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Analele Ştiinţifice ale Universităţii „Al. I. Cuza” Iaşi
s. II a. Biologie vegetală, 2014, 60, 2: 11-19
http://www.bio.uaic.ro/publicatii/anale_vegetala/anale_veg_index.html
ISSN: 1223-6578, E-ISSN: 2247-2711
EVALUATION OF BIOACTIVE COMPOUNDS FROM COMMERCIAL
LAVENDER PRODUCTS AND COMPARATIVE HISTO-ANATOMICAL STUDY
Cristina LUNGU
1
, Andreia CORCIOVA
2
*, Adrian SPAC
3
,
Constantin CIOBANU2, Bianca IVANESCU1
Abstract: This study focuses on the assessment of biologically active compounds from commercial
samples of lavender tea and the comparison of anatomical characteristics of two species of lavender used
in phytotherapy. The volatile oils from two Lavandulae flos tea samples were analysed using gas
chromatography coupled with mass spectrometry. Quantitative evaluation of rosmarinic and chlorogenic
acids was performed using spectrophotometrical methods. The presence of heavy metals in samples was
determined by atomic absorption spectroscopy in order to assess the safety use of products. The histo-
anatomical analysis of leaf and stem of Lavandula angustifolia Mill. and Lavandula latifolia Medik. was
carried out by usual techniques applied in plant anatomy. Although the main compounds of volatile oils
were in both cases linalool and linalyl acetate, a major difference was observed in the yield of essential
oil, with 1.05%, respectively 2.75% in the two commercial samples. Significant amounts of rosmarinic
(1.19 - 4.74 g%) and chlorogenic acid (2.53 - 10.06 g%) were observed, thus explaining some of the
pharmacological effects of the vegetal product. A very low content of heavy metals was determined in
plant and infusion samples. The two species exhibit only small anatomical differences that are mainly of
quantitative nature.
Key words: Lavender, essential oil, hydroxycinnamic acids, heavy metals.
Introduction
Lavender is an aromatic and medicinal plant used since ancient times as sedative,
anxiolytic, carminative, cholagogue, spasmolytic, antimicrobial, wound healing and anti-
inflammatory remedy. Also, it possesses antioxidant effect, is an insect deterrent and shows
positive effects on learning, memory and nociception (Rabiei et al., 2014). The main active
principles in lavender flowers, responsible for pharmacological properties, are the volatile
oil and hydroxycinnamic acids, especially rosmarinic and chlorogenic acids, both found in
significant amounts in plant. Lavender oil is highly valuable in perfumery and cosmetics,
and used successfully in aromatherapy. Hydroxycinnamic acids are powerful antioxidants
and can be used in treating free-radical mediated diseases. Furthermore, rosmarinic acid
manifests antiviral, antibacterial, anticarcinogenic and anti-inflammatory activity. Recent
studies have shown that rosmarinic acid breaks-up amyloid-beta conglomerates of
Alzheimer's disease (Park et al., 2008; Reichling et al., 2008).
The quality of the vegetal products used in therapy is regulated by the European
Pharmacopoeia where both lavender flower and lavender oil have monographs. According
to the European Pharmacopoeia, the vegetal product of pharmaceutical interest, Lavandulae
1
Department of Plant and Animal Biology, Faculty of Pharmacy, University of Medicine and Pharmacy “Grigore
T. Popa”, 16 Universitatii Street, 700150, Iaşi
2
Department of Drug Analysis, Faculty of Pharmacy, University of Medicine and Pharmacy “Grigore T. Popa”,
16 Universitatii Street, 700150, Iaşi
3
Department of Physical Chemistry, Faculty of Pharmacy, University of Medicine and Pharmacy “Grigore T.
Popa”, 16 Universitatii Street, 700150, Iaşi
* corresponding author - Andreia Corciova, e-mail: acorciova@yahoo.com
Lungu, C. et al. 2014/ Analele Stiint. Univ. Al. I. Cuza Iasi, Sect. II a. Biol. veget., 60, 2: 11-19
12
flos, is represented by the dried flowers of Lavandula angustifolia Mill. and the volatile oil,
Lavandulae aetheroleum, is defined as the essential oil obtained by steam distillation from
the flowering tops of L. angustifolia (European Pharmacopoeia 7.0, 2010).
However, the commercial lavender oil is extracted from different species and
hybrids by distillation: lavender flower oil from Lavandula angustifolia (common
lavender), lavender spike oil from L. latifolia Medik. (Spike lavender) and lavandin oil
from L. x intermedia hybrids (lavandins, group of hybrids of L. angustifolia and L.
latifolia).
This paper aims to assess the quality of two commercial tea products found on
Romanian market, sold as Lavandulae flos. To this purpose, the quality and quantity of
essential oil will be evaluated, as well as the quantity of hydroxycinnamic acids. In order to
assess the safety in using these products, the heavy metal content will be determined.
Finally, a comparison of the histo-anatomical characteristics of two species of lavender (L.
angustifolia and L. latifolia), commonly cultivated in Romania, will be performed.
Materials and methods
Plant material
The commercial samples of Lavandulae flos tea, codified L1 and L2, were obtained
from two different manufacturers. The leaves and stem parts of two lavender species
(Lavandula angustifolia Mill. and L. latifolia Medik.) were collected from a garden in Iasi,
in August 2012. The voucher specimens were deposited in the Herbarium of Department of
Plant and Animal Biology, Faculty of Pharmacy, UMF „Grigore T. Popa”, Iaşi, Romania.
The vegetal material for histo-anatomical study was preserved in 70% ethylic alcohol.
Essential oil isolation
The plant material (40 g) was subjected to hydro-distillation with 1000 mL water in
a Clevenger type apparatus for 2 h according to the official method described in European
Pharmacopoeia 7th Edition. The oil was collected and dried over anhydrous sodium
sulphate and stored at 4°C until analysis.
Histo-anatomical study
The sections through leaf and stem were carried out by usual techniques using a
microtome and a botanical razor. The cross sections were stained with alum carmine and
iodine green and photographed through a Nikon Eclipse E400 microscope, equipped with a
Nikon camera.
Quantitative evaluation of rosmarinic and chlorogenic acid
Rosmarinic and chlorogenic acids were quantified in alcoholic and aqueous extracts
by UV-VIS spectrophotometric method officialised by the European Pharmacopoeia 7th
Edition. A Jasco V530 double beam UV-VIS spectrophotometer was used. All the
measurements were made in 1.0 cm quartz cells at a scan speed of 1000 nm min-1 and scan
range of 400 - 600 nm, fixed slit width of 2 nm.
For alcoholic extracts, samples of 10 g of powdered plant material were extracted
with 100 mL of 70% v/v ethanol at 40°C for 1 hour, then the extracts were filtered in flasks,
the filter washed with the same solvent to 100 ml. The aqueous extracts (infusions) were
obtained in accordance with package instructions.
Lungu, C. et al. 2014/ Analele Stiint. Univ. Al. I. Cuza Iasi, Sect. II a. Biol. veget., 60, 2: 11-19
13
1mL sample was mixed with 2 mL of 0.5 M hydrochloric acid, 2 mL of a solution
(prepared by dissolving 10 g of sodium nitrite and 10 g of sodium molybdate in 100 ml of
water) and 2 mL of dilute sodium hydroxide solution and diluted to 10 ml with distilled
water. The absorbance was measured immediately at 505 nm for rosmarinic acid and at 525
nm for chlorogenic acid, using as compensation liquid a solution prepared from 1 mL
sample diluted at 10 mL with distilled water. Hydroxycinnamic acid derivatives content has
been calculated by the following formula: Conc g % = Ap x dilution x 100/
1
1
A
x m
where: Ap = sample absorbance;
1
1
A
= specific absorbance of rosmarinic acid – 400
or
1
1
A
= specific absorbance of chlorogenic acid – 188; m = mass of sample (g) (European
Pharmacopoeia 7.0, 2010).
The results were expressed in rosmarinic acid, respectively in chlorogenic acid.
Rosmarinic and chlorogenic acids were purchased from Sigma Aldrich, USA.
Atomic absorption spectroscopy method
The presence of heavy metals in samples was determined by atomic absorption
spectroscopy. The analyses were performed on an atomic absorption spectrometer ContrAA
300: HR-CS AAS (High Resolution Atomic Absorption Spectrometry Source Continuum)
(Analytic Jena, Germany). The wavelengths at which the absorbances of the solutions were
recorded were: 213.85 (Zn) nm, 217.00 nm (Pb), 228.80 nm (Cd) and 324.75 nm (Cu). The
instrumental parameters have been optimized in accordance with the manufacturer's
recommendations. All the reagents used to perform the analyses were of analytical grade:
65% nitric acid (Chemical Company, Romania), 30% hydrogen peroxide (Chemical
Company, Romania), and deionized water. In order to carry out the calibration curves,
dilutions of a multi-element standard solution Multi Element ICP Standard Solution (0.1
mg/L) (Merck, Germany) were used. The samples were mineralized by using a mixture of
65% nitric acid and 30% hydrogen peroxide. The limits of detection and quantification
were calculated for 0.5 g sample and a final volume of 25 mL. The infusions were prepared
in accordance to the manufacture`s instructions.
Results and discussions
In order to evaluate the bioactive compounds of Lavandulae flos commercial
products, the chemical composition of the essential oil was determined by GC-MS. Hydro-
distillation of two commercial products Lavandulae flos yielded pale yellow oils. Sample
L1 contains 2.75% essential oil while sample L2 has a lower yield of 1.05%. The minimum
content of volatile oil in Lavandulae flos stated by European Pharmacopoeia is 1.3%, so
only sample L1 fulfils the official requirements. The results of GC-MS analyses of the
essential oils are given in Table I. A number of 30 compounds were identified in sample L1
representing 85.19% of the total oil content and 34 compounds were identified in sample
L2 which accounted for 87.81% of the total oil composition.
The chromatographic analyses highlighted differences between the major
constituents of the two samples. In sample L1 the main components were: linalool
(16.51%), camphor (12.62%), linalyl acetate (10.99%) and 1,8-cineole (10.88%), while the
chief constituents of sample L2 were linalool (24.79%) and linalyl acetate (11.03%).
Lungu, C. et al. 2014/ Analele Stiint. Univ. Al. I. Cuza Iasi, Sect. II a. Biol. veget., 60, 2: 11-19
14
Oxygenated monoterpenes predominate in both volatile oils (52.06%, respectively 47.52%).
Other components identified in the oils were: caryophyllene oxide, α-cadinol, endo-
borneol, α-terpineol. The esters, known as compounds with a significant importance for
aromatherapy, that give a special note to the oil, are found in the range of 16.99 % (L2) –
17.77 % (L1).
The European Pharmacopoeia 7th Edition sets some limits regarding the composition
of Lavandulae aetheroleum: limonene (maximum 1%), 1,8-cineol (maximum 2.5%), 3-
octanone (0.1-5%), camphor (maximum 1.2%), linalool (20-45%), linalyl acetate (25-47%),
terpinen-4-ol (0.1-8%), lavandulyl acetate (minimum 0.2%), lavandulol (minimum 0.1%),
α-terpineol (maximum 2%). The results show that none of the two commercial samples
meets completely the conditions of European Pharmacopoeia concerning the qualitative and
quantitative composition of the volatile oil.
Based on scientific literature data we observe a resemblance between the chemical
composition of sample L1 and that of lavender spike oil, the essential oil from Lavandula
latifolia which contains high amounts of camphor and 1,8-cineole (Chatzopoulou et al.,
2003; Verma et al., 2010; Herraiz-Peñalver et al., 2013).
Table 1. Chemical composition (%) of volatile oils from Lavandulae flos commercial samples
Retention
time (min.)
Compound
L1
L2
5.659
α-pinene
0.46
0.34
5.931
camphene
0.84
0.21
6.262
sabinene
0.15
0.11
6.369
β-pinene
0.77
1.05
6.466
myrcene
1.05
0.88
6.826
(+)-3-carene
-
0.15
6.952
α-terpinene
0.19
-
7.079
p-cymene
0.23
0.65
7.234
1,8-cineole
10.88
6.98
7.361
trans- β-ocimene
0.74
0.81
7.584
γ-terpinene
0.26
0.08
8.041
cis-linalool oxide
1.91
2.48
8.284
linalool
16.51
24.79
8.644
3-methylene 1,5,5-
trimethyl cyclohexene-1
-
0.28
9.072
camphor
12.62
2.55
9.198
lavandulol
-
1.88
9.276
pinocarvone
-
0.51
9.431
endo-borneol
3.89
2.35
9.519
terpinen-4-ol
1.97
0.75
9.636
cryptone
-
0.80
9.743
α-terpineol
3.45
3.92
9.976
eucarvone
-
0.34
10.093
trans-geraniol
0.83
0.92
10.443
linalyl acetate
10.99
11.03
10.880
lavandulyl acetate
3.13
2.27
Lungu, C. et al. 2014/ Analele Stiint. Univ. Al. I. Cuza Iasi, Sect. II a. Biol. veget., 60, 2: 11-19
15
10.997
bornyl acetate
0.23
-
11.094
p-cymene-7-ol
-
0.05
11.522
hexyl tiglate
0.38
-
11.911
neryl acetate
1.03
1.36
12.173
geranyl acetate
2.01
2.33
12.825
santalene
0.37
0.30
12.912
trans-caryophyllene
1.14
2.20
12.990
trans-α-bergamotene
0.17
-
13.175
β-farnesene
0.43
1.43
13.369
α-humulene
-
0.12
13.680
germacrene D
0.19
0.13
14.974
caryophyllene oxide
5.64
7.14
15.606
α-cadinol
2.73
6.62
Monoterpene hydrocarbons
4.69
4.56
Oxygenated monoterpenes
52.06
47.52
Sesquiterpene hydrocarbons
2.3
4.18
Oxygenated sesquiterpenes
8.37
13.76
Others
17.77
17.79
Total identified
85.19
87.81
Rosmarinic and chlorogenic acids are other bioactive compounds from lavender
flowers. The present study compared the differences between the level of hydroxycinnamic
acids obtained by alcoholic extraction and aqueous extraction (infusions) performed
according to package instructions. Quantitative evaluation was performed by
spectrophotometric method officialised by the European Pharmacopoeia 7th Edition. Thus,
Ash leaf and Melissa leaf monographs were used for determination of chlorogenic acid and
rosmarinic acid, respectively. The principle of the method is based on the formation of
oxymes in the presence of sodium nitrite and sodium molybdate in alkaline medium.
The extraction method influences the amount of rosmarinic and chlorogenic acid,
which was reduced in aqueous extracts compared to the alcoholic extracts. As can be seen
from Table 2, the amount of chlorogenic acid varied between 2.53-5.91 g % in aqueous
extract and 7.95-10.06 g % in alcoholic extract. For rosmarinic acid, the amount ranged
between 1.19-2.79 g % in aqueous extract and 3.75-4.74 g % in alcoholic extract. Our
results are similar to those in the specialty literature that reports concentrations of
rosmarinic acids in plants from Lamiaceae family ranging from 0 to 58.5 mg/g, quantified
by HPLC methods (Shekarchi et al., 2012). In wild plants of L. angustifolia, rosmarinic
acid content varies between 2.31 and 4.04 mg/L depending on the solvent and method of
extraction (Komes et al., 2010).
Table 2. The content of chlorogenic and rosmarinic acids in Lavandulae flos commercial samples
Samples
Chlorogenic acid (g %)
Rosmarinic acid (g %)
Alcoholic extract
Aqueous extract
Alcoholic extract
Aqueous extract
Sample L1
10.06
5.91
4.74
2.79
Sample L2
7.95
2.53
3.75
1.19
Lungu, C. et al. 2014/ Analele Stiint. Univ. Al. I. Cuza Iasi, Sect. II a. Biol. veget., 60, 2: 11-19
16
Quantification of metals in plant products and infusions showed that the levels of
cadmium and lead were smaller than the quantification limits for both metals (Table 3).
Therefore, the concentrations of these two toxic metals is below the maximum permissible
limits of 10 µg/g for lead and 0.3 µg/g for cadmium in medicinal plants set by WHO, 2005.
The concentrations of copper in the analysed plant material ranged between 10.25 µg/g
(L1) and 10.78 µg/g (L2). For medicinal plants, the maximum permissible limits of copper
has not been yet established, however, several countries such as China and Singapore set
these limits to 20 and 150 µg/g, respectively (WHO, 2005). Our results showed that the
levels of copper were well below these limits. The concentrations of zinc in the analysed
plants varied between 23.43 µg/g (L2) and 31.39 µg/g (L1). With respect to the
concentrations of zinc in the analysed infusions, the values varied from 0.143 µg/mL (L2)
to 0.181 µg/mL (L1) and for copper from 0.063 µg/mL (L2) to 0.085 µg/mL (L1). If we
compare the obtained values with the WHO’s guideline values for drinking water, one can
be seen that our concentrations are below these values (2 µg/mL for copper and for zinc
WHO does not specify any value) (WHO, 2011).
Table 3. Zinc, copper, lead and cadmium concentrations
Sample
Concentration
Zn
Cu
Pb
Cd
Sample L1
Vegetal material (µg/g)
31.39
10.25
< LOQ
< LOQ
Infusion (µg/mL)
0.181
0.085
< LOQ
< LOQ
Sample L2
Vegetal material (µg/g)
23.43
10.78
< LOQ
< LOQ
Infusion (µg/mL)
0.143
0.063
< LOQ
< LOQ
Limit of quantification for lead = 0.0294 μg/mL; Limit of quantification for cadmium = 0.0065 μg/mL
Limit of quantification for zinc = 0.0149 µg/mL; Limit of quantification for copper = 0.0014 µg/mL
Based on the results of GC study and in relation to literature data, it is possible that
Lavandula species in the two vegetal products might be different. Taking into account the
possibility that one of the commercial samples derives from L. latifolia, and not L.
angustifolia as required by European Pharmcopoeia, we considered of interest to make a
histo-anatomical comparison between the two species, most frequently cultivated in our
country. The two species show differences both in terms of histo-anatomy and morphology.
Thus, it is necessary for plant products to be identified by specialists or to have clear
specifications on the label regarding the origin of plant material.
Regarding the leaf structure, the two species have the same equifacial structure of
the leaf blade. The mesophyll presents only palisade parenchyma on both sides. The
epidermis has one layer of cells with exterior walls covered with cutine and slightly toothed
cuticle. The midrib vein contains a singular vascular bundle of collateral type and two areas
of angular collenchyma beneath the two epidermises. On both leaf surfaces there are
protective and glandular trichomes. Glandular trichomes are of different types, most
common with one-celled stalk and one, two or eight-celled gland. Protective trichomes are
typically branched with one-celled stalk and three or more branches at the top (Nikolakaki
and Christodoulakis, 2006).
Lungu, C. et al. 2014/ Analele Stiint. Univ. Al. I. Cuza Iasi, Sect. II a. Biol. veget., 60, 2: 11-19
17
The stem cross-sections of L. angustifolia and L. latifolia have a square shape with
four prominent ridges, typical for plants of Lamiaceae family. The epidermis has one layer
of cells and shows protective and glandular trichomes of the same type as observed in the
leaf structure. The four ridges present under the epidermis a well-developed angular
collenchyma and big vascular bundles with a sclerenchyma cap over the phloem. Between
the main vascular bundles there are smaller ones and the stem centre contains pith of
parenchyma type.
Conclusions
The comparative anatomical study of leaves and stems revealed only small
differences, mainly of quantitative nature, between L. angustifolia and L. latifolia.
Numerous glandular and protective trichomes were observed on the epidermis of both
species. One of the two commercial samples has a higher quantity of bioactive compounds:
volatile oil and hydroxycinnamic acids. Also the essential oils differ quantitatively and
qualitatively, one of the samples resembling more to the composition of spike lavender oil
reported in literature. The amount of heavy metals in plants and tea preparations is reduced
so the plant products are safe to use in phytotherapy.
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Herraiz-Peñalver, D., Cases, M.A., Varela, F., Navarrete, P. et al., 2 013. Chemical characterization of Lavandula
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Nikolakaki, A., Christodoulakis, N., 2006. Histological investigation of the leaf and leaf-originating calli of
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Rabiei, Z., Rafieian-Kopaei, M., Mokhtari, S., Alibabaei, Z., Shahrani, M., 2014. The effect of pretreatment with
different doses of Lavandula officinalis ethanolic extract on memory, learning and nociception,
Biomedicine & Aging Pathology 4, 1: 71-76.
Reichling, J., Nolkemper, S., Stintzing, F.C., Schnitzler, P., 2008. Impact of ethanolic Lamiaceae extracts on
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Shekarchi, M., Hajimehdipoor, H., Saeidnia, S. et al., 2012. Comparative study of rosmarinic acid content in some
plants of Labiatae family, Pharmacognosy Magazine. 8, 29: 37-41.
Verma, R., Rahman, L., Chanotiya, C., Verma, R., Chauhan, A., Yadav, A., Singh, A., Yadav, A.K., 2010.
Essential oil composition of Lavandula angustifolia Mill. cultivated in the mid hills of Uttarakhand, India.
J. Serb. Chem. Soc. 75, 3: 343-348.
WHO, 2005. Quality Control Methods for Medicinal Plant Materials, Revised, Geneva.
WHO, 2011. Guidelines for drinking water-quality. 4th edition.
***European Pharmacopoeia 7.0, Strassbourg, Council of Europe, 2010, 1163-1164.
Lungu, C. et al. 2014/ Analele Stiint. Univ. Al. I. Cuza Iasi, Sect. II a. Biol. veget., 60, 2: 11-19
18
Figures 1, 2. Cross section through the leaf of Lavandula angustifolia Mill.; Figure 3. Glandular and
protective trichomes of L. angustifolia leaf; Figure 4. Protective trichome of L. angustifolia leaf;
Figures 5, 6. Cross section through the leaf of L. latifolia Medik.; Figure 7. Glandular and protective
trichomes of L. latifolia leaf; Figure 8. Protective trichome of L. latifolia leaf
Lungu, C. et al. 2014/ Analele Stiint. Univ. Al. I. Cuza Iasi, Sect. II a. Biol. veget., 60, 2: 11-19
19
Figures 9, 10. Cross section through the stem of Lavandula angustifolia Mill.; Figure 11. Glandular
trichome of L. angustifolia stem; Figure 12. Glandular and protective trichomes of L. angustifolia
stem; Figure 13. Cross section through the stem of L. latifolia Medik.
