In Vitro Screening of Natural Drug Potentials for Mass Production

Methanolic extracts of 16 plant species, used in herbal and traditional medicinal preparations, collected from the same geographic region (Golcuk-Seben/Bolu, Turkey) were evaluated for their relative total phenolic contents, total antioxidant activities and their mineral contents in both dried plant samples and methanolic extracts. These species included Hypericum perforatum L., Thymus sipyleus Boiss., Equisetum arvense L., Achillea millefolium L., Tanacetum parthenium L., Thymus leucostomus Hausskn. & Velen., Usnea longissima, Herniaria incana Boiss., Teucrium polium L., Stachys bithynica Boiss., Anthemis nobilis L., Sambucus ebulus L., Berberis vulgaris L., Malus communis Desf., Matricaria chamomilla L. and Scorzonera laciniata L. Spectrophotometric Folin-Ciocalteu and DPPH free radical scavenging activity methods were performed to detect the total phenolic contents and total antioxidant activities of the methanolic extracts, respectively. The results show that the fruits of Berberis vulgaris L., have the highest antioxidant activity and aerial parts of Tanacetum parthenium L. have the highest total phenolics (92.62%). Mineral contents were determined by inductively coupled – mass spectrometry (ICP-MS). All dried plant samples and methanolic extracts contained high amounts of Si, Cu, Fe and Ca. The highest levels of Si, Cu, Fe and Ca were found in Equisetum arvense L., Anthemis nobilis L., and Thymus sipyleus Boiss. dried plant samples, whereas Se was only detected in Usnea longissima. The highest levels of Si, Cu, Fe and Ca were found in the methanolic extracts of Usnea longissima, Anthemis nobilis L. and Thymus sipyleus Boiss. respectively.


Introduction
Medicinal plants have a growing economic value in clinical, pharmaceutical, cosmeceutical, and other wide areas of international trade, although this varies widely among countries (Schmidt, 2012).
The plant extracts and phytochemicals isolated from these medicinal plants have been shown to exert biological activity in vitro and in vivo, which justified further research on traditional medicine focused on the characterization of their mostly antimicrobial and antioxidant activities (Deliorman Orhan et al., 2012).It is well accepted that, reactive oxygen compounds such as singlet oxygen, superoxide radicals, hydrogen peroxide, hydroxyl radicals and nitric oxide are unstable and extremely reactive (Ames et al.,1993).Oxidative stress-induced reactive oxygen species are considered to be the indicators of the development and progress of various diseases.Antioxidants prevent the negative impacts of free radicals and reactive oxygen species and protect the body (Gavamukulya et al., 2014).The widely known synthetic antioxidants are butylated hydroxyltoluene (BHT), butylated hydroxylanisole (BHA), propyl gallate and tertiary butylhydroquinone.However, the reliability of these synthetic antioxidants has been debated due to their toxic and carcinogenic effects, which can result in liver injury.Therefore, discovery of new and reliable antioxidants from natural resources has become a prominent research topic (Birman, 2012).
Flora of Gölcük-Seben, 100 km distance from Istanbul and Black sea region, which has been a little studied by scientific means before.This study provides an identification of potential, bioactive species that can be used as raw materials for plant derived products in several industries.The objectives of this study were to determine the relative total phenolic contents and antioxidant activity of 16 medicinal plant species including H. perforatum L., T. sipyleus Boiss., E. arvense L., A. millefolium L., T. parthenium L., T. leucostomus Hausskn.& Velen., U. longissima, H. incana Boiss., T. polium L., S. bithynica Boiss., A. nobilis L., S. ebulus L., B. vulgaris L., M. communis Desf., M. chamomilla L. and S. laciniata L. found in the Gölcük-Seben/Bolu area.The healing properties of these plants are very well known and used by local people in treatments of various diseases.Furthermore, some minerals, minutes at room temperature and their absorbance was measured with a spectrophotometer at 415 nm.According to calibration curves, total flavonoids were calculated as quercetin equivalents using the absorbance of the samples (Woisky and Salatino, 1998).

Determination of the concentration of minerals
The mineral content in plant samples was determined using inductively coupled -mass spectrophotometry (Perkin Elmer Nexion 350 D / ICP-MS) according to the NMKL 186 method.Sample preparation was conducted by adding 8 mL HNO3 (65.0%) and 2 mL hydrogen peroxide (35.0%) to a 0.25 g extract/plant sample and completing up to 50 mL by distilled pure water, in a microwave system (Berghof MWS4).The measurements were calculated as dry weight in mg/kg according to the following formula: W = [(a×V×F) / m×1000], where: a -is the content of the element in test solution in microgram per litre; V -is the volume of the digestion solution after being made up, in milliliter; F -is the dilution factor of the test solution; m -is the initial sample mass, in gram.

Mineral substance content
The mean of the level of minerals determined in 16 medicinal plant samples is summarized in table 5-6.Se has important role on thyroid metabolism, improving and sustaining of nervous system (Georgieff, 2007;Skröder et al., 2015).Neurological problems have occurred due to deficiency known to be useful for human health at certain dose levels, and which have a number of physiological functions in living organisms, were quantitatively determined both in dried plants and methanolic extracts.All these data will be used to help select the plant species, to be cultured in that region, if commercialization is warranted.

Site characterization
Collection site is located in an area of Euro-Siberian, Mediterranean and Irano-Turanian Floristic Regions together with Sub-Euxine and Xero-Euxine zones.The coordination of research area is given in Table 2.The soil type of the research area is mainly brown forest soil, which is extremely calcified.The structure of the soil is fine-pored or granulated and the pH of the soil is alkaline and rarely neutral.The study site, dating back to upper-Eocene (35-40 mya) of the Tertiary in the Cenozoic era, consists of different types of rock, including basalt, andesite, turf and aglomera of a variety of colours from pink to black to claret red.The surface morphology, formed by erosion and tectonic activities, is elaborated by metamorphite and volcanic rocky types (Ikinci and Guneri, 2007).According to the data obtained from the Bolu Meteorology Station, the mean annual temperature of the area is expected to be between 7.1 °C and 9.6 °C.Mean annual precipitation of the research area is expected to be between 599.6 and 869.6 mm.Of this precipitation 32% is in winter, 29% is in spring, 21% is in autumn and 18% is in summer.The rainiest months of the year are December, January and February whereas the least rainy month is August.

Plant materials and extraction of the plants
A list of the studied plants, including the botanical name, voucher specimen and data related to therapeutic usage and the plant parts used are listed in Table 1.Voucher specimens were identified by Dr. Mecit Vural and they were deposited in the Herbarium of Field Crop Department, Abant İzzet Baysal University (Bolu, Turkey).Plant samples were air dried at room temperature and 20 g powdered plant materials extracted by chloroform and evaporated till dryness.Then 80% methanol (2×250 mL) was added to the remaining material after chloroform filtration and again vaporized.Chloroform and methanolic extracts were used for further analyses.

Determination of total phenolic compounds
Total phenolic contents of sixteen extracts were determined by using Folin-Ciocalteau Method (Singleton and Rossi, 1965).From each extract, 2 mg mL -1 sample was weighed and dissolved in 75% ethanol.Reference gallic acid dilutions and each extract samples were mixed with 100 µL Folin-Ciocalteau reagent, sodium carbonate (20%) and distilled water.Mixtures were incubated for 30 minutes at 40ºC and their absorbances were measured at 765 nm.According to the calibration curve, the total phenolic concentration was calculated as gallic acid equivalents using the absorbance of the samples.

Determination of total flavonoids
To determine total flavonoids, 2 mg mL -1 of each extract samples and the reference quercetin solutions were mixed with 75% EtOH, 10% AlCl3, 1M sodium acetate and 2,800 µL distilled water separately.Mixtures were incubated for 30  of it (Georgieff, 2007).Only U. longissima dried plant sample contained Se (220.11± 44.02 ppm) The WHO has advised iron's grade in plants as 20 mg kg -1 (Shah et al., 2011), which has positive effects on strengthening the immune system and cognitive performance (Domellöf et al., 2001;Kon et al., 2010).Low levels of iron gives rise to inflammation, pain, loss of appetite, dysphagia, exhaustion, cold limbs and impairment in nails, on the other hand, high iron intake causes lethargy, increasing blood flow of vessel and hypertension.(Berridge, 1975), and has role on skeletal structure.Deficiency of Ca causes convulsion on muscles, and lumbago (Passwater, 1983), however, excess intake of Ca triggers gastrointestinal diseases such as dyspepsia, mouth dryness caused by hyperkalemia; weakens of physical activity (Watts, 1990).Total Ca concentrations in the dried plants ranged from 0.08 ± 0.01 to 4.74 ± 0.66, with the highest value in E. arvense and the lowest value in M. communis.Copper has an antioxidant activity (Tsuji et al., 2016) and its Daily recommended intake is 10 mg kg -1 (Hassan et al., 2012).Excessive amount of Cu uptake gives rise to pain, irritation, anemia, diarrhea and organ damage (brain, kidney, liver etc.), (Water Treatment Solutions (Cu), 1998;Adnan Iqbal et al., 2011;Mebrahtu and Zerabruk, 2011;Aenab et al., 2013) however, deficiency of copper causes arthrosis and cardiovasculer problems and, susceptibility to infection (Passwater, 1983;Klevay, 1975;Passwater, 1983;Campbell, 2001).A. nobilis, T. parthenium, S. bithynica and S. potentilla also contained significant amounts of Cu (1.84 ± 0.24 -13.06 ± 1.70).In addition, A. nobilis L. methanolic extract included more copper than dried plant sample.
In metanolic plant extract, T. leucostomus and U. longissima contained selenium (386.78 ± 77.36 -746.02 ± 149.20 ppm) respectively.Although, dried plant samples demonstrated lower levels of Se, methanolic extracts had higher levels of Se that might due to the water solubility of the Se mineral.The highest Fe content had T. sipyleus (596.35 ± 77.53 ppm) and it was respectively followed by E. arvense (135.23 ± 17.58 ppm), S. bithynica (121.14 ± 15.75 ppm), H. incana (79.99 ± 10.40 ppm).The amount of Si content in exctract plant samples changed from 568.54 ± 85.28 to 27.92 ± 4.19 ppm; with the highest value in U. longissima and the lowest value in B. vulgaris.Total Ca concentrations in the extract of plants ranged from 0.023 ± 0.003 to 0.93 ± 0.13, with the highest value in T. sipyleus and the lowest value in B. vulgaris.In addition, A. nobilis, T. polium and T. parthenium also contained significant amounts of .Furthermore, dry plant samples contained higher mineral elements than methanolic plant extracts.

Discussion
In previous literatures, total flavonoid, phenolic content and antioxidant activity of H. incana and T. parthenium reported are in agreement with the result of the present study (Rabiei et al., 2014;Wu et al., 2006).Studies on A. millefolium demonstrated the presence of flavonoids such as apigenin, luteolin and rutin, which show powerful antioxidant properties, in this plant (Tuberoso et al., 2009;Kocevar et al., 2012).Hanachi and Sh (2009) reported that the antioxidant activity of the ethanolic extract of B. vulgaris fruit has the highest (27.26 ± 1.07%).Our data indicate that T. polium and U. longissima have moderate levels of antioxidant activity in agreement with the fact that polyphenols and chemical composition are affected by different factors, such as genotype, environmental conditions and extraction procedures (Halici et al., 2005;Sharififar et al., 2009;Stankovic et al., 2012;Andrè et al., 2012).Ahmed et al. (2016) reported that the species such as A. millefolium L., Equisetum arvense L., H. perforatum L., S. aromaticum L., M. chamomilla L. exhibited high antioxidant properties.Kukric et al. (2013) evaluated the antioxidative effect of dried ethanolic extract of E. arvense, which had an IC50 and AAI (antioxidant activity index) of 13.5 and 3.9, respectively.Although present findings are somehow similar to results of those earlier studies, differences in extraction and activity methods, climate, soil, environmental factors, diseases, and pesticide treatments, harvest time, drying and storage methods and plant parts used in analyses may significantly affect the antioxidant activity of plants (Bergonzi et al., 2001;Wang and Zheng, 2001).In previous scientific studies, all over the world on nutritive composition of wild plants high quantities of minerals, especially K, Na, Ca, P, Mg (Guil Guerrero et al., 1998;Agrahar-Murugkar and Subbulakshmi, 2005) are indicated.Iron values were reported between 10 and 981 mg kg -1 with highest value determined in sage (Zengin et al., 2008).Se values were reported between 0.0015 and 0.4 mg kg -1 with the highest value determined in Solenostemma arghel (Sheded, 2006).Average values for copper content of some edible plants varied from 0.05-18.4mg kg -1 (Yildirim et al., 2001;Turan et al., 2003).Equisetum arvense had minerals content such as silica, calcium, magnesium, selenium, iron, potassium, zinc (Veit et al., 1995;Beckert et al., 1997).Ozcan (2004) indicated that the highest levels of Ca, Fe, K, Mg and S were found in T. vulgaris, L. officinalis L., A. graveolens L., O. basilicum L. and S. alba L., respectively while Bi, Cd, Li, Pb and Se contents of condiments were found to be very low.Our studies on mineral content of the plants were also in accordance with the previous findings.

Conclusions
In the present study, it was determined that some plants in Bolu/Turkey contained abundant phenolic compounds and mineral constituents.Antioxidant activities were also evaluated from methanolic extracts of these plants.From the experimental results, T. sipyleus and Stachys bithynica Boiss with A. nobilis L., E. arvense L., T. parthenium L, T. polium L., T. leucostomus Hausskn.& Velen were of particular interest.If these plants are protected by culturing, then, mass production of natural drug potential can be carried out easily and efficiently.

Table 1 .
Chemical constituents of the 16 extracts with their traditional usage in Turkey

Table 2 .
Collection sources of the plant material

Table 4 .
Total flavonoid and phenolic contents

Table 5 .
Mineral content of dried plant samples

Table 6 .
Mineral content of the methanolic extracts