Comparative Analysis of Biologically Active Substances in Trifolium Pratense and Trifolium Repens Depending on the Growing Conditions

Journal of Environmental Treatment Techniques

2019, Special Issue on Environment, Management and Economy, Pages: 874-877

J. Environ. Treat. Tech.

ISSN: 2309-1185

Journal web link: http://www.jett.dormaj.com

Comparative Analysis of Biologically Active

Substances in Trifolium Pratense and Trifolium

Repens Depending on the Growing Conditions

Alexander L. Mikhailov*, Olga A. Timofeeva Uliana A. Ogorodnova, Nikita S. Stepanov

Institute of Fundamental Medicine and Biology, Kazan Federal University, Kazan, Russia

Received: 05/08/2019

Accepted: 20/11/2019

Published: 20/12/2019

Abstract

In our studies, we studied the content of pharmaceutically promising biologically active substances, namely phenolic

compounds, flavonoids, ascorbic acid, provitamin A (b-carotene) in plants of meadow clover and creeping clover (Trrifolium

repens). The effect of the plant growth site on the content of the studied compounds was also studied. The obtained data showed

that the content of biologically active compounds in white clover (Trifolium repens L.) is similar to those of red clover (Trifolium

pratense L.) and white clover is a source of a whole set of pharmacologically active compounds, as well as red clover. The

influence of the place of plant growth on the content of the studied compounds was revealed, which may be important for solving

production problems. For example, in clover plants of both species studied, the flavonoid content collected in meadow steppes is

higher than in other places. Regarding vitamin C, a significant content is shown for plants from floodplain meadows, also for

both species. The content of provitamin A and phenolic compounds, according to our data, depended more on the type of clover.

So the content of provitamin A was found to be higher in white clover, and phenolic compounds - in red clover.

Keywords: Red clover, White clover, Biologically active substances, Favonoids, Isoflavonoids, Phenolic compounds

therapeutic properties of clover are due to the content in the

Introduction

aerial part of Trifolium pratense of a number of phenolic

compounds, including flavonoids, in particular, quercetin,

rutin (3, 5). In addition, isoflavonoids are of particular

importance. Also, in the aerial part of red clover, depending

on the place of growth, isoflavonoids are represented by

biochanin A, genistein, ononin, formononetin, prunetin,

daidzein and their derivatives (3, 6). In this regard,

Trifolium pratense L. attracts the attention of

pharmacologists for experimental studies. Creeping Clover

Trifolium repens L., a no less widespread species compared

to meadow clover, has not been practically studied,

although it can also be of biological value. In addition, it

should be borne in mind that the phytochemical

composition of plants largely depends on various

environmental factors.

Throughout the world, interest in herbal medicine is

increasing every year, including in our country. The

process of growing medicinal plant materials is

economically viable, which ultimately has a positive effect

on the cost of production and has a slight negative impact

on the environment.

The most common plant in our country, characteristic

of meadow and pasture biocenoses, is considered clover

Trifolium L.), which genus includes about 65 species (1).

In total, this genus has about 300 species. Red meadow

Trifolium pratense L. is considered the most studied

species, which is an excellent fodder and honey plant, and

therefore has widespread use in agriculture (2, 19).

There is a significant amount of data in the literature

that the herb Trifolium pratense L. has a whole set of

biologically active substances (BAS): triterpenoids,

sesquiterpenoids, carotenoids, coumarins, cumestanes,

flavonoids, etc. (3). In folk medicine, Trifolium pratense

has long been used as a medicine for inflammatory

processes in the bladder, for various bleeding, and as a

diuretic for edema of cardiac and renal origin (4, 18). These

(

The objective of this work is to show the possibility of

using white clover (Trrifolium repens L.) as a source of

plant material containing biologically active substances.

Methods

The objects of our research were red clover (Trifolium

prätense L.) and white clover (Trifolium repens L.). In

order to identify the influence of the plant growth on the

content of biologically active substances, plant material

was collected in the steppe, which is characterized by water

Corresponding author: Alexander L. Mikhailov, Kazan

Federal

University,

Kazan,

Russia.

E-mail:

almihailov@bk.ru. Tel.: 89274455173.

874

Journal of Environmental Treatment Techniques

2019, Special Issue on Environment, Management and Economy, Pages: 874-877

deficiency, in a meadow (in the floodplain of the river),

where there is plenty of moisture and lighting, and forest

edge, where there is also a moisture deficit and lighting.

For experiments, the aboveground parts of plants were

collected and dried to constant weight.

The quantitative content of ascorbic acid in the aerial

part of the studied plants was determined using potassium

hexacyanoferrate. Dry plant material weighing 50 mg was

triturated using 1.5 ml of 0.1 M citrate buffer (pH 3.69),

then transferred to eppendorf tubes and heated in a water

bath (20 min at 40°C). After it was centrifuged for 5 min at

ml volumetric flask, was used. The content of the total

flavonoids in was converted to absolutely dry raw materials

and quercetin in percent was calculated by the formula.

Identification of flavonoids was carried out by high

performance liquid chromatography (HPLC), which is

characterized by high sensitivity and accuracy, allowing to

identify the composition of the studied group of

biologically active substances. The peaks of compounds

detected in the chromatogram were identified using

working standards (WS) of quercetin.

2500 g. The supernatant was used for further reactions.

Results and Discussion

The optical density was measured at a wavelength of 680

nm relative to the control solution.

Trifolium prаtense grows in moderately humid and

upland meadows, in forests and forest edges, along river

valleys and banks, on mountain meadow slopes, in fields,

along field roads. The chemical composition and medicinal

properties are studied relatively well. It contains various

aromatic compounds, essential oils, fatty acids, steroids,

coumarins, flavonoids and many other compounds of

pharmacological value.

White clover grows in floodplain meadows, in the

steppes, in forests, along river banks, on the side of roads,

in wastelands. There is relatively little information about

the chemical composition of clover in the literature.

Perhaps that is why this type of clover is not popular in

pharmacy, unlike red clover.

The vitamin complex of red clover Trifolium pratese is

represented by a wide variety of vitamins. There are

vitamins with a high content, namely vitamins of group B,

C, E, K, as well as provitamin A or β-carotene. The above

vitamins are highly important for the human body,

especially in childhood. As shown by our studies of

provitamin A, it was most found in Trifolium repens, which

is consistent with the literature (7,11,15,16) and in all three

locations (Fig. 1). It is also important to note that of the

three places of growth, plants collected in the steppe

meadows have the highest rate.

The content of provitamin A was determined by the

number of carotenoids isolated from the aerial parts of the

studied plants. To obtain an acetone extract, a sample of

dry plant material (100 mg) was ground with 2 ml of

acetone in a porcelain mortar. The extract was filtered

through a paper filter, the extraction was repeated in small

portions of the solvent. Then the filtrate was adjusted to 25

ml and used for determination on a spectrophotometer at

wavelengths of 662, 644, 440.5 nm. The pigment

concentration was calculated using the Rebbelen formula.

To determine the total content of soluble phenolic

compounds, they were extracted with water from the dried

ground part of plants at the rate of 1.5 ml of distilled water

per 50 mg of material for 45 minutes at a temperature of

70°C. The supernatant was centrifuged to separate from the

precipitate.

113 μl of Folin-Denis reagent was added to 113 μl of

the aqueous extract and stirred, after 3 minutes 180 μl of

NaCHO3 solution (10%) was poured, mixed, and 1.8 ml of

water was added. After 45 minutes, the solution was

centrifuged at 16 thousand rpm for 2 minutes. Then, the

optical density in the supernatant was determined at a

wavelength of 725 nm. For a control solution, 113 μl of

solvent was used instead of the extract.

In order to determine the amount of flavonoids in terms

of quercetin, the raw materials were crushed to particles

with a diameter of 1 mm. Then the raw material was placed

in a 50 ml flask weighing 0.2 g, 30 ml of 90% alcohol

containing 1% concentrated hydrochloric acid was added.

The flask was heated in a boiling water bath for 30 minutes

using a reflux condenser. The flask was then cooled to

room temperature and filtered through a paper filter into a

50 ml volumetric flask. The extraction was repeated once

again in the above manner.

The extracts were filtered through the same filter into

the same volumetric flask, the filter was washed with 90%

alcohol and the filtrate volume was adjusted with 90%

alcohol to the mark (solution A).

For spectrophotometry, 2 ml of solution A was placed

in a 10 ml volumetric flask, 1 ml of a 1% solution of

aluminum chloride in 95% alcohol was added, and the

solution volume was adjusted to the mark with 95%

alcohol. After 20 minutes, the optical density of the

solution was measured on a spectrophotometer at a

wavelength of 430 nm in a cell with a layer thickness of 10

mm. As a comparison solution, a solution consisting of 1

ml of solution A, brought 95% alcohol to the mark in a 10

Figure 1: The content of provitamin A in plants Trifolium pratеnse

and Trifolium repens from different places of growth

Perhaps this is due to the fact that carotenoids perform

protective function, protecting various organic

substances, primarily chlorophyll molecules, from

destruction in the light during photooxidation, since plants

in the steppe zone are more susceptible to direct light rays.

Ascorbic acid has strong antioxidant properties, thereby

acting as an important component of the human body's

immune system. Together with bioflavonoids, tocopherol

and retinol, ascorbic acid acts as

a direct-acting

875

Journal of Environmental Treatment Techniques

2019, Special Issue on Environment, Management and Economy, Pages: 874-877

bioantioxidant. This complex of compounds with

antioxidant properties causes a low level of free radical

states of lipids and biopolymers in the cell. Vitamin C and

bioflavonoids with simultaneous exposure enhance the

effect and complement each other, in connection with

which, they often occur together in dosage forms.

The results of the study of vitamin C content were

identical for both types of clover (Fig. 2). In this case, the

maximum values were found in plants from floodplain

meadows. There is evidence that the accumulation of

vitamin C in plants is significantly affected by the

conditions of their growth. In plants of the northern regions,

the content of ascorbic acid is much higher, compared with

plants growing in the southern regions. Also, the type of

soil on which plants grow affects the content of vitamin C.

It has been shown that on heavy soils the synthesis of

ascorbic acid in plants is weaker than on light soils (8, 13).

The maximum value of the studied parameter for

meadow clover was found in raw materials from a

floodplain meadow, and the minimum value was found

from the forest edge, which is opposite to the data obtained

for creeping clover. Possibly, these results can be explained

by the fact that most of the secondary metabolites are

represented by phenolic compounds, which are formed in

large quantities when plants are exposed to stress factors.

The phenolic compounds contained in red clover are

mostly represented by flavonoids. The chemical structure

of flavonoids presents a phenylpropane skeleton with the

formula C6-C3-C6. These compounds are interesting in

that they exhibit diverse biological activity (10, 12). This is

probably due to the presence of an oxygen atom in the ring

of these compounds.

Given the wide range of pharmacological effects of

flavonoids, namely immunomodulatory, antispasmodic,

anti-stress, capillary-strengthening, anti-toxic, anti-

atherosclerotic, anti-carcinogenic, etc., the value of

Trifolium pratense as a medicinal plant becomes clear.

Among the huge variety of Trifolium prenens flavonoids,

some of them can be noted: flavones (apigenin, baicalein

tricetin, and luteolin), isoflavones (biohanin-A-7-glucoside,

prinetin, formononetin, and ononin), and flavonols (rutin,

kempferol, isoquercetin, etc.) (14).

Our data on the study of flavonoid content depending

on the place of growth were approximately similar for each

of the studied clover species (Fig. 4). The highest values

were obtained from the raw materials of meadow steppes,

and the lowest - from the edges of the forest. If neglecting

the significance of differences between the variants, for the

studied compound, namely for flavonoids, white clover is

not much inferior to red clover.

Figure 2: The content of vitamin C in plants Trifolium pratеnse and

Trifolium repens from different places of growth.

Phenolic compounds of plants are usually the products

of secondary metabolism, which are represented by a large

group differing in structure, chemical properties and

biological activity. Phenols in plants are found in all

organs, especially a significant concentration is found in

actively functioning organs. The content and concentration

of phenolic compounds also depends on the type of plant.

Plants of the same genus can differ greatly in the content

and concentration of phenolic substances and in their

qualitative composition.

Plants of Trifolium pratens compared to Trifolium

repens showed a higher total content of soluble phenolic

compounds (Fig. 3). Apparently, this indicator depends on

the type of plants, as in the case of provitamin A, since the

results are higher in all three locations.

Figure 4: The content of flavonoids in terms of quercetin in plants

Trifolium pratеnse and Trifolium repens from different places of

growth.

Summary

The obtained data showed that the content of

biologically active compounds in white clover (Trifolium

repens L.) is similar to those of red clover (Trifolium

pratense L.). The influence of the place of plant growth on

the content of the studied compounds was revealed, which

may be important for solving production problems. For

example, in clover plants of both species studied, the

flavonoid content collected in meadow steppes is higher

than in other places. Regarding vitamin C, a significant

content is shown for plants from floodplain meadows, also

Figure 3: The content of phenolic compounds in plants Trifolium

pratеnse and Trifolium repens from different places of growth.

876

Journal of Environmental Treatment Techniques

2019, Special Issue on Environment, Management and Economy, Pages: 874-877

for both species. The content of provitamin A and phenolic

compounds, according to our data, depended more on the

type of clover. So the content of provitamin A was found to

be higher in white clover, and phenolic compounds - in red

clover.

11. Sajjadifar S, Hamidi H, Pal K. Revisiting of Boron Sulfonic

Acid Applications in Organic Synthesis: Mini-Review. Journal

of Chemical Reviews. 2019 Jan 1;1(1, pp. 1-77.):35-46.

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of cadmium sulfide nanoparticles (CdSNPs) using Panicum

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The results of analysis of the obtained data of the study

of the phytochemical composition of two types of clover

show that white clover is a potential source of a number of

pharmacologically active compounds, like red clover,

which creates the basis for a deeper, more detailed study of

it as a possible object of phytotherapy methods and

treatment directions, since the species under study refers to

the sources of natural herbal preparations.

Acknowledgments

The work is performed according to the Russian

Government Program of Competitive Growth of Kazan

Federal University, as well as with the financial support of

the Russian Foundation for Basic Research and the

Government of the Republic of Tatarstan in the framework

of the research project No. 18-44-160015.

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Management Position and Role in Iran Oil Industry. Journal of

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Farid HM. Thermodynamic and Theoretical solvation

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