FLAVonoiDS eXtrACtion From AlPiniA zeRumbet ( PerS . ) Burtt et SmitH LeAVeS uSing DiFFerent ProCeDureS

The current study aims to verify the best method for a rapid and efficient extraction of flavonoids from Alpinia zerumbet. Dried leaves were extracted using distillated water and ethanol 70% by extraction methods of shaking maceration, ultrasonic, microwave and stirring. By the application of TLC and reversed-phase HPLC techniques the rutin and kaempferol-3O-glucuronide were detected. Ethanol 70% was more efficient for flavonoids extraction than water. No significant yielding variation was verified for ultrasonic, microwave and stirring methods using ethanol 70% (11 to 14%). Relative concentration of rutin and kaempferol-3-Oglucuronide, respectively, was higher by ultrasonic (1.5 and 5.62 mg g-1 dried leaves) and by microwave (1.0 and 6.64 mg g-1 dried leaves) methods using 70% ethanol. Rapid and simplified extraction proceeding optimize phytochemical work and acquisition of secondary metabolites.


introduction
Alpinia zerumbet (Pers.)Burtt et Smith (Zingiberaceae) is an herbaceous perennial plant with wide use in Brazil.This plant is among the most cited in folk medicine [1].It is indicated to treat arterial hypertension [2].The flavonoids and essential oils of this species present remarkable medicinal properties and flavonoids are related to its main biological activity [3][4][5][6][7][8].Mpalantinos et al. [4] isolated and identified the flavonoids rutin and kaempferol-3-O-glucuronide by NMR, which have a high medicinal value in therapeutic uses [4].
Parameters as time, solvent, temperature and extraction technique influence secondary metabolites extraction.For this reason different pro-cedures have been used for acquisition of biologically active compounds from crude plant extracts.Recent studies mentioned the ultrasonic and microwave methods as efficient in secondary metabolites extraction [9][10][11].Solvent type and method of extraction are fundamental factors to consider for optimizing yield extraction [12].The purpose of this work was to develop and evaluate efficient and simple procedures for extraction of flavonoids from A. zerumbet leaves in short time.

Materials
Samples of Alpinia zerumbet (Pers.)Burtt et Smith leaves were collected in Rio de Janeiro (Rio de Janeiro state, Brazil), in the "Núcleo de Pesquisas de Produtos Naturais", in the Universidade Federal do Rio de Janeiro.Voucher specimens were identified and are deposited at the Herbarium of Rio de Janeiro Botanical Garden, accession number RB 433485.
All chemicals used in analysis as methanol and phosphoric acid were of HPLC grades and were purchased from Merck.MilliQ water was utilized to HPLC mobile phase and sample preparation.Kaempferol-3-O-glucuronide was isolated from Alpinia zerumbet and identified by Nuclear Magnetic Resonance (NMR) [4].Rutin was purchased from Merck  .

Preparation of extracts
Leaves of A. zerumbet were collected from adult plants, in the morning, then plant material was dried for 3 d in stove (60 o C) and macerated in 70% ethanol or distillated water, in the same proportion of 1 g dried leaves/20 mL solvent.
It was evaluated four extraction methods from dried leaves: maceration in shaker at 100 rpm, ultrasonic bath (40 kHz, Thornton Unique, model 1400 USC), microwave (PANASONIC  -Auto Sensor Diet, full power) and stirring (Table 1).In the microwave extraction, the suspensions were irradiated under microwaves in pre-setting procedures (3 s power on, 60 s off) for three times to the desired temperature about 60 and 70 o C. The temperature was measured after turn off the microwave using a thermometer into extracts in Becker.For the ultrasonic extraction, the 30 mL flask containing 1 g of dried leaves plus 20 mL of one of extracting solvents was partially immersed into the ultrasonic bath and temperature was controlled.Crude extracts were filtered in vacuum through a Whattman ® filter (110 mm ø, 1).Aqueous extracts were frozen, liophylizated and the hydroalcoholic extracts were evaporated to dry under reduced pressure at 60 o C. The dried weight was measured.The yielding was defined as follows: (crude extract weight/plant material weight) x 100.The crude extract obtained by each extraction technique was analyzed directly by TLC and HPLC.

HPLC analysis
Crude extracts were dissolved in methanol (70%) at 10 mg.mL -1 , filtered in vacuum, and HPLC analyses were performed on a Shimadzu apparatus equipped with SPD-M10A diode array detector, LC-10AD pump and CBM-10 interface, UV-vis detector.Data were acquired and processed by a reversed-phase column (Lichrosorb RP-18, 25 cm x 5 mm), ambient temperature.The solvent system used was a gradient of MilliQ water + 0.1% phosphoric acid (A) and methanol (B).The gradient was as follows: 1-10 min (30% B); 1-20 min (30 a 40% B); 20 to 60 min (40 a 100% B).The flow rate was 1 mL.min -1 .The prepared mobile phase was degassed using ultrasonic agitation.The elution was monitored at 254 nm and 360 nm.Flavonoids were identified by comparison of HPLC retention times, UV spectra and co-elution with authentic samples analyzed in the same conditions.Standards were dissolved in methanol 70% at 1 mg.mL -1 and analyzed in the same elution.For co-elution, it was prepared a mixture (1:1, v/v) of extracts at 10 mg.mL -1 and standard at 1 mg.mL -1 .The injections were repeated three times.Determination of the content of the flavonoids was performed by the external standard method, using authentic standards.Linearity was observed in concentration range of 0.0078 -0.0625 mg.mL -1 of rutin (y= 3.10 -7 x -31152, R= 0.9991, n= 9) and 0.01325 -0.25 mg.mL -1 of kaempferol-3-O-glucuronide (y= 1.10 -7 x -51000, R= 0.9951, n= 9).Each determination was carried out in triplicate.Quantification of flavonoids in the extracts was obtained against these calibration curves of standards, where y is peak area and x concentration in mg.mL -1 .

results and Discussion
Preliminary results obtained by different extraction time 15 and 30 min for ultrasonic and 1 and 2 days for maceration techniques did not indicate significant differences in relation to extraction time presented in this study.
Table 2 shows the extraction yielding obtained for each extraction technique.Regarding the extractive techniques, the most yielding was obtained after ethanol extraction in comparison with distillated water.The lowest yielding was found by maceration technique for both solvents.Hydroalcoholic crude extracts analysis using HPLC revealed six main compounds, among them the peak corresponding to rutin (RT: 31.42 min) and kaempferol-3-O-glucuronide (RT: 34.49min) can be observed right after 30 min, without interference of other components (Figure 1).These flavonoids were also verified through TLC technique.The results indicated that the highest extraction of flavonoids was obtained using 70% ethanol, by all techniques extraction (Figure 2).Aqueous, alcoholic and hydroalcoholic extracts are commonly used in researches with plant crude extracts [12].The results in Figure 2 also indicate that these four extraction techniques reach significant differences in content of flavonoids in the same material content.The use of ultrasonic and microwave techniques provide a high flavonoid extraction, followed by stirring (Figure 2).By comparison with the conventional techniques, ultrasonic and microwave techniques resulted in high levels of flavonoid extracted with the advantage of saving time and solvent.These methods have been reported by the efficiency in extracting secondary metabolites [6,7,13].Yang and Zhang [14] verified the efficiency and short extraction time of flavonoids using ultrasonic technique.The use of microwave for extracting biologically compounds is recent.Some reports have shown its positive results for extracting phenolic compounds, essential oil, flavonoids and alkaloids, more effective than conventional extraction methods [9,[15][16].
Extraction methods, involving heating, raised the efficiency of solvents.Microwave, ultrasonic and stirring may improve the flavonoid extraction using as water as 70% ethanol.These techniques present in common, besides higher temperature, a reduced extraction time in comparison with maceration in shaker.
TLC and HPLC chromatographic techniques are wide used and favorable for flavonoid detection [17].Reversed-phase HPLC is one of the most employed techniques for the analysis of flavonoids [17].Some results using HPLC were reported for Alpinia officinarum and A. purpurata species [1,19].The described HPLC procedures can be useful for the qualitative and quantitative analysis of flavonoids in crude extracts, especially from Zingiberaceae family that has a pronounced presence of flavonoids in its species verified through chemotaxonomic studies [20].

ConCLuSion
From the results obtained in the current study, the relative proportion of these flavonoids was reduced by maceration conventional technique, while microwave and ultrasonic techniques in combination with 70% ethanol solvent were the most efficient.It may suggest that microwave and ultrasonic methods using 70% ethanol are suitable for fast extraction of flavonoids in a simple way, also considering extraction yield and extraction time.These methods also permitted the acquisi-tion of flavonoids from reduced raw plant material.

Figure 2 .
Figure 2. Flavonoid contents in the aqueous and hydroalcoholic extracts of Alpinia zerumbet obtained by HPLC, comparing different extraction techniques.Each value consisted of average ± SD.Equal letters indicate no statistical differences among extraction techniques considering 70% ethanol as solvent, p<0.05.Comparing the extractor solvents, *p<0.05,**p<0.01 and ***p<0.001indicate statistical differences for each isolated technique; #p< 0.05 considering aqueous extract in relation to maceration technique (Bonferroni, n= 3).

table 1 .
Specifications of extraction methods.
a Data are related to water and ethanol 70%, respectively.

table 2 .
Yielding from dried leaves extraction of Alpinia zerumbet.
a Values indicate the averages of the three replicates.