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    • Abstract: European Journal of Scientific ResearchISSN 1450-216X Vol.29 No.1 (2009), pp.47-54© EuroJournals Publishing, Inc. 2009http://www.eurojournals.com/ejsr.htmUsing HPLC to Determination the Composition and Antioxidant

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European Journal of Scientific Research
ISSN 1450-216X Vol.29 No.1 (2009), pp.47-54
© EuroJournals Publishing, Inc. 2009
Using HPLC to Determination the Composition and Antioxidant
Activity of Berberis Vulgaris
Parichehr Hanachi
Women Research Centre, Biomedical Unite, Alzahra University
Tehran, Iran
E-mail: [email protected]
Tel: +989125426316; Fax: +982177498112
Golkho SH
Women Research Centre, Biomedical Unite, Alzahra University
Tehran, Iran
The Berberis vulgaris (BV) plant has been extensively used as a traditional
medicine in some part of Asian region especially in Western Asia. The BV fruits is claimed
also to have anti-viral activities, and as a treatment for chronic candidiasis, indigestion and
parasites. The present study was conducted to evaluate the antioxidative activity, nutritional
and antinutritional composition of Berberis vulgaris fruit. The antioxidant activity was
determined by using thiobarbituric acid (TBA method). Absolute ethanol, absolute
methanol and aqueous solution (distilled water) were used as a solvent in this study. Result
shows significantly differences (P< 0.05). The antioxidant activity of ethanol extract was
the highest (27.26 ±1.07 %), followed by BHT (20.29 ±0.23 %), methanol extract (16.80
±0.23 %), Vitamin E (6.68 ±0.25 %) and the lowest was the aqueous extract (6.53 ±0.29
%). Besides antioxidant activity, Berberis vulgaris also contains nutritional and
antinutritional composition namely vitamin C, malic acid and Tannin. Vitamin C and malic
acid also shows antioxidative properties. Vitamin C and malic acid also showed
antioxidative properties and act synergistically to each other as antioxidants. The presence
of antinutritional such as Tannin which can act as a medium to prevent nutritional
composition from being absorbed by the body also has been detected. The amount of
nutritional and antinutritional detected in 100g of berberry were 11102.81μg ± 2.01 of
Vitamin C, 116.03μg ± 1.12 of Malic acid and 20.51μg ± 0.59 of Tannin.
Keywords: Antioxidant, Berberis vulgaris, Thiobarbituric acid, Vitamin C, Malic acid,
Tannin, HPLC.
Medicinal plant research includes much more than the discovery of new drugs. This field has been
expanding to also include such diverse subjects as negotiation of power based on medicinal plant
knowledge (Garro, 1986) and the co-evolution of humans and plants (Alcorn, 1981). The field also
provides opportunities to study how human interaction with biological diversity is influenced by
human psychology, cognition, and evolution. Therefore the identification of active plant chemicals is
Using HPLC to Determination the Composition and Antioxidant Activity of Berberis Vulgaris 48
an essential component of modern pharmacognosy and medical effects are not necessarily restricted to
a single plant chemical. The biological activity and clinical value of the whole plant, as in medicinal
herbalism, is also being pursued (Pasquale, 1984).
The importance of identification of active plant chemicals does not limit itself to local
Malaysian plants but also sparks an inquisitive knowledge to foreign plants. This effort leads to study a
non-native plant called Berberis vulgaris which predominantly found in Northern Asia and Europe
(Chevallier, 2001).
The determination of nutritional and anti-nutritional compounds in Berberis vulgaris or
berberry fruits is very important as this fruit in a part of daily diet of certain people. Their presence and
relative ratio, in fact, can affect the chemical and sensorial characteristics of the matrix (e.g., pH, total
acidity, microbial stability, sweetness, global acceptability) and can provide precious information on
food wholesomeness or on how to optimize some selected technological processes (Romero, et al
1992). The nutritional values such as vitamin components, antioxidant compounds in this plant might
be invaluable for treating diseases as more secrets within this plant are yet to be discovered. Hanachi et
al 2005 reported that Berberis vulgaris exhibited vary degree of antioxidant properties Recently
(Maznah, et al, 1999) had shown that antioxidant capacities varied according to the plant extracts and
the comparison between different solvents for the extraction method could be done especially between
water and organic solvents to investigate which extract had the highest antioxidant activities.
The identification of certain alkaloids and phenolic compounds in Berberis vulgaris somehow
provide an alternative method for medicine and remedies as many studies have proved to alleviate
certain ailments in treating liver cancer, gall bladder problems, kidney stones, menstrual pains and etc
(Hanachi et al, 2008). All these problems have lead to the development of new drugs derived from
plants, which is believed to be safer and more effective (Wargovich, 1999). The development of drugs
from plants continues with the research of screening on herbs (Vickers and Zollman, 1999). This
shows that herbal medicine has created a great deal of public interest. The objective of this study was
to Using HPLC to determination the composition and antioxidant activity of Berberis vulgaris.
Material and Methods
The fruits of Berberis vulgaris were purchased from Iranian market in Kuala Lumpur. The fruits were
bought in the dried form and were prepared to be extracted.
Preparation of Plant Extract
Barberry fruit extract was prepared according to method modified Shamsa et al. 310 g BV fruit that
was free from fungus, bacteria and any other plant diseases were selected randomly. The fruits were
dried in an oven (Memmert SLM 400, GmbH, Germany) for 3 days at a constant temperature of 65˚C.
The fruits were cut into small pieces and were grounded into fine powder using a dry grinder. The
grounded samples were sieved to get uniform particle size, then were kept in air-tight container and
stored for further extraction. The sample was extracted by boiling in water for 20-30 minutes at a
temperature of 75˚C.Water and ethanol were added in the ratio of 1:10 and stirred at 250 rpm in an
orbital shaker (Unimax 1010, Heidolph Instruments GmbH & Co.KG,Germany) for 1 h at room
temperature. The extract was then separated from the residue by filtration through Whatman No.1 filter
paper. The remaining residue was re-extracted twice, and then the two extracts were combined. The
residual solvent of ethanolic extract was removed under reduced pressure at 50˚C using a rotary
evaporator (BÜCHI Rotavapor R-200, Germany) until thick syrup was collected. The thick syrup was
evaporated completely using freeze drying system (FreeZone 77520, LABCONCO, USA) for the
determination of total antioxidant activity and phenolic content.
49 Parichehr Hanachi and Golkho SH
Absolute Methanol and Aqueous Extractions
For absolute methanol and aqueous extractions, the extraction was done according to Endrini (2002)
with slight modification. The extraction process was carried out by soaking about 100 g of dried
powder in 1000 ml absolute methanol for 24 hours. The supernatant was filtered using Whatman filter
paper no. 1. The supernatant was then evaporated at 45°C under reduced pressure and subsequently air-
dried. The above steps were repeated to obtain aqueous extraction.
Determination of Antioxidant Activity
The method of Ottolenghi (1959) was used to determine the TBA values of the samples. In brief, a
mixture of 4 mg of Berberis vulgaris extracts, BHT and α- tocopherol acetate (vitamin E) in 4 ml
absolute ethanol, 4.1 ml of 2.52% linoleic acid in absolute ethanol, 8 ml of 0.05 M phosphate buffer
(pH 7.0), and 3.9 ml of distilled water was placed in a vial with a screw cap and then placed in a
shaking water bath (100 rpm) at 40°C in the dark for 24 hours. A control was set up by replacing the
sample with 4 ml ethanol. After the incubation period, 2 ml of 20% trichloroacetic acid (TCA) solution
and 2 ml thiobarbituric acid (TBA) solution were added to 1 ml of sample solution. The mixture was
placed in a boiling water bath for 10 minutes. After cooling, it was centrifuged at 3,000 rpm for 10
minutes and the absorbance of the supernatant was measured at 532 nm. The antioxidant activities
were calculated as below:
% Antioxidant Activity = (ODcontrol - ODsample/ODcontrol) X 100 %
In which ODsample is the optical density of sample and ODcontrol is optical density of control.
Determination of Nutritional and Antinutritional Component
Standards of nutritional and anti-nutritional for ascorbic acid, malic acid and tannin (Sigma Chemicals,
Co, USA) were used. The sample preparation techniques must be done before using the HPLC for the
purpose of eliminating contamination and to get the best outcome of the experiment. Sample
preparation of Vitamin C determination was prepared with a mixture of KH2PO4, methanol and
TBAOH with ratio of 97:3:0.05. The following sample preparation for Tannin was used. Samples of
berberry fruits were dissolved in 100ml of a mixture of 100 volumes of 10% methanol and 0.1 volume
of phosphoric acid. Different steps were used to prepare Malic acid. About 3 grams of berberry
samples were diluted into 3 ml of distilled or purified water. Therefore the concentrations must be
50:50, v/v.
Determination of Vitamin C, Malic acid and Tannin
Hewlett Packard series 1100 consist of Hewlett Packard 1049A programmable electrochemical
detector, degasser G1322A, UV/ visible detector was used to detect Vitamin C, Malic acid and Tannin.
The method of Abdulnabi et al, 1997 with a slight modification was used to determine Vitamin C using
HPLC. The content of organic acid of Malic acid can be determined by using method from Rebecca,
(2000). Meanwhile the presence of Tannin is adopted from Hagerman et al., 1992 with slight changes.
This method is particularly useful for separating the constituents of tannic acid in a simple isocratic
system. Better resolution can be obtained with reversed phase system especially if gradient HPLC is
available. Typical run takes about 30 minutes to ensure elution of all peaks although longer runs could
also be necessary if a sample had very high molecular weight Tannin. The conditions for Vitamin C,
Malic acid and Tannin are listed in Table 1
Using HPLC to Determination the Composition and Antioxidant Activity of Berberis Vulgaris 50
Statistical Analysis
The results obtained were analyzed using one-way ANOVA for mean differences. The Statistical
Package for Social Science for windows version (12.0) was used to analyze the data. The (p

Use: 0.1335