INTRODUCTION

The most important source of natural drugs used in conventional treatment methods is plants. Research conducted worldwide during the recent years claim that almost 72,000 (corresponding to about 17%) of the 422,000 flowering plants which are spreading across the world have a therapeutic value¹. In the 18th century, nearly 8000 plant species were classified systematically by Carolus Linnaeus (1707-1778) and this classification not only facilitated the works of naturalists but also the pharmaceutical chemists². The science of ethnobotany, which deals with serious research recognized by all the world today, was born as a result of the bond that has existed between humans and plants for centuries. Ethnobotanic knowledge contributes greatly in the scientific evaluation of plants with its content that reflects very valuable information which was acquired by trial and error and has reached today via transfer from generation to generation over a long period of time³. The term "phytotherapy", that means "therapy with medicinal plants" was used by the French doctor Henri Leclerc (1870-1955) for the first time. Phytotherapy is one of the oldest natural therapy methods known in the human history, which aims to prevent and cure diseases with natural medications prepared by using all or some parts of the plants⁴. In this way, it was considered a strong possibility that bioactive substances which a plant species of well-known medical value contains, might be present in other plant species that are related with the former one and thus, the diversity of plants that can be used as herbal medicine has increased rapidly⁵. Plants convert, in their own metabolism, the water and minerals they get from the soil to nutrients that can be digested by the human body. Examples of the main nutrients include carbohydrates, proteins, lipids, vitamins and minerals⁶. These are commonly used active macromolecules that are produced in the plant’s metabolism.

They increase the power of the defense of the body, support the functions of organs and/or accelerate recovery. Thus, they have a positive effect on the functions of specific tissues and organs in the organism⁷. Throughout human history, many diseases (diabetes, jaundice, shortness of breath etc.) have been tried and are still being tried to be cured by using plants. Scientists have conducted various kinds of studies for centuries to identify these therapeutic aspects⁸. The World Health Organization (WHO) has reported that about 4 billion humans around the world (80% of the world’s population) try to cure their health problems with herbal remedies at the first step⁹. Furthermore, herbal active substances (vimbilastin, reserpine, kinin, aspirin etc.) make up around 25% of the prescription drugs in developed countries¹⁰. It was found in the literature that thousands of phytochemicals obtained from plants are reliable and have very few adverse effects. Many plants have been reported to exhibit anticancer, antimicrobial, antioxidant, antidiarrheal, analgesic and wound healing benefits^11,12. There are many reasons behind the increase in studies conducted on plants. These reasons include frequent use of antibiotics that leads to pathogen micro-organisms’ developing resistance and consequently, the reduction in the effects of antibiotics, the desire of developing countries that do not have a competent chemical industry of gaining an easy and economic treatment possibility, harmful adverse effects of the synthetic compounds used for therapeutic purposes, the fact that some substances that can be obtained from herbal drugs can be extracted more economically, easily and multiple effects of drugs¹³. These facts support the supplementary effect of herbal medicines on the organism when used in combination with synthetic drugs that are toxic and expensive and their use as an alternative means of treatment. Recently, detailed studies have been conducted to identify the bioactivity of extracts obtained from medical and aromatic plants with the aim of researching new compounds that control the oxidative DNA damage causing cancer¹⁴.

In a study conducted by Moaket et al.¹⁵ to identify the bioactive components of the endemic yellow iris plant, it was found that the plant had active components with antioxidant, antibacterial and DNA protecting properties. Plants with antimicrobial effects are of great importance in terms of controlling the organism species that develops resistance against the commonly used antibiotics¹⁶. The primary factor causing food spoilage and the loss of nutritional values is the microbial activity. Essential oils vary in their biological effects since they are complex compounds with different components¹⁷. While their effects change depending on their active substances, many essential oils have effects including antimicrobial, carminative, choleretic, sedative and diuretic effects¹⁸. Besides, essential oils of the aromatic plants, most of which belong to the Labiatae family have been shown to have antimicrobial activity¹⁹. For instance, it has been found that the essential oils of basil, laurel, clove, thyme and rosemary shows antibacterial activity against Listeria monocytogenes (L. monocytogenes) and other pathogens. It is claimed that garlic, cinnamon, curry, mustard, basil, ginger and some other plants exhibit antimicrobial effects²⁰. In this study, the application of some chemical methods used in isolation, extraction and characterization of the secondary metabolites, the most important bioactive components of plants, have been examined as well as some experimental methods used in identification of the bioactive component contents of the extracts obtained by using either biochemical or molecular biological methods.

Secondary metabolites of plants and how to obtain them: Plants produce proteins, lipids, carbohydrates and chlorophyll as the primary metabolic products after photosynthesis: Primary metabolites (carbohydrates, lipids, proteins, etc.) are quite common in nature and found quite a lot in the seeds and vegetative tissues of tall plants^21,22.

They are necessary for the physiological development of the plant due to their essential roles in the cell metabolism. Secondary metabolites, on the other hand are the chemical components that are not responsible for the growth and development of the plant but are generally believed to have roles such as adaptation to environmental conditions, chemical defense against micro-organisms, insects and other predators (hunters) and competition with other plants¹⁷(Fig. 1). The secondary metabolites that were previously assumed to have no role and be waste matter produced by the plants were discovered to be quite complex mechanisms developed by plants for defense, protection, adaptation, survival and continuity of the family purposes, in the 19th century²³. These metabolites enable the plants to adapt to the biotic and abiotic stress conditions (against infection, injury, water, stress, cold and high intensity light)²².

Secondary metabolites are hard to extract and purify since they are synthesized in specialized cell types of plants and different growth stages of plants²⁵. Extraction, pharmacological screening, isolation and characterization of the bioactive substances with beneficial biological activity is extremely important. One of these process steps applied to obtain these metabolites from the plant under the most suitable conditions is as shown in the below Fig. 2²⁶.

Since the secondary metabolites are found in trace quantities in the plant, various biological and chemical technologies have been developed in recent years to obtain them. Extraction is the first step used in the process of drug research carried out on plants. In this very critical first step, efficient extraction of the chemical components from the plant material is necessary for their identification and proper separation in the following stages²⁷.

These extracts have various types of phytochemical or bioactive component combinations with various polarities, which allows serious efforts in the characterization of bioactive components. There are several separation techniques used commonly in practice to separate these bioactive components smoothly²⁸.

Some of these include column chromatography, sephadex chromatography, flash chromatography and HPLC. Apart from these, non-chromatographic methods such as immuno-assay and phytochemical imaging assay might also be used.

Biological methods used in the identification of bioactive characteristics of plants: Most of the bioactivity assessments are related to antimicrobial, antitumoral effects and the immune system²⁹. There are various biological and biochemical and molecular biology based methods used in the identification of bioactive characteristics of plants.

Fig. 1:	Primary and secondary metabolites of plants24

Table 1:	Antioxidant assay methods

Fig. 2:	A diagram of general approach on isolation, characterization and extraction of bioactive components from the plant extracts

Both in vivo and in vitro assessments are used in the identification of the biological activities of plant extracts³⁰. All in vivo methods involve the use of micro-organisms and tests are applied on animals (mice, rats etc.) with proper doses and methods, followed by the assessment of blood or tissue samples. In vitro methods use sub-cellular systems such as enzymes and receptors that are isolated from animal or cell cultures³¹. The most commonly used one among these is the evaluation of the oxidant states of plants. Identification of the antioxidant activity in plant extracts is both an easy and cost-efficient method to be used in the elimination of the possible effects of the free radicals. Free radical types are required for cellular activities such as phagocytosis, regulation of cell proliferation, signal transmission, active component transfer and production of ATP³². Free radicals also have harmful effects. These harmful effects occur as a result of the irregularities between over-accumulation of free radicals and the anti-oxidant defense system. Therefore, free radicals must be neutralized³³. Antioxidants are molecules that generally carry a phenolic function in their structure and protect the cell against damage by preventing the formation of free radicals or removing the existing radicals. Antioxidants significantly inhibit or delay oxidation of the substrate that is started with pro-oxidants in lower concentrations as compared to the substrates that can be oxidized^34,35.

Therefore, study of oxidant states in the extractions obtained after the required purification process while identifying the bioactivities of the plants is among the important biological and biochemical methods. There are 29 different methods in literature, including both in vivo and in vitro ones, which are used in the identification of antioxidant levels. As shown in Table 1, 19 of them are in vitro and 10 are in vivo assay methods^36,37.

According to the results of the study conducted by Alam et al.³⁸ the most commonly used 4 in vitro assays among these methods are DPPH>hydroxyl radical inhibitor>SOD>b-carotene>Linolate, whereas, the most common in vivo assay is LPO, followed by CAT, GSHPx. It is highly important to evaluate the DNA protection activities of plants while determining their antioxidant capacities. The methods to identify the DNA protection activity of plant extracts are among the most essential molecular biology methods. It is known that UV rays that reach the world due to the destruction of stratosphere layer have negative effects on the living beings. Antioxidants also provide protection against the harmful effects of UV rays. UV rays lead to severe diseases that result in skin cancer and aging. Topical application (on the skin) of enzymatic and non-enzymatic antioxidants is an effective approach in protection of skin against the harmful effects of UV rays^39,40. Actually, human skin has a set of mechanisms that reduces the harmful effects of VIS (visible rays) and UV rays on human skin. But exposure to UV rays at a high level might lead to a decrease in the amount of cellular antioxidants and consequently UV-related oxidative DNA damage caused by reactive oxygen types.

Free radicals might also lead to DNA damage as well as the UV rays. For instance, hydrogen peroxide as a type of free radical, leads to DNA damage, by converting the guanine to 8 hydroxyguanine⁴¹. Recently, detailed studies have been conducted on extracts obtained from medical and aromatic plants with the aim of researching new compounds that control the oxidative DNA damage causing cancer⁴².