CC BY
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Zdoryk Oleksandr, Candidate of pharmaceutical science, Associate professor, Pharmaceutical chemistry department, National University of Pharmacy, Pushkinska str., 53, Kharkiv, Ukraine, 61002 E-mail: oleksandr_zdoryk@ukr.net
Georgiyants Viktoria, Doctor of pharmaceutical sciences, Professor, head of the department, Pharmaceutical chemistry department, National University of Pharmacy, Pushkinska str., 53, Kharkiv, Ukraine, 61002 E-mail: vgeorg@ukr.net
УДК 615.322
DOI: 10.15587/2313-8416.2016.61495
DEVELOPMENT OF METHODS FOR DETERMINATION OF PHENOLIC ACIDS AND FLAVONOIDS IN CAPSULES CONTAINING CORYLUS AVELLANA L. DRY EXTRACT
© N. Blyznyuk, Yu. Prokopenko, V. Georgiyants
The questions of standardization and quality control of both herbs and herbal remedies remain relevant, because it is well-known that product quality standards are essential, whether consumer using herbs or drugs. The necessity of the standardization methods development for the initial herbal material and capsule dosage form for the further quality control under manufacturing conditions remains relevant.
Aim. The aim of our research was to develop simple, specific, accurate and reproducible methods for identification offlavonoids and phenolic acids in capsule dosage form containing Corylus avellana L. dry extract. Methods. The samples of gelatine capsules containing Corylus avellana L. dry extract for oral administration were analyzed. The analysis was carried out using Camag HPTLC system.
The absorption spectroscopy determination of the sum of flavonoids was carried out using THERMO Scientific Evolution 60S Spectroscope in wavelength range of300-600 nm.
Results. As a result of HPTLC research rutine and quercitrine have been identified in capsule dosage form containing Corylus avellana L. dry extract. Among phenolic acids, neochlorogenic and chlorogenic acids have been identified.
Under the given conditions, the spectrum of the test solution had a maximum absorption at wavelength 406 nm. The analysis of flavonoids total content in gelatine capsules containing Corylus avellana L. dry extract calculated as rutine has shown the content of 1,7 %.
Conclusion. Effective HPTLC and absorption spectroscopy methods for determination offlavonoids and phenolic acids in capsule dosage form containing Corylus avellana L. dry extract have been developed. It has been found that described methods are promising enough for standardization of capsules with Corylus avellana L. dry extract and may be suggested for the quality control of the dosage form under manufacturing conditions Keywords: capsules, Corylus avellana L., extract, HPTLC, absorption spectroscopy, phenolic acids, flavonoids
Питання стандартизацИ та контролю якостi якрослин, так i лжарських 3aco6ie рослинного походжен-ня набувае актуальностi, враховуючи той факт, що стандарти якостi продукцИ е вкрай важливими, не-залежно вiд того, чи вживае споживач лжарсьт рослини або лжарсьт засоби. Необхiднiсть розробки методик стандартизацИ для вихiдноi рослинно'1' сировини та капсульовано лiкарськоi форми для пода-льшого контролю якостi в умовах виробництва залишаеться актуальною.
Мета. Метою нашого до^дження була розробка просто'1] сnецифiчноi, точно'1' та вiдmворюваноi методики iдентифiкацii флавоноiдiв та фенольних кислот у капсульовант лiкарськiй формi з сухим екст-рактом Corylus avellana L.
Методи. Для до^дження використовували зразки желатинових капсул з сухим екстрактом Corylus avellana L. для орального застосування. Аналiз проводили з використанням системи Camag для ВЕТСХ. Визначення вмiсmу суми флавоноiдiв методом абсорбцiйноi спектроскопа здшснювали за допомогою спектрометра THERMO Scientific Evolution 60S у дiапазонi хвиль 300-600 нм.
Результати. В резульmаmi ВЕТСХ аналiзу у капсульовант лжарськт формi з сухим екстрактом Corylus avellana L. були iденmифiкованi рутин та кверцитрин. Серед фенольних кислот були iденmифiкованi не-охлорогенова та хлорогенова кислоти.
В умовах проведення спектрофотометричного до^дження спектр випробовуваного розчину мав максимум поглинання за довжини хвилi 406 нм. Вмкт суми флавоноiдiв у желатинових капсулах з сухим екстрактом Corylus avellana L. у перерахунку на рутин становив 1,7 %.
Висновки. З метою визначення флавоно'Шв та фенольних кислот у капсульовант лжарськт формi з сухим екстрактом Corylus avellana L. були розроблеш ефективш методики ВЕТСХ та абсорбцтно'1 спектрофотометра.
Було визначено, що описан методики е достатньо перспективними для стандартизацИ капсул з сухим екстрактом Corylus avellana L. та можуть бути запропонованi для проведення контролю якостi лкар-сько! форми в умовах виробництва
Ключовi слова: капсули, Corylus avellana L., екстракт, ВЕТСХ, абсорбцшна спектрофотометрiя, фено-льнi кислоти, флавоно'1'ди
1. Introduction
The use of herbs with medical purpose is the oldest form of healthcare known to humanity and it has been used in all cultures throughout human's history [1]. Herbal remedies occupy a leading position in global pharmaceutical markets.
That's why one of the priority areas of modern pharmaceutical science and practice is the development of new herbal remedies, due to their low toxicity, the breadth of therapeutic action, and low risk of side effects during prolonged use. Generally, all medicines, irrespective of their origin, should meet the basic requirements of being at least safe and effective.
2. Formulation of the problem in a general way, the relevance of the theme and its connection with important scientific and practical issues
Nevertheless, questions of standardization and quality control of both herbs and herbal remedies remain relevant, because it is well-known that product quality standards are essential, whether consumer using herbs or drugs [2]. Therefore, the given question of the search, development and standardization is still pressing and requires solutions.
3. Analysis of recent studies and publications in which a solution of the problem and which draws on the author
Standardization of any herbal remedy involves regulating the given remedy to a defined content of a constituent or a group of substances with established and proved therapeutic activity [1, 2].
Earlier, the anticonvulsant properties of Corylus avellana L. dry extract have been determined; it was the reason to develop the original dietary supplement for nerve health. The screening of the role of biologically active substances or fractions from different Ukrainian flora herbs in realization of the anticonvulsant activity has shown that one of the main groups of compounds responsible for mechanisms of anti-seizure activity were flavonoids [3, 4]. Besides, wide spectrum of pharmacological activity of flavonoids gives certain reasons to analyze their content in dosage forms without reference to application of the given remedy [5, 6].
Analytical methods used in drug analysis are diversified and are being constantly improved to find better solutions to satisfy manufacturers and institutions that test drug quality [7]. According to the literature data, there are several analytical techniques for the identification and quantification of maker compounds in herbal dosage forms [8, 9].
As a rule, pharmaceutical manufacturers recommend various analytical techniques, but spectroscopy and
chromatography methods still playing a significant role in pharmaceutical analysis. Due to the complex compound of herbal remedies, given methods are commonly used for the quality control and standardization of both herbs and herbal products [7, 8].
HPTLC (High performance thin layer chromatography) method has been widely used for the standardization and qualitative analysis of herbs and herbal dosage forms due to its simplicity, high speed, and relatively low cost.
The use of HPTLC plates in combination with automated sample applicators and development chambers, high resolution cameras, and computing software allows more control over experimental conditions and greater data analysis capabilities [10]. Moreover, HPTLC method can run many samples simultaneously, requires small volumes of solvents, and gives rather instant visual results [11]. For example, individual bands of different active compounds may act as additional characteristics for their better identification: compounds with native fluorescence are observed as bright zones on a dark background under UV light. While natural substances with no fluorescence (no chromophore groups) or colour can be visualized after post-chromatographic derivatiza-tion [2, 12, 13].
Absorption spectroscopy is another effective method for identification and assay of flavonoids [14]. As a rule, AlCl3 reagent is used. Adding AlCl3 reagent allows eliminating the influence of another biologically active substances having polyphenol structure. Reference compounds for recalculations are being chosen generally after TLC or HPTLC identification tests. Subsequently, the content of the sum of flavonoids in the given herb or remedy will be calculated and regulated as the chosen reference standard [5, 14].
Thus, both HPTLC and absorption spectroscopy methods remain the most flexible and reliable, methods ideally suited for the analysis of herbs and herbal remedies. When used with standardized procedures, described methods guarantee reproducible results, a vital element in the routine analysis of plant extracts and pharmaceutical products during manufacturing process.
4. Allocation of unsolved parts of the general problem, which is dedicated to the article
The necessity of the standardization methods development for the initial herbal material and capsule dosage form for the further quality control under manufacturing conditions remains relevant.
5. Formulation of goals (tasks) of Article
Considering the facts given above, the aim of our research was to develop simple, specific, accurate and
reproducible methods for identification of flavonoids and phenolic acids in capsule dosage form containing Corylus avellana L. dry extract.
6. Statement of the basic material of the study (methods and objects) with the justification of the results
HPTLC identification of phenolic acids and flavonoids. The samples of gelatine capsules containing Corylus avellana L. dry extract for oral administration were analyzed.
The capsules filling was removed and placed into a volumetric flask, and the ultrasound extraction was allowed to run using methanol as a solvent in a ratio 1 to 10 at room temperature for 20 minutes. After centrifuga-tion, the supernatant was collected and directly applied onto HPTLC plates.
The analysis was carried out using Camag HPTLC system equipped with a semi-authomatic TLC sampler Linomat 5, Visualizer, integrated software Win-CATS 1.4.9., and Videoscan.
TLC plates Si 60 F254 (Merck, Germany) of 0.2 mm layer thickness were used. TLC separation was carried out using mobile phase of water, ethyl acetate, anhydrous formic acid and anhydrous acetic acid (17,5:67,5:7,5:7,5 V/V/V/V).
After the mobile phase draw up the plate until it is approximately 0,5 cm from the end, the plate was dried, proceed with 2-aminoethyl diphenylborinate and macro-gol, and after that, examined in UV-light at 365 nm.
Flavonoids and phenolic acids reference solutions were used.
The scheme of the obtained chromatogram is presented on Fig. 1.
Fig. 1. HPTLC chromatogram scheme obtained from capsule dosage form containing Corylus avellana L. dry extract (rutine (1), quercitrine (2), test solution from capsule dosage form (3), neochlorogenic acid (4), chlorogenic acid (5)).
As a result of HPTLC research rutine (1) and quercitrine (2) have been identified in capsule dosage form containing Corylus avellana L. dry extract (3). Among phenolic acids, neochlorogenic (4) and chloro-genic (5) acids have been identified.
Considering the necessity of the reference compound chose for the further quantitative analysis of the sum of flavonoids, rutine reference standard was chosen, which is consistent at obtained HPTLC research results and the literature data.
The absorption spectroscopy determination of the sum of flavonoids. The analysis was carried out using THERMO Scientific Evolution 60S Spectroscope.
Test solution for determination of the sum of fla-vonoids has been prepared as follows: the capsules filling was removed and placed into a volumetric flask. Ethanol 50% was added, and the ultrasound extraction was allowed to run at room temperature for 30 minutes. After centrifugation, the obtained solution was filtered.
1 ml of the obtained solution was put into a volumetric flask; ethanol 50 % and then AlCl3 ethanol solution 50 g/l were added. Then, in 10 minutes the acetic
acid solution 50 g/l was added, and the solution was brought up to the mark with ethanol 50 %.
The mixture was left for 10 min at room temperature and then subjected to spectral analysis in the range of 300-600 nm against the blank, where AlCl3 solution was substituted by ethanol solution 50 %.
Rutine reference sample solution of 100 ^M was used.
The content of the sum of flavonoids was calculated acoording to the formula:
x=A-
m„ -1
A {100 - 25
(50 - 50^
m - i
100
(100 - d )
where Ai - the test solution absorption coefficient;
Ao - the reference solution absorption coefficient; mo - reference standard sample weight to prepare reference solution;
mi - the capsules filling sample weight to prepare test solution;
d - loss on drying, %.
Under the given conditions, the spectrum of the test solution had a maximum absorption at wavelength
406 nm (Fig. 2). The similar spectrum of rutine reference solution had a maximum absorption at wavelength 408 nm.
Fig. 2. The UV-spectrum of the test solution obtained from capsule dosage form containing Corylus avellana L. dry extract
The analysis of flavonoids total content in gelatine capsules containing Corylus avellana L. dry extract calculated as rutine has shown the content of 1,7 %.
7. Conclusion
Effective HPTLC and absorption spectroscopy methods for determination of flavonoids and phenolic acids in capsule dosage form containing Corylus avellana L. dry extract have been developed. As a result of research work, neochlorogenic and chlorogenic acids have been identified in capsule dosage form containing Corylus avellana L. dry extract, as well as flavonoids rutine and quercitrin. The analysis of flavonoids total content in gelatine capsules containing Corylus avellana L. dry extract calculated as rutine has shown the content of 1,7 %.
It has been found that described methods are promising enough for standardization of capsules with Corylus avellana L. dry extract and may be suggested for the quality control of the dosage form under manufacturing conditions.
References
1. Bauer, R. Quality criteria and standardization of phy-topharmaceuticals: can acceptable drug standards be achieved? [Text] / R. Bauer // Therapeutic Innovation & Regulatory Science. - 1998. - Vol. 32, Issue 1. - P. 101-110. doi: 10.1177/ 009286159803200114
2. Wani, M. Herbal medicine and its standardization [Text] / M. Wani // Pharmaceutical information. - 2007. -Vol. 1. - P. 6.
3. Tsyvunin, V. V. Experimental defining of anticonvul-sant action of perspective phytogenic anticonvulsants [Text] / V. V. Tsyvunin, S. Yu. Shtrygol', Yu. S. Prokopenko, E. L. To-ryanic // Ukrainian biopharmaceutical journal. - 2014. - Vol. 3, Issue 32. - P. 45-49.
4. Tsyvunin, V. V. Screening research of the anticonvulsant action of dry extracts from 8 species of herbs members of the Solanaceae, Papaveraceae, Lamiaceae and Polemoniaceae families [Text] / V. V. Tsyvunin, S. Yu. Shtrygol', Yu. S. Prokopenko, V. A. Geprgiyants // Clinical pharmacy. - 2012. -Vol. 16, Issue 4. - P. 47-50.
5. Mishchenko, V. A. A targeted search of the anticon-vulsant substances from herbs members of the Solanaceae family [Text]: PhD thesis / V. A. Mishchenko - Zaporizhzhya, 2013. - 160 p.
6. Bluznyuk, N. A. A comparative research of the fla-vonoids content in leaves of decorative shrubs - members of Ukrainian flora [Text] / N. A. Bluznyuk, Yu. S. Prokopenko, V. A. Geprgiyants // Collection of scientific works of Shupyk NMA. - 2015. - Vol. 24, Issue 4. - P. 321-325.
7. Tistaert, C. Chromatographic separation techniques and data handling methods for herbal fingerprints: a review [Text] / C. Tistaert, B. Dejaegher, Y. V. Heyden // Analytica Chimica Acta. - 2011. - Vol. 690, Issue 2. - P. 148-161. doi: 10.1016/j.aca.2011.02.023
8. Attimarad, M. High-performance thin layer chroma-tography: A powerful analytical technique in pharmaceutical drug discovery [Text] / M. Attimarad, K. K. Mueen Ahmed, B. E. Aldhubaib, S. Harsha // Pharmaceutical Methods. - 2011. -Vol. 2, Issue 2. - P. 71-75. doi: 10.4103/2229-4708.84436
9. Sherma, J. Review of HPTLC in drug analysis: 1996-2009 [Text] / J. Sherma // Journal of AOAC International. - 2010. - Vol. 93, Issue 3. - P. 754-764.
10. Heyden, Y. V. Extracting information from chroma-tographic herbal fingerprints [Text] / Y. V. Heyden // LCGC Europe. - 2008. - Vol. 21, Issue 9. - P. 438-443.
11. Blatter, A. Qualitative and quantitative HPTLC methods for quality control of Stephaniatetrandra [Text] / A. Blatter, E. Reich // Journal of liquid chromatography and related technologies. - 2004. - Vol. 27, Issue 13. - P. 20872100. doi: 10.1081/jlc-120039420
12. Fang, C. Comparison of HPTLC and HPLC for determination of isoflavonoids in several kudzu samples [Text] /
C. Fang, X. Wan, C. Jiang, H. Cao // Journal of Planar Chromatography - Modern TLC. - 2005. - Vol. 18, Issue 101. - P. 7377. doi: 10.1556/jpc.18.2005.1.13
13. Spangenberg, B. Quantitative thin-layer chromatography: a practical survey [Text] / B. Spangenberg, C. Poole, C. Weins. - Berlin: Springer, 2011. - 373 p. doi: 10.1007/9783-642-10729-0
14. Selivanchikova, I. B. A quantitative determination of flavonoids in gomeopathic Thuja tinctures by the absorption spec-troscopy method [Text] / I. B. Selivanchikova, M. N. Lyakina, Z. P. Kostennikova // Pharmacy. - 2001. - Vol. 6. - P. 14-16.
References
1. Bauer, R. (1998). Quality Criteria and Standardization of Phytopharmaceuticals: Can Acceptable Drug Standards be Achieved? Therapeutic Innovation & Regulatory Science, 32 (1), 101-110. doi: 10.1177/009286159803200114
2. Wani, M. (2007). Herbal medicine and its standardization. Pharmaceutical information, 1, 6.
3. Tsyvunin, V. V., Shtrygol', S. Yu., Prokopenko, Yu. S., Toryanic, E. L. (2014). Experimental defining of anticon-vulsant action of perspective phytogenic anticonvulsants. Ukrainian biopharmaceutical journal, 3 (32), 45-49.
4. Tsyvunin, V. V., Shtrygol', S. Yu., Prokopenko, Yu. S., Geprgiyants, V. А. (2012). Screening research of the anti-convulsant action of dry extracts from 8 species of herbs members of the Solanaceae, Papaveraceae, Lamiaceae and Polemoniaceae families. Clinical pharmacy, 16 (4), 47-50.
5. Mishchenko, V. A. (2013). A targeted search of the anticonvulsant substances from herbs members of the Sola-naceae family. Zaporizhzhya, 160.
6. Bluznyuk, N. A., Prokopenko, Yu. S., Geprgiyants, V. A. (2015). A comparative research of the flavonoids con-
tent in leaves of decorative shrubs - members of Ukrainian flora. Collection of scientific works of Shupyk NMA, 24 (4), 321-325.
7. Tistaert, C., Dejaegher, B., Heyden, Y. V. (2011). Chromatographic separation techniques and data handling methods for herbal fingerprints: A review. Analytica Chimica Acta, 690 (2), 148-161. doi: 10.1016/j.aca.2011.02.023
8. Attimarad, M., Mueen Ahmed, K. K., Aldhubaib, B. E., Harsha, S. (2011). High-performance thin layer chromatography: A powerful analytical technique in pharmaceutical drug discovery. Pharmaceutical Methods, 2 (2), 71-75. doi: 10.4103/ 2229-4708.84436
9. Sherma, J. (2010). Review of HPTLC in drug analysis: 1996-2009. Journal of AOAC International, 93 (3), 754-764.
10. Heyden, Y. V. (2008). Extracting information from chromatographic herbal fingerprints. LCGC Europe, 21 (9), 438-443.
11. Blatter, A., Reich, E. (2004). Qualitative and Quantitative HPTLC Methods for Quality Control of Stephania tetrandra . Journal of Liquid Chromatography & Related Technologies, 27 (13), 2087-2100. doi: 10.1081/jlc-120039420
12. Fang, C., Wan, X., Jiang, C., Cao, H. (2005). Comparison of HPTLC and HPLC for determination of isoflavo-noids in several kudzu samples. Journal of Planar Chromatography - Modern TLC, 18 (101), 73-77. doi: 10.1556/ jpc.18.2005.1.13
13. Spangenberg, B., Poole, C., Weins, C. (2011). Quantitative thin-layer chromatography: a practical survey. Berlin: Springer, 373. doi: 10.1007/978-3-642-10729-0
14. Selivanchikova, I. B., Lyakina, M. N., Kostenniko-va, Z. P. (2001). A quantitative determination of flavonoids in gomeopathic Thuja tinctures by the absorption spectroscopy method. Pharmacy, 6, 14-16.
Дата надходження рукопису 26.01.2016
Blyznyuk Nataliya, Quality, standardization and certification department, Institute of Pharmacy Professionals Qualification Improvement of National University of Pharmacy, Pushkinskaya str., 53, Kharkiv, Ukraine, 61002 E-mail: natasha.bliznyuk@mail.ru
Prokopenko Yuliya, PhD, Associate Professor, Department of quality, standardization and certification of medicines, National University of Pharmacy, Pushkinskaya str., 53, Kharkiv, Ukraine, 61002 E-mail: yuliya.prok@gmail.com
Georgiyants Victoriya, Doctor of pharmaceutical sciences, Professor, head of Department, Department of Pharmaceutical chemistry, National University of Pharmacy, Pushkinskaya str., 53, Kharkiv, Ukraine, 61002 E-mail: vgeor@ukr.net
