BHST. 2015, 13 (1) : 29-37 Charoenchai et al.

Bulletin of Health, Science and Technology

BHST ISSN 0858-7531

Volume 12, Number 2, 2015 : 29-37

EVALUATION OF THE ANTIOXIDANT ACTIVITIES, TOTAL PHENOLIC AND

FLAVONOID CONTENTS OF MELIENTHA SUAVIS PIERRE

Laksana Charoenchai

1,2*, Sukunya Settharaksa

1,2, Fameera Madaka

1,2, Lukman Sueree

1,2

1Faculty of Pharmacy, Rangsit University, Pathum Thani, THAILAND.

2The Herbal Medicinal Products Research and Development Center (Cooperation between Rangsit University,

Harbin Institute of Technology and Heilongjiang University of Chinese Medicine), Pathum Thani 12000,

THAILAND.

*Corresponding author: Laksana Charoenchai; E-Mail: laksana.c@rsu.ac.th

Abstract: Melientha suavis Pierre (pak-wan pa) is a wild food plant found in Thailand and

Southeast Asia. The aim of this research was to examine its antioxidant activities, total phenolic

and flavonoid contents of pak-wan pa in Thailand. Pak-wan pa extracts were prepared by solvent

extraction using soxhlet apparatus. The antioxidant activities of the extracts were determined using

2,2-diphenyl-1-picryhydrazyl radical scavenging (DPPH) assay and ferric reducing antioxidant

power (FRAP) assay. The results found that the water extract of pak-wan pa exhibited antioxidant

activity with an IC50 of 900-1150 g/mL in DPPH assay and 20 g/mL in FRAP assay, compared

with that of gallic acid (2.84+0.008 g/mL) and trolox (0.94 ± 0.224), respectively. The highest

total phenolic and flavonoid contents of pak-wan pa was found in the methanolic extract (265-

1,978 mgGE/100 g dry extract and 21-459 mgTE/100g dry extract) and in water extract (183-296

and 17-20 mgCE/100g dry extract), respectively. The results also showed a strong correlation

between the FRAP scavenging activities and the flavonoid contents (Pearson correlation = 1.000)

with significantly difference (p-value=0.013).

Keywords: Melientha suavis Pierre, Antioxidant activity, Flavonoid content, Phenolic content

บทคัดย่อ: ผกัหวานป่าเป็นพืชอาหารท่ีพบไดใ้นป่าของประเทศไทยและภูมิภาคเอเชียตะวนัออกเฉียงใต ้ จุดม่งหมายของงานวิจยัเร่ืองน้ี คือ ตรวจสอบฤทธ์ิตา้นอนุมูลอิสระของผกัหวานป่าในประเทศไทย ส่วนสกดัของผกัหวานเตรียมโดยใช้ชุดสกดัแบบซอกเลตสกดัดว้ยตวัท าละลาย น าส่วนสกดัมาศึกษาฤทธ์ิตา้นอนุมูลอิสระดว้ยวธีิ 2,2-diphenyl-1-picryhydrazyl radical scavenging assay (DPPH), ferric reducing antioxidant power assay (FRAP) and nitric oxide assay (NO) ผลการวิจยัพบวา่ ส่วนสกดัน ้ าของผกัหวานป่ามีฤทธ์ิยบัย ั้งอนุมูลอิสระแสดงเป็นค่า IC50 เท่ากบั 900-1150 มคก./มล. เปรียบเทียบกบัสารมาตรฐานแกลลิคแอซิดท่ีมีค่า IC50 เท่ากบั 2.84 ± 0.008 มคก./มคล. เม่ือทดสอบดว้ยวธีิ DPPH และ 20 มคก./มล. เปรียบเทียบกบัสารมาตรฐานโทรลอกซ์ท่ีมีค่า IC50 เท่ากบั 0.94 ± 0.224 มคก./มคล. เม่ือทดสอบดว้ยวธีิ FRAP ปริมาณสารฟีนอลลิกรวมและปริมาณฟลาโวนอยด์สูงสุดพบในส่วนสกดัเมทานอลและส่วนสกดัน ้ าของผกัหวานป่า มีค่าเป็น 265-1,978 มก.สมมูลแกลลิคแอซิดต่อ 100 กรัมของสารสกดัแห้ง และ 21-459 มก.สมมูลของแคททีชินต่อ 100 กรัมของสารสกดัแห้งส าหรับส่วนสกดัเมทานอล และ 183-296 มก.สมมูลแกลลิคแอซิดต่อ 100 กรัมของสารสกดัแห้ง และ 17-20 มก.สมมูลของแคททีชินต่อ 100 กรัมของสารสกดัแห้งส าหรับส่วนสกดัน ้ าตามล าดบั ผลการศึกษาน้ียงัแสดงความสัมพนัธ์อยา่งมีนยัส าคญัทางสถิติระหวา่งฤทธ์ิตา้นอนุมูลอิสระโดยวธีิ FRAP และปริมาณสารฟลาโวนอยด ์(Pearson correlation = 1.000, p-value = 0.013) ค ำส ำคัญ: ผกัหวานป่า, ฤทธ์ิตา้นอนุมูลอิสระ, ปริมาณสารฟลาโวนอยด,์ ปริมาณสารฟีนอลลิกรวม

BHST. 2015, 13 (1) : 29-37 Charoenchai et al.

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INTRODUCTION

Melientha suavis Pierre (pak-wan pa) is an edible plant which can be found in the

forests of Thailand and Southeast Asia. Pak-wan pa is a deciduous tree in the Opiliaceae

family and variable genetic strains grow in different locations in Thailand (Prathepha, 2000).

However, it is quite difficult to locate this plant due to deforestation. As it is more preferable

to use pak-wan pa than pak-wan ban (Sauropus androgynus L. Merr) for cooking, pak-wan pa

is cultivated particularly in the Saraburi (SB) province of Thailand. Therefore, the young

leaves and stems are collected and sold as vegetables in the local markets. Pak-wan mao

(Urobotyra siamensis Hiepko) is another plant in the Opiliaceae family which confused

people in the local areas due to its similar name and appearance. Both plants show similar

botanical characteristics, leaves, stems and flowers, but have slightly different fruits. When

ripe, fruit of pak-wan pa is yellow, whereas the fruit of pak-wan mao is red (Semsuntud,

2009). Moreover, there were some reports which mentioned the toxicity of pak-wan mao

(Sawasdeemonkol, 1980; Tourdjman, 2009).

Although the phytochemical components of pak-wan pa are not well understood,

results have found alkaloids, carbohydrates, coumarins, flavonoids, saponins and tannins in

the extracts of pak-wan pa (Charoenchai et al., 2013). Some studies have reported the

nutritional values of pak-wan pa (Bureau of Nutrition. 1992; Tianpech, 2008) compared with

pak-wan ban (Benjapak et al., 2008). Pak-wan pa is high in beta carotene, vitamin C,

phosphorus and calcium (Bureau of Nutrition. 1992; Somsub et al, 2008). One study claimed

pak-wan pa’s potential to reduce the mutant spots induced by ethyl carbamate in Drosophila

melanogaster somatic cells (Sukprasansap, 2010). In addition, hexane, dichloromethane and

ethanol soxhlet extraction of Melientha suavis showed high inhibitions of Fusarium

oxysporum and Xanthomonas campestris, with average clear zones of 7-9 mm and more than

10 mm, respectively (Hatthakitpanichakul, 2007). Since there is less information about the

medicinal properties of pak-wan pa, one approach is to use bioassay screening. As plant-

derived antioxidants have become popular alternatives to synthetic medicines, it would be

interesting to investigate the antioxidant activities of this plant.

Many diseases occur due to free radical damage, for example, cancer, cardiovascular

disease and neurodegenerative diseases (Valko et al., 2007). Antioxidants have been shown to

prevent cellular damage, resulting in a lower onset of some degenerative diseases (Sailaja et

al., 2011). There are many articles reviewing medicinal plants with antioxidant properties

(Wong et al., 2006 ; Surveswaran et al., 2007; Krishnaiah et al., 2011). Micronutrients such

as beta carotene, vitamin C and vitamin E, which are found in plants and in the diet, can act as

antioxidants in the body. These antioxidants can safely interact with free radicals, which

terminate the chain reaction and stop cellular damage. There are many in vitro antioxidant

tests available to evaluate antioxidant activities. In this research study, the DPPH activity and

FRAP assay are compared. In addition, the total phenolic and flavonoid contents are

examined. This study focused on the antioxidant activities of pak-wan pa. The results are

addressed in detail in later section.

MATERIALS AND METHODS

Chemicals 2,2-diphenyl-1-picryhydrazyl (DPPH), 2, 4, 6-tris ( 2-pyridyl )-triazine (TPTZ), trolox,

gallic acid, catechin, and Folin-ciocalteu phenol reagent were purchased from Sigma Aldrich,

USA. Ascorbic acid, sodium carbonate anhydrous, aluminium chloride, sodium acetate,

sodium nitroprusside, and sulphanilamide were purchased from Carlo Erba, UK.

Iron(III)chloride hexahydrate and sodium hydroxide were purchased from Merck, USA.

Ferrous sulfate heptahydrate and sodium nitrite was purchased from Fluka, USA.

BHST. 2015, 13 (1) : 29-37 Charoenchai et al.

31

Hydrochloric acid, hexane, ethyl acetate, and methanol were purchased from Mallincrokt,

USA.

Plants and sample preparation

Pak-wan pa was purchased from Saraburi (SB) and Uthai Thani (UT) provinces in

Thailand. All pak-wan plants were examined according to their botanical characteristics to

identify the right species. The fresh leaves and stems were cleaned and dried in a hot air oven

at a temperature of 50-60 OC for 6-24 hours. Then, the dried plants were ground and weighed.

Each 500 mL of three solvent systems were used consecutively in the extraction process:

hexane, ethyl acetate, and methanol, using soxhlet apparatus. The crude extracts were filtered

and dried in vacuo. The samples were then boiled in 500 mL of distilled water for 3 hours, left

to cool down, and filtered. The water extracts were freeze dried using a lyophilizer.

Phytochemical screening

All of the extracts were tested for specific phytochemical groups: alkaloids,

carbohydrates, coumarin, flavonoids, saponin, sterols, terpenoids and tannins. Qualitative

chemical test methods and reagents were used (Harborne, 1998; Sofowora, 2008). The

detailed procedures are described previously (Charoenchai, 2013).

Antioxidant activities

DPPH radical scavenging assay: The DPPH activity evaluates the antioxidant

potential through free radical scavenging of the test samples, by monitoring the change in

optical density of DPPH radicals (Brand-Williams, 1995). DPPH solution was prepared in a

concentration of 2x10-4

M in ethanol. The pak-wan extracts were prepared in water and

diluted to concentration in the micromolar range. Both 100 L of the sample solution and 100

L of the DPPH solution was added into a 96-well plate and mixed well. All determinants

were performed in triplicate. After leaving in the dark and at room temperature for 30

minutes, absorbance was measured at the 517 nm wavelength using a Biorad microplate

reader. Gallic acid in the concentration range of 0-5 g/mL was used as the standard, and

assessment of the antioxidant activity was expressed in milligram gallic acid equivalent (GE)

per 100 gram of dry weight extract. %DPPH radical scavenging was calculated from the

equation as shown below. Percentage inhibition was calculated from the inhibition curve.

%DPPH radical scavenging = [(Abscontrol - Abssample)/( Abscontrol)] x 100

FRAP assay: FRAP measures the ability of the antioxidants to reduce the complex of

ferric ion and 2, 4, 6-tri (2-pyridyl )-tri-azine (TPTZ) to the ferrous form at low pH (Benzie,

1996). FRAP reagent was prepared in 20 mM concentration from the mixture of TPTZ,

FeCl3.6H2O and sodium acetate buffer. The 30 L aliquot of the sample solution was added to

the 96-well plate and 270 L of FRAP reagent. All determinants were performed in triplicate.

After 30 minutes of dark light at room temperature, the reduction was monitored by the

change in absorption at 595 nm wavelength using a Biorad microplate reader. Trolox in the

concentration range of 0-250 g/mL was used as the standard, and the assessment of the

antioxidant activity was expressed in milligram trolox equivalent (TE) per 100 gram of dry

weight extract. %FRAP radical scavenging was calculated from the equation as shown below.

Percentage inhibition was calculated from the inhibition curve.

%FRAP radical scavenging = [(Abssample - Abscontrol)/(Abssample)] x 100

Determination of total phenolic content: Total phenolic content of pak-wan pa was

determined by Folin-Ciocalteu assay (Slinkard, 1997). A 12.5 L aliquot of the sample

solution was diluted with 50 L distilled water in a 96-well plate, and 12.5 L of Folin-

Ciocalteu solution was then added and mixed well. The plate was kept in the dark at room

temperature for 6 minutes. Following this, 125 L of 7% w/v Na2CO3 solution was added and

mixed, then another 100 L of distilled water was added immediately. All determinants were

performed in triplicate. After they were kept at room temperature with no light for 90 minutes,

the absorbance was measured at 760 nm wavelength using a Biorad microplate reader. Gallic

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32

acid in the concentration range of 0-200 g/mL was used as the standard, and the assessment

of the antioxidant activity was expressed in milligram gallic acid equivalent (GE) per 100

gram of dry weight extract.

Determination of flavonoid content: The flavonoid content of pak-wan pa was

determined by spectrophotometric method in a 96-well plate (Chen, 2007). The 25 L aliquot

of sample solution was diluted with 125 L distilled water; and 10 L of 5% sodium nitrite

solution was then added and mixed well. All determinants were performed in triplicate. The

plate was kept in the dark at room temperature for 6 minutes. Then 15 L of 10% w/v

aluminium chloride was added and the plates were left in the dark at room temperature for 90

minutes. After 50 L of 1 M sodium hydroxide was added and mixed, the absorbance was

measured at 510 nm wavelength using a Biorad microplate reader. Catechin in the

concentration range of 0-200 g/mL was used as a standard, and the assessment of the

antioxidant activity was expressed in milligram catechin equivalent (CE) per 100 gram of dry

weight extract.

Statistical analysis: All measurements were performed in triplicates. The results were

expressed as mean ± standard deviation. Experimental results were analyzed for Pearson

correlation coefficient (r) between total phenolic, flavonoid, DPPH radical scavenging and

FRAP assays using IBM SPSS 21 software. The values were considered to be significantly

difference at 95% confidence.

RESULTS AND DISCUSSION

The dry weight of 217.69 g pak-wan pa from SB province and 193.55 g of pak-wan pa

from UT province were extracted consecutively with four different solvent systems using

soxhlet apparatus: hexane, ethyl acetate, methanol, and water. The total yields were 136.44 g

(62.68%) and 75.51 g (39.01%) for pak-wan pa SB, and UT, respectively. The crude extracts

of each solvent system are shown in Table 1. All pak-wan pa samples which showed the

highest percent yields (21.76-43.61%) were the methanolic extracts. In comparison, it has

been reported that the crude extract of Melientha suavis gave the highest percent yield

(5.10%) in ethanol soxhlet extract (Hatthakitpanichakul, 2007).

The phytochemical screening results are summarized in Table 2. Most phytochemicals

found in pak-wan pa extracts were alkaloids, carbohydrates and flavonoids although some

results were not clear due to the intense color of the extracts. Sterols and terpenoids were

found in pak-wan pa UT but were not present in pak-wan pa SB. Besides the source variations

of samples, this may be because young leaves and stems of pak-wan pa SB were used, while

those of pak-wan pa UT were old. Solvent extractions were used in according to increasing

polarities from hexane to water. Methanol was a high polarity solvent and miscible with

water. Some phytochemical compounds, such as polyphenols, flavonoids, and glycosides, can

be soluble in polar solvents (Alam, 2013). Preliminary phytochemical screening results for

pak-wan pa SB and UT, especially in the methanolic and water extracts, showed positive

results in alkaline reagent test for flavonoids (Table 2). These results were consistent with the

highest total phenolic and flavonoid contents, which were also found in the methanolic

extracts. Thus, solvent polarity has influenced the antioxidant activities of the pak-wan pa

extracts.

Table 1 Percent yields of pak-wan pa extracts using different solvent systems

Extracts g (%w/w) Hexane Ethyl acetate Methanol Water

Pak-wan pa Saraburi (SB) 5.59% 3.17% 43.61% 10.31%

Pak-wan pa Uthai Thani (UT) 1.99% 1.89% 21.76% 13.38%

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Table 2 Phytochemical screening results of pak-wan pa extracts

Phytochemicals Alkaloids Carbohydrates Coumarin Flavonoids Saponin Sterols and

Terpenoids

Tannins

Pak-wan pa SB Hexane + + -- + -- -- +

Ethyl

acetate

++ + -- + -- -- --

Methanol + + + + -- -- +

Water ++ + + +/- + -- --

Pak-wan pa UT Hexane ++ +/- -- +/- -- + --

Ethyl

acetate

++ + + + -- + +

Methanol ++ + -- +/- -- +/- +

Water ++ + + + + -- --

+ = positive; -- = negative; +/- = not clear

The free radical scavenging potentials of the samples expressed as the 50 percent

inhibitory concentration (IC50) are presented in Table 3. The water extract of pak-wan pa SB,

showed the highest inhibition (900±16 g/mL), followed by the ethyl acetate extracts of pak-

wan pa UT (1040±49 g/mL). In comparison with the standard (gallic acid), the scavenging

activity was 2.84±0.008 g/mL. The FRAP assay showed similar results: the methanolic and

water extracts of both pak-wan pa SB and UT exhibited high antioxidant activities. In

particular, the ethyl acetate extract of pak-wan pa UT exhibited the highest antioxidant

activities (2.5±0.3 g/mL) compared with the standard trolox (0.94±0.224 g/mL). The

antioxidant capacity of pak-wan pa SB has been reported previously using a DPPH assay and

2-deoxyribose assay

(Tianpech, 2008). Its antioxidant capacity was shown as IC50 value of

20.64±0.07 M (3.6 g/mL) and 21.94±0.73 M for vitamin C and pak-wan pa, respectively,

determined by DPPH assay. Another study reported an IC50 value of 1.31±0.09 mg/mL of the

water extract of pak-wan pa determined by DPPH assay in methanol solution (Noipa, 2011)

and was consistent with the IC50 of the water extract of two samples of pak-wan pa (0.90-

1.15 mg/mL) in this study. The high IC50 reported by Noipa et al. may be due to the plant

sample preparation, as the dry plant sample was extracted with deionized water, allowed to

stand for 1 hour in the dark and then filtered. However, in this study, any interfering

substances were removed from the sample using low polarity solvents such as hexane and

ethyl acetate before water extraction.

Table 3 Inhibition concentration (IC50) of pak-wan pa extracts using two different assays

Extracts DPPH (g/mL) FRAP (g/mL)

Gallic acid 2.84 ± 0.008 ND

Trolox ND 0.94 ± 0.224

SB

- Hexane 3,260 ± 389 4,060 ± 690

- Ethyl acetate 1,130 ± 18 690 ± 96

- Methanol 1,940 ± 73 20 ± 3

- Water 900 ± 16 20 ± 2

UT

- Hexane 4,150 ± 951 2,040 ± 238

- Ethyl acetate 1,040 ± 49 2.5 ± 0.3

- Methanol 1,980 ± 63 70 ± 14

- Water 1,150 ± 44 20 ± 14

The values are mean ± SD (n=3) ND = not determined

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In this study, the methanolic extracts of the pak-wan samples showed the highest

contents of total phenolic and flavonoid compounds (Table 4). The highest phenolic content

was the methanolic extract of pak-wan pa SB (1,978.06±25.96 mg GE/100 g dry extract). The

highest value of flavonoid content was also found in the methanolic extract of pak-wan pa SB

(459.54±14.42 mg CE/100 g dry extract). In addition, Tianpech and colleagues (2008)

reported the phenolic content of pak-wan pa juice extract, which was 370.69±8.74 mg GE/100

mL. But in this study, the water extract of pak-wan pa SB had 296.44±14.07 mg GE/100 g dry

extract. These results may be due to the different methods of extraction used. In this research,

the extraction method of dry plant was prepared at a high temperature of between 80 and 90 OC using soxhlet apparatus, but Tianpech et al. (2008) prepared the sample at room

temperature using homogenized fresh plants. Although it is not directly comparison, it implies

that polar solvent extraction of pak-wan pa seems to yield relatively high antioxidant activity

than non-polar solvent.

Table 4 Total phenolic and flavonoid contents of pak-wan pa extracts

Extracts

Phenolic content

(mg GE/100 g dry

extract)

Flavonoid content

(mg CE/100 g

dry extract)

SB

- Hexane ND ND

- Ethyl acetate 58.69 ± 4.34 12.67 ± 4.58

- Methanol 1,978.06 ± 25.96 459.54 ± 14.42

- Water 296.44 ± 14.07 17.59 ± 2.32

UT

- Hexane ND ND

- Ethyl acetate 99.62 ± 1.82 7.00 ± 0.72

- Methanol 265.63 ± 2.13 20.81 ± 3.60

- Water 183.98 ± 1.51 20.32 ± 2.21

The values are mean ± SD (n=3) ND = not determined

In this paper, antioxidant activity from the DPPH assay exhibited similar results to that

of the FRAP assay. However, the models cannot be directly compared due to the differences

in each method. Results show some correlations between DPPH and FRAP activities (Pearson

correlation = 0.889-0.901). Since the hexane extracts of pak-wan SB were not very soluble,

they were not determined in the DPPH assay or in the total phenolic and flavonoid contents. A

strong correlation was found between the FRAP scavenging activities and the flavonoid

contents, compared to the total phenolic contents (Table 5). The results imply that both

phenolic and flavonoid compounds may contribute to the DPPH activities of pak-wan pa

extracts, but the phenolic compounds may have significant effects.

Table 5 Correlation between the antioxidant activities of DPPH, FRAP and the total

phenolic and flavonoid contents

Extracts Pearson’s correlation (Sig. 2-tailed)

DPPH-Phenolic DPPH-Flavonoid FRAP-Phenolic FRAP-Flavonoid

SB 0.934 (0.232) 0.829 (0.299) 0.996 (0.054) 1.000 (0.013)

UT 0.994 (0.070) 0.829 (0.378) 0.883 (0.373) 0.481 (0.680)

In comparison, the percentage inhibition of all pak-wan pa SB and UT extracts are

shown in Figure 1 and Figure 2. At a concentration of 1 mg/mL, the methanolic and water

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35

extracts of pak-wan pa SB exhibited the highest scavenging activity in FRAP (87.43-89.17%)

assays, while the water (63.35%) and ethyl acetate (36.52%) extracts of pak-wan pa SB

exhibited moderate scavenging activity in DPPH assay. In similar, the water, methanol and

ethyl acetate extracts of pak-wan pa UT exhibited high scavenging activity in FRAP (77.44-

91.64%) assays.

Figure 1 Percentage inhibition of the pak-wan pa extracts from DPPH assays

Figure 2 Percentage inhibition of the pak-wan pa extracts from FRAP assays

0

10

20

30

40

50

60

70

SB UT

%Inhibition of pak-wan pa extracts (DPPH)

Hexane

Ethyl acetate

Methanol

Water

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

100.00

SB UT

%Inhibition of pak-wan pa extracts (FRAP)

Hexane

Ethyl acetate

Methanol

Water

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36

CONCLUSION

This research found that pak-wan pa samples exhibited similar antioxidant activities in

two in vitro test models: DPPH and FRAP assays. Overall, the extracts of pak-wan pa SB

exhibited higher values of antioxidant activities and higher contents of total phenolic and

flavonoids than pak-wan pa UT. These results suggest that in vitro evidence supports the

antioxidant activities of pak-wan pa. In particular, high DPPH activities of the methanolic

extracts of pak-wan pa resulted from high content of phenolic and flavonoid compounds.

Further phytochemical research of pak-wan pa is needed to understand its composition and

reveal what parts are related to antioxidant activity.

ACKNOWLEDGMENTS

The author would like to thank the Research Institute of Rangsit University for

funding this research (Grant number: 63/2554). The author is thankful to Mr. Nirun

Vipunngen for his help in acquiring pak-wan pa from Uthai Thani province. The author

greatly appreciated Ms. KI Tull for her assistance with the English language used in this

paper.

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