DEVELOPMENT AND OPTIMIZATION OF ULTRASOUND EXTRACTION OF CHAMOMILE FLOWERS � CHAMOMILLAE RECUTITA

Статья посвящена разработке технологии ультразвукового экстрагирования ромашки аптечной цветков. Целью работы являлась разработка и оптимизация технологии ультразвукового экстрагирования суммы флавоноидов из цветков ромашки аптечной с определением содержания эфирного масла в полученных извлечениях. Материалы и методы. Предметом исследования являлось лекарственное растительное сырье (ЛРС), приобретенное через аптечную сеть – ромашки аптечной цветки. Способ экстрагирования – двухступенчатая мацерация с ультразвуковой обработкой ЛРС. Экстрагирование проводили в ультразвуковой ванне «Сапфир» ТТЦ (РМД) 9,5 л. Для определения оптимальных параметров технологического процесса экстрагирования суммы флавоноидов строили ортогональный факторный план типа 3. Результаты и обсуждение. Изучено влияние следующих факторов на процесс экстрагирования суммы флавоноидов из ромашки аптечной цветков: размер частиц ЛРС, количество ступеней экстракции, содержание спирта этилового в спирто-водной смеси, соотношение ЛРС – экстрагент, время экстрагирования, температура среды. Получены уравнения регрессии, описывающие процесс ультразвукового экстрагирования флавоноидов из ромашки аптечной цветков на первой и второй ступенях мацерации. Определены параметры технологического процесса ультразвукового экстрагирования суммы флавоноидов из ромашки аптечной цветков для первой ступени: время экстрагирования – 22 мин, содержание спирта этилового в спирто-водной смеси – 52% при соотношении ЛРС – экстрагент 1:7 и комнатной температуре; для второй: время экстрагирования – 27 мин, температура среды – 61°С при соотношении ЛРС – экстрагент – 1:5 (экстрагент – спирт этиловый 52%). Общий выход суммы флавоноидов составил 97%, эфирного масла – 81%. Заключение. Таким образом, разработана и оптимизирована технология ультразвукового экстрагирования суммы флавоноидов из ромашки аптечной цветков, позволяющая получить извлечения с высоким выходом эфирного масла. Ключевые слова: ромашка аптечная, цветки, флавоноиды, эфирное масло, экстрагирование, ультразвук

В связи с этим представляет интерес разработка технологии ультразвукового экстрагирования флаво-factorial plan of Type 3 2 was built.Results and discussion.The following factors' in uence on the extraction process of the sum of chamomile owers avonoids have been studied: the size of particles of (MRPM), the quantity of extraction steps, ethanol content in alcohol-water mixture, ratio (MRPM) /extragent, extraction time, media temperature.The regression equations describing the process of ultrasound extraction of chamomile owers avonoids at the rst and second steps of maceration have been obtained.The following parameters of the technological process of chamomile owers sum of avonoids ultrasound extraction have been worked out: for the rst step the extraction time was 22 min., the content of ethanol in alcoholwater mixture was 52% at the ratio MRPM/extragent 1:7 and the room temperature; for the second step: the extraction time was 27 min., the media temperature was 61°C at the ratio MRPM/extragent 1:5 (52% ethanol was used as an extragent).The general yield of the avonoids sum was 97% and that of the essential oil was 81%.Thus, the ultrasound extraction technology of the sum of chamomile owers avonoids has been developed and optimized, allowing to obtain extracts with a high yield of the essential oil.Keywords: chamomile owers, avonoids, essential oil, extraction, ultrasound DOI:10.19163/2307-9266-2018-6-2-151-166 Количественное определение суммы флавоноидов в ЛРС и полученных извлечениях проводили в соответствии с требованиями фармакопейной статьи ФС.2.5.0037.15«Ромашки аптечной цветки» ГФ XIII: в пересчете на рутин с использованием дифференциальной спектрофотомерии по реакции комплексообразования с алюминия хлоридом [5].

Фармацевтическая технология и биотехнология Pharmaceutical Technology and Biotechnology
Anti-in ammatory and spasmolytic actions of chamomile owers are is associated with avonoids and essential oils presence [2][3][4][11][12][13].According to the literature data, avonoids of different classes are able to inhibit the induction of separate groups of tissue mediators of in ammation.So, for example, three-, tetra-and pentaalkyl avons (kaempferol, quercetin) are inhibitors of lipoxygenase [14].One of the chamomile owers essential oil components -hamazulen -performs an anti-in ammatory effect by inhibition of leykotriyen B4 formation [2,4,[11][12][13].According to the literature data, the greatest contribution to miotrophy spasmolytic action of chamomile owers are made by apigenin, α-bisabolol and also by cis-spiroether [4,11].Thus, two speci ed groups of BAS provide anti-in ammatory and spasmolytic actions of this MRPM to a greater extent.
Studying of chamomile owers BAS extraction is a subject of many researches.Extraction of the essential oil, avonoids, coumarines, the sum of extractive substances, lipophilic fraction, etc. with various extragents, including nonaqueous (propylene glycol, rape oil, r oil, lique ed gases) has been described in different ways [15][16][17][18][19]. Hereby joint extraction of two BAS groups -avonoids and the essential oil -with the use of modern ways of intensi cation of the extraction process is still of scienti c and practical interest.
One of the perspective methods of BAS extraction is ultrasound processing of MRPM.Ultrasound processing of the media is followed by effects of its blending, heating and cavitation -forming, pulsation and collapsing of cavitational bubbles ensemble.Ultrasound accelerates processes of dissolution of rm particles, intensi es a mass transfer into liquid volume, exerts insigni cant impact on surface thickness, accelerates a mass transfer in porous bodies with larger sizes of pores and increases permeability of vegetable membranes [20].
Today there are suf cient data about ultrasound extraction of compounds of different classes without any change of structure of these substances -avonoids, terpenic compounds, fat oil, phenolic compounds, etc. [21].There was a research on studying the ultrasound extraction of chamomile owers essential oil in order to develop its quantitative de nition method, where r oil was an extragent [16].
In this regard the development of ultrasound extraction technology of avonoids and essential oils from chamomile owers is of great interest.
It should be noted that avonoids in comparison with terpenic substances are more stable compounds, therefore they are more preferable as marker connections during working out and optimization of ultrasound extraction process.
THE AIM OF THE RESEARCH is working out and optimization of ultrasound extraction technology of the sum of avonoids from chamomile owers with further determination of the essential oil content in the extraction obtained.
MATERIALS AND METHODS.In the research, chamomile owers bought in pharmacy are used ("Krasnogorskleksredstva, JSC", series number 130616).
Extraction was performed in the ultrasound bath of "Sapphire" of Technical and Commercial Center (TCC) (DME) of 9.5 l, operating frequency -35 kHz, at the supersonic generator power of 180 W. The extraction method was maceration with ultrasound processing of MRPM.
The result of the research of chamomile owers ultrasound extraction are the parameters of the technological process providing the maximum yield of avonoids.For the determination of the speci ed parameters, series of experiments with variation of certain factors were made.
The orthogonal factor plan of the second order being a special case of the composition plan of the second order of Type 3 2 with value of a "star" shoulder α=1 was built.The plan included a variation of two factors at three levels [22,23].
In each point of the plan two experiences were performed.The yield of avonoids percentage in MRPM was selected as a process response function.
The response function characterizing extraction process was described by the following equation (1) [22]: (1) determination of the sum of avonoids in MRPM and the extractions obtained was performed according to the requirements of the pharmacopoeial entry "Chamomile owers (Chamomillae recutita ores)" : in terms of rutin with the use of differential spectrophotometry according to reaction of complex formation with aluminum chloride [5].
The statistical processing of the results was performed according to the general pharmacopoeia entry 1.1.0013.15"Statistical processing of results of a chemical experiment" with the use of Microsoft Of ce Excel software [25].
In preliminary researches the in uence of the following evaluated: particle size of MRPM, steps quantity of the extraction, the content of ethanol in alcohol-water mixture, extraction time, media temperature.In the obtained extractions the content of the sum of avonoids In the technological process the following parameters of were used: 45% ethanol was used as extragent (here and elsewhere the ethanol ratio in alcohol-water mixtures is speci ed in volume percent), ratio MRPM/extragent was 1:10 [2-4], the time of ultrasound processing was 15 min.(single maceration) at room temperature [16,21,27].The speci ed conditions of preliminary researches coordinate with traditionally applied ones in pharmacy and medicine and scienti c literature data.
At the rst stage, the in uence of the particle size of MRPM on the yield of avonoids at ultrasound extraction was investigated.The results are presented in Table 1.It has been established that the highest yield of avonoids was observed in the process of MRPM fraction with the particle size of 1.0-2.0mm (Table 1).It is possible to explain this fact by the structure heterogenety of the MRPM different fractions.The fraction with ticle size of 1.0-2.0mm was a mixture of parts of thalamus, ligulate and tubular orets of an anthodium while in smaller fractions tubular owers prevailed.It should be noted that fractions with a larger particle size contain larger anthodium parts which complicated receiving a homogeneous sample.
For the choice of extraction quantity steps the yield of avonoids was evaluated when performing one-, twoand triple maceration with ultrasound processing.
It was established that in various conditions at the third step of maceration the yield of avonoids was in the range from 4% to 13%, therefore its performance could be considered inexpedient.As at single maceration the full extraction of the sum of avonoids [26] was not reached, a two-level maceration with ultrasound processing of MRPM was chosen for chamomile owers extraction.
Earlier the in uence of heating on the yield of avonoids at ultrasound processing at two steps of maceration had been studied.It had been established that raising temperature increases the content of the sum of avonoids in extraction [28].
As far as the joint extraction of both, avonoids and the essential oil, was one of technological tasks of this research, ultrasound extraction of chamomile owers was performed without additional heating [27].At the same time, the media temperature was determined: it was established that depending on its volume in the chosen conditions the values of temperature were in the range of 20-25ºС.
The study of the in uence of the factor "ratio MRPM/ extragent" onto the process of avonoids ultrasound extraction was performed in the range from 1:4 to 1:30.It was established that the yield of avonoids raises with the increase in this ratio.
Hereby the choice of values of this parameter is limited by the requirements to the nal volume of obtained extraction; that is meant for dosage forms and should not be diluted.In this regard the expedient choice was the smallest ratio of MRPM/extragent.
It was established that by reducing the ratio of MRPM -extractant, starting with the value 1:6 at the rst step of ultrasound maceration and with 1:4 at the second, the yield of avonoids decreases.Therefore for ultrasound extraction of chamomile owers the following ratios of MRPM /extragent were chosen: 1:7 -at the rst step and 1:5 at the second.
The decrease in the yield of BAS, apparently, is connected with necessity of suf cient content of a free liquid phase presence for effective in uence of ultrasound in the media.This fact is also con rmed by the fact that the studied MRPM has a low value of bulk density (0.11±0.03 g/cm 3 ) and rather a high absorption coef cient (from 2.0 to 3.4 in alcohol-water mixtures).
When studying the in uence of time of ultrasound processing (5-60 min.)on the yield of avonoids at the rst step it was established that in the range from 5 up to 25 min.the yield increases from 8-12% to 35-45%.Further increase in processing time did not lead to signi cant increase in the yield of avonoids.In this regard the interval of time of ultrasound processing from 5 to 25 min.was chosen for the purpose of optimization.
When studying in uence of the content of ethanol in the alcohol-water mixture, it was established what the yield of avonoids changes mostly in the range from 20% to 80%.When using ethanol at the concentration higher than 80% it was established that the yield of avonoids became less than 20%.At the same time the use of ethanol at a lower concentration (lower than 20%) was inexpedient because of the structure of the target BAS groups.
Thus, for the rst step of ultrasound extraction the following conditions were chosen: the extraction timefrom 5 to 25 minutes, the content of ethanol in alcoholwater mixture from 20% to 80%, the ratio MRPM/ extragent -1:7, at the room temperature.The levels and intervals of the factors variation are presented in Table 2.When using orthogonal plans of the second order taking into account linear transformation of square coef cients (2): (2) the matrix of experiment planning for two variation factors with one experience in the center of the plan takes the following form (Table 3, [22]).Coef cients calculation of the regression equation was performed by the following formulas (3) and ( 4): (3) (4) Dispersions of the coef cients were calculated by the formula (5): (5) By virtue of the obtained results, the coef values of the regression equation (Tab.4) were calculated.By virtue of the analysis of data in Table 5 it was established that the model obtained re ects the process of ultrasound extraction of avonoids of chamomile owers at the rst step of maceration in the set intervals of factors variation.The coef cient re ecting factors interaction -B 12 -is insigni cant.Thus, the required regression equation for the rst step of ultrasound maceration looks as follows ( 6): (6) It was established that Y value is at its maximum of 54.67% at X 1 =1.70 and X 2 =0.06, with ultrasound processing time of 22 min.and 52% ethanol in alcoholwater mixture.The yield of avonoids in the experimental conditions was 55.02±0.91%.
Thus, the optimal parameters for the rst step of the maceration technological process are: the extraction time is 22 min., 52% ethanol in alcohol-water mixture with ratio MRPM/extragent1:7 and at the room temperature.
The yield of the essential oil in the extractions obtained is about 69%.
It was de ned that at the second step of ultrasound maceration the yield of avonoids at the room temperature is low (less than 20%).Correspondingly it is expedient to perform the second step of extraction upon the application of heat.By virtue of the preliminary research results analysis and also on the experimental data obtained at the rst step, at the second step the following factors were selected for variation of avonoids ultrasound extraction: the media temperature (from 30 to 60°C) and the extraction time (from 5 to 25 min.).As an extragent 52% ethanol was used, with ratio MRPM/extragent1:5.The levels and intervals of factors variation are displayed in Table 6.For the extraction, MRPM obtained after the step of ultrasound maceration in experimentally selected conditions was used The experiment planning matrix and the implemented experiments for the second step are displayed in Table 7.
Фармацевтическая технология и биотехнология Pharmaceutical Technology and Biotechnology The calculated values of the regression equation coef cients are displayed in Table 8.The results of the statistical analysis of the experimental data are presented in Table 9.By virtue of the data analysis presented in Table 9 it has been established that the received equation reflects the technological process of the sum of flavonoids ultrasound extraction from a chamomile flowers at the second step of maceration in the set intervals of factors variation.All equation coefficients are significant.
Thus, the regression equation describing the second step of ultrasound maceration process of extracting sum of avonoids from chamomile owers looks as follows ( 7): (7) It was established that Y value is at its maximum of 42.67% at X 1 =1,70 and X 2 =0.75, with ultrasound processing time of 27 min.and the temperature of 61°C.
The yield of avonoids in the experimental conditions was 42.10±0.53%.
Thus, optimal parameters for the second step of maceration technological process were: the extraction time was 27 min., the temperature was 61°C at the chosen ratio MRPM/extragent 1:5 (52% ethanol was used as an extragent).The yield of the essential oil in the extractions obtained at second step is 12%.
CONCLUSION.The parameters for the technological process of the sum of avonoids and essential oil ultrasound extraction from chamomile owers have been determined.At the rst step the extraction time was 22 min., the content of ethanol in alcoholwater mixture was 52% at the ratio MRPM/extragent 1:7 and the room temperature.At the second step the extraction time was 27 min., the media temperature was 61°C at the ratio MRPM/extragent 1:5 (52% ethanol was used as an extragent).
The general yield of the sum of avonoids was 97%, the yield of the essential oil was 81%.
Thus, the extraction technology of ultrasound extraction of the sum of avonoids from chamomile owers allowing to obtain high essential oil yield has been worked out and optimized.

Table 1 -
The yield of avonoids depending on chamomile owers fraction

Table 2 -
Levels and intervals of the factors variation for the rst step of ultrasound maceration of chamomile owers

Table 3 -
Matrix of experiment planning and the implemented experiences at the rst step of ultrasound maceration of chamomile owers

Table 4 -
Values of coef cients of the regression equation for the rst step of chamomile owers ultrasound extractionThe results of the statistical analysis of the experimental data are displayed in Table5.

Table 5 -
Statistical analysis of the experimental data for the rst step of chamomile owers ultrasound extraction

Table 6 -
Levels and intervals of factors variation for the second step of chamomile owers ultrasound maceration

Table 7 -
Experiment planning matrix and the implemented experiments of the second step of chamomile owers ultrasound maceration

Table 8 -
Values of regression equation coef cients for the second stage of chamomile owers ultrasound extraction

Table 9 -
Statistical analysis of the second step of chamomile owers ultrasound extraction experimental data