Evalution of Antioxidant Activity of Various Herbal Folk Medicines

The leaves of some plants are flamed and used to dress injured skin, to stimulate healing and to ward off infection. This study aims to determine antioxidant activities of Plantago lanceolata L., Plantago major L. ssp. major, Rubus hirtus Waldst& Kit., Sambucus ebulus L., Morus alba L. and Hedera helix L., which grown in Düzce and its surroundings and are used for anti-inflammatory purposes, and especially for wound-healing. The total phenolic compound of these plants was also evaluated. In order to determine the antioxidant compounds and antioxidant capacity of these plants, different in vitro methods. Resulting from our study, while P. lanceolata, R. hirtus, S. ebulus and P.major ssp. major had similarities in terms of phenolic compounds content, M. alba and H. helix were found to have relatively low content of phenolic compounds. In general, Plantago species and R. hirtus showed high antioxidant activities.


Introduction
It has been established that oxidative stress is among the major causative factors in induction of ageing and immunosupression, and many chronic and degenerative diseases including atherosclerosis, ischemic heart diseases, diabetes mellitus, cancer, neurodegenerative diseases and others [1]. Natural antioxidants can protect the human body from free radicals and retard the progress of many chronic diseases as well as retarding the lipid oxidative rancidity in foods [2]. Recent studies have investigated the potential of plant products as antioxidants against various diseases induced by free radicals [3].
There is an increasing interest in natural antioxidants, e.g, polyphenols, present in medicinal and dietary plants, which might help preventing oxidative damages [4]. It has been determined that the antioxidant effect of plant products is mainly attributed to phenolic compounds such as flavonoids and phenolic acids [5].
Rubus hirtus is commonly called as blackberry belonging to family Rosaceae. It is present and widespread in the Turkey. The leaves of R.hirtus are used for abscess and to stop bleeding in cuts [7] and contains quercetin, kaempferol, caffeic acid and chlorogenic acid), phenolic acids, tannins, amino acids, sugars, pectins, carboxylic acids, anthocyanins, catechins, vitamin C and saturated or unsaturated fatty acids [8][9][10][11].
leaves of plants, infusion, decoction, poultice and expressed juice from fresh plants.
The plants in this study were selected from among those used especially for wound-healing in Düzce and its surroundings. The study was designed to examine antioxidant characteristics of Plantago lanceolata L., Plantago major L. ssp. major, Rubus hirtus Waldst & Kit., Sambucus ebulus L., Morus alba L. and Hedera helix L. which used as anti-inflammatory. In addition, through assessing total phenolic contents of the plants aimed to investigate whether there is a relationship between phenolic content and antioxidant activity. Plant leaves were extracted with 50% (w/v) ethanol and methanol and bidistilled water. A mass of 1 g of dry herb was added to 15 ml of solvent and was stirred up in a glass flask tightly closed for 45 minutes. Then, 5 ml of solvent was added and stirred up for another 45 min;. Finally 5 ml of solvent was added and stirred up for a final 15 min (totalling 105 min). First, the herb extracts were filtered out using a fine textured cloth. Then, the extracts were filtered out of a filter paper. All studied plant extracts were portioned and stored at -80°C [22].

Folin-Ciocalteu method for total phenolic content
The amount of total soluble phenolics was determined according to the Folin-Ciocalteu method [23], modified for use by a plate reader.
Folin Ciocalteu's phenol reagent was diluted to a volume ratio of 1:3 with 96% deionised water before used. Lowry A solution was prepared from sodium carbonate so that the strength (w/v) of Na 2 CO 3 in 0.1 M NaOH solution was 2%. Lowry B solution was prepared from copper(II) sulphate so that the strength (w/v) of CuSO 4 in 1% sodium potassium tartrate (NaKC4H4O6) solution was 0.5%. Lowry C solution was prepared by freshly mixing 50 ml Lowry A with 1 ml Lowry B. To 100 µl of herb extract, 125 µl Lowry C was added. The mixture was mixed on a microplate shaker at 250 rpm. Then, 12.5 µl Folin reagent was added and 30 min was allowed for stabilization and to turn blue in colour. The absorbance against a reagent blank was measured at 750 nm.

CUPRAC assay
Cuprac assay is based on reduction of Cu(II) to Cu(I) by antioxidants (reductant) which are present in the sample [24]. We modified this assay to work with 96-well plates. CuCl 2 solution, 1.0×10 -2 M, was prepared by dissolving 0.17 g CuCl 2 .2H 2 O in water, and diluting to 100 mL. Ammonium acetate buffer at pH 7.0, 1.0 M, was prepared by dissolving 19.27 g NH 4 Ac in water and diluting to 250 mL. Neocuproine (Nc) solution, 7.5×10 -3 M, was prepared daily by dissolving 0.039 g Nc in 96% ethanolic. 50 µl CuCl 2 solution (10 -2 M), 50 µl neocuproine alcoholic solution (7.5×10 -3 M) and 50 µl NH 4 Ac buffer solution, 27.5 µl sample and 27.5 µl water were added to wells. The mixture is then mixed well and incubated the microwell strips at room temperature (18° to 25°C) for about 30 min in dark. After that, absorbance was measured against a reagent blank at 450 nm.

DPPH assay
The free radical scavenging activity of these plants were measured by 1,1-diphenyl-2-picryl-hydrazyl (DPPH.) method, modified for working with 96 well plate. With this method it was possible to determine the antiradical power of an antioxidant by measuring the decrease in the absorbance of DPPH at 517 nm. As a result of the color changing from purple to yellow the absorbance is decreased when the DPPH radical is scavenged by an antioxidant through donation of hydrogen to form a stable DPPH-H molecule [25]. This method was applied to ethanolic, methanolic and aqueous extracts of plants (1000-500-250-125 µg/ml). First, 0.1 mM solution of DPPH . in MeOH was prepared. Then, 50 µl of this solution was added to 150 µl of extracts of plants. The mixture was shaken vigorously and allowed to stand at room temperature for 30 min. Then the absorbance was measured at 517 nm in an ELISA reader. Lower absorbance of the reaction mixture indicated higher free radical scavenging activity [26]. The DPPH . scavenging effect calculated using following equation:

DMPD assay
DMPD assay modified for use by a plate reader. DMPD (100 mM) was prepared by dissolving 209 mg of DMPD in 10 ml of deionised water. This solution (1 ml) was added to 100 ml of 0.1 M acetate buffer, pH 5.25, and the coloured radical cation (DMPD+) was obtained by adding 0.2 ml of a 0.05 M solution of ferric chloride (the final concentration was 0.01 mM). This solution (225 µl) was transferred directly to the microwell and its absorbance at 505 nm was measured (A0). Samples (15 µl) were added to all wells. Then added 210 µl of DMPD+ to all samples and, stirred and left to stand for 10 min. After this period, a decrease in absorbance was measured (A1). The DMPD. solution was used as control [27].
The DMPD + scavenging effect calculated using following equation:

ABTS assay
ABTS was dissolved in water to a 7 mM concentration. ABTS radical cation (ABTS.) was produced by reacting ABTS stock solution with 2.45 mM potassium persulphate (final concentration) and allowing the mixture to stand in the dark, at room temperature for 12-16 h before use. The kept solution was diluted with 96% ethanolic at a ratio of 1: 30. Respectively 10 µl sample, 200 µl ABTS. was added to wells. 210 µl of ABTS. solution was used as control (A0). Then, the change in absorbance during 5 min was recorded and measured at 734 nm by an ELISA reader [28].
The ABTS . scavenging effect calculated using following equation:

Ferric thiocyanate assay
The reaction mixture consisted of 0.28 g linoleic acid, 0.28 g of Tween 20 and 50 ml of phosphate buffer (0.05 M, pH 7.4). To 1.25 ml of the above linoleic acid emulsion, 0.1 ml of test sample and 1.25 ml of phosphate buffer (0.2 M, pH 7.0) were added and incubated at 40°C for 24 h. The mixture prepared, as above, without the test sample, was the control. After 24 h, 0.1 ml of the mixture was taken and mixed with 4.7 ml of 75% ethanolic, 0.1 ml of 30% ammonium thiocyanate and 0.1 ml of 20 mM ferrous chloride in 3.5% HCl and allowed to stand at room temperature. Precisely 3 min after the addition of ferrous chloride to the reaction mixture, the absorbance at 500 nm was measured [29].

Scavenging of superoxide
The reaction mixture, which contained 50 µL of sample, 50 µL of NADH (936 µM) in phosphate buffer (100mM, pH 7.4), 50 µL of NBT (300 µM) in phosphate buffer and 50 µL of PMS in phosphate buffer was incubated at room temperature for 5 min. The absorbance was read at 560 nm by an ELISA reader, against blank samples (A 1 ). The reaction mixture, without the extract was used as control (A 0 ) [30].
The superoxide scavenging effect calculated using following equation:

Scavenging of hydrogen peroxide
A solution (43 mM) of hydrogen peroxide was prepared in phosphate buffer (0.1 M pH 7.4). 400 µL of sample was mixed with 960 µL phosphate buffer. Then, 240 µL of hydrogen peroxide solution (43 mM) was added to the mixture. Absorbance was read at 230 nm by a spectrophotometer against blank samples (A 1 ). The reaction mixture, without the extracts was used as control (A 0 ) [31].
The hydrogen peroxide scavenging effect calculated using following equation:

Total Antioxidant Response (TAR)
To 5 µl herbal extract, 200 µl R1 (o-dianisidine) was added. The first absorbance was taken before mixing R1 and R2 (as sample blank). After 4,5 min, 10 µl of R2 (H 2 O 2 ) was added the mixture and the last absorbance was taken when the reaction trace drew a plateau line (about 3-4 min after the mixing). Measurements were taken by an Elisa reader at 444 nm [32].

Results
On the basis of these mechanisms, the present study was designed to examine the antioxidant capacity by eight commonly used methods of some plants, which are considered to have anti-inflammatory effects. Determination of total phenolic content, DPPH and DMPD radical scavenging, cupric ion (Cu(2+)) reducing, inhibition lipid peroxidation, hydroxyl radical scavenging capacity were applied to the plants which are collected from Düzce and its surroundings and used as folk medicine. As far as we know, Folin, Cuprac, and DPPH methods were modified for the first time for our study, to work with 96 well plates.

Folin-Ciocalteu method for total phenolic content
The total phenolic contents of the plants were determined from the regression equation of gallic acid calibration curve (y=0.4685 ln(x)-1.131) (Figure 1). The concentrations of total phenolic content of extracts of plants are shown in Table 2. The highest amount was found in the P. lanceolata, followed by R. hirtus, S. ebulus, P. major ssp. major, H. helix and M. alba, respectively.

CUPRAC assay
Cuprac is one of the assays to determine the total antioxidant activity, which uses cupper II as an oxidant. This method is based on the reduction of Cu(II) to Cu(I) by antioxidants (reductant), which present in the sample. This method measures the capacity of hydrophilic and lipophilic antioxidants. On the first trial, standards gave good results and the same results were obtained from extracts. Trolox (a watersoluble tocopherol analogue), gallic acid and vitamin C were used as standard solutions. We observed a good correlation between the concentration and percentage inhibition(y=0.0032 ln (x)-0.0379, R 2 =0.9985 for trolox, y=0.4685 ln (x)-1.131, R 2 =0.9893 for gallic acid, y=0.0024 ln (x)-0.2221, R 2 =0.9695 for vitamin C). According to the results of the cupper reducing antioxidant capacity (CUPRAC) assay, R. hirtus and P. lanceolata showed the highest antioxidant activity, M. alba showed the lowest activity in aqueous and methanolic extracts ( Figure  1a and 1b). In ethanolic extracts, while P. lanceolata exhibited the

DPPH assay
DPPH free radical scavenging is one of the generally accepted mechanisms against lipid oxidation. Difference between DPPH free radical binding method and the other method is the short run time allowing rapid determination of the radical scavenging. The effect of antioxidants on DPPH radical scavenging was thought to be due to their hydrogen donating ability. The antioxidant activity of plant extracts were calculated according to the percentage inhibition in DPPH assay. Gallic acid was used as the reference standard (y=0.9221 ln (x)+01.039, R 2 =0.9925). The DPPH free radical scavenging activity of the plant extracts are shown in Figure 2a-2c. While R. hirtus had the highest activity in aqueous and methanolic extracts, P. lanceolata showed the highest DPPH free radical scavenging activity in ethanolic extracts. M. alba showed the lowest DPPH free radical scavenging activity in aqueous, methanolic and ethanolic extracts.

DMPD assay
DMPD method is suitable for fast and sensitive measurement of antioxidant activity of hydrophilic compounds. In our study, although we observed high correlations in DMPD radical and Trolox standard studies, we couldn't reach the same consistency in all plant extracts. However, similar results to those in other methods were observed in methanolic extracts gave the most uniform results. Different concentration of DMPD was carried out (y=0.1339 ln (x)-0,083, R 2 =0.9875 ) and trolox was used as standard (y=17.771 ln (x)-63.376, R 2 =0.9901). The antioxidant activity of plant extracts were calculated according to the percentage inhibition in DMPD assay. According to our results R. hirtus gave the highest antioxidant activity in methanolic extracts, P. major ssp. major gave the highest antioxidant activity in aqueous extracts and P. lanceolata gave the highest antioxidant activity in ethanolic extracts (Figure 3a and 3c). While S. ebulus had the lowest antioxidant capacity in ethanolic extracts, M. alba showed the lowest antioxidant capacity in methanolic and aqueous extracts (Figure 3b).

ABTS assay
ABTS method is commonly used to assess radical scavenging or antioxidant activity of the sample on the inhibition of ABTS radical (ABTS . ), which is a synthetic compound and is produced by oxidation of ABTS. ABTS assay is based on the decolourization of blue-green ABTS radial cation, which is reduced by antioxidant compounds, plant, and fruit extracts. Trolox, which was observed to have a good correlation between the concentration and percentage inhibition (y=-0.0001 x 2 +0.2374x-3.2115, R 2 =0.9953), was used as standard. According to our results, P. lanceolata, P. major ssp. major, R. hirtus, and S. ebulus gave almost the same antioxidant activity, M. alba showed the lowest activity in aqueous extracts (Figure 4a). Outside the small differences, we observed roughly similar results in methanolic and ethanolic extracts (Figure 4b and 4c).

Ferric thiocyanate assay
In our study, inhibition of lipid peroxidation capacity of aqueous plants were investigated by ferric thiocyanate assay, which is based on the oxidation of fats and oxidizing Fe +2 to Fe +3 by peroxides and Fe +3 ions which occur in this reaction, making it rather complex with SCN. Decrease in the absorbance indicates an increase in the free radical scavenging activity. In this assay, gallic acid was used as standard (y=-0.224 ln (x)+1.6333, R 2 =0.9673). The results showed that P. lanceolata had the highest antioxidant activity followed by P.major ssp. major, R. hirtus, H. helix, and S. ebulus. The present work demonstrated that the aqueous extract of M. alba leaves did not show remarkable activity ( Figure 5).

Scavenging of superoxide
Superoxide (O 2¯• ) is accepted to be a very harmful radical that can be converted into more reactive species, such as hydroxyl radical or peroxynitrite, contributing to tissue damages and various diseases. The superoxide radical assay of samples was measured according to the method reported by Robak and Gryglewski [30]. The scavenging activity of superoxide radical of plant extracts was measured and the percentage inhibition was calculated. Different concentration of vitamin C and gallic acid used as standard (y=-6E-05 x 2 +0.109x+25.947 for vitamin C, R 2 = 0.9994 and y=12.485 ln (x)+2.186, R 2 =0.9848 for gallic acid). According to the data R. hirtus gave the highest antioxidant activity and M. alba showed the lowest activity in aqueous and ethanolic extracts (Figure 6a and 6c). In methanolic extracts, while R. hirtus exhibited the highest antioxidant activity, H. helix showed the lowest activity ( Figure  6b).

Scavenging of hydrogen peroxide
Hydrogen peroxide (H 2 O 2 ) is a weak oxidizing agent but it can cross cell membranes rapidly. Once inside the cell, H 2 O 2 can react with Fe +2 ions to generate the hydroxyl radical and this may be the origin of many of its toxic effects [33]. H 2 O 2 is relatively stable in the absence of reducing compounds. Scavenging of H 2 O 2 by plant extracts may be attributed to their electron donating abilities [34]. Hydrogen peroxide scavenging activity of the plant extracts is presented in Figure 7; In this method, we used only one concentration (50 μg/mL) of all extracts. In aqueous extracts, M. alba showed the highest activity and P. lanceolata showed the lowest activity. While S. ebulus gave the highest antioxidant activity, P. lanceolata showed lowest activity in methanolic and ethanolic extracts.

Total antioxidant response (TAR)
In the last method, to measure total antioxidant response, the  radical is produced by the Fenton reaction and oxidize colourless substrate O-dianisidine to the dianisyl radical, which is bright yellowish-brown in colour. This method is based on suppression the colour formation by antioxidants, which are present in the sample. Results were expressed as trolox equivalent in mg of Trolox per ml (y=-0.0028 ln (x)+0.8247, R 2 =0.9971). When we examined the Troloxequivalent antioxidant capacity of the samples, R. hirtus and P. lanceolata showed the highest activity, M. alba showed the lowest activity in aqueous extracts. While Plantago species showed the highest activity, S. ebulus and M. alba did not show any activity in ethanolic extracts. In methanolic extracts, R. hirtus showed the highest activity, H. helix and S. ebulus showed lowest activity. The ethanolic extracts of S. ebulus and M. alba did not give any result (Figure 8), this might be due to insufficient amount of the kit, therefore experiment cannot be replicate. The results, which are obtained by TAC method, are consistent with other experiments.

Discussion
The process of inflammation may be initiated by an infection or cell damage that stimulates the secretion of interleukins or tumour necrosis factor by macrophages. Activated neutrophils produce large amounts of ROS, thus causing the production of toxic reactive species to increase. Neutrophils also release myeloperoxidase (MPO). Myeloperoxidase (MPO) catalyzes the reaction of hydrogen peroxide with chloride ion to produce hypochlorous anion. Also reaction of hypochlorous anion with hydrogen peroxide yields singlet oxygen, which is a reactive species. The interaction of superoxide radical with hydrogen peroxide and iron is causing the formation of hydroxyl radical, which is highly reactive, and the reaction of superoxide with nitric oxide is resulting in the formation of peroxynitrite. These reactive species kill pathogens by halogenations or by protein oxidation and lipid peroxidation. If the balance between oxidants and antioxidants shifts in the direction of oxidants during the inflammation, healthy cells can get damaged [35,36].
Phenolic compounds are herbal substances whose chemical structures may range from quite simple compounds to highly polymerized substances. The capacity of phenolics to scavenge free radicals may be due to many phenolic hydroxyl groups they posses. Phenols play an important role in antioxidant activity, because they transfer hydrogen to radicals and produce phenoxide radical, which is stabilized. Therefore, it is important to determine the total amount of phenolics to determination of antioxidant capacity of plants. In our study, we found higher phenolic content in P. lanceolata, R. hirtus, S. ebulus and P. major ssp. major than those in H. helix and M. alba.
Plantago genus contains 275 species throughout the world and has been used for medical purposes since ancient times. In recent years, more research has been devoted to the study of antioxidants for these plants, which are astringent, anti-toxic, antimicrobial, antiinflammatory, expectorant and diuretic. Some of the compounds in Plantago sp. such as ursolic acid, plantamajoside R, aceteoside R and plantagonin have been shown to have both an antioxidant and antiinflammatory effect [6]. Numerous studies have shown that Plantago species had a high phenolic content and antioxidant activity [39][40][41][42][43][44][45][46][47][48]. There are few studies carried out in Turkey showing high antioxidant activity for P. lanceolata [40][41][42][43][44][45][46][47][48][49][50] and P. major [51,52]. In our study, we also observed high content of phenolic compounds and antioxidant activity for P. major ssp. major and P. lanceolata in some of the methods.
In several studies, very high antioxidant activities were observed in different types of Rubus (Rubus urticifolius Poir., Rubus parviflorus Nutt.) [53][54][55][56][57]. Although anti-inflammatory and antinociceptive effects of R. hirtus have shown in previous studies [58,59], only one study has specifically investigated the antioxidant activity of its fruit [60]. As far as we know, this is the first study showing antioxidant activity in the leaves of R. hirtus. According to our results, its antioxidant activities were mostly higher than other plants studied and we think that these high antioxidant activities of R. hirtus may contribute to its antiinflammatory effects.
S. ebulus has shown high antioxidant activity in generally and a positive correlation was observed between antioxidant activity and phenolic content [61,62]. According to our results, although the phenolic content of S. ebulus was close to P. lanceolata and R. hirtus, their antioxidant properties were not similar. However, high levels of thiocyanate found in S. ebulus indicate its ability to inhibit lipid peroxidation. According to our knowledge, this is the first study, which has investigated the antioxidant activity of S.ebulus in Turkey.
It has been shown that alpha hederin and hedera saponin C component of H. helix had a strong total antioxidant activity and inhibits lipid peroxidation [63][64][65][66][67]. However, H. helix showed lower total phenolic content and lower antioxidant activity when compared
It is thought to be due to the different time and collection zone of study materials. In a previous study, Sivaci and Sökmen showed seasonal changes in antioxidant activity and total phenolic contents in stems of Morus species [77]. According to their results, Morus species had the highest antioxidant activity and total phenolic contents in October whereas the lowest in February, even in June, which is when we collected them. The similar seasonal changes in the contents of H. helix leaves have been shown by Fischer and Feller [78].
In conclusion, the aim of this study was to examine the antioxidant properties of some plant species chosen due to their anti-inflammatory uses and to obtain results can be useful for further studies. In our study, showing high antioxidant activity of the plant species with high phenolic content supports many other studies, which have shown an association between phenolic compounds and antioxidant activities. Because seasonal changes affect the contents of plants too, it is difficult to compare the activities of different plants. Observation of different antioxidant activities of some plant species which have been demonstrated their anti-inflammatory effect in the literature and used by people for this purpose can be interpreted in different ways. Because of their use for different purposes other than anti-inflammatory effects, and knowing their major effects makes it easy to comment on the results. For example, S. ebulus has been mostly used as a pain killer, as an antiinflammatoir and has showed a relatively lower antioxidant activity in our study. In addition, if we had looked for their anti-inflammatory activities, it could be useful for the interpretation of such results.
Although it is not our main goal, we modified Folin, Cuprac, and DPPH methods to work with 96 well plates. This would be useful for those who will work with these methods.