Detailed literature research into the phenolic compounds present in Cistus incanus
was carried out. There are no data available concerning kinetic studies of the selected drug by total polyphenol and flavonoid content, antioxidant power, and total dry residue, except the extraction kinetics presented by Dimcheva and Karsheva [18
] for Bulgarian Cistus incanus
leaves with 50% ethanol in water solution. In addition, the influence of seasonality on the presence of the examined bioactive components and thus the AOC of the wild herb has never been studied. There are no data in the literature concerning the polyphenol content and the AOC of the aerial parts (stalks, buds, hard-coated seeds) of Cistus incanus
3.1. Effect of the Solvent Used
The conventional method of polyphenol recovery from the plant is based on solid–liquid solvent extraction. It is generally known that the yield of extracted polyphenols depends on the chemical composition and physical characteristics of the samples as well as on the type of solvents used, their different polarity, extraction manner, contact time and temperature. The results can vary even by one order of magnitude when one or another procedure is used for the same sample. Thus, it is necessary to adjust the extraction method for each new crude drug.
Solvents such as methanol, ethanol, acetone, ethyl acetate and combinations of them are most commonly used to extract phenolics from plants, often in different ratios with water. Choosing the right solvent is essential for the industry, it must be safe, cheap and non-toxic. Ethanol is a good solvent for the extraction of polyphenols and preferable for the extraction of Cistus incanus
according to a patent publication for Cistus incanus
]. Therefore, the ethanol was chosen as the solvent in the present investigation.
To be evaluated, the appropriate solvent composition was used a deionized water or an ethanol in water solution (10–50%, v
) to establish the optimal yield of total polyphenols, flavonoids, and antioxidant capacity. The extractions were done by a magnetic stirring for 80 min. The results for TPC, TFC, and IC50
are shown in Figure 1
The pure deionized water (ET0) showed the worst values for the desired components at the expense of the medium polar mixtures, such as ET30 and ET40. It can be seen from Figure 1
that the IC50
rests constant with the decrease of the polarity of the extracting solvent. Of these, the high values of the extracted antioxidant substances and the highest value of the IC50
, the ET30, was chosen as the optimal solvent concentration and used in the further examinations.
The present investigation evaluated an extraction parameter—the extraction time (0–500 min)—on total polyphenols, total flavonoids and the scavenging activity of the Cistus incanus leaves, stalks, and buds. The temperature and the solid-to-solvent ratio were kept constant during the whole extraction kinetics procedure, which was carried out through magnetic stirring extraction and with ET30 as a solvent. In conventional methods, sampling is manual at chosen time intervals, which are not precise, as there is always a time gap between sampling and analysis that may lead to errors during the kinetic measurements. Nevertheless, in the present study, we tried to make the interval between the various extractions relatively small. On the other hand, the measured raw material was kept as far as possible to the same ratio of leaves, stalks, and buds. The evaluation of the extraction time was investigated on TPC, TFC, IC50, and the total dry residue, all shown below.
3.2. Total Polyphenols
Phenolic compounds act as essential metabolites for plant growth and reproduction, and as protecting agents against pathogens. These compounds involve a large group of about 8000 compounds with different structures and chemical properties [20
]. In general, these substances contain one or more aromatic rings with one or more hydroxyl groups and can be classified into three main categories: simple phenols, which include phenolic acids; polyphenols, constituted by flavonoids and tannins; and a miscellaneous group that comprises compounds such as coumarins, stilbenes, and lignans.
The total polyphenol content for the 30% ethanol extracts was estimated by the Folin–Ciocalteau method using gallic, pyrogallic and tannic acids as standards.
Gallic acid is commonly used in the pharmaceutical industry to determine the total phenol content by Folin–Ciocalteau assay [21
]. Phenolic acid is mostly used to express the content of phenolic compounds in most foods [22
]. On the other hand, pyrogallic acid is used as a standard for the determination of the total polyphenols according to the Eur. Ph. [23
]. Similarly, to previous compounds, tannic acid was proved to possess antioxidant [24
], antimutagenic [25
] and anticarcinogenic properties [26
According to the obtained results, a statistically significant effect of the time of each extract is presented in Table 1
, where the content of total polyphenol compounds is shown, expressed by the phenolic acids represented above.
The total amount of polyphenols, expressed as the gallic acid in the extracts, varies between 36.26 and 115.32 mg GAE/g dw as a function of time. The quantity of the polyphenols expressed as tannic acid equivalents ranged from 71.88 to 228.56 mg TAE/g dw and from 27.30 to 86.81 for the PGAE/g dw. Lower phenolic contents were detected at the 5th min and the highest at the 390th min, as shown in Table 1
. However, it can be concluded that the equilibrium was achieved at the 180th min because the obtained values for the desired polyphenols were only 4.7% less than those obtained after a 3.5 h extraction and 0.41% less than those obtained after 5.3 h of stirring.
The used Folin–Chiocalteau assay is specific not only to polyphenols but to any other substance that could be oxidized by the Folin reagent; many non-phenolic compounds like ascorbic acid and saccharides can reduce the amount of reagent [14
3.3. Total Flavonoids
Flavonoids are low molecular weight polyphenolic secondary metabolic compounds, universally distributed in the green plant kingdom [27
]. Flavonoids represent a broad family of more than 4000 secondary plant metabolites such as 4-isoflavonoids (flavones and flavonols), isoflavones, anthocyanins, and flavan-3-ols derivatives (tannins and catechins) [28
]. For centuries, preparations that contain flavonoids have been applied as the primary physiologically active components used for treating human diseases [29
Quercetin, rutin and catechin are important bioflavonoids present in more than twenty plant materials and are known for their anti-inflammatory, antihypertensive, and vasodilator effects, as well as their antiobesity, antihypercholesterolemic and antiatherosclerotic activity [30
The total flavonoid content of the ethanolic extracts was measured using an aluminum chloride colorimetric assay using quercetin, rutin and (+)-catechin as standards. Aluminum chloride forms acid stable complexes with the C-4 keto groups and either the C-3 or C-5 hydroxide group of the flavones and flavonols. In addition, it also forms liable complexes with ortho dihydroxide groups in the A/B rings of flavonoids.
The extraction kinetics data for different species are shown in Table 2
The total flavonoids, expressed as the quercetin equivalent of the extracts, show higher values varying between 40.80 and 119.20 mg QE/g dw from the 5th to the 500th min extraction time. The lower quantities of the flavonoids were calculated as the (+)-catechin equivalent ranging from 6.0 to 17.53 mg CE/g dw and the middle ones ranged from 20.40 to 59.60 mg RE/g dw for the flavonoids calculated as the rutin equivalent. In Table 2
, the presented kinetics show the same tendency as the total polyphenol equilibrium achieved at the 180th min, but the difference here is their decrease after the 390th min. Probably, this decrease is due to their unstable nature or to error due to the time between experiments and other random factors.
No previous study of the kinetics of the total content of the polyphenols and flavonoids in Cistus incanus exists, including Bulgarian Cistus incanus, as was already mentioned. Hence, the data obtained can only be compared with that found for the Cistus incanus species grown in different regions and extracted using different extraction procedures and different conditions to those used in the present study.
For example, for the aqueous extracts of Cistus ladanifer
and Cistus populifolius
from Spain, the TPC values at levels of 229.3 mg GAE/g dw and 318.9 mg GAE/g dw, respectively, were found. The TFC values in these plants were found to be 30.4 mg QE/g dw and 59.5 mg QE/g dw, respectively [34
Similarly, the results for TPC obtained for aqueous extracts of Turkish Cistus laurifolius
were 289.9 mg GAE/g extract [35
Lower levels of TPC and TFC were reported for methanol and ethanol extracts of Moroccan Cistus ladanifer
: 18.43 mg GAE/g extract and 64.33 mg RE/g extract; and 11.87 mg GAE/g extract and 61.40 mg RE/g extract, respectively [36
In another study of the extracts obtained from Cistus incanus
grown in Turkey and Cyprus, the following values for the valuable components were obtained: 258.42 mg GAE/g dw and 202.95 mg GAE/g dw for the aqueous extracts, and 105.02 and 114.18 mg GAE/g dw for the hydromethanolic extracts for the total polyphenols content. The total flavonoids for the same extracts were 4.27 and 3.97 mg QE/g dw and 2.39 and 2.27 mg QE/g dw, respectively [37
From the research it can be concluded that the Bulgarian Cistus incanus
contain the greatest total flavonoid content (138.44 mg QE/g dw and 69.22 mg RE/g dw) in comparison not only with Cistus ladanifer
from Morocco and Cistus populifolius
from Spain and Turkey, but in comparison with the Turkish and Cyprian Cistus incanus
leaf extracts. The results found in the literature for the total polyphenols of the Cistus
species are higher than those obtained in this study for the Cistus incanus
leaf, stalk, and bud hydroethanolic extracts. The quantities of extracted polyphenolic compounds in the plants depend on the differences in extractive parameters and the solvent used. The various biological and environmental factors in which the plant grew also contribute to the plant antioxidant power [38
3.4. Antioxidant Capacity
It is well established that the flavonoids and phenolic acids have antioxidant activities due to the presence of structural hydroxyl groups significantly contributing to protection against the oxidative damage due to endogenous free radicals [39
]. Many of them are reported to have high levels of antioxidant activity [41
]. Due to their redox properties, these compounds contribute to the overall antioxidant activity of plants. Usually, the antioxidant activity neutralizes lipid free radicals and prevents the decomposition of hydroperoxides into free radicals [42
and TEAC are presented in Figure 2
. Expressed as the concentration of the extract, they vary from 305.71 to 122.16 μg/mL; expressed as a Trolox equivalent, they vary from 303.88–747.13 μmol TEAC/g dw. The best values for the IC50
and TEAC of the Cistus incanus
leaves, stalks, and buds were obtained at the 390th min and were 768.44 μmol TEAC/g dw or 119.25 μg/mL of the extract can reduce 50% of the free radicals.
The literature study found data on 15 different samples of Cistus incanus from different countries. The results showed that the values of DPPH for hydromethanolic and aqueous extracts were varied in the range of 20.06–96.69 μmol TEAC/g dw and 1.52–96.85 μmol TEAC/g dw, respectively.
These results are much lower than those obtained in the present study. That means that the Bulgarian Cistus incanus is a rich source of antioxidants and the environmental factors of Strandja Mountain are obviously suitable for their formation.
3.5. Total Dry Residue
In the evaluation of plant extracts, it is good to know the kinetics of the process also by total dry residue when equilibrium is achieved, not least for a better understanding of the plant material extraction. Using the gravimetric method described above, the kinetics of the total dry residue (TDR) of the Cistus incanus
leaves, stalks, and buds picked during the summer harvest season in the liquid phase and their total dry mass were studied. Extracts and exhausted plant materials from the extraction kinetic with 30% ethanol and a 0.05 g/mL solid-to-solvent ratio were used. The quantity of the extracts after the hand pressing of the plant material were measured and plotted in the graph. The results for TDR and total dry mass were expressed in grams of dry weight per liter v/s extraction time. The measured volumes of the received extracts were expressed in liters. The yield of the extracts was studied because it is an essential parameter for the industrial production of extracts. The kinetics curves obtained are shown in Figure 3
In the kinetics presented, the water contents (9.70%) was not recalculated and thus the presence of volatile substances is quite probable. As shown, the kinetic curves have three parts with different characters. The increase of TDR in the liquid phase (extract) corresponds to the decrease of the total dry mass. The initial steep part of the graphic corresponds to the dissolution of the readily available substances on the surface of the sample particles. The second curved part could be explained by the simultaneous dissolution of the rest from the surface and from inside the sample particle (the mixed zone control). Based on the yield of extract kinetic, the plateau or the extraction equilibrium was achieved after the 180th min, where the quantity of the dry extract was 5.38 g in 413 mL extract, but increased to 6.7 g in 425 mL extract at the 390th min. Likewise, there was an increase after the 180th min illustrated by the TDR kinetic responsible for the liquid phase, and the highest results for this kinetic can be seen at the 390th min. However, based on the total dry mass, the plateau was reached approximately at the 80th min, where the quantity of total dry residue of the extract was 1.4135 g and slowly decreased with 1.0% up to the 500th min. These results may be due to the uneven plant material used or measurement errors. Based on the kinetics by total polyphenols, flavonoids, and AOC, it can be concluded that the 390th min, or 6.5 h, is the optimal extraction time also in relation to the yield of the extract and the TDR in the liquid phase. The long extraction time probably shows that magnetic stirring is not the best way to extract the examined mixtures of drugs or that there are bioactive substances in the hard buds and stalks which need more time for their discharging. In both cases, further extraction optimization is required, maybe by increasing the extraction temperature or changing the applied extraction manner.
3.6. Evaluation of Cistus incanus Aerial Parts
In this study different aerial parts were used, as follows: hard-coated seeds and young buds, as well as a mixture of stalks and leaves (50:50%, w/w). They were extracted for 80 min with 30% ethanol in water solution.
The total polyphenols and flavonoids in the buds and in the hard-coated seeds provided good results. The buds should have contained much more of the desired components than the seeds, because they were picked during the plant’s flowering, when it is in its polyphenol power. It is known that the woody parts of the aromatic herbs also contain flavonoids and polyphenols, which play an important role in protecting the plant. [43
] As shown in Figure 4
, the mixture of leaves and stalks in a ratio 50:50 provided the best results, which is normal because the main quantities of polyphenols are concentrated in the leaves. The obtained results show that the hard-coated seeds, buds, and stalks can also be used as a raw material for the production of antioxidants in the nutraceutical industry or for making a tea (infusion) at home.