 512 | 64 | 512 | 1024 | – | 1024 | – | 1024 | – | – | – |
Agrimonia eupatoria (whole plant) | Wat | 1024 | – | – | – | 1024 | – | 1024 | – | 1024 | – | 1024 | – | – | – |
Met | 256 | – | 128 | – | 256 | 1024 | 512 | – | 1024 | – | 1024 | – | – | – |
CF | 256 | 512 | 256 | 1024 | 512 | 1024 | 512 | – | 512 | – | 256 | 1024 | 512 | – |
Ace | 256 | – | 128 | 1024 | 128 | 512 | 512 | – | 512 | – | 256 | – | 512 | – |
Hex | 256 | 1024 | 256 | 1024 | 128 | 512 | – | – | 512 | – | 512 | 1024 | 1024 | – |
Lilium armenum (bulb) | Wat | 1024 | – | 1024 | – | 1024 | – | – | – | 1024 | – | – | – | – | – |
Met | 512 | – | 512 | 1024 | 256 | 512 | 1024 | – | – | – | 1024 | – | – | – |
CF | 512 | – | 1024 | – | 128 | 512 | 1024 | – | 512 | – | 512 | 1024 | 1024 | – |
Ace | 512 | – | 512 | – | 128 | 512 | 512 | – | 512 | – | – | – | – | – |
Hex | 1024 | – | 1024 | – | 256 | – | – | – | 1024 | – | – | – | – | – |
Rumex obtusifolius (seed) | Wat | 512 | – | 1024 | – | 256 | 1024 | – | – | 1024 | | 1024 | – | 1024 | – |
Met | 256 | – | 128 | – | 128 | 512 | 512 | – | 512 | | 512 | – | – | – |
CF | 1024 | – | 1024 | – | 512 | – | – | – | 1024 | – | 1024 | – | – | – |
Ace | 256 | – | 128 | – | 128 | 1024 | 512 | – | 512 | | 512 | – | – | – |
Hex | 512 | – | 512 | – | 512 | 1024 | – | – | 1024 | – | 1024 | – | 1024 | – |
Sanguisorba officinalis (aerial part) | Wat | – | – | 1024 | – | 512 | – | – | – | – | – | – | – | – | – |
Met | 256 | – | 128 | 512 | 256 | 512 | 512 | – | 512 | – | 512 | – | 512 | – |
CF | 512 | – | 512 | – | 128 | 256 | 1024 | | 1024 | | 512 | 1024 | 512 | |
Ace | 128 | – | 128 | 1024 | 64 | 256 | 256 | – | 256 | – | 512 | – | 512 | – |
Hex | 256 | – | 512 | 1024 | 256 | 512 | 1024 | – | 1024 | – | 256 | – | 512 | – |
| PCd
| 0.25 | 0.5 | 0.25 | 0.5 | 0.25 | 0.5 | 0.5 | 2 | 1 | >2 | 2 | >4 | 2 | 4 |
Comparison of obtained MIC values of the tested plant materials with literature data showed both similaraities and differences. Wegiera et al. [
27] showed that ethanol extracts of
Rumex confertus seeds had 62.5–125 μg ml
−1 MIC values against
S. aureus strains which is in coincidence with our data. The results were also similar in case of
E. coli. However, in case of
P. aeruginosa and
C. albicans the MIC values obtained by Wegiera et al. [
27] were different from our results. In our experiments methanol and acetone extracts of
Rumex obtusifolius had 128 μg ml
−1 MIC value against
P. aeruginosa, whereas its MIC was above the 500 μg ml
−1 in the other research [
27]. The MIC against
C. albicans were above 1024 μg ml
−1 in our experiments. In contrast, it was 250 μg ml
−1 in research done by Wegiera et al. [
27]. These differences could be explained by differential susceptibility of the used test strains. Copland et al. [
28] in their research showed that only hexane extracts of
Agrimonia eupatoria seeds (they tested methanol and dichloromethane extracts as well) showed activity against
B. subtilis with 750 μg ml
−1 MIC value at tested concentrations. They did not find antimicrobial activity against tested
P. aeruginosa and
S. aureus strains. In contrast, Watkins et al. [
29] who used water, red wine, as well as 25% and 75% ethanol as solvents, showed that
Agrimonia eupatoria had moderate antimicrobial activity against
S. aureus, B. subtilis and
E. coli. Antimicrobial activity of ethanol extract of
Agrimonia eupatoria against
Helicobacter pylori was also shown [
30]. Kokoska et al. [
25] showed that ethanol extracts of
Sanguisorba officinalis had 62.5–250 μg ml
−1 MIC values against
E. coli, S. aureus and P. aeruginosa which partially coincides with our data. There was no evidence in literature about antimicrobial activity of
Lilium armenum and
Hypericum alpestre.
Determination of MBC/MFC values of the selected plant materials
MBC/MFC of selected five plant materials was determined. Three of the tested five plant species
Agrimonia eupatoria, Sanguisorba officinalis and
Lilium armenum possessed fungicidal activity against
C. guilliermondii (see Table
3)
. Only
Hypericum alpestre and
Agrimonia eupatoria extracts killed
S. aureus cells. In contrast, all five plant materials expressed bactericidal activity against
P. aeruginosa: some of them even at 256 μg ml
−1 concentrations (chloroform and acetone extracts of
Sanguisorba officinalis). Within tested five plant species only
Rumex obtusifolius had no bactericidal activity against
B. subtilis at tested concentrations. This is particularly interesting as tested
B. subtilis strain could have high resistance according to our previous work [
17]. Cidal activity of almost all tested plant materials toward this endospore forming bacteria also allowed us to assume that they possessed activity against endospores as well. All tested plant materials showed only static activity against
E. coli, S. typhimurium and C. albicans till the concentration of 1024 μg ml
−1. The obtained data indicated that MBC/MFC concentrations were two-three times higher than respective MIC values.
We did not find any research about bactericidal/fungicidal activity of all tested five plant species. Therefore, bactericidal/fungicidal activity of these plants was investigated for the first time.
Evaluation of tested solvents’ efficiency
Since we used five solvents in our screening protocol, the collected data allowed us to evaluate solvents for their efficiency to solubilize antimicrobial compounds from plant materials as well as their other properties. The obtained results (see Tables
2 and
3) showed that acetone extracts demonstrated the highest antimicrobial activities for almost all tested plant materials. Methanol extracts showed the second strongest antimicrobial activity, followed by chloroform, hexane and water. Methanol was the best solvent in regard of quantity of extracted materials, followed by water, acetone, chloroform and hexane. During extraction with hexane, only small amount of dry material was harvested. From the point of easy handling (quick evaporation, easy harvesting and weighing, high further solubility in DMSO, absence of residual moisture) we considered methanol as the best solvent, followed by acetone, water, chloroform and hexane. Our considerations about solvent efficiency agree with literature data. According to Eloff [
9], acetone received the best overall rating for yielding antimicrobials from plant materials among the tested six common solvents (methylene dichloride, methanol, ethanol, water, acetone, and a mixture of chloroform, methanol and water). However, Eloff [
9] conducted his study only on two plant materials, therefore, there was a necessity for more data for generalization of effectiveness of acetone as an extractant. Mothana and Lindequist [
31] showed that methanol extracts were more active than chloroform extracts which confirms our data. In our study aqueous extracts have shown poor antimicrobial activities at tested concentrations which coincides with literature data [
3,
10,
31‐
33].