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Atanassova,
Journal of the University of ChemicalS. Technology
Georgieva, and
K. Ivancheva
Metallurgy, 46, 1, 2011, 81-88
1
National Center of Public Health Protection, Department Received 02 November 2010
of Foods and Nutrition, 15 Akad. Ivan Ev. Geshov blvd, Accepted 15 February 2011
1431, Sofia, Bulgaria
2
Institute of Chemical Engineering, Bulgarian Academy
of Sciences, Acad. Bonchev St., Block 103,
1113 Sofia, Bulgaria
E-mail: stefanova@myway.com
ABSTRACT
Herbs are an ancient source of flavouring, aromatic compounds and medicines, not only for culinary application.
The increasing interest in the powerful biological activity of plant phenolics and flavonoids outlined the necessity of
determining their content in medicinal herbs. In this present study, a comparative evaluation of the polyphenol composi-
tion, antioxidant capacity and biological contaminants (microbes and other organisms) as major common contaminants in
medicinal herbs from the Lamiaceal family to which belong: lemon balm (Melissa officinalis), sage (Salvia officinalis) and
mint (Mentha piperita) were carried out. The total phenolic and total flavonoid contents in medicinal herbs ware evalu-
ated using the Folin-Ciocalteu method, were determined with an aluminum chloride colorimetric assay. The 2,2-diphenyl-
1-picryl hydrazyl (DPPH) radical scavenging effect of the herbs was measured also spectrophotometrically, like the total
phenolic and total flavonoid contents. Microbiology was investigated using the current ISO methods. The present paper
shows by the results of total phenolic and total flavonoid contents, antioxidant activity and biological contaminants in
medicinal herbs that they must be relatively safe for the patient (consumer).
Keywords: lemon balm (Melissa officinalis), sage (Salvia officinalis) and mint (Mentha piperita), total phenolics,
total flavonoids, 2,2-diphenyl-1-picryl hydrazyl (DPPH), biological contaminants.
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Journal of the University of Chemical Technology and Metallurgy, 46, 1, 2011
high quality requirements for medicinal plants and re- derived from the Latin salvere, to be saved, attesting to its
lated products on the market are shared at the global curative powers [17]. According to Zheng and Wang [18],
level in pharmacopoeias, while legal frameworks exist common sage ranked towards the lower end of the scale in
at the national or regional level [1]. terms of phenolic content and antioxidant activity. An in-
For a long period of time, plants have been a vari- teresting area of research with respect to sage, has been in
able source of natural products for maintaining human relation to brain function, particularly Alzheimers dis-
health, especially in the last decade, with more intensive eases. An animal study found that a sage leaf extract im-
studies devoted to natural therapies [5]. The World Health proved memory retention, possibly through an observed
Organization [6] has recommended that this should be effect upon the cholergenic system in the brain [19]. In
encouraged especially in places where access to conven- addition, the extract showed anti-inflammatory, estrogenic
tional treatment is not adequate. Studies have shown that and sedative effects, which are also relevant to the treat-
many plants have chemical components and biological ment of Alzheimers diseases [20, 21].
activities that produce definite physiological actions in Mint also belongs to the Lamiaceae family and
the body and, therefore, could be used to treat various includes 25-30 species, the most popular of which is
ailments [7]. The most important of these bioactive con- common mint [22]. Along with a close relative, pepper-
stituents of plant are alkaloids, tannins, flavonoids and mint (Mentha piperita), it has been used as a folk-popu-
phenolic compounds [8, 9]. lar remedy, particularity for complaints of the digestive
Flavonoids, a group of pholyphenolic compounds tract, including nausea, indigestion, flatulence and even
with known properties, such as free radical scavenging hiccups [16]. In the fourteenth century it was used to
activity, inhibition of hydrolytic and oxidative enzyme whiten teeth and today it is still used to flavor tooth-
and anti-inflammatory action [10, 11] have been iso- paste [17, 22]. Some of the health benefits attributed to
lated from plants [7]. mint include anti-fungal, anti-viral, anti-microbial, in-
Several investigations have shown that many of secticidal, antioxidant, anti-amoebic, and antihaemolytic
these plants have antioxidant activities that could be activities, but is also cited as a central nervous system
therapeutically beneficial and it has been mentioned that suppressant (sedative) and allergen [16, 22].
the antioxidant potential of plants might be due to their Free radicals and other reactive oxygen species
phenolic components [12]. (ROS), such as superoxide anions, hydroxyl radicals,
It is well known that oxidative stress included by and hydrogen peroxide are an entire class of highly re-
oxygen-free radicals and resultant tissue damage are the active spieces derived from the normal metabolism of
hallmarks of several chronic disorders and cell death oxygen or from exogenous factors and agents. Oxidative
[13]. The therapeutic potentials of medicinal plants as damage to crucial cellular molecules, induced by ROS,
natural antioxidants in reducing such free radical in- has been implicated as a possible factor in the etiology
duced tissue damage [14] and in the maintenance of of several human diseases, including cancer, cardiovas-
health and protection from some age-related degenera- cular disease, and aging [23]. In recent years, there is an
tive disorders such as cancer and coronary heart dis- increasing interest in finding antioxidant
eases is established [8]. phytochemicals, because they can inhibit the propaga-
A less common herb, lemon balm (Melissa tion of free radical reactions and protect the human
officinalis) is used to give a citrus flavour and aroma to body from diseases [24]. The oxidation of lipids in foods
foods and beverages, though it has also been used as a is responsible for the formation of off-flavours and un-
herbal medicine to treat headaches, gastrointestinal disor- desirable chemical compounds which may be detrimental
ders, nervousness and rheumatisms [14]. Like many herbs, to health. Antioxidants are used by the food industry to
the essential oil of lemon balm (Melissa officinalis) which delay the oxidation process [25]. Antiradical antioxi-
is rich in aldehydes and terpenic alcohols [15], is reported dants act by donating hydrogen atoms to lipid radicals.
to have anti-microbial properties as well as a strong pro- Radicals obtained from antioxidants with molecular
tective ability against lipid peroxidation [14, 16]. structures such as phenols, are stable species and will
Sage is another member of the Lamiaceae or mint then stop the oxidation chain reaction [26]. To evaluate
family and the botanical name of this genus, Salvia, is the antioxidative activity of specific compounds or ex-
82
M. Atanassova, S. Georgieva, K. Ivancheva
tracts, the latter are allowed to react with a stable radi- NaOH, rutin, ammonium molybdate, Indigo carmine,
cal, 2,2-diphenyil-picrylhydrazyl (DPPH) in a metha- 0.1 N water solution of KMnO4, 96-98 % H2SO4, 2,2-
nol solution [25]. diphenyl-1-picryl hydrazyl (DPPH) and ascorbic acid
Biological contamination refers to impurities in were purchased from Sigma Chem. Co. All other chemi-
medicinal herbs and their preparations and products, cals were of analytical grade.
and may involve living microbes such as bacteria and
their spores, yeasts and moulds, viruses, protozoa, in- Sample preparation
sects (their eggs and larvae), and other organisms [1]. A ground dried sample of 0.5 g was weighted
Microbial contamination of herbs and/or products may and phenolic and flavonoid compounds were extracted
result from improper handling during production and with 50 ml 80 % aqueous methanol on an ultrasonic
packaging. The most likely sources of contamination bath for 20 min. An aliquot (2 ml) of the extracts was
are microbes from the ground and processing facilities ultracentrifugated for 5 min at 14000 rpm [27].
(contaminated air, microbes of human origin) [1]. Cross
contamination is also possible from extraneous materi- Determination of the total phenolic assay
als such as plastics, glass, and other materials which The total phenolic content of the dry herbs was
come in to contact with medicinal herbs, herbal prepa- determined with the Folin-Ciocalteau assay. An aliquot
rations or products [1]. World Health Organization (1 ml) of extracts or a standard solution of gallic acid
(WHO) contaminant guidelines [4] propose that con- (20, 40, 60, 80 and 100 mg/l) was added to a 25 ml
tamination should be avoided and controlled through volumetric flask, containing 9 ml of distilled deionised
quality assurance measures such as good agricultural water (dd H2O). A reagent blank using dd H2O was also
and collection practices (GACP) for medicinal plants, prepared. One milliliter of the Folin-Ciocalteus phe-
and good manufacturing practices (GMP) for herbal nol reagent was added to the mixture and shaken. After
medicines. Today, only a small percentage of the me- 5 min, 10 ml of 7% Na2CO3 solution was added to the
dicinal plants are collected from the wild, and there are mixture. The solution was diluted to 25 ml with dd H2O
too few data to compare biological contamination be- and mixed. After incubation for 90 min at room tem-
tween wild and cultivated medicinal herbs [1]. perature, the absorbance against the prepared reagent
The focus of the present study is a comparative blank was determined at 750 nm with an UV-VIS Spec-
evaluation of the total phenolics and flavonoids, anti- trophotometer Lambda 5. The data for the total phe-
oxidant capacity (DPPH) and biological contaminants nolic contents of white birch leaves were expressed as
in medicinal herbs as souces for human health. milligrams of gallic acid equivalents (GAE) per 100
grammes dry mass (mg GAE/100 g dw). All samples
EXPERIMENTAL were analysed in duplicates [27].
83
Journal of the University of Chemical Technology and Metallurgy, 46, 1, 2011
pressed as milligrams of (+)-catechin equivalents (CE) lemon balm (Melissa officinalis), sage (Salvia officinalis)
per 100 grammes dry mass (mg CE/100 g dw). All and mint (Mentha piperita).
samples were analyzed in duplicates [27].
Microbiology (biological contaminants)
DPPH assay Sample preparation
The most commonly used antioxidant methods The samples were prerared according to ISO 6887
are those with 2,2-Azinobis (3-ethyl-benzothiazoline- 4. Microbiology of food and animal feeding stuffs
6-sulfonic acid) - ABTS and with 2,2-diphenyl-1-picryl Preparation of test samples, initial suspension and deci-
hydrazyl (DPPH). Both of them are characterized by mal dilutions for microbiological examination Part
excellent reproducibility under certain assay conditions, 4: specific rules for the preparation of products other
but they also show significant differences in their re- than milk and milk products, meat and meat products,
sponse to antioxidants. The DPPH free radical (DPPH) and fish and fishery products [31].
does not require any special preparation, while the ABTS Microbiological examination
radical cation (ABTS) must be generated by enzymes or The following standard methods were used:
chemical reactions [28]. In the DPPH free radical method ISO 4833 Microbiology of food and animal feed-
antioxidant efficiency is measured at ambient tempera- ing stuffs Horizontal method for the enumeration of
ture and thus the risk of thermal degradation of the microorganisms Colony-count technique at 30oC [31].
molecules tested is eliminated [26]. The hydrogen atom ISO 4831 Microbiology of food and animal feed-
or electron donation abilities of the corresponding ex- ing stuffs Horizontal method for the detection and
tracts and some pure compounds were measured from enumeration of coliforms Most probable number tech-
the bleaching of the purple-colored methanol solution nique [31].
of 1,1-diphenly-2-picrylhydrazyl (DPPH). This spectro- ISO 7251 Microbiology of food and animal feed-
photometric assay uses the stable radical DPPH as a ing stuffs Horizontal method for the detection and
reagent. One thousand microlitres of various concen- enumeration of presumptive Esherichia coli - Most prob-
trations of the extracts in ethanol were added to 4 ml of able number technique [31].
0.004 % methanol solution of DPPH. After a 60 min ISO 6579 Microbiology of food and animal feed-
incubation period at room temperature, the absorbance ing stuffs Horizontal method for the detection of Sal-
was read against a blank at 517 nm. All spectrophoto- monella spp [31].
metric data were acquired using a Varian UV-Vis spec- ISO 6888-3 Microbiology of food and animal
trophotometer. Disposable cuvettes (1 cm l cm x feeding stuffs Horizontal method for the enumeration
4.5cm) from Ratiolab (Dreieich, Germany) were used of coagulase-positive staphylococci (Staphylococcus
for visible absorbance measurements. aureus and other species) Part 3: detection and MPN
technique for low number [31].
Calculations ISO 7954 Microbiology of food and animal feed-
Inhibition of free radical by DPPH in percent (I %) ing stuffs General guidance for enumeration of yeasts
was calculated in following way: and moulds - Colony-count technique at 25 C [31].
where: Ablank is the absorbance of the control reaction Phenolic and flavonoid contents
(containing all reagents except the test compound), Asample Different phytochemicals have various protective
is the absorbance of the test compound. and therapeutic effects which are essential to prevent
The extract concentration providing 50 % inhi- diseases and maintain a state of well being. Methanolic
bition (IC50 %) was calculated from an inhibition per- extract of lemon balm (Melissa officinalis), sage (Salvia
centage against extract concentration graph [29, 30]. The officinalis) and mint (Mentha piperita) were analyzed
objectives of this study were to evaluate and compare for their phytoconstituents. The quantitative estimation
the total antioxidant capacity of the medicinal herbs- of the phytochemical constituents of lemon balm (Mel-
84
M. Atanassova, S. Georgieva, K. Ivancheva
Table 1. Total phenolics and total flavonoids in the studied medicinal herbs.
Microbiological analysis
In the Table 4 are shown the microbial limits or
the absence of specified microorganisms in medicinal
herbs. The total aerobic microbial count and the total
yeast and mould count (presented as colony-forming units
per gram CFU/g of dry herbal material), the absence of
salmonellae, Esherichia coli and Gram-negative bacterial
species have been used as indicators of microbiological
quality. Microbial count is just one of medicinal herbs
quality indicators. All medicinal herbs must be clear of
bacterial pathogens such as Salmonella secies.
CONCLUSIONS
Fig. 1. Free radical scavenging capacity of the extracts On the basis of the results obtained from the
measured by the DPPH assay.
evaluation of phenolic and flavonoid contents and the
DPPH values of the medicinal herbs, we conclude that
(IC50%)AA/( IC50%)extract are shown in Table 3. They rep- it is important to educate consumers on the benefits of
resent the ascorbic acid equivalent of the extracts anti- varying medicinal herbs consumption, choosing those
oxidant capacity (AOCEAA), i.e. the amount of ascorbic that have the highest antioxidant capacity in order to
acid in grams equivalent to one liter extract. promote a healthy diet.
lemon < 10 < 0.30 Absent in 1.0 Absent in Absent in 1.0 g < 10 < 10
balm g 25.0 g
(Melissa
officinalis)
sage < 10 < 0.30 Absent in 1.0 Absent in Absent in 1.0 g < 10 < 10
(Salvia g 25.0 g
officinalis)
mint < 10 < 0.30 Absent in 1.0 Absent in Absent in 1.0 g < 10 < 10
(Mentha g 25.0 g
piperita)
86
M. Atanassova, S. Georgieva, K. Ivancheva
The analysis of the differences in phenolic and 11. B. Pourmorad, S.J. Hosseinimehr, N. Shanabi Majd,
flavonoid contents and the antioxidant capacity in me- Afric. J. Biotech., 5, 11, 2006, 1142-1145.
dicinal herbs are an important but underestimated di- 12. N.C. Cook, S. Samman, J. Nutr. Biochem., 7, 1996,
etary component, providing some protective/preventa- 66-76.
tive health effect along with very few calories and inter- 13. M.J. Mates, M. Ortiz-Lombardia, B.Boitel, A. Haouz,
esting flours. D. Tello, S.A. Susin, J. Penninger, G. Kroemer, P.M.
Absence of specified microorganisms in medici- Alzari, Nat. Struct. Mol. Biol., 9, 2002, 44-46.
nal herbs has been used as an indicator of microbio- 14. N. Mimica-Dukic, B. Bozin, M. Sokovic, N. Simin,
logical quality. Lemon balm (Melissa officinalis), sage J. Agric. Food Chem., 52, 9,2004, 2485-2489.
(Salvia officinalis) and mint (Mentha piperita) improve 15. M.A. Robeiro, M.G. Bernardo-Gil, M.M. Esquivel,
the microbiological safety with maintaining or even J. Supercritical Fluids, 21, 1, 2004, 51-60.
enhancing the antioxidative activity. The use of medici- 16. L.J. Hedges, C.E. Lister, Nutritional attributes of
nal herbs as the first choice in self-treatment of minor herbs. Crop and Food Research Confidential Re-
conditions continues to expand rapidly across the world. port 1891, A report prepared for Horticulture
This makes the safety of medicinal herbs an important New Zealand, 2007.
public health issue. 17. M. Grieve, A Modern Herbal, Harmondsworth, Pen-
The results can be used in public health cam- guin, 1931, p. 912.
paigns to stimulate the consumption of lemon balm 18. W. Zheng, S.Y. Wang, Agric. Food Chem., 49, 11,
(Melissa officinalis), sage (Salvia officinalis) and mint 2001, 5165-5170.
(Mentha piperita) which are able to provide signifi- 19. M. Eidi, A. Eidi, M. Bahar, Nutrition, 22, 3, 2006,
cant health protection in order to prevent chronic 321-326.
diseases. 20. N.S.L. Perry, P.J. Houghton, J. Sampson, A.E.
Theobald, S. Hart, M. Lis-Balchin, J.R.S. Hoult, P.
REFERENCES Evans, P. Jenner, S. Milligan, E.K. Perry, J. Pharm.
Pharmac., 53, 10, 2001, 1347-1356.
1. I. Kosalec, J. Cvek, S. Tomi, Arh. Hig. Rad. Toksikol., 21. N.S.L. Perry, C. Bollen, E.K. Perry, C. Ballard,
60, 2009, 485-501. Pharmac. Biochem. Behavior, 75, 3, 2003, 651-
2. Association Europenne des Spcialits Pharma- 659.
ceutiques Grand Public (AESGP) 15th ed., 2009, 22. R.P. Choudhury, A. Kumar, A.N. Garg, J. Pharm.
Brussels. Biochem. Anal., 41, 3, 2006, 825-832.
3. World Health Organization, 2005. 23. B. Halliwell, J.M.C. Gutteridge, Meth. Enzym., 186,
4. World Health Organization, 2007. 1990, 185.
5. R.S. Kumar, T. Sivakuma, R.S. Sunderem, M. Gupta, 24. J.E. Kinsella, E. Frankel, B. German, J. Kanner,
K. Murugesh, Y. Rajeshwa, M.S. Kumar, K.A. Kumar, Food Tech., 47, 1993, 8589.
Braz. J. Med. Biol. Res., 38, 2005, 1015-1024. 25. W. Brand-Williams, M.E. Cuvelier, C. Berset,
6. World Health Organization, 1980. Lebensmittel-Wissenschaft und Tech., 28, 1995, 25-
7. J. Omale, P.N. Okafor, Afric. J. Biotech., 7, 17, 2008, 30.
3129-3133. 26. V. Bondet, W. Brand-Williams, C. Berset,
8. A.F. Hill, Economic Botany, A textbook of useful Lebensmittel-Wissenschaft Tech., 30, 1997, 609
plants and plant products, Company Inc, New York, 615.
1952. 27. D. Marinova, F. Ribarova, M. Atanassova, J. Univ.
9. H.O. Edeoga, D.E. Okwu, B.O. Mbaebie, Afric. J. Chem. Tech. Met.(Sofia), 40, 3, 2005, 255-260.
Biotech., 4, 7, 2005, 685-688. 28. A. Wojdyo, J. Oszmianski, R. Czemerys, Food
10. E. Frankel, Nutritional benefits of flavonoides. In- Chem., 105, 2007, 940949.
ternational Conference of food factors: Chemistry 29. K. Gezer, M.E. Duru, I. Kivrak, A. Turkoglu, N.
and cancer prevention, Hamamatsu, Japan, Abstract Mercan, H. Turkoglu, S. Gulcan, Afric. J. Biotech.,
C6-2, 1995. 5, 20, 2006, 1924-1928.
87
Journal of the University of Chemical Technology and Metallurgy, 46, 1, 2011
30. L. Bektas Tepe, A. Sihoglu-Tepe, D. Daferera, M. Polissiou, 34. F. Shahidi, J.P.D. Wanasundara, Crit. Rev. Food Sc.
A. Sokmen, Food Chem., 103, 2007, 766770. Nutr., 32, 1992, 67-103.
31. ISO Standards 35. A. Sofowora, Medicinal plants and traditional medi-
32. B. Havesteen, Biochem. Pharm., 30, 1983, 1141- cine in Africa, Spectrum books, Ind, Ibadan, Nigeria,
1148. 1993, p. 289.
33. V.S. Deepa, P.S. Kumar, S. Latha, P. Selvamani, S. 36. N.P. Das, T.A. Pereira, J. Am. Oil Chem. Soc., 67,
Srinivasan, Afric. J. Biotech., 8, 8, 2009. 1630-1636. 1990, 255-258.
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