1. No category

BIHAREAN BIOLOGIST 9 (1): 55-58
Article No.: 141132
©Biharean Biologist, Oradea, Romania, 2015
http://biozoojournals.ro/bihbiol/index.html
A comparative study of dry and fresh Hibiscus trioni Herba tinctures
Simona G. BUNGĂU1, Gabriela VONHAZ2, Delia M. ŢIŢ1 and Lucian COPOLOVICI3,*
1. University of Oradea, Faculty of Medicine and Pharmacy, P-ta 1 Decembrie, no.1, Oradea, Romania.
2. University of Chester, Exton Park, Chester,Cheshire, CH1 4AR, United Kingdom.
3. Institute of Technical and Natural Sciences Research-Development-Innovation of “Aurel Vlaicu” University, 2 Elena Dragoi St., 310330, Arad, Romania.
* Corresponding author, L. Copolovici, E-mail: [email protected], Phone: 0040745259816, Fax: 0040257369091
Received: 9. July 2014 / Accepted: 4. October 2014 / Available online: 13. April 2015 / Printed: June 2015
Abstract. A qualitative and quantitative study has been performed using the chromatographic method associated with
photodensitometry of the tincture obtained from fresh and dry Hibiscus trioni Herba. Using thin layer chromatography followed by
photodensitometry it have been shown that both tincture are rich in polyphenols (flavonoids and carboxylic compounds), and free
amino acids in significant quantities. The results indicate that fresh Hibiscus trioni Herba have higher concentrations of active
ingredients which support the hypothesis that plants can not be kept dry for a long time without loosing their properties.
Key words: Hibiscus trioni herba, ethanolic extract, thin layer chromatography, flavonoids.
Introduction
Medicinal plants have been an available source of drugs
since ancient times and even today almost 50% of the new
drugs have been patterned after phytochemicals (Saleem et
al. 2008). Plants have almost limitless ability to synthesize
aromatic substances, most of which being secondary metabolic compounds (as flavonoids, terpens, etc.) (Saleem et al.
2008, Papuc et al. 2008, Papuc et al. 2010, Vallverdu-Queralt
et al. 2014).
In 1997, World Health Organization (WHO) recognized
in its guideline the medicinal significance of indigenous
plants and states that “effective locally available plants can
be used as substitutes for drugs” (Govindarajan et al. 2003).
These metabolites are very interesting, due to their biological properties, which present benefits for human health
(González-Laredo et al. 2003, Parkhomenko et al. 2006,
Hayet et al., 2008, Sarr et al. 2009, Pereira et al. 2009, Ahmed
et al. 2010, Xu et al. 2010, Policegoudra et al. 2011, Annegowda et al. 2012, ): anti-oxidant (Xu et al. 2010), antibacterial (Hayet et al. 2008, Annegowda et al. 2012), antiviral (Xu
et al. 2010, Annegowda et al. 2012), antitumoral (Ahmed et
al. 2010, Papuc et al. 2010) vasodilatator (Sarr et al. 2009),
neurological decease (Pereira et al. 2009), antifungal and anitimutagenic, medicinal metabolite (González-Laredo et al.
2003), hepatoprotective (Parkhomenko et al. 2006). Some
polyphenolic products are already used in curative or preventive purposes, single or associated with other treatments.
Among this interesting poliphenolic compounds are
found: cumaric acid, cafeic acid, quercitin, rutin, polymers of
procyanidins, etc (Parkhomenko et al. 2006, Hayet et al.
2008, Vukics et al. 2008, Medić-Šarić et al. 2009, Pereira et al.
2009, Policegoudra et al. 2011 ).
The importance of a high dietary content of phenolic
compounds, such as flavonoids and hydroxycinnamic acids,
has been growing due to their apparent multiple biological
effects such as free-radical scavenging, metal chelation process, inhibition of cellular proliferation, and signal transduction pathways (Del Rio et al. 2013).
Standard pharmaceutical products derived from plants
are herbal, fruits, root and flower tea or ethanolic extracts.
Less used in current practice but also mentioned in the literatures have been methanol (Hayet et al. 2008, Vukics et al.
2008), buthanol (Hayet et al. 2008, Sarr et al. 2009, Xu et al.
2010) ethyl acetate extracts (Hayet et al. 2008). The composition of those pharmaceutical products depends on various
factors as: plant species, plant botanical part, place and time
of harvest (Perez-Gregorio et al. 2014), climatic conditions
(Nour et al. 2013) during the plant growth and not at the
least the preparation recipe.
Chromatography analysis have been used to identify
phenolic/flavonoid compounds from plants including: thinlayer chromatography(TLC) (Nikitina et al. 2000, Dmitrienko
et al. 2012), high-performance HPTLC densitometric determination (Medić-Šarić et al. 2009, Pereira et al. 2009, Annegowda et al. 2012) and high performance liquid chromatography (HPLC) analysis (Akyuz et al. 2014; Markeb & ElMaali 2014, Riffault et al. 2014).
In the Romanian flora the Hibiscus genus is represented
by Hibiscus trionum L. (Flower of an Hour – rom. Zǎmoşiţǎ)
as native species with four subspecies: H. longilobus, H. prostratus, H. cordifolius, H. ternatus. Among them, Hibiscus
trionum L. is the only species with known therapeutic use. In
the pharmacology for tinctures from plant have been used
Hibiscus trioni herba harvested in early flowering. The literature mentions as well therapeutic use for underground organ Hibisci radix (Tămas et al. 2005). Hibiscus trionum appears
in different blends of tea, but so far there are only a few
standard pharmaceutical products derived from plants.
Our experiments have been going in two directions: (i) to
identify the secondary metabolites compounds from tinctures of Hibiscus trionum plants and (ii) to compare the relative contents of them for fresh and dry plants.
Materials and methods
Preparation of tincture by maceration
The Hibiscus plant material have been harvested in 2011 respectively
2012, in Oradea area, during the flowering season. The Hibiscus
trionum Herba harvested in 2011 have been subject to dry immediately after harvesting at ambient temperature and have been grinded
by spraying. The plants harvested in 2012 have been used fresh for
preparation of the tinctures.
The vegetable powder have been moistened with ethanol 70°,
using 1 ml ethanol 70% per gram of dry or fresh plant. Wetting was
achieved at room temperature for 2-3 minutes, and then were added
alcohol 70° in relation to 1 part to 5 parts solvent/herba. Maceration
was performed for 10 days with daily shaking two times for 10 min-
S.G. Bungău et al.
56
utes. After maceration the obtained tinctures were decanted and the
remaining plant material was manually pressed into the cloth and
pressed extract was added to the decanted tincture. Tincture thus obtained was kept to rest and then filtered and brought to the corresponding amount of 70° alcohol. The symbols of the tinctures obtained are: sample A - tincture of herba harvested in 2012 – fresh
samples sample B - tincture of herba harvested in 2011-dry samples,.
Qualitative and quantitative analysis of tincture
To characterize the tincture obtained the following determinations
have been made: organoleptic characteristics according to Romanian
Pharmacopoeia (Farmacopeea Română, 1993), determining the relative density with an Anton Paar digital densitometer - DSM35, quantitative determination of the residue obtained by evaporation, the determination of the concentration of alcohol by distillation and the determination of the relative density of hydroalcohol distillate obtained. For both tinctures were studied, by using thin layer chromatography: polyphenols and free amino acids .
The thin layer chromatographic analysis of polyphenols was
performed in the following experimental conditions: analyzed solutions: A and B; standard solutions: rutozide methanolic solution 1.25
mg/mL (Roth), chlorogenic acid 0.75 mg/mL (Fluka) - applied for 10
mL, -Stationary phase: Kieselgel 60F254 (Merck); mobile phase: ethyl
acetate (Merck) - methyl ethyl ketone (Merck) - formic acid (Merck)water (50:30:10:10, %V); migration distance 8 cm; development time
45 minutes. The application of solutions in the start was done in
band 1 cm at a distance of 1.5 cm from the bottom of the plate. Revelation of the plates was performed with reagents 1 and 2, as follows:
1. NEU (2-aminoethyl diphenylborate 1% in methanol), 2. PEG 400
(polyethylene glycol 1% in ethanol).
The analysis of chromatograms was done under UV light at 254
nm, and vizualization with NEU + PEG in fluorescence. In the case
of flavonoids compounds, when it is used two revealing reagents the
fluorescent spots yellow are obtained, yellow-green or yelloworange and polyphenolcarboxylic acids fluorescent blue, yellow or
blue-green (Wagner et al. 1996, European Pharmacopoeia 2004).
Chromatographic plate has been scanned with a CD Desaga 60
photodensitometer. The photodensitometry parameters were: the reflection mode, deuterium lamp, λ = 254 nm. Device parameters: deuterium and tungsten lamp, λ = 200-500 nm.
The determination of free amino acid by thin layer chromatography was performed in the following experimental conditions: solutions analyzed, standard solutions: solutions in ethanol 50% vol of
serine 1.1 mg/mL (Fluka), leucine 1.8 mg/mL (Fluka) and phenylalanine 1 mg/mL (Fluka); stationary phase: Kieselgel 60F254 (Merck);
mobile phase: n-buthanol (Merck) - acetone (Merck) - acetic acid
(Merck) - water (35:35:10:20, % vol); migration distance 10 cm; development time 40 minutes.
The application of solutions in the start was done in band 1 cm,
1.5 cm from the bottom of the plate. The plate revelation was per-
formed with ninhydrin at 120 ºC for 10 minutes. Analysis of chromatograms was made in visible light. In the case of amino acids vizualization of the revealing reagent have been violet-colored blue or violet-red spots ..
To confirm the presence of amino acids in the chemical composition of the species Hibiscus trionum was carried out on thin layer
chromatography coupled with photodensitometry. The photodensitometry parameters were: reflective mode, tungsten lamp, λ = 500
nm.
Results
On the basis of chromatograms, and densitograms of Rf values in the tinctures of Hibiscus plant have been identified an
ester of caffeic acid - chlorogenic acid and a flavonoid - rutin
(Fig. 1), and three amino acids: serine, leucine and phenylalanine (Fig. 2).
The results of organoleptic and physical characterization
of the tinctures of Hibiscus trioni herba preparations are presented in Table 1.
Rf values of the compounds in tinctures separated
through thin layer chromatography are presented in Table 2.
Discussions
Even that from the figures can be seen that there are more
compounds in the samples, chlorogenic acid and rutin are
the most important ones. Rutin have been identified as well
from Tripodanthus acutifolius extracts (Soberon et al. 2007)
and have been demonstarted their antibacterian properties.
A high concentrations of those aminoacids have been found
in tinctures of Pedilanthus tithymaloides with antiinflammatory and antioxidant properties used in traditional
Cuban medicines. From Table 1 can be seen that both tinctures have the same organoleptic characteristics. As well the
Rf values are the same which suggested that even that the
extractions have been done from dry plants, it can not be
seen only from visual exploration. From the quantitative
point of view, the tincture extracted from fresh plant material has been demonstrated to be superior.
By photodensitometric evaluation was performed the
quantitative determination of compounds identified by
comparing drop areas corresponding to the total drop area
and a separate calibration curve.
Figure 1. Rutin and chlorogenic acid densitograms from the tincture of fresh (Sample A) and dry (Sample B) Hibiscus trioni herba.
Study of dry and fresh Hibiscus trioni Herba tinctures
57
Figure 2. Free aminoacids densitogram from the tincture of fresh (Sample A) and dry (Sample B) Hibiscus trioni herba.
Table 1. Characterization of the tinctures.
Characteristics
Dry Hibiscus trioni herba
Fresh Hibiscus trioni herba
Organoleptic
Clear liquid, brown – transparent green,
aromatic smell, characteristic taste
Clear liquid, brown – green incolour,
aromatic smell, characteristic taste
Density (g/L)
0.891
0.894
Residues after vaporization (%)
1.76
2.26
Alcoholic concentration (% m/m)
61.5
60.4
Table 2. The Rf values of the compounds separated from tinctures.
Dry Hibiscus trioni
herba
Fresh Hibiscus trioni
herba
Rutin
0.28
0.28
Chlorogenic acid
0.46
0.45
Serine
0.23
0.25
Leucine
0.57
0.55
Phenylalanine
0.62
0.62
Separated compound
Table 3. The results of quantitative determination through thin layer
chromatography – densitometry for 5 different samples.
Compound
Dry Hibiscus trioni
herba (± SE)
Fresh Hibiscus trioni
herba (± SE)
mg/mL
mg/mL
Rutin
0.130 ± 0.007
0.309 ± 0.009
Chlorogenic acid
0.077 ± 0.009
0.093 ± 0.014
%
%
Serine
4.00 ± 0.21
17.2 ± 2.1
Leucine
5.44 ± 0.45
9.17 ± 0.32
Phenylalanine
1.98 ± 0.17
1.42 ± 0.19
The results of quantitative determinations by thin layer
chromatography-densitometry are given in Table 3.
Our results have been shown that fresher herba has
higher concentrations of active ingredients (in some cases 3
times more than dry). The same results have been observed
in fresh and dry extracts of Tulbaghia violacea plant where in
case of fresh plants the total flavonoids are 50% higher than
for dry plants (Olorunnisola et al. 2011). This can be explain
by a oxidation of flavonoids. Flavonoids and phenolcarboxilic compounds have a higher efficiency in extraction in a
hydroalcoholic mixture of 70°. Hydroalcoholic extracts obtained by natural maceration of the plant are the pharmaceutical tinctures with a concentration of standard active sub-
stances having stability between 1 and 3 years. These considerations underlie the extraction method used; the method
is simple and reproducible.
Following the qualitative and quantitative chromatographic study contained in the tincture of Hibiscus trioni
herba harvested in 2011 and 2012 it was noted that the tincture is rich in flavonoids and phenocarboxilic compounds
and free amino acids in significant quantities. The results indicate that fresh herba has higher concentrations of active
ingredients (rutoside and chlorogenic acid), indicating that
the plant can not be kept dry for long, if it will be used in
medical purposes.
These studies come to underline the need for standardization with simply and inexpensive methods of analysis of
the polyphenolic compounds found in plants in order to use
this data for some functional medicinal or food products.
The bioactive components from the plant material were
extracted using friendly environmentally solvents, as ethanol and water which made them compatible for both food
and non-food industrial use.
Thin layer chromatography coupled with photodensitometry allowed a quantitative determination of amino acids
concentration in tincture, Our results have been shown that
also the serine concentrations vary between 4 and 17.6
mg/mL, leucine concentration vary between 5.44 and 9.17
mg/mL while phenylalanine concentrations have been
found between 1.42 mg/mL and 1.98 mg/mL.
Acknowledgements. This work was supported by project co-funded
by European Union through European Regional Development Funds
Structural Operational Program “Increasing of Economic
Competitiveness” Priority axis 2, operation 2.1.2. Contract Number
621/2014.
58
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