APPLICATION OF HIGH PERFORMANCE THIN-LAYER CHROMATOGRAPHY TO SEPARATION OF OLEANOLIC, URSOLIC AND BETULINIC ACIDS

A.    Abstract

High performance thin-layer chromatography on silica was used for the separation of three triterpenic acids. Because of the similarity of chemical structures of oleanolic and ursolic acid, isocratic chromatography using a mixture of two or three solvents was not successful; however, the multiple gradient development (MGD) technique was more suitable.

Oleanolic and ursolic acid were separated after five steps of development with mixtures of petroleum ether/ethyl acetate in concentrations 20%,18% and 15%. The mixture of cyclohexane/ethyl acetate/formic acid (7.5:2.5:0.05 v/v) was used in the last step to improve the shape of the chromatographic bands. The progress of separation was controlled after each step of development by use of densitometer Desaga CD 60. The measurements were carried out after derivatization of 10% H₂SO₄ in ethanol at λ = 515 nm in absorbance mode.

B.     Key words

High performance thin-layer chromatography (HPTLC), oleanolic acid, ursolic acid, betulinic acid, multiple gradient development.

 

C.    Introduction

Oleanolic, ursolic and betulinic acids belong to pentacyclic triterpenes and are common constituents of many medicinal herbs and plants [1]. These terpenes may exist in the form of free acids or as aglycones for triterpenoid saponins. They have many important pharmacological effects. In the literature there are numerous data on their anti-inflammatory, hepatoprotective, anti-tumour, anti-HIV, anti-microbial, antifungal, anti-ulcer, gastroprotective, hypoglycemic and antihyperlipidemic properties [2-8]. They are relatively non-toxic and have been used in cosmetics and health products, e.g. oleanolic acid is marketed in China as an oral drug for human liver disorders, and ursolic acid is used in anti-tumour therapy in Korean traditional medicine.

The most explored method in the determination of these compounds is HPLC [9-14] but TLC is still an important tool of phytochemical investigations. Its advantages are the low amount of organic solvent used in the separation process and possibility of application of samples without any pretreatment.

These triperpenes, especially oleanolic and ursolic acids, have a similar molecular structure (Fig. 1) which makes their separation by thin-layer chromatography very difficult.

Oleanolic acid	Ursolic acid
Figure 1 Structures of investigated compounds

This paper presents results of the application of HPTLC to the separation of oleanolic, ursolic and betulinic acids on silica gel.

D.    Materials And Methods

All solvents were pro analysis grade from Polish Reagents (POCh, Gliwice, Poland). Triterpenic acids standards of the highest grade were purchased from Sigma (St. Louis, MO, USA). All samples were prepared as 0.05 % solutions in methanol.

Extracts of folium Salviae (Salvia officinalis L.), folium Plantaginis lanceolatae (Plantago lanceolata L.) and flos Lamii albi (Lamium album L.) were prepared by 24 h extraction with diethyl ether at room temperature. The ether extracts were evaporated to dryness and the residues were dissolved in 2 mL of acetone.

In the experiments, HPTLC plates 10 × 10 cm coated with silica gel (Merck, Darmstadt, Germany) were used. The plates were washed with methanol and dried in a stream of hot air before use.

2 μL of standard solutions and extracts were spotted using an automatic applicator Desaga AS 30 (Heidelberg, Germany) under nitrogen at 2.5 atm as streaks 6 mm long. Chromatograms were developed in horizontal Teflon DS chambers (Chromdes, Lublin, Poland).

To separate the mixtures of oleanolic, ursolic and betulinic acid multiple development was used according to the programme shown in Table 1. The mobile phase was removed by evaporating in a stream of warm and then cold air for 10 min after each step of development.

The plates were sprayed with 10% m/m H₂SO₄ in ethanol, dried, and then heated to 1100 C for 2-3 min. After derivatization the chromatograms were observed in daylight or in UV light at λ = 366 nm. The densitograms were obtained using Desaga CD-60 densitometer (Heidelberg, Germany) controlled by a Pentium computer in absorbance/reflectance mode at λ = 515 nm.

E.     Results And Discussion

Triterpenic acids are an important group of natural compounds with confirmed pharmacological activity. They occur simultaneously in many medicinal herbs and plants.

The closeness of their chemical structures, especially oleanolic and ursolic acids, makes their TLC separation very difficult. There are some chromatographic systems for analysing triterpenes, given in the literature [15-18] but none of them enable the separation of oleanolic and ursolic acids.

To find the most suitable eluent, numerous tests using various organic solvents were performed. The mixtures of weakly polar solvents: heptane, cyclohexane, petroleum ether, toluene, dichloromethane with acetone, diisopropyl ether, diethyl ether, ethyl acetate, methanol, 2-propanol, butanol in various ratios were tested.

In most investigated chromatographic systems betulinic acid was well separated but hRF values for oleanolic and ursolic acids were identical. Small differences of hRF values were observed in mixtures of cyclohexane/ethyl acetate, petroleum ether/diisopropyl ether and petroleum ether/ethyl acetate, but the separation of the mixture of oleanolic and ursolic acids was poor.

In several publications, Matysik et al. demonstrated that the multiple gradient development technique (MGD) is useful for separating compounds with similar chemical structures [19,20]. MGD technique requires evaporation of the mobile phase from the plate before the next step of development; therefore, due to their volatility, the mixtures of petroleum ether/diisopropyl ether and petroleum ether/ ethyl acetate were chosen for further investigations.

To optimize the gradient programme of chromatogram development the relationships between hRf values and mobile phase composition were determined. The differences of hRF values were observed in the range of concentrations 40-60% of diisopropyl ether and 15-20% of ethyl acetate in petroleum ether. The example of plots of hRF against % of modifier are presented in Figure 2.

The best result was obtained using the gradient programme shown in Table 1. The presented chromatographic system is

Table 1 Programme of gradient elution
Step No.	Solvents	Distance of development (cm)	Time of development (min.)
1 2 3 4 5	Petroleum ether/ethyl acetate (8:2 v/v) Petroleum ether/ethyl acetate (1.8:7.2 v/v) Petroleum ether/ethyl acetate (1.5/8.5 v/v) Petroleum ether/ethyl acetate (1.5/8.5 v/v) Cyclohexane/ethyl acetate/HCOOH (2:8:0.05 v/v)	7 8 9 9 9	15 15 17 17 25
After each step, the plate was dried for 10 min in a stream of warm and then cold air

80

70

60

50

40

30

20

10

0

     10%                       15%             20%            25%                       30%             35%           40%

% of ethyl acetate in petroleum ether

Figure 2 Relationships between hRF values and concentration of ethyl acetate in petroleum ether; OA – oleanolic acid, UA – ursolic acid, BA – betulinic acid.

 

 125,0                                                          ^UA _OA

AU                                                                             ^{UA           UA} _OA

 100,0                                                                              _OA

                                                                                               

   75,0

   50,0

                                                 I          II         III        IV        V

   25,0

     0,0

        10,0      15,0        20,0       25,0        30,0         35,0        40,0        45,0        50,0   mm

Figure 3 Densitogram of mixture of oleanolic acid (OA) and ursolic acid (UA) obtained after each step of development. Measurements were carried out at λ=515 nm after derivatisation of 10 % (v/v) methanol H2SO4.

Figure 4 Chromatogram of herbal extract obtained using gradient programme

according Table I. I – extract of folium Plantaginis lanceolatae; II – extract of folium Salviae; III mixture of standards (1 – oleanolic acid, 2 – ursolic acid, 3 – betulinic acid); IV – flos Lamii albi; V – standard of ursolic acid. Documentation was obtained after derivatisation of 10 % (v/v) methanol H2SO4 in daylight.

Journal of Pre-Clinical and Clinical Research, Vol 1, No 2 not suitable for quantitative analysis of oleanolic and ursolic acids because the resolution of peaks is insufficient (Fig. 3) but enables their identification in plant materials. The presented methods were successfully applied to the identification of triterpenic acids in extracts of Salvia officinalis herba, Lamium album flos, Plantaginis lanceolatae folium (Fig. 4).

F.     Conclusion

The similarity of chemical structures of triterpenic acids makes their separation on silica very difficult. Isocratic planar chromatography was not successful, however, the MGD technique permitted satisfactory separation of all investigated compounds after five steps of development using mixtures of ethyl acetate/ petroleum ether (steps 1-4) and a mixture of ethyl acetate/cyclohexane/formic acid 2.5:7.5:0.5 v/v (step 5).

The presented gradient programme can be used for the identification of oleanolic, ursolic and betulinic acids in plant material.

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