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Optimization of Lyophilization Technology for Purification and Stabilization of Anthocyanins from Elderberry Fruits

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Natural medicinal compounds of plant origin have numerous perceived advantages in selected therapeutical applications compared with the synthetic chemistry. With regard to general isolation of these compounds (secondary metabolites of plants), distillation and extraction methods are most often used. The technique of lyophilization (freeze drying) has certain advantages over distillation or extraction technologies, especially with unstable compounds such as anthocyanins. The aim of this research was the optimization of a lyophilization process with acetone extracts of elderberry (Sambucus nigra) fruits. The device Lyophil SMART LYO 2 from the German producer GEA was used for the lyophilization. The work consisted of two parts: optimizing the dilution of samples by deionized water for mechanical bottle line filling (which also affected final product quality), and optimizing the lyophilization procedure to achieve the final product in the form of a dry powdered tablet at the end of the process. The optimal dilution for the elderberry extract was 4.7:1. The program for the lyophilization of elderberry extract was optimized in 11 steps, where temperature and pressure in time were changed. The basic research in its various stages confirmed the assumption that isolated natural substances (anthocyanins) can be extracted from plant material by freeze-drying, thus stabilizing their biological activities. INTRODUCTION Anthocyanins (also anthocyans; from Greek: ἀνθός (anthos) = flower + κυανός (kyanos) = blue) are water-soluble vacuolar pigments that may appear red, purple, or blue depending on the pH of the solution. These natural components can occur in all tissues of most higher plants, including leaves, stems, roots, flowers, and fruits. Generally they are known to provide health benefits when consumed by humans, and have anti-inflammation, antimicrobial, anti-tumor, anti-mutagenic and antioxidant pharmacological properties (Prasad and Aggarwal, 2011). Anthocyanins are unstable in changing pH, high temperatures and prolonged light exposure (He et al., 2012). The renewed interest by industrial countries in traditional herbal medicines and the development of 'functional foods' are stimulating the need for more information regarding the bioavailability and efficacy of plant polyphenols. Anthocyanins, potent flavonoid antioxidants widely distributed in fruits, vegetables and red wines, normally occur in nature as glycosides, a form not usually considered as bioavailable (Milbury et al., 2002). Various plant species, including elderberry (Sambucus nigra L.), contain different amounts and types of anthocyanins (Lee and Finn, 2007; Fejer et al., 2015). Anthocyanins can be used as dietary supplements, but they must be prepared, stored, packaged, and transported using methods that do not contribute to their degradation. Distillation methods (hydro distillation or water vapor) are often used to extract natural compounds and secondary metabolites from plants. Distillation can extract volatile oils and solids to produce liquid and dry extracts. In both methods, a variety of solvents and high temperatures are used, which can directly affect the stability, and frequently the breakdown of some unstable natural compounds. Extraction of anthocyanins from elderberry fruits using acidified ethanol-water solutions has been
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Optimization of Lyophilization Technology for Purification and
Stabilization of Anthocyanins from Elderberry Fruits
I. Salamon1, D. Grulova1, M. Hancianu2 and O. Cioanca2
1 University of Presov, Department of Ecology, 01, 17th November St., SK-081 16 Presov,
Slovak Republic
2 University of Medicine and Pharmacy, Department of Pharmacognosy, 16 University
St., RO-700 115 Iasi, Romania
Keywords: Sambucus, extract, freeze-drying, dilution, conditions
Abstract
Natural medicinal compounds of plant origin have numerous perceived
advantages in selected therapeutical applications compared with the synthetic
chemistry. With regard to general isolation of these compounds (secondary
metabolites of plants), distillation and extraction methods are most often used. The
technique of lyophilization (freeze drying) has certain advantages over distillation or
extraction technologies, especially with unstable compounds such as anthocyanins.
The aim of this research was the optimization of a lyophilization process with
acetone extracts of elderberry (Sambucus nigra) fruits. The device Lyophil SMART
LYO 2 from the German producer GEA was used for the lyophilization. The work
consisted of two parts: optimizing the dilution of samples by deionized water for
mechanical bottle line filling (which also affected final product quality), and
optimizing the lyophilization procedure to achieve the final product in the form of a
dry powdered tablet at the end of the process. The optimal dilution for the
elderberry extract was 4.7:1. The program for the lyophilization of elderberry
extract was optimized in 11 steps, where temperature and pressure in time were
changed. The basic research in its various stages confirmed the assumption that
isolated natural substances (anthocyanins) can be extracted from plant material by
freeze-drying, thus stabilizing their biological activities.
INTRODUCTION
Anthocyanins (also anthocyans; from Greek: ἀνθός (anthos) = flower + κυανός
(kyanos) = blue) are water-soluble vacuolar pigments that may appear red, purple, or blue
depending on the pH of the solution. These natural components can occur in all tissues of
most higher plants, including leaves, stems, roots, flowers, and fruits. Generally they are
known to provide health benefits when consumed by humans, and have anti-
inflammation, antimicrobial, anti-tumor, anti-mutagenic and antioxidant pharmacological
properties (Prasad and Aggarwal, 2011). Anthocyanins are unstable in changing pH, high
temperatures and prolonged light exposure (He et al., 2012).
The renewed interest by industrial countries in traditional herbal medicines and the
development of ‘functional foods’ are stimulating the need for more information
regarding the bioavailability and efficacy of plant polyphenols. Anthocyanins, potent
flavonoid antioxidants widely distributed in fruits, vegetables and red wines, normally
occur in nature as glycosides, a form not usually considered as bioavailable (Milbury et
al., 2002). Various plant species, including elderberry (Sambucus nigra L.), contain
different amounts and types of anthocyanins (Lee and Finn, 2007; Fejer et al., 2015).
Anthocyanins can be used as dietary supplements, but they must be prepared, stored,
packaged, and transported using methods that do not contribute to their degradation.
Distillation methods (hydro distillation or water vapor) are often used to extract
natural compounds and secondary metabolites from plants. Distillation can extract
volatile oils and solids to produce liquid and dry extracts. In both methods, a variety of
solvents and high temperatures are used, which can directly affect the stability, and
frequently the breakdown of some unstable natural compounds. Extraction of
anthocyanins from elderberry fruits using acidified ethanol-water solutions has been
Proc. Is
t
IS on Elderberry
Ed.: A.L. Thomas
Acta Hort. 1061, ISHS 2015
246
developed (Salamon et al., 2015), however the resulting products remain unstable.
Lyophilization may be more suitable for isolating natural substances that are more
sensitive to solvents and high temperatures. The lyophilization process is primarily used
in pharmaceutical industries to produce dry injectable products which are diluted in
different solutions before use.
Lyophilization (freeze-drying) is performed using a simple principle of physics
called sublimation. Sublimation is the transition of a substance from the solid to the vapor
state, without first passing through an intermediate liquid phase. This technology is
important in pharmaceutical, food and cosmetic industries. The process of lyophilization
consists of two standard steps (Deluca, 1985; Salamon, 2012): 1) freezing at atmospheric
pressure, and 2) evacuation in reducing pressure. On a larger scale, freezing is usually
done directly in a freeze-drying machine. In this step, it is important to cool the material
below its triple point, the lowest temperature at which the solid and liquid phases of the
material can coexist. This ensures that sublimation rather than melting will occur in the
subsequent steps. Larger crystals are easier to freeze-dry. To produce larger crystals, the
product should be frozen slowly or the temperature can be cycled up and down
(Willemer, 1991). Primary drying is the sublimation action which turns solid to gas and
its dissipation from space of lyophilization. Secondary drying is the process of removing
residual moisture at increased temperatures, and its dissipation from space of
lyophilization. The secondary drying step aims to remove unfrozen water molecules,
since the ice is removed in the primary drying phase. This part of the freeze-drying
process is governed by the material’s adsorption isotherms. In this step, the temperature is
increased, and can even be above 0°C, to break any physical-chemical interactions that
have formed between the water molecules and the frozen material. Usually the pressure is
also lowered in this stage to encourage desorption (typically in the range of microbars, or
fractions of a Pascal). However, there are products that benefit from increased pressure as
well. After the freeze-drying process is complete, the vacuum is usually broken with an
inert gas, such as nitrogen, before the material is sealed. At the end of the operation, the
final residual water content in the product is extremely low, around 1-4% (Pregnolo and
Curto, 1992).
The process of lyophilization is a unique process which differs from one solution
and product to another. Optimizing the lyophilization of a particular compound usually
consists of two parts: first, optimization of sample dilution in order to obtain an ideal
initial and final consistency, and second, optimization of the lyophilization program.
There are a few reports about the extraction, content, composition, and properties of
anthocyanins from elderberry (He et al., 2012; Lee and Finn, 2007; Thole et al., 2006;
Milbury et al., 2002; Inami et al., 1996) but none describe the purification and
stabilization of anthocyanins by lyophilization. The aim of the research, therefore, was to
optimize the lyophilization technology for processing acetone extracts of elderberry fruits
and isolation of pure anthocyanins.
MATERIALS AND METHODS
Plant Material
Ripe fruits of the elderberry cultivar ‘Hachsberg’ were collected from commercial
production fields in the area of Lesne, district of Michalovce, in Eastern Slovakia, with
the geographical coordinates 48°78’39”N, 21°81’87”E in 2012.
Acetone Extraction
Freeze-drying followed the extraction method developed by Salamon et al. (2015)
to obtain pure anthocyanins from elderberry fruits. One kg of fresh fruits was macerated 5
times in an excess volume of acetone (double weight of plant material). The filtrate was
separated by vacuum aspiration, transferred to a separator funnel, mixed with a double
volume of chloroform and shaken several times. The solution was stored overnight at
4°C. The aqueous phase was then separated off by the influence of gravitation and
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physical properties of two non-mixing solutions – water and chloroform. Additionally the
residues of acetone and chloroform were evaporated off of the extracts in a vacuum
evaporator at 38°C. Extracts were then frozen in aliquots of about 400 ml until transferred
to Medicproduct, Co. (Lipany, Slovakia) for lyophilization.
Dilution
In order to utilize the industrial-scale lyophilization equipment available at
Medicproduct, which included an automated vial-filling line (Fig. 1), it was necessary to
thaw the extracts and dilute them to a consistency that could be used in the equipment.
After defrosting the original extracts, they had a dense consistency which could not be
used in the equipment. This dilution also affected the quality and consistency of the final
lyophilized product. The extracts were diluted in an experimental manner with purified
deionized water that complies with the European Pharmacopoeia (Aqua purificata
PhEur). The experiment was conducted and refined in two steps. First we studied
dilutions (water:extract) in ratio 1:1, 2:1, 3:1, 4:1 and 5:1. After determining that a
satisfactory dilution rate should be between 4:1 and 5:1, we refined the dilution by
conducting a second experiment using dilutions in ratio 4.1:1, 4.3:1, 4.5:1, 4.7:1 and 4.9:1
(Tables 1 and 2). These dilution experiments were repeated three times in order to
determine the optimum dilution rate for lyophilization, including automated vial-filling,
and the consistency and quality of the final product.
Lyophilization Program
Frozen extracts were defrosted at room temperature, diluted (as above), and
mechanically loaded into small vials in amounts of 4 ml for lyophilization. Hundreds of
combinations of lyophilization steps and procedures (temperature, time, and pressure
variables) were tested in order to obtain the most desirable final product in the form of a
dry powdered tablet (Tables 3 and 4). In many experiments we tested changes of the
temperatures in steps 2, 3 and 4 of the lyophilization process, ranging from -30 to -40°C
in 1°C intervals. The fourth step (evacuation) started the process of drying the sample. In
step 5, we tested the time for the initial drying in the range 400-800 min in 10 min
intervals. Steps 6-9 tested the changes in temperature (from -5 to 35°C) and time of
continued drying process. For the final drying steps (9-11) we experimented with pressure
and time. We tested increase of pressure from 0.1 to 0.2 mBar at step 9, no pressure
changes (0.1 mBar) at step 10 and decrease of pressure from 0.1 to 0.05 mBar at final
step. The time of drying in mentioned steps varied from 180 to 270 min.
Lyophilization Process
Lyophilization of anthocyanin extracts was performed by a Lyophil SMART LYO
SL2 (GEA, Düsseldorf, Germany) at the Research and Development Department,
Medicproduct, Co., Lipany, Slovakia. The liquid material (defrosted extract) was filled on
an automatic filling line (Flexicom Watson Marlow FPC 50W, Denmark) into vials (size
10R, diameter 24 mm, height 45 mm) in approximate volume of 4 ml. Vials were capped
after filling, with rubber stoppers (type no. V9355), placed to the position needed for
lyophilization and loaded into the lyophilizer. After lyophilization, the stoppers were
closed and capped on the line (edging aluminum stopper type no. 25345).
RESULTS AND DISCUSSION
Dilution
The extract dilution experiments were conducted in two steps. The results of the
initial (less precise) step consistently produced a thick, powder-like porous mass with
dilution 4:1, and a very dense powdered tablet with dilution 5:1 (Table 1), suggesting that
an ideal dilution would be between these levels. The next, more refined, dilution trials
therefore were focused between these two rates. The ratio of 4.7:1 consistently proved
optimal, working well in the automated filling line and ultimately yielding a desired dry
248
powdered tablet of pure anthocyanins (Table 2, Fig. 2). More concentrated extract
finished as a sticky fluid mass reminiscent of “jam” but not the desired powdered tablet.
Lyophilization Program
Hundreds of combinations of temperature, time, and pressure steps were evaluated
in the lyophilization program, with experimental programs ranging from 33 to 37 h (Table
3). The optimum lyophilization program required 36 h, where the temperature was
lowered to -35°C (Table 4). Usually in pharmaceutical industry for producing
medicaments, the freezing temperatures are between -50 and -80°C (Deluca and
Lachman, 1995). This program was refined into eleven timed steps where the temperature
and pressure changed. This included four overall stages – loading, freezing, evacuating
and drying.
The initial freezing step is the most critical in the whole freeze-drying process,
because the product can be spoiled at this stage. In this case, the freezing was done
rapidly, in order to lower the material to below its eutectic point quickly, thus avoiding
the formation of ice crystals. Amorphous materials do not have a eutectic point, but they
do have a critical point, below which the product must be maintained to prevent melt-
back or collapse during primary and secondary drying (Deluca and Lachman, 1995). The
amount of heat necessary can be calculated using the sublimating molecules’ latent heat
of sublimation. In the initial drying phase, about 95% of the water in the material is
sublimated. This phase may be slow (sometimes several days in the industry), because, if
too much heat is added, the material’s structure could be altered.
Pressure is controlled through the application of partial vacuum. The vacuum
speeds up the sublimation, making it useful as a deliberate drying process. Furthermore, a
cold condenser chamber and/or condenser plates provide a surface(s) on which the water
vapor can re-solidify. This condenser plays no role in keeping the material frozen; rather,
it prevents water vapor from reaching the vacuum pump, which could degrade the pump’s
performance. Condenser temperatures are typically below -50°C (Deluca, 1985; Salamon,
2012). It is important to note that, in this range of pressure, the heat is applied mainly by
conduction or radiation; the convection effect is negligible due to the low air density.
Freeze-drying is a relatively expensive process. The equipment is about three
times as expensive as the equipment used for other separation processes, and the high
energy demands lead to high production costs. Furthermore, freeze-drying also has a long
process time, because the addition of too much heat to the material (to speed the process)
can cause melting or structural degradation. The low operating temperature of the process
leads to minimal damage of these heat-sensitive products – in our case the anthocyanins.
Therefore, freeze-drying is often reserved for materials that are heat-sensitive, such as
proteins, enzymes, microorganisms, and blood plasma (Timothy, 1990; William, 1984).
Our successful experiments yielded a pure and stable anthocyanin product.
Anthocyanins extracted from elderberry stored as a powder are protected against
degradation. This product could be used for many different purposes in dietary
supplements. These results set the stage for additional research on the use of
lyophilization to isolate and stabilize beneficial plant metabolites in elderberry and other
plants or fruits.
CONCLUSION
The pharmaceutical industry uses freeze drying to produce tablets or wafers which
require less excipient and result in better absorption of easily administered dosage forms.
In chemical synthesis, products are often freeze-dried to make them more stable or easier
to dissolve in water for subsequent use. In bioseparations, freeze-drying can be used as a
late-stage purification procedure, because it can effectively remove solvents.
Furthermore, it is capable of concentrating substances with low molecular weights that
are too small to be removed by a filtration membrane (William and Polli, 1984). The
pharmaceutical company Medicproduct, Co., in Lipany (Slovakia) uses freeze-drying to
increase the shelf life of products such as vaccines and other injectables. By removing the
249
water and sealing the material in a vial, it be easily stored, shipped, and later reconstituted
to its original form for injection, or for direct or indirect consumption as a high-quality,
stable natural plant product with health benefits.
ACKNOWLEDGEMENT
This research was supported by the Slovak Research and Development Agency
(SRDA), the project: SK-RO-0002-12 “Monitoring of Anthocyanins Content in Selected
Plant Species and Determination of their Antioxidant Activity”
Literature Cited
Deluca, P. 1985. Fundamentals of Freeze-Drying Pharmaceuticals. I.I.R.-Com.C1, Tokyo,
Japan.
Deluca, P. and Lachman. K. 1995. Lyophilization of pharmaceuticals IV: determination
of eutectic temperatures of inorganic salts. J. Pharm. Sci. 54(10):1411-1415.
Fejer, J., Salamon, I., Grulova, D., Michalek S. and Zvalova, M. 2015. Elderberry
(Sambucus nigra) cultivation in Slovak Republic and identification and quantification
of anthocyanins. Acta Hort. 1061:253-258.
He, F., Liang, N., Mu, L., Pan, Q.-H., Wang, J., Reeves, M.J. and Duan, Ch.-Q. 2012.
Anthocyanins and their variation in red wines I. Monomeric anthocyanins and their
color expression. Molecules 17(2):1571-1601.
Inami, O., Tamura, I., Kikuzaki, H. and Nakatani, N. 1996. Stability of anthocyanins of
Sambucus canadensis and Sambucus nigra. J. Agric. Food Chem. 44(10):3090-3096.
Lee, J. and Finn, C.E. 2007. Anthocyanins and other polyphenolics in American
elderberry (Sambucus canadensis) and European elderberry (S. nigra) cultivars. J. Sci.
Food Agric. 87(14):2665-75.
Milbury, P.E., Cao, G., Prior, R.L., Blumberg, J. 2002. Bioavailablility of elderberry
anthocyanins. Mech. Ageing Dev. 123(8):997-1006.
Prasad, S. and Aggarwal, B.B. 2011. Turmeric, the golden spice from traditional medicine
to modern medicine. In: I.F.F. Benzie and S. Wachtel-Galor (eds.), Herbal Medicine:
Biomolecular and Clinical Aspects. 2nd ed., CRC Press, Boca Raton, FL.
Pregnolato, F. and Curto, P. 1992. Parenteral lyophilization facilities: an innovative
approach to loading and unloading operations. Proceedings of International Congress:
Advanced Technologies for Manufacturing of Aseptic and Terminally Sterilized
Pharmaceuticals and Biopharmaceuticals. Parenteral Drug Assoc., Basel. p.4-30.
Salamon, I. 2012. Správna poľnohospodárska, zberová a výrobná prax liečivých rastlín. 1.
vyd., Prešov: Centrum excelencie ekológie živočíchov a človeka PU v Prešove,
Grafotlač Prešov, s.r.o., ISBN 987-80-89561-11-7.
Salamon, I., Mariychuk, R. and Grulova, D. 2015. Optimal extraction of pure
anthocyanins from fruits of Sambucus nigra. Acta Hort. 1061:73-78.
Thole, J.M., Kraft, T.F.B, Sueiro, L.A., Kang, Y.-H., Gills, J.J., Cuendet, M., Pezzuto,
J.M., Seigler, D.S. and Lila, M.A. 2006. A comparative evaluation of the anticancer
properties of European and American elderberry fruits. J. Med. Food 9(4):498-504.
Willemer, H. 1991. Loading: a Critical Step in Freeze-Drying of Sensitive
Pharmaceuticals. I.I.R.-C1, Tokyo, Japan.
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Tables
Table 1. First step of extract dilution: testing the optimal water:elderberry extract ratio
(v/v) needed for mechanical bottle line filling prior to lyophilization.
Water Elderberry extract Result – after lyophilization
1 1 Sticky fluid mass
2 1 Sticky fluid mass
3 1 Sticky dense mass
4 1 Thick porous mass
5 1 Very dense powdered tablet
Table 2. Second step of extract dilution: refining the water:elderberry extract ratio (v/v)
for mechanical bottle line filling prior to lyophilization.
Water Elderberry extract Result – after lyophilization
4.1 1 Thick porous mass
4.3 1 Thick porous mass
4.5 1 Dry powder
4.7 1 Dry powdered tablet
4.9 1 Dense powdered tablet
Table 3. Range of experimental protocols evaluated for lyophilization of elderberry
anthocyanins; total 33 to 37 h.
Step Temp. (°C)* Vacuum (mBar) Time (min) Stage
1 5 0 1 Loading
2 -30 to -40 0 60-120 Freezing
3 -30 to -40 0 60-120 Freezing
4 -30 to -40 0.2 1-30 Evacuation
5 -5 0.2 400-800 Drying
6 -5 0.2 100-250 Drying
7 5 0.2 300-350 Drying
8 5 0.2 100-250 Drying
9 35 0.1-0.2 200-240 Drying
10 35 0.1 200-240 Drying
11 35 0.05-0.1 180-270 Drying
* Temperature was evaluated in intervals of 1°C in steps 2-4; time was evaluated in intervals of 1 min in
steps 2-4, and 10 min in steps 5-11; pressure (vacuum) was evaluated in intervals of 0.05 mBar.
251
Table 4. Optimal lyophilization program for the anthocyanin extract of elderberry.
Step Temp. (°C) Vacuum (mBar) Time (min) Stage
1 5 0 1 Loading
2 -30 0 60 Freezing
3 -30 0 120 Freezing
4 -30 0.2 30 Evacuation
5 -5 0.2 420 Drying
6 -5 0.2 240 Drying
7 5 0.2 330 Drying
8 5 0.2 210 Drying
9 35 0.2 240 Drying
10 35 0.1 240 Drying
11 35 0.05 270 Drying
Figures
Fig. 1. Industrial-scale elderberry extract lyophilization process in vials.
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A B C
Fig. 2. Examples of lyophilized anthocyanin-rich elderberry extract products resulting
from varying experimental protocols. A: undesirable sticky fluid mass, B:
undesirable thick porous mass, C: desired dry powdered tablet of pure
anthocyanins.
... By lowering the water activity of the matrix through the sublimation of water molecules at low temperatures, lyophilization reduces the reactivity of anthocyanins, including their conversion to colorless hemiketal and chalcone forms that occur naturally in aqueous environments [16]. This freeze-drying method has already been used successfully by others to preserve the anthocyanin content of other plant matrices for 6 months, including extracts of sweet cherry [17] and elderberry [18]. Therefore, although the most efficient extraction process required a solvent containing 50% ethanol, the presence of ethanol limits the postextraction stability of anthocyanins over time when stored as pure extracts, concentrates, or lyophilized powder. ...
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