The polyphenol composition of Cistus incanus L., Trachystemon orientalis L. and Melissa officinalis L. infusions by HPLC-DAD method

Because of plants, health benefi ts consumption of herbal infusions as a dietary additive has increased in the last years. The aim of the present study was to establish the individual phenolic profi les using highperformance liquid chromatography with diode-array detection (HPLC-DAD) method of Bulgarian Cistus incanus leaves harvested in summer and winter seasons, Cistus incanus hard-coated seeds, Greek Cistus incanus leaves harvested in summer, Bulgarian Trachystemon orientalis, and Melissa offi cinalis leave and stems extracts prepared as infusions. Cistus incanus L. has played a very interesting role in the historical medicine of the Mediterranean region where is well known. Trachytsemon orientalis L. is wild borage typical for Caucasus nature. Both wild plants are widely distributed in Strandja mountain, Bulgaria and the ethnomedicinal description of them as anti-infl ammatory and antioxidant herbs are missing. The Melissa offi cinalis is wildly used as an aromatic, culinary and medicine remedy and its medicinal properties are well known of Bulgarian folk medicine. In the present study, the newly established HPLC-DAD method developed and validated previously for simultaneous separation of 9 natural substances common in plants tree major catechins, four major fl avonoids, gallic and vanillic acid was used. The method was successfully applied to the determination of the specifi c bioactive substances in the investigated herbs and especially Cistus incanus species. Analyses under the method applied revealed that in the Bulgarian Cistus incanus summer leaves infusion, the values of (-) epicatechin (3460.54 ± 1.89 μg/mL) and (-) epigallocatechin (7369.23 ± 2.56 μg/mL) were found to be signifi cantly higher (p > 0.05) than values of the two catechins found in the other infusions. However, the winter Cistus incanus leaves contains 640.03 ± 1.58 μg/mL (-) epigallocatechin and 489.98 ± 2.89 μg/mL rutin which are signifi cantly higher concentrations (p > 0.05) almost 2 times more than these present in the Greek Cistus incanus leaves infusion 386.69 ± 3.85 μg/mL and 273.07 ± 0.96 μg/mL, respectively. Nevertheless, the Greek Cistus incanus leaves contains the highest concentration of gallic acid (219.04 ± 1.06 μg/mL, p > 0.05) among all analyzed infusions. The highest polyphenol concentration found in Melissa offi cinalis leaves and stems infusion was (-) epigallocatechin (188.99 ± 2.08 μg/mL) follow by quercetin, gallic acid, and kaempferol. In Trachystemon orientalis were found only the fl avonoids rutin (56.46 ± 0.31 μg/mL) and myricetin (630.23± 1.63 μg/mL). The investigation has led to a better understanding of the polyphenolic profi les of the analyzed plant infusions, especially the non-traditional for the Bulgaria herbs – Cistus incanus and Trachystemon orientalis and has clearly supported the further utilization of the two species in the Bulgarian traditional medicine. Not least the Cistus incanus leaves harvested in the winter season and Cistus incanus hard-coated seeds could be used as an antioxidant infusion with high added value. Research Article The polyphenol composition of Cistus incanus L., Trachystemon orientalis L. and Melissa offi cinalis L. infusions by HPLC-DAD method Vanya Dimcheva*, Nikolay Kaloyanov and Maria Karsheva Department of Chemical Engineering, University of Chemical Technology and Metallurgy, 8 Kl. Ohridski bul,1756 Sofi a, Bulgaria Received: 11 July, 2019 Accepted: 06 August, 2019 Published: 07 August, 2019 *Corresponding author: Vanya Dimcheva, Department of Chemical Engineering, University of Chemical Technology and Metallurgy, 8 Kl. Ohridski bul,1756 Sofi a, Bulgaria, E-mail:


Introduction
The research interest towards active compounds, especially polyphenols, of a natural source has greatly increased in re cent years [1]. Plants containing natural antioxidants are used as an important source of new drugs formulations and the plant extracts have been used to treat many diseases and their mode of action may well have been based on the phenolic compound content [2,3]. Phenolic compounds are the most widely occurring groups of phytochemicals and are of considerable physiological and morphological importance in plants as well as having strong antioxidant properties [4].
The wild herb Cistus incanus is not traditional in Bulgarian ethnomedicine but is widespread in all Strandja Mountain [5].
The Bulgarian Cistus incanus is a rich source of polyphenols and antioxidants [6]. For the "Pamukliyka" (Strandja local name) it is reported that the high values of polyphenols, fl avonoids, and infusions by HPLC-DAD method. Open J Anal Bioanal Chem 3(1): 031-038. DOI: http://dx.doi.org/10.17352/ojabc.000008 the high antioxidant capacity occurred not only in the leaves but were also found in the Cistus incanus hard-coated seeds [5]. The history of the Holy rose in the world began an ancient time [6]. The wild herb is wild spread in all Mediterranean.
Some of Cistus species are well-known around the world and they are studied in detail. Cistus incanus infusions and products containing extracts are notable examples for such polyphenolrich food supplements because many research studies have demonstrated that the main biologically active components of the wild herb refer to polyphenolic compounds such as gallic acid, rutin, quercetin, kaempferol, glycoside compounds belonging to the fl avonol family, fl avan-3-ols as well as catechin, epicatechin [7], gallocatechin and gallocate chin-3gallate [8]. Traditional Mediterranean folk medicine has used Cistus species for anti-infl ammatory, antiulcerogenic, wound healing, antimicrobial, cytotoxic and vasodilator remedies [9]. of monomers such as gallic acid, epigallocatechin, catechin, and epicatechin [10].
Trachystemon orientalis L. is commonly known as "Abraham-Isaac-Jacob" or "Oriental Borage" in Turkey. The medicinal plant is all edible and all parts of it are consumed as a vegetable in Istanbul and different parts of the black sea [11,12].
Phytochemical constituents are phenolic compounds, tannins, essential oils, mucilages, saponins, nitrates and resins among others. Trachystemon orientalis recorded folkloric usages are as a diuretic, blood purifi er, emollient, antipyretic, etc. [13]. In Bulgaria, the "Eastern Lopuh" (Strandja local name) is not popular and is used as food only from population lives where it is distributed -Eastern Central Balkans and all of Strandja.
Melissa offi cinalis L., commonly known as lemon balm or balm mint, is a well-known medicinal plant of Lamiaceae family. For more than 2000 years, fragrant leaves of this plant have been widely used in cooking to add fl avour to dishes [14].
Melissa offi cinalis L. is a medicinal plant that has long been used in different ethnomedical systems especially in the European Traditional Medicine and the Iranian Traditional Medicine for the treatment of several diseases [15]. Phytochemical investigations revealed that this plant contains volatile compounds, triterpenoids, phenolic acids and fl avonoids [16].
Crude extracts and pure compounds isolated from Melissa offi cinalis exhibited numerous pharmacological effects, from which only anxiolytic, antiviral and antispasmodic activities of this plant as well as its effects on mood, cognition, and memory have been shown in clinical trials [17]. In traditional medicine, lemon balm is commonly administered as a tea infusion that is used to treat maladies such as gastrointestinal complaints, headache, and fever [15]. The phenolic content of Melissa offi cinalis varies in different regions and is signifi cantly affected by cultivar [15,18]. Nevertheless, there are no reports about the phenolic profi le of garden cultivated in Northeastern Bulgaria Melissa offi cinalis.
Therefore, identifi cation and quantifi cation of phenols from different sources is becoming increasingly important due to their potential application for treating diseases.

HPLC Analysis
The analyses were performed with an Agilent 1100 HPLC

Infusions preparation
All samples were kept in a dry place for two years before being ground in the grinder and sieved. The leaves and seeds were used contained water, not more than 10 %. For the experiments, a fraction of 0.5 -1.0 mm particle size was used.
The extractions were prepared with the aid of the infusion technique. The solid-to-solvent ratio was fi xed at 1:30 (1 g leaves/seeds in 30 mL solvent) for all samples. The infusions were carried out with deionized water for 15 minutes time.
The infusions were left to stay at room temperature without additional heading. After each extraction, the exhausted plant material was carefully pressed, and the extract was fi ltered through cotton, fi lter paper, and after that was used 0.45 μm Nylon-membrane syringe fi lter (Acrodisc, Sigma-Aldrich, Bulgaria). The fi ltered samples were introduced in the 2 mL vials and stored in the fridge before the examination.      together with gallic acid, vanillic acid, (-) -epigallocatechin, rutin, myricetin, and kaempferol. The concentration of rutin was the richest in the winter leaves infusion with the value of 489.98 ± 2.89 μg/mL, p > 0.05. The Cistus incanus winter leaves polyphenol profi le wasn't studied before and they are not used as an infusion for human consumption according to the literature. Data are missing, perhaps, because it is well known that plants produce polyphenols in higher concentrations only in summer. The present study on the Cistus incanus leaves harvested in winter season explore their potential as an antioxidant infusion with high-added value.

Discussion
In the Greek Cistus incanus summer leaves (-) -epicatechin and kaempferol weren't found, unlike Bulgarian Cistus incanus summer leaves. The differences in the content of the same plants growing at different latitudes could be attributed to environmental and harvesting conditions which can lead to a loss of bioactive compounds in plants.
Plants can also contain a lot of phenolic acids such as gallic and vanillic acids. Gallic acid is a well-known natural antioxidant that is basically a secondary polyphenolic metabolite. Gallic acid is a very important common antioxidant tea formulation [28]. The gallic acid presented in the highest amount in the Greek wild herb -219.04 ± 1.06 μg/mL, (p > 0.05) among all herb infusions where the compound also consists. Gallic acid wasn't quantifi ed only in Trachystemon orientalis leaves and stems infusion and was determined with the lowest amounts in the Bulgarian Cistus incanus hard-coated seeds infusion (18.89 ± 0.86 μg/mL). Vanillic acid was quantifi ed only in the Cistus incanus leaves extracts with the highest concentration found in the Bulgarian Cistus incanus summer leaves (47.13 ± 0.92 μg/ mL, p > 0.05).
Identifi ed fl avonoids in the plant extracts were rutin, myricetin, quercetin, and kaempferol. Rutin was found in all herbal infusions without Melissa offi cinalis infusion in signifi cant quantities between 26.74 ± 0.33 μg/mL, (p > 0.05) in the Bulgarian Cistus incanus hard-coated seeds and 489.98 ± 2.89 μg/mL, (p > 0.05) in the Bulgarian Cistus incanus winter leaves. Rutin is a common dietary fl avonoid glycoside that is consumed in fruits, vegetables, and plant-derived beverages [29].
Myricetin was found in the infusions prepared from the Cistus incanus leaves in the range of 4.32 ± 0.11 to 7.79 ± 0.84 μg/ mL and in the Trachystemon orientalis leaves and stems where was found with a higher concentration of 30.23 ± 1.63 μg/mL, p > 0.05. Myricetin is a naturally occurring fl avonoid in tea, berries, fruits, vegetables, and medicinal herbs [29].
Quercetin was quantifi ed only in the one extract -mint balm infusion with the amount of 58.16 ± 2.66 μg/mL. Quercetin is on e of the important biofl avonoids present in more than twenty plants material and which is known for its anti-infl ammatory, antihypertensive, vasodilator effects, antiobesity, antihypercholesterolemic and antiatherosclerotic activities [30].
In the Cistus incanus, summer and winter leave infusions were found similar concentrations of kaempferol 0.48 ± 0.17 and 0.47 ± 0.19 μg/mL, respectively. In the Melissa offi cinalis leaves and stems' infusion, the presence of kaempferol was with the highest concentration -0.67 ± 0.19 μg/mL. Kaempferol is a naturally occurring fl avonol that has been isolated from edible plants such as the tea as well as in plants or botanical products commonly used in traditional medicine [29].
Cistus incanus is called a medicine herbal plant in the Mediterranean due to its antimicrobial, anti-infl ammatory, cytotoxic and antiulcerogenic properties [5,6,31]  This work allows the use of the selected infusions with high added value antioxidants to be used such as dietary additives.
The remaining undefi ned high-added constituents of the investigated herbs infusions must be evaluated in the future for better confi rmation of their bioactive properties.
Our research highlights the need the herbal teas to be studied through from an ethnopharmacological perspective. It is required greater consideration of traditional knowledge in the pharmacological research. Product safety and sustainability issues should also be considered, so the traditional applications of herbal teas can be transformed into effi cient health-boosting functional products.