Pharmacognostic studies of Origanum L. species Medicinal plant raw materials

 

Bokov D.O.^1,2*, Nizamova L.A.³, Morokhina S.L.⁴, Marakhova A.I.⁵, Bobkova N.V.¹,

Sergunova E.V.¹, Kovaleva T.Yu.¹, Balobanova N.P.¹, Prostodusheva T.V.¹, Klyukina E.S.¹, Chevidaev V.V.¹, Samylina I.A.¹, Lazareva N.B.¹, Krasnyuk I.I.¹

¹Sechenov First Moscow State Medical University, 8, Trubetskaya St., bldg. 2, 119991, Russian Federation.

²Federal Research Center of Nutrition, Biotechnology and Food Safety, 2/14, Ustyinsky pr., Moscow,109240, Russian Federation.

³Bashkir State Medical University, 3, Lenina street, Ufa, 450008, Republic of Bashkortostan, Russian Federation

⁴Financial University under the Government of the Russian Federation (Financial University), 55,

Leningradsky Prospekt, Moscow, 125057, Russian Federation.

⁵Рeoples’ Friendship University of Russia (RUDN University), 6, Miklukho-Maklaya Street, Moscow, 117198, Russian Federation.

 

ABSTRACT:

Objective: Russian Oregano (Origanum vulgare L.) and Turkish oregano (Origanum onites L.) are pharmacopoeial plants that are used for producing medicinal plant raw materials (crude herbal drugs). The aim of the study was to determine the composition and content of biologically active substances in Origanum crude herbal drugs. Materials and methods: Complex of modern physicochemical methods was used in pharmacognostical research. Thin-layer chromatography, gas-liquid chromatography was applied for the determination of crude herbal drugs composition. The content of total flavonoids in terms of cynaroside was determined by differential UV spectrophotometry, total tannins content – by redox titration, essential oil – by distillation. Microscopic analysis was performed on a Biomed-C2 microscope with 5×, 10× eyepieces, 4×, 10× and 40× objectives, photographies – were taken with a Canon Power Short SX 210 IS digital camera. Results: Thymol is a dominant component in essential oil. Two species contain a cynaroside (flavonoid). The content of essential oil in O. vulgare and O. onites herb was 0.12±0.03% and 2.60±0.02% respectively; the total flavonoids in terms of cinaroside – 0.98±0.02% and 1.25±0.01%; total polyphenols – 11.54±0.35% and 19.43± 0.72% (condensed polyphenols precipitated by gelatin – 4.94±0.15% and 13.62±0.21%); ascorbic acid – 0.35± 0.05% and 0.40±0.06%. Existing ones were modified and a number of new unified procedures were developed, allowing qualitative and quantitative analysis of phenolic compounds in O. vulgare and O. onites herb. A complex of anatomical and diagnostic features (the structure of the stomatal complex, simple hairs, ethereal-oil glands) was established to carry out CHD identification. Conclusion: Composition and content of the polyphenolic complex and hydrophilic biologically active compounds of Origanum vulgare L. and Origanum onites L. crude herbal drugs (CHD) were determined. The obtained data will be used to improve the existing regulatory documentation for Origanum crude herbal drugs standardization.

 

 

 

INTRODUCTION:

Russian Oregano (Origanum vulgare L. ssp. vulgare, Figure 1A) and Turkish oregano (Origanum onites L., Figure 1B), (syn.: Smyrna oregano (Origanum smyrnaeum Sibth. and Sm.)–herbaceous perennial plants of the Lamiaceae Lindl. family, 35-80cm, with a characteristic strong aromatic odor^[1].

 

A

 

B

Figure 1: A – Origanum vulgare L. in flowering (East Kazakhstan, Glubokovsky district, the neighborhood of the village Tarkhanka, the slope of the hill)^[3]; B – Origanum onites L. in the beginning of flowering (Greece, the Aegean Sea, Paros island, the neighborhood of Lefkada city, Byzantine trail)^[2].

 

O. vulgare is common in Western and Eastern Europe, in the mountainous regions of Central Asia, the Caucasus, Kazakhstan, South Siberia, Сoastal zone, Amur, as the introduced plant is found in the Far East; in Europe, its range extends from south to north from the Mediterranean to Norway and Southwest Asia reaches the Himalayas^[4]. Turkish oregano is widely represented mostly in the Mediterranean (Greece, Turkey, and other countries)^[5], O. vulgare and O. onites are cultivated as an aromatic and medicinal plants in the U.S., France, Germany, Russia, and some other countries^[6]. In Russia Oregano grows everywhere (except the Far North): in meadows, clearings, forest edges, forest clearings, river valleys, in bushes, on the slopes of hills, birch, and light coniferous forests^[4].

 

Essential oil (EO) and its derivatives are undoubtedly the main products, obtained from plants of the Origanum L. genus^[7]. It is widely used in various branches of industry: medical, foods, perfumes, cosmetics, and alcoholic beverages^[8]. The components, that are most characteristic for Oregano EO, according to various authors^[9], are α-thujene, linalool, α-pinene, ß-pinene, myrcene, camphene, borneol, selinen, ß-phellandrene, sabinene, ocimene, limonen, α-terpinene, undecanon-2, 1,8-cineole, α-terpineol, ß-caryophyllene, α-muurolene, thymol, thymol acetate, carvacrol, methyl esters of carvacrol and thymol^[10]. O. vulgare and O. onites number several chemotypes (chemorace) due to the wide area, as well as a variety of growing conditions. Chemotypes differ mainly by the accumulation of one dominant component in the EO – thymol, carvacrol, or sesquiterpenes^[9,11]. The total content of phenols in the EO, in terms of thymol, can reach 65-80%.

 

O. vulgare herb contains a range of biologically active compounds (BAC), in addition to the EO: flavonoids, such as luteolin, luteolin-7-glucoside, luteolin-7-glucuronide, cosmosiin (apigenin-7-glucoside), chrysin-7-glucuronide, 5-oxyflavone, hydroxycinnamic acids – rosemary, chlorogenic acids, tannins, and some other phenolic compounds. Phenolic compounds are dominant in the O. vulgare and O. onites herbs, this group of BAC provides the main expected pharmacological effect^[6]. O. vulgare leaves possess antioxidant action^[12].

 

An earlier set of pharmacological studies has shown that the Oregano herb has a strong stimulating effect on the secretory and motor function of the gastrointestinal tract and bronchi. Taken orally preparations from Oregano herb possess expectorant activity for respiratory diseases (acute respiratory viral infection, acute and chronic bronchitis), also they improve digestion and increase appetite, used in treating of intestinal atony, enterocolitis, which are accompanied by flatulence and constipation, applied externally in the complex therapy for atopic dermatitis (diathesis) and pyoderma. O. vulgare EO is used as a tonic, anti-inflammatory, antifungal, and antibacterial agent, enters into the composition of complex preparations registered in Russia^[13]. In homeopathic practice, Oregano is used as a drug curing hypertension and atherosclerosis, calming the nervous system and having a soporific effect^[14]. Also, herbal drugs based on Oregano medicinal plant raw materials or crude herbal drugs (CHD) can be used in adjuvant therapy of cancer^[15].

 

Based on the mentioned information, both types of oregano have a wide range of pharmacotherapeutic action, which is caused by the presence of different groups of BAC. In connection with this statement, there is a great interest in more in-depth study to retain the future prospects of CHD standardization. Phytochemical study of O. vulgare and O. onites CHD has shown that the dominant group of BAC were: condensed tannins, simple phenols (in the EO), and flavonoids^[16]. This work is devoted to the study of the hydrophilic and phenolic compounds, as well as the development of sample preparation procedure and selective procedures for reliable determination of the BAC studied groups using modern physicochemical methods of analysis.

 

MATERIALS AND METHODS:

The objects of study were air-dry samples of O. vulgare herbs harvested in June 2012 in Moscow at the All-Russian Research Institute of Medicinal and Aromatic Plants (VILAR) and O. onites harvested on the plantation, located on the outskirts of the city of Izmir (Turkey) in 2012 during the phase of mass flowering. The essential oil was obtained by hydro distillation^[17] according to the method no.1 of SP ed. XI (2a. without the addition of decalin)^[18]. Refractometric analysis of the obtained oil was carried out using an IRF-454 B2M refractometer according to the requirements of SP ed. XII.

 

Essential oil GC-FID (gas chromatography with flame ionization detector) analysis:

Currently, GC-MS analysis is widespread when analyzing the composition of the essential oils^[19-21] and other thermostable volatile BAC^[22,23]; nevertheless, it can be analyzed by GC-FID method for standardization purposes. The composition of the essential oil of O. vulgare and O. onites was studied with a Carlo Erba Instruments gas-liquid chromatograph (Italy) equipped with a flame ionization detector. Detector temperature – 220°C; injector temperature – 220°C. Сolumn temperature (programming): initial temperature – 100°C. Further temperature increases at a rate of 5°С per minute to 220°С for 20 min. A ZB-FFAP capillary column was used (polyethylene glycol-modified by nitrotereftalate, size 30m x 0.53 mm, film thickness 1 mm). The flow rate of carrier gas (nitrogen) was 2ml/min, the hydrogen flow rate was 30ml/min, the speed of air was 350 ml/min. The sample injection volume was 1ml of 2% essential oils in hexane.

 

Microscopic analysis:

It was performed on a Biomed-C2 microscope with 5×, 10× eyepieces, 4×, 10× and 40× objectives, photographies – were taken with a Canon Power Short SX 210 IS digital camera. Microscopic preparations^[24,25] of leaf surface were prepared by the procedure listed in SP ed. XI^[18].

 

Qualitative reactions^[26-29] were carried out in an alcohol extract (prepared for TLC procedure) of O. vulgare and O. onites CHD, flavonoids were detected. 0.1g of magnesium powder is added to 1ml of the solution, and 1ml of concentrated hydrochloric acid is heated in a water bath for 5 minutes until red color gradually appears.

 

Water extracts from the samples were prepared according to the General Pharmacopoeial Monograph (GPM) of SP ed. XI, “Infusions and decoctions”^[18]. Qualitative detection and quantification of the major groups of BAC were carried out using this extract.

 

A detailed study of the chemical composition of the O. vulgare and O. onites herbs was necessary for the development of procedures for the quantitative analysis of the BAC dominant groups. Initially, the presence of flavonoid compounds in plant aerial parts was necessary to detect and to confirm by qualitative reactions (Shinoda reaction, boric citric reaction, 0.5% alcoholic solution of iron chloride (III), ammonia solution). Further flavonoid complex was studied more detailed by thin-layer chromatography (TLC).

 

Currently, the well-known pharmacopoeia permanganometric method is used for the quantitative determination of total tannins in herbal drugs ^[24]. However, it is necessary to note the following fact: in an acid medium, potassium permanganate shows strong oxidizing properties. During the titration, all substances, that are capable to oxidize, enter into the reaction. Simple phenols, vitamins, dihydro flavonoids, and other compounds are such substances; since this reaction is not specific unambiguously, the result is often inflated^[30].

 

TLC analysis of flavonoids:

Material sample, weighing about 5g, was placed in a 100 ml flask, 80ml ​​of 60% ethanol was added to the flask, which was heated on a steam bath for 25 minutes; after this extract was cooled, filtered through the cotton-gauze filter, concentrated to 2ml and 4ml of 96% ethanol was added. Then 0.09ml of the extract and 0.03ml of rutoside and cynaroside state standard samples (SSS) 0.5% alcohol solution and deposited by microsyringe on the plate “Silufol UV-254”, size 20 x 20cm. Plate with the specimens, dried in air and then finally dried in the oven at 65-70°C, then the plate was placed in a chamber (previously saturated for 1 hour) with the solvent system: ethyl acetate - formic acid - water (88:6:6), was chromatographed by ascending method. After the solvent front has passed about 10cm, the plate was removed from the chamber, dried and viewed under 254 nm.

 

Determination of total flavonoids content in terms of cymaroside:

Spectrophotometric analysis is widely used in pharmacognosy practice for determining the flavonoids (sum of BAC)^[31-39] along with modern HPLC-DAD-MS^[39,40] and HPLC-DAD^[41]. The main advantages of this method are the high sensitivity and selectivity, the low analytical error of measurement, efficiency, and also simplicity and speed of execution. Absorption spectrophotometry of flavonoids is studied thoroughly, and identified patterns are widely used to determine the content of many compounds in herbal drugs and medicines based on it^[24]. UV spectrophotometric method has been proposed as the main method in the quantitative development of this substances group, absorbing in the UV region of the spectrum^[24]. Differential ΔE-spectrophotometric method was used for the analysis of complex phenolic compounds. This method has a number of positive characteristics: high sensitivity and accuracy, which allow reliable judgment on the qualitative and quantitative composition of the BAC studied group. The experiments were performed on a “Cary 50 Scan” Company “Varian” with subsequent computer processing of the results (the program “Cary WinUV Analysis Pack ver. 3.1” for “Windows”). An optimized procedure for quantifying the total flavonoids content in terms of cynaroside was developed during the study.

 

Accurately weighed sample (about 1g) of crushed material, passing through the sieve with openings of 2 mm (Russian National Standard 214-83), was placed in a 100ml round-bottom flask with ground glass neck, 60ml of 70% ethanol was added, the flask was connected to a reflux condenser, then heated in a boiling water bath for 45-50 minutes. After cooling, the flask contents were filtered through a filter paper into a 100ml volumetric flask. 40ml of 70% ethanol was added to the flask containing the material, the flask was connected to a reflux condenser and also heated for 15-20 min. After cooling, the solution was filtered through the same filter to the same flask, the volume of solution in the flask adjusted to the specific mark. Then 2.5ml of the resulting solution was transferred into a 25ml volumetric flask, 6 ml of an aluminum chloride solution (AlCl₃ – pure for analysis) was added, placed for 3-5 minutes in a boiling water bath, then quenched with, 2ml of acetate buffer solution (рН = 4.0) was added and 70% ethanol was adjusted to the specific mark, then waited another 30 minutes to complete the reaction complex. Further, absorbance measuring of the test solution was performed at a wavelength of 396±3nm in a cuvette with 10mm layer thickness. A reference solution consisted of 2.5ml of extract from CHD, 2ml of acetate buffer solution (pH 4.0), placed in a 25ml volumetric flask, and 70% ethanol was added to the specific mark.

 

At the same time, the absorbance of a standard solution, containing 1ml cynaroside SSS, treated the same way as test solution, was measured using a reference solution that consisted of 1ml cynaroside SSS solution and 2ml of buffer solution (pH 4.0), placed in a 25ml volumetric flask, 70% ethanol was added to the specific mark. The total flavonoids content (X) in terms of cynaroside and absolutely dry CHD in CHD, in%, was calculated using the formula:

 

where D – optical density of the test solution; D₀ – optical density of the cynaroside SSS standard solution; m₀ – cynaroside sample weight in grams; a – the CHD sample weight in grams; W – loss on drying,%.

 

Preparation of the cynaroside SSS solution:

Approximately 0.025g (accurately weighed) cynaroside sample, a preliminary dried at 130-135°C for 3 hours, dissolved in 50ml of 70% ethanol in a 100ml volumetric flask by heating in a boiling water bath and then the resulting solution cooled to room temperature, 70% ethanol was added to the necessary volume and mixed thoroughly.

 

Preparation of a 3% aluminum chloride solution:

Approximately 3g of aluminum chloride was placed in a 100 ml volumetric flask, then 30ml of 70% ethanol was added, thoroughly stirred to dissolve. After that 70% ethanol adjusted to a label volume of the solution, and then again mixed thoroughly.

 

Preparation of the samples water extracts was carried out according to the general pharmacopoeial monograph (GPM) of the State Pharmacopoeia XI (SP XI) “Infusions and decoctions”^[24]. Qualitative detection (gelatin precipitation, 5% potassium dichromate solution, basic lead acetate solution, the Stiasni reaction (40% formaldehyde solution, and concentrated HCl), 1% ferric alum solution). The amount of tannic substances was determined by a modified permanganometric titration method based on the SP XI^[18]. The indicator shows the content of total polyphenols extracted with water. 1% gelatin solution was added to water extract, the precipitate was filtered off, the titration was carried out in the filtrate to determine the content of tannic substances.

 

The amount of ascorbic acid was determined by titration with sodium 2,6 - dichlorophenolindophenol in water extract obtained by extracting 20 g of ground CHD in 300 ml of purified water^[18].

 

RESULTS AND DISCUSSION:

The main diagnostic features that allow to determine the species of plant are the essential oil glands and simple trichomes. O. vulgare has 8-celled ethereal-oil glands, located mainly on the underside of leaves, epidermal cells often form a rosette at the attachment points of the essential oil glands. Rosettes can consist of 25 radially elongated cells. O. onites can have 16-celled essential oil glands, and the size of cells may differ significantly.

 

Figure 2: A – preparation O. vulgare leaf surface (A) and O. onites leaf surface (B). SW. x 200 (1 – stomatal complex, 2 – ethereal-oil glands); B – O. vulgare leaf surface (A) and O. onites leaf surface SW. x 200 (1 – stomatal complex, 2 – simple hairs, 3 – prismatic crystals of calcium oxalate).

 

Both species have numerous,1-5-celled, sharply-tapered, slightly-, warty-, simple trichomes reaching a size up to 680 microns and capitate trichomes on the stem with the oval unicellular head (head diameter 21-25 microns). In O. onites there are easily defined inclusions of prismatic calcium oxalate crystals, which are absent in O. vulgare. It is the main diagnostic feature that enables one to distinguish two species even in the CHD powder (Figure 2).

 

The mass fraction of the essential oil (MFEO), obtained by distillation of the Oregano sample, amounted to 0.12% in terms of absolutely dry CHD that met the requirements of SP ed. XI (in whole material – at least 0.1% essential oil). MFEO of O. onites sample amounted to 2.6% in terms of absolutely dry CHD, which also met the pharmacopoeia requirements (for EP 7 – MFEO not less than 2.5% in terms of absolutely dry CHD). The refractive index (n_d²⁰) of O. vulgare essential oil was 1.451, the refractive index (n_d²⁰) of O. onites essential oil was 1.455. There are 14 peaks on the chromatogram for O. vulgare essential oil obtained in the experimental conditions, one of them is identified (Figure 3A). The dominant component of essential oil made from O. vulgare, growing in the Moscow region, is thymol. Thus, the O. vulgare sample can be attributed to the first chemorace with a high content of thymol according to the classification of E. Werker^[9]. There are 16 peaks on the chromatogram for O. onites essential oil. During the investigation one of them was identified as thymol (Figure 3B).

 

Figure 3: Chromatogram of O. vulgare (A; peak No14 – thymol) and O. onites (B; peak No16 – thymol) essential oil obtained during the experiment.

 

Flavonoids were detected in an alcohol extract of O. vulgare and O. onites CHD, the same intensity of the color indicated roughly similar amounts of the analyte BAC. In a series of test-tube reactions with appropriate reagents, it was established that tannins in O. vulgare and O. onites CHD belonged to one group – catechins (condensed tannins). Three adsorption zone with Rf = 0.35; 0.5; 0.7 in two extracts (more adsorption zone with Rf = 0.85 in O. onites extract sample), which are characteristic for flavonoids, have been observed on the plates during thin-layer chromatography of O. vulgare and O. onites herbs concentrated water-alcohol extract. And the adsorption zone with Rf = 0.7 belongs to the cynaroside SSS, indicating its presence in the studied samples. Corresponding spots in the samples across the rutoside SSS adsorption zone were not found, indicating the absence of that flavonoid glycoside in the studied materials. Scheme of chromatogram shown in Figure 4.

 

Figure 4: Chromatogram scheme (Plate “Silufol UV-254” 20 x 20 cm). The solvent system: ethyl acetate-formic acid-water (88:6:6): 1 – water-alcohol extract of O. vulgare herb; 2 – water-alcohol extract of O. onites herb, 3 – cynaroside SSS; 4 – rutoside SSS

It is known that a major problem in the development of procedures for the quantitative determination for CHD is the study of the basic hydrodynamic factors of the extraction process. Thus, the great attention of this study was paid to estimate the influence of the extractant nature and other factors (the ratio CHD: extractant, CHD grinding, the duration and frequency of extractions) on the yield of BAC. Information representing the selection of optimal conditions is shown in Table. 1.

 

Table 1: The dependence of the complete extraction of the total flavonoids content from the O. vulgare and O. onites by extraction conditions.

 

Figure 5: A – dependence of the optical density from the time of the O. onites flavonoids complexation reactions with aluminum chloride (1); B – absorption spectra obtained during the investigation of reaction products of cynaroside SSS solution(1), O. onites flavonoids (2), O. vulgare flavonoids (3) with 3% aluminum chloride solution.

 

Measurement of the resulting complex optical density was carried out at regular intervals to determine the optimal period of time to reaction progress and produce stable complex flavonoids and aluminum chloride. The results are shown in Figure 5A.

 

It was found that the BAC maximum yield from the O. vulgare and O. onites CHD was observed under the same conditions: the fineness of CHD – 2 mm, two-time sequential extraction with 70% ethanol, the ratio of CHD and extraction agent 1:100, heated in a boiling water bath for 60 minutes. The optimal time to obtain complex of flavonoids (O. vulgare and O. onites extracts) with aluminum chloride is 30 min. The selection of analytical wavelength was based on data obtained as a result of the UV spectrophotometric study of CHD water-alcohol extraction and cynaroside SSS. The maxima of the absorption spectra of the objects are the same and located at 396 ± 3 nm (Figure 5B).

 

Cynaroside SSS selection as a standard sample was due to its quantitative dominance in the CHD and also there was a “shoulder” (300-450 nm) in the spectrum UV region. Thus, the experimental parameters were established for the quantification procedure of the total flavonoids content in terms of cynaroside. Check of the procedure reproducibility was done by carrying out certain total flavonoids content in 7 replications. The metrological characteristics of a quantitative procedure for determining the total flavonoids content in terms of cynaroside are presented in Table 2.

 

Table 2: The metrological characteristics of the quantitative determination procedure of the total flavonoids content in terms of cynaroside in the O.vulgare and O. onites the Turkish herb water-alcoholic extract

 

Table 3: Chemical composition of the phenolic compounds in O. vulgare and O. onites herbs (CHD)

 

CONCLUSION:

The quality of products from the CHD can be guaranteed only in those cases where the composition of the CHD is clearly defined. Characteristics of the herbal drugs include a detailed description of botanical and phytochemical plant properties, analytical testing, and manufacturing process of medicine. Standard documents (pharmacopoeial monographs) are used to assess the final product. In these documents, a list of tests, references to analytical and biological methods, and acceptance criteria (margins, spacing, or description) are contained. There are several parameters, regulating the quality of the CHD and herbal drugs based on it, for the intended use. Identification and justification specifications, testing, and acceptance criteria should be based on information from pharmaceutical development (the study of the composition, developing of procedures of analysis, stability studies, validation experiments), the literary sources, and historical data^[42,43].

 

The basic anatomical and diagnostic features allowing to identify the producing Origanum plant were defined (the structure of the stomatal complex, simple hairs, ethereal-oil glands). Refractive index and chemical composition were defined for O. vulgare (n_d²⁰ = 1,451) and O. onites (n_d²⁰ = 1,455.) essential oil. Established dominant component – thymol. The content of essential oil in the O. vulgare herb was 0.12±0.03%, and in O. onites – 2.60 ± 0.02%. A procedure of quantifying the total flavonoids content in the O. vulgare and O. onites herbs by differential spectrophotometry (analytical wavelength of 396 ± 3 nm) was developed. The feasibility of using cynaroside SSS in the analysis was justified in view of the spectral flavonoid characteristics study of the O. vulgare and O. onites herbs extracts. Total flavonoids content in the O. vulgare herb samples reached 0.98 ± 0.02%, O. onites herb samples 1.25±0.01% (in terms of cynaroside). Amount of total polyphenols in the O. vulgare herb sample was 11.54±0.35%, in O. onites herb sample was 19.43±0.72%. The content of condensed polyphenols precipitated by gelatin in the O. vulgare was 4.94±0.15%, in the O. onites was 13.62±0.21%. The ascorbic acid was found in the water extract of CHD. The amount of ascorbic acid in O. vulgare – 0.35±0.05% and in O. onites 0.40±0.06%.

 

CONFLICTS OF INTEREST:

None.

 

AUTHOR’S CONTRIBUTIONS:

Bokov D.O., Nizamova L.A. contributed equally to this work.

 

ACKNOWLEDGMENTS:

This paper was financially supported by “Russian Academic Excellence Project 5-100” (Sechenov University). The publication has been prepared with the support of the “RUDN University Programm 5-100”.

 

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Received on 08.01.2019           Modified on 21.04.2019

Accepted on 19.06.2019         © RJPT All right reserved