Flavonol glycosides of berries of three major sea huckthorn subspecies, Hippophaë rhamnoides ssp rhamnoides, ssp sinensis and ssp mongolica Heikki Kallioa, Baoru Yanga,b and Teemu Halttunena

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Flavonol glycosides of berries of three major sea huckthorn subspecies, Hippophaë rhamnoides ssp. rhamnoides, ssp. sinensis and ssp. mongolica

Heikki Kallioa, Baoru Yanga,b and Teemu Halttunena

aDepartment of Biochemistry and Food Chemistry, University of Turku, FIN-20014, Turku, Finland

bAromtech Ltd, Veturitallintie 1, FIN-95410 Tornio, Finland

Flavonol glycosides of sea buckthorn (Hippophaë rhamnoides L.) berries were separated by RP-HPLC-DAD analysis and identified by reference compounds. Glycoside fractions were hydrolyzed by -glycosidase and the aglycones identified, again, by HPLC and reference compounds. All the three subspecies investigated (ssp. mongolica, ssp. rhamnoides, ssp. sinensis) contained the same major flavonoid glycoside species, the lowest total contents existing regularly in the mongolica berries. The major aglycon was isorhamnetin followed by quercetin with minor amounts of kaempferol. The flavonol glycosides, such as flavonol 3-O-rutinosides, 3-O-glucosides and 3-O-sophoroside-7-rhamnosides were determined to distinguish between the three subspecies.

Key words: Flavonol glycosides, Hippophaë rhamnoides, isorhamnetin, kaempherol, quercetin, sea bucktorn


Flavonols and flavons protect plants against UV and visible light and damages caused by bursts of free radicals (Takahama, 1983). The compounds also have anti-viral and anti-microbial potential (Parr and Bolwell, 2000). Quercetin seems to be the most common flavonol in the plant kingdom, being abundant in tee, apples, onion, broccoli and many other vegetables. In some cases also red wine may have high contents of flavonols, even up to 50 mg/l (Frankel et al., 1995).

Statistically, quercetin and, in some extent, also kaempferol were shown to reduce the risk of dying in iscemic heart disease (Hertog et al., 1995; Knekt et al., 2002). There are also evident indications of beneficial effects of flavonols on cancer, especially on lung cancer (Stefani et al., 1999; Knekt et al., 2002).

Materials and methods


Wild berries of sea buckthorn, Hippophaë rhamnoides ssp. rhamnoides were picked on Raippaluoto island at west-coast of Finland, ssp. sinensis in Wenshui county in Shanxi province in China, and cultivated berries of ssp. mongolica in Novosibirsk in Russia. The berries were frozen within a day.

Reagents and reference compounds

-glucosidase was purchased from Novozyme. The reference compounds isorhmnetin, quercetin, kaempferol, isorhamnetin 3-O-rutinoside, isorhamnetin 3-O-glucoside, quercetin 3-O-rutinoside and quercetin 3-O-glucoside were from Extrasynthése (Genay, France) and the internal standard floridzin from Sigma-Aldrich (Steinheim, Germany). Isorhamnetin 3-O-ß-D-sophoroside-7-O--rhamnoside verified by NMR-analysis (Rösch et al., 2003) was donated by Kroh and Rösch at the Technical University of Berlin.

Extraction of flavonol glycosides

Methanol and acetic acid (99.8 %) were added in ratio 14:1 on thawed berry samples (20 g) to reach the total volume of 60 ml. After homogenizing for two minutes the mixture was filtered and the filtrate was collected. The residue was re-extracted twice with 50 ml of methanol:water:acetic acid (70:30:5) by homogenizing for one minute and filtered. After evaporation the residue was dissolved in 25 ml methanol and a volume of 10 ml of the methanol solution was spiked with 2 ml of the internal standard, floridzine solution (1.00 mg/ml). The solution was evaporated to dryness and dissolved in water (10 ml).

Purification of flavonol glycosides

The extract was purified in one-gram polyamide cartridge activated with methanol (20 ml) and water (60 ml). After removal of the polar compounds by water, the flavonol glycoside fraction was collected with 40 ml of methanol. The fraction was evaporated to dryness, dissolved in methanol, filtrated and stored at – 20 oC. Each berry sample was extracted and purified in duplicates and analyzed twice with HPLC-DAD.

HPLC-DAD analysis of flavonol glycosides

The HPLC instrument consisted of Shimadzu SIL-10A auto injector, sample cooler, two CTO-10A pumps, CTO-10A column oven, SPD-M10AVP diode array detector and SCL 10AVP central unit (Shimadzu Ltd, Kyoto, Japan). A Phenomex Prodigy ODS 5 (3) column (250 x 4.60 mm, particle size 5 m) was applied. The effluent consisted of a mixture of water – tetrahydrofuran (THF) – trifluoroacetic acid (TFA) (98:2:0.1) (solvent A) and acetonitril (solvent B). The gradient profile used in glycoside analysis is presented in Table 1. Flow rate of the effluent was 1 ml/min, the detector wavelengths 270 nm and 370 nm and the volume of injection 10 l. Identification was based on co-injections of reference compounds and comparisons of absorption spectra.

Table 1. Gradients used in HPLC-DAD analysis

Glycoside gradient

Aglycone gradient

T / min

% B

T / min

% B

































Fractionation of flavonol glycosides and enzymatic hydrolysis

The five major glycoside peaks of the HPLC-DAD analysis were isolated and re-analyzed with the same method to verify the purity of the fractions. The glycosidic fractions were evaporated to dryness under dry N2 stream and the residues dissolved in 1 ml of 0.1 M acetate buffer (pH 4.6). A volume of 10 l of -glucosidase solution was added (activity 640 nkat/ml) and the sample was incubated for 5 h at 45 o C. The sample hydrolyzed was evaporated to dryness under dry N2 and the aglycones of the residue dissolved in 1 ml of methanol and filtered.

Analysis of flavonol aglycones

The aglycones were analyzed by HPLC as defined above, but by using the aglycone gradient (Table 1.)

Results and discussion

Figure 1 shows a chromatogram of flavonoid glycosides of sea buckthorn ssp. rhamnoides berries. The five major glycosides numbered from 1 to 5, which were taken into account in the HPLC analyses, did separate well from other peaks. Identification of the compounds was based on retention times, co-injection with the reference compounds, DAD-spectra of the glycosides, and HPLC analysis of the aglycones isolated from the fractionated and enzyme hydrolyzed glycosides. The DAD-spectra of the flavonol glycosides together with the aglycone analysis indicated the reasonable purity of all the the five glycoside peaks taken into account.

The compounds numbered in Figure 1 as peaks 1 to 5 were identified as isorhamnetin 3-sophoroside-7-rhamnoside, quercetin 3-O-rutinoside, quercetin 3-O-glucoside, isorhamnetin 3-O-rutinoside and isorhamnetin 3-O-glucoside, respectively. Trace amounts of kaempferol glycosides may have been hidden behind the isorhamnetin 3-O-glucoside and isorhamnetin 3-O-rutinoside peaks.


  1. 6


2 3

Figure 1. HPLC-DAD chromatogram of flavonol glycosides of sea buckthhorn ssp. rhamnoides berries. 1 = isorhamnetin 3-sophoroside-7-rhamnoside, 2 = quercetin 3-O-rutinoside, 3 = quercetin 3-O-glucoside, 4 = isorhamnetin 3-O-rutinoside, 5 = isorhamnetin 3-O-glucoside and 6 = phloridzin (internal standard)

In order to verify the purity of the aglycon moieties of the five glycoside peaks, each peak was isolated by the analytical HPLC column. A thorough investigation with ssp. rhamnoides was carried out. Each fraction collected was re-analyzed with the normal procedure to control that the collection had been successful. After enzymatic hydrolysis, the aglycones were analyzed by HPLC. The glycoside peaks 1, 4 and 5 yielded isorhamnetin, 100 %, 100 % and > 95 %, respectively, and the glycoside peaks 2 and 3 quercetin, > 98 % and > 95 %, respectively, as expected.

Floridzine was used as internal standard in quantitative analysis of the glycosides. Correction factors for the glycosides in HPLC analysis were 1.0 for isorhamnetin 3-O-rutinoside, 0.70 for isorhamnetin 3-O-glucoside, 0.90 for quercetin 3-O-rutinoside and 0.72 for quercetin 3-O-glucoside In case of isorhamnetin 3-sophoroside-7-rhamnoside, correction factor 1 was applied due to the lack of the isolated glycoside for the correction factor determination.

Contents of the flavonol glycosides in different subspecies are summarized in Table 2. In the subspecies rhamnoides and sinensis the five compounds were always the most abundant flavonol glycosides. In the berries of ssp. mongolica isorhamnetin 3-sophoroside-7-rhamnoside existed in trace amounts only, and some other glycosides not investigated in this study were found in more abundance. The proportions of unknown flavonol glycosides seen in Figure 1. could not be defined due to the HPLC-DAD analysis without known response factors.

Table 2. Content of flavonol glycosides in sea buckthorn berries in mg / kg and % of total flavonols (in parentheses).







252  22

(31,6 ± 1,9)

84,4 ± 6,1

(11,1 ± 0,6)

17,3 ± 3,3

(4,9± 0,7)


47,2  3,0

(5,9± 0,2)

55,6 ± 3,0

(7,3 ± 0,6)

33,6 ± 5,7

(9,4 ± 1,1)


30,2  2,1

(3,8 ± 0,2)

44,5 ± 3,0

(5,9 ± 0,3)

36,9 ± 5,8

(10,3± 0,8)


69,7 ± 5,5

(8,7 ± 0,5)

63,9 ± 2,8

(8,4 ± 0,7)

23,3 ± 2,9

(6,6 ± 0,9)


121  8,0

(15,1 ± 0,7)

167 ± 6,1

(22,0 ± 2,2)

50,1 ± 7,0

(14,1 ± 2,1)


799 ± 30

763 ± 81

362  85

The ssp. rhamnoides berries always containes high amounts of isorhamnetin 3-sophoroside-7-rhamnoside regardless of the origing and time of harvesting (unpublished results).

As early as sixty years ago Biehlig showed the existence of isorhamnetin in sea buckthorn berries (Biehlig, 1944), and in 1963 Friedrich reported isorhamnetin-3-glucoside and isorhamnetin-3-rutinoside as typical components of the fruit (Friedrich, 1963). It is of common knowledge that in addition to isorhamnetin and quercetin glycosides also kaempferol and myricetin glycosides exist in sea buckthorn berries (Hoerhammer et al., 1966; Rösch et al, 2003). One of the most reliable investigations is that of Rösch et al. (2003) where proper NMR-data are presented and e.g. the structure of a major compound isorhamnetin 3-sophoroside-7-rhamnoside is verified. According to our knowledge, a comparison between flavonol glycosides of the three, commercially most important sea buckthorn subspecies, ssp. rhamnoides, ssp. mongolica and ssp. sinensis has not been published earlier.


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