Chemical Composition and Antioxidant Activity of Tánara Ótó (Dracocephalum palmatum Stephan), a Medicinal Plant Used by the North-Yakutian Nomads

Dracocephalum palmatum Stephan (Lamiaceae) is a medicinal plant used by the North-Yakutian nomads. From the crude ethanolic extract of the aerial parts of this plant, 23 compounds (phenylpropanoids, coumarins, flavonoids, and triterpenes) were isolated. Among these, eight compounds (salvianolic acid B, caftaric acid, cichoric acid, umbelliferone, aesculetin, apigenin-7-O-β-d-glucuronopyranoside, isorhoifolin, and luteolin-4'-O-β-d-glucopyranoside) were detected for the first time in the genus Dracocephalum. Their structures were elucidated based on chemical and spectral data. The levels of most of the compounds detected in the cultivated sample were close to that of the wild sample, indicating the reproducibility of the biologically active compounds of D. palmatum through cultivation. Investigation into the biological activity of D. palmatum under in vitro conditions demonstrated that its extracts have a strong antioxidant effect due to the presence of high concentrations of phenolic compounds.


Introduction
Yakutia (Sakha), a federal subject of Russia spanning 3,083,523 km 2 , is the largest subnational governing body by area in the World. If the federal subjects of Russia were compared with other countries, it would the eighth largest territory in the World. More than 40% of the territory is lies within the Arctic Circle. Due to its geographical location Yakutia is characterised by a variety of habitats and natural resources. Despite the uniqueness and diversity of the Yakutian flora, the scale of bioprospecting has been extremely low. As a part of the scientific programme on the investigation of plant diversity, we are now carrying out comprehensive studies of the chemical composition of Yakutian plants.
Palmate dragonhead Dracocephalum palmatum Stephan (D. schelechowii Turcz. ex Ledeb., Ruyschiana palmata (Stephan ex Willd.) House; Subsection Keimodracontes Briq., Section Buguldea Benth., Subgenus Eudracocephalum Briq., Genus Dracocephalum L., Family Lamiaceae), is a perennial rhizomatous plant with numerous stems and ovate-rounded, pinnatifid leaves, and purple flowers on short stalks gathered in false whorls at the end of the stems in an oblong inflorescence ( Figure 1). It grows on cliffs and sandy deposits, on gravelly and rocky slopes. It is endemic to the Arctic tundra (Chukotka, Anadyr), East Siberia (Yakutia), and the Russian Far East region [1]. The Nomads of North Yakutia call this plant tánara ótó (таңара ото, the herb devoted to the supreme god Tánara). Young shoots and flowers have ethnobotanical uses as diuretic and chloretic remedy, for fumigation, treatment of gastro-intestinal tract disorders and alcoholism [2]. There are no scientific data about the chemical components and biological activity of D. palmatum herb.
Dracocephalum is a large genus of Lamiaceae family that includes about 60 species as perennial herbs (rarely semishrubs), growing in the territory of the extra-tropical Asia, Europe, and Russia. Ethnopharmacological information about species of this genus allows describing some of them as well-known and valuable sources of drugs. Among them, the most prominent species is D. moldavica L., traditionally used in the ethnomedicine of European countries for the treatment of hypertension and heart disease [3]. The aerial part of D. heterophyllum Benth. was used in Xinjiang (China) for treatment of asthma and gastropathy [4]. The best drug for diseases of the stomach and liver in Tibetian medicine was the herb of D. nutans [5]. In Buryatia (Russia) decoctions from D. ryushiana L. and D. argunense Fisch. ex Link. were recommended by ancient doctors (lamas) as choleretic remedies [6]. Despite a long history of human use of Dracocephalum species, scientific data about the chemical composition are known just for 16 species. Approximately a hundred compounds were isolated and identified as derivatives of terpenoids, flavonoids, alkaloids, and others [7]. These facts demonstrated the necessity of expanding scientific information about the species of the genus Dracocephalum. In this study, we present the results of a phytochemical investigation of D. palmatum herb of both wild (collected from Yakutia) and cultivated varieties and the antioxidant activity data of the ethanolic extracts from D. palmatum determined by in vitro methods.
Despite insufficient chemical information of the phenolic compounds of Dracocephalum genus, available data suggest that flavonoid compounds, particularly derivatives of apigenin and luteolin, i.e., flavones with 5,7,4' and 5,7,3',4' types of substitution are specific to this genus. These substances were found in the majority of Dracocephalum species studied, thus reflecting their important chemosystematic character. The systematic function of rosmarinic acid, a specific marker for Lamiaceae family, is not clear because its presence has not been detected in all Dracocephalum species. However, it should be noted that caffeic acid and its derivatives are an essential part of the Dracocephalum genus extracts.

HPLC-UV Analysis of the Main Phenolic Compounds in D. palmatum
A quantitative analysis of the phenolic compounds found in D. palmatum was performed using microcolumn HPLC with ultraviolet detection (HPLC-UV), which allowed separation of 15 dominant components ( Figure 3). The predominant phenolic compounds from the wild sample of D. palmatum are flavonoids (20.987 mg/g) and the main group of the flavonoids is flavones (20.844 mg/g), with cynaroside (12.075 mg/g) and cosmosiin (5.683 mg/g) as dominating compounds (Table 1). Luteolin and its derivatives accounted for about 70% of the total flavonoids and apigenin and its derivatives accounted for less than 30%. The amount of flavanones did not exceed 1% of the total flavonoids. The content of glycosides was 11 times more than that of aglycones. Among glycosides, monoglycosides were dominant (19.420 mg/g) and the biosides (rutinosides) represented about 3% of the total amount of flavonoids. The content of phenylpropanoids in D. palmatum did not exceed 15% of the identified phenolic compounds. The dominant components of this group of metabolites were rosmarinic acid (1.614 mg/g) and salvianolic acid B (1.456 mg/g). The comparative analysis of the phenolic compounds in wild and cultivated samples of D. palmatum showed that the total content of identified compounds in the cultivated sample was 28% lower (17.571 mg/g vs. 24.503 mg/g in wild sample) ( Table 2).
It should be noted that the set of compounds detected in the cultivated sample was close to those of the wild sample, except for a significantly higher content of biosides in the cultivated samples (2.735 mg/g). This indicates the reproducibility of D. palmatum composition under artificial culture conditions.
Chromatographic study of the aerial parts of D. palmatum showed an uneven distribution of the phenolic compounds in the plant. The maximum content of identified compounds was observed in the leaves (37.825 mg/g), minimum in the flowers (11.251 mg/g) and stems contained an intermediate level (20.959 mg/g) ( Table 1).
The flavonoids were concentrated in the leaves (35.482 mg/g) and phenylpropanoids accumulated in the stems of plant (3.982 mg/g). The leaves showed a high content of all flavonoidal groups except aglycone and flavonol-biosides, which were concentrated in the flowers (2.843 mg/g) and stems (5.565 mg/g), respectively. The dominating flavonoids in flowers were cynaroside (5.062 mg/g), luteolin (1.998 mg/g) and cosmosiin (1.410 mg/g); in leaves-cynaroside (25.172 mg/g), cosmosiin (7.874 mg/g) and scolymoside (1.240 mg/g); in stems-cynaroside (6.727 mg/g), scolymoside (4.188 mg/g) and cosmosiin (1.640 mg/g). A high content of such rare flavonoids such as isorhoifolin, luteolin-7-O-glucuronide, and luteolin-4'-O-glucoside were detected in the stems of plants (1.377, 0.580, and 0.376 mg/g, respectively). The organ with maximal phenylpropanoid content was the stem (3.982 mg/g) characterised by the highest concentration of rosmarinic acid (2.791 mg/g, that is 4.1 times more than in the leaves and 9 times higher than in the flowers. Unlike rosmarinic acid, salvianolic acid B accumulates in leaves (1.662 mg/g). The detected concentrations of 3-O-caffeoylquinic acid, caffeic acid and cichoric acid were found only in the stems. The distribution of phenolics in the organs of the cultivated sample of D. palmatum was similar to that of the wild sample (Table 2). However, for a number of compounds, higher levels were found in cultivated samples. For example, stems of the cultivated sample were enriched with caffeic acid, scolymoside, and eriodictyol-7-O-glucoside. In the leaves of wild sample, trace amounts of cichoric acid were observed, whereas in the cultivated plant, the concentration of this compound was up to 0.356 mg/g. The above results demonstrate that D. palmatum is a plant with a high content of flavonoids.

Antioxidant Activity of D. palmatum
Experimental investigations of the ethanolic extracts from wild (WSE) and cultivated samples (CSE) of D. palmatum herb were conducted using the traditional assays: total antioxidant capacity; 2,2-diphenyl-1picrylhydrazyl radical (DPPH • ) scavenging activity; 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) radical (ABTS •+ ) scavenging activity, bromine radical (Br • ) scavenging activity; carotene bleaching assay; nitric oxide (NO) inactivating activity; hydrogen peroxide (H 2 O 2 ) inactivating activity; ferrous (II) ions (Fe 2+ ) chelating activity; ferric reducing antioxidant power; erythrocyte membrane stabilising activity (Table 3). All experiments include the determination and comparative estimation of the same antioxidant factors for cynaroside, the predominant component of D. palmatum with known antioxidant activity [10]. The values of total antioxidant capacity of WSE and CSE were 312.44 and 284.63 mg caffeic acid per gram of extract, respectively. These values indicate a high antioxidant potential of the studied extracts. The radical scavenging activity of WSE and CSE against radicals of different nature (organic, inorganic, neutral, and charged) was much expressed, and in some cases was higher than the same parameter of cynaroside (DPPH • and ABTS •+ scavenging activities). This data classifies D. palmatum extracts as a radical scavenger. The examination of the influence of WSE and CSE on the oxidative destruction of β-carotene in the oleic acid-DMSO-H 2 O 2 system demonstrated a high value of antioxidant activity, with IC 50 = 1.64 and 3.38 μg/mL, respectively. The feature of this system is the ability to investigate the influence of sample on the presence of a complex of damaging factors, including H 2 O 2 , O 2 •− , OH •− , and alkyl-radicals that form in this in vitro system. The efficiency of cynaroside in this assay was slightly lower (10.28 μg/mL). The activity of WSE in the NO and H 2 O 2 inactivating assays and both the extracts in Fe 2+ chelating activity and FRAP assays were characterised as very high because the activity of cynaroside was quite low. The final stage of the biological study was to investigate the ability of the extracts to protect living cells (RBCs) from oxidative damage caused by the influence of Fenton's reagent. The presence of Fenton's reagent causes a cascade of negative reactions leading to loss of integrity of erythrocyte membranes, which in turn lead to cellular death [11]. The WSE has successfully demonstrated high protective properties (IC 50 = 14.07 μg/mL) exceeding those of the reference substance (IC 50 = 25.67 μg/mL).
The obtained chemical information about D. palmatum herb allows characterizing the isolated phenolic compounds as responsible factors stipulated the antioxidant properties of the total extract. Earlier, the expressed antioxidant activity of the aglycone and glucosides of luteolin and apigenin [12], as well as derivatives of caffeic acid [13], have been shown by various researchers. To confirm the leading role of flavonoids and phenylpropanoids found in D. palmatum herb in formation of the antioxidant effect of the total extracts, we applied the original methodical approach. The identification of antioxidants present in the extracts realized after HPLC-separation of the extracts samples pretreated with excess of DPPH • or ABTS •+ radicals (DPPH-HPLC or ABTS-HPLC). The reaction between an antioxidant and a radical results in the oxidation of the antioxidant, that leads to a decrease of the corresponding peak areas in the chromatograms. Comparison of the HPLC chromatograms of untreated and radical-treated samples allows to determine the most active compounds. Previously, some plant species, including Lonicera japonica Thunb. [14], Selaginella sinensis (Desv.) Spring. [15], Artemisia gmelinii Webb. ex Stechm. [16], Pueraria lobata (Wild.) Ohwi [17], Arachis hypogaea L. [18] and Eucommia ulmoides Oliv. [19], were successively investigated by this method.
Chromatograms of ethanolic extract from D. palmatum herb (wild sample) spiking with DPPH • and ABTS •+ radicals are shown in Figure 4a,b, respectively, which present obviously reduced peak areas for some compounds in comparison with untreated sample. Therefore, seven compounds, caffeic acid (peak 2), scolymoside (peak 4), cynaroside (peak 7), cosmosiin (peak 11), rosmarinic acid (peak 12), salvianolic acid B (peak 13) and luteolin (peak 14), in extract of D. palmatum herb possessed antioxidant activity. It should be noted that the peaks of luteolin, cynaroside, cosmosiin, rosmarinic acid and salvianolic acid B decreased more sharply than the other peaks, so it may be concluded that they are the major active compounds.  ) and NO molecules, inactivate hydrogen peroxide, and chelate Fe 2+ ions i.e., exhibit a high antioxidant potential in processes involving hydrogen atom transfer reactions, electron-transfer reaction and other mechanisms. Comparative analysis of the data indicates that in some cases, the antioxidant activity of the extracts was similar to or exceeded the activity of reference antioxidant, cynaroside.
The unique feature of this plant species is its ability to simultaneously accumulate active compounds such as cynaroside and cosmosiin, which are known to be compounds with high biological activity. In particular, cosmosiin possesses anti-inflammatory [20], insulin-mimetic [21], and cancer prevention activity [22]; cynaroside possesses anti-atherosclerotic [23], anti-inflammatory [24], anti-diabetic [25], and cardioprotective activity [26]. Recently, the effects of cosmosiin against liver injury caused by CCl 4 were investigated [27]. It was shown that the application of this phytocomponent not only suppressed the elevation of hepatic stress-indicators, glutamic pyruvic transaminase, glutamic oxaloacetic transaminase, malonic dialdehyde and hydroxydeoxyguanosine, and inhibited the decrease of the reduced glutathione level, but also reduced hepatocyte damage. In addition, in a model of hepatic oxidative injury the normalizing action of cosmosiin on the concentrations of aspartate transaminase, alanine transaminase, alkaline phosphatase, glutamate, total bilirubin, lactate dehydrogenase and total serum protein was demonstrated [28]. The results obtained thus suggest that cosmosiin has protective effects against chemical-induced hepatic damages. Cynaroside is the strongest bioactive component responsible for the detoxification of bromobenzene-induced hepatic lipid peroxidation [29]. A significant protective effect of cynaroside reflects in its reduction of lipid peroxide levels and enhanced activity of epoxide hydrolase, a toxicant agent-removing enzyme, rather than by acting on the epoxide-producing system. The modern data of pharmacological activity of cosmosiin and cynaroside are in good agreement with the ethnoscientifical information concerning the use of D. palmatum as a hepatoprotective agent.

Plant Material
The For analytical HPLC total probes of the flowers, leaves, stems and herb from 15 specimens of D. palmatum were used.

Preparation of the Extracts WSE and CSE
The total herb of wild or cultivated samples of D. palmatum were air-dried and powdered in a mechanical grinder. The powdered total herb was weighted accurately (100 g), extracted twice with 60% ethanol (1.5 L) in an ultrasonic bath for 90 min at 45 °C. The extracted solutions were filtered through cellulose filter and evaporated in vacuo until dryness using rotary evaporator. The extracts yields are 35.63% (w/w; from wild sample, WSE) and 29.15% (w/w; from cultivated sample, CSE).

Statistical Analysis
Statistical analyses were performed using a one-way analysis of variance (ANOVA), and the significance of the mean difference was determined by Duncan's multiple range test. Differences at p < 0.05 were considered statistically significant. The results were presented as mean values ± SD (standard deviations) of the three replicates.

Conclusions
Dracocephalum palmatum, a medicinal plant used by the North-Yakutian nomads, was investigated chemically for the first time. The presence of phenylpropanoids, coumarins, flavonoids and triterpenes in D. palmatum herb was shown. A comparative study of wild and cultivated samples demonstrated the lower level of the phenolic compounds in cultivated plants, possibly due to the fact that extreme environmental conditions increased the amount of these active compounds. However, both wild and cultivated plants could be used. The organ-specific distribution of phenolic compounds and the finding of eight new compounds specific to D. palmatum, which are not found in other species of Dracocephalum, suggest that this herb is one of the best sources of these phenolic compounds for humans. The high level of the antioxidant activity of the crude extract and isolated compounds was revealed using different in vitro methods. Obtained results confirmed the ethnopharmacological use of D. palmatum as a natural phytotherapeutic agent.