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 1 Faculty of Pharmacy, Rangsit University, Pathum Thani, THAILAND. 2 The 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: [email protected]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 IC 50 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: ผักหวานป่าเป็นพืชอาหารที่พบได้ในป ่าของประเทศไทยและภูมิภาคเอเชียตะวันออกเฉียงใต้ จุดม่งหมายของงานวิจัยเรื่องนี ้ คือ ตรวจสอบ ฤทธิ ์ต้านอนุมูลอิสระของผักหวานป่าในประเทศไทย ส่วนสกัดของผักหวานเตรียมโดยใช้ชุดสกัดแบบซอกเลตสกัดด้วยตัวทาละลาย นาส่วนสกัดมา ศึกษาฤทธิ ์ต้านอนุมูลอิสระด้วยวิธี 2,2-diphenyl-1-picryhydrazyl radical scavenging assay (DPPH), ferric reducing antioxidant power assay (FRAP) and nitric oxide assay (NO) ผลการวิจัยพบว่า ส่วนสกัดน ้าของผักหวานป่ามีฤทธิ ์ยับยั ้งอนุมูลอิสระแสดงเป็นค่า IC 50 เท่ากับ 900-1150 มคก./มล. เปรียบเทียบ กับสารมาตรฐานแกลลิคแอซิดที่มีค่า IC 50 เท่ากับ 2.84 ± 0.008 มคก./มคล. เมื่อทดสอบด้วยวิธี DPPH และ 20 มคก./มล. เปรียบเทียบกับสารมาตรฐาน โทรลอกซ์ที่มีค่า IC 50 เท่ากับ 0.94 ± 0.224 มคก./มคล. เมื่อทดสอบด้วยวิธี FRAP ปริมาณสารฟีนอลลิกรวมและปริมาณฟลาโวนอยด์สูงสุดพบในส่วน สกัดเมทานอลและส่วนสกัดน ้าของผักหวานป่า มีค่าเป็น 265-1,978 มก.สมมูลแกลลิคแอซิดต่อ 100 กรัมของสารสกัดแห้ง และ 21-459 มก.สมมูลของ แคททีชินต่อ 100 กรัมของสารสกัดแห้งสาหรับส่วนสกัดเมทานอล และ 183-296 มก.สมมูลแกลลิคแอซิดต่อ 100 กรัมของสารสกัดแห้ง และ 17-20 มก. สมมูลของแคททีชินต่อ 100 กรัมของสารสกัดแห้งสาหรับส่วนสกัดน ้าตามลาดับ ผลการศึกษานี ้ยังแสดงความสัมพันธ์อย่างมีนัยสาคัญทางสถิติระหว่าง ฤทธิ ์ต้านอนุมูลอิสระโดยวิธี FRAP และปริมาณสารฟลาโวนอยด์ (Pearson correlation = 1.000, p-value = 0.013) คำสำคัญ: ผักหวานป่า, ฤทธิ ์ต้านอนุมูลอิสระ, ปริมาณสารฟลาโวนอยด์, ปริมาณสารฟีนอลลิกรวม
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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,
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
BHST. 2015, 13 (1) : 29-37 Charoenchai et al.
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%
BHST. 2015, 13 (1) : 29-37 Charoenchai et al.
33
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
BHST. 2015, 13 (1) : 29-37 Charoenchai et al.
34
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