One of the biggest problems the humanity is cur- rently facing is human-environmental interactions in the aspects of human health and homeostasis. Polluted environment damages  immune  status  irrespectively of social stratum. This problem is  especially  rele- vant for regions with bad ecology and harsh climate [5]. Functional foods and dietary supplements with immunostimulant plant agents have  become  a  popu- lar preventive action against immunodeficiency [20]. Functional foods with targeted properties possess a high  degree  of  usefulness  and  safety,  which  allows

them to substitute pharmaceutical products, to a certain extent [1–3].

The alarming health status of population demands that Russian scientists started using medicinal plants since they are one of the largest domestic resources in the sphere of medicine. Biologically active substances obtained from medicinal plants can be used to treat even severe diseases. Medicinal plants have become a popular source of raw materials for preventive medical treatment. All these make studies of biologically active substances, extracts, and botanical medicines extremely relevant.

Unlike their artificial analogues, botanical medicines

produce a mild therapeutic effect and no pronounced

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side effects [13]. Despite the growing consumers’ inte- rest, the share of botanical medicines and herbal repe- rtories on the Russian pharmaceutical market remains small: 11–12 million US dollars, or 0.5–1.5% [16]

Botanical medicines are getting more popular in Eu- rope as well. For instance, sales of galenicals (mostly ex- tracts) grew by 2% (440 million Euros) in France between 2009 and 2010. And if compared to 2005, the sector of painkillers grew by 12.3%, sedatives and soporifics – by 21.5%, cough mixtures, cold remedies, and antiallergic drugs – by 0.5%, and digestants – by 7.6% [17].

Medicinal plant materials contain a variety of biologi- cally active compounds from various chemical classes of natural substances, i.e. terpenoids, polysaccharides, phe- nolic compounds, alkaloids, etc. Each class and group of BAS has a specific spectrum of biological activity, which is typical of the whole group. However, this spectrum may be variable for each subgroup of biologically active compounds. In some cases, there may also be fundamen- tal differences for each individual substance, depending on its specific chemical structure [7, 8, 34].

The list of advantageous Siberian medicinal plants includes butterfly orchid (Platanthera bifolia L.), yellow seet-clover (Melilotus officinalis L.), skullcap (Scutellaria moniliorhiza L.), roseroot (Rhodiola rosea L.), maral root (Rhaponticum carthamoides L.), various sorts of milk- vetch (Astragalus turczaninowii L., Astragalus danicus L., etc.), hedisarum (Hedysarum turczaninóvii L.), etc.

To create new efficient and safe substances and func- tional foods, one has to use techniques based on the most recent knowledge about life and living systems.  The basic idea behind all biotechnologies is how to use bio- objects in order to produce efficient and safe functional products. The share of vegetative BAS on the market of functional foods is 60–65% [1].

To study a group of biologically active substances and predict its toxic and pharmaceutical properties, one should start with a phytochemical analysis. A study of chemical formula of vegetative organic compounds be- gins with a series of identification tests that make it pos- sible to define groups of biologically active substances [19]. The thin layer chromatography (TLC) method helps to refer a micronutrient to a particular group. It is often used to identify medicinal plant materials. Hence, the method defines the further research, as well as the choice of solvent and stationary phases [11, 13]. The method of high performance liquid chromatography (HPLC) makes it possible to perform a complete chemi- cal analysis of plant samples, including identification and potency assay [6, 18].

Purple Echinacea (Echinacea purpurea L.) pos- sesses immunomodulatory, anti-inflammatory, antivi- ral, and tonic properties [23]. It is a valuable medicinal plant from the Asteraceae family. The plant originated in North America, where it grows in the wild on fields, limestone wastelands, stony hills, and dry prairies of central and southern states. Some sources define it as Rudbесkiа рurрurеа, although modern botany separates these two species [21].

Echinacea   purpurea   is   a   perennial   herb.   It   is

50–100 cm tall and has one or several cylindrical ribbed

ramose caulis. For medicinal purposes, the plant is har- vested during its blooming stage, while its roots are usu- ally dug in autumn [15, 24].

One of the main groups of biologically active sub- stances found in Echinacea is phenylpropanoids, name- ly, derivatives of cinnamic acids. Another typical component is chicory acid, which is responsible for the immunomodulating and antiviral properties of Echi- nacea-based pharmaceuticals. The amount of chicory acid depends on the age of the plant [12, 14]. Phenyl- propanoids contained in Echinacea include caffeic and chlorogenic acids. Virus-neutralising and immunosti- mulating properties are due to the presence of saponins. Inulin can be found in roots and, in lesser amounts, in leaves and stems. It possesses anti-inflammatory prop- erties. The alkamides  are  responsible  for  the  analge- tic effect and improve the immune system. The same is true for vitamins A, E, and C. In spite of the fact that the pharmaceutical effect of separate components is rela- tively low, the medicinal effect of cumulative prepara- tions, e.g. potions, extracts, or juices, is rather high [9, 10, 12]. Hydroalcohol potions, alcoholates, extracts, and juices are used to boost immune system by improving phagocytosis, bacericyte and cytotoxic properties of macrofags, and antibody synthesis.

Chemical industry produces various artificial substi- tutes, e.g. flavouring agents, preparations, active compo- nents, etc. Still, natural plant extracts remain in demand in food, cosmetics, and pharmaceutical industries [22, 25]. Echinacea purpurea makes part of many herbal im- munoamplifiers.

Chemical composition of various plant parts de- pends on the climate and soil of the region where the plant grows. Hence, the research objective was to study the profile of the biologically active substances found in Echinacea purpurea that grows in the Kemerovo region. A set of physical and chemical methods helped to sub- stantiate their use in functional food production.

STUDY OBJECTS AND METHODS

The research featured medicinal herbs of Есhinасеа purpurea. The averaged samples originated from the vil- lage of Novostroyka near the city of Kemerovo (Keme- rovo region, GPS coordinates: 55°15’14”N, 86°13’05”E). The local soil can be characterized as black, leached, and argillaceous, with enough macroelements  for  medici- nal herbs. The amount of humus was found to be 7.7%, nitrate nitrogen  (N-NO3)  –  45  mg/100  g;  labile  phos-

142 mg/100 g of soil. To identify soil contamination and plant mass material with heavy metals, the averaged samples were sorted according to the state standards and approved techniques [28].

The atomic absorption method was used to deter- mine heavy metals (Zn, Pb, Co, Ni, Cd) for an avera- ged soil sample [26, 27]. The samples were taken from the roots at the depth of 0–20 cm, from the rhizomes, and from the herbs. Heavy metals were extracted for 24 hours using an ammonium acetate buffer with a pH of

1. 8. The soil-solution ratio was 1:10. The sample prepa-

ration of rhizomes, leaves, stems, and flowers was con- ducted separately and took 24 hours. Dry ashing was followed by extraction with diluted nitric acid (1:1).

In the Russian Federation, there are no current stan- dards for the toxicological assessment of medicinal raw materials for heavy metals. Hence, most researchers have to use regulations adopted for plant-based dietary supplements [30, 31].

The research featured various parts of Есhinасеа, as well as components extracted from it with the help of a 70% ethanol solution. The latter was chosen for its uni- versal properties in extracting of a wide range of biologi- cally active substances.

The methods included high performance liquid chro- matography (HPLC), thin layer chromatography (TLC), and IR spectroscopy.

Statistical data processing was performed with the help of Microsoft Excel. The assay content of biologically active substances was defined with the help of standard curves. The concentration range was 0.5–150.0 mkg/ml.

The research involved five consecutive stages.

1. 1. 1. Extraction of the samples and preparation for screening and analysis of biologically active substances (secondary metabolites).

To define the sum of biologically active substances, the field test samples were air-dried (0.5 kg). An avera- ged sample of herbs (shoots, leaves, flowers) and foot ends (rhizomes and roots) were extracted and ground. After that, the plant material underwent a complete ex- traction with ethanol with the ratio of 1:10 at 10°С for 48 hours. The extract was a green-brown transparent li- quid with a specific smell. The extracts were kept in the dark at 4–6°С.

1. 1. 2. Preparation  of  samples  for  HPLC  analysis. The ground plant material (0.5 kg) underwent ex- traction with 70% ethanol in ratio  1:10  in  a  sonica- tion bath (100 W, 35 kHz) at 40°С. The process lasted 30 min and was conducted twice. The extract was fil- tered through 0.2 micron membranes. Then,  a  vac- uum rotary evaporator was used to concentrate the permeate in order to get water residue. After that, the permeate underwent a liquid-phase extraction with hex- ane (fraction 1) and an ethylacetat-ethanol mix (5:1). Fraction 2 was chromatographed with sorbent LH-20 with column of 10×350 mm by using a chromatograph (Biо-Rаd, USA). The elution was conducted with isopropylalcohol at the gradient of 20–90%.

The extract  was  filtered  and  condensed  with  an 8 vacuum rotary evaporator at 72 Mbar to a thick con- sistency. The thickened suspension was then diluted four times with water and left for 12 hours at 40°С. The tarry residue was removed by filtration. After that, the perme- ate was treated with chloroform and ethyl acetate. The extract was then drained with anhydrous sodium sul- phate. It was concentrated with the help of a rotary evap- orator at 400°С, 240 mbar. The fractions were applied to column with an LH-20 sephadex (Рhаrmасiа,). The frac- tions were mixed with a small amount of sorbent, loaded into the column, and eluted with aqueous alcohols at a ratio of 5:5; 6:4; 7:3; 8:2; and 9:1, as well as with absolute ethyl alcohol. The fractions were collected by 10–15 ml.

86

The composition of the eluate was controlled with the help of TLC. If the fractions contained the same com- ponents, they were put together and condensed using a vacuum rotary evaporator.

After that, we defined the substances that could be classified as biologically  active  substances  according to qualitation tests and chromatograms. Their structure was defined according to UV and IR Fourier spectra. UV-spectra were measured using СФ-2000 spectropho- tometer both as pure components and with chemical reagents to specify the location of hydroxyl groups and clycosidation.

1. 1. 3. Preparation of samples for IR Fourier-transform spectrometry.

Two mg of a dried sample was ground in an agate mortar together with potassium bromide at a ratio of 1:100 (Fluka, Germany). A disk was formed in a press at 4,000 рsi. IR spectra were measured by a single-beam interferometer with a ФСМ-1202 Fourier spectrometer (Infraspek, St-Petersburg, Russia). The spectra were re- gistered in the range of 4.000–400 cm1 with the reso- lution of 4 cm. The FSрес software 4.0.0.2 was used to process the data.

1. 1. 4. TLC stage. TLC analysis was performed on TLC aluminium foil analytical plates. It was followed by den- sitometry using a Sony densitometer ( HDR-CH 405, OOO IMID, Russia). Photofixation was  conducted  at the waves of 254 and 365 nm and at a visual band after specific derivatization. The elution involved the follow- ing fluid systems: n-butanol – glacial acetic acid – water at the ratio of 60:15:25 and ethyl acetate – formic acid – glacial acetic acid – water at the ratio of 100:11:11:26.

In the preparative variant the chromatographic zones were cut out and subjected to further analysis.

1. 4. 5. HPLC conditions. The substances were sepa- rated using a Shimаdzu –20 Prominence chromato- graph with a photodiode array and a Shimаdzu re- fractometric  detector.  The  Krоmаsil  –18  column was 250×4.6 mm, particle size – 5 µm. A mix of sol- vents was used as eluent components, namely methyl syanide MECN (solvent A) and 0.1%  aqueous  for- mic  acid  (solvent  B).  During  separation,  a  gra- dient elution mode was used with  the  following isocratic components: 0 min – 20% A, 4 min – 55% A, 14 min – 55% A, 16 min – 20% A. The flow rate was
   5. ml/min, the column temperature was 24°С, the sam- ple volume was 20 μl, reference wave lengths were 254 and 330 nm.

Two approaches were used for identification.

1. 1. 1. UV spectra and retention time of peaks were com- pared with the reference samples. The chromatograms were developed using programme.

2. HPLC and/or TLC were used together with IR Fourier-transform sepctrometry. The column tempera- ture was 40°С, while the volumetric flow rate of the eluent phase was 0.4 ml/min. A 0.1% water solution of formic acid (solvent A, v/v) and a 0.1% solution of for- mic acid in MECN (solvent B, v/v) were used as eluent. HPLC separation was conducted by gradient eluation. The eluent composition was as follows (solvent B, by volume): 0–1 min - 15%, 1–5 min – 30%, 5–15 min –

Table 1. Content of heavy metals in soil and in plant raw material of Есhinасеа, mg/100 g

Elements                                     Soil *                                                                        Plant raw material

MPC, TPC

Assigned value     MPC for dietary

supplements [32]

Assigned value

roots and rhizomes      stems and leaves          flowers

Zink (Zn)                 23.0           2.00 ± 0.12           –                                0.93 ± 0.09                  0.25 ± 0.007                0.09 ± 0.003

Lead (Pb)                3.2             0.82 ± 0.08           6                                0.46 ± 0.06                  0.18 ± 0.007                0.02 ± 0.002

Cobalt (Co)             5.0             1.15 ± 0.08           –                                0.44 ± 0.06                  0.21 ± 0.008                0.02 ± 0.002

Nickel (Ni)              4.0             1.78 ± 0.12           –                                0.55 ± 0.06                  0.34 ± 0.05                  0.02 ± 0.002

Cadmium (Cd)         1.0             0.56 ± 0.06           1                                0.22 ± 0.008                0.11 ± 0.007                –

Note: content of heavy metals measured in the active form

30 → 38%, 15–15.5 min – 48 → 45%, 15.5–23 min –

45%, 23–23.5 min – 45 → 95%. Each chromatographic

fraction was analyzed after accumulation.

A quantitative assay of the secondary metabolites (flavones) was conducted with the help of standard curves at the concentration range of 1.9–235.0 mkg/ml. The equation for the standard curves was as follows:

у = а × х × Х,                                  (1)

where Х is standard concentration (mkg/ml); y is HPLC

peak response (cu); and a is proportionality factor.

Formula (2) connects the peak response and the dry weight unit:

С = S / (а × m × 1000),                            (2)

where C is dry flavones in the portion of dry material (mg/g); m is mass of dry weight (g); a is proportionality factor from the standard curve equation. A coefficient of 1,000 is necessary to transform C into mg/g dimension.

The concentration of chlorophylls a and b and caro- tinoids was measured using a UV 1800 Shimadzu spec- trophotometer. The stems and leaves were used as as- similative bodies. A 0.1 g portion of plant material was ground, rubbed, put into a vile with 10 ml of 98% etha- nol and was stored in a dark place. After 12 hours it was measured for pigments using spectrophotometry at the wave length of 649 nm, 665 nm, and 440.5 nm. The con- centrations of chlorophylls a and b and carotinoids were measured as follows (mg/g):

Table 1 shows that the content of heavy metals (Zn, Pb, Co, Ni, Cd) in the soil does not exceed the maximum permissible level. For food plants, including medicinal ones, the MPC content for heavy metals (HM) is stated in Sanitary Regulations and Norms 2.3.2.1078-01* for die- tary supplements. However, the document features only lead and cadmium, which belong to technogenic metals and are of no biological significance for plants [31].

According to the assigned value analysis of the heavy metals, they are mostly accumulated by roots and rhi- zomes, and to a lesser degree – by flowers. However, the content of standardized elements (Pb, Cd) in diffe- rent parts of the plant is significantly below the permis- sible level (Table 1). In general, the results indicate that the content of HM in the raw material corresponds to the standard indicators and can be used to obtain biological- ly active substances for dietary supplements and food.

Photosynthesis plays the key role in plant growth and development. Therefore, it determines the formation of the secondary metabolites, including those with biolo- gically active properties. Chlorophylls a and b, as well as carotenoids, are involved in photochemical reactions. A high content of chlorophyll a and the chlorophyll a/b ra- tio may indicate a high potential photochemical activity of the leaves, and, consequently, a more active accumu- lation of biologically active substances [33].

Hence, it seemed important to investigate the quan in the leaves of Echinacea purpurea (Table 2).

The experimental data indicate that Echinacea leaves

have the highest amount of chlorophyll a in their pig-

а = 13.7·D

665

– 5.76·D

649,

(3)

ment complex. Apparently, chlorophyll a has the grea-

b = 25.8·D

649

– 7.6·D

665,

(4)

test stability among  other pigments of  photosynthesis

= 4.695·D

440.5

– 0.268·(С

Table 2. Content of photosynthetic pigments in leaves of Echi- nacea purpurea

b),                               (5)

Indicator                                                           Assigned value

where  D

440,5

are  absorbency  at  the  wave

Chlorophyll а, mg/g                                       0,700 ± 0.01

lengths of 649 nm, 665 nm, and 440.5 nm, respectively.

RESULTS AND DISCUSSION

As a rule, pollutants enter the plant tissue from soil

Chlorophyll b, mg/g                                       0,282 ± 0.01 Chlorophyll amount (а + b), mg/g        0,990 ± 0.02 Chlorophyll ratio а/ b       2.480 ± 0.01

Carotinoids, mg/g                                           0.190 ± 0.01

through the root system. They also enter the plant from

the dust that settles down on the surface of its aerial or- gans and penetrates into the intercellular spaces through natural passages, e.g. stomata, pores, or lenticels [29, 32].

Ratio of chlorophylls and carotinoids, chlorophyll/carotinoid

Note: mean values of triple consecutive tests

5.210 ± 0.01

Therefore, herbal raw materials must be environmental- ly pollution-free. The analysis of soil samples and plant material confirmed this requirement (Table 1).

87

* Sanitary Regulations and Norms 2.3.2.1078-01. Sanitary rules and Regulations 2.3.2. 1078-01. Hygienic requirements for safety and nu- tritional value of food.

150

100

50

0

0         5        10        15       20       25       30       35        40

Min

100

75

 

 

50

 

25

 

0

 

0         5        10        15        20       25       30       35       40

Min

Fig. 1. HPLC chromatogram of the ethanol extract from Echi- nacea purpurea stems and leaves.

Fig. 2. HPLC chromatogram of the ethanol extract from Echi-

nacea purpurea rhizomes and roots.

Table 3. Residence time Content of the main biologically ac- tive substances in the ethanol extracts from stem and leaves

Table 4. Content of the main biologically active substances in the ethanol extracts of Echinacea purpurea stem and leaves

Component                     Concentration

range mkg/ml

Correlation

coefficient

Residence

time

Component                                                           Content, mg/g

of dry weight

and optimizes the photosynthetic processes. As the data show, the amount of chlorophylls (a + b) significantly exceeds the content of carotenoids – by 5.2 times. The pigment complex of Echinacea leaves has a rather high ratio of chlorophylls a/b. High ratios of chlorophyll a/b are characteristic of chloroplasts. The proportion of stro- mal thylakoids in chloroplasts prevails, and they have a

greater light absorption and a better membrane protec- tion of from photodamage.

Thus, a  high  content  of  chlorophyll  a  and  a high chlorophyll a/b ratio indicate a high potential for photo- synthesis of Echinacea leaves. Indirectly, it may indicate a more intensive synthesis of secondary metabolites, in particular, those with biologically active properties.

The main active biologically active substances of Echinacea plants are hydroxy acids and polysaccha- rides. For a more complete assessment of the Echinacea biologically active substances, we conducted a physi- co-chemical study of its main components, i.e. rhizomes, roots, stems, leaves, and flowers.

Table 5. Residence times of the main biologically active substances in the samples of chromatographic fractions of ethanol extracts of Echinacea purpurea rhizomes and roots

Table 6. Contents of the main biologically active substances in the ethanol extract of Echinacea purpurea rhizomes and roots

Table 7. Residence times of the main biologically active sub- stances in the samples of chromatographic fractions of ethanol

                                                                                                                extracts of Echinacea purpurea flowers

Component                                                            Content, mg/g                                                                                                                 

of dry weight 8-hydroxy-tetradec-8E-en-11,13-diyn-2-one             2.67 ± 0.01

Tetradec-8Z-en-11,13-diyn-2-one                                3.31 ± 0.01

8-hydroxy-tetradec-9E-en-11,13-diyn-2-one                  15.84 ± 0.01

Cynarine                                                                23.68 ± 0.01

Echinacoside                                                          16.65 ± 0.01

Chlorogenic acid                                                 3.38 ± 0.01

Chicoric acid                                                       3.43 ± 0.01

Component                               Concentra- tion range mkg/ml

8-hydroxy-tetradec-8E-en- 11,13-diyn-2-one

8- hydroxy-pentadec-9Z-en- 11,13-diyn-2-one

Cor- relation coeffi- cient

Resi- dence time

6.03

Isobutylamide dodeca-2E,4E,8Z,10Z-tet- raenic acid

3.51 ± 0.01

Tetradec-8Z-en-11,13-di-

yn-2-one

7.87

8-hydroxypentadien-9E-en-11,13-diyn-2-one  3.40 ± 0.01

3,4-dioxybenzoic acid             1.03–150.00        0.9800    19.01

Isobutylamide undeca-2E-en-8,10-diyn- ic acid

Methylbutylamide dodeca-2E,4Z-di- en-8,10-diynic acid

8.88 ± 0.01

15.19 ± 0.01

Echinacoside                              0.50–5.03         0.9881      21.78

Chlorogenic acid                     0,103–150.05   0.9881    24.25

Chicoric acid                           0.51–50.01       0.9891    26.98

Vanillic acid                                                                   29.70

For the phytochemical characteristics of the extracts, we chose those groups of compounds that were more like-

8- hydroxy-pentadec-9E-en- 11,13-diyn-2-one Methylbutylamide do-

deca-2E,4Z-dien-8,10-di-

33.19

36.56

ly to be present in the hydrophilic extracts in question.

To study the content of biologically active substan- ces, we analyzed the extract obtained by using a 70% ethanol solution of the stem and leaves. To determine the content of the main groups of biologically active sub- stances, we used HPLC and TLC, accompanied with an IR Fourier-transform spectrometry.

Fig. 1 and Tables 3 and 4 present a detailed analy- sis of the composition of the extract obtained from the stems and leaves of Echinacea purpurea.

The analysis showed that the leaf extract of Echina- cea purpurea contained such biologically active sub- stances as acetylene and alkene derivatives, as well as phenylpropanoids, which are derived from caffeic acid. The fact that the extracts from leaves and stems contain phenylpropanoids confirms the biological value of the plant. This group of organic compounds is a rich source of substances with adaptogenic, tonic, immunomodula- ting, hepatoprotective, and antioxidant properties.

The next stage involved an analysis of the extract obtained from the rhizomes and roots of Echinacea pur- purea. The extraction was performed with a 70% etha- nol solution.

To determine  the  content  of  the  main  biological- ly  active  substances,  we  used  HPLC  and  TLC,  ac-

 ynic acid                                                                                        

companied with IR Fourier-transform spectrometry. Fig. 2 and Tables 5 and 6 show the results of the che- mical and physical analysis of the extract from rhi- zomes and roots.

Tables 5 and 6 and Fig. 2 show that the extract ob- tained from Echinacea rhizome and roots contain alky- lamides and phenylpropanoids.

Alkilamides demonstrate a great variety. Despite their relatively simple molecular structure, these sub- stances have a wide spectrum of biological  activity. They have an immunomodulating, antimicrobial, antivi- ral, insecticidal, diuretic, and antioxidant properties. In addition, they can potentiate antibiotics and inhibit pros- taglandin synthesis.

The final stage of the study featured the extract ob- tained from Echinacea flowers. The extraction was per- formed with a 70% ethanol solution.

For the physical and chemical evaluation of the flo-

wer extracts, we used HPLC and TLC, accompanied

Table 8. Content of the main biologically active substances in the ethanol extract of Echinacea purpurea flowers

100

75

50

25

0

Component                                                          Content, mg/g

Fig. 3. HPLC chromatogram of the ethanol extract from Echi-

Methylbutylamide dodeca-2E,4Z-di-

19.19 ± 0.01

nacea flowers.

 en-8,10-diynic acid