Lucrări Ştiinţifice-Seria Zootehnie, vol.

60

ANTIOXIDANT ACTIVITY
IN EXTRACTS FROM SEA BUCKTHORN
Cristina Damian1*, Ana Leahu1, M. Oroian1, M. Avramiuc1, N. Carpiuc2
1
Stefan cel Mare University of Suceava, Romania
2
Paltinoasa Secondary School, Suceava, Romania

Abstract
The antioxidant components of sea buckthorne (Hippophaë rhamnoides L.) stored at 0.5, 10 and
20ºC for 4 days were studied. Overall fruit quality declined more rapidly at 20ºC. Weight loss of
fruit was negligible for 2 days at all temperatures, it increased rapidly from day 3 at 20ºC. Total
phenolic compounds were slightly higher at 20ºC than at other temperatures. Total ascorbic acid
concentrations of the fruit remained similar for the first 2 days of storage, then declined in fruit
stored at 0.5 and 20ºC, but remained unchanged at 10ºC. The total antioxidant activity of fruit was
higher at 10ºC than at 0.5 and 20ºC on day 3. In conclusion, while the best temperature for long-
term storage is 0.5ºC, quality could be maintained at 10ºC for acceptable periods of time for
marketing and may be associated with better nutritional quality.
Key words: sea buckthorn, total phenols, antioxidant activity

INTRODUCTION1 (Hippophaë rhamnoides) fruits are of special

Sea buckthorn is a hardy plant, drought interest because they are characterized by
and cold tolerant, useful for land reclamation two different types of oil accumulation,
and farmstead protection [1]. The genus which are characteristic for the mesocarp and
belongs to the family Elaeagnaceae, which seeds, respectively [5]. Sea buckthorn
consists of six species and 10 subspecies, mesocarp oil includes not only the residues
among which the most economically of widely occurring fatty acids, such as
important one is Hippophaë rhamnoides Linn., palmitic, stearic, oleic, linoleic, and linolenic,
commonly known as sea buckthorn [2]. In but also an unusual hexadecenoic (16:1) acid;
summer, plentiful round yellow-orange berries in most cultivars of this plant species, this
cover the female plants. SB berries consist of a acid serves as a main fatty acid component of
fairly tough skin and juicy pulp enveloping a their oils [6]. Reverse-phase HPLC allowed
small, hard, oval seed. Ripe berries from H. detection of triglycerides (containing
rhamnoides are orange-red and have a palmitoleic acid radicals) in sea buckthorne
diameter of 10–15 mm and a soft outer fleshy oil extracted from the parenchyma of the fruit
tissue and hard seed. Soft parts of the berries, on the background of the conventional
or pulp, contain 3–5% oil and seed contains 6– triglycerides used as extractants. This allows
15% oil (1). SB berries are an excellent source rapid verification of the authenticity of the oil
of bioactive phytochemicals such as and detection of adulteration [7].
carotenoids, tocopherols, vitamin C, organic Sea buckthorn (Hippophaë rhamnoides L.)
acids, and polyphenols [3]. berries are a rich source of flavonoids, mainly
Sea buckthorn (Hippophaë rhamnoides flavonol glycosides and proanthocyanidins [8].
L.) is increasingly recognized as an important Sea buckthorn berries can be processed into
potential source of bioactive ingredients for jams, juices, yellow pigments, and seed oils [9].
functional foods, nutraceuticals, and Juice extraction from sea buckthorn berries leads
personalcare products [4]. Sea buckthorn to a residual press cake, which can be used for
seed separation following extraction of seed oil.
The remaining pulp is reported to contain
*Corresponding author: [email protected] mainly flavones [10]. Berries of sea buckthorn
The manuscript was received: 22.03.2013 have a unique aroma not comparable to any
Accepted for publication: 03.10.2013

- 151 -
University of Agricultural Sciences and Veterinary Medicine Iasi

other common fruit [11]. The flavor of sea Ascorbic acid determination
buckthorn (Hippopha¨e rhamnoides L.) berries Determination of vitamin C content in sea
is perceived as sour and astringent mainly due to buckthorn berries was cut by titration with 2.6-
the high acid content, most importantly malic diclorophenolindophenol (reagent Tillmans).
acid [12]. Due to the high acidity, the berry does The method is based on color change of the
not always attract consumers [13]. reagent, oxidation or reduction. Thus, the
The plant has also been scientifically ionized form of 2,6-diclorophenolindophenol
analyzed and reported to have antioxidative, is red in acid and blue in basic medium.
Dehydroascorbic acid is obtained through
immunomodulatory, cyto-protective, hepato-
reaction with vitamin C, and after reducing the
protective and tissue regenerative properties.
identification reactive, 4-(p-
The efficacy of sea buckthorn leaf extract, hidroxiphenilamino)-2.6 dichlorophe-nol. This
fruit and seed oils has been shown in method is commonly used, due to the fact that
dermalwound healing, burns of different it is easy to use and to the reagent sensitivity.
etiology, skin radiation lesions, gastric and
duodenal ulcers [14]. Moreover, sea Total phenols
buckthorn has demonstrated high ecological Total phenols were determined using the
value; it has been planted on a large scale to Folin-Ciocalteau reagent. 100 mL sample
combat soil erosion, particularly in China and was transferred to a volumetric flask, to
Canada [15]. which 500 mL undiluted Folin-Ciocalteau
reagent was subsequently added. After 1 min,
1.5 mL 20% (w.v-1) Na2CO3 was added and
MATERIAL AND METHOD the volume made up to 10.0 mL with H2O.
Plant material: After 2 h incubation at 25ºC, the absorbance
Sea buckthorn (Hippophaë rhamnoides was measured at 760 nm and compared to a
L.) berries were obtained from shrubs grown gallic acid calibration curve. Total phenols
in Suceava county, România. The berries were determined as gallic acid equivalents.
were cleaned manually to remove all foreign
2,2-Di (4-tert-octylphenyl)-1-pycrilhydra-
material and broken seeds. Sea buckthorn zyl (DPPH) scavenging capacity assay
(Hippophaë rhamnoides L.) berries were
divided in three groups and stored at 0.5, 10 The method used for determining the
and 20ºC for 4 days. antioxidant activity of sea buckthorn extracts
is based on scavenging 2,2-Di (4-tert-
Extraction procedure octylphenyl)-1-picrylhydrazyl (DPPH)
Plant materials were sieved and 30 g were radicals. The decrease in absorbance was
extracted with ethyl acetate in a Soxhlet measured at 515 nm against a blank without
apparatus. All the extracts were freeze-dried extract, using a spectrophotometer. Using a
after evaporation in vacuum and stored at 0.5, calibration curve with different amounts of
10 and 20ºC. A further 30 g ground material DPPH, the IC50 was calculated. The IC50 is the
was macerated with 200 mL of 70% aqueous concentration of an antioxidant that is required
methanol for 24 h and then filtered. This to quench 50% of the initial DPPH radicals
maceration procedure was repeated with fresh under the experimental conditions given.
solvent for 5 days. All the macerates were Statistics
combined and freeze-dried after removal of Samples were assayed in triplicate and
methanol and stored at 0.5, 10 and 20ºC. results are given as averages ± SD. Student’s t
Weight loss determination test was used for the statistical evaluation and P
Weighed samples are placed in an oven < 0.05 was considered statistically significant.
for 4 days at 0.5, 10 and 20ºC.
RESULTS AND DISCUSSIONS
Determination of soluble solids Weight loss determination
In Table 1 weight loss values for sea
concentrations
buckthorn berries samples at temperatures of
The soluble solid concentration was 0.5, 10 and 20ºC are presented.
determined by refractometry.

- 152 -
 Lucrări Ştiinţifice-Seria Zootehnie, vol. 60

As seen from the data presented in for 2 days at all temperatures it increased
Table 1, weight loss of fruit was negligible rapidly from day 3 at 20ºC.

Table 1 Weight loss of sea buckthorn berries

Weight loss (%)
1 day 2 day 3 day 4 day
0
0.5 C 0.45 ± 0.01 0.43 ± 0.02 0.72 ± 0.1 1.13 ± 0.11
0
10 C 0.55 ± 0.2 0.65 ± 0.01 2.11± 0.02 3.84 ± 0.01
0
20 C 0.64 ± 0.02 0.84 ± 0.1 5.22 ± 0.01 9.62 ± 0.02

Ascorbic acid determination

Ascorbic acid concentrations of the fruit
remained similar for the first 2 days of
storage, then they declined in fruit stored at
0.5 and 20ºC, but remained unchanged at
100C (Figure 1).
The variation of ascorbic acid throughout
the storage period (Figure 1) showed that the
vitamin content can be affected by
temperature. Storage at 0.5ºC and 10ºC
temperature was an effective way to maintain Figure 2 Total phenols of sea buckthorn berries
( – 0.5ºC; – 10ºC; – 20ºC)
the initial level of total ascorbic acid for
additional days, but it did not increase the 2,2-Di (4-tert-octylphenyl)-1-pycrilhydra-
vitamin content significantly. zyl (DPPH) scavenging capacity assay
There are reports [16] of the positive
influence of low temperature in the In Figure 3 the IC50 values are indicated.
maintenance of vitamin C content during The lowest IC50 value indicates the highest free
storage of fruit and vegetables. radical scavenging activities. Free radicals are
involved in the process of lipid peroxidation
and are considered to play a cardinal role in
numerous chronic pathologies, such as cancer
and cardiovascular diseases among others, and
are involved in the aging process. Therefore, the
extracts were assessed against DPPH radicals to
determine their free radical scavenging
properties. In this assay, the DPPH radical
serves as the oxidizing substrate, which can be
reduced by an antioxidant compound to its
hydrazine derivative via hydrogen donation,
and as the reaction indicator molecule.
Figure 1 Ascorbic acid concentrations of sea
buckthorn berries
( – 0.5ºC; – 10ºC; – 20ºC)

Total phenols
Total phenols content of sea buckthorn
berries is not altered by storage (Figure 2).
The total phenols content was assayed using
the Folin-Ciocalteau reagent method and is
expressed as ggallic acid.kg-1extract.
Figure 3 Free Radical Scavenging Activities of
extract of sea buckthorn berries
( – 0.5ºC; – 10ºC; – 20ºC)

- 153 -
University of Agricultural Sciences and Veterinary Medicine Iasi

The total antioxidant activity of fruit was L.) Seeds, Russian Journal of Plant Physiology,
higher at 10ºC than at 0.5 and 20ºC on day 3. Vol. 50, No.3, 2003, 381-389
[6] Pchelkin, V.P., Kuznetsova, E.I., Tsydendambaev,
V.D., Vereshchagin, A.G., Distribution of Unusual
CONCLUSIONS Fatty Acids in the Triacylglycerols of Sea Buckthorn
Sea buckthorn has various bioactive Mesocarp Oil, Russian Journal of Plant Physiology,
components with potential health benefits, Vol. 53, No. 3, 2006, 346-354
including anti-diabetic, anti-obese, anti- [7] Deineka, V.I., Deineka, L.A., Sorokopurov,
oxidant and anti-inflammatory. V.N., Reverse-phase HPLC in the analysis of plant
In conclusion, the data presented in this oils. Monitoring of authenticity and detection of
paper indicated that the storage temperatures adulteration of sea buckthorn oil, Pharmaceutical
affect the antioxidant capacity of sea Chemistry Journal, Vol. 43, No. 1, 2009, 51-54
[8] Larmo, P.S., Yang, B., Hurme, S.A.M., Alin,
buckthorn berries. The content of total J.A., Kallio, H.P., Salminem, E.K., Tahvonen,
phenolics was not affected by the temperature R.L., Effect of a low dose of sea buckthorn berries
lowering, while ascorbic acid was affected by on circulating concentration of cholesterol,
temperature. The total antioxidant activity of triacylglycerols and flavonols in healthy adults,
fruit was higher at 10ºC than at 0.5 and 20ºC Eur J Nutr, 48, 2009, 277-282
on day 3. The free radical scavenging property [9] Zadernowski, R., Naczk, M., Czaplicki, S.,
may be one of the mechanisms by which this Rubinskiene, M., Szalkiewicz, M., Composition of
drug is useful as health food. Phenolic Acids in Sea Buckthorn (Hippophaë
rhamnoides L.) Berries, JAOCS, vol. 82, no.3,
While the best temperature for long-term
2005, 175-179
storage is 0.5ºC, quality could be maintained at [10] Rösch, D., Krumbein, A., Kroh, L.W.,
10ºC for acceptable periods of time for marketing Antioxidant gallocatechins, dimeric and trimeric
and may be associated with better nutritional proanthocyanidins from sea buckthorn Hippophaë
quality. This finding is not only of sensorial or rhamnoides L.) pomace, Eur Food Res Technol,
health relevance, but also economical, because of 219, 2004, 605-613
the additional costs involved in the maintenance [11] Tiitinen, K., Hakala, M., Kallio, H.,
of lower temperatures. Headspace volatiles from frozen berries of sea
buckthorn (Hippophaë rhamnoides L.) varieties,
Eur Food Res Technol, 223, 2006, 455-460
REFERENCES [12] Tiitinen, K., Vahvaselkä, M., Laakso, S.,
[1] Périno-Issartier, S., e-Huma, Z., Abert-Vian, Kallio, H., Malolactic fermentation in four
M., Chemat, F., Solvent Free Microwave-Assisted varieties of sea buckthorn (Hippophaë rhamnoides
Extraction of antioxidants from Sea Buckthorn L.), Eur Food Res Technol, 224, 2007, 725-732
(Hippophaë rhamnoides L.) Food By-Products, [13] Tiitinen, K., Vahvaselkä, M., Hakala, M., Laakso,
Food Bioprocess Technol, 4, 2011, 1020-1028 S., Kallio, H., Malolactic fermentation in sea buckthorn
[2] Yoon, M.Y., Oh, J.S., Kang, H., Park, J.-K., (Hippophaë rhamnoides L.) juice processing, Eur Food
Antioxidant and antibacterial behavior for Res Technol, 222, 2006, 686-691
sediment removed ethanol extract from sea [14] Ozerinina, O.V., Tsydendambaev, V.D.,
buckthorn seed, Korean J. Chem. Eng., 2011, 1-5
Effect of Pre-Sowing γ-Irradiation of sea
[3] Ranjith, A., Savin Kumar, K., Vernigopalan, V.V.,
buckthorn Seeds on the Content and Fatty Acid
Arumughan, C., Sawhney, R.C., Singh, V., Fatty
Composition of Total Lipids in the Seeds of the
Acids, Tocols and Carotenoids in Pulp Oil of Three
First Plant Generation, Russian Journal of Plant
Sea Buckthorn Species (Hippophaë rhamnoides L.,
Physiology, Vol. 58, No. 2, 2011, 370-374
H. salicifolia and H. tibetana) Grown in the Indian
[15] Vernet, A., L’argousier (Hippophaë
Himalayas, JAOCS, Vol. 83, no. 4, 2006, 359-364
rhamnoides L.), Phytothérapie, 3, 2006, 125-129
[4] Yang, B., Kallio, H., Analysis of
[16] Cordenunsi BR, Genovese MI, Oliveira do
Triacylglycerols of Seeds and Berries of Sea
Nascimento JR, Hassimoto NMA, dos Santos RJ,
Buckthorn (Hippophaë rhamnoides L.) of Different
Lajolo FM. Effects of temperature on the chemical
Origins by Mass Spectrometry and Tandem Mass
composition and antioxidant activity of three
Spectrometry, Lipids, Vol. 41, no. 4, 2006, 381-392
strawberry cultivars. Food Chemistry 91, 2003,
[5] Vereshchagin, A.G., Tsydendambaev, V.D.,
113-121.
Changes in Triacylglycerol Composition in
Maturing Sea Buckthorn (Hippophaë rhamnoides

- 154 -