Quantification of Valerenic Acid and its Derivatives in Some Species of Valeriana L. and Centranthus longiflorus Stev.
Ekhteraei Tousi Samaneh,
Bashiri Sadr Zeinalabedin,
The present study aims to characterize some Iranian wild species of Valerianaceae with respect to their contents of valerenic acid and its derivatives. Identification of these compounds which are known as reliable markers in Valerianaceae family was achieved using TLC and UV-spectrophotometry methods. Valerenic acid makes substantial contribution to the sedative effect of valerian. Separation of valerenic acid and its derivatives was performed by TLC using a ternary mobile phase of hexane-ethyl acetate-glacial acetic acid (65:35:0.5 v/v) on silica gel HF254+366 plates. Spots were detected at 254 and 366 nm and then revelation of them was carried out with HCl-acetic acid reagent followed by anisaldehyde-sulphuric acid reagent spraying at visible light. Quantitative analysis of valerenic acid and its derivatives was performed using UV-spectrophotometry. The calibration curve of authentic valerenic acid was linear in the range of 2-51 mg L-1. Additionally, for a more accurate determination of total valerenic acid derivatives (including valerenic acid, hydroxyvalerenic acid and acetoxyvalerenic acid) extinction coefficient of each compound in ethanol was used. This is the first report for the identification and quantification of valerenic acid derivatives in all organs of Valeriana sisymbriifolia, Valeriana alliariifolia and Centranthus longiflorus in comparison with those of commercial Valeriana officinalis. Total valerenic acid derivatives content in different organs of these Iranian wild species ranged from 0.06-1.11% (D.W.). Present results showed that Iranian wild species of Valeriana and Centranthus DC. may be used as worthy sources of valerenic acid derivatives.
to cite this article:
Ebrahimzadeh Hassan, Radjabian Tayebeh, Ekhteraei Tousi Samaneh, Bashiri Sadr Zeinalabedin, Niknam Vahid and Zarrei Mehdi, 2008. Quantification of Valerenic Acid and its Derivatives in Some Species of Valeriana L. and Centranthus longiflorus Stev.. Asian Journal of Plant Sciences, 7: 195-200.
Valerianaceae contains about 40 genera and 400 species of almost cosmopolitan distribution, mostly at high elevations (Hidalgo et al., 2004; Bell and Donoghue, 2005). Six species of Valeriana L. and one species of Centranthus DC. grow in Iran (Moussavi-Allashlou, 2001).Valerian root has already been used by the Greek and the Roman physician as a diuretic, anodyne and spasmolytic agent. In the 17th century it was used to treat epilepsy. Its current use as a mild sedative dates back to the 18th century. Nowadays, valerian preparations are used primarily to treat light forms of neurasthenia and emotional stress. Further indications are disturbances in falling asleep and cramping pains in the gastro-intestinal tract, as a consequence of tension (Bos, 1997). Valerian is the 8th top-selling herbal supplement in North America and in Australia; it is in the top 10 selling retail herbs (Singh et al., 2006).
The roots and rhizomes of valerian contain several compounds with demonstrable
pharmacological activity. These include the essential oil and its sesquiterpenoids
(valerenic acid derivatives), epoxy iridoid esters (valepotriates) and their
decomposition products such as baldrinal and homobaldrinal, amino acids (arginine,
GABA, glutamine, tyrosine) and alkaloids (Upton et al., 1999). The main
non-volatile cyclopentane sesquiterpenes are valerenic acid, acetoxyvalerenic
acid, hydroxyvalerenic acid and valerenal. The first two compounds are specific
for V. officinalis, while the third one is probably an artifact formed
during unfavorable storage conditions, e.g. high humidity. Hydroxyvalerenic
acid may be produced from acetoxyvalerenic acid by hydration (Bos, 1997). It
is yet unclear which group of valerian active substances is responsible for
the sedative effect of the plant. In the last two decades, the essential oil
and valerenic acid derivatives have become more important, not only from a pharmacological
point of view, but also for the quality control and standardization of valerian
phytomedicines (Hendriks et al., 1981, 1985). Recently several clinical
studies have presented some biological activities of valerian root extracts
and introduced the plant as anti-HIV (Murakami et al., 2002), sleep
aid (Fernandez et al., 2004), antidepressants (Miyasaka et
al., 2006), anticoronaryspastic, antihypertensive and antibronchospastic
(Circosta et al., 2007) agent.
A simple, rapid, cost-effective and accurate thin layer chromatographic method has been developed for separation of valerenic acid (Singh et al., 2006). In this study, valerenic acid derivatives were detected by Thin-Layer Chromatography (TLC) and quantified by UV-spectrophotometry in different organs of V. sisymbriifolia Vahl, V. alliariifolia Adams and Centranthus longiflorus Stev. and the results compared to those reported in the literature for a commercial cultivar of V. officinalis.
MATERIALS AND METHODS
Plant materials: Three populations of V. sisymbriifolia were
collected from different Iranian geographical locations at north and west regions
(locations 1, 2 and 3). Table 1 summarizes the characteristics
of the analyzed species, including geographic coordinate, altitude and harvesting
time. Organ samples including root, rhizome, stem, leaf and inflorescence were
separated and dried at 20±3°C in a ventilated dryer for 12-15 h.
All samples were powdered and stored at -20°C, until analysis. The dried
roots and rhizomes of V. officinalis were prepared from a valerian grower.
Sample preparation for TLC analysis: Freshly powdered plant organs including
root, rhizome, stem, leaf, inflorescence (0.2 g) were extracted with 5 mL dichloromethane
(DCM) for 1 min in a laboratory test tube. After 5 min standing, samples were
filtered and filter papers washed with 2 mL DCM fraction. The final collected
filtrate was dry evaporated on a water bath at 40°C. The residue was collected
in 0.2 mL DCM and transferred into a small sample vial. A standardized sample
of valerenic acid (HPLC grade, Fluka) was extracted with 5 mL DCM for 1 min
in a laboratory test tube and processed as mentioned above for plant material.
One aliquot (10 μL) from all prepared samples was spotted in 5 mm band
width on the same silica gel HF254+366 plate (Merck, Germany). Separation
was achieved using a ternary mobile phase containing hexane: ethyl acetate:
glacial acetic acid (63: 35: 0.5 v/v). Distance of development was 10 cm. Spots
were detected at 254 and 366 nm and then revelation of them was carried out
with HCl-acetic acid reagent followed by anisaldehyde-sulphuric acid reagent
spraying at visible light (Upton et al., 1999).
Sample preparation for UV-spectrophotometry: Each 0.2 g plant sample
was first extracted with 5 mL DCM then evaporated. After volume adjustment to
1 mL with DCM, organic acids were transferred into 2 mL 2% NaOH. The alkaline
aqueous phase was acidified up to pH~2 using 1% HCl and the organic acids (including
valerenic acid derivatives) retransferred into 1 mL petroleum ether: diethyl
ether (2:1) organic phase (Bos, 1997). The obtained organic phase was divided
into two equal fractions and processed as follow:
||Fraction 1 was used to determine valerenic acid derivatives
content using their respective aliquot extinction coefficients (ε)
found in the literature reference (Bos, 1997). Thus a volume of 0.5 mL was
dry evaporated then the residue collected in 1 mL ethanol. After absorbance
measurement at 212 nm (hydroxyvalerenic acid), 217 nm (acetoxyvalerenic
acid) and 218 nm (valerenic acid), each valerenic acid derivatives content
(Ci) was determined using the following formula:
||Fraction 2 was used to determine the total valerenic acid
derivatives (including valerenic acid, hydroxyvalerenic acid and acetoxyvalerenic
acid) content using a 10 points calibration curve. Each calibration curve
point corresponded to a dilution obtained from the valerenic acid concentration
stock solution. The stock solution was prepared using the valerenic acid
standardized sample. Thus, a 0.5 mL of fraction 2 was dry evaporated then
collected in 1 mL methanol and its absorbance measured at 225 nm.
||Characteristics of the analyzed species
|a Indicates these vouchers were deposited in Central
Herbarium of Shahed University
Calibration curve: Stock solution (51 mg L-1) of valerenic
acid was prepared in methanol. Ten points from different valerenic acid concentrations
in the range of 2-50 mg L-1 were obtained. The regression equation
for valerenic acid at 225 nm was y = 0.0125+0.019x (y is absorbance and x defines
concentration) and the correlation coefficient (r) was 0.9993.
All lab works have been performed during Autumn-Winter 2006 at the Plant Physiology laboratories of University of Tehran and Shahed University (Tehran, Iran).
Data analysis: The data obtained in our laboratory represent the average±SE of triplicate determinations. Data obtained from UV-spectrophotometry were subjected to one-way ANOVA. Post hoc multiple comparisons between species for valerenic acid derivatives contents were made using Tukey´s test. Significance was reported starting at the 0.05 level.
RESULTS AND DISCUSSION
Valerenic acid with Rf = 0.49 appeared following application of
the HCl-acetic acid reagent as a very faint violet colored band in visible light
and as a weak fluorescent band under 366 nm. Subsequent application of anisaldehyde-sulphuric
acid reagent (Wagner et al.,1984), valerenic acid appeared as a strong
violet band (Fig. 1).
Table 2 shows Rf values and colors of separated
spots on TLC plates for rhizome extracts of V. officinalis. Valerenic
acid derivatives spots order arrangement and colors were similar to those reported
by the literature references (Wagner et al., 1984; Singh et al.,
2006; Upton et al., 1999). Values of Rf were similar to slightly
different depending on the TLC stationary phase polarity.
Table 3 shows the results obtained from the TLC detection
of the main sesquiterpenes and some other components present in different organs
of Iranian wild species of Valerianaceae and commercial V. officinalis.
Our qualitative TLC results confirmed the presence of valerenic acid derivatives
in all organs of studied plants.
After sample preparation, absorbance of methanolic and ethanolic extracts, were determined at 212, 217, 218 and 225 nm. Valerenic acid, hydroxyvalerenic acid and acetoxyvalerenic acid contents were calculated using their respective extinction coefficients.
Total valerenic acid derivatives content (Table 4) was determined
through the regression equation of the calibration curve for valerenic acid
at 225 nm based on the assumption that the extinction coefficients of these
compounds are almost similar. In Fig. 2, variation of valerenic
acid derivatives content is shown for each plant organ.
Measurement of total valerenic acid derivatives is more perfect than determination
of valerenic acid alone. Additionally, some analytical laboratories calculate
total valerenic acid derivatives using specific reference
standards, while others calculate the total valerenic acid derivatives content
based on the assumption that the extinction coefficients of these compounds
are almost similar. However, calculating valerenic acid derivatives content
using their respective extinction coefficients provides more accurate results
than total valerenic acid derivatives values or valerenic acid quantification
alone by plotting calibration curve (Upton et al., 1999).
||Characteristic thin-layer chromatography of some components
in V. officinalis roots
||TLC plate viewed valerenic acid derivatives and other main
components at visible light with HCl-acetic acid reagent followed by anisaldehyde-
sulphuric acid reagent spraying in plant rhizomes
||Comparative determination of the main sesquiterpenes and some
other components in different organs of Iranian wild species of Valerianaceae
in comparison to V. officinalis by TLC
|HVA (Hydroxyvalerenic acid), AVA (Acetoxyvalerenic acid),
VA (Valerenic acid), BL (Baldrinal), CP (Cryptofauronol), PA (Patchouli
alcohol), VL (Valerenal), VN (Valerenone)
||Mean values contents ± SE of valerenic acid derivatives
(g/100 g DW) in different organs of Iranian wild species of Valerianaceae
and commercial V. officinalis
|a All ε values were extracted from literature
reference (Bos, 1997)
||Comparative analysis of valerenic acid derivatives in different
organs of the plant samples; root (A), rhizome (B), stem (C), leaf (D) and
inflorescence (E). All results are based on dry weight
Valerenic acid content in commercially available roots from plants of the Anthos
cultivar, the current preferred industry cultivar of V. officinalis in
Australia, was reported to be about 3 mg g-1 (dry weight basis) as
an average (Wills and Shohet, 2003). A survey of some valerian commercial manufactured
products showed considerable variation in concentration of valerenic acid derivatives
from <0.01 to 6.32 mg g-1 (Li et al., 2006). The content
of valerenic acid derivatives in V. officinalis ranges from 0.05% (in
wild plants) to 0.9% (in cultivated strains) (Bos, 1997).
Our quantitative analysis showed that acetoxyvalerenic acid was the dominant fraction in all samples, compared to valerenic acid and hydroxyvalerenic acid. The survey of valerenic acid derivatives content in the studied samples showed a considerable variation from 0.06% in stem of V. sisymbriifolia from location 2 to 1.11% in inflorescence of V. sisymbriifolia from location 3.
Total valerenic acid derivatives content of roots in V. sisymbriifolia plants from locations of 1, 2, 3 and V. alliariifolia was 0.11, 0.12, 0.12 and 0.11%, respectively, which showed lower amounts compared to that from roots of Anthos cultivar (0.3%) (Significantly mean difference, p<0.05). The same results were found for rhizomes of V. sisymbriifolia from locations 1 (0.14%), 2 (0.15%), 3 (0.14%), V. alliariifolia (0.10%) and C. longiflorus (0.15%) compared to that of commercial V. officinalis (0.28%) (Significantly mean differences, p<0.001).
In spite of different geographical distribution and habitat altitude for three studied populations of V. sisymbriifolia, data did not show any considerable differences in total amounts of valerenic acid derivatives in their roots and rhizomes. Among the different organs of wild plants, inflorescence of V. sysimbriifolia from location 3 (1.11%) and C. longiflorus (0.5%) had higher amounts of total valerenic acid derivatives.
Valerenic acid derivatives content of valerian root and rhizome is considered as an essential quality factor regarding the health benefit to consumers (Bos, 1997). Thus, we were very interested to get some comparing data between some Iranian wild species and commercial valerian figuring in the European Pharmacopoeia. Present results showed that, studied Iranian wild species of Valeriana L. and also wild species of Centranthus DC. may be used as worthy sources of valerenic acid derivatives.
The authors wish to thank to Dr. V. Mozzaffarian and Dr. Sh. Zarre for collection and identification of the wild plants and Mr. Rashet for kindly providing us with dried rhizome and root samples of V. officinalis. This work was supported by University of Tehran and Shahed University and Chemical Industries Institute of the Iranian Research Organization for Science and Technology.
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