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. . 1, . . 1, . . 1, . . 1,2 1 , , . 139, 4000 2 , , . 26, 4000

A. S. Marchev1, I. G. Ivanov1, V. G. Georgiev1, A. I. Pavlov1,2 1 Laboratory of Applied Biotechnologies Plovdiv, The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Science, 139 Ruski Blvd, 4000 Plovdiv, Bulgaria 2 University of Food Technologies, Department of Organic Chemistry and Microbiology, 26 Maritza Blvd, 4002 Plovdiv, Bulgaria Abstract
A rapid and accurate HPLC method for quantitative determination of bioactive di- and triterpenoids (carnosic acid, betulin, betulinic, oleanolic and ursolic acids) in Sage in vitro cultures was developed. The method was precise and the linearity of the calibration curves ranged between 25-400 g/mL (r2=0.9934 to 0.9979). It showed very high reliability, which was proved by running reproducibility test with terpenoid extracts from in vitro cell suspension culture of Salvia tomentosa Mill. The method could be used for precise qualitative and quantitative analyzes of di- and triterpenes in biological samples producing complex mixtures of terpenes in low concentrations such as plant in vitro systems are.

Keywords: Sage, in vitro, ursolic acid, oleanolic acid, betulin, betulinic acid, carnosic acid, HPLC Introduction
Betulin (1), betulinic (2), oleanolic (3) and ursolic (4) acids are pentacyclic triterpenes of natural origin and are common constituents of many medicinal plants [9]. Recently the compounds have gained considerable interest because of their multiple pharmacological effects: anti-inflammatory, hepatoprotective, anti-tumour, anti-HIV, antimicrobial, anti-ulcer, gastro protective, hypoglycemic and anti-hyperlipidemic properties [12; 13; 3; 8; 18; 1; 17; 20; 10; 27]. 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 [23]. Carnosic acid (5) is abietane diterpenoid with antioxidant, antileukemic, anticancer, anti-angiogenic, antiinammator, anti-metabolic disorder, anticlastogenic, hepatoprotective and neuroprotective activities [7; 15; 19; 16; 14]. The above-mentioned compounds are exclusively found in plants, belonging to the genus Salvia [6]. About 19 native Salvia species are found in Bulgaria. Salvia tomentosa Mill. (Lamiaceae) is widespread in the old World. It is a popular herb for tea preparation named balsamic sage in Europe. It is also used in traditional medicine for treatment of open cuts in Turkey [2]. Since S. tomentosa Mill. occurs in very limited populations on the territory of Bulgaria, its collection for commercial purposes has been prohibited [22]. Development of in vitro systems of this protected species presents an attractive alternative for both studying and producing biologically active compounds without endangering their natural habitats.


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22 21 23 20 19 26


24 23 22

12 13 28 5 0 4 8 9 27 10 11

17 16



20 14 19


15 3 14 7 24 8 29

16 28 12 11

0 18


17 27

1 3 2 6 7


4 6 5 9 10




Betulin (1)
33 27


Betulinic acid (2)




13 19 8 14 10

12 13 25 5 0 4 8 9 24 11

17 18 16


4 7 3

22 15 25 23

17 32 19 21 16 11

OH 2
1 5

10 14 28


1 26 HO 2 6 3 7

9 15

30 24 26 22 20


32 31 28

Oleanolic acid (3)

Ursolic acid (4)

5 0 4 3 2 6 12


16 11 17

19 9 8


22 21

Carnosic acid (5)

The main analytical methods of triterpene acids are thin layer chromatography (TLC) [26], gas chromatography (GC) [9; 21], high-performance liquid chromatography (HPLC) [24; 4], capillary zone electrophoresis (CZE) [25] and micellar electrokinetic chromatography (MEKC) [11]. HPLC and MEKC are the most common methods for quantitative analysis of the triterpene acids with high separation efficiency and short analytical time [5]. However, the resolution of the acids is not satisfied due to their similar structures. OA and UA are hydrophobic position isomers. The only difference between them is the configuration of the methyl group on the ring E [5]. The present study is focused on the development of a rapid and accurate HPLC method for the quantitative analysis of the above-mentioned di- and triterpenoids in multiple samples of S. tomentosa Mill. plant cell suspension culture.

Material and Methods

Reagents and standards. All reagents and solvents used were of HPLC grade. Methanol, acetic, formic and phosphoric acids were from Merck (Germany). K2HPO4 (99.99%) and analytical standards of betulin (>98%), betulinic (>98%), ursolic (>90%), oleanolic (>97%) and carnosic (>97%) acids were purchased from Sigma (St. Louis, MO, USA). Deionised water was purified through Ultrapure Water Systems Arium 611DI (Sartorius AG , Goettingen, Germany).

Preparation of calibration solutions. Standard stock solutions from each compound were prepared by dissolving them in methanol. Nine calibration solutions were prepared: 10.0; 25.0; 50.0; 100.0; 200.0; 400.0; 600.0; 800.0 and 1000 g/mL. Cultivation of S. tomentosa Mill. plant cell suspension. S. tomentosa suspension was grown in Linsmayer-Skoog (LS) nutrient medium, supplemented with 30 g/L sucrose and 0.2 mg/L 2,4dichlorophenoxyacetic acid (2,4-D). The cultivation was conducted in 100 ml Erlenmeyer flasks with 20% (v/v) 10-days old suspension as inoculum, on shaker (11.6 rad/sec.), at 26 C in darkness. Preparation of sample solutions. Lyophilized biomass of S. tomentosa cell suspension (0.1 0.2 g) was extracted in triplicates with acetone (1:10 w/v) at 45 C under sonification. The acetone fractions were filtrated through filter paper and evaporated to dryness at 60 C. The residues were dissolved in 500 L methanol, filtrated by 22 m filter and analyzed by HPLC. Apparatus and conditions. The triterpenes were analyzed by HPLC system consisting of Waters 1525 Binary pump (Waters, Milford, MA, USA), Waters 2487 Dual Absorbance Detector (Waters, Milford, MA, USA), controlled by Breeze 3.30 software. Supelco Discovery HS C18 column (5 m, 25 cm 4.6 mm) operated at 26 C was used for separation. The compounds were detected by monitoring at 210 nm. Several mobile phases at different conditions were tested: 1) Methanol : 0.01 M KH2PO4 (pH=2.8) = 88 : 12 2) Methanol : 0.03 M KH2PO4 (pH=2.8) = 88 : 12 Both experiments were performed under gradient flow rate as follows: 0 min 18 min (0.8 L/min); 18 min-19 min (decrease to 0.6 ml/min); 19 min 30 min (0.6 ml/min); 30 min 31 min (rising back to 0.8 l/min) and 31 min 40 min (0.8 mL/min). 3) Methanol : 0.5% H3PO4 = 88 : 12; flow rate 0.8 mL/min 4) Methanol : 1.0% H3PO4 = 88 : 12; flow rate 0.8 mL/min 5) Methanol : H2O : CH3COOH = 85 : 15 : 0.3; flow rate 0.8 mL/min 6) Methanol : 0.1% HCOOH = 92 : 8; flow rate 0.4 mL/min

Results and discussions

To find the optimal elution conditions, several reported mobile phase systems were investigated. Since OA and UA are more difficult to separate our efforts were focused on those compounds. Different acidic aqueous buffers have been used as mobile phase in most HPLC studies. However, we have established that buffers containing inorganic acids,

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such as phosphoric acid were not able to consistently and completely separate oleanolic acid and ursolic acid. The increase in ionic strength of the used buffer did not improve compounds separation as well, but rather resulted in incensement of their retention times (figure 1).

Figure 2. HPLC profile of oleanolic and ursolic acids using mobile phase: A - Methanol : 0.5% H3PO4 = 88 : 12, (1) OA Rt=22.655 min, (2) UA Rt=23.967 min; B - Methanol : 1.0% H3PO4 = 88 : 12, (1) OA Rt=22.292 min, (2) UA Rt=23.552 min.

Figure 1. HPLC profile of oleanolic and ursolic acids using mobile phase: A - Methanol : 0.01 M KH2PO4 (pH=2.8) = 88 : 12, (1) OA Rt=23.798 min, (2) UA Rt=25.491 min; B - Methanol : 0.03 M KH2PO4 (pH=2.8) = 88 : 12, (1) OA Rt=26.881 min, (2) UA Rt=28.775 min. Aqueous solutions containing 0.5% and 1.0% H3PO4, to keep the acidic compounds in their neutral form, were tested as well. The results were similar as those obtained with KH2PO4 buffer (pH=2.8) and no complete resolution was achieved (figure 2). The pH value of mobile phase also had effect on triterpenes separation. It is considered that the addition of acetic acid in the mobile phase could have positive effect on separation of OA and UA by restraining the ionization of -COOH group and enhancing the effect of different configuration of the methyl group on the ring E [5]. In our experiments a mobile phase consisted of methanol : H2O : CH3COOH = 85 : 15 : 0.3 was tested. Unfortunately the separation of OA and UA was not satisfying enough and the peaks did not reach the base line as expected (figure 3 A). However, a significant improvement in resolution and separation to base line was achieved when the acetic acid was replaced with the stronger formic acid. In this case the mobile phase consisted of methanol : 0.1% HCOOH = 92 : 8 (figure 3 B).

Figure 3. HPLC profile of oleanolic and ursolic acids using mobile phase: A - Methanol : H2O : CH3COOH = 85 : 15 : 0.3, (1) OA Rt=33.933 min, (2) UA Rt=35.841 min; B - Methanol : 0.1% HCOOH = 92 : 8, (1) OA Rt=27.115 min, (2) UA Rt=28.591 min. After the successful separation of oleanolic and ursolic acids the same method was applied for all investigated terpenoids. The obtained chromatogram had good resolution and the compounds were separated very well (figure 4 A). Extracts of S. tomentosa cell suspension culture were analyzed in the same manner and the peaks were identified by comparison of the retention time and UV spectrum with those corresponding to the standards. The separation efficiency could be considered as satisfactory (figure 4 B).


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oleanolic acids. The r2 values were in the range from 0.9934 to 0.9979 that confirmed the linearity of the method (Table 1). Method reproducibility was evaluated by five replicate analyses of S. tomentosa cell suspension extracts. Mean values and relative standard deviations (RSD) between the measurements are presented in Table 2. The cell suspension culture produced only oleanolic and ursolic acids. The compounds were well discriminated in all investigated samples. Conclusions The similarity of chemical structures of triterpenic acids makes their separation very difficult. Several HPLC methods for that purpose were investigated. Complete separation on reverse phase C18 column (5 m, 25 cm 4.6 mm) operating at 26 C was achieved only when methanol : 0.1% HCOOH = 92 : 8 was used as a mobile phase at flow rate 0.4 mL/min. In these conditions, the developed method allows simultaneous determination of di- and triterpenoids in S. tomentosa Mill. plant cell suspension culture. The validation data indicates that this method is reliable. Table 1

Figure 4. Application of methanol : 0.1% HCOOH = 92 : 8 mobile phase for separation of: A terpenoid standards; B extract of S. tomentosa cell suspension culture. (1) carnosic scid; (2) betulin; (3) betulinic acid; (4) oleanolic acid; (5) ursolic acid. Under the above mentioned conditions linear relationships between the peak areas and concentrations were established in the range of 25400 g/mL for betulin, betulinic and ursolic acids and between 25-200 g/mL for carnosic and

Linearity of calibration curves of the investigated terpenoids

Compound Carnosic acid Betulin Betulinic acid Oleanolic acid Ursolic acid Retention time, min 15.518 22.402 23.924 27.115 28.591 Equation Y=2.88x105X-9.18x105 Y=1.21x104X-3.84x104 Y=1.48x104X-5.67x104 Y=2.48x104X-2.81x104 Y=2.66x104X-1.08x105 r2 0.9967 0.9963 0.9964 0.9934 0.9979 Table 2 Precision and reproducibility test for determination of terpenoids in S. tomentosa suspension culture Compound Oleanolic acid, g/mL Ursolic acid, g/mL Replicates 1 2 71.006 73.161 254.427 254.419 Mean SD 3 70.766 253.617 4 73.293 260.417 5 71.263 258.513 71.8921.23 256.2792.68 RSD, % 1.71 1.05

We could state that the presence the organic acids in the mobile phase had better influence on the separation efficiency of oleanolic and ursolic acid. Probably formic acid maintained stronger the effect of the different configurations between both acids.

Acknowledgements This research was supported by the Bulgarian Science Foundation, Bulgarian Ministry of


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Education and Science under Projects DMU 02/9, 2009 and DNTS 02/5-2010. References
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