The Study of the Composition of Chloroform Fraction of Caltha palustris

 

Viktoriia Karpiuk1, Roksolana Konechna1, YulianKonechnyi2, Wieczorek Piotr Pawel3,

Jasicka Misiak Izabela4, Lesia Zhurakhivska1, Lilia Bolibrukh1

1Department of Technology of Biologically Active Substances, Pharmacy and Biotechnology,

Lviv Polytechnic National University, Stepan Bandera Street, 12, Ukraine.

2Danylo Halytsky Lviv National Medical University, Department of Microbiology, Lviv 79013, Ukraine.

3University of Opole, Faculty of Chemistry, Department Analytical Chemistry, Opole, Poland.

4University of Opole, Department of Pharmacy and Ecological Chemistry, Opole, Poland.

*Corresponding Author E-mail: viktoriia.r.liakh@lpnu.ua

 

ABSTRACT:

The analysis presented in this article is a continuation of a broader qualitative study examining the composition of the chloroform fraction of Caltha palustris. The study discovered 29 compounds, and 26 of them have been identified:paraffinic hydrocarbons, esters, fatty aldehydes, morphine derivatives, thiazole derivatives, benzodiazepines, naphthalene, and a small amount of terpene. It was figured out that the investigated lipophilic extract has an antioxidant impact. For the investigation, there were used such assays as the DPPH radical and the ABTS  radical cation assays. Moreover the investigated lipophilic extract has an antimicrobial activity both in terms of gram-positive (Staphylococcus aureus (ATCC 25923 (F-49)), Bacillus cereus 34 (non-MDR), Enterococcus faecalis 26(MDR) and gram-negative, (Raoultella terrigena (ATCC 33257), Escherichia coli (ATCC 25922), Escherichia coli 168) microorganisms and yeast Candida spp. In addition, the significant content of biologically active substances in the lipophilic volatile fraction of Caltha palustris indicates the prospects for further study.

 

KEYWORDS: Caltha palustris, Сhloroform fraction, Gas chromatography, Lipophilic extract, Antioxidant effect, Antimicrobial activity.

 

 


INTRODUCTION: 

Over the recent years, we can observe an increasing interest in herbs. Due to this, it is necessary to pay attention to understanding different aspects of phytoremedies production and their practical implementation.

 

One of the most topical and promising species of the Ukrainian flora to be used in contemporary medicine and pharmacology is Caltha palustris in the buttercup family (Ranunculaceae).

 

This plant is widely used in complementary and alternative medicine as an anti-inflammatory, antispasmodic, bactericidal, antimicrobial, analgesic, and diuretic agent.

 

 

To the main biologically active compounds, there belong tannins, glycosides (protoanemonin and anemonin γ-lactones) saponins, berberine, bitterance, vitamin C, choline, carotene, flavonoids, and alkaloids1,2.

 

The main area for Caltha palustris spread is the highlands of the Ukrainian Carpathians. At the same time, the plant is rarely grown in the other administrative territories of Ukraine3.

 

Taking into consideration the broad experience of the plant implementation in conventional and alternative medicine, and partially official medicine4-6, its wide spectrum of implementation in pharmacology, its content, and biologically active compounds7-9 together with the results of phytochemical and pharmacological investigations, it is possible to understand that the further use of Caltha palustris is an up-to-day task both for pharmacology and pharmaceutic biotechnology due to the perspective of investigations on new phytoremedies development and implementation10.

The primary objective of the research is the investigation and determination of the chloroform fraction of Caltha palustris and the study of its antioxidant effectsand antimicrobial activity11.

 

MATERIALS AND METHODS:

Plant material and preparation of extract:

The object of research was the herb of Caltha palustris that was gathered in 2018 in an ecologically clean area of the Carpathians (Ivano-Frankivsk region, Ukraine).  The shade-dried parts of the plant were powdered and extracted by chloroform with help of Soxhlet extractor. Then, the solvent was removed with the help of the vacuum until the condition of dry extract.

 

Chromatographic analysis:

The lipophilic fractions of biologically active substances were analyzed usingchromatograph Agilent Technology 6890 GC System with mass spectrometry detector HP 5973 Mass Selective Detector. The components were separated in a fused silica column Restek Rtx-5MS being 30 m long and an internal diameter of 0.32 mm.12,13

 

The stationary liquid phase was 0.25μm thick and consisted of 95% polydimethylsiloxane and 5% phenyl polysiloxane. Helium was used as the carrier gas. The speed of the carrier gas was 2.0ml/min. The thermostat of the column was heated in the following mode: the initial temperature of 60°C was maintained for 10 minutes, and then the temperature was increased with the speed of 20°C/min to 280°C. This temperature remained constant for 30min, the temperature of the detector and the evaporator was 280°C. Biologically active substances were identified by the time of their retention, compared with standards and the library of mass spectra WILEY 2007 and NIST 05, where the total number of spectra is more than 470000.14-16

 

Antioxidant effect determination:

a) DРРH radical scavenging impact:

The research of the antioxidant impact was held with the help of the modified method. First of all, there was obtained a freshly prepared solution of 0.1mM DРРH. Then, 500μL of the extract together with 4.5mL of DРРH solution were mixed in a test tube. The obtained solution remained incubated for 30 minutes at a dark place and room temperature. Lastly,Hitachi U-2810 spectrophotometer was used for measuring the optical density of samples with the length of the wave of 517. Ethanol was used as a reference sample.

 

There was implemented the following formula aimed at calculating the percentage of inhibition of the radicals:

 

%inhibition = (Acontrol − Asample)/Acontrol × 100%.

 

In the formula, Acontrol means the absorbance of DРРH solution without extract and Asample determines the absorbance of the sample with the added DРРH solution. There has been carried out a 3-fold measurement to ensure the correctness and accuracy of the data obtained17-20.

 

b) The scavenging effect of ABTS radical:

The extract`s free radical scavenging effect was also determined by the decolorization assay ofthe ABTS  radical cation. The concentration of ABTSin water was dissolved to 0.014mM21,22.

 

The interaction of the original ABTS solution with the solution of potassium persulfate (prepared by dissolving 0.0135g K2S2O8 in 10ml of water) allowed to obtain a radical cation ABTS (ABTS*), and then the mixture was stirred in the dark at room temperature for 20h.

 

After that, 1ml of thesolution was diluted to 100ml. There was taken reagent blank reading (Acontrol). The absorbance ratio was measured after adding 1.0mL of diluted ABTS*+solution to 100μL of the extract precisely 6 minutes after the initial mixing at 30°C (Asample).

 

Blanks of appropriate solvent were run in each assay. In addition, there has been carried out a 3-fold measurement to ensure the accuracy of the data23,24.

 

Antibacterial and antifungal bioassay:

Four Gram-negative bacteria (clinical pandrug- resistant (PDR) Pseudomonas putida 182, Raoultella terrigena (ATCC 33257), Escherichia coli (ATCC 25922), andclinical multi-drug resistance (MDR) Escherichia coli 168), three gram-positive bacteria (Staphylococcus aureus (ATCC 25923 (F-49)), Bacillus cereus 34 (non-MDR), Enterococcus faecalis 26 (MDR) and clinical methicillin-resistant Staphylococcus aureus (MRSA) 23) and two strain of yeast (reference strains Candida albicans (ATCC 885-653) and clinical MDR strain Candida albicans 169 were used for antibacterial and antifungul activity (disc diffusion and serial dilutions assay)24,25.

 

After adjusting turbidity of each broth culiture of bacteria with saline (0.5 McFarland), inoculation of Mueller Hinton and Sabouraud agar plates was performed by using cotton swabs (6mm diameter and containing 100μL of extract in concentration 1,4 mg/mL)26.

 

RESULTS AND DISCUSSION:

Determination of chemical compounds of extract of Caltha palustris:

The first set of analyses examined the chromatogram of the extracts under study (Figure 1).As the result of the study, the quantitative content of 29 volatile compounds was established, 26 of which were identified The identification of the main lipophilic extractives and the corresponding quantification isv summarized in Figure 1 and Table 1.


 

Table 1. Identified compounds of Caltha palustris chloroform fraction

№ з/п

Retention time

Substance

Content, %

№ з/п

Retention time

Substance

Content, %

1.

7.317

D-Limonen

0,41

28.

46.616

 Petroselinic acid

0,04

2.

8.846

Оleic acid

0,09

29.

46.716

Petroselinic acid

0,04

3.

19.249

Нeneycosan

0,34

30.

46.901

Deoxyestradiol

0.18

4.

20.050

Нeneycosan

0,66

31.

47.087

Deoxyestradiol

0.27

5.

23.265

Tricosane

0,27

32.

47.416

androst-5-en-3-one

0.96

6.

24.894

Hexadecane

0,73

33.

48.202

Pyridine-3- carboxamide)

7.29

7.

25.823

Docosane

0,34

34.

48.388

Nordekstrometorfan (2-metoksimorfinan)C17H23NO

2.70

8.

30.453

Ethyl palmitate

1.14

35.

48.531

Nordekstrometorfan (2-metoksimorfinan)

2.46

9.

31.868

Нeneycosan

0.50

36.

48.631

Nordekstrometorfan (2-metoksimorfinan)

1.51

10.

32.168

Phytol

0.85

37.

48.773

5,10- dimetilbenzakridin C19H15N

2.13

11.

32.268

Unidentified

1.62

38.

48.816

5,10- dimetilbenzakridin C19H15N

1.09

12.

32.582

Isopropyl linoleate

0.64

39.

49.031

Nordekstrometorfan (2-metoksimorfinan)

2.55

13.

32.654

Еthyl linoleate

1.58

40.

49.088

1,5 - dimetilbenzakridin C19H15N

2.50

14.

32.954

Еthyl stearate

0.55

41.

49.274

1,5 - dimetilbenzakridin C19H15N

0.92

15.

34.083

Octacosane

0.50

42.

49.331

Nordekstrometorfan (2-metoksimorfinan)

1.24

16.

34.540

L-Valine, N-Penta-fluoropropionyl-Heptyl Ester, C15H24F5NO3

1.53

43.

49.631

Nordekstrometorfan (2-metoksimorfinan)

1.17

17.

34.912

Нeneycosan

1.20

44.

49.702

Unidentified

1.26

18.

35.498

Tricosane

0.47

45.

49.788

5,10- dimetilbenzakridin C19H15N

1.02

19.

35.783

Tricosane

0.89

46.

50.060

Unidentified

1.03

20.

35.926

Docosane

1.06

47.

50.203

Geranyl acetone

2.23

21.

36.241

Docosane

1.26

48.

50.588

Fumaric acid, 2-decyl tridecyl ester

1.26

22.

36.312

Nonadecane

0.86

49.

50.960

Unidentified

1.84

23.

36.612

Octadecyl chloride

1.85

50.

51.346

Nordekstrometorfan (2-metoksimorfinan)

1.69

24.

36.969

Icosane

1.31

51.

51.946

Nordekstrometorfan (2-metoksimorfinan)

0.27

25.

37.670

Нeneycosan

0.79

52.

52.189

Nordekstrometorfan (2-metoksimorfinan)

0.22

26.

37.998

Icosane

0.79

53.

52.332

Nordekstrometorfan (2-metoksimorfinan)

0.29

27.

46.315

Androstenediol diacetate

0.05

 

 

 

 

 


As it can be seen from these data, in thestudy of the component composition of theCaltha palustris chloroform fraction,26compounds were identified, being: paraffinic hydrocarbons, esters, fatty aldehydes, morphine derivatives, thiazole derivatives, benzodiazepines, naphthalene, and a small amount of terpene.

 

 

Figure 1. Chromatogram of Caltha palustris chloroform fraction

 

Further analysis showed that the dominant component in the sample was Nordekstrometorfan– 14, 1%.

 

The following alkanes were detected: heneycosan - 3.49%, tricosanе - 1.63%, docosanе - 2.66%, icosanе - 2.1%, nonadecane - 0.86%, and others.

 

Also, there was detecteda small amount of Deoxyestradiol - 0.45%. together with small amounts of oleic (0.09%) and petroselinic (0.08%) acids.

 

Antioxidant effectof extract of Caltha palustris:

For the investigation, there were used such assays as the DPPH radical and the ABTS radical cation assays, purposed at determining the freeradicalscavenging features of extracts of Caltha palustris.

 

Diagram 1. DPPH and ABTS radical scavenging activity of Caltha palustris  extracts

 

The effect of Caltha palustris extract compared to quercetin and vitamin C on the inhibition of DPPH radical and the ABTS radical cation are shown in Diagram 1.

 

Caltha palustris extract showed almost the same antioxidant activity as ascorbic acid and quercetinum.

 

Antibacterial and antifungal bioassay of extract of Caltha palustris:

For the first time, the antimicrobial and antifungal properties of the extract of Caltha palustriswere investigated The antibacterial activityand antifungalactivity of extracts of Caltha palustris is represented in Table 2.


Table 2. Antibacterial and antifungul activity of chloroform fraction of Caltha palustris

Tipe of species

Species of bacteria and fungi

Diameter of inhibitory zones (mm ± SD)

MinimumInhibitory (MIC)

Extract of Caltha palustris

Water

Vancomycin

Clotrimazole

Ciprofloxacin

Extract of Caltha palustris (mg/mL)

1.

Gram-

negative bacteria

Escherichia coli (ATCC 25922)

NA

NA

-

-

35,0 ± 0,3

-

2.

Escherichia coli 168 (MDR)

NA

NA

-

-

20,0 ± 0,2

-

3.

Pseudomonas putida 182 (PDR)

8,9± 0,2

NA

-

-

14,0 ± 0,4

0.7

4.

Raoultella terrigena ATCC 33257

NA

NA

-

-

30,0 ± 0,5

-

5.

Gram-positive bacteria

Staphylococcus aureus (MRSA) 23

NA

NA

11.2 ± 0,2

-

9,0 ± 0,2

-

6

Staphylococcus aureus (ATCC 25923 (F-49)

NA

NA

32 ± 0,4

-

35,0 ± 0,5

-

7.

Bacillus cereus 34 (non-MDR)

6,8 ± 0,3

NA

25,1 ± 0,5

-

8,0 ± 0,2

-

8.

Enterococcus faecalis 26 (MDR)

9,0 ± 0,4

NA

12,0 ± 0,3

-

10,0 ± 0,2

0.7

9.

Fungi

Candida albicans169 (MDR)

7,1 ± 0,2

NA

-

10,0 ± 0,3

-

-

10.

Candida albicans (ATCC 885-653)

12,6 ± 0,4

NA

-

19,0 ± 0,5

-

0.35

NA: no activity; ‘-‘ not tested; Vancomycin 30 μg (inhibition zone 17-21 mm for S.aureus); Ciprofloxacin 5 μg (inhibition zone 25-33 mm for P.aeruginosa, 22-30 mm for S.aureus, 30-40 mm for E.coli); Clotrimazole 10 μg (inhi-bition zone 12-17 mm for Candida spp;

 


The results obtained of the study biological activity of Caltha palustrischloroform fraction suggest that the lipophilic extract under study has antimicrobial and antifungal activity. The extract showed the highest antimicrobial activity against clinical Pseudomonas putida 182(PDR) and the highestantifungal activity against clinical MDR strain Candida albicans 169.

 

The raw materials of Caltha palustris is rich in D-Limonen so it could be potential sources of this compound.

 

This compound has antioxidant properties, which have been confirmed by the results of research on the herb extract of Caltha palustris. Detection of this compound indicates the prospect of using the extract of of Caltha palustrisas one of the components of medical and cosmetic products.

 

Not all substances could be identified during the chromatographic study of raw materials, as they were not available in the library that was used.According to the received results of the studies, further screening of phar-macological studies, and development of parameters for standardization ofCaltha palustris herb are planned.

 

CONCLUSIONS:

This study set out to analyze thecomposition of the Caltha palustris chloroformfraction. The Caltha palustris lipophilicfraction was obtained using the method ofexhaustive extraction with chloroform in the Soxhlet extractor.

 

The yield was 6,93%. For the first time, the chloroform fraction of the Caltha palustris was obtained, and the component composition of the volatile matter was discovered using chromatography (mass spectrometry).

 

29 substances were discovered, with 26 of them being identified. The extract was found to have the same antioxidant effect as vitamin C and quercetinum.

 

This study has shown that the Caltha palustris lipophilic extraction has anantimicrobial action on clinical MDR/PDR strains of both gram-positive, gram-negative microorganisms and Candida spp. Of particular note is the activity against PDR Pseudomonas, which is promising for further research.

 

Based on the data obtained from the research, Caltha palustris can be considered a promising medicinal plant for herbal remedy development, and further research of the plant.

 

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Received on 27.05.2022            Modified on 04.08.2022

Accepted on 28.09.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(3):1254-1258.

DOI: 10.52711/0974-360X.2023.00207