# Phytochemical Composition and Biological Activities of Selected Wild Berries (Rubus moluccanus L.,R. fraxinifolius Poir., andR. alpestris Blume)

Authors: Mohd Fadzelly Abu Bakar; Nur Amalina Ismail; Azizul Isha; Angelina Lee Mei Ling
Journal: Evidence-Based Complementary and Alternative Medicine
 (2016)

---

## Abstract

Berries, from the genusRubus, are among the vital components in a healthy diet. In this study, 80% methanol extracts from the three wildRubus species (Rubus moluccanus L.,Rubus fraxinifolius Poir., andRubus alpestris Blume) were evaluated for their phytochemical contents (total phenolics, flavonoid, anthocyanin, and carotenoid content), antioxidant (DPPH, FRAP, and ABTS assays), antiacetylcholinesterase, and antibacterial activities. GC-MS was used for quantification of naturally occurring phytochemicals. The results showed thatR. alpestris contained the highest total phenolic [24.25
±
0.1 mg gallic acid equivalent (GAE)/g] and carotenoid content [21.86
±
0.63 mg β-carotene equivalents (BC)/g], as well as the highest DPPH scavenging and FRAP activities. The highest total flavonoid [18.17
±
0.20 mg catechin equivalents (CE)/g] and anthocyanin content [36.96
±
0.39 mg cyanidin-3-glucoside equivalents (c-3-gE)/g] have been shown byR. moluccanus. For antibacterial assays,R. moluccanus andR. alpestris extracts showed mild inhibition towardsBacillus subtilis,Staphylococcus aureus,Escherichia coli, andSalmonella enteritidis. Anticholinesterase activity for all extracts was in the range of 23–26%. The GC-MS analysis revealed the presence of at least 12, 21, and 7 different organic compounds in 80% methanol extracts ofR. alpestris,R. moluccanus, andR. fraxinifolius, respectively, which might contribute to the bioactivity.


---

## Body

## 1. Introduction

Berries have been traditionally used by many cultures to treat various ailments. In traditional Chinese medicine, combination of Chinese raspberry (Rubus chingii) and “yang” tonic was used to treat infertility, impotence, low backache, poor eyesight, and frequent urination. According to aboriginal people in Australia, berries are considered as mild laxative if eaten in large quantities [1]. Berries are among the vital components in healthy diet. Their small, soft-fleshed fruits are usually consumed in fresh, frozen, dried, and product form [2]. They have received significant attention due to their potential benefit to human health [3].Berries extracts have been demonstrated to exert anti-inflammatory, antioxidant, anticancer, antimicrobial, anthelminthic, and anti-Alzheimer activities [4–7]. Examples of berries or brambles in Rosaceae family are yellow Himalayan raspberry (Rubus ellipticus), hill raspberry (Rubus niveus), Korean black raspberry (Rubus coreanus), cloudberry (Rubus chamaemorus), and red raspberry (Rubus idaeus) [8, 9]. The bioactivities shown by these species are mainly due to the occurrence of their phytochemicals. Even though these berries fall under the same genus, the phytochemicals content and biological activities exerted by the closely related species are different [6]. Phenolics profile and concentration were affected by genetic (genus, species, and cultivar/genotype) and environmental (plant maturity, plant age, growing season, and field location) factors [9–12]. Previous research has shown that black raspberries displayed the highest amount of total polyphenols, flavonoid, and anthocyanin when compared to Korean raspberries and blackberries. It is also reported that the high antioxidant and anti-inflammatory activities shown by black raspberries were contributed by their high polyphenols and anthocyanin content [4].In Malaysia Borneo, genusRubus could be found on highland area of Sabah. Generally, this genus comprises 12 subgenera and consists of 500 species [13], including few domesticated species. Based on the record by Corner and Beaman [14], more than 8 species ofRubus can be found above 1200 m on Mount Kinabalu, for instance,Rubus lineatus, Rubus benguetensis, Rubus elongates, andRubus rosifolius. Other species such asRubus moluccanus L.,Rubus fraxinifolius Poir., andRubus alpestris Blume could be found in Mount Alab, Crocker Range Park, Sabah, Borneo. The diversity of wildRubus species in this area has attracted our attention to further investigate the bioactivities and their chemical components. Therefore, the current study aims to investigate the antioxidant, antiacetylcholinesterase, and antibacterial activities of the selected wildRubus species fruits (R. moluccanus L.,R. fraxinifolius Poir., andR. alpestris) as well as their phytochemicals content.


## 2. Materials and Method

### 2.1. Plant Materials and Sample Preparation

All samples [R. moluccanus (Figure 1),R. alpestris (Figure 2), andR. fraxinifolius (Figure 3)] were collected from Mount Alab, Crocker Range Park, Sabah, Malaysian Borneo, on February 2014. These plant materials were identified by a botanist, Mr. Johnny Gisil. Voucher specimens of the three samples were deposited at BORNEENSIS, Institute for Tropical Biology & Conservation, Universiti Malaysia Sabah. The fruits were cleaned, weighed, and cut into smaller pieces. Then, the fruits were kept in a freezer (−80°C) overnight before being freeze-dried for 3 days. The freeze-dried samples were ground into fine powder by using a dry grinder. The samples were sieved to get a uniform particle size and kept in an airtight container and stored in a freezer (−20°C) for further analysis.Figure 1
R. moluccanus.Figure 2
R. fraxinifolius.Figure 3
R. alpestris.


### 2.2. Sample Extraction

Extraction method was adapted from the method described previously [15] with slight modification. About 0.1 g of freeze-dried sample was added to 30 mL of 80% (v/v) methanol. The mixture was shaken for 2 hours by using an orbital shaker set at 200 rpm at room temperature. The supernatant was decanted into a vial for further analysis.


### 2.3. Determination of Phytochemicals Content in Wild Rubus Extracts

#### 2.3.1. Total Phenolic Content

Total phenolic content was determined using Folin-Ciocalteu method [15]. About 100 µL of sample extract was mixed with 0.75 mL of Folin-Ciocalteu reagent (prediluted 10 times with distilled water). The mixture was vortexed for 15 seconds. After 5 minutes, 0.75 mL of sodium bicarbonate (60 g/L) solution was added to the mixture and allowed to stand at 22°C for 90 minutes. The absorbance value was measured at 725 nm by using microplate reader. Gallic acid was used as a standard in the range of 0 to 100 µg/mL and the results were expressed as mg of gallic acid equivalent in 1.0 g of dried sample (mg GAE/g). Analyses were done in triplicate for each sample.


#### 2.3.2. Total Flavonoid Content

Aluminium colorimetric method [16] was used to determine flavonoid content. Briefly, 1 mL of sample extract was added to the beaker with 4.0 mL distilled water and 0.3 mL of (5% w/v) sodium nitrite was added to the mixture. Then, 0.6 mL of (10% w/v) aluminium chloride hexahydrate was added after 5 minutes. After 6 minutes, 2.0 mL sodium hydroxide (1 M) was added to the solution and vortexed for 15 seconds. The absorbance values were measured at 510 nm by using spectrophotometer. Catechin (20–100 µg/mL) was used as a standard. Results were expressed as mg catechin equivalent/g (mg CE/g) sample.


#### 2.3.3. Total Anthocyanin Content

Total anthocyanin content was measured by using a spectrophotometric pH differential protocol [17] with slight modification. Briefly, 0.5 mL extract was mixed thoroughly with 3.5 mL of potassium chloride buffer (0.025 M; pH 1.0). The mixture was mixed well with vortex and allowed to stand for 15 minutes. The absorbance values were measured at 515 and 700 nm against distilled water blank. The extract was mixed with 3.5 mL of sodium acetate buffer (0.025 M; pH 4.5) and allowed to stand for 15 minutes. The absorbance values were measured at the same wavelength. The total anthocyanins content was calculated by using the following formula:(1)
T
o
t
a
l
a
n
t
h
o
c
y
a
n
i
n
c
o
n
t
e
n
t
m
g
/
g
o
f
d
r
i
e
d
s
a
m
p
l
e
=
A
×
M
w
×
D
F
×
10
ε
×
C
,where A is absorbance = (A
515
-
A
700) pH 1.0 − (A
515
-
A
700) pH 4.5, Mw is molecular weight for cyanidin-3-glucoside = 449.2, DF is a dilution factor of the samples, ε is the molar absorptivity of cyanidin-3-glucoside = 26,900, and C is the concentration of the buffer in mg/mL.Results were expressed as mg cyanidin-3-glucoside equivalents (c-3-gE)/g of dried sample.


#### 2.3.4. Total Carotenoid Content

Carotenoid content in the extract was determined based on the method described previously [18]. About 150 µL extract, with 150 µL distilled water and 600 µL methanol, was mixed in a centrifuge tube. The mixture was extracted with 300 µL hexane solution and centrifuged at speed 2000 ×g for 5 minutes at 4°C. Two layers of solution were formed. The absorbance value was measured at 350 nm by using organic layer solution. β-Carotene was used as a reference. The result was expressed in mg BC/g sample.


#### 2.3.5. Gas Chromatography-Mass Spectroscopy (GC-MS)

Sample extracts were analysed by gas chromatography equipped with mass spectrometry (GC-MS-2010 Plus-Shimadzu). The column temperature was set to 50°C for 4 min, then increased to 320°C at the rate of 7°C/min, and then held for 20 min. The injector temperature was set at 280°C (split mode with the ratio being adjusted to 20 : 1, injection volume = 0.1 L). The flow rate of the helium carrier gas was set to 1 mL/min with a total run time of 60 min. Mass spectra were obtained from the rangem/z 40 to 700 and the electron ionization at 70 eV. The chromatograms of the sample were identified by comparing their mass spectra with the library data (NIST 11 Library and Wiley Library) and the GC retention time against known standards.


### 2.4. Determination of Antioxidant Activities in Wild Rubus Extracts

#### 2.4.1. DPPH (2,2-Diphenyl-1-picrylhydrazyl) Radical Assay

The scavenging activity of the extract was estimated by using DPPH as a free radical model [19]. Firstly, 0.3 mM DPPH was prepared by dissolving 0.0118 g DPPH powder into 100 mL absolute methanol. Then, 1.0 mL from 0.3 mM DPPH methanol was added to 2.5 mL sample extract with the different concentration and allowed to stand for 30 minutes at room temperature in dark room. The solution was transferred into cuvette and absorbance value was measured at 518 nm by using a spectrophotometer. The blank and control absorbance value were also taken. The free radical scavenging activity was calculated by using the following formula: (2)
S
c
a
v
e
n
g
i
n
g
e
f
f
e
c
t
%
=
100
-
A
b
s
s
a
m
p
l
e
-
A
b
s
b
l
a
n
k
A
b
s
c
o
n
t
r
o
l
×
100
,where Abs blank = 1 mL 80% (v/v) methanol + 2.5 mL extract and Abs control = 1 mL 0.3 mM DPPH methanol + 2.5 mL 80% (v/v) methanol.The calibration curve for scavenging activity against concentration was plotted and the IC50 (half maximal inhibitory concentration) value was determined.


#### 2.4.2. FRAP (Ferric Reducing/Antioxidant Power) Assay

The ability of the extract to reduce ferric ion (Fe3+) to ferrous ion (Fe2+) was determined according to the previous method [20] with slight modification. FRAP reagent was prepared first. Briefly, 300 mM acetate buffer (pH 3.6) was mixed with 10 mM TPTZ and 20 mM FeCl3
·6H2O with ratio 10 : 1 : 1. Then, FRAP reagent was used as a blank and was measured at 593 nm by using spectrophotometer. About 100 µL sample extract and 300 µL distilled water were added to the blank in test tube. After 4 minutes, second reading was taken. Fe(II) was prepared as a standard using several concentrations from 0 to 100 µg/mL. A standard curve was prepared by plotting the FRAP value of each standard versus its concentration. The results were expressed as the concentration of antioxidant having a ferric reducing ability in 1 gram of sample (mM/g).


#### 2.4.3. ABTS [2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)]

The ABTS decolorization assay was adapted from [21] with slight modification. Briefly, 7 mM of ABTS solution and 2.45 mM potassium persulfate were added to a beaker to produce blue-green color of ABTS·+. The mixture was allowed to stand for 16 hours in a dark room to prevent incomplete oxidation process. The mixture was diluted with 80% methanol in order to obtain absorbance of 0.7 ± 0.2 units at 734 nm. Then, 200 µL extract was added to 2.0 mL ABTS·+ solution. The mixture was vortexed for 45 seconds and was transferred into cuvette. The absorbance value was measured at 734 nm by using a spectrophotometer. Ascorbic acid was used as a standard in the concentration range 0 to 60 µg/mL. The final results were expressed as mg ascorbic acid equivalent antioxidant capacity in 1 g of sample (mg AEAC/g).


### 2.5. Determination of Acetylcholinesterase Inhibition Activity

Acetylcholinesterase (AChE) inhibition activity was determined spectrophotometrically using acetylcholine as substrate according to the method described previously [22] with slight modification. The samples with several tested concentrations were prepared separately (0–5 mg/mL). In this assay, 250 µL phosphate buffer (200 mM; pH 7.7) was added to 10 µL fruit extract sample. Following that, 80 µL of DTNB (3.96 mg of DTNB and 1.5 mg sodium bicarbonate dissolved in 10 mL phosphate buffer pH 7.7) and 10 µL enzyme (2 U/mL) were added to the mixture. The mixture was incubated for 5 minutes at room temperature (25°C). Finally, the reaction was started by adding 15 µL of ATCI (the substrate that contained 10.85 mg acetylthiocholine iodide in 5 mL sodium phosphate buffer) and incubated for 5 minutes at room temperature (25°C). The color developed was measured in microtiter plate by microplate reader at 412 nm. The hydrolysis of acetylthiocholine was determined by monitoring the formation of yellow 5-thio-2-nitrobenzoate anion as a result of the reaction with DTNB with thiocholines which is catalysed by enzymes at a wavelength of 412 nm. The solvent 80% methanol was used as negative control. Donepenzil dissolved in methanol was used as standard drug at 0–5 mg/mL concentration. The percent of inhibition was calculated by using the formula below.The percentage inhibition of acetylcholinesterase was calculated using the following formula:(3)
A
C
h
E
I
n
h
i
b
i
t
i
o
n
%
=
A
0
-
A
1
A
0
×
100
,where A
0 is the absorbance of the control (without extract) and A
1 is the absorbance of the tested extract.


### 2.6. Determination of Antibacterial Activity

The antibacterial activities of the extracts were evaluated by using disc diffusion assay [23] with slight modification. A total of 0.4 mL of bacterial culture was inoculated and spread on the agar. Two Gram-positive bacteria were tested, which wereStaphylococcus aureus andBacillus subtilis and two Gram-negative bacteria,Escherichia coli andSalmonella enteritidis, were used in this study. 100 µL of each sample extract was pipetted into filter paper discs (diameter: 6 mm). After drying, the filter paper discs were placed on the agar plate. Methanol was used as negative control, whereas kanamycin was used as positive control. The inhibitory activity was determined by a clear zone around the disc after incubation at 37°C for 24 h. The zone of inhibition was measured in millimeters (mm) including the disc diameter.


### 2.7. Statistical Analysis

All experiments (except for GC-MS analysis) were carried out in 3 replicates in 3 independent experiments. The result was presented as mean ± standard deviation. The data was statistically analysed by using one-way ANOVA with a significance value ofp
<
0.05 to test the significant difference between the samples. Pearson’s correlation was used to determine the relationship between phytochemicals and antioxidant activity.


## 2.1. Plant Materials and Sample Preparation

All samples [R. moluccanus (Figure 1),R. alpestris (Figure 2), andR. fraxinifolius (Figure 3)] were collected from Mount Alab, Crocker Range Park, Sabah, Malaysian Borneo, on February 2014. These plant materials were identified by a botanist, Mr. Johnny Gisil. Voucher specimens of the three samples were deposited at BORNEENSIS, Institute for Tropical Biology & Conservation, Universiti Malaysia Sabah. The fruits were cleaned, weighed, and cut into smaller pieces. Then, the fruits were kept in a freezer (−80°C) overnight before being freeze-dried for 3 days. The freeze-dried samples were ground into fine powder by using a dry grinder. The samples were sieved to get a uniform particle size and kept in an airtight container and stored in a freezer (−20°C) for further analysis.Figure 1
R. moluccanus.Figure 2
R. fraxinifolius.Figure 3
R. alpestris.


## 2.2. Sample Extraction

Extraction method was adapted from the method described previously [15] with slight modification. About 0.1 g of freeze-dried sample was added to 30 mL of 80% (v/v) methanol. The mixture was shaken for 2 hours by using an orbital shaker set at 200 rpm at room temperature. The supernatant was decanted into a vial for further analysis.


## 2.3. Determination of Phytochemicals Content in Wild Rubus Extracts

### 2.3.1. Total Phenolic Content

Total phenolic content was determined using Folin-Ciocalteu method [15]. About 100 µL of sample extract was mixed with 0.75 mL of Folin-Ciocalteu reagent (prediluted 10 times with distilled water). The mixture was vortexed for 15 seconds. After 5 minutes, 0.75 mL of sodium bicarbonate (60 g/L) solution was added to the mixture and allowed to stand at 22°C for 90 minutes. The absorbance value was measured at 725 nm by using microplate reader. Gallic acid was used as a standard in the range of 0 to 100 µg/mL and the results were expressed as mg of gallic acid equivalent in 1.0 g of dried sample (mg GAE/g). Analyses were done in triplicate for each sample.


### 2.3.2. Total Flavonoid Content

Aluminium colorimetric method [16] was used to determine flavonoid content. Briefly, 1 mL of sample extract was added to the beaker with 4.0 mL distilled water and 0.3 mL of (5% w/v) sodium nitrite was added to the mixture. Then, 0.6 mL of (10% w/v) aluminium chloride hexahydrate was added after 5 minutes. After 6 minutes, 2.0 mL sodium hydroxide (1 M) was added to the solution and vortexed for 15 seconds. The absorbance values were measured at 510 nm by using spectrophotometer. Catechin (20–100 µg/mL) was used as a standard. Results were expressed as mg catechin equivalent/g (mg CE/g) sample.


### 2.3.3. Total Anthocyanin Content

Total anthocyanin content was measured by using a spectrophotometric pH differential protocol [17] with slight modification. Briefly, 0.5 mL extract was mixed thoroughly with 3.5 mL of potassium chloride buffer (0.025 M; pH 1.0). The mixture was mixed well with vortex and allowed to stand for 15 minutes. The absorbance values were measured at 515 and 700 nm against distilled water blank. The extract was mixed with 3.5 mL of sodium acetate buffer (0.025 M; pH 4.5) and allowed to stand for 15 minutes. The absorbance values were measured at the same wavelength. The total anthocyanins content was calculated by using the following formula:(1)
T
o
t
a
l
a
n
t
h
o
c
y
a
n
i
n
c
o
n
t
e
n
t
m
g
/
g
o
f
d
r
i
e
d
s
a
m
p
l
e
=
A
×
M
w
×
D
F
×
10
ε
×
C
,where A is absorbance = (A
515
-
A
700) pH 1.0 − (A
515
-
A
700) pH 4.5, Mw is molecular weight for cyanidin-3-glucoside = 449.2, DF is a dilution factor of the samples, ε is the molar absorptivity of cyanidin-3-glucoside = 26,900, and C is the concentration of the buffer in mg/mL.Results were expressed as mg cyanidin-3-glucoside equivalents (c-3-gE)/g of dried sample.


### 2.3.4. Total Carotenoid Content

Carotenoid content in the extract was determined based on the method described previously [18]. About 150 µL extract, with 150 µL distilled water and 600 µL methanol, was mixed in a centrifuge tube. The mixture was extracted with 300 µL hexane solution and centrifuged at speed 2000 ×g for 5 minutes at 4°C. Two layers of solution were formed. The absorbance value was measured at 350 nm by using organic layer solution. β-Carotene was used as a reference. The result was expressed in mg BC/g sample.


### 2.3.5. Gas Chromatography-Mass Spectroscopy (GC-MS)

Sample extracts were analysed by gas chromatography equipped with mass spectrometry (GC-MS-2010 Plus-Shimadzu). The column temperature was set to 50°C for 4 min, then increased to 320°C at the rate of 7°C/min, and then held for 20 min. The injector temperature was set at 280°C (split mode with the ratio being adjusted to 20 : 1, injection volume = 0.1 L). The flow rate of the helium carrier gas was set to 1 mL/min with a total run time of 60 min. Mass spectra were obtained from the rangem/z 40 to 700 and the electron ionization at 70 eV. The chromatograms of the sample were identified by comparing their mass spectra with the library data (NIST 11 Library and Wiley Library) and the GC retention time against known standards.


## 2.3.1. Total Phenolic Content

Total phenolic content was determined using Folin-Ciocalteu method [15]. About 100 µL of sample extract was mixed with 0.75 mL of Folin-Ciocalteu reagent (prediluted 10 times with distilled water). The mixture was vortexed for 15 seconds. After 5 minutes, 0.75 mL of sodium bicarbonate (60 g/L) solution was added to the mixture and allowed to stand at 22°C for 90 minutes. The absorbance value was measured at 725 nm by using microplate reader. Gallic acid was used as a standard in the range of 0 to 100 µg/mL and the results were expressed as mg of gallic acid equivalent in 1.0 g of dried sample (mg GAE/g). Analyses were done in triplicate for each sample.


## 2.3.2. Total Flavonoid Content

Aluminium colorimetric method [16] was used to determine flavonoid content. Briefly, 1 mL of sample extract was added to the beaker with 4.0 mL distilled water and 0.3 mL of (5% w/v) sodium nitrite was added to the mixture. Then, 0.6 mL of (10% w/v) aluminium chloride hexahydrate was added after 5 minutes. After 6 minutes, 2.0 mL sodium hydroxide (1 M) was added to the solution and vortexed for 15 seconds. The absorbance values were measured at 510 nm by using spectrophotometer. Catechin (20–100 µg/mL) was used as a standard. Results were expressed as mg catechin equivalent/g (mg CE/g) sample.


## 2.3.3. Total Anthocyanin Content

Total anthocyanin content was measured by using a spectrophotometric pH differential protocol [17] with slight modification. Briefly, 0.5 mL extract was mixed thoroughly with 3.5 mL of potassium chloride buffer (0.025 M; pH 1.0). The mixture was mixed well with vortex and allowed to stand for 15 minutes. The absorbance values were measured at 515 and 700 nm against distilled water blank. The extract was mixed with 3.5 mL of sodium acetate buffer (0.025 M; pH 4.5) and allowed to stand for 15 minutes. The absorbance values were measured at the same wavelength. The total anthocyanins content was calculated by using the following formula:(1)
T
o
t
a
l
a
n
t
h
o
c
y
a
n
i
n
c
o
n
t
e
n
t
m
g
/
g
o
f
d
r
i
e
d
s
a
m
p
l
e
=
A
×
M
w
×
D
F
×
10
ε
×
C
,where A is absorbance = (A
515
-
A
700) pH 1.0 − (A
515
-
A
700) pH 4.5, Mw is molecular weight for cyanidin-3-glucoside = 449.2, DF is a dilution factor of the samples, ε is the molar absorptivity of cyanidin-3-glucoside = 26,900, and C is the concentration of the buffer in mg/mL.Results were expressed as mg cyanidin-3-glucoside equivalents (c-3-gE)/g of dried sample.


## 2.3.4. Total Carotenoid Content

Carotenoid content in the extract was determined based on the method described previously [18]. About 150 µL extract, with 150 µL distilled water and 600 µL methanol, was mixed in a centrifuge tube. The mixture was extracted with 300 µL hexane solution and centrifuged at speed 2000 ×g for 5 minutes at 4°C. Two layers of solution were formed. The absorbance value was measured at 350 nm by using organic layer solution. β-Carotene was used as a reference. The result was expressed in mg BC/g sample.


## 2.3.5. Gas Chromatography-Mass Spectroscopy (GC-MS)

Sample extracts were analysed by gas chromatography equipped with mass spectrometry (GC-MS-2010 Plus-Shimadzu). The column temperature was set to 50°C for 4 min, then increased to 320°C at the rate of 7°C/min, and then held for 20 min. The injector temperature was set at 280°C (split mode with the ratio being adjusted to 20 : 1, injection volume = 0.1 L). The flow rate of the helium carrier gas was set to 1 mL/min with a total run time of 60 min. Mass spectra were obtained from the rangem/z 40 to 700 and the electron ionization at 70 eV. The chromatograms of the sample were identified by comparing their mass spectra with the library data (NIST 11 Library and Wiley Library) and the GC retention time against known standards.


## 2.4. Determination of Antioxidant Activities in Wild Rubus Extracts

### 2.4.1. DPPH (2,2-Diphenyl-1-picrylhydrazyl) Radical Assay

The scavenging activity of the extract was estimated by using DPPH as a free radical model [19]. Firstly, 0.3 mM DPPH was prepared by dissolving 0.0118 g DPPH powder into 100 mL absolute methanol. Then, 1.0 mL from 0.3 mM DPPH methanol was added to 2.5 mL sample extract with the different concentration and allowed to stand for 30 minutes at room temperature in dark room. The solution was transferred into cuvette and absorbance value was measured at 518 nm by using a spectrophotometer. The blank and control absorbance value were also taken. The free radical scavenging activity was calculated by using the following formula: (2)
S
c
a
v
e
n
g
i
n
g
e
f
f
e
c
t
%
=
100
-
A
b
s
s
a
m
p
l
e
-
A
b
s
b
l
a
n
k
A
b
s
c
o
n
t
r
o
l
×
100
,where Abs blank = 1 mL 80% (v/v) methanol + 2.5 mL extract and Abs control = 1 mL 0.3 mM DPPH methanol + 2.5 mL 80% (v/v) methanol.The calibration curve for scavenging activity against concentration was plotted and the IC50 (half maximal inhibitory concentration) value was determined.


### 2.4.2. FRAP (Ferric Reducing/Antioxidant Power) Assay

The ability of the extract to reduce ferric ion (Fe3+) to ferrous ion (Fe2+) was determined according to the previous method [20] with slight modification. FRAP reagent was prepared first. Briefly, 300 mM acetate buffer (pH 3.6) was mixed with 10 mM TPTZ and 20 mM FeCl3
·6H2O with ratio 10 : 1 : 1. Then, FRAP reagent was used as a blank and was measured at 593 nm by using spectrophotometer. About 100 µL sample extract and 300 µL distilled water were added to the blank in test tube. After 4 minutes, second reading was taken. Fe(II) was prepared as a standard using several concentrations from 0 to 100 µg/mL. A standard curve was prepared by plotting the FRAP value of each standard versus its concentration. The results were expressed as the concentration of antioxidant having a ferric reducing ability in 1 gram of sample (mM/g).


### 2.4.3. ABTS [2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)]

The ABTS decolorization assay was adapted from [21] with slight modification. Briefly, 7 mM of ABTS solution and 2.45 mM potassium persulfate were added to a beaker to produce blue-green color of ABTS·+. The mixture was allowed to stand for 16 hours in a dark room to prevent incomplete oxidation process. The mixture was diluted with 80% methanol in order to obtain absorbance of 0.7 ± 0.2 units at 734 nm. Then, 200 µL extract was added to 2.0 mL ABTS·+ solution. The mixture was vortexed for 45 seconds and was transferred into cuvette. The absorbance value was measured at 734 nm by using a spectrophotometer. Ascorbic acid was used as a standard in the concentration range 0 to 60 µg/mL. The final results were expressed as mg ascorbic acid equivalent antioxidant capacity in 1 g of sample (mg AEAC/g).


## 2.4.1. DPPH (2,2-Diphenyl-1-picrylhydrazyl) Radical Assay

The scavenging activity of the extract was estimated by using DPPH as a free radical model [19]. Firstly, 0.3 mM DPPH was prepared by dissolving 0.0118 g DPPH powder into 100 mL absolute methanol. Then, 1.0 mL from 0.3 mM DPPH methanol was added to 2.5 mL sample extract with the different concentration and allowed to stand for 30 minutes at room temperature in dark room. The solution was transferred into cuvette and absorbance value was measured at 518 nm by using a spectrophotometer. The blank and control absorbance value were also taken. The free radical scavenging activity was calculated by using the following formula: (2)
S
c
a
v
e
n
g
i
n
g
e
f
f
e
c
t
%
=
100
-
A
b
s
s
a
m
p
l
e
-
A
b
s
b
l
a
n
k
A
b
s
c
o
n
t
r
o
l
×
100
,where Abs blank = 1 mL 80% (v/v) methanol + 2.5 mL extract and Abs control = 1 mL 0.3 mM DPPH methanol + 2.5 mL 80% (v/v) methanol.The calibration curve for scavenging activity against concentration was plotted and the IC50 (half maximal inhibitory concentration) value was determined.


## 2.4.2. FRAP (Ferric Reducing/Antioxidant Power) Assay

The ability of the extract to reduce ferric ion (Fe3+) to ferrous ion (Fe2+) was determined according to the previous method [20] with slight modification. FRAP reagent was prepared first. Briefly, 300 mM acetate buffer (pH 3.6) was mixed with 10 mM TPTZ and 20 mM FeCl3
·6H2O with ratio 10 : 1 : 1. Then, FRAP reagent was used as a blank and was measured at 593 nm by using spectrophotometer. About 100 µL sample extract and 300 µL distilled water were added to the blank in test tube. After 4 minutes, second reading was taken. Fe(II) was prepared as a standard using several concentrations from 0 to 100 µg/mL. A standard curve was prepared by plotting the FRAP value of each standard versus its concentration. The results were expressed as the concentration of antioxidant having a ferric reducing ability in 1 gram of sample (mM/g).


## 2.4.3. ABTS [2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)]

The ABTS decolorization assay was adapted from [21] with slight modification. Briefly, 7 mM of ABTS solution and 2.45 mM potassium persulfate were added to a beaker to produce blue-green color of ABTS·+. The mixture was allowed to stand for 16 hours in a dark room to prevent incomplete oxidation process. The mixture was diluted with 80% methanol in order to obtain absorbance of 0.7 ± 0.2 units at 734 nm. Then, 200 µL extract was added to 2.0 mL ABTS·+ solution. The mixture was vortexed for 45 seconds and was transferred into cuvette. The absorbance value was measured at 734 nm by using a spectrophotometer. Ascorbic acid was used as a standard in the concentration range 0 to 60 µg/mL. The final results were expressed as mg ascorbic acid equivalent antioxidant capacity in 1 g of sample (mg AEAC/g).


## 2.5. Determination of Acetylcholinesterase Inhibition Activity

Acetylcholinesterase (AChE) inhibition activity was determined spectrophotometrically using acetylcholine as substrate according to the method described previously [22] with slight modification. The samples with several tested concentrations were prepared separately (0–5 mg/mL). In this assay, 250 µL phosphate buffer (200 mM; pH 7.7) was added to 10 µL fruit extract sample. Following that, 80 µL of DTNB (3.96 mg of DTNB and 1.5 mg sodium bicarbonate dissolved in 10 mL phosphate buffer pH 7.7) and 10 µL enzyme (2 U/mL) were added to the mixture. The mixture was incubated for 5 minutes at room temperature (25°C). Finally, the reaction was started by adding 15 µL of ATCI (the substrate that contained 10.85 mg acetylthiocholine iodide in 5 mL sodium phosphate buffer) and incubated for 5 minutes at room temperature (25°C). The color developed was measured in microtiter plate by microplate reader at 412 nm. The hydrolysis of acetylthiocholine was determined by monitoring the formation of yellow 5-thio-2-nitrobenzoate anion as a result of the reaction with DTNB with thiocholines which is catalysed by enzymes at a wavelength of 412 nm. The solvent 80% methanol was used as negative control. Donepenzil dissolved in methanol was used as standard drug at 0–5 mg/mL concentration. The percent of inhibition was calculated by using the formula below.The percentage inhibition of acetylcholinesterase was calculated using the following formula:(3)
A
C
h
E
I
n
h
i
b
i
t
i
o
n
%
=
A
0
-
A
1
A
0
×
100
,where A
0 is the absorbance of the control (without extract) and A
1 is the absorbance of the tested extract.


## 2.6. Determination of Antibacterial Activity

The antibacterial activities of the extracts were evaluated by using disc diffusion assay [23] with slight modification. A total of 0.4 mL of bacterial culture was inoculated and spread on the agar. Two Gram-positive bacteria were tested, which wereStaphylococcus aureus andBacillus subtilis and two Gram-negative bacteria,Escherichia coli andSalmonella enteritidis, were used in this study. 100 µL of each sample extract was pipetted into filter paper discs (diameter: 6 mm). After drying, the filter paper discs were placed on the agar plate. Methanol was used as negative control, whereas kanamycin was used as positive control. The inhibitory activity was determined by a clear zone around the disc after incubation at 37°C for 24 h. The zone of inhibition was measured in millimeters (mm) including the disc diameter.


## 2.7. Statistical Analysis

All experiments (except for GC-MS analysis) were carried out in 3 replicates in 3 independent experiments. The result was presented as mean ± standard deviation. The data was statistically analysed by using one-way ANOVA with a significance value ofp
<
0.05 to test the significant difference between the samples. Pearson’s correlation was used to determine the relationship between phytochemicals and antioxidant activity.


## 3. Results and Discussions

### 3.1. Total Phenolic, Flavonoid, Anthocyanin, and Carotenoid Contents

Phenolics, flavonoids, anthocyanins, and carotenoids are the phytochemicals that normally presented in berries, known to possess antioxidant, anti-inflammatory, anticancer, antihypertension, antimutagenic, antineurodegenerative, and other bioactivities [4, 6, 24, 25]. Naturally, these phytochemicals are vital components for plant’s physiological functions such as for pollination and protection against UV light, pathogens, and herbivore [26]. Therefore, the occurrence of the total phenolic, flavonoid, anthocyanin, and carotenoid content in the selectedRubus species was investigated in this study. The result (Table 1) displayed the significant differences (p
<
0.05) among theRubus species except for total carotenoid betweenR. moluccanus andR. fraxinifolius. The highest phenolic (24.25 ± 0.12 mg GAE/g) and carotenoid contents (21.86 ± 0.63 mg BC/g) were observed inR. alpestris crude extract. The highest flavonoid (18.17 ± 0.20 mg CE/g) and anthocyanin contents (36.96 ± 0.39 mg c-3-gE/g) were displayed byR. moluccanus fruit.Table 1
Phytochemicals content of selected wildRubus species.
Sample
Total phenolic1
Total flavonoid2
Total anthocyanin3
Total carotenoid4
R. moluccanus
20.76 ± 0.24b
18.17 ± 0.20a
36.96 ± 0.39a
9.69 ± 0.58b
R. fraxinifolius
11.09 ± 0.10c
5.82 ± 0.02c
23.82 ± 0.77c
10.49 ± 1.01b
R. alpestris
24.25 ± 0.12a
8.88 ± 0.53b
33.62 ± 1.39b
21.86 ± 0.63a
Values are presented as mean ± standard deviation (SD) (n
=
3) which, with different letters (within column), are significantly different at p
<
0.05.
1Total phenolic content was expressed as mg gallic acid equivalents in 1 g of dried sample (mg GAE/g).
2Total flavonoid content was expressed as mg catechin equivalents in 1 g of dried sample (mg CE/g).
3Total anthocyanin content was expressed as mg of cyanidin-3-glucoside equivalents in 1 g of dried sample (mg c-3-gE/g).
4Total carotenoid content was expressed as mg of β-carotene in 1 g of dried sample (mg BC/g dried sample).Previous studies have demonstrated the occurrence of phenolics such as ellagic acid (which normally presented as polymer of glycosylated derivative), gallic acid, chlorogenic acid, and caffeic acid inRubus species [3, 8]. Both total phenolic and flavonoid contents obtained in the current study were relatively higher than otherRubus species in previous studies [8, 27], but slightly lower thanR. ulmifolius methanolic extract [7]. The dark red color of fruits indicates that it might contain high level of anthocyanin and flavonoid [28] as can be seen inR. moluccanus. Netzel et al. [29] stated that cyanidin-3-glucoside, cyanidin-3-rutinoside, and pelargonidin-3-rutinoside are among the anthocyanins that could be found inR. moluccanus. The range of total anthocyanins (23.82 to 36.96 mg c-3-gE/g) of selectedRubus species is similar to the previously published data by Krauze-Baranowska et al. [30] on hydroethanolic extract of raspberries with the total anthocyanins between 13.0 and 88.0 mg/g dry weight. Jung et al. [4] demonstrated thatRubus fruticosus,Rubus coreanus, andRubus occidentalis exerted antioxidant and anti-inflammatory activities in hydrogen peroxide and lipopolysaccharide treated RAW264.7 cells which are probably attributed to the anthocyanin content inRubus species. The anthocyanins could be found on the external layer of fruit’s skin cells (hypodermis), whereas the small amount of granular-form anthocyanin is deposited in the vacuole [24].The total carotenoid content ofR. alpestris was much higher than the quantity of carotenoid inRosa canina andRosa rugosa (Rosaceae family) as reported by Razungles et al. [31]. β-Carotene might be the major carotenoid compound inRubus species as displayed byR. chamaemorus [32]. The variation in the phytochemicals content is due to the genetic and environmental factors [9, 10]. The species that contained most abundant polyphenols may not necessarily possess great bioactivities. This is due to the fact that polyphenols might have lower intrinsic activity and might be poorly absorbed from intestine, highly metabolized, or rapidly eliminated [27].


### 3.2. Secondary Metabolite Profiling Using GC-MS

GC-MS profiling was performed to identify the bioactive compounds presented inRubus species. The gas chromatogram of 80%Rubus methanolic extracts is shown in Figures 4–6. The analysis separated and identified a total of 21 known compounds forR. moluccanus, 7 known compounds forR. fraxinifolius, and 12 compounds forR. alpestris (Table 2). The major compounds inRubus moluccanus included hydroxymethylfurfural (21.642%), 1,1,2-triacetoxyethane (17.908%), 2,4-dihydroxy-2,5-dimethyl-3(2H)-furan-3-one (10.345%), and 2-propenoic acid, 2-propenyl ester (6.002%). ForR. fraxinifolius, the major compounds are 2(1H)-pyridinone, 6-hydroxy- (14.589%), 1,1,2-triacetoxyethane (10.370%), 2,4-dihydroxy-2,5-dimethyl-3(2H)-furan-3-one (8.283%), and 2-propenoic acid, 2-propenyl ester (3.589%). The major compounds inR. alpestris are 5-hydroxymethylfurfural (38.142%), 2(1H)-pyridinone, 6-hydroxy- (25.430%), furfural (6.6372%), and 2,4-dihydroxy-2,5-dimethyl-3(2H)-furan-3-one (5.438%).Table 2
Chemical composition of differentRubus species, that is, R. moluccanus, R. fraxinifolius, and R. alpestris.
Sample
Number
Name of the compound
Concentration (%)
Retention time
R. moluccanus
1
2-Propenoic acid, 2-propenyl ester
6.002
3.693
2
Pyruvate
2.998
3.969
3
Furfural
3.185
5.272
4
1,3-Butadiene-1-carboxylic acid
0.913
5.714
5
Propenoic acid, 2-methyl-, methyl ester
2.857
6.403
6
dl-Glyceraldehyde dimer
0.934
7.041
7
2(1H)-Pyridinone, 6-hydroxy-
5.716
8.949
8
2,4-Dihydroxy-2,5-dimethyl-3(2H)-furan-3-one
2.127
10.049
9
Pentanoic acid, 4-oxo-
2.891
13.002
10
2-Hydroxy-3-methyl-4- pyrone
7.549
14.370
11
Isopropylmethylnitrosamine
0.922
17.391
12
2,4-Dihydroxy-2,5-dimethyl-3(2H)-furan-3-one
10.345
17.604
13
Hydroxy methyl furfural
21.642
21.970
14
1,1,2-Triacetoxyethane
17.908
22.569
18
Butanedioic acid, 2-hydroxy-2-methyl, (S)-
1.493
27.358
20
Benzeneacetic acid, 4-hydroxy-, methyl ester
1.504
28.634
21
Succinic acid, 3-methylbutyl pentyl ester
1.193
29.396
23
β-D-Glucopyranoside, methyl
3.800
37.574
24
Quinic acid
1.981
39.498
25
β-Tocopherol
0.322
80.794
26
γ-Sitosterol
0.204
87.214
R. fraxinifolius
3
2-Propenoic acid, 2-propenyl ester
3.589
3.685
5
Furfural
2.503
5.261
6
1,3-Butadiene-1-carboxylic acid
0.505
5.703
9
2(1H)-Pyridinone, 6-hydroxy-
14.589
8.940
16
2,4-Dihydroxy-2,5-dimethyl-3(2H)-furan-3-one
8.283
17.560
22
1,1,2-Triacetoxyethane
10.370
22.514
32
3-Deoxy-d-mannoic lactone
0.508
38.612
R. alpestris
3
Furfural
6.637
5.259
6
2(1H)-Pyridinone, 6-hydroxy-
25.430
8.990
9
2,4-Dihydroxy-2,5-dimethyl-3(2H)-furan-3-one
1.517
10.035
11
Furaneol
3.325
12.979
13
1H-Imidazole-4-carboxylic acid, methyl Ester
3.287
14.681
18
2,4-Dihydroxy-2,5-dimethyl-3(2H)-furan-3-one
5.438
17.572
21
5-Hydroxymethylfurfural
38.142
22.107
27
Butane, 1,1′-1-[(Isopentyloxy)methoxy]-3-methylbutane
0.968
29.364
28
Rhamnose
2.277
34.260
31
5,5′-Oxy-dimethylene-bis(2-furaldehyde)
0.249
52.683
35
β-Tocopherol
0.140
80.799
36
Stigmast-5-en-3-ol
0.117
87.228Figure 4
A typical gas chromatogram of the chemical constituents ofRubus moluccanus crude extract.Figure 5
A typical gas chromatogram of the chemical constituents ofRubus fraxinifolius crude extract.Figure 6
A typical gas chromatogram of the chemical constituents ofRubus alpestris crude extract.The similar compounds that could be found in the threeRubus species are furfural and 2,4-dihydroxy-2,5-dimethyl-3(2H)-furan-3-one. Furfural is a precursor of furan which could be formed naturally in fruits or in processed food during thermal storage. It also acts as indicator for the occurrence of Maillard reaction [33, 34]. Furfural also is highly concentrated in berry cactus [2]. Hydroxymethylfurfural or 5-hydroxymethylfurfural (HMF) is a major compound that could be found inR. moluccanus andR. alpestris. HMF might be presented naturally in the fruit or produced at high temperature processes such as drying or during GC-MS analysis [34]. Previous study showed that 5-HMF is an antioxidative agent fromAlpinia oxyphylla which could serve as novel therapeutic agent for Alzheimer’s disease treatment and prevention [35]. In addition, HMF also has been reported to possess anticancer properties [34].


### 3.3. DPPH, FRAP, and ABTS Assays

The antioxidant capacities ofRubus species were investigated using three differentin vitro antioxidant assays. In DPPH assay, purple color of DPPH solution turns into yellow color in the presence of antioxidant compound [19]. The antioxidant effect of extracts on DPPH free radical was due to its hydrogen-donating ability. The DPPH scavenging activity of selectedRubus species and standard (ascorbic acid) was depicted in Figure 7.Figure 7
Graph of DPPH scavenging activity against concentration.The highest percentage of scavenging activity (94.36 ± 1.33%) was observed inR. alpestris at 100 μg/mL concentration, followed byR. moluccanus (87.72 ± 0.71%) andR. fraxinifolius (59.78 ± 3.79%). However, the activities of all extracts are lesser than the ascorbic acid. The trend for IC50 values (Table 3) of DPPH radical scavenging activity is as follows: ascorbic acid (10.00 ± 0.58 μg/mL) >R. alpestris (29.00 ± 3.07 μg/mL) >R. moluccanus (38.00 ± 1.63 μg/mL) >R. fraxinifolius (86.00 ± 3.65 μg/mL). The DPPH radical scavenging activity of our investigatedRubus species is similar to or even better than the previous study [36]. As reported by Ahmad et al. [36], at 100 µg/mL concentration,Rubus ulmifolius was able to inhibit 87.62%, whereasR. ellipticus andR. niveus were able to inhibit 54.82% and 74.54% DPPH free radical. This indicates that the investigatedRubus species in current analyses are more effective to inhibit DPPH free radical compared to otherRubus species.Table 3
Antioxidant activities of selectedRubus species.
Samples
DPPH assay1
FRAP assay2
ABTS assay3
R. moluccanus
38.00 ± 1.63c
50.37 ± 5.28b
0.73 ± 0.03a
R. fraxinifolius
86.00 ± 3.65d
26.34 ± 4.79c
0.75 ± 0.03a
R. alpestris
29.00 ± 3.07b
70.93 ± 6.26a
0.79 ± 0.05a
Ascorbic acid
10.00 ± 0.58a
—
—
Values are presented as mean ± SD (n
=
3) which, with different letters (within column), are significantly different at p
<
0.05.
1DPPH free radical scavenging activity represented by IC50 was expressed as μg/mL.
2FRAP was expressed as mM ferric reduction to ferrous in 1 g of dry sample.
3ABTS free radical scavenging activity was expressed as mg ascorbic acid equivalent antioxidant capacity (AEAC) in 1 g of dry sample.FRAP method is the assay to determine the antioxidant capacity which involves Single Electron Transfer (SET) mechanism. In this method, ferric ion is reduced to ferrous ion at low pH, which caused colored ferrous tripyridyltriazine complex to be formed [20]. The reducing ability ofR. alpestris (70.93 ± 6.26 mM Fe2+/g) is the highest compared toR. moluccanus (50.37 ± 5.28 mM Fe2+/g) andR. fraxinifolius (26.34 ± 4.79 mM Fe2+/g). Our results showed higher FRAP value as compared toRubus ellipticus (3.43 mM ascorbic acid equivalent (AAE)/100 g) andRubus niveus (2.06 AAE/100 g) in previous research [8].Principally, ABTS decolorization assay is quite similar to DPPH assay, which involves the scavenging activity of the free radicals. However, the ABTS salt must be generated by enzymatic or chemical reaction first [21]. TheRubus fruit extracts exerted lower scavenging effects against ABTS radicals. There are no significant differences (p
<
0.05) between the samples for ABTS assay. In comparison to the previous literature, ABTS assay in current study displayed lower value compared toR. idaeus cultivar which are “Autumn Bliss” and “Polka” in the range 1.94 to 2.12 mg AEAC/g [26]. In summary,R. alpestris revealed the highest content for phenolics and carotenoid which is in agreement with its highest antioxidant activity.The phytocompounds (phenolics, flavonoid, anthocyanin, and carotenoid) might contribute to the antioxidant activity of the extract. Hence, correlation analyses were performed to investigate the relationship between the phytochemical compounds and antioxidant activity. Strong positive correlation has been displayed by total phenolics and DPPH scavenging activity (r
=
0.972). The total carotenoid and anthocyanin content showed moderate positive correlation with DPPH scavenging assay (r
=
0.761 and r
=
0.764). However, no correlation existed between total flavonoid and DPPH scavenging activity which is in contrast with past research [7, 8, 11]. These results corroborate the previous report that proved the presence of positive correlation between phenolic and antioxidant capacity in the extracts [3, 7].FRAP assays showed significant correlation with total phenolic, carotenoid, and anthocyanin (p
<
0.05) with value r
=
0.949, 0.770, and 0.745. Findings by Pantelidis et al. [27] and Deighton et al. [37] are supporting this study. Analysis on various berries species indicated that there was a strong correlation between phenol content with the FRAP assay [27, 37]. However, total anthocyanins have less linear correlation with total antioxidant capacity (r
=
0.635) in previous research. The correlation between phytochemicals investigated in this study with ABTS assay was not significant (p
>
0.05). Current result is in contrast with the previous study that displayed strong positive correlation between the total phenolic, flavonoid, and anthocyanin with ABTS assay [7, 8, 26].


### 3.4. Acetylcholinesterase Inhibition Activity

Several drugs for memory loss and cognitive deficits improvement are available in the market. However, these drugs possess some side effects. Phytochemicals from plants might be an alternative to be developed as a source of acetylcholinesterase inhibitors [38]. The acetylcholinesterase enzyme activity is measured based on the reduction of yellow color produced from thiocholine when it reacts with dithiobisnitrobenzoate ion [22]. The results were expressed in percentage. At the highest concentration (5 mg/mL), the fruit extracts displayed weak anticholinesterase activity. The highest anticholinesterase activity was shown byR. moluccanus (26.42 ± 1.41%), followed byR. alpestris (25.30 ± 1.56%) andR. fraxinifolius (23.06 ± 1.12%). Donepenzil (positive control) showed complete acetylcholinesterase inhibition activity (100%) when tested at 1 mg/mL. The inhibition of acetylcholinesterase noted in this study is very similar to theSanguisorba minor aerial part andRosa Damascene floret (Rosaceae family), which showed a weak inhibitory effect on acetylcholinesterase enzyme [39]. Kim et al. [38] demonstrated thatRubus coreanus ethanolic extract (1 mg/mL concentration) exhibits 36.60 ± 1.25% inhibitory effects on acetylcholinesterase enzyme. Active compound that was isolated fromR. coreanus was identified as 3,4,5-trihydroxybenzoic acid (gallic acid). Thus, these phenolic acids might also contribute to the acetylcholinesterase activity of the investigatedRubus species in current study.


### 3.5. Antibacterial Activity

SinceRubus species displayed numerous secondary metabolites, the effects of the extract against some common pathogenic bacteria were investigated in this study. The result of our current research demonstrated thatR. moluccanus andR. alpestris were effective against Gram-positive and Gram-negative bacteria (Table 4).R. alpestris showed the highest activity againstS. enteritidis (8.50 ± 1.80 mm) followed byB. subtilis (7.83 ± 1.26 mm). Rauha et al. [40] have reported thatR. chamaemorus andR. idaeus displayed only slight antibacterial effects againstS. aureus andE. coli, whereas the inhibition towardsB. subtilis was found to be moderate. In our study,R. fraxinifolius showed no inhibition towardsB. subtilis. The variation in the inhibition activity ofRubus species is due to the differences in cell surface structure between the Gram-negative and Gram-positive bacteria [41].Table 4
Antibacterial activities ofRubus species against common pathogenic bacteria.
Species
Gram positive
Gram negative
B. subtilis
S. aureus
E. coli
S. enteritidis
R. moluccanus
7.33 ± 0.29b
7.67 ± 0.58b
7.00 ± 0.50b
7.17 ± 0.58b
R. fraxinifolius
0.00c
6.67 ± 1.15b
7.00 ± 0.87b
7.17 ± 1.15b
R. alpestris
7.83 ± 1.26b
7.33 ± 0.76b
7.67 ± 0.58b
8.50 ± 1.80b
Kanamycin
12.67 ± 1.53a
11.83 ± 0.76a
12.67 ± 0.58a
11.83 ± 0.76a
Values are presented as mean ± SD (n
=
3) which, with different letters (within column), are significantly different at p
<
0.05.Phenolic compounds such as flavone, quercetin, and naringenin were the potential compounds that contribute to the antibacterial activities againstS. aureus,B. subtilis, andE. coli [40]. In addition, anthocyanidin, pelargonidin, delphinidin, cyanidin, and cyanidin-3-glucoside in berry extracts were able to inhibit the growth ofE. coli [41]. Our findings suggested that all of the investigatedRubus species contained high amount of anthocyanin which might contribute to the inhibition towardsE. coli. Puupponen-Pimiä et al. [41] demonstrated that Finnish berry extracts were more effective to inhibit the growth of Gram-negative bacteria as compared to Gram-positive bacteria. Generally,R. alpestris is more beneficial in treating the Gram-positive and Gram-negative bacteria. It is worth noting that the present study is only a preliminary attempt to assess the antibacterial potential of theRubus species. Hence, further detailed bioassay needs to be applied for assessing the antibacterial activity.


## 3.1. Total Phenolic, Flavonoid, Anthocyanin, and Carotenoid Contents

Phenolics, flavonoids, anthocyanins, and carotenoids are the phytochemicals that normally presented in berries, known to possess antioxidant, anti-inflammatory, anticancer, antihypertension, antimutagenic, antineurodegenerative, and other bioactivities [4, 6, 24, 25]. Naturally, these phytochemicals are vital components for plant’s physiological functions such as for pollination and protection against UV light, pathogens, and herbivore [26]. Therefore, the occurrence of the total phenolic, flavonoid, anthocyanin, and carotenoid content in the selectedRubus species was investigated in this study. The result (Table 1) displayed the significant differences (p
<
0.05) among theRubus species except for total carotenoid betweenR. moluccanus andR. fraxinifolius. The highest phenolic (24.25 ± 0.12 mg GAE/g) and carotenoid contents (21.86 ± 0.63 mg BC/g) were observed inR. alpestris crude extract. The highest flavonoid (18.17 ± 0.20 mg CE/g) and anthocyanin contents (36.96 ± 0.39 mg c-3-gE/g) were displayed byR. moluccanus fruit.Table 1
Phytochemicals content of selected wildRubus species.
Sample
Total phenolic1
Total flavonoid2
Total anthocyanin3
Total carotenoid4
R. moluccanus
20.76 ± 0.24b
18.17 ± 0.20a
36.96 ± 0.39a
9.69 ± 0.58b
R. fraxinifolius
11.09 ± 0.10c
5.82 ± 0.02c
23.82 ± 0.77c
10.49 ± 1.01b
R. alpestris
24.25 ± 0.12a
8.88 ± 0.53b
33.62 ± 1.39b
21.86 ± 0.63a
Values are presented as mean ± standard deviation (SD) (n
=
3) which, with different letters (within column), are significantly different at p
<
0.05.
1Total phenolic content was expressed as mg gallic acid equivalents in 1 g of dried sample (mg GAE/g).
2Total flavonoid content was expressed as mg catechin equivalents in 1 g of dried sample (mg CE/g).
3Total anthocyanin content was expressed as mg of cyanidin-3-glucoside equivalents in 1 g of dried sample (mg c-3-gE/g).
4Total carotenoid content was expressed as mg of β-carotene in 1 g of dried sample (mg BC/g dried sample).Previous studies have demonstrated the occurrence of phenolics such as ellagic acid (which normally presented as polymer of glycosylated derivative), gallic acid, chlorogenic acid, and caffeic acid inRubus species [3, 8]. Both total phenolic and flavonoid contents obtained in the current study were relatively higher than otherRubus species in previous studies [8, 27], but slightly lower thanR. ulmifolius methanolic extract [7]. The dark red color of fruits indicates that it might contain high level of anthocyanin and flavonoid [28] as can be seen inR. moluccanus. Netzel et al. [29] stated that cyanidin-3-glucoside, cyanidin-3-rutinoside, and pelargonidin-3-rutinoside are among the anthocyanins that could be found inR. moluccanus. The range of total anthocyanins (23.82 to 36.96 mg c-3-gE/g) of selectedRubus species is similar to the previously published data by Krauze-Baranowska et al. [30] on hydroethanolic extract of raspberries with the total anthocyanins between 13.0 and 88.0 mg/g dry weight. Jung et al. [4] demonstrated thatRubus fruticosus,Rubus coreanus, andRubus occidentalis exerted antioxidant and anti-inflammatory activities in hydrogen peroxide and lipopolysaccharide treated RAW264.7 cells which are probably attributed to the anthocyanin content inRubus species. The anthocyanins could be found on the external layer of fruit’s skin cells (hypodermis), whereas the small amount of granular-form anthocyanin is deposited in the vacuole [24].The total carotenoid content ofR. alpestris was much higher than the quantity of carotenoid inRosa canina andRosa rugosa (Rosaceae family) as reported by Razungles et al. [31]. β-Carotene might be the major carotenoid compound inRubus species as displayed byR. chamaemorus [32]. The variation in the phytochemicals content is due to the genetic and environmental factors [9, 10]. The species that contained most abundant polyphenols may not necessarily possess great bioactivities. This is due to the fact that polyphenols might have lower intrinsic activity and might be poorly absorbed from intestine, highly metabolized, or rapidly eliminated [27].


## 3.2. Secondary Metabolite Profiling Using GC-MS

GC-MS profiling was performed to identify the bioactive compounds presented inRubus species. The gas chromatogram of 80%Rubus methanolic extracts is shown in Figures 4–6. The analysis separated and identified a total of 21 known compounds forR. moluccanus, 7 known compounds forR. fraxinifolius, and 12 compounds forR. alpestris (Table 2). The major compounds inRubus moluccanus included hydroxymethylfurfural (21.642%), 1,1,2-triacetoxyethane (17.908%), 2,4-dihydroxy-2,5-dimethyl-3(2H)-furan-3-one (10.345%), and 2-propenoic acid, 2-propenyl ester (6.002%). ForR. fraxinifolius, the major compounds are 2(1H)-pyridinone, 6-hydroxy- (14.589%), 1,1,2-triacetoxyethane (10.370%), 2,4-dihydroxy-2,5-dimethyl-3(2H)-furan-3-one (8.283%), and 2-propenoic acid, 2-propenyl ester (3.589%). The major compounds inR. alpestris are 5-hydroxymethylfurfural (38.142%), 2(1H)-pyridinone, 6-hydroxy- (25.430%), furfural (6.6372%), and 2,4-dihydroxy-2,5-dimethyl-3(2H)-furan-3-one (5.438%).Table 2
Chemical composition of differentRubus species, that is, R. moluccanus, R. fraxinifolius, and R. alpestris.
Sample
Number
Name of the compound
Concentration (%)
Retention time
R. moluccanus
1
2-Propenoic acid, 2-propenyl ester
6.002
3.693
2
Pyruvate
2.998
3.969
3
Furfural
3.185
5.272
4
1,3-Butadiene-1-carboxylic acid
0.913
5.714
5
Propenoic acid, 2-methyl-, methyl ester
2.857
6.403
6
dl-Glyceraldehyde dimer
0.934
7.041
7
2(1H)-Pyridinone, 6-hydroxy-
5.716
8.949
8
2,4-Dihydroxy-2,5-dimethyl-3(2H)-furan-3-one
2.127
10.049
9
Pentanoic acid, 4-oxo-
2.891
13.002
10
2-Hydroxy-3-methyl-4- pyrone
7.549
14.370
11
Isopropylmethylnitrosamine
0.922
17.391
12
2,4-Dihydroxy-2,5-dimethyl-3(2H)-furan-3-one
10.345
17.604
13
Hydroxy methyl furfural
21.642
21.970
14
1,1,2-Triacetoxyethane
17.908
22.569
18
Butanedioic acid, 2-hydroxy-2-methyl, (S)-
1.493
27.358
20
Benzeneacetic acid, 4-hydroxy-, methyl ester
1.504
28.634
21
Succinic acid, 3-methylbutyl pentyl ester
1.193
29.396
23
β-D-Glucopyranoside, methyl
3.800
37.574
24
Quinic acid
1.981
39.498
25
β-Tocopherol
0.322
80.794
26
γ-Sitosterol
0.204
87.214
R. fraxinifolius
3
2-Propenoic acid, 2-propenyl ester
3.589
3.685
5
Furfural
2.503
5.261
6
1,3-Butadiene-1-carboxylic acid
0.505
5.703
9
2(1H)-Pyridinone, 6-hydroxy-
14.589
8.940
16
2,4-Dihydroxy-2,5-dimethyl-3(2H)-furan-3-one
8.283
17.560
22
1,1,2-Triacetoxyethane
10.370
22.514
32
3-Deoxy-d-mannoic lactone
0.508
38.612
R. alpestris
3
Furfural
6.637
5.259
6
2(1H)-Pyridinone, 6-hydroxy-
25.430
8.990
9
2,4-Dihydroxy-2,5-dimethyl-3(2H)-furan-3-one
1.517
10.035
11
Furaneol
3.325
12.979
13
1H-Imidazole-4-carboxylic acid, methyl Ester
3.287
14.681
18
2,4-Dihydroxy-2,5-dimethyl-3(2H)-furan-3-one
5.438
17.572
21
5-Hydroxymethylfurfural
38.142
22.107
27
Butane, 1,1′-1-[(Isopentyloxy)methoxy]-3-methylbutane
0.968
29.364
28
Rhamnose
2.277
34.260
31
5,5′-Oxy-dimethylene-bis(2-furaldehyde)
0.249
52.683
35
β-Tocopherol
0.140
80.799
36
Stigmast-5-en-3-ol
0.117
87.228Figure 4
A typical gas chromatogram of the chemical constituents ofRubus moluccanus crude extract.Figure 5
A typical gas chromatogram of the chemical constituents ofRubus fraxinifolius crude extract.Figure 6
A typical gas chromatogram of the chemical constituents ofRubus alpestris crude extract.The similar compounds that could be found in the threeRubus species are furfural and 2,4-dihydroxy-2,5-dimethyl-3(2H)-furan-3-one. Furfural is a precursor of furan which could be formed naturally in fruits or in processed food during thermal storage. It also acts as indicator for the occurrence of Maillard reaction [33, 34]. Furfural also is highly concentrated in berry cactus [2]. Hydroxymethylfurfural or 5-hydroxymethylfurfural (HMF) is a major compound that could be found inR. moluccanus andR. alpestris. HMF might be presented naturally in the fruit or produced at high temperature processes such as drying or during GC-MS analysis [34]. Previous study showed that 5-HMF is an antioxidative agent fromAlpinia oxyphylla which could serve as novel therapeutic agent for Alzheimer’s disease treatment and prevention [35]. In addition, HMF also has been reported to possess anticancer properties [34].


## 3.3. DPPH, FRAP, and ABTS Assays

The antioxidant capacities ofRubus species were investigated using three differentin vitro antioxidant assays. In DPPH assay, purple color of DPPH solution turns into yellow color in the presence of antioxidant compound [19]. The antioxidant effect of extracts on DPPH free radical was due to its hydrogen-donating ability. The DPPH scavenging activity of selectedRubus species and standard (ascorbic acid) was depicted in Figure 7.Figure 7
Graph of DPPH scavenging activity against concentration.The highest percentage of scavenging activity (94.36 ± 1.33%) was observed inR. alpestris at 100 μg/mL concentration, followed byR. moluccanus (87.72 ± 0.71%) andR. fraxinifolius (59.78 ± 3.79%). However, the activities of all extracts are lesser than the ascorbic acid. The trend for IC50 values (Table 3) of DPPH radical scavenging activity is as follows: ascorbic acid (10.00 ± 0.58 μg/mL) >R. alpestris (29.00 ± 3.07 μg/mL) >R. moluccanus (38.00 ± 1.63 μg/mL) >R. fraxinifolius (86.00 ± 3.65 μg/mL). The DPPH radical scavenging activity of our investigatedRubus species is similar to or even better than the previous study [36]. As reported by Ahmad et al. [36], at 100 µg/mL concentration,Rubus ulmifolius was able to inhibit 87.62%, whereasR. ellipticus andR. niveus were able to inhibit 54.82% and 74.54% DPPH free radical. This indicates that the investigatedRubus species in current analyses are more effective to inhibit DPPH free radical compared to otherRubus species.Table 3
Antioxidant activities of selectedRubus species.
Samples
DPPH assay1
FRAP assay2
ABTS assay3
R. moluccanus
38.00 ± 1.63c
50.37 ± 5.28b
0.73 ± 0.03a
R. fraxinifolius
86.00 ± 3.65d
26.34 ± 4.79c
0.75 ± 0.03a
R. alpestris
29.00 ± 3.07b
70.93 ± 6.26a
0.79 ± 0.05a
Ascorbic acid
10.00 ± 0.58a
—
—
Values are presented as mean ± SD (n
=
3) which, with different letters (within column), are significantly different at p
<
0.05.
1DPPH free radical scavenging activity represented by IC50 was expressed as μg/mL.
2FRAP was expressed as mM ferric reduction to ferrous in 1 g of dry sample.
3ABTS free radical scavenging activity was expressed as mg ascorbic acid equivalent antioxidant capacity (AEAC) in 1 g of dry sample.FRAP method is the assay to determine the antioxidant capacity which involves Single Electron Transfer (SET) mechanism. In this method, ferric ion is reduced to ferrous ion at low pH, which caused colored ferrous tripyridyltriazine complex to be formed [20]. The reducing ability ofR. alpestris (70.93 ± 6.26 mM Fe2+/g) is the highest compared toR. moluccanus (50.37 ± 5.28 mM Fe2+/g) andR. fraxinifolius (26.34 ± 4.79 mM Fe2+/g). Our results showed higher FRAP value as compared toRubus ellipticus (3.43 mM ascorbic acid equivalent (AAE)/100 g) andRubus niveus (2.06 AAE/100 g) in previous research [8].Principally, ABTS decolorization assay is quite similar to DPPH assay, which involves the scavenging activity of the free radicals. However, the ABTS salt must be generated by enzymatic or chemical reaction first [21]. TheRubus fruit extracts exerted lower scavenging effects against ABTS radicals. There are no significant differences (p
<
0.05) between the samples for ABTS assay. In comparison to the previous literature, ABTS assay in current study displayed lower value compared toR. idaeus cultivar which are “Autumn Bliss” and “Polka” in the range 1.94 to 2.12 mg AEAC/g [26]. In summary,R. alpestris revealed the highest content for phenolics and carotenoid which is in agreement with its highest antioxidant activity.The phytocompounds (phenolics, flavonoid, anthocyanin, and carotenoid) might contribute to the antioxidant activity of the extract. Hence, correlation analyses were performed to investigate the relationship between the phytochemical compounds and antioxidant activity. Strong positive correlation has been displayed by total phenolics and DPPH scavenging activity (r
=
0.972). The total carotenoid and anthocyanin content showed moderate positive correlation with DPPH scavenging assay (r
=
0.761 and r
=
0.764). However, no correlation existed between total flavonoid and DPPH scavenging activity which is in contrast with past research [7, 8, 11]. These results corroborate the previous report that proved the presence of positive correlation between phenolic and antioxidant capacity in the extracts [3, 7].FRAP assays showed significant correlation with total phenolic, carotenoid, and anthocyanin (p
<
0.05) with value r
=
0.949, 0.770, and 0.745. Findings by Pantelidis et al. [27] and Deighton et al. [37] are supporting this study. Analysis on various berries species indicated that there was a strong correlation between phenol content with the FRAP assay [27, 37]. However, total anthocyanins have less linear correlation with total antioxidant capacity (r
=
0.635) in previous research. The correlation between phytochemicals investigated in this study with ABTS assay was not significant (p
>
0.05). Current result is in contrast with the previous study that displayed strong positive correlation between the total phenolic, flavonoid, and anthocyanin with ABTS assay [7, 8, 26].


## 3.4. Acetylcholinesterase Inhibition Activity

Several drugs for memory loss and cognitive deficits improvement are available in the market. However, these drugs possess some side effects. Phytochemicals from plants might be an alternative to be developed as a source of acetylcholinesterase inhibitors [38]. The acetylcholinesterase enzyme activity is measured based on the reduction of yellow color produced from thiocholine when it reacts with dithiobisnitrobenzoate ion [22]. The results were expressed in percentage. At the highest concentration (5 mg/mL), the fruit extracts displayed weak anticholinesterase activity. The highest anticholinesterase activity was shown byR. moluccanus (26.42 ± 1.41%), followed byR. alpestris (25.30 ± 1.56%) andR. fraxinifolius (23.06 ± 1.12%). Donepenzil (positive control) showed complete acetylcholinesterase inhibition activity (100%) when tested at 1 mg/mL. The inhibition of acetylcholinesterase noted in this study is very similar to theSanguisorba minor aerial part andRosa Damascene floret (Rosaceae family), which showed a weak inhibitory effect on acetylcholinesterase enzyme [39]. Kim et al. [38] demonstrated thatRubus coreanus ethanolic extract (1 mg/mL concentration) exhibits 36.60 ± 1.25% inhibitory effects on acetylcholinesterase enzyme. Active compound that was isolated fromR. coreanus was identified as 3,4,5-trihydroxybenzoic acid (gallic acid). Thus, these phenolic acids might also contribute to the acetylcholinesterase activity of the investigatedRubus species in current study.


## 3.5. Antibacterial Activity

SinceRubus species displayed numerous secondary metabolites, the effects of the extract against some common pathogenic bacteria were investigated in this study. The result of our current research demonstrated thatR. moluccanus andR. alpestris were effective against Gram-positive and Gram-negative bacteria (Table 4).R. alpestris showed the highest activity againstS. enteritidis (8.50 ± 1.80 mm) followed byB. subtilis (7.83 ± 1.26 mm). Rauha et al. [40] have reported thatR. chamaemorus andR. idaeus displayed only slight antibacterial effects againstS. aureus andE. coli, whereas the inhibition towardsB. subtilis was found to be moderate. In our study,R. fraxinifolius showed no inhibition towardsB. subtilis. The variation in the inhibition activity ofRubus species is due to the differences in cell surface structure between the Gram-negative and Gram-positive bacteria [41].Table 4
Antibacterial activities ofRubus species against common pathogenic bacteria.
Species
Gram positive
Gram negative
B. subtilis
S. aureus
E. coli
S. enteritidis
R. moluccanus
7.33 ± 0.29b
7.67 ± 0.58b
7.00 ± 0.50b
7.17 ± 0.58b
R. fraxinifolius
0.00c
6.67 ± 1.15b
7.00 ± 0.87b
7.17 ± 1.15b
R. alpestris
7.83 ± 1.26b
7.33 ± 0.76b
7.67 ± 0.58b
8.50 ± 1.80b
Kanamycin
12.67 ± 1.53a
11.83 ± 0.76a
12.67 ± 0.58a
11.83 ± 0.76a
Values are presented as mean ± SD (n
=
3) which, with different letters (within column), are significantly different at p
<
0.05.Phenolic compounds such as flavone, quercetin, and naringenin were the potential compounds that contribute to the antibacterial activities againstS. aureus,B. subtilis, andE. coli [40]. In addition, anthocyanidin, pelargonidin, delphinidin, cyanidin, and cyanidin-3-glucoside in berry extracts were able to inhibit the growth ofE. coli [41]. Our findings suggested that all of the investigatedRubus species contained high amount of anthocyanin which might contribute to the inhibition towardsE. coli. Puupponen-Pimiä et al. [41] demonstrated that Finnish berry extracts were more effective to inhibit the growth of Gram-negative bacteria as compared to Gram-positive bacteria. Generally,R. alpestris is more beneficial in treating the Gram-positive and Gram-negative bacteria. It is worth noting that the present study is only a preliminary attempt to assess the antibacterial potential of theRubus species. Hence, further detailed bioassay needs to be applied for assessing the antibacterial activity.


## 4. Conclusions

Our results indicate there is a high variability in the phytochemicals content ofRubus species investigated which might be due to the genetic and environmental factors. Methanol-water extracts ofR. moluccanus, R. fraxinifolius, andR. alpestris fruits showed a significant amount of phytochemicals, which contribute to antioxidant, antibacterial, and antiacetylcholinesterase activities.R. alpestris displayed the highest potential as a natural source of tested activities. Utilization ofRubus fruits in diet could offer health benefit to our body. Since there is less previous research on these particular species, this study might contribute to the additional data on phytochemistry and bioactivities of the genusRubus.



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*Source: 2482930-2016-06-28.xml*