CC BY
37PHARMACEUTICAL SCIENCES
PREPARATION AND RESEARCH OF OREGANO HERB DRY EXTRACT
Chernetska S.
I. Horbachevsky Ternopil National Medical University,
Ternopil, Ukraine Beley N.
I. Horbachevsky Ternopil National Medical University,
Ternopil, Ukraine
ABSTRACT
The article presents the results of technological, phytochemical, microbiological and biological studies of oregano herb dry extract are given in the article. The influence of the extractant type on the degree of extraction of biologically active substances from oregano herb has been studied. It has been experimentally established that the optimal extractant for obtaining a dry extract of oregano is ethanol solution with concentration 40 %. The antimicrobial and anti-inflammatory properties of the obtained extract have been proved.
Keywords: dry extract, oregano herb, extraction, biologically active substances, flavonoids, microbiological studies, anti-inflammatory action.
Acute respiratory disease has traditionally been considered a collective term, but such a diagnosis is made in patients with acute respiratory disease of the upper respiratory tract, which is caused by a bacterial agent or of unknown etiology. An inflammatory process in the pharynx, tonsils or nasopharynx often causes acute sore throat. Pharyngeal infections may be caused by viral pathogens and bacterial pathogens. These pathogens include Streptococcus pyogenes [1]. However, fungi, viruses and other bacterial agents can also cause sore throats: Staphylococcus aureus, Diplococcus pneumoniae, Cory-nebacterium diphtheriae, Bor-detella pertussis, Haemophilus influenzae, Neisseria species [2]. Drugs used to the mucous membrane of the upper respiratory tract must have a wide range of antimicrobial action (antiviral, antibacterial and antifungal activity), they should not have toxic effect and ability to cause allergies, have not to irritate the mucous membrane. High rate of absorption from mucous membranes, pleasant taste and convenient dosage form are requirements for these drugs.
Herbal drugs are promising in the treatment and prevention of many diseases, as they are characterized by several advantages: they contain a complex of biologically active substances, have low toxicity, almost no side effects, they are not addictive when used for a long time [3, 4, 5].
Origanum vulgare, L has anti-inflammatory, wound-healing, hemostatic and antimicrobial properties [6-9]. In folk medicine of different countries, it is used for treatment of neuralgia, cuts, rheumatism, paralysis, toothache and ear pain[12]. The biologically active substances of oregano include flavonoids, ascorbic
The effect of ethanol content in the extractant on the
acid, tannins, essential oil, the main components of which are thymol, carvacrol and several other compounds [10-11].
We have done an analysis of the Ukrainian pharmaceutical market of medicines for the treatment of throat diseases and we found that the solid dosage form occupies the largest share of the market for drugs used to the mucous membrane of the upper respiratory tract [13]. Therefore, it is advisable to develop a drug based on oregano extract common for the treatment of oral diseases in solid dosage form.
The aim of the work. Investigation of the effect of ethanol content in the extractant on the extraction efficiency in the manufacture of oregano herb dry extract, study of antimicrobial activity and anti-inflammatory effect of dry extract of oregano as an intermediate in the production of solid dosage form based on it for the treatment of throat diseases.
Materials and Methods. Material for research -liquid extracts obtained by fractional maceration with stirring of oregano herb with alcoholic solutions containing 40-90 % of the ethanol, the ratio of raw material to extractant 1:10, multiplicity extraction - 3, one extraction duration - 24 hours.
The study of the thymol was performed by chro-matographic method; the quantitative content of thymol and flavonoids was determined by spectrophotometric method, the content of the dry residue - by gravimetric method.
The results of the study of the effect of ethanol content in the extractant on the extraction of biologically active substances from the herb oregano are shown in table 1.
Table 1
The ethanol content in the extractant, % The content of biologically active substances in extracts of oregano vulgare, %
flavonoids Thymol Dry residue content
40 0,147±0,0003 0,0301±0,0002 2,90±0,0377
50 0,147±0,0003 0,0224±0,0001 2,87±0,0087
60 0,137±0,0003 0,0252±0,0001 2,76±0,0029
70 0,116±0,0005 0,0176±0,0003 2,61±0,0058
80 0,064±0,0003 0,0125±0,0003 1,84±0,0029
90 0,025±0,0001 0,0043±0,0001 1,01±0,0058
From the above results it is seen that the extractant with an ethanol content of 40 % (v/v) provided the highest content of biologically active substances in the extracts of oregano herb (thymol - 0,0301±0,0002 and flavonoids - 0,147±0,0003 mg/ml in terms of luteolin), as well as the degree of extraction (dry residue -2,90±0,0377 %). Therefore, it was decided to use this extractant in the production of oregano herb dry extract. Alcohol extracts were concentrated and dried in a vacuum oven at a temperature of 45-50 0C.
The antimicrobial activity of oregano herb dry extract was investigated in the experiments in vitro. For this purpose, 50 % solutions of the obtained dry extract were prepared[7].
In this study, museum strains of Staphylococcus aureus, Escherichia coli, Candida albicans, Bacillus subtilis, and Pseudomonas aeruginosa were used as test cultures. Bactericidal and bacteriostatic properties were determined by the presence and nature of growth of cultures of microorganisms of the studied extract[8]. Methods of serial dilutions and diffusion into agar (method of wells) were used.
Tubes with two consecutive dilutions of dry extract were prepared by the method of serial dilutions in a liquid nutrient medium.
The method of diffusion into agar is the ability of the active substances to diffuse into the agar medium, which contains cultures of microorganisms. The diameter of the growth retardation zone of the test cultures was measured in millimeters, including the diameter of the well.
To assess the antibacterial activity of the oregano herb dry extract used the following criteria:
• the absence of zones of growth retardation of microorganisms around the well, as well as zones of delay up to 10 mm, indicate that the microorganisms are not sensitive to the drug introduced into the well or its concentration;
• zones of growth retardation with a diameter of 11-15 mm, indicate a low sensitivity of the culture to the test concentration of antibacterial substance;
• growth retardation zones with a diameter of 16-25 mm are regarded as an indicator of the sensitivity of the microorganism to the test substance;
• zones of growth retardation, the diameter of which exceeds 25 mm, indicate a high sensitivity of microorganisms to the test substance.
The research results are shown in tables 2 and 3.
Table 2.
The results of the study of antimicrobial activity of oregano herb dry extract by diffusion into agar
Diameters of the growth retardation zone, mm
Dilution of the extract Staphylococus aureus Escherichia coli Candida albicans Bacillus subtilis Pseudomonas aeruginosa
Native solution 25 11 22 - 20
1:2 22 - 16 - 15
Table 3.
The results of the study of antimicrobial activity of the oregano herb dry extract by serial dilutions
50% solution of dry extract of oregano
Strain of microorganisms Native solution 1:2 1:4 1:8 Control
Staphylococcus aureus ++ ++ + + -
Escherichia coli - - - - -
Candida albicans ++ ++ + + -
Bacillus subtilis - - - - -
Pseudomonas aeruginosa + + - - -
Note: «++» - the presence of bactericidal properties,
«+» - the presence of bacteriostatic properties,
«-» - lack of bactericidal and bacteriostatic properties.
According to the study, it can be concluded that the oregano herb dry extract in relation to Staphylococ-cus aureus and Candida albicans in a dilution of 1:2 has a bactericidal effect, and in a dilution of 1: 4 - bac-teriostatic effect.
Experimental studies of anti-inflammatory activity were performed on nonlinear white rats of both sexes weighing 180-220 g. General edema was induced by injection under aseptic conditions of 0.1 ml of 2 % carrageenan solution under the aponeurosis of the sole of the hind limb of the rat.
The presence of an inflammatory reaction was determined by the change in limb volume by the oncomet-ric method at the beginning of the experiment and 4
hours after the introduction of the phlogogenic agent. In 40 minutes prior to administration of the carrageenan solution, the test substances were administered intra-gastrically to the animals. For comparison, the antiexu-dative effect of known anti-inflammatory drugs was studied under similar conditions: diclofenac sodium, ketanol and quartcetin in moderate therapeutic doses.
The anti-exudative activity of the drugs was determined by the degree of reduction of edema in experimental animals compared with control and expressed as a percentage. The test extract was administered intra-gastrically at doses of 50, 100, 150, 200 mg/kg. The results of the experiments are shown in table 4.
Table 4.
Anti-inflammatory activity of oregano herb dry extract in the model of carrageenan edema
The test compound Dose, mg/kg The percentage increase in paw volume on the 4th hour,% The rate of suppression of the inflammatory reaction,%
1 2 3 4
Control 134.3 -
Diclofenac intraperi-toneally 8 75.7 43.6
Ketanol intraperitoneal^ 10 82.4 38.6
Quercetin (intra-gastrically) 50 123.1 -
Control 130.4 -
Oregano extract 200 mg/ g (intra-gastrically) 104.0 20.2
Studies have shown that the highest degree of anti-edematous activity in a model of carrageenan edema in white rats was found when animals were administered an oregano herb dry extract at a dose of 200 mg/kg. The experiment shows that the dry extract of oregano has anti-inflammatory effects.
Conclusions. The extractant with the ethanol content of 40 % is the most effective for the extraction of biological active substances in the manufacture of oregano dry extract. Antimicrobial activity and the anti-inflammatory effect of the obtained dry extracts of oregano herb as an intermediate in the production of solid dosage form based on it for the treatment of throat diseases have been proved.
References
1. Pelucchi C., Grigoryan L., Galeone C., Esposito S., Huovinen P., Little P., Verheij T. Guideline for the management of acute sore throat ESCMID. Sore Throat Guideline Group. Clinical Microbiology and Infection. 2012; 18: 1-27.
2. Angotoeva I.B. [Differentsial'naya diagnostika boley v gorle]. Meditsinskiy sovet. 2015; 15: 42-46. Russian.
3. Voloshin O.I., Garnik T.P., Voloshin L.O., Vlasyuk V.L. [Liky roslynnoho pokhodzhennya v klin-itsi vnutrishnikh khvorob - odyn iz vazhlyvykh shlyakhiv vyrishennya problem komorbidnosti (ohlyad literatury ta vlasni doslidzhennya)]. Fitoter. Chas. 2013; 1: 4-9. Ukrainian.
4. Mikheev A.O. [Perspektyvy zastosuvannya roslynnykh oliy yak protyhrybkovykh zasobiv]. Zaporozhye medical journal. 2017; 2: 221-226. Ukrainian.
5. Boyko E.F. [Origanum vulgare i Origanum Tyttanthum Gontsch kak lekarstvennyye, efiromaslich-nyye, pryano-aromaticheskiye i dekorativnyye ras-teniya]. Tavricheskiye zapiski natsional'nogo universi-teta im. I.V. Vernadskogo, seriya "Biologiya, khimiya". 2009; 2(68): 9-15. Russian.
6. Kocic-Tanackov S., Dimic R., Pejin D., Mojo-vic L., Pejin J. Antifungal activity of Oregano (Origanum vulgare, L) extract on the growth of Fusarium and Penicillium species isolated from food. Faculty of Technical Sciences. NoviSad Serbia. 2012; 3: 2-9.
7. Yoshino K., Higashi N., Koga K. Antioxidant and Antiflammatory Activities of Oregano Extract. Journal of HealthScience.2006; 52(2): 169-173.
8. Chernetska S.B., Beley N.M. Prospects for a creation of new drugs based on Oregano (Literature review). Fitoter. Chas. 2018; 1: 25-28. Ukrainian.
9. S. Saed, P. Tario Antibacterial Activiti of Oregano (Origanum vulgare Linn.) Against Gram positive bacteria. Pac. J. Pharm. Sci. 2009; 4: 421-424.
10. Voloshin O.I., Bachuk-Ponch N.V., Kardash A.V. [Materynka zvychayna v narodniy medytsyni ta klinichniy praktytsi, perspektyvy vykorystannya (Ohlyad literatury)]. Fitoter. Chas. 2015; 1: 10-13. Ukrainian.
11. Alyakin A.A., Efremov A.A., Kachin S.V. [Fractional composition of oregano essential oil of the Krasnoyarsk Territory]. Chemistry of vegetable raw materials. 2010; 1: 99-104. Russian.
12. Kotyuk L.A., Rakhmetov D.B. [Biolohichno aktyvni rechovyny Origanum vulgare L]. Fiziologiya rasteniy i genetika. 2016; 1: 20-25. Russian.
13. Chernetska S.B., Beley N.M. [Analysis of the pharmaceutical market of drugs for the treatment of throat diseases]. Fitoter. Chas. 2019; 1: 34-37.
