Nature has blessed aerobic organisms with an inner defense system thet resist against [600024]

Gheorghe Asachi Technical University of Iași
Faculty of Chemical Engineering and
Environmental Protection

Polyphenol s Extraction and
Encapsulation

Master Student: [anonimizat]: Control and Processing F ood

Iași
2015 -2016

Introduction
Nature has blessed aerobic organisms with an inner defense system thet resist against
oxidative damage due to reactive oxygen species. However, supplementing the natural
defense mechanism with dietary antioxidants might offer better protection against the risk of
certain diseases.
Lipid peroxidation that results from the reaction between unsaturated fatty acids and
molecular oxygen is a severe problem for the oil and fat i ndustry. It not only deteriorates the
quality of fats and fatty foods, bringing about chemical spoilage, but also produces free
radicals and reactive oxygen specues (ROS) which are reportedly associated with
carcinogenesus, mutagenesis, inflammation, agin g and cardiovascular diseases [1]. In the
order to overcome the stability problems of oils and fats, synthetic antioxidants such as
butylatedhydro xyanisole (BHA), butylatedhydroxytoluene (BHT), ter -butyl hydroquinone
(TBHQ) have been used as food additive s.
But recent reports reveal that these compounds may be implicated in many health risk,
including cancer and carcinogenesis [2]. On the other hand, the use of plant -based antioxidant
compounds in foosa and preventive medicine is gaining a great deal of i nterest because of the
potential health benetits [3]. It is well accepted that plants are the richest source of
antioxidants. Among plants, spices are prominent because they contain a wide array of
antioxidants including some vitamins, flavonoids, terpenoi ds, carotenoids, phytoestrogens,
minerals, etc. Which render them or their antioxidant components as preservative agents in
food [4].
Polyphenolic compounds are commonly bound in both edible and inedible plants and they
have been reported to have multiple biological functions such as antioxidant, anti -inflamatory,
anti-cancer and anti -microbial activities [5].

MATERIAL AND METHODS
Plant materials:
1. Clove (Syzygium aromaticum ) a tree belonging to family Mirataceae is one of the
most valuable spices that has been used for centuries as food preservative and for
many madicinal purpouses. Clove is native of Indonesia but nowadays is cultures in
several parts of the world including Brasil. This plant represents one of the r ichest
source of phenolic compounds such as eugenol acetate and gallic acid and posses great
potential for pharmaceutical, cosmetic, food and agricultural applications. Clove
represents one of the major vetegal sources of phenolic compounds as flavonoids,
hidroxibenzoic acids, hidroxicinamic acids and hidroxiphenyl propens. Eugenol is the
main bioactive compound of clove.

With regad to the phenolic acids, gallic acid is the compound found in higher
concentration. However, other gallic acid derivates as hi drolizable tannins are present
in higher concentrations. Other phenolic acids found in clove are the caffeic, ferulic,
elagic and salicylic acids. Flavonoids as kaempferol, quercitin and its derivates are
also found in clove in lower concentrations. Unopen ed flowering buds are used as
spice in food preparations all over the world. Clove oil is traditionally used as
antimicrobial, antiviral, anti -inflammatory, antidiabetic and hepatoprotective agent [6 –
9].
2. Leaves of Salvia officinalis L. (Lamiaceae ) are use d for both culinary and theapeutic
purposes. They possess hypoglycemiant, spasmolytic, stomahic, estrogenic chololetic
[10-14], antioxidant, antiproliferative [15], anti -inflammatory [16] and
gastroprotective properties [17].
The phytochemical contituents of sage leaves include:
– floavonoids (luteolin, apigenin, quercitin -glycosides) [18],
– phenolcarboxylic acids (caffeic, chlorogenic, rosmarinic, ferulic),
– volatile oil (monoterpenic derivates)
– tannins,
– carnosol [17],
– bitter and triterpenic substances [10 -14].
Among phenolic compounds, caffeic and chlorogenic acids are well known for
their hepatoprotective and hypolidaemic activity, through inhibition of lipid
peroxidation and antioxidant properties [19]. Rosmarinic acid (2 -hydroxi –
dihydrocaffeic acid) has a wide spectrum of biological activities including anti –
inflammatory, anti -microbial, antioxidant and immunomodulatory properties [20,
21]. Moreover, rosmarinic adic and ceffeic acid have antidepressant and anxiolytic
effect, throught inhibition of monoamino xidase activity and modulatory properties
[22, 23].
3. Hyper icum perforatum (HP) is a perennial herb og Hypericeae family native to
Europe, West Asia and North Africa [24]. The herb has beem used in folk medicine as
healing and anti -inflammatory agent since a ntiquity. Nowadays, HP is widely used as
an antidepressant remedy in Europe and the United States. HP contains many
bioactive compounds:
– The antidepressant activity is attributed to phloroglucinols (hyperforin,
adhyperforin) located in the unripe fruit, al so contains polyphenolic acids and a
broad range of flavonoids like hyperoside, rutin, quercitin, quercitrin, isoquercitrin
and amentoflavone [25] , while sesquiterpenes and monoterpenoids are major
components of HP essential oil [26].
Flavonoids are most know for their antioxidant, anti -inflammatory and
antimicrobial propeties [27] and their health benefits in chronic diseases, such as
heart disease and cancer [28].

Extraction Procedure

Methods: The extraction procedure was carried out in an ultrasonic bath (Hielscher
Ultrasonics).
Hielscher Ultrasonics specializes in the design and manufacturing of high power
ultrasonic homogenizers for lab, and production level. Ultrasonic power is an effective
and energy -efficient means to apply high shear and intense stress to liquids,
powder/liquid mixtures and slurrie. This makes it a strong alternative to high shear
mixers, hight oressure homogenizers and agitated bead mills.
The applications span mixing, dispersing, particle size reduction, extraction a nd
chemical reactions. [29]

Raw materials
– Plant material
o Clove – 2g
o Salvia officinalis – 2g
o Hypericum perforatum -2g
Solvents
Method I : water – 50 ml
Method II: Ethanol 80% – 50 ml
Extraction Conditions
Amplitude: 100%
Cycle: 1 (10 minutes)
Cycle: 0,5 (20 minutes)

Duration of Extraction : 30 minutes in an ice bath.
Centrifugation (MIRKO spin – Hettich) – 10 minutes, 6500 rpm

Sample dilution 1:10 (0,5 ml of sample + 4,5 ml of distilled water)

Determination of Total Phenolics Content – (TPC)
Total phenols content was determined using the Folin -Ciocalteu colorimetric method as
described by Gao et al. [30]
– 20 µL plant extracts (dilution 1:10) were mixed in a test tube with:
– 200 µL of Folin -Ciocalteu reagent and
– 2 ml of distilled Water
And incubated at room temperature for 3 minutes.
Following by:
– 1 ml of 20% sodium carbonate was added to the mixture, and re -incubated for 2 h
at room temperature.

The absorbance of the resulting blue color was measured using a quartz cuvet at 765 nm.
Gallic acid was us ed as standard and total phenols were expressed as gram gallic acid
equivalents per 100g of extracts. [31]
T80+ UV/VIS Spectrometer, PG Instruments Ltd.

Plant Materials Absorbance
Water Extraction Absorbance
Ethanol 80% Extraction
Clove 0,548 0,972
Salvia officinalis 0,237 0,315
Hypericum perforatum 0,063 0,110

The calibration curve for galic acid used to interpret the results was realised by the
same method as for the analysis of polyphenols (Determination of TPC)
Calibration Curve

Abs Gallic acid
concentration,
mg/L
0.12 100
0.203 200
0.29 300
0.401 400
0.459 500

The results obtained from the spectrophotometer were then analysed using the
calibration curve:

Absorbance Total polyphenols c oncentration TPC
RESULTS: mg/L mg/g Recalculation taking into
account the dilution 1/10
Water -Cloves 0.548 587.3958 14.6849 146.849
Ethanol 80% -Cloves 0.972 1068.254 26.70636 267.0636
Water – Salvia 0.237 234.6907 5.867268 58.67268
Ethanol 80% -Salvia 0.315 323.1505 8.078763 80.78763
Water -Hypericum
perforatum 0.083 60.0393 1.500983 15.00983
Ethanol -Hypericum
perforatum 0.11 90.66 2.2665 22.665

In conclusion, clove contain a higher amount of polyphenols .

REFERENCES
[1] Sikwese, F.E. and Duodu, K. G. (2007) Antioxidant effects of crude phenolic extract from
sorghum bran in sunflower oil in the presence of ferric ions . Food Chemistry .104, 324 -331.
doi:10.1016/j.foodchem.2006.11.042
[2] Prior, R.L. (2004) Absorption and metabolism of anthocy anins: Potential healt effects. In:
Meskyn, M.S., Bidlack, W. R., Davies, A.J., Lewis D.S. and Randolph R.K.,Eds,
Phytochemicals: Mechanisms of Action, CRC Press, Boca Raton, 1 -19.
[3] Hussain, A.I., Anwar, F., Sherazi, S.T.T. and Przybylski, R. (2008) Che mical
composition, antioxidant and antimicrobial activities of basil ( Ocimum basilicum ) essential
oils depends on seasonal variations. Food Chemistry , 108, 986 -995.
doi:10.1016/j.foodchem.2007.12.010
[4] Calucci, L., Pinzono, C., Zandomeneghi, M, and Capoc hi, A. (2003) Effect if gamma
irradiation on the free radical and antioxidant contents in nine aromatic herbs and soices.
Journal of Agricultural and Food Chemistry , 51, 927 -934. doi.10.1021/jf020739n
[5] Jhon, A. M. And Grohmann, K. (2001) phenols in xitr us peel by products, concentration
of hydroxycinnametes and polymethoxylated flavones in citrus peel molasses. Journal of
Agricultural and Food Chemistry , 49, 3268 -3273. doi.10.1021/jf010011r
[6] Ramadan MF, Asker MMS, Tadros M. Lipid profile, antiradical power and antimicrobial
properties of Syzygium aromaticum oil. Grasasy Aceites 2013; 64 (5) :509-520.
Http://dx.doi.org/10.3989/gya.011713
[7] Suganya RA, Jothi GJ. Preliminary phytochemical screening, antibacterial and antioxidant
activies of Commelina mudiflora (C0mmelinaceae). International Research Journal of
Pharmacy. 2014;5(11):651 -855. http://dx.doi.org/107897/2230 -8407.0511174
[8] Dua Anita, Grupta SK, Mittal A. Mahajan R. Bioreactive compound and antioxidant
properties of methanolic extract of Fannel. International Research Journal of Pharmacy 2014;
4 (5):445 -450 http://dx.doi.org.10.7897/2230 -8407.04551
[9] Milind P, Khanna D. Clove; A champion Spice, International Journal of Research in
Ayureda and Pharmacy 2011; 2(1):47 -54
[10] Dumitrescu C., Olteany R.L., Bumbac M., Gorghiu L.M., Antioxidant effect of some
flavonoids on organic substrate. Rev. Chim.,2009; 60(4):329 -331.
[11] Istudor V., Farmacognozie, Fitochimie, Fitoteraoei, ed. Medical[, Buc., 2001; vol. II,
115-119.
[12] Manicula D., Roba C., Oprean I., Misca -Barbu R., Sy nthesis of ursoluc acid dipeptide
derivates with potential bilogical ctivity. Rev. Chim, 2013; 64(12):1454 -1458
[13] Stanescu U., Hancianu M., Miron A., Aprotosoaie C., Plante medicinale de la A la Z,
monografii ale produselor de interes terapeutic, ed. Gr . T. Popa Iasi. 2004; vol II, 503 -505.

[14] Teuscher E., Anton R., Lobstein A., Plantes aromatiques, epices, aromates, condiments et
huiles essentielles, TEC & DOC, Paris, 2005; 445 -448.
[15] Kontogiani V.G., Tomic G., Nikoloc I., Nerantzaki A., Sayyad N., Stosic -Grujicic S.,
Stonovic I., Gerothanassis I.P., Tzakos A.G., Phytochemical profile of Rosmarinus
officinalis and Salvia officinalis extracts and correlation to their antioxidant and
antiproliferative activity. Food Chemistry, 2013; 136(1); 120 -129.
[16] Bauer J., Kuehnl J., Rollinger J.M., Scherer O., Northoff H., Stuppner H., Werz O.,
Koeberle A., Carnasol and carnosic acids from Salvia officinalis inhibit microsomal
prostagaldin E2 synthase -1. The journal of Pharmacology and Experimantal Thera peutics,
2012; 342(1); 169 -176.
[17] Mayer B., Baggio C.H., Freitas C.S., Dos Santos A.C., Twardowschy A., Horst H.,
Pizzolatti M.G., Micke G.A., Heller M., Dos Santos E.P., Otuki M.F., Marques M.C.A.,
Gastroprotective constituents of Salvia officinalis L . Fitoterapia , 2009; 80: 421 -426.
[18] Roby M.H., Sarhan M.A., Selim K.A.H., Khael K.I., Evaluation of antioxidant activity,
total polyphenols and phenolic compounds in thyme ( Thymus vulgaris L.), sage ( Salvia
officinalis L .) and marjoram ( Origanum majora na L.) extracts . Industrial Crops and
Products , 2013; 43: 827 -831.
[19] Karthikesan K., Pari L., Menon V.P., Antihyperlipidemic effect of chlorogenic acid and
tetrahydrocurcumin in rats subjected to diabetogenic agents. Chemico -Biological Interactions ,
2010; 188(3); 643 -650.
[20] Bruneton J., Pharmacognoise, Phytochimie, Plantes medicinales, TEC & DOC, Paris,
2005; 249 -250.
[21] Harborne J., Baxter H., Moss G.P., Phytochemical dictionary, Taylor&Francis Group ,
1999, second edition, 533.
[22] Takeda H., Ca ffeic acid produces antidrepressive – and/or anxiolytic -like effects through
indirect modulation of the [alpha]1A -adrenoceptor system in mice. Clinical Neuroscience and
Neuropathology , 2003; 14(7): 1067 -1070.
[23] Takeda H., Tsuji M., Inazu M., Egashira T. , Matsumiya T., Rosmarinic acid and caffeic
acid produce antidepressive -like effect in the forced swimming test in mice. Biological &
Pharmaceutical Bulletin, 2002; 449(3): 261 -267.
[24] Greeson, Sandord, & Monti 2001
[25] Butterweck and Schmidt, 2007, Tat sis et al., 2007 and Umek et. Al., 1999)
[26] Schwob, Bessiere, & Viano, 2002
[27] Boots et al., 2008, Cowan, 1999, Cushnie and Lamb, 2005 and Pietta, 2000
[28] Boots et al., 2008 and Clifford and Brown, 2006.

[29] www.hielscher.com/ultrasonic -homogenizers -for-liquid -processing -3.htm
[30] Gao, X., Ohlander, M., Jeppsson, N., Bjork, L, and Rrajkovski, V. (2000) changes in
antioxyd ant effects and their relationship to phytonutrients in fruits of sea buckthorn
(hippophaerhamnoides L) during maturation. Journal of Agricultural and Food Chemistry,
48, 1485 -1490. doi:10.1021/jf991072g
[31] Hilaire Macaire Womeni, Fabrice Tonfack Djikeng , Bernard Tiencheym Micher Linder,
Advances in Biological Chemistry, 2013, 3, 304 -313.
http://dx.doi.org/10.4236/abc.2013.33034