Determination of Triterpenoids in Some Lamiaceae Species
Mariia Shanaida1, Antonina Pryshlyak2, Olena Golembiovska3
1Department of Pharmacognosy with Medical Botany, I. Horbachevsky Ternopil State Medical University, Ternopil, Ukraine
2Department of Human Anatomy, I. Horbachevsky Ternopil State Medical University, Ternopil, Ukraine
3Institute of Organic Chemistry, National Academy of Sciences of Ukraine, Kyiv, Ukraine
*Corresponding Author E-mail: shanayda-mi@ukr.net
ABSTRACT:
The present study was designated to analyze the triterpenoids presence and content in ethanolic extracts of three Lamiaceae species gathered under the condition of West Podillya region (Ukraine). The presence of triterpenoids in Dracocephalum moldavica L., Lophanthus anisatus (Nutt.) Benth and Satureja hortensis L. aerial parts was proved by the identification procedures. The chemical composition and content of triterpenoids was investigated using HPLC. The revealed triterpenoids were the derivates of α-amyrin (ursolic, euscaphic and tormentic acids), β-amyrin (oleanilic acid) and lupane (lupeole and betulin). It was established that high concentrations of ethanol (96 % and 70 %) removed from plants more considerable amount of triterpenoids than 50 % ethanol. The comparative HPLC-analysis showed the significant differences in content of pentacyclic triterpenoids in the investigated species. Ursolic and euscaphic acids were the most abundant components in the ethanoic extracts of all three plants while the content of tormentic acid and lupeol were the smallest. The obtained data can be used in the planning of pharmacological studies and in the chemotaxonomy of Lamiaceae Family.
KEYWORDS: Dracocephalum moldavica, Lophanthus anisatus, Satureja hortensis, pentacyclic triterpenoids, HPLC.
INTRODUCTION:
Medicinal plants are extremely relevant nowadays despite the progress in the creation of synthetic drugs. They are effective healing agents due to their enormous structural and chemical diversity(1). Phytopreparations, as a rule, have a multipurpose impact because in addition to the influence on the target organs they also activate the regulatory systems of the organism (2).
Triterpenoids are quite common in the plant world and exhibit a wide range of biological activity and low toxicity(1,3). Medicinal plants of the Lamiaceae Family accumulate mainly pentacyclic that exhibit anti-inflammatory, hepatoprotective, antioxidant, anticancer, antiviral and antimicrobial properties(4-9). Previously we have studied such groups of secondary metabolites of some Lamiaceae species as essential oils and polyphenols which manifest a valuable pharmacological activity(10,11). Despite the using in folk medicine and nutritional value such species as Dracocephalum moldavica L., Lophanthus anisatus (Nutt.) Benth and Satureja hortensis are till not investigated phytochemically well enough, in particular, regarding the content and composition of triterpenoids. They are not also used in the official medicine of Ukraine (12).
The purpose of this study was to identify and quantificate the triterpenoids in the ethanoic extracts of some Lamiaceae species (Dracocephalum moldavica, Lophanthus anisatus and Satureja hortensis) aerial parts under the condition of West Podillya region, Ukraine.
MATERIALS AND METHODS:
Chemicals:
Methanol, ammonium acetate, petroleum ether, chloroform, acetic acid, ursolic acid, oleanolic acid, lupeole and betulin were purchased from Merck (Germany); euscaphic acid and tormentic acid were from Wuxi Gorunjie Natural-Pharma co. LTD (China). All chemicals were of analytical grade.
Plant material:
The aerial parts of plants were collected at the flowering stage from the experimental plots in West Podillya region (Ukraine) in 2017. Herbs were dried in shadow. A voucher specimens of plants were deposited at the Department of Pharmacognosy and Medical Botany, I. Horbachevsky Ternopil State Medical University.
Extraction procedure:
Herbal extracts were prepared with the addition of ethanol in different concentration (50 %, 70% and 96 %). At first, the plant material was macerated with ethanol at room temperature for 3 hours with periodic stirring, then we carried out the heating of extracts on water bath for 1 h.
Plant material (5.0 g), grinded to 1 mm of particle size, was placed in a 250 ml round bottom flask and 100 ml of solvent was added. The cooled extract was filtered through a paper filter in a 100 ml volumetric flask and, if necessary, ethanol of a suitable concentration was added to the mark. Aliquot of each extract was evaporate to the dry residues, then dissolved in methanol and used for HPLC investigation. The certain amount of each extract was evaporated to 1/4-1/5 of initial volume and then used for conducting the identification and TLC-analysis of triterpenoids.
Identification of triterpenoids:
Foam test(1):
About 1 ml of extract is diluted to 5 ml with distilled water and then vigorously shaked in test tube for 1 min. Formation of a stable foam above the level of the liquid in test tube indicates the presence of triterpenic glycosides (saponins).
Reaction of sedimentation(1):
To about 1 ml of each extract is added several drops of 10 % Pb(CH3COO)2 solution. The appearance of precipitate is the result of reaction with triterpenoids.
Color reaction(1):
To about 2 ml of extract were added 1 drop of 10 % CuSO4 and 1 ml of H2SO4 concentrated; then this mixture is carefully heated. The appearance of blue-green colour indicates the presence of triterpenoids.
Thin layer chromatography (TLC) analysis(13):
About 0.05 ml of each evaporated extract was used for TLC identitfication of triterpenic acids. Тhe "Sorbfil" plates and petroleum ether-chloroform-acetic acid (10:4:0.4) solvent systems were used in TLC-analysis. The procedure was carried out with the triterpenoids standards. After removal the TLC plates from chromatographic chamber they were sprayed with 20 % of H2SO4 solution, then heated in a drying oven a for 8-10 min at a temperature of 105 °C. The appearance a pink-red color turning into a blue on chromatograms in absorption zones witnesses the presence of triterpenoids.
High performance liquid chromatography (HPLC) analysis of triterpenoids:
The qualitative and quantitative analysis of triterpenoids were performed with Shimadzu LC20 Prominence system according to (14, 15) with slight modifications. Сhromatograph was equipped with diode array detector (SPDM20A), PC software ChemStation (LC20), autosampler (SIL20A), thermostat of column (СТО20А) and four-channel pump of low pressure gradient (LC20AD). The separation was achieved by a X-Bridge C18 column (250 x 4.6 mm i.d., 5 µm particle size) at 30 °C. The mobile phase consisted of methanol and ammonium acetate (0.2 %, рН=6.75) at 1.0 ml/min flow rate, volume injection was 5 μL. Solvents were filtered through a 0.45 μm millipore filter before use and degassed in an ultrasonic bath. To prepare standard solutions, standards was dissolved in methanol for analysis. All solutions were filtered through 0.2 μm millipore filter before injection.The elution mode was isocratic.
The UV absorption spectra of the standards as well as the samples were recorded in the range of 190-800 nm. Identification of compounds was based on comparing their retention times with the reference standards characteristics.
RESULTS AND DISCUSSION:
Various solvents are known in extracting of triterpenes but using different concertation of ethanol is the most effective and environmentally justified(4). It was found that 96 % and 70 % ethanol extracts of the investigated plants showed a positive result about the presence of triterpenoids in the foam test, by the color reaction, reaction of sedimentation and after using the TLC method.
It should be noted that α-amyrin derivates (ursolic and euscaphic acids, fig. 5) are the most abundant compounds of all investigated plants. This data correlate with the literature data about Lamiaceae species(2-4, 13, 16). Ursolic and euscaphic acids were found in many plants and demonstrate several important biological activities such as anti-inflammatory, antioxidant, antiseptic, hepatoprotective and antitumor properties(3, 4, 17, 18). The highest content of ursolic acid was found in the 96 % ethanolic extract of D. moldavica (0.36 %), while euscaphic acids–in 70 % ethanolic extract of S. hortensis (0.56 %).
According to Silva et al.(16) such representative of Lamiaceae family as Ocimum basilicum harvested in Brasil accumulates the highest amount of ursolic acid (0.3 %) which was extracted with methanol. Among several Dracocephalum species the highest content of this acid measured by the gas chromatography method (GC) was found in D. diversifolium (0.81 %)(5). It was found(7) that Satureja montana leaves accumulate 0.49 % of ursolic acid (revealed by GC). Amounts of ursolic acid in the range of 0.06 to 0.21 % are specific for Prunella spica samples collected in China and Korea and extracted with ethanol(15). It was revealed 0.12 % of this triterpenoid in Ziziphora clinopodioides raw material after harvesting in China(8). Information about the content of euscaphic acid in plants of Lamiaceae family in literature sources is much less common(19).
Oleanolic acid as a β-amyrin derivate coexists with α-amyrin derivates in many medicinal plants of Lamiaceae Family as a free acid form or as aglycone for triterpenoid saponins(3-5). They may occur in their free acid form, as shown in fig. 5, or as aglycones for triterpenoid saponins which are comprised of a triterpenoid aglycone linked to one or more sugar moieties. 96 % ethanol was the best solvent for this acid extraction. Oleanolic acid has similar biological activity to ursolic acid(3, 17). As we can see from Table 1, among investigated species D. moldavica and L. anisatus accumulate a higher content of triterpenoids than S. hortensis. According to(9) Melissa officinalis leaves contain 0.16 % of oleanolic acid. Razboršek et al.(7) revealed with using the GC that content of this acid in Satureja montana leaves equals 0.14 %.
|
А) |
D) |
|
B) |
E) |
|
C) |
F) |
Fig. 1: UV-spectra of triterpenoids: A – ursolic acid; B – euscaphic acid; C - tormentic acid; D – oleanolic acid; E - betulin; F- lupeol.
Table 1: Content of pentacyclic triterpenoids in the aerial parts of some Lamiaceae species revealed by HPLC
Species |
Concentration of ethanol, % |
Content, % |
|||||
Ursolic acid |
Euscaphic acid |
Tormentic acid |
Oleanolic acid |
Betulin |
Lupeol |
||
|
D. moldavica |
96 |
0.36 |
0.16 |
0.01 |
0.15 |
0.12 |
0.04 |
|
70 |
0.14 |
0.26 |
0.02 |
0.14 |
0.13 |
<0.01 |
|
|
50 |
0.03 |
0.07 |
<0.01 |
0.02 |
0.07 |
<0.01 |
|
|
L. anisatus |
96 |
0.32 |
0.44 |
0.01 |
0.16 |
0.11 |
0.04 |
|
70 |
0.21 |
0.39 |
0.03 |
0.13 |
0.12 |
<0.01 |
|
|
50 |
0.02 |
0.08 |
0.01 |
0.02 |
0.09 |
<0.01 |
|
|
S. hortensis |
96 |
0.14 |
0.49 |
0.03 |
0.05 |
0.06 |
0.01 |
|
70 |
0.13 |
0.56 |
0.04 |
0.04 |
0.07 |
<0.01 |
|
|
50 |
0.03 |
0.22 |
0.01 |
<0.01 |
0.02 |
<0.01 |
|
Fig. 2: HPLC chromatogram of D. moldavica 96 % ethanolic extract
Fig. 3: HPLC chromatogram of L. anisatus 96 % ethanolic extract
Fig. 4: HPLC chromatogram of S. hortensis 96 % ethanolic extract
Ursolic acid Tormentic acid Euscaphic acid
Oleanolic acid Betulin Lupeol
Fig. 5: The chemical structures of revealed triterpenoids
Betulin is the lupane derivate(1) and revealed the anticancer activity(20); it can be converted after oxidation to the betulinic acid which is biologically more active than itself. The best solvent for betulin extraction was 70 % ethanol. Lupeol is another lupane derivate(1) and its content in the investigated species is the smallest.
Analysis of literature date has shown that the HPLC method is the most often used to determine the component composition and content of triterpenoids in plants(6, 8, 9, 14). Some scientists have been investigated triterpenoids in Lamiaceae species by GC method after derivatization(5, 7) or HPTLC-densitometric method(21). Their research proved that ursolic acid is the predominant component among triterpenoids in aerial parts of Lamiaceae species.
As it is known, the component composition of plant constituents is affected by climatic factors, growth conditions, and their genetic chemotypes; іt depends also of the choice of a solvent, an extraction procedures and method of analysis. In this regard, the content of triterpenoids in the same species from the different region can differ significantly(5, 9, 13, 16, 17).
CONCLUSION:
The presence and composition of triterpenoids in the aerial parts of D. moldavica, L. anisatus and S. hortensis (Lamiaceae) species growing under the condition of West Podillya region (Ukraine) was determined in this study. It was revealed that high concentrations of ethanol (96 % and 70 %) removed from plants the most considerable amount of triterpenoids. Using the HPLC-analysis, the content of 6 triterpenoids was identified, i.e. ursolic, euscaphic, tormentic and oleanilic acids, lupeole and betulin. It was revealed the significant differences in their contents in plants. Ursolic and euscaphic acids were the most abundant components of all investigated species.
CONFLICT OF INTERESTS:
None Declared.
REFERENCES:
1. Trease and Evans' Pharmacognosy: 16th Edition. Saunders Ltd.; 2009: 603 р.
2. Lu J-J, Pan W, Hu Y-J, Wang Y-T. Multi-target drugs: the trend of drug research and development. The Trend of Drug. 2012; 7 (6): 1-6.
3. Liu J. Pharmacology of oleanolic acid and ursolic acid. J. Ethnopharmacol. 1995; 49(2): 57-68.
4. Jäger S, Trojan H, Kopp T, Laszczyk MN, Scheffler A. Pentacyclic triterpene distribution in various plants – rich sources for a new group of multi-potent plant extracts. Molecules. 2009; 14(6): 2016-2031.
5. Kakasy AZ. New phytochemical data on Dracocephalum species. Theses of the PhD dissertation. Budapest, 2006. 14 p.
6. Leng G. Determination of oleanolic acid and ursolic acid in different parts of Mesona Chinensis Benth (Lamiaceae) by RP–HPLC. Chin. J. Spectrosc. Lab. 2011; 28: 2111–2114.
7. Razboršek MI, Vončina DB, Doleček V, Vončina E. Determination of oleanolic, betulinic and ursolic acid in Lamiaceae and mass spectral fragmentation of their trimethylsilylated derivatives. Chromatographia. 2008; 67: 433-440.
8. Tian S, Shi Y, Yu Q, Upur H. Determination of oleanolic acid and ursolic acid contents in Ziziphora clinopodioides Lam. by HPLC method. Pharmacogn Mag. 2010; 6: 116-119.
9. Zhang Y, Xue K, Zhao EY, Li Y, Yao L, Yang X, Xie X. Determination of oleanolic acid and ursolic acid in Chinese medicinal plants using HPLC with PAH polymeric C18. Pharmacognosy Mag. 2013; 9(Suppl. S1):19-24.
10. Golembiovska O. Simultaneous determination of flavonoids and phenolic acids in different parts of Prunella vulgaris L. by HPLC-DAD. Int. J. of Pharmacognosy and Phytochemistry. 2014; 29(1): 1248-1254.
11. Shanaida M, Ivanusa І, Kernychna І. Phytochemical analysis of secondary metabolites of Satureja hortensis L. Int J Pharm Pharm Sci. 2017; 9(2): 315-318.
12. Derzhavnyi reiestr lіkarskyh zasobiv Ukrainy (2017) [Elektronnyi resurs]. Rezhym dostupu: http://www.drlz.com.ua
13. Chumakova VV, Popova OI, Dmitriyev AB, Mezenova TD. Triterpenic acids in Giant Hyssop (Lophanthus anisatus Benth) herb. Pharmacy. 2013; 4: 35-39.
14. Chen JH, Xia ZH, Tan RX. High-performance liquid chromatographic analysis of bioactive triterpenes in Perilla frutescens. J. Pharm. Biomed. Anal. 2003; 32: 1175-1179.
15. Lee MK. Development of a validated liquid chromatographic method for the quality control of Prunellae Spica: determination of triterpenic acids. Analytica Chimica Acta. 2009; 633: 271–277.
16. Silva MGV, Vieira ÍGP, Mendes FNб Albuquerque IL, Santos RN, Silva FO, Morais SM. Variation of ursolic acid content in eight Ocimum species from Northeastern Brazil. Molecules. 2008; 13: 2482-2487.
17. Shi QQ, Dang J, Wen HX, Yuan X, Tao YD, Wang QL. Anti-hepatitis, antioxidant activities and bioactive compounds of Dracocephalum heterophyllum extracts. Bot Stud. 2016; 57(1):16-22.
18. Csuk R, Siewert B, Dressel C, Schäfer R. Tormentic acid derivatives: Synthesis and apoptotic activity. Eur. J. of Medicinal Chemistry. 2012; 56: 237-245.
19. Woo KW et al. Triterpenes from Perilla frutescens var. acuta and Their Cytotoxic Activity. Natural Product Sciences. 2014; 20(2): 71-75.
20. Król SK, Kiełbus M, Rivero-Müller A, Stepulak A. Comprehensive review on betulin as a potent anticancer agent. BioMed Research International. 2015. ID 584189: 11 p.
21. Wójciak-Kosior M, Krzaczek T, Matysik G, Skalska A. HPTLC-densitometric method of determination of oleanolic acid in the Lamii albi flos. J Sep Sci. 2005; 28: 2139-2143.
Received on 07.06.2018 Modified on 17.07.2018
Accepted on 27.08.2018 © RJPT All right reserved
Research J. Pharm. and Tech 2018; 11(7): 3113-3118.
DOI: 10.5958/0974-360X.2018.00571.1