pre-extraction and extraction of hypericin in hypericum ...€¦ · (hypericum perforatum) using...

015/2009

SpeedExtractor E-916

Pre-Extraction and Extraction of Hypericin in St. John’s Wort (Hypericum perforatum) using the SpeedExtractor E-916

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SHORT NOTE Pre-Extraction and Extraction of Hypericin in St. John’s Wort

(Hypericum perforatum) using the SpeedExtractor E-916 St. John’s Wort herb and capsules with dry extract are widely used in herbal medicine. For quality control reason the amount of hypericin in the products is determined by extraction and photometric quantification. The quantification by photometry is inter-fered by co-extracted chlorophyll. Classically, chlorophyll is removed by a time-consuming manual procedure. A convenient removal of chlorophyll followed by quantitative extraction of hypericin is presented, based on pressurized solvent extraction using the SpeedExtractor E-916.

Introduction

In herbal medicine St. John’s Wort herb and capsules with dry extract are widely used for the treatment of depressions. In this products hypericin is determined for quality reasons. The determination of the total amount of hypericin can be done by extraction and photometric quantification at 590 nm. The quantification by photometry is interfered by co-extracted chlorophyll. Removal of the interfering chlorophyll was achieved by a pre-extraction with dichloromethane. The remainings were then extracted with methanol to quantify hypericin.

Experimental

Instrumentation: SpeedExtractor E-916, Photometer: Thermo Ηelios , Ultrazentrifugal mill: Retsch, ZM 200 with distance sieve 0.5 mm. Samples: Dried and cut St. John’s Wort herb and capsules containing 425 mg dry extract of St. John’s Wort Approx. 0.6 g of the ground herb or approx. 0.05 g of the dry extract was mixed with quartz sand and extracted in two consecutive extractions with the SpeedExtractor using the parameters shown in Table 1. The samples were ex-tracted in triplicate. Table 1: Extraction method of the SpeedExtractor E-916

Pre-extraction Main-extraction

Temperature 80 °C 80 °C

Pressure 100 bar 100 bar

Solvent Dichloromethane Methanol

Cells 10 ml 10 ml

Vials 240 ml 240 ml

Cycles 2 4

Heat-up 1 min 1 min

Hold 4 min 2 min

Discharge 2 min 2 min

Flush with solvent 5 min 5 min

Flush with gas 4 min 4 min

After completing to 200 ml, photometric quantification at 590 nm was done.

Absorption coefficient: 870=g/100mlcmE

Results

By performing a pre-extraction with dichloromethane the chlorophyll can be efficiently removed from the samples without affecting the hypericin content. Only a negligible amount of chlorophyll remains, with insignificant interference with hypericin at 590 nm (Fig. 1 and 2). Found

concentrations in herb and capsules correspond with the declared values (Table 2).

Figure 1: Spectrum without pre-extraction; 2: hypericin, 3: chlorophyll

Figure 2: Spectrum with pre-extraction; 2: hypericin

Table 2: Determined content of hypericin, n=3

Hypericin

[rsd]

Declared value of supplier

Herb

0.8 mg/g

[1.6 %]

min. 0.8 mg/g

Capsules

1.18 mg / Capsule [1.9 % ]

min. 0.75 mg / Capsule

Conclusion

Application of two consecutive extractions is a fast and reliable way for the determination of total hypericin in St. John’s Wort herb and capsules.

References

[1] PhEur 6.2, 07/2008:1438, Hyperici herba SpeedExtractor E-916 operation manual For more detailed information refer to Application note 015/2009

015/2009 SpeedExtractor E-916

Application Note 015/2009 Version A, Copyright © 2009 Büchi Labortechnik AG 3/9

1 Introduction

Most natural product samples comprise many ingredients which sometimes interfere in the analysis of the component of interest. It is therefore desirable to separate this component already during an extraction. In the following, an application is presented which shows a separation of the interfering components just by pre-extracting with a different solvent.

St. John’s Wort contains the red-colored anthraquinone-derivates hypericin and pseudohypericin. For quality control reasons the total amount of hypericins is deter-mined.

Figure 1: Hypericin: R=H; Pseudohypericin: R=OH

The total amount of hypericin and pseudohypericin can be determined by extraction and photometric quantification at 590 nm. The total value of hypericins is expressed as hypericin. The quantification by photometry is interfered by co-extracted chlorophyll a and chlorophyll b (Fig. 2). The traditional determination [1] is done by a time consuming manual extraction to remove chlorophyll. Alternative removal of chlorophyll and extraction of hypericin can be done in a fast and reliable way in two consecutive extractions [2] by pressurized solvent extraction using the SpeedExtractor E-916.

Figure 2: The absorption spectrum of chlorophyll a and b


2 Equipment

− SpeedExtractor E-916 with 10 ml cells − 6 additional cells for SpeedExtractor E-916 (any size possible; used to flush the

lines between the two extractions) − Analytical balance, reading precision 0.1 mg − Volumetric flasks 200 ml − Photometer, Thermo Helios − Ultrazentrifugal mill, Retsch, ZM 200 with distance sieve 0.5 mm

3 Chemicals and materials

− Methanol, Sigma-Aldrich (Order No. 32213) − Dichlormethane, Sigma-Aldrich (Order No. 66740) − Quartz sand, particle size 0.3-0.9 mm, Buchi (Order No. 037689) − Glas-Cuvettes, 4 and 5 cm (depending on hypericin content)

4 Samples

4.1 Dried an cut herb Hyperici herba conc, Hänseler AG (Order No. 09-4231-01)

Total hypericin content declared by the supplier according to PhEur 6.2, 07/2008:1438 [1] at least 0.8 mg/g in dry sample. Dry matter: 85.0 %

4.2 Dry extract of hypericum perforatum in capsules Lucilium 425, Sandoz Pharmaceuticals AG

Containing 425 mg dry extract 3.5-6.0:1, containing at least 0.75 mg total hypericin per capsule. Due to the highly variable concentration of hypericin in raw material the content of hypericin in the capsules can be much higher than the minimal value.

5 Procedure

The extraction and analysis of hypericin in St. John’s Wort and dry extract capsules includes the following steps:

− Preparation of sample − Preparation of cells − Pre-extraction and removal of chlorophyll with SpeedExtractor E-916 − Change of solvent − Main-Extraction to determine the content of hypericin with SpeedExtractor E-916 − Quantification with photometry

5.1 Preparation of the samples − Total herb: Grind the herb with am ultrazentrifugal mill to a uniform powder.

(0.5 mm grid) − Dry extract in capsules: Open the capsule and discard the gelatine capsule.

Grind the containing dry extract in a mortar.

5.2 Preparation of the cells for the extraction of herb − Give approx. 3.5 g of sand into the cells. − Weigh in approx. 0.6 g of herb and mix with approx. 6 g of sand. − Fill in the mixture into the 10 ml cells using a funnel. − Fill up any void volume to 10 mm to the upper filter with sand.


5.3 Preparation of the cells for the extraction of the dry extract

− Give approx. 6 g of sand into the cells. − Weigh in approx. 50 mg of dry extract and mix with approx. 3 g of sand. − Fill in the mixture into the 10 ml cells using a funnel. − Fill up any void volume to 10 mm to the upper filter with sand.

5.4 Extraction with SpeedExtractor E-916 − Carry out the pre-extraction, using the parameters of Table 1. − Remove the cells from the SpeedExtractor and insert empty cells. − Carry out a flush of 30 s with methanol to the waste port. − Change the vials to collect the second fraction separately. − Insert the cells with the sample again. − Carry out the main-extraction, using the parameters of Table 2. Table 1: Pre-extraction method, extraction time 35 min, extract volume approx. 35 ml

Parameter Value

Temperature 80 °C

Pressure 100 bar

Solvent Dichloromethane

Cells 10 ml

Vials 240 ml

Cycles 2

Heat-up 1 min

Hold 4 min

Discharge 2 min

Flush with solvent 5 min

Flush with gas 4 min

Table 2: Main-extraction method, total extraction time 51 min, extract volume approx. 60 ml

Parameter Value

Temperature 80 °C

Pressure 100 bar

Solvent Methanol

Cells 10 ml

Vials 240 ml

Cycles 4

Heat-up 1 min

Hold 2 min

Discharge 2 min

Flush with solvent 5 min

Flush with gas 4 min


5.5 Quantification by photometry Transfer the extracts quantitatively to 200 ml flasks and complete the flasks to volume with methanol. Let the solutions stand at daylight for at least one hour.

Quantify by measuring the absorption maxima at 590 nm and calculating the value by using the absorption coefficient of hypericin.

[ ]dE m

10 VAmg/g w

g/100mlcmSample

FlaskHerb

⋅⋅

⋅⋅= (1)

[ ]100dE m

425 VAmgm

g/100mlcmSample

FlaskCapsule

⋅⋅⋅

⋅⋅= (2)

A: measured absorbance VFlask: volume of the flask [ml] mSample: sample weight [g]

g/100mlcmE : absorption coefficient of hypericin: 870

d: pathlength [cm] wHerb: weigth fraction hypericin in herb mCapsule: amount of hypericin per capsule

6 Results

6.1 Verification of the pre-extraction performance Without doing a pre-extraction, there is a strong interference in the measurement of hypericin by different chlorophyll peaks (Figure 3). By doing a pre-extraction with dichloromethane, the chlorophyll can be efficiently removed from the samples without removing the hypericin. Only an insignificant amount of chlorophyll is left in the sample and the measurement of hypericin at 590 nm is not influenced anymore. The spectra of the pre-extraction solution corresponds to the spectra of pure chlorophyll (Figure 4). Post-extraction of the samples shows that the extraction of the hypericin is complete.

Figure 3: Spectrum of extract without pre-extraction, peak max: 1: 547 nm, 2 = 590 nm, 3 = 665 nm


Figure 4: Spectrum pre-extraction of St. John’s Wort herb, peak max: 1 = 414 nm, 2 = 508 nm, 3 = 538 nm, 4 = 610 nm, 5 = 668 nm

Figure 5: Spectrum main-extraction of St. John’s Wort herb, peak-max: 1 = 547 nm, 2 = 590 nm


Figure 6: Spectrum post-extraction of St. John’s Wort herb with methanole

Figure 7: Green solution: pre-extract, containing chlorophyll; red solution: main extract, containing hypericin

6.2 Hypericin content Table 3: Content of hypericin in St. John’s Wort herb (Abbreviations: see chapter 5.5)

mSample [g] A Vtot [ml] d [cm] w [mg/g]

1 0.5151 0.595 200 4 0.67

2 0.6010 0.720 200 4 0.69

3 0.6255 0.732 200 4 0.67

Mean value 0.68

rsd [%] 1.6

Mean value referring to dry matter1 0.8

rsd [%] 1.6

The determined content of 0.8 mg/g is in accordance to the value of PhEur 6.2. of at least 0.8 mg/g in dried herb.

1 Dry matter: 85%


Table 4: Content of hypericin in capsules containing dry extract of St. John’s Wort (Abbreviations: see chapter 5.5)

msample [g] A V [ml] d [cm] mcapsule [mg]

4 0.0511 0.314 200 5 1.20

5 0.0561 0.338 200 5 1.18

6 0.0544 0.322 200 5 1.16

Mean value 1.18

rsd [%] 1.9

The determined content of 1.18 mg per capsule is in accordance to the declared value of at least 0.75 mg per capsule.

7 Conclusion

The extraction of St. John’s Wort herb for the determination of total hypericin content can be done in a easy and powerful way by using the SpeedExtractor E-916. The applied pre-extraction removes the chlorophyll in an easy and reliable way. Found concentrations of hypericin are in accordance with the declared values of the suppliers.

The short extraction times and the small solvent volumes used are further benefits of this procedure.

8 References

[1] PhEur 6.2, 07/2008: 1438, Hyperici herba

[2] Jana Richter, Analytische Untersuchungen zum Einfluss der Mykorrhizierung auf Ertrag, Inhaltsstoffzusammensetzung und Pathogentoleranz von Heil- und Gewürzpflanzen, Dissertation, Martin-Luther-Universität, Halle-Wittenberg (2007)

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pre-extraction and extraction of hypericin in hypericum ...€¦ · (hypericum perforatum) using...

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