ANTICANCER COMPOUNDS FROM MEDICINAL PLANTS


BIOLOGICA NYSSANA  4 (1-2)  December 2013: 81-85  Stamenković, J. et al.  Optimization of HPLC method for the… 

  

 81 

 

 

 

Original Article 

 

Optimization of HPLC method for the isolation of Hypericum 
perforatum L. methanol extract 
 

Jelena Stamenković, Ivana Radojković, Aleksandra Đorđević, Olga Jovanović, Goran 

Petrović, Gordana Stojanović 

 

Department of Chemistry, Faculty of Science and Mathematics, University of Niš, Serbia 

 

* E-mail: jelena.stamenkovic@pmf.edu.rs 

 
 

Abstract:  

Stamenković, J., Radojković, I., Đorđević, A., Jovanović, O., Petrović, G., Stojanović, G.: Optimization 

of HPLC method for the isolation of Hypericum perforatum L. methanol extract. Biologica Nyssana, 4 (1-

2), December 2013: 81-85. 

St. John's Wort (Hypericum perforatum L.) is one of the most studied plant species in the family 

Hypericaceae. The aim of this study was the identification of the constituents of methanol extract of H. 

perforatum and optimization of conditions for their isolation. The main components of the methanol extract 

were isolated on preparative ZORBAX Eclipse XDB C18 column with solvent system consisting of methanol 

and 1x10
-2

 M ammonium acetate in water. Constituents of the extract were identified by comparing their 

retention times with the retention times of the standards, with the literature data and the UV spectra. By 

varying the conditions of chromatography, the optimal conditions for isolation of the methanol extract 

constituents were determined: mobile phase consisting of methanol and 1x10
-2

 M ammonium acetate in water 

in ratio 1 : 1, sample concentration 100 mg/mL, sample volume 30 µL, flow 2 mL/min. Under these 

conditions 7 components of the methanol extract were isolated. 

 

Key words: Hypericum perforatum L., methanol extract, isolation, preparative HPLC 

. 

 

 

Introduction 
 

St. John’s Wort (Hypericum perforatum) is a 

herbaceous perennial plant of the Hypericaceae 

family, which is distributed in Europe, Asia, and 

Northern Africa, and naturalized in the US. The 

plant grows approximately one metre high with 

opposite and paired branches. The leaves are 

opposite and sessile, up to 2 cm long, oblong and 

contain numerous translucent glandular dots, which 

are visible against light. The yellow flowers contain 

5 petals with many stamens protruding. These 

flowers contain a group of reddish fluorescent 

dianthrone pigments with biological activity 

(V a t t i k u t i  & C i d d i , 2005). 

Hypericum species are medicinal plants 

known as healing herbs due to their various 

medicinal properties for the last two hundred years. 

Oily Hypericum preparations may be applied 

externally to treat minor burns, wounds, 

inflammation of the skin, and nerve pain 

(B l u m e n t h a l  et al., 1998). Internally, the herbal 

preparation is indicated for the treatment of anxiety 

and depression. 

St John’s Wort (Hypericum perforatum L.) 

has a chemical composition well studied: 

anthraquinone derivatives naphthodianthrones – 

hypericin, pseudohypericin and isohypericin, 

protohypericin and protopseudohypericin 

(biosynthetic precursors of hypericin and 

pseudohypericin, respectively); flavonoids – 

flavonols (kaempferol, quercetin), flavones 

(luteolin) and glycosides (hyperoside, isoquercitrin, 

quercitrin, and rutin), biflavonoids including 

biapigenin (a flavone) and amentoflavone (a 

biapigenin derivative) and catechins (flavonoids 

11
th

 SFSES • 13-16 June 2013, Vlasina lake 

 

4 (1-2) • December 2013: 81-85 

 



BIOLOGICA NYSSANA  4 (1-2)  December 2013: 81-85  Stamenković, J. et al.  Optimization of HPLC method for the… 

 

 82 

often associated with condensed tannins); 

prenylated phloroglucinols – hyperforin and 

adhyperforin; tannins; procyanidins (condensed 

type); other phenols – caffeic, chlorogenic, p-

coumaric, ferulic, p-hydroxybenzoic and vanillic 

acids; volatile oils, carotenoids, choline, β-sitosterol 

etc. (C r e t u  et al., 2011). 

Naphthodianthrones, namely hypericin and 

pseudohypericin, are found in the flowering portions 

of the plant. Until recently, these two compounds 

were considered responsible for the purported 

antidepressant effect of Hypericum. Numerous 

flavonoid compounds, including hyperoside, 

quercitrin, isoquercitrin, rutin, quercetin, campferol, 

luteolin, and myricetin, as well as biflavonoids 

I3,II8-biapigenin and I3′,II8-biapigenin 

(amentoflavone) are found in the aboveground 

portions of the plant, including the leaves, stalk, 

flowers, and buds. Phloroglucinol compounds, 

including hyperforin and adhyperforin, are present 

in the flowers and buds (G r e e s o n  et al., 2001). 

Several methods for the analysis of H. 

perforatum by TLC, photometry, HPLC, HPLC-MS 

and HPLC-NMR have been reported, but most of 

these focus on the determination of 

naphthodianthrones and/or hyperforin only (Gray et 

al., 2000; Liu et al., 2000; Mulinacci et al., 1999). 

The aim of this study was to develop the new 

simple and efficient method for isolation and 

identification of the Hypericum perforatum L. 

methanol extract main constituents. For this 

purpose, the following methods were used: 

 Maceration of dried plant material with 
methanol to obtain the extract; 

 High-pressure chromatography (HPLC) method 
for the isolation, identification and 

determination of purity of isolated compounds. 

 

Materials and methods 
 

Plant material was collected in the vicinity of 

Leskovac in July 2011. Voucher specimen is 

deposited at the Herbarium of the Department of 

Biology and Ecology, Faculty of Sciences and 

Mathematics, University of Niš. 

Naphthodianthrones and hyperforin are 

known to be thermolabile and especially 

photosensitive; thus, the extraction process was 

performed at room temperature and protected from 

the light (W i r z  et al., 2001; O r t h  et al., 1999). 

Dry aerial part of the plant was chopped and 

powdered. 60 g of dry material was transferred in a 

dark bottle and submeged in 600 mL (1:10, v/v) 

methanol (Sigma Aldrich, p.a.), and left in the dark 

for 48 hours. The methanol extract was filtered 

through filter paper (white bar) into the pre-

measured flask. Methanol was evaporated under 

reduced pressure in a rotary evaporator and 12.10 g 

of dry extract was obtained. 

Re-extraction of the previously treated 

material was performed with another 500 mL of 

methanol. After 5 days, the methanol extract was 

again filtered through filter paper (white bar) into 

the same flask. The extract was evaporated and 

16.28 g of dry extract was obtained in whole after 

repeated extraction.  

The dry extract was extracted with 100 mL of 

hexane (Sigma-Aldrich, p.a.) in the ultrasonic bath 

for 30 min. The crude hexane extract was 

evaporated to dryness at reduced pressure by 

rotoevaporation and 0.28 g of dry material was 

obtained. 

The respective yields were calculated as 

percentage using the formula: (crude extract 

weight/plant material weight) x 100 and were: 

20.16%, 27.13% and 1.72% respectively. 

For HPLC analysis a weighed amount of 

extract was dissolved in HPLC grade methanol. All 

samples were filtered through a 0.45 μm filter 

before undertaking HPLC analysis. Each sample 

solution was injected in triplicate and standard 

deviations were below 2.2 % for all experiments, 

indicating that the method is precise and 

reproducible. 

HPLC analysis was performed on HPLC 

chromatography with DAD detector (HPLC system 

1200 series, Agilent Technologies, USA). 

Separation and purification of the extract 

components was performed on Zorbax Eclipse XDB 

C18 column with dimensions (5 μm, 150 mm × 4.6 

mm, analytical and 5 μm, 250 mm x 9.4 mm, 

semipreparative). Solvent system consisting of 

methanol and 1x10
-2

 M ammonium acetate in water 

was used as the mobile phase in isocratic and 

gradient mode. The ratio of the solvents was varied 

in order to determine the optimum conditions for 

separation. All experiments were carried out at room 

temperature (25±2°C). Chromatograms were 

recorded at different wavelengths. The constituents 

of the extract were identified by comparing their 

retention times with the retention times of standards, 

based on literature data and comparing the UV 

spectra. Fractions were isolated and purified using 

the fraction collector. 

 
Results and Discussion 

 

There are numerous literature data on the 

HPLC analysis of different extracts H. perforatum 

L. (B r o l i s  et al., 1998; R o e m p p  et al., 2004). 

The fact common to all of the researched methods is 

that thay were developed primarily for analytical 



BIOLOGICA NYSSANA  4 (1-2)  December 2013: 81-85  Stamenković, J. et al.  Optimization of HPLC method for the… 

  

 83 

purposes in order to achieve a better separation of 

the constituents of the extract (L i  & F i t z l o f f , 

2001). Described methods are based on the usage of 

the system with different solvents in gradient and 

isocratic mode. Therefore they are not suitable to be 

applied in preparative purposes primarily due to 

long duration of analysis. 

In order to determine the optimal conditions 

for isolating components, preliminary investigations 

were carried out on the analytical column, varying 

the amount of injected sample, eluent polarity and 

flow rate of mobile phase. Work on the analytical 

column was carried out in isocratic and gradient 

mode and chromatograms were recorded at different 

wavelengths. 

The methanol extract first was eluated on the 

analytical column under isocratic conditions. The 

mobile phase was run at flow rate of 0.5 mL/min 

and consisted of methanol : water (1 % HCOOH) = 

80 : 20.5 µL of sample (concentration 5 mg/mL) 

was injected in all experiments performed on 

analytical column. 

Since the separation in isocratic mode was not 

obtained in satisfactory manner, gradient elution 

was performed by using the following solvent 

gradient: 0-5 min – 50 % MeOH; 5-10 min – 70 % 

MeOH; 10-30 min – 90 % MeOH. The flow-rate 

was kept at 0.5 mL/min. 

As gradient conditions did not provide good 

results, the possibility of using isocratic method 

with the changed polarity of the mobile phase was 

tested. A mixture of methanol : water (1x10
-2

 M 

CH3COONH4) = 50 : 50 was used as mobile phase. 

The resulting chromatogram is shown in Fig. 1. 
 

  
Fig. 1. HPLC chromatogram obtained in isocratic conditions, methanol : water (1x10

-2 
M CH3COONH4) = 

50 : 50. 

 

  

Fig. 2. HPLC chromatogram with the sample volume of 20 µl, at a flow rate of 2 mL/min. 

 

This system was the basis for the transition 

from the analytical to semipreparative column, by 

varying the flow rate and injected sample volume, in 

order to obtain optimal results. All samples that 

were injected to semipreparative column were 

significantly more concentrated (100 mg/mL), 

aimed to increase method efficiency. 

Fig. 2 shows the chromatogram of HPLC 

analysis that was performed on semipreparative 

column with the optimal composition of mobile 



BIOLOGICA NYSSANA  4 (1-2)  December 2013: 81-85  Stamenković, J. et al.  Optimization of HPLC method for the… 

 

 84 

phase (methanol and 1x10
-2

 M ammonium acetate in 

water, ratio (1:1). This system was chosen because 

of good separation and short runtime. Injection 

volume of the sample was 20 µl, with a flow rate of 

2 mL/min. The peaks in the HPLC chromatogram 

were identified based on literature data (Hansen et 

al.,1999), by comparing the retention time and UV 

spectra (Hillwing et al., 2008) with reference 

standards. 

 

  

Fig. 3. HPLC chromatogram with the sample volume of 30 µl, at a flow rate of 2 mL/min. 

Table 1. Retention time of identified and isolated compounds, mass of isolated compounds and their purity. 

No. Compound  Rt (min) Isolated mass (mg) Purity % 

1. Chlorogenic acid 4.033 / / 

2. Gallic acid 4.213 / / 

3. Trans- p-coumaric acid 4.535 / / 

4. Unidentified compound 1 5.600 1.4 87.65 

5. Rutin  7.425 / / 

6. Hyperoside 7.425 / / 

7. Isoquercitrin 7.631 16.8 79.84 

8. Quercitrin  8.467 / / 

9. Quercetin 9.444 5.5 92.71 

10. Pseudohypericin 10.634 0.8 96.66 

11. Hypericin 12.860 1.0 97.12 

12. Unidentified compound 2 16.464 0.9 97.43 

13. Hyperforin  20.974 1.4 98.26 

 

Table 1 shows the retention times of 

identified and isolated components, mass of isolated 

components and their purity, based on the integral 

of the peak areas. The purity was determined by the 

integration of HPLC spectra of the isolated 

compounds and it was directly proportional to the 

achieved resolution. 

In order to increase method efficiency, 40 μl 

of sample was injected, but this led to the reduced 

resolution. As a compromise solution, 30 μl of 

sample volume was injected at the same flow rate of 

2 mL/min (Fig. 3). Based on obtained 

chromatograms, it was concluded that the best 

results for the separation and isolation of 

constituents of H. perforatum L. methanol extract 

on the semipreparative column, were obteined by 

injecting the volume of 30 µl of the sample 

(concentration 100 mg/mL) at a flow rate of 2 

mL/min. 

 



BIOLOGICA NYSSANA  4 (1-2)  December 2013: 81-85  Stamenković, J. et al.  Optimization of HPLC method for the… 

  

 85 

Conclusion 
 

The results showed that the optimal 

conditions for isolation of the constituents of 

Hypericum perforatum L. methanol extract on the 

semipreparative column were: mobile phase 

methanol : water (1x10
-2

 M CH3COONH4) = 50 : 

50, sample concentration 100 mg/mL, injection 

sample volume 30 µl and the flow rate of 2 mL/min. 

Seven compounds were isolated from the extract 

with purity : unidentified compound (peak 4) 87.65 

%, isoquercitrin (peak 7) 79.84 %, quercetin (peak 

9) 92.71 %, pseudohypericin (peak 10) 96.66 %, 

hypericin (peak 11), 97.12 %, unidentified 

compound (peak 12) 97.43 %, hyperforin (peak 13) 

98.26 %. According to obtained results a new 

simple isocratic HPLC method was developed, 

allowing the baseline separation of thirteen main 

Hypericum perforatum standard compounds in less 

than 25 min, which means an improved separation 

25-50 min less than other published systems. 

 
Acknowledgements. The results obtained in this study 

are part of the research project 172047, funded by 

Ministry of Education, Science and Technological 

Development of Serbia. 

 

References 
 

Blumenthal, M., Busse, W.R., Goldberg, A., 

Grüenwald, J., Hall, T., Riggins, C.W., Rister, 

R.S.(eds), 1998: The complete German 

Commission E monographs: therapeutic guide to 

herbal medicines. American Botanical Council, 

Boston. 

Brolis, M., Gabetta, B., Fuzzati, N., Pace, R., 

Panzeri, F., Peterlongo, F. 1998: Identification 

by high-performance liquid chromatography-

diode array detection-mass spectrometry and 

quantification by high-performance liquid-

chromatography-UV absorbance detection of 

active constituents of Hypericum perforatum. 

Journal of Chromatography A, 825: 9–16. 

Cretu, R., Mihailescu, R., Mitroi, G., Iacob, E., 

Verdes, R., Tebrencu, C. 2011: Phytochemical 

investigation of Crataegi folium cum flos 

(hawthorn leaves and flowers) and Hyperici 

herba (St. Johns wort aerial parts) hydroalcoholic 

extracts., Annals of the ''Alexandru Ioan Cuza'' 

University Sect.II a. Genetics and Molecular 

Biology, 12 (4): 133-138. 

Gray, D.E., Rottinghaus, G.E., Garrett, H.E., 

Pallardy, S.G. 2000: Simultaneous 

Determination of the Predominant Hyperforins 

and Hypericins in St. John's Wort (Hypericum 

perforatum L.) by Liquid Chromatography. 

Journal of AOAC International, 83 (4): 944-949.  

Greeson, J. M., Sanford, B.,  Monti, D.A. 2001: St. 

John's wort (Hypericum perforatum): a review of 

the current pharmacological, toxicological, and 

clinical literature. Psychopharmacology, 153 (4): 

402-414. 

Hansen, S.H.,. Jensen, A., Cornett, C., Bjørnsdottir, 

I., Taylor, S., Wright, B., Wilson, I.D. 1999:  

High-Performance Liquid Chromatography On-

Line Coupled to High-Field NMR and Mass 

Spectrometry for Structure Elucidation of 

Constituents of Hypericum perforatum L. 

Analytical Chemistry, 71 (22): 5235–5241. 

Hillwig, M.L., Hammer, K.D.P.,  Birt, D.F., 

Wurtele, E.S. 2008: Characterizing the Metabolic 

Fingerprint and Anti-inflammatory Activity of 

Hypericum gentianoides. Journal of Agricultural 

and  Food Chemistry, 56 (12): 4359–4366. 

Li, W., Fitzloff, J.F. 2001: High performance liquid 

chromatographic analysis of St. John’s Wort 

with photodiode array detection. Journal of 

Chromatography, 765 (1): 99–105.   

Liu, F.F., Ang, C.Y., Heinze, T.M., Rankin, J.D., 

Beger, R.D., Freeman, J.P., Lay, J.O. 2000: 

Evaluation of major active components in St. 

John’s Wort dietary supplements by high-

performance liquid chromatography with 

photodiode array detection and electrospray mass 

spectrometric confirmation. Journal of 

Chromatography A, 888 (1–2): 85–92. 

Mulinacci, N., Bardazzi, C., Romani, A., Pinelli, P., 

Vincieri, F.F., Costantini, A. 1999: HPLC-DAD 

and TLC-Densitometry for quantification of 

hypericin in Hypericum perforatum L. extracts. 

Chromatographia, 49 (3-4): 197-201. 

Orth, H.C.J., Rentel, C., Schmidt, P.C. 1999: 

Isolation, purity analysis and stability of 

hyperforin as a standard material from 

Hypericum perforatum L. Journal of Pharmacy 

and Phamacology, 51: 193-200. 

Römpp H., Seger C., Kaiser C.S., Haslinger E., 

Schmidt P.C. 2004: Enrichment of hyperforin 

from St. John’s Wort (Hypericum perforatum) by 

pilot-scale supercritical carbon dioxide 

extraction. European Journal of Pharmaceutical 

Sciences, 21: 443-451. 

Vattikuti U.M.R., Ciddi, V. 2005: An overview on 

Hypericum perforatum Linn. Natural Product 

Radiance, 4 (5): 368-381. 

 

http://www.ncbi.nlm.nih.gov/pubmed?term=Gray%20DE%5BAuthor%5D&cauthor=true&cauthor_uid=10995119
http://www.ncbi.nlm.nih.gov/pubmed?term=Rottinghaus%20GE%5BAuthor%5D&cauthor=true&cauthor_uid=10995119
http://www.ncbi.nlm.nih.gov/pubmed?term=Garrett%20HE%5BAuthor%5D&cauthor=true&cauthor_uid=10995119
http://www.ncbi.nlm.nih.gov/pubmed?term=Pallardy%20SG%5BAuthor%5D&cauthor=true&cauthor_uid=10995119