The Qualitative Analysis of Ethanol Extracts of Herbal Raw Materials by Method of High Pressure Liquid Chromatography

8/6/2019 The Qualitative Analysis of Ethanol Extracts of Herbal Raw Materials by Method of High Pressure Liquid Chromatogr

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Acta Poloniae Pharmaceutica Drug Research, Vol. 64 No. 4 pp. 327333, 2007 ISSN 0001-6837Polish Pharmaceutical Society

At present, the use of complex liquid herbal

preparations has been increasing. Taking into con-sideration the needs of Lithuanian consumers, wechose to produce a new sedative, indigestion-reliev-ing complex phytopreparation. For the preparationof the multi-componential herbal extract, we select-ed herbal raw material (ginger rhizomes, cinnamonbark, rosemary leaves, and lavender flowers) withsedative and indigestion-relieving effects of its bio-logically active substances. During the selection ofthe raw material, attempts were made to achieve thatthe biologically active substances in the compositionof herbal raw materials, i.e. essential oils (borneol,bisabolene, cineol, camphor, linalool, terpineol,eugenol, cinnamaldehyde, gingerol, shogaol, geran-iol) and flavonoids (hyperforin, quercitrin, iso-quercitrin, rutin), would be targeted at particularsymptoms and would complement each otherseffect. Four herbal raw materials with positiveeffects of their active substances on digestive tractdisorders were selected. According to literature, themain active substances of the component raw mate-rials, i.e. ginger rhizomes, rosemary leaves, cinna-mon bark, and lavender flowers are essential oils (1-

8). All these raw materials can be used for the treat-

ment and prophylaxis of digestion disorders.The herbal raw material of St. Johns wort(Hyperici herba) with sedative and anti-depressiveaction of its biologically active substances wasselected. It is one of the most popular herbs inLithuania, which is attributed to the traditionalgroup of medicinal plants. St. Johns wort accumu-lates up to 13 per cent of enzymes, 0.1 1.25 percent of essential oils, up to 8 per cent of flavonoids(rutin, quercetin, quercitrin, hyperoside) as well asantrachinones (hypericin and its derivatives), hyper-phorin, carotinoids, and resins (2, 9, 10).Flavonoids, i.e. quercetin and quercitrin are distin-guished by their sedative and antidepressive effectsof monoamine oxydase A type. Xantones also act asmonoamine oxydase A inhibitors: they inhibit anxi-ety. Biflavonoids possess great affinity to benzodi-azepine receptors. They relieve anxiety and stimu-late sleep (11-15). R. F. Weiss recommended St.Johns wort for the treatment of somatogenic andpsychogenic forms of depression (16). In theRepublic of Lithuania, the following indications ofSt. Johns wort are approved: the complementary

THE QUALITATIVE ANALYSIS OF ETHANOL EXTRACTS OF HERBAL RAWMATERIALS BY METHOD OF HIGH PRESSURE LIQUID

CHROMATOGRAPHY

ZENONA KALVNIEN, SAUL VELIEN, KRISTINA RAMANAUSKIEN*, ARNASSAVICKAS, LIUDAS IVANAUSKAS1 and VALDAS BRUSOKAS2.

Department of Pharmaceutical Technology and Social Pharmacy, 1Department of Analytic andToxicological Chemistry, 2Department of Pharmaceutical Chemistry and Pharmacognosy, Kaunas

University of Medicine, A. Misckeviiaus g. 9 LT 44307 Kaunas, Lithuania

Abstract: The article describes the qualitative analysis of ethanol extracts from St. Johns wort, cinnamon bark,

rosemary leaves, lavender flowers, and ginger rhizomes, and the extract of their mixture using the method ofhigh pressure liquid chromatography in order to identify flavonoids. The aim of the study was to identify theamounts of rutin, hyperoside, quercetin, and other flavonoids in the multi-component extract as well as in thecontrol extracts of its compositional parts using the method of high pressure liquid chromatography, by evalu-ation of the correlation between the amounts of flavonoids in the extracts. The evaluation of the results of thestudy showed that flavonoid amount in the control extracts of the herbal material and flavonoid amount in theethanol extracts of the mixture of raw material did not differ significantly. It was found that the largest amountof flavonoids was extracted from St. Johns wort. The results of the analysis confirmed the assumption that theyield of flavonoids from St. Johns wort has no influence on the active substance of other medicinal herbal rawmaterials.Keywords: St. Johns wort, ginger, cinnamon, lavender, rosemary, flavonoids

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* Correspondence: Kristina Ramanauskien , tel +370 37 327255, +370 601 02567, e-mail [email protected];


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328 ZENONA KALVNIEN et al.

remedy for the treatment of neurotic depressions,

sleep disorders, and anxiety; the anti-inflammatoryand wound-healing remedy (17). For the study ofherbal extracts, the same methods of analysis asthose for chemical drugs are applied, and theirresults are evaluated according to analogous criteria.High pressure liquid chromatography (HPLC) wasselected as a reliable method of analysis. Thismethod allows for a qualitative and quantitativeanalysis of components of herbal extracts (18-20). Itseems relevant to analyze the chemical compositionof the prepared extracts and to prove that St. Johnswort is a major source of flavonoids.

The aim of the study was to identify theamounts of rutin, hyperoside, quercetin and other

flavonoids in a multi-component extract using the

method of high pressure liquid chromatography, byestimating the correlation among the amounts offlavonoids in the extract.

MATERIALS AND METHODS

The object of the analysis is a multi-componentherbal extract containing: herbs of St. Johns wort,ginger rhizomes, cinnamon bark, rosemary leaves,lavender flowers, and 70 % ethanol (Table 1).

For the qualitative and quantitative identifi-cation of flavonoids in the extracts of the crude

drug, control extracts were prepared, with the ratioof crude drug and the vehicle matching the ratio of

Composition of a multi-componentextract

St. Johns wort herb (4 parts),

Ginger rhizome (3 parts),

Cinnamon bark (1 part),

Rosemary leaves (1 part),

Lavender flowers (1 part)

1 part of crude drug mixturerequires 5 parts of ethanol

Ratio of crude drug and ethanol ina multi-component extract

1. St. Johns wort herb 1 part,ethanol 12.5 parts

2. Ginger rhizome 1 part, ethanol16.7

3. Cinnamon bark 1 part, ethanol50 parts

4. Rosemary leaves 1 part, ethanol50 parts

5. Lavender flowers 1 part, ethanol50 parts

Ratio of crude drug and ethanol incontrol extract

1. St. Johns wort herb extract, ratio1:12.5

2. Ginger rhizome extract, ratio1:16.7

3. Cinnamon bark extract, ratio1:50

4. Rosemary leaves extract, ratio1:50

5. Lavender flowers extract, ratio1:50

Table 1. The composition of herbal extracts.

Table 2. The quantitative identification of active compounds in herbal extracts.

Active Extract of Extract of Extract of Extract of Extract of Extract of compounds, St. Johns ginger cinnamon lavender rosemary the mixture of g/mL wort rhizomes bark flowers leaves herbal raw materials

Quercetin 700.04 1.11 568.1

Hyperoside 477.82 39.9 11.42 664.78

Rutin 531.49 11.68 658.22

Chlorogenicacid 169.58 1.96 171.86

Caffeic acid 0.83

Astragalin

Keampferol 0.62 84.0

Quercitrin 1.90

Apigenin-7-glycosyde 0.49

Vitexinrhamnoside 19.5

Vicenin 20.68

Orientin 0.43


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The qualitative analysis of ethanol extracts of herbal raw... 329

the multi-component extract (Table 1). Themethod of the preparation was percolation (21-23).The solvent of the crude herbal materials is 70 %

ethanol. Identification of flavonoids was per-formed using high pressure liquid chromatogra-phy; the equipment used was a chromatographWaters 2690 with the spectrophotometric detectorWaters 2487; chromatographic column X-TerraRP18 3,5 m, column dimensions 3.0 150 mm;the volume of injected sample was 10 l. Elutionis gradient, i.e. the composition of the gradientchanges with time. Eluent A: acetonitrile and tri-fluoroacetic acid (99.9:0.1, v/v). Eluent B: dis-tilled water and trifluoroacetic acid (99.9:0.1, v/v).

Gradient shift: from 0 min 5% of eluent A and95% of eluent B; from 45.0 min 45% of eluent Aand 55% of eluent B; from 60.0 min 5% of elu-

ent A and 95% of eluent B. The eluent flow ratewas 0.4 mL/min. Preparation of the tested solu-tion: the extract was filtered through a membranefilter with pore diameter of 5 m and diluted with70% vol. ethanol solution, ratio 1 : 1. Flavonoidswere identified at 360 nm wavelength. The com-pounds were identified and their amounts inextract calculated according to the length of reten-tion of the extracted components, peak areas, andratios between individual peak areas and those ofstandard peaks.

Figure 1. The chromatogram of flavonoid identification by HPLC in the extract of the multi-component herbal mixture.1 chlorogenic acid; 2 caffeic acid; 3 vicenin; 4 orientin; 5 vitexin-4-rhamnoside; 6 vitexin; 7 isovitexin; 8 rutin; 9 hyper-oside; 10 apigenin-7-glucoside; 11 astragalin; 12 quercitrin; 13 luteolin; 14 quercetin; 15 apigenin; 16 kaempferol.

Figure 2. The chromatogram of flavonoid identification by HPLC in the control extraction of St. Johns wort.

1 chlorogenic acid; 2 caffeic acid; 3 vicenin; 4 orientin; 5 vitexin-4-rhamnoside; 6 vitexin; 7 isovitexin; 8 rutin; 9 hyper-oside; 10 apigenin-7-glucoside; 11 astragalin; 12 quercitrin; 13 luteolin; 14 quercetin; 15 apigenin; 16 kaempferol .


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RESULTS

In the prepared multi-component extract(MCE), the largest amounts of the followingflavonoids were identified: hyperoside, quercetin,rutin; chlorogenic acid was also identified (Table 2).Figure 1 shows a chromatogram in which otheractive substances in addition to the above-men-tioned flavonoids in the multi-component herbalextract were found. In the control extract of St.

Johns wort (SJWCE), similar flavonoids were iden-tified and their amounts measured (Table 2).

Furthermore, not only flavonoids but also two phe-nolic acids caffeic acid and chlorogenic acid were identified in SJWCE (Fig. 2). The amount ofchlorogenic acid was measured (Table 2). In thecontrol extract of ginger (GCE), phenolic acids, i.e.caffeic acid and chlorogenic acid were identifiedand their amounts measured (Table 2). The results ofthe analysis have shown a small amount offlavonoids in ginger rhizome extract, which is con-gruent with the data in the literature (1-3, 5, 11).

Flavonoids were not detected in this extract (Fig. 3).In the control extract of cinnamon bark (CCE), very

Figure 3. The chromatogram of flavonoid identification by HPLC in the control extraction of ginger rhizomes.1 chlorogenic acid; 2 caffeic acid; 3 vicenin; 4 orientin; 7 isovitexin; 8 rutin; 10 apigenin-7-glucoside; 11 astragalin; 12 quercitrin; 13 luteolin; 14 quercetin; 16 kaempferol.

Figure 4. The chromatogram of flavonoid identification by HPLC in the control extract of cinnamon bark.1 chlorogenic acid; 2 caffeic acid; 3 vicenin; 4 orientin; 5 vitexin-4-rhamnoside; 6 vitexin; 7 isovitexin; 8 rutin; 9 hyper-oside; 10 apigenin-7-glucoside; 11 astragalin; 12 quercitrin; 13 luteolin; 14 quercetin; 15 apigenin; 16 kaempferol.


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small amounts of the following substances were

identified: quercetin, apigenin-7-glycoside,quercitrin, and kaempferol (Table 2). In the chro-matogram (Fig. 4), hyperoside, rutin, luteolin,chlorogenic and caffeic acids were identified; how-ever, their amounts were not significant. The largestamount of biologically active substances in crudecinnamon belongs to essential oils (1-3, 11), and theamount of the identified flavonoids is not pharma-cologically relevant (Table 2). In the control extractof lavender flowers (LCE), hyperoside, vitexin,rhamnoside, vicenin, and rutin were identified(Table 2). In addition to these, luteolin, apigenin,

and apigenin-7-glycoside were also found in LCE(Fig. 5). In the control extract of rosemary leaves

(RCE), the following compounds and their amounts

were identified: caffeic acid, isovitexin, luteolin(Fig. 6). A big part of the area under peaks belongsto some unidentified compounds released during 31and 34-36 min., and the length of their release sig-nificantly differs from the release of the standardsfrom the column (Fig. 6).

The study showed that the highest amount offlavonoids (hyperoside, rutin, quercetin) and chloro-genic acid was released from the control extract ofSt. Johns wort (Fig. 7). Figure 7 shows that the con-trol extract of St. Johns wort yields the largestamount of quercetin and the smallest amount of

chlorogenic acid. The identified amounts offlavonoids in the control extract of lavender, cinna-

Figure 5. The chromatogram of flavonoid identification by HPLC in the control extract of lavender flowers.1 chlorogenic acid; 2 caffeic acid; 3 vicenin; 4 orientin; 5 vitexin-4-rhamnoside; 6 vitexin; 7 isovitexin; 8 rutin; 9 hyper-

oside; 10 apigenin-7-glucoside; 11 astragalin; 12 quercitrin; 13 luteolin; 14 quercetin; 15 apigenin; 16 kaempferol

Figure 6. The chromatogram of flavonoid identification by HPLC in the control extraction of rosemary leaves.2 caffeic acid; 5 vitexin-4-rhamnoside; 6 vitexin; 7 isovitexin; 8 rutin; 9 hyperoside; 10 apigenin-7-glucoside; 11 astragalin;12 quercitrin; 13 luteolin; 14 quercetin; 16 kaempferol


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mon and rosemary are relatively small if comparedto the control extract of St. Johns wort (Fig. 7).Therefore, it can be stated that the largest amount offlavonoids in the multi-component extract is extract-ed from crude herbs of St. Johns wort. This coin-cides with the data in the literature, in particular thatthe main flavonoids in the herbs of St. Johns wortare hyperoside, rutin and quercetin (2, 9, 10). Thecomparison of the findings in control extract and in

the extract of the mixture of the herbal raw materi-als showed that the amount of flavonoids differedinsignificantly, which supports our assumption thatother components of the herbal mixture have noinfluence on the yield of flavonoids. The method ofhigh pressure liquid chromatography has confirmedthe compatibility of biologically active substancesthat were extracted from the mixtures of herbal rawmaterials.

CONCLUSION

Using the method of high pressure liquid chro-matography it was found that the largest amount offlavonoids is extracted from crude herbs of St.Johns wort; therefore, the amount of flavonoids ina multi-component extract differs insignificantlyfrom the flavonoid amount in the extract of St.Johns wort.

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Figure 7. Correlation of flavonoid amounts in extracts.


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Received: 5.01.2007

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The Qualitative Analysis of Ethanol Extracts of Herbal Raw Materials by Method of High Pressure Liquid Chromatography