Study on standardization of herb melissa
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L. V. Vronska, A. Ye. Demyd STUDY ON STANDARDIZATION OF HERB MELISSA Ternopil State Medical University by I. Ya. Horbachevsky FORMULATION OF THE PROBLEM The problem of quality drugs from medicinal plants is partly solved now by the introduction of monographs on herbal drugs in the State Pharmacopoeia of Ukraine [1-3]. National requirements for medicinal plants harmonize with the European Pharmacopoeia (EP) [4]. However, a number of difficulties related. For example, how different types of raw materials: such as in Europe - Melissa leaves, in Ukraine - herb lemon balm; the differences phytochemical composition of national samples of raw materials and of the need to introduce national monographs on herbal drug types are absent in EP. ANALYSIS OF RECENT RESEARCH AND PUBLICATIONS The leaves of Melissa officinalis are officinal herbal drug in many countries and the corresponding monograph included in EP [5-7]. Approaches to standardization of the raw materials have changed: in 5th edition of EP amount of total hydroxycinnamic acids content standardized calculated on rosemary acid (not less than 4%), while starting from the 6th edition - content only acid rosemary quantitatively standardized (at least 1 %). In Ukraine, the leaves of lemon balm is not registered as a drug. Melissa officinalis herb was registered (registration certificate number R.09.03/07437, duration - from 30.09.2003 to 30.09.2008), at present it is not included in any of the supplements SPU. Research on compliance of Melissa officinalis leaves of national origin and the possibility of harmonizing the requirements of the EP being in the direction of study of the essential oil composition and rosemary acid content [8-11]. RELEASE NO EARLIER SOLVED PART OF THE PROBLEM The existing regulatory documentation on the Melissa officinalis herb provides by TLC-identification of coffee acid derivatives (indicating Rf individual compounds). Earlier, we proposed to identify the Melissa officinalis herb applied detection of luteolin-7-glucoside, chlorogenic, rosmarinic and a coffeic acids by TLC [12] with the use of standard samples and the processing of plates by selective reagents (diphenylboric acid aminoethyl ester and macrogol 400). This allows to provide the reliability of the identification of raw materials in the use of different types of plates, when using plates "Sorbfyl" and Silica gel 60 F254 (by "Mersk", Germany) Rf values will be different. Quantitative indicators of quality Melissa officinalis herb, under the existing analytical regulatory documentation, are the amount of acid derivatives calculated on a coffee acid. their content is determined by direct spectrophotometry and normalized - not less than 4%. The using of direct measurement of alcohol solution absorbance provides a measurement of the total absorption of various substances present in alcoholic extracts of herb. This makes the obtained results are not entirely reliable, because even if the explicit maximum absorption in the electronic spectrum, which is not always observed in extracts of herbal drugs, dealing with overlapping absorption spectra of substances that contribute to the maximum of the other classes of biologically active substances (BAS). Validation and application of different methods for systematic errors detecting provide an opportunity to improve the reliability of the result, but the complexity of herbal drugs as an object of study - namely, the variability of qualitative and quantitative ratio of BAS in raw materials, necessitates frequent validation and application of the additives method to calculate the content. Last reception is also not always acceptable, due to the availability and cost of multiple reference standards of "primary standard" qualification.
The purpose of this work is the scientific study and development of methods of quantification of hydroxycinnamic acids amount in Melissa officinalis herb. PRESENTATION ON THE MAIN MATERIAL The Melissa officinalis herb production by "Liktravy" used in this work. Spectrophotometric measurement and electronic absorption spectra recording were performed on Cary 50 spectrophotometer. To develop of quantitative determination method of hydroxycinnamic acids in Melissa officinalis herb spectrophotometric method is selected as the pharmacopoeia available, selective and weighty enough - an express method of analysis; the calculation of quantitative content of proposed spending on acid rosemary. The possibility of using direct spectrophotometric determination of hydroxycinnamic acids in grass balm examined. Absorption curves and a maximum position on them for ethanol extracts of herbs and standard samples of hydroxycinnamic acids are similar (Figs. 1 and 2). This allows the use of optical density measurements to calculate their contents in the studied raw materials. 
Fig. 1. Electronic absorption spectra of the test solution in the hydroxycinnamic acids quantification conditions (in order of decreasing absorbance at 325 nm wavelength) for samples 2, 1, 4, 3, 5, 6. 
Fig. 2. Electronic absorption spectra of standard samples solutions (in order of increasing absorbance at 325 nm wavelength): chlorogenic acid, acid, rosmarinic and caffeic acid. Method of quantitative determination of the amount hydroxycinnamic acids in Melissa officinalis herb by direct measurement of the optical density of the alcoholic extracts solution of herbal substances. Stock solution. Place 0.2000 g of the powdered drug in a conical flask 250 ml, add 100 of ethanol (50 % V/V) R. Heat the mixture under a reflux condenser on a water-bath for 30 minutes. Cool and filter, extract was decanted into a volumetric flask of 200 ml. Extraction is repeated twice, each using 40 ml of ethanol (50 % V/V) and heat the mixture under a reflux condenser on a water-bath for 10 minutes. These extracts filtered into the same volumetric flask. Rinse the filter and the residue with a few millilitres of ethanol (50 % V/V) R, dilute to 200 ml with ethanol (50 % V/V) R. Test solution. Dilute 5.0 ml of stock solution to 25.0 ml with ethanol (50 % V/V) R. Reference solution. 50.0 mg of rosmarinic acid R dissolve in 100.0 ml of ethanol (50 % V/V) R (solution A). Dilute 5.0 ml of solution A to 50.0 ml with ethanol (50 % V/V) R (solution B). Dilute 5.0 ml of solution B to 25.0 ml with ethanol (50 % V/V) R.
Measure the absorbance of the test solution, reference solution at 325 nm by comparison with the compensation solution. Calculate the content of hydroxycinnamic acids, calculated on rosmarinic acid, using the following expression:
Х – content of hydroxycinnamic acids, %; А – absorbance of test solution; А0 – absorbance of reference solution; m – mass of drug sample, in grams; m0 – mass of rosmarinic acid, in grams; W – loss of drying, %. The results of hydroxycinnamic acids amount determining in the studied series of herb give in Table 1. In the 5th edition EP [7] for spectrophotometric quantification of the hydroxycinnamic acids amount was offered photometric reaction. It is based on the hydroxycinnamic acids properties react with nitrite molybdenum reagent and the ability to form a red color compound with maximum absorption at 505 nm wavelength. The given technique we tried to analyze the Melissa officinalis herb. Extracts of the Melissa officinalis herb obtained with alcohol (50% V/V), because, as shown by previous studies, it allows you to remove the maximum of determined biologically active substances. Hydroxycinnamic acids with nitrite molybdenum reagent form a red color solution with maximum absorption at a wavelength of 505 ± 2 nm. The corresponding absorption spectrum is shown in Fig. 3. 
Fig. 3. The electronic absorption spectrum of solution of the herb balm extract with nitrite molybdenum reagent. This reaction can be used to quantify the amount of hydroxycinnamic acids in herb balm. Additionally, it was found that getting alcohol extraction is necessary to use a sample of material to 0.4 g. This will work in optical densities range of with minimal measurement error. Quantitative calculation proposed spending using specific absorbance. Specific absorbance for rosmarinic acid in the conditions of the reaction with nitrite molybdenum reagent at a wavelength of 505 nm is 400 [7].
Stock solution. Place 0.4000 g of the powdered drug in a conical flask 250 ml, add 100 of ethanol (50 % V/V) R. Heat the mixture under a reflux condenser on a water-bath for 30 minutes. Cool and filter, extract was decanted into a volumetric flask of 200 ml. Extraction is repeated twice, each using 40 ml of ethanol (50 % V/V) and heat the mixture under a reflux condenser on a water-bath for 10 minutes. These extracts filtered into the same volumetric flask. Rinse the filter and the residue with a few millilitres of ethanol (50 % V/V) R, dilute to 200 ml with ethanol (50 % V/V) R. Test solution. To 1.0 ml of the stock solution in a volumetric flask of 10.0 ml add 2.0 ml of 0.5 M hydrochloric acid, 2.0 ml of a solution prepared by dissolving 10 g of sodium nitrite R and 10 g of sodium molybdate R in 100 ml of water R and then add 2.0 ml of dilute sodium hydroxide solution R and dilute to 10.0 ml with water R and mix. Compensation solution. In another volumetric flask of 10.0 ml place 1.0 ml of the stock solution, 2.0 ml of 0.5 M hydrochloric acid, 2.0 ml of dilute sodium hydroxide solution R and dilute to 10.0 ml with water R. Immediately measure the absorbance of the test solution at 505 nm by comparison with the compensation solution. Calculate the total hydroxycinnamic acids content, expressed as rosmarinic acid, from the expression:
Х – total hydroxycinnamic acids content, %; А – absorbance of test solution; 400 – specific absorbance for rosmaranic acid at 505 nm; m – mass of the substance to be examined, in grams; W – loss of drying, %. Herb balm of “Liktravy” six series was analyzed according to this method. The results of the quantitative determination of the total hydroxycinnamic compounds by this method are given in Table 1. Table 1
RESULTS OF DETERMINATION OF HYDROXYCINNAMIC ACIDS IN MELISSA OFFICINALIS HERB (n = 5, P = 0,95)
Content amount of hydroxycinnamic acids is different: for the first 4 series obtained similar values, and for series 5 and 6, there is a significant difference from the previous four, and from each other. It should be noted that the content on the second method is much lower. The general correlation results between the two methods: 1-4 series characterized by higher content of hydroxycinnamic acids, and 5 and 6 differ among themselves and from 1-4. Quantitative differences contents determined by the two techniques can be linked to the difference of quantitative calculation methods - the standard method is in the first technique and the method of specific absorbance – in the second. On the other side, the first technique is based on direct spectrophotometric of test solution containing alcoholic extract of herb, and the second - on the specific reaction of hydroxycinnamic acids with nitrite molybdenum reagent. In the first case, by measuring the absorption of the solution directly, you can get overestimated absorbance values due to the absorption of other substances that are also optically active in the range of 325 nm.
Quantitative determination of the hydroxycinnamic acids amount suggested to standardization of Melissa officinalis herb transfering their contents to rosmarinic acid. Spectrophotometric method using nitrate molybdenum reagent developed for this purpose. The application of the photometric reaction allowed increasing the selectivity of determination compared to the existing spectrophotometric method, based on the hydroxycinnamic acid absorption measurements in multicomponent solution which is non-selective. Specific absorbance method have been proposed for the quantitative calculation that avoids the need to use inaccessible standard samples, and thus provide an opportunity for the introduction of real quality Melissa officinalis herb at various levels laboratories. The application of the proposed spectrophotometric method, in our opinion, is more appropriate than the definition of rosmarinic acid content, as suggested new editions for EP balm leaves, for several reasons. At first, manufacturers of herbal drugs in Ukraine are not able enforce the Guidelines for Good Agricultural Practice (GAP) of Medicinal and Aromatic Plants, through its individual component structure is not strictly reproducible, whereas the total content of compounds class is significantly reproducible. Secondly, the use of HPLC is often complicated by the absence of appropriate equipment in laboratories for drugs quality control, whereas spectrophotometers are widespread analytical equipment, which are equipped with all manufacturers of drugs laboratories and quality control laboratory at various levels. Establishing of criteria for quality in "quantification of hydroxycinnamic acids" terms is possible after examining a larger number of samples (with varying geography of cultivation and harvest) and studying the dynamics of change in this indicator during storage. REFERENCES
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