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LYTHRUM SALICARIA (PURPLE LOOSESTRIFE).
MEDICINAL USE, EXTRACTION AND
IDENTIFICATION OF ITS TOTAL PHENOLIC
COMPOUNDS
SUHAD S. HUMADI1, VIORICA ISTUDOR2*
1
University of Baghdad, Faculty of Pharmacy
University of Medicine and Pharmacy “Carol Davila”, Faculty of
Pharmacy, Traian Vuia 6, sect. 2, 020956, Romania
*corresponding author: [email protected]
2
Abstract
Lythrum salicaria L., known as purple loosestrife (Lythraceae) has a wide range
of beneficial health effects. It is well known as a medicinal plant from ancient Greek and
Roman times and it has been an important drug for centuries. Its pharmacological activity is
mostly due to its phenolic compounds, mainly tannins. Therefore obtaining an extract with
a high percent of total phenolic compounds would be interesting for studying the biological
potential of the plant. The dried herbal parts of Lythrum salicaria L. were sequentially
extracted with different solvents: 96% ethanol, 70% ethanol, and distilled water, in order to
select the best solvent exhibiting the maximum percent of total phenolic compounds; the
measurements were performed using modified Folin-Ciocâlteu method according to
Makkar et al. In addition, the total flavonoid content was also analyzed in the different
extracts using Chang et al method. The quantitative analysis of the examined chemical
compounds showed that 70% ethanol solution was the best solvent used in order to obtain
the highest phenolic content.
Rezumat
Lythrum salicaria L., răchitan (Lythraceae) are multiple efecte benefice asupra
sănătăţii. Specia este bine cunoscută ca plantă medicinală încă din antichitate, de greci şi
romani. A fost considerată de secole ca produs valoros datorită proprietăţilor sale
farmacologice imprimate în special de compuşii fenolici (majoritar taninuri). Prin urmare,
obţinerea unui extract cu un înalt conţinut de compuşi fenolici va fi valoros, întrucât ar
putea prezenta o activitate farmacologică interesantă (hipoglicemiantă, hipolipemiantă,
antiseptică, antioxidantă). În acest scop vârfurile florale uscate ale speciei Lythrum
salicaria L. au fost extrase selectiv cu diferiţi solvenţi : etanol (96% şi 70%) şi apă distilată,
pentru a alege solventul potrivit, care extrage o cantitate maximă de compuşi fenolici (acizi
fenolici, taninuri, flavone). Aceştia au fost determinaţi prin metoda Folin-Ciocâlteu
modificată de Makkar şi colaboratori. Conţinutul în flavone a fost analizat prin metoda
Chang şi colaboratori. Rezultatele obţinute au demonstrat că etanolul 70% a fost solventul
adecvat pentru a obţine un extract de cea mai bună calitate.
Keywords: Lythrum salicaria; medicinal plant; tannins; phenolic compounds;
modified Folin-Ciocâlteu method.
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Introduction
The genus Lythrum (Lythraceae) is spread throughout the world. It
is represented by almost 30 species, 10 of which are found in Europe. The
generic name comes from the Greek "luthron," blood, possibly referring to
the color of the flowers or to one of its herbal uses as an astringent. L.
salicaria is originally Eurasian, but during the 19th century it was spread via
the ballast of European ships not only to Europe but also into North and
South America, as well as Australia. Its English name is “blooming sally”,
“purple willow-herb”, “rainbow weed” and “purple loosestrife”. It is known
in German as “Blutweiderich”, in French “Salicaire”, in Romanian
“răchitan” and in Swedish “fackelblomster” [1- 4]. L. salicaria is known as
a medicinal plant from the ancient Greek and Roman times and it has been
an important drug for centuries.
The whole flowering plant and the flowering branch tips of this
plant are used not only in folk medicine but also in pharmaceuticals. It is
used internally for diarrhea, chronic intestinal catarrh, in the form of a
decoction or a fluid extract. Externally, it is used to treat varicose veins,
venous insufficiency, bleeding of the gums, hemorrhoid and eczema [5- 7].
The phytochemical examination carried on this plant reported that
tannins were the main compounds in Lythrum salicaria. It contains a
notable amount of flavons represented by flavon C-glycosides (vitexin,
isovitexin, orientin and isoorientin) and anthocyanins, also. In addition,
vescalagin, pedunculagin, vanoleic acid dilactone, 1,6-di-O-galloylglucose,
1-O-galloylglucose and 6-O-galloylglucose were identified. Sterols as βsitosterol were also detected in this plant [7- 10].
Antioxidant, antimicrobial, and hypoglycemic effects of Lythrum
salicaria have been reported [11-14]. An ethanolic extract of this plant
showed concentration-dependent superoxide anion radical scavenging
activity and inhibitory effect on lipid peroxidation [14]. Aqueous
methanolic extract showed a moderate antioxidant activity regarding the
auto-oxidation of methyl-linoleate [12] and against the ABTS radical [2, 2’
aziono bis (3-ethylbenzoline)-6-sulfonic acid radical] [13]. The effect of
Lythrum salicaria on the growth of Candida albicans, Staphyllococcus
aureus and Escherichia coli and its antifungal activity have been reported
[6]. Stems and flowers of Lythrum salicaria show hypoglycemic activity in
epinephrine-induced hyperglycemia [11].
Tannins (the main component of Lythrum salicaria) are extracted
from the plant matrix depending on their physical properties (solubility in
water, alcohol and acetone). These solvents can be used alone or as a
mixture [15]. Some solvents are incompatible with some methods of
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analysis, in particular acetone which inhibits all protein precipitation assays
except the radial diffusion assay [16].
The addition of water to methanol (or ethanol) was found to
increase the total phenolic (TP) recovery, since water allows the plant tissue
to swell allowing the solvent to better penetrate the sample matrix [17].
In this study, different solvent extracts of Lythrum salicaria were
quantitatively analyzed for their total phenolic compounds content via
modified Folin-Ciocâlteu method [18] according to Makkar et al [19]
measured as tannic acid equivalent, in order to select the best solvent able to
extract the highest percent of total phenolic compound exhibiting the best
pharmacological activity.
Materials and methods
Plant material
The plant samples were provided by Phytotherapy S.C., Romania.
Instruments
UV/Vis spectrophotometer Jasco V-530 (Jasco, Japan) with PC-HP
845 x UV-Visible System (Jasco, Japan) and 1 cm quartz cells were used for
all absorbance measurements.
Reagents and solutions
Ethyl acetate, 96% ethanol, sodium carbonate, sodium tungstate,
sodium molybdate, phosphoric acid, lithium sulfate, aluminum chloride
hexahydrate, and potassium acetate, were provided by Fluka-chemika; we also
used during the study bromine solution and concentrated hydrochloric acid.
For standard chemicals: tannic acid and quercetin (Sigma/
Germany) were used.
Preparation of plant extracts
Two grams of the powder plant samples were refluxed separately
with 50 mL of the three different solvents: 96% ethanol (E-1), 70% ethanol
(E-2), and distilled water (E-3), for 30 minutes at 700C, followed by
successive re-extraction with the same extracting solvent until the
disappearance of the brown extract color.
The resultant filtrates were made off up to 50 mL with the same
extracting solvent (solution A = S.A).
Estimation of the total phenolic content
Determination of total phenolic content (TPC), measured as tannic
acid equivalent, was performed using the modified Folin-Ciocâlteu method
according to Makkar et al [19].
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To perform this analysis three reagents were used: Folin-Ciocâlteu,
sodium carbonate (20% solution), and standard tannic acid solution.
Folin-Ciocâlteu reagent was obtained according to the literature
procedure [20].
Preparation of standard calibration curve
The tannic acid standard solution (0.1 mg/ mL) was prepared by
dissolving 10 mg of tannic acid in 10 mL of distilled water, and then the
volume was completed to 100 mL with the same dissolving solvent (we
used freshly prepared solutions).The standard calibration curve was then
developed as mentioned in the proceeding table.
All the flasks were mixed well and left in the dark at room
temperature for 40 minutes, then the absorbance was read at λ = 725 nm [19].
Flask
Blank
Test Stand. 1
Test Stand. 2
Test Stand. 3
Test Stand. 4
Test Stand. 5
Tannic acid sol.
(0.1mg/ mL)
Volume taken
in mL
0.00
0.02
0.04
0.06
0.08
0.10
Table I
Preparation of tannic acid standard curve
Sod. Carbonate
Distilled water
Folin Reagent
solution.
Volume taken
Volume taken
Volume taken
in mL
in mL
in mL
0.50
0.25
1.25
0.48
0.25
1.25
0.46
0.25
1.25
0.44
0.25
1.25
0.42
0.25
1.25
0.40
0.25
1.25
The volumes of the standard tannic acid solution together with the
volumes of the reactive substances used in the preparation of the standard
calibration curve are mentioned in table (I) whereas the resulted series of
tannic acid concentrations with their corresponding absorbance are showed
in table II. The obtained tannic acid standard curve is shown in figure 1.
Analysis of the total phenolic compounds in plant extracts
Sample extracts were prepared by diluting 25 mL of sol. A of each
of E-1, E-2, and E-3 separately to a final volume of 50 mL with 96 %
ethanol (sol. SC: stock solution of 1g dried plant /50 mL). To proceed with
the Folin-Ciocâlteu method, 0.1 mL of sol. SC (sample extract) was diluted
with 0.4 mL of distilled water, followed by the addition of 0.25 mL of
Folin-Ciocâlteu reagent and 1.25 mL sodium carbonate solution. The blank
was prepared using the same chemical reagents excluding the extract.
The flasks were mixed well and left in the dark, at room
temperature for 40 minutes, then the absorbance was read at λ = 725 nm.
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Quantification of tannins in salicaria extracts
The determination of tannins in the tested extracts was performed
using the modified Makkar et al method [19].
The modification here was the use of hide powder instead of PVPP
(Polyvinyl poly-pyrrolidone). To carry out the test, 100 mg of hide powder was
weighed, then 1.0 mL of distilled water followed by 1.0 mL of the tannincontaining extract were added. This mixture was shaken for 60 minutes,
afterwards centrifuged for 10 min and finally the supernatant was collected.
The supernatant has only simple phenolic compounds other than tannins (the
tannins would have been precipitated along with the hide powder).
The phenolic content of the supernatant was then measured
following the same procedure described above, taking at least double the
volume (preferably three times i.e. 0.3 mL) that was used for total phenol
estimation, because the extract had already been diluted, in addition to the
lose of tannin-phenols through binding with the hide powder.
The content of non-tannin phenols was expressed on a dry plant
basis (y%), and the percentage of tannins was calculated as tannic acid
equivalent, estimated as grams per 100 grams dried plant as follows:
Tannin percentage = (x %) – (y %)
where x % is the percentage of the total phenolic compounds (g/100g dried
plant), y % is the content of the non-tannin phenols (g per 100 grams dried plant)
Estimation of the total flavonols content
The total flavonoid content in Salicaria extracts was measured using
Chang et al method [21, 22] with simple modifications applied to the sample
preparation. The same standard curve equation was applied in order to
calculate the flavonol percent which was expressed as quercetin equivalent.
Sample preparation
The total flavonol content in Salicaria extracts was measured using
Chang et al method with simple modifications applied to the sample
preparation as such: 25 mL of S.A for each of E-1, E-2, and E-3 were first
hydrolyzed with a solution of 4 N HCl for 30 minutes, then the filtrates were
partitioned with ethyl acetate (15 mL x 3) after that evaporated to dryness
and re-dissolved in 96% ethanol to a final volume of 25 mL, solution D
(S.D). The test was applied on 1 mL of S.D to which 3 mL of 96% ethanol
(V/V), 0.2 mL 10% aluminium chloride (m/V), 0.2 mL of 1 mol/ L
potassium acetate and 5.6 mL distilled water were added. A volume of 10 %
(m/V) aluminum chloride was substituted by the same volume of distilled
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water in the blanks. After incubation at room temperature for 30 minutes,
the absorbance of the reaction mixture was measured at λ = 415 nm.
Statistical analysis
The results of all experiments were expressed as the Mean ±
standard deviation (SDOM) upon three independent trials.
Results and discussion
The total phenolic content in different Salicaria extracts was
expressed as tannic acid equivalent using the standard curve equation:
Ab = A + B x Conc. (A= 0.0055; B= 0.2666); r2 = 0.9999
0 .6 2 3 1
0 .4
Abs
0 .2
0
0
1
C o n c .[ % ]
2
2 .1 6
Figure 1
Tannic acid standard curve
Table II
Tannic acid calibration curve
Expression: Abs = A + B x Conc.( Factors: A= 0.0055; B = 0.2666
Coefficient r 2 = 0.9999)
Sample No.
Conc.% (mg /100 mL)
Absorbance
1
0.400
0.1119
2
0.800
0.2178
3
1.200
0.3270
4
1.600
0.4321
5
2.000
0.5379
Then the final test sample concentration was obtained using the
following formula:
(R x DF x V x100)/W
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where R= result obtained from the standard curve equation; DF= dilution
factor; V= volume of stock solution; 100= for 100 grams dried plant; W=
weight of plant used throughout the experiment in (g).
Flavonols were estimated by means of Chang et al method using
the same standard curve equation (y = 0.0603 x + 0.0007, r2 = 0.9997) and
values were calculated as g % (g Quercetin per100g of dried plant material).
Modifications of the original Chang et al method were performed
due to the presence of turbidity which was noted after the addition of
potassium acetate to the unhydrolyzed test sample that could be attributed to
a reaction between one or more extract constituents with the applied
potassium acetate. Therefore, a hydrolysis process to the test sample was
needed in order to exclude the interference of other plant chemicals with the
applied reagents, thus the analysis was applied to the aglycone part of the
extracts.
The quantities of each of the examined chemicals are represented in
the table bellow.
Extract
E-1
E-2
E-3
Table III
Quantitative analysis of the chemical constituents in different Salicaria extracts
Total phenolic compounds Tannins% (g/100g
Total flavonols percent
percent expressed as tannin
of dried plant)
expressed as quercetin
equivalent (g /100 g dried
equivalent (g /100 g dried
plant)
plant)
9.2 g % ± 0.22
3.062 g% ± 0.220
0.632 g % ± 0.012
17.5 % ± 0.111
6.153 g% ± 0.213
0.594 g % ± 0.142
14.0 g % ±0.031
7.579 g % ± 0.301
0.214 g % ± 0.223
The presented data indicates that the order of increasing phenolic
content, expressed as tannic acid equivalent (g) per gram of dry plant was:
70% aqueous ethanol >water > ethanol. The addition of water to ethanol
was found to increase the total phenolic recovery, since water allows the
plant tissue to swell, allowing the solvent to better penetrate the sample
matrix. It is clear from the results obtained that a mixture of ethanol: water
in a ratio of 70:30 (v/v) evoked the best results.
An interesting difference in tannin content was noted from the
above data showing the independency of tannins percentage as related to the
total phenolic content. This may be attributed to the fact that alcohol
solvents have a harmful effect on the recovery of hydrolysable tannins
(represented by ellagitannins in Salicaria extracts). Viriot et al. found that
ellagitannins undergo hydrolysis to give ellagic acid in aqueous ethanol
solution [23], where as Puech and coworkers demonstrated that the
hydrolysis of ellagitannins increase with the ethanol concentration [24].
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199
They also found that hydrolysis was not the only degradation process
associated with ellagitannins in aqueous ethanol solutions. Oxidation also
occurs, giving ellagitannins derivatives containing ethoxy groups [24].
Methanol was also found to cleave ester bonds in gallotannins at room
temperature through methanolysis, suggesting that methanol was also not a
suitable solvent for extracting ellagitannins [25].
The aqueous extracts show a higher percent of recovery of tannins
than the other examined solvents.
Conclusions
This study assured that Lythrum salicaria extracted with 70%
ethanol solution showed the highest percent of total phenolic compound,
whereas the maximum tannin percent was obtained in aqueous extracts of
this plant.
References
Rauha J.P. - The research for biological activity in Finnish plant extracts
containing phenolic compounds; PhD Thesis, Pharmacognosy Department,
Pharmacy Faculty of Science, University of Helsinki, 2001, 31–45
2. Baytop T. - Therapy with Plants in Turkey; Istanbul University Publications,
Istanbul, 1999, 3255: 374
3. Davis P.H. - Flora of Turkey and the East Aegean Islands; University Press,
Edinburgh, 1982, 4: 174–176
4. Al-Borza – Dicţionar Etnobotanic; Editura Academiei Republicii Socialiste,
Romania, 1968, 104
5. Mantle D., Eddeb, F., Pickering, A.T. - Comparison of relative antioxidant
activities of British medicinal plant species in vitro; Journal of
Ethnopharmacology, 2000, 72: 47–51
6. Rauha J.P., Remes, S., Heinonen, M., Hopia, A.I., Kahkonen, M., Kujala, T.S.,
Pihlaja, K., Vuorela, H.J., Vuorela, P.- Antimicrobial effects of Finnish plant
extracts containing flavonoids and other phenolic compounds; International
Journal of Food Microbiology, 2000, 56: 3–12
7. Thomson physical desk reference: - PDR for Herbal Medicine; 2nd Ed. 2000, 394395, 666
8. Rauha J.P., Wolfender J.L., Salminen J.P., Pihlaja K., Hostettmann K., Vuorela
H. - Characterization of the polyphenolic composition of purple loosestrife
(Lythrum salicaria). Zeitschrift fur Naturforschung, 2001, C 56: 13–20
9. Bruneton J., - Pharmacognosie, phytochimie, plantes medicinales; TEC&DOCLaveisier, 2005, 3rd Ed., 375
10. Bisset N. G., Wichtl M.- Herbal Drugs and Phytopharmaceuticals; A Hand Book
for Practice on a scientific basis; CRC Press, Boca Raton, Ann Arbor, London,
Tokyo, 1994, 266-267, 663
11. Lamela M., Cadavid I., Calleja J.M., Effects of Lythrum salicaria extracts on
hyperglycemic rats and mice; Journal of Ethnopharmacology, 1986, 15: 153–160
1.
200
FARMACIA, 2009, Vol. 57, 2
12. Kahkonen M.P., Hopia A.I.,Vuorela H.J., Rauha J.P., Pihlaja K.,Kujala T.S.,
Heinonen M., - Antioxidant activity of plant extracts containing phenolic
compounds; Journal of Agricultural Food Chemistry, 1999, 47: 3954–3962
13. Mantle D., Eddeb F., Pickering A.T. - Comparison of relative antioxidant activities
of British medicinal plant species in vitro; Journal of Ethnopharmacology, 2000,
47–51
14. Coban T., C¸ Itoglu G.S., Sever B., Isan M. - Antioxidant activities of plants used
in traditional medicine in Turkey; Pharmaceutical Biology, 2003, 41: 608–613
15. Istudor V. - Farmacognozie, Fitochimie, Fitoterapie; Editura Medicală, Bucureşti,
1998, 1: 194-195
16. Ann E. Hagerman- Tannin handbook, 2002, 3-5
17. Mukhopadhyay S., Luthria D. L., and Robbins R. - Optimization of Extraction
Process for Phenolic Acids from Black Cohosh (Cimicifuga racemosa) by
Pressurized Liquid Extraction; J. Sci. Food Agric., 2006, 86: 156–162
18. Farmacopeea Română ediţia a X-a, Editura Medicală, Bucureşti, 1993, 1063-1064
19. Makkar H.P.S., Bluemmel, M., Borowy N.K., Becker K.- Gravimetric
determination of tannins and their correlations with chemical and protein
precipitation methods; J. Sci. Food Agric., 1993, 61: 161–165
20. Krebs K.G. et al., Thin Layer Chromatography, Stahl E., Springer-Verlag, New
York, NY, 1969, 878
21. Chang C.C., Yang M.H., Wen H.M., and Chern J.C. - Estimation of total
flavonoid content in propolis by two complementary colorimetric methods; J.
Food Drug Analysis, 2002,10: 178-182
22. Humadi S., Istudor V.– Quantitative analysis of bio-active compound in Hibiscus
sabdariffa L. extracts: I, Quantitative analysis of flavonoids; Farmacia, 2008, 56,
6: 699-706
23. Viriot C.,Scalbert A., Lapierre C.and Moutounet M. – Ellagitannin and lignins in
aging of spirits in Oak Barrels; J. Agric. Food Chem., 1993, 41:1872-1879
24. Puech J.-L., Mertz C., Michon C.,Guerneve C.L., Doco T.,and Herve du Penhoat
C.– Evolution of castalagin and vescalagin in ethanol solutions. Identification of
new derivatives; J. Agric. Food Chem.,1999, 47: 2060-2066
25. Mueller-Harvey I., Analysis of hydrolysable tannins; Animal Feed Sci. and
Technol., 2001, 91: 3-20.
Manuscript received: 29.11.2008