ANTIOXIDANT PROPERTIES OF DIFFERENT PORTIONS OF ORGANIC Anoectochilus formosanus HAYATA WITH DIFFERENT DRYING TREATMENTS

Anoectochilus formosanus HAYATA (Orchidaceae) is a valuable herb used as herbal medicine in Taiwan and other Asian countries. In order to enhance the economic efficiency of organic cultivation and understand how the processing affects plant properties, organic A. formosanus extracts were used as the investigative material in this study. The entire plant, the roots, stems and leaves from organic A. formosanus were freeze-dried or hot air-dried; all samples used methanol to extract. The antioxidant properties and active components were analyzed. The results indicated that antioxidant properties of both the freeze-dried and hot air-dried leaves of organic A. formosanus have better performance, including reducing power and α,α-diphenyl-β-picrylhydrazyl (DPPH) radical scavenging activity. The contents of phenols and flavonoids showed positive relationships with reducing power, DPPH radical scavenging activity and trolox equivalent antioxidant capacity. The contents of carotenoids positively correlated with trolox equivalent antioxidant capacity and the contents of all active components were negatively correlated with the ferrous ion chelating ability. To summarize the results, freeze-dried leaves of organic A. formosanus have better antioxidant properties at a suitable concentration. This study may raise the economic values of A. formosanus by the organic cultural technique.


INTRODUCTION
Changes in the global market environment show that the aging population is increasing yearly.Most consumers agree that a particular food or drink intake can achieve the purpose of promoting health and disease prevention, thus resulting in the rise of the market of herbal health products used as complementary and alternative medicine (CAM) (CHAUHAN et al., 2013).Therefore, the development of new and functional herbal health products is a good way to promote people's health.
Reactive Oxygen Species (ROS) are the most abundant free radicals in cells, which are the unavoidable products during normal intracellular metabolism.However, a lot of cell components are believed to be damaged by ROS, including DNA damage, lipid peroxidation and also protein oxidation (CENCIONI et al., 2013;HAN;CHEN, 2013;CRUZ PADUA et al., 2014).There are two main antioxidant systems in cell (namely, enzymatic and non-enzymatic antioxidants), which perform systematically to scavenge the free radicals.Among them, vitamins (such as vitamin C and E) are the most well-known non-enzymatic antioxidants.In addition to vitamins, various functional small molecules also act as the non-enzymatic antioxidants (CZEIZEL et al., 2013;PENG et al., 2014).For example, phenolics, flavonoids and carotenoids are naturally produced in plants with similar structural basics.These antioxidants can be acquired from the daily diets, belonging to a group of food-derived phytochemicals named as nutraceuticals (PETCHETTI et al., 2007).
The genus Anoectochilus (Orchidaceae) is a perennial herb found in many countries including Taiwan, China, India and other Asian countries (ZHANG et al., 2013).Among them, four species are found in Taiwan, including Anoectochilus formosanus HAYATA, Anoectochilus koshunensis HAYATA, Anoectochilus inabai, and Anoectochilus lanceolatus (TSENG et al., 2006).A. formosanus is a valuable herb used as herbal tea in Taiwan and China.It is also known as "King Medicine" due to the various biological effects, such as its antioxidant, anti-inflammation, hepato-protective, anticancer and immunomodulatory activities (LIN et al., 1993;WANG et al., 2002;TSENG et al., 2006;LAI et al., 2015).Therefore, A. formosanus is a potential phytomedicine that can be used to develop the herbal health products.However, to the best of our knowledge, no study has discussed the relationships between the different drying treatments and the antioxidant activities of different parts of A. formosanus.
The cultivation environment is very important for the mass ratio in different parts of A. formosanus; for the plant, illumination, temperature, humidity and fertilizing has significant relatedness.Besides, the preparation processes may also influence the characterization of plant extract.Therefore, in order to enhance the cultivation of the economic efficiency and understand how the processing affects plant properties, the organic, A. formosanus was used as study material.The entire plant, the roots, the stems, and the leaves, from organic A. formosanus were freeze-dried or hot airdried; all samples used methanol as extraction solvent.The antioxidant properties, including reducing power, ferrous ion chelating ability, α,αdiphenyl-β-picrylhydrazyl (DPPH) radical scavenging activity, and trolox equivalent antioxidant capacity were analyzed.The active components measurement items included phenols, flavonoids, ascorbic acid, and carotenoids.The correlation of active components and antioxidant properties in the different parts of the plant were also analyzed in this presented study.

Preparation of extracts
The entire plant, the roots, the stems and the leaves from organic A. formosanus were freeze-dried (-45°C) or hot air-dried (55°C) overnight to remove water from the samples.In this study, methanol was used as the extraction solvent.Then 50 mL of methanol was added to 500 mg of the dried organic A. formosanus, the ratios of sample weight to solvent volume are 1, 2, 4, 6, 8 and 10 mg/mL; samples were then placed at a stirrer for 30 min.After the extraction procedure, the filtrates were collected and lyophilized.

Reducing power
Each 10 mL of the extracts with different concentrations and each antioxidants BHA and αtocopherol were individually mixed with 5 mL sodium phosphate buffer (0.2 M, pH 6.6) and 1 mL 1% potassium ferricyanide, and then the mixture was incubated at 50°C for 20 min.After trichloroacetic acid (1 mL, 10%, w/v) was added, the mixture was centrifuged at 4°C, 3000 rpm for 10 min.The 5 mL supernatant was mixed with 5 mL ddH 2 O and 1 mL ferric chloride (0.1%).After 10 min at 25°C, the absorbance of mixture was determined at 700 nm (A 700 nm ) (Oyaizu, 1986).

Chelating effect on ferrous
Each 5 mL of the extracts with different concentrations and EDTA were individually mixed with 3.7 mL sodium phosphate buffer (0.05 M, pH 7.4), 0.1 mL ferrous chloride (2 mM) and 0.2 mL ferrozine (5 mM).After 10 min at 25°C, the absorbance of the mixture was measured at 562 nm.The lower absorbance indicates the higher chelating effect (DECKER E WELCH, 1990).

Scavenging effect on 2,2-diphenyl-1picrylhydrazyl (DPPH) radicals
Each 5 mL of extracts with different concentrations and each antioxidants BHA and αtocopherol were individually mixed with 1 mL of methanolic solution containing DPPH radicals and the final concentration of DPPH was 1 mM.The mixture was shaken vigorously and left for 30 min at 25°C in the dark, and the absorbance was then determined at 517 nm (SHIMADA et al., 1992).

Trolox equivalent antioxidant capacity (TEAC)
The mixture of 7.0 mM ABTS and 2.45 mM potassium persulfate was used at room temperature in the dark to form the stable radical cation ABTS + .The solution was diluted with ethanol and absorbance was measured at 734 nm.An aliquot of each sample was mixed with the solution of the radical cation ABTS + (0.5 mL) and the decrease of absorbance was measured at 734 nm after 6 min.Different concentrations of trolox were used as positive reference.Therefore, higher TEAC level indicates higher antioxidant effect (ARNAO et al., 1996).

Quantification of total phenolic compounds
The samples and standard gallic acid solutions with 0.5 mL were diluted with 7 mL water.Subsequently, 0.5 mL Folin-Ciocalteu reagent was added and the solution was incubated at 100°C for 1 min.The absorbance was measured at 750 nm and the results were calibrated using the well-known phenolic compound gallic acid and expressed as the equivalents of gallic acid in mg/g (AINSWORTH E GILLESPIE, 2007).

Quantification of flavonoids
The samples and standard quercetin solutions with 1 mL were added to 1 mL of 2% methanolic AlCl 3 ．6H 2 O.After 10 min incubation at room temperature, the absorbance was measured at 430 nm.The results were expressed as the equivalents of quercetin in mg/g (QUETTIER-DELEU et al., 2000).

Quantification of ascorbic acid
The sample with 0.5 g was extracted by 50 mL of 1% metaphosphoric acid.Subsequently, 1 mL of extract solution was added to 9 mL DCIP (15 mg/L).The absorbance was measured within 15 s at 515 nm.The level of ascorbic acid was quantified by the standard curve (KLEIN; PERRY, 1982).

Quantification of carotenoids
According to the standard method for carotenoids quantification from A.O.A.C., 2 g samples were individually extracted by 100 mL acetone/n-hexane (6:4) solution with 0.1 g MgCO 3 for 5 min.The solutions were than extracted with MgO-Celite column and eluted by a 50 mL acetone/n-hexane (1:9) elution solution.The absorbance was measured at 430 nm and the level of carotenoids was quantified by the standard curve(HORWITZ E AOAC INTERNATIONAL., 1980).

The reducing power of the freeze-dried and the hot air-dried methanolic extracts of different portions from organic A. formosanus
To prepare the extracts, the entire plant, the roots, the stems and the leaves of organic A. formosanus were individually freeze-dried or hot air-dried to remove water from the samples; methanol was then used as the solvent to extract the active compounds from organic A. formosanus.The prepared extracts were subsequently used to analyse their antioxidant properties and active components.

Statistical analysis
Statistical analysis was performed using the variance (one-way ANOVA), Scheffe's method and the correlation analyses of Statistic Analysis System (SAS) to confirm the correlations between each sample and the antioxidant activity.

RESULTS
As shown in Figure 1, the reducing power of the freeze-dried and hot air-dried methanolic extracts of different portions from organic A. formosanus was examined.For freeze-dried extracts, the freeze-dried leaves extract has a better reducing power than the other parts from the concentrations of 1 to 10 mg/mL (Figure 1A).The extract of freeze-dried roots has a lower reducing power.Although the reducing powers of all tested freeze-dried extracts are relatively different when compared to those of the BHA and α-tocopherol groups (control groups) at all tested concentrations, we found that all freeze-dried extracts have moderate reducing powers (Figure 1A).In contrast, in Figure 1B, hot air-dried extracts have similar pattern with freeze-dried extracts.The hot air-dried leaves extract has better reducing power than those of the other parts at all tested concentrations.However, both the extracts of freeze-dried roots and stems have the lowest reducing power.Moreover, 10 mg/mL hot air-dried leaves extract has the greatest reducing power (about A 700 = 1.5) in all prepared extracts except in the control groups (Figure 1B).Besides, results in Figure 1 also indicated that both freeze-dried and hot air-dried leaves extracts have better reducing power than other extracts.
In the case of the ferrous ion chelating ability, results are shown in Figure 2 and EDTA is used as a control.In Figure 2A, all freeze-dried extracts of organic A. formosanus revealed obvious ferrous ion chelating ability.At lower concentrations (1 to 2 mg/mL), the freeze-dried leaves extract has the best ferrous ion chelating ability than other extracts; the ability can achieve up to 78% at 2 mg/mL (Figure 2A).However, at higher concentrations, the ferrous ion chelating ability of freeze-dried leaves extract is reduced with the increasing dosage.At 10 mg/mL, freeze-dried stem extract has the greatest ferrous ion chelating ability higher than 80% (Figure 2A).Besides, EDTA in all tested concentrations has the highest ferrous ion chelating ability.In the case of the hot air-dried extracts, results are shown in Figure 2B.The extract from hot air-dried entire plants has the most potent ferrous ion chelating ability than other extracts if the concentrations are higher than 4 mg/mL.The ferrous ion chelating ability can be up to 83% at the concentration of 8 mg/mL in hot air-dried entire plants extract treated group (Figure 2B).Similar to the results in Figure 2A, the ferrous ion chelating ability of hot air-dried leaves extract increased if between 1 and 4 mg/mL conditions, but decreased if the concentrations are higher than 4 mg/mL (Figure 2B).Therefore, the results revealed that the extracts of freeze-dried stem and air-dried entire plants from organic A. formosanus have the greatest ferrous ion chelating abilities at higher concentrations The DPPH radical scavenging activity of the freeze-dried and hot air-dried methanolic extracts of different portions from organic A. formosanus In the case of DPPH radical scavenging activity, results are shown in Figure 3; the BHA and α-tocopherol are used as control groups.For freezedried extracts of organic A. formosanus, the DPPH radical scavenging activities are all higher than 50% at the highest concentration (10 mg/mL).The freeze-dried leaves extract has the greatest DPPH radical scavenging activity than other extracts at the concentration of 10 mg/mL and higher than both used control compounds (Figure 3A).In additions, the extracts from freeze-dried roots and stems are exhibiting the lowest DPPH radical scavenging activities at all tested concentrations.For hot air-dried extracts, in Figure 3B, hot air-dried leaves extract has the greatest DPPH radical scavenging activity than other extracts in all used concentrations.Although all hot air-dried extracts have clear DPPH radical scavenging activities, however, results showed that the DPPH radical scavenging activities of hot air-dried extracts are evidently poorer than that of freeze-dried extracts.Therefore, we can suggest that the freezedried extracts of different portions from organic A. formosanus have better DPPH radical scavenging activities than the hot air-dried counterpart.
The trolox equivalent antioxidant capacity (TEAC) of the freeze-dried and hot air-dried methanolic extracts of different portions from organic A. formosanus In the case of the trolox equivalent antioxidant capacity (TEAC) assay, results are shown in Figure 4.The results showed that every freeze-dried organic A. formosanus extracts have clear TEAC values around 2.5 mmol trolox at concentrations from 1 to 10 mg/mL (Figure 4A).Equally, all hot air-dried organic A. formosanus extracts also exhibited approximately TEAC values of 2.5 mmol trolox in all tested concentrations (Figure 4B).Therefore, the results proposed that two dried processes have no obvious effect on the TEAC of all parts of organic A. formosanus extract (Figure 4).

The contents in active components of freezedried and hot air-dried methanolic extracts of different portions from organic A. formosanus
The contents of active components of freeze-dried organic A. formosanus extracts are shown in Table 1.On the other hand, the contents of the active components of hot air-dried organic A. formosanus extracts are shown in Table 2.The analytical results of the content of active components in different portions from freeze-dried and hot air-dried organic A. formosanus were performed using the Statistic Analysis System (SAS) with one-way ANOVA.Our results showed that hot air-dried process has great effects on the contents of phenols, ascorbic acid and carotenoids in parts of organic A. formosanus.Among them, leaves part has a greatest difference (Table 1 and 2).In contrast, freeze-dried process can obtain abundant flavonoids; the part of leaves has the highest variance (Table 1 and 2).This might be that the two different drying treatments have diverse temperatures in each process.Besides, flavonoids are relatively thermostable than the other antioxidants and active compounds.Therefore, the flavonoids are not as much of different between two processes.The correlation coefficients between the content of active components of different portions and the antioxidant properties indices from freezedried and hot air-dried organic A. formosanus The analytical results of the correlations between each sample and the antioxidants were shown in table 3 and 4. The results are performed through the assessment of a single concentration (10 mg/mL) in all samples with all four-antioxidant activity examined.Our results showed that carotenoids positively correlated with the reducing power and DPPH radical scavenging activity in hot air-dried organic A. formosanus.Moreover, carotenoids positively correlated with the ferrous ion chelating ability in the freeze-dried organic A. formosanus.In addition, the contents of phenols and ascorbic acid in all parts of both freeze-dried and hot air-dried organic A. formosanus positively correlated with the reducing power, the DPPH radical scavenging activity and the TEAC.

DISCUSSION
In the study, all parts of organic A. formosanus were individually freeze-dried or hot air-dried to remove water.The methanol extracted from the plant extracts were used to analyse the antioxidant properties and active components.These results indicated that antioxidant properties of both the freeze-dried and hot air-dried leaves of organic A. formosanus have better performance, including reducing power and α,α-diphenyl-β-picrylhydrazyl (DPPH) radical scavenging activity.
In addition, TEAC assay is a standard method to evaluate the antioxidant activity on the scavenging of radical cation ABTS + .The ABTS + radical is also one of the compositions used in the method of measuring total antioxidant activity (TAA) for foods, drugs and chemicals (ARNAO et al., 2001).In this study, all freeze-dried and hot airdried organic A. formosanus extracts exhibited the TEAC values of 2.5 mmol trolox in all tested concentrations (Figure 4B).For the reason, we supposed that all the used extraction processes have no effects on the ABTS + radicals scavenging activity of the extracts from every part of organic A. formosanus.
In tables 1 and 2, hot air-dried process is better for the contents of total phenols, ascorbic acid and carotenoids.When compared, the variance in leaf part is the most obvious, freeze-dried by lyophilisation can retain plentiful flavonoids; this also noticeable in the results of the leaf part.Therefore, in contrast with the results shown in tables 3 and 4, we can assume that using different processes to extract active components from all parts of organic A. formosanus may affects their antioxidant properties.Some studies have observed that the functional constituents may affect (positively or negatively) the antioxidant activities of plant extract.The phenomenon may be caused by the synergistic effect of these active compounds (LOU et al., 2010;RANJBAR NEDAMANI et al., 2015).
During the drying process of plant materials, drying methods and drying temperatures significantly influenced the extraction efficacy as well as the properties of the extract (WU, 2015).
Although the lower extraction temperature may retain the stability of functional constituents, the higher extraction temperature can remove water quickly and also enhance the dissolving of active compounds from plant tissues (GAUR E RAO, 2016).Therefore, the examination for different drying methods in the extraction process is fairly important; this is also conducted in the presented study.

CONCLUSIONs
The antioxidant properties were improved with increase in sample concentrations of the freeze-dried leaves of organic A. formosanus; it has better reducing power and DPPH radical scavenging activity.
For the trolox equivalent antioxidant capacity, the TEAC values in different parts of organic A. formosanus were close to that of 2.5 mM Trolox.
The contents of phenols and flavonoids showed positive relationships with reducing power, DPPH radical scavenging activity and trolox equivalent antioxidant capacity.Besides, the contents of carotenoids positively correlated with TEAC and the contents of all active components negatively correlated with the ferrous ion chelating ability.
Therefore, freeze-dried leaves of organic A. formosanus have better antioxidant properties at proper concentrations.This study may also raise the

Figure 1 .
Figure 1.The reducing power of the methanolic extracts of different portions from organic A. formosanus HAYATA, BHA and alpha-tocophenol.(A) Freeze-dried and (B) Hot air-dried.

Figure 2 .
Figure 2. The ferrous ion chelating ability of the methanolic extracts of different portions from organic A. formosanus HAYATA and EDTA.(A) Freeze-dried and (B) Hot air-dried.

Figure 3 .
Figure 3.The DPPH radical scavenging activity of the methanolic extracts of different portions from organic A. formosanus HAYATA, BHA and alpha-tocophenol.(A) Freeze-dried and (B) Hot airdried.

Figure 4 .
Figure 4.The equivalent trolox antioxidant capacity (TEAC) of the methanolic extracts of different portions from organic A. formosanus HAYATA.(A) Freeze-dried and (B) Hot air-dried.

Table 1 .
The contents in active components of different portions from freeze-dried A. formosanus HAYATA.

Table 2 .
The contents in active components of different portions from hot air-dried A. formosanus HAYATA.Each value is expressed as the mean ± S.D. (n=3); Means with identical letter in the same column are not significantly different at p＜ 0.05.

Table 3 .
The correlation coefficients between the content of active components of different portions and the antioxidant properties indices from freeze-dried A. formosanus HAYATA.

Table 4 .
The correlation coefficients between the content of active components of different portions and the antioxidant properties indices from hot air-dried A. formosanus HAYATA.RP: reducing power, FICA: ferrous ion chelating ability, DRSA: DPPH radical; scavenging activity, TEAC: Trolox equivalent antioxidant capacity; *Correlation is significant at p= 0.05 level; ** Correlation is significant at p= 0.01 level. a