Dietary fiber has shown beneficial effects in the prevention of several diseases, such as cardiovascular diseases, diverticulosis, constipation, irritable colon, colon cancer, obesity, and diabetes. Some dietary fibers can retard starch digestion, absorb glucose, reduce glucose absorption, and control postprandial serum glucose levels [1]. Nephelium lappaceum L. is native to South East Asia. It is commonly known as rambutan (King of Fruits) which belongs to the family of Sapindaceae and is an attractive tropical fruit widely distributed in Myanmar. Myanmar's name is Kyet-mout. The present research work aimed to investigate the phytochemical constituents and some biochemical properties of the peel of Nephelium lappaceum L. (rambutan).

Plant Materials

In the present work, the peel of rambutanwas chosen as a sample to be studied. The peel was collected from MawlamyineTownship, Myanmar. The dried powder of rambutan peel was used to investigate their chemical and biological properties.

Phytochemical Screening

Preliminary phytochemical tests such as alkaloids,α-amino acids, carbohydrates, cardiac glycosides, cyanogenic glycosides, flavonoids, glycosides, organic acids, phenolic compounds, polyphenols, proteins, reducing sugars, saponins, steroids, tannins, and terpenoids on the sample were carried out according to the appropriate reported methods [2].

Estimation of Phytochemical Constituents

The phytochemical investigation of sample powderwas carried out qualitatively and quantitatively.

Determination of Total Phenol Content by Folin-Ciocalteu Reagent (FCR) Method

One of the anti-oxidative factors, total phenol content (TPC) was measured spectrophotometrically according to the Folin-Ciocalteu method. The total phenolic content (TPC) in each sample was estimated by Folin-Ciocalteu method according to the procedure described by Song [3], (2010). Each extract solution (100 mg/mL) was mixed with 5 mL of F-C reagent (1:10) and incubated for about 5 minutes. To each test tube, 4 mL of 1 M sodium carbonate was added and the test tubes were kept at room temperature for 15 minutes. UV absorbance of the reaction mixture was read λ _max 765 nm. The blank solution was prepared as the above procedure by using distilled water instead of the sample solution. Total phenolic content was estimated as milligram gallic acid equivalent per gram (mg GAE/g) of extract.

Determination of Total Flavonoid Content by Aluminium Chloride Method

The total flavonoid content (TFC) in each sample was estimated by aluminium chloride method according to the procedure described by Song et al., (2010). Each extract solution (100mg/mL) was mixed with 1.5 mL of methanol, 0.1 mL of 1 % AlCl₃ solution, and 2.8 mL of distilled water. The absorbance of the reaction mixture was readatλ_max 415 nm. The blank solution was prepared as the above procedure by using distilled water instead of the sample solution. Total flavonoid content was estimated as microgram quercetin acid equivalent per 100 milligrams (μg GAE/mg) of extract.

Determination of Total Steroid Content by Zak’s Method

The total steroid content (TSC) in each sample was estimated by Zak’s method according to the procedure described by Zak [4]. Each extract solution (100mg/mL) was prepared by a ferric chloride diluting agent. The test sample solution (5 mL) was added 4.0 mL of concentrated sulphuric acid in each tube.  After 30 minutes of incubation, the intensity of the colour developed was read at 540 nm. The blank solution was prepared as the above procedure by using ferric chloride diluting agent instead of the sample solution. Total steroid content was estimated as milligram cholesterol equivalent per gram (mg CE/g) of extract.

Determination of Total Condensed Tannin Content by Broadhurst’s Method

The tannin contents or Proanthocyanidin were determined by the method of Broadhurst and Jones (1978) with slight modification, using tannic acid as a reference compound. A volume of 0.4 mL of the extract is added to 3 mL of a solution of vanillin and 1.5 mL of concentrated hydrochloric acid. After 15 minutes of incubation, the absorbance was read at 500 nm. The blank solution was prepared as the above procedure by using methanol instead of the sample solution. The condensed tannin was expressed as milligrams of tannic acid equivalent per gram of extract.

Determination of Total Cardiac Glycoside Content by Baljet’s Method

The cardiac glycoside contents were determined by the method of Baljet [5] using digitoxin as a reference compound. A volume of 10 mL of the extract is added to 10 mL of Baljet’s reagent is taken and allowed to stand for one hour and diluted the solution with 20 mL of distilled water.  After that, the absorbance was read at 495 nm. The blank solution was prepared as the above procedure by using methanol instead of the sample solution.  The cardiac glycoside content was expressed as milligram of digitoxin equivalent per gram of extract.

Determination of Biological Activities of Ethanol and Watery Extracts of Peel of Rambutan

Screening Of Antimicrobial Activities of the Ethanol and Watery Extracts of the Peel of Rambutan

The antimicrobial activity of ethanol and watery crude extracts of the peel of rambutan was determined against six strains of microorganisms such as Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus pumilus, Candida albicans,and Escherichiacoli by employing agar well diffusion method [6]. The crude extract (0.5 g), peptone (0.5 g), and sodium chloride (0.25 g) were mixed with distilled water and made up to 100 mL with distilled water.  The pH of this solution was adjusted at 7.2 with 0.1 M sodium hydroxide solution and 1.5 g of agar was added. Nutrient agar was prepared according to the method described by Cruick (1975).  Briefly, nutrient agar was boiled and 20-25 mL of the medium was poured into a test tube and plugged with cotton wool, and autoclaved at 121 ºC for 15 minutes.  Then the tubes were cooled down to 60 ºC and poured into sterilized petri-dish and 0.1 mL of spore suspension was also added into the dishes.  The agar was allowed to be set for 30 minutes after which a 10 mm plate agar well was made with the help of a sterilized cork border.  After that, about 0.1 mL of the sample was introduced into the agarwell and incubated at 37 ºC for 24 hours.  The inhibition zone (clear zone) appeared around the agar well indicating the presence of antimicrobial activity. The extent of antimicrobial activity was measured from the zone of inhibition diameter.

Determination of the Antioxidant Activity of Ethanol and Watery Extracts of the Peel of Rambutan

There are several methods for screening of antioxidant activity of plant extracts. DPPH (2, 2- diphenyl-1-picryl hydrazyl) free radical scavenging assay and ferric reducing antioxidant power assay were chosen to assess the antioxidant activity of the samples.  DPPH (2, 2- diphenyl-1-picryl hydrazyl) free radical scavenging assay has been widely used to evaluate the free radical scavenging effects of various flavonoids and polyphenols in the food system. In this experiment, the antioxidant activity of ethanol and watery extracts of two selected plant samples was determined by DPPH free radical scavenging assay [7]; [8].The control solution was prepared by mixing 1.5 mL of 0.002 % DPPH solution and 1.5 mL of ethanol in the brown bottle.  The sample solution was also prepared by mixing 1.5 mL of 0.002 % DPPH solutions and 1.5 mL of test sample solution.  These bottles were incubated at room temperature and were shaken on a shaker for 30 min.  After 30 minutes, the absorbance of each solution was measured at 517 nm by using a UV-visible spectrophotometer.  The percent radical scavenging activity was calculated by the following equation.

% RSA   =                                [A_DPPH-(A_Sample - A_Blank)/ A_DPPH] × 100

where,    % RSA                     =      % radical scavenging activity

                A_DPPH                        =      absorbance of DPPH in EtOH solution

                A_sample                       =      absorbance of sample + DPPH solution

                A_Blank                         =      absorbance of sample + EtOH solution

The antioxidant activity (IC₅₀) is expressed as the test substances concentration (µg/mL) that results in a 50 % oxidative inhibition of the substance.  IC₅₀ (50% inhibitory concentration) values were calculated by linear regressive excel program. The standard deviation was also calculated by the following equation.

Standard Deviation (SD) =

where,                                           =    average % inhibition

x₁ + x₂ + …. + x_n                =              % oxidative inhibition of test sample solution

n              =      number of times

The ferric-reducing antioxidant power was measured by using the cyanoferrate method [9]; [10]. In this experiment, Fe (III) reduction is often used as an indicator of electron-donating activity, which is an important mechanism of phenolic antioxidant action.  The reducing power of extracts was determined by using the cyanoferrate method. The different concentrations of 1 mL sample extracts and positive control solutions were mixed with 2.5 mL of phosphate buffer (pH 6.6) and 2.5 mL of 1 % potassium ferricyanide.  The mixture was incubated at 50 ºC for 20 minutes.  A portion (2.5 mL) of 10 % trichloroacetic acid was added to the mixture to stop the reaction, which was then centrifuged at 3,000 rpm for 10 minutes.  2.5 mL of upper layer solution was mixed with 2.5 mL of distilled water and 0.5 mL of
0.01 % FeCl₃ solution and the absorbance measured at 700 nm were recorded.  Each experiment was carried out at least three times and the data were presented as an average of three independent determinations. The ferric-reducing antioxidant activity was calculated by the following equation.

% FRAP                                   =      [A_control-(A_Sample - A_Blank)/ A_control] × 100

where,   

                % FRAP                   =      % ferric reducing antioxidant power

                A_control                       =      absorbance without sample solution

                A_sample                       =      absorbance of the sample

                A_Blank                         =      absorbance of sample + distilled water solution

The ferric-reducing antioxidant power (IC₅₀) is expressed as the test substance concentration (µg/mL) that results in a 50% reducing power of the sample.  The IC₅₀ values were calculated by linear regressive excel program.   The standard deviation was also calculated by the following equation.

Standard Deviation (SD) =

where,                                           =    average % inhibition

x₁ + x₂ + …. + x_n                =              % reducing power of test sample solution

n       =              number of times

Determination of the Antidiabetic Activity of Ethanol and Watery Extracts of the Peel of Rambutan

The a-amylase and a-glucosidase enzymes were used to determine the inhibition effect of ethanol and watery extracts of the peel of rambutan. The a-amylase and a-glucosidase inhibitory activity was determined using a modified assay of that described in the Worthington Enzyme Manual [11]. The a-amylase enzyme inhibitory activity was expressed as a decrease in units of maltose liberated. A modified dinitrosalicylic acid (DNS) method was adopted to estimate the maltose equivalent. The tested samples (1 mL) were pre-incubated with 1 mL of phosphate buffer and 2 mL of α-amylase at 37 °C for 20 minutes and thereafter 0.4 mL (1 % w/v) starch solution was added. The mixture was further incubated at 37 °C for 30 minutes. Then the reaction was stopped by adding 2 mL of DNS reagent and the contents were heated in a boiling water bath for 10 minutes. A blank was prepared without plant extracts and another without the amylase enzyme, replaced by equal quantities of the buffer. The absorbance was measured at 540 nm. The reducing sugar released from starch was estimated as maltose equivalent from a standard graph. Acarbose was used as standard. The anti-diabetic activity was determined through the inhibition of α-amylase which was expressed as a percentage of inhibition and calculated by the following equations:

% Inhibition                            =      [A_control-(A_Sample - A_Blank)/ A_control] × 100

where,    % Inhibition            =      % a-amylase inhibition

                A_control                       =      absorbance without sample solution

                A_sample    =                 absorbance of the sample

                A_Blank       =              absorbance of sample + distilled water solution

The a-amylase inhibition (IC₅₀) is expressed as the test substance concentration (µg/mL) that results in a 50% a-amylase inhibition of the sample.  The IC₅₀ values were calculated by linear regressive excel program.   The standard deviation was also calculated by the following equation.

Standard Deviation (SD)

=

Where,    = average % inhibition

              x₁ + x₂ + …. + x_n                               =              % a-amylase inhibition of test sample solution

              n = number of times

The a-glucosidase inhibitory activity was measured by the procedure described by McCue [12].  The a-glucosidase was assayed using 0.4 mL of sample extracts and 1 mL of 0.1 M phosphate buffer (pH 6.9) containing 2 mL of a-glucosidase solution, which was then incubated at 25 °C for 10 minutes. After the pre-incubation period, 0.5 mL of 0.005 M p-nitrophenyl-a-D-glucopyranoside solution was added to each well at timed intervals. The reaction mixtures were incubated at 25 °C for 5 minutes.  After incubation, absorbance readings of the samples were recorded at 405 nm and compared with a control that had 0.4 mL of buffer solution in place of the extract. Acarbose was used as standard. The anti-diabetic activity was determined through the inhibition of a-glucosidase which was expressed as a percentage of inhibition and calculated by the following equations:

% Inhibition                            =      [A_control-(A_Sample - A_Blank)/ A_control] × 100

Where,  

                % Inhibition            =      % a-glucosidase inhibition

                A_control                       =      absorbance without sample solution

                A_sample                       =      absorbance of the sample

                A_Blank                         =      absorbance of sample + distilled water solution

The a- glucosidase inhibition (IC50) is expressed as the test substance concentration (µg/mL) that results in a 50% a-glucosidase inhibition of the sample.  The IC50 values were calculated by linear regressive excel program.   The standard deviation was also calculated by the following equation.

Standard Deviation (SD)

 =

where,                                           =    average % inhibition

x₁ + x₂ + …. + x_n                =              % a-glucosidase inhibition of test sample solution

      n        =              number of times

Determination of Cytotoxicity of Ethanol and Watery Extracts of the Peel of Rambutan by Brine Shrimp Lethality Bioassay

The ethanol and watery extracts of the peel of rambutanwere investigated by brine shrimp lethality bioassay according to the procedure described by Dockery and Tomkins, [13]. The brine shrimp (Artemia salina) was used in this study for cytotoxicity bioassay [1]. Brine shrimp cysts were purchased from a pet shop, Baho Road, Hlaing Township, Yangon Division. Brine shrimp cysts (0.5 g) were added to the 1.5 L of the artificial seawater bottle. The suspension was aerated by bubbling air into the funnel and kept for 24 hours at room temperature. After aeration had been removed, the suspension was kept for 1 hour undisturbed, whereby the remaining unhatched eggs dropped. To get a higher density of larvae, one side of the separating funnel was covered with aluminium foil and the other illuminated with a lamp, whereby the phototropic larvae were gathered at the illuminated side and could be collected by pipette. The shrimp larvae were transferred to an agar well filled with 9 ml of salt water and the dead larvae counted (number N). A solution of crude extracts (31.25 - 1000 ppm) (1 mL) was added and the plate was kept at room temperature in the dark. After 24 hours, the dead larvae were counted in each well under the microscope (number A). The still-living larvae were killed by the addition of ca. 0.5 ml methanol so that subsequently the total number of the animals could be determined (number G). The mortality rate M was calculated in %. Each test row was accompanied by a brine solution (number B). The mortality rate M was calculated using the following formula:

          M       =      percent of dead larvae after 24 hours

                A     =      number of dead larvae after 24 hours

                B     =      average number of dead larvae in the brine solution after 24 hours

                N     =      number of dead larvae before starting the test.

                G     =      total number of brine shrimps

                The control solution was prepared as the above procedure by using distilled water instead of the sample solution.

Determination of the antiproliferative activity of ethanol and watery extracts of thepeel of rambutanon Agrobacterium tumerfaceins cell

In vitroantiproliferative activity of EtOH and watery extracts of the peel of rambutan was determined against the tumor cellAgrobacteriumtumerfaceinsassay [14] at the Department of Chemistry, University of Yangon. The diluted cell solution (9 mL) was mixed with the sample solution (1 mL) and incubated in an incubator for 12 hours.  After incubation, absorbance readings of the samples were recorded at 600 nm and compared with a control that had the diluted cell culture, and 1 mL of fresh nutrient medium was used in place of the extract as the blank. Fluorouracil (5-FU) was used as the standard. The anti-tumoractivity was determined through the inhibition of Agrobacteriumtumerfaceins cell which was expressed as a percentage of inhibition and calculated by the following equations:

% Inhibition                            =      [A_control-(A_Sample - A_Blank)/ A_control] × 100

Where,  

                % Inhibition            =      % Agrobacteriumtumerfaceinscell inhibition

                A_control                       =      absorbance without sample solution

                A_sample                       =      absorbance of the sample

                A_Blank                         =      absorbance of sample + fresh nutrient broth medium

The Agrobacteriumtumerfaceinscell inhibition (IC₅₀) is expressed as the test substance concentration (µg/mL) that results in a 50% Agrobacteriumtumerfaceins cell inhibition of the sample. The IC₅₀ values were calculated by linear regressive excel program. The standard deviation was also calculated by the following equation.

Standard Deviation (SD)

 =  

where,                             =        average % inhibition

                x₁ + x₂ + …. + x_n   =  % Agrobacteriumtumerfaceins cell inhibition of test sample solution

                                    n                = number of times

Determination of the Antiproliferative Activity of Ethanol and Watery Extracts of the Peel of Rambutan by Using CCK – 8 Assays

In in vitro antiproliferative activity of the ethanol and watery extracts of the peel of rambutan was determined against three human cancer cell lines such as A 549 (lung cancer), MCF 7 (human breast cancer), and Hela (human cervix cancer) [15]. These tests were done at the Department of Natural Products Chemistry, Institute of Natural Medicine, and University of Toyama, Japan. After the cell growth, the 70-100 % cell in the medium was aspirated with an aspirator. The cell was washed with PBS (5 mL) about 2 times. The cells are trypsinased with trypsin (4 mL) and incubated for 2–3 minutes. And then the medium (1 mL) was added to stop trypsinization. The cell suspension was transferred to a 15 mL centrifuge tube. The tube (cell suspension) was centrifuged in the refrigerated centrifuge machine (3000 rpm) with the same centrifuge tube for 3 minutes. After the centrifugation, the supernatant was carefully removed without disturbing the cell pellet and the cell was found at the bottom of the centrifuge tube. The cell in the centrifuge tube was added with fresh medium (2 mL) gently to the side of the tube and slowly pipetted up and down 2 to 3 times to re-suspend the cell pellet. The number of cells was counted with Haemacytometer.The cell solution (30 µL) was mixed in the Tryphan blue (120 µL). The chamber and the covered slip were cleaned with alcohol (70 % EtOH). The chamber was dried and the overslip was fixed in position. The cell was harvested and10 µL of the cell was added to the Haemacytometer (do not overfill). And then the chamber was placed in the inverted microscope under a 10X objective and phase contrast was used to distinguish the cell. The cell was counted in the large, central gridded square (1 mm²). The gridded square was circled and multiplied by 10⁴ to estimate the number of cells per millimeter. The number of cells was counted by the following equation, No. of cell in stock = counted cell/4 × 10⁴ × dilution factor × volume of stock cell solution After the cell counting, the cell was added with 50 mL (500 µL) of medium for 6 plates.  10 mL (100 µL) medium of the cell was filled in 96 well plates.  The cell in 96 well plates was incubated in an incubator for 24 hours. After the incubation, the medium was removed by an absorption machine (very carefully) and washed with 100 µL PBS solution. And then 100 µL of the different concentrations of sample and control solution was added to the 96 well plates.  The sample solutions in 96 well plates with cells were incubated in an incubator for 72 hours. The sample solution with cell and medium was added with 100 µL CCK-8 reagent.  And then the 96 well plates were incubated in an incubator for 3 hours.  After the incubation, the absorbance of each solution was measured at 450 nm by using a UV-visible spectrophotometer. The percent cell viability activity was calculated by the following equation.  

% Cell viability = [(Abs(test sample)−Abs(blank))/(Abs(control)−Abs(blank))]× 100 

Where,   Abs_{(test sample)}           =  absorbance of test sample solution

                                Abs_(control)               =  absorbance of DMSO solution

                                Abs_(blank) =  absorbance of CCK-8 reagent

 IC₅₀ (50% inhibitory concentration) values were calculated by linear regressive excel program.  The standard deviation was also calculated by the following equation.

Standard Deviation (SD)    =             

Where,  X             =              average % inhibition

x₁, x₂,.. …., x_n            =              % cell inhibition of test sample solution

n              =      number of times

Results and Discussion

Collection and Preparation of the Sample

In the present work, the peel of rambutan was chosen as the sample to be studied. The peels were collected from Mawlamyine Township, Myanmar.  The dried powder of peel was stored in an air-tight container and used for the chemical and biological activity determination.

The Results of Preliminary Phytochemical Tests of Peel of Rambutan

To know the type of phytochemical constituents present in the sample, preliminary phytochemical tests were carried out as shown in Table 3.1. According to the experimental results, alkaloids,α-amino acids, carbohydrates, flavonoids, glycosides, organic acids, phenolic compounds, polyphenols, protein, saponins, steroids, tannins, and terpenoids were observed to be present in the peel of rambutan.  But cyanogenic glycosides, reducing sugars, and starch were not detected in this sample. Due to the presence of bioactive phytochemical constituents such as saponins, steroids, flavonoids, terpenoids, alkaloids, and glycosides present in the peel of rambutan may possess biological activities such as antidiahorreal, antimicrobial, anti-inflammatory, antioxidant, anticancer and antiviral properties.

Figure 2.1: Rambutan.

                                  Figure 3.1: Preparation of air-dried sample powder.

Table 3.1: Phytochemical Screening of the Peel of Rambutan.

Estimation of Phytochemical Constituents

The total phenol contents of ethanol and watery extracts of the peel of rambutan were determined by Folin-Ciocalteu Reagent (FCR) method.  One of the total phenolic compounds, gallic acid (3,4,5-trihydroxybenzoic acid) was used to construct a standard curve. The ethanol extract was found to have a higher TPC value (125.98±0.93mg of GAE per gram of extract) and the water extract was found to be 109.34±1.10 mg of GAE per gram of crude extract. The total flavonoid contents of the water and ethanol crude extracts of the peel of rambutan were evaluated with the spectrophotometric method using aluminium chloride reagent.  The content of phenolic was significantly different between ethanol and watery extracts. The ethanol extract was found to have 144.85±4.1mg of quercetin equivalence (QE) per gram of crude extract and the watery extract was found to be 113.33±4.3 mg of quercetin equivalence (QE) per gram of crude extracts. The total steroid contents in the ethanol and watery extracts of the peel of rambutan estimated by Zak’s method were found to be 68.58±13.72 mg and 39.54±10.91 mg of cholesterol equivalence (CE) per gram of crude extracts, because of the higher solubility of steroid in ethanol. The total tannin contents of the ethanol and watery extracts of the peel of rambutanwere estimated by Broadhurst’s method (acidified vanillin method). A high level of tannin was found in both extracts. It was found in the ethanol extract (270.92±14.2 mg of tannic acid equivalence per gram of ethanol extract).  The watery extract showed 225.08±2.9 mg of tannic acid equivalence per gram of watery extract. The concentrations of cardiac glycoside in the ethanol and watery extracts of the peel of rambutan were determined by Baljet’s method.  It was observed that the cardiac glycosides concentrations of ethanol extract (250.00±9.6 mg of digitoxin equivalence per gram of crude extract) and watery extract (26.33±5.03 mg of digitoxin equivalence per gram of crude extract). It is due to the higher solubility of cardiac glycosides in ethanol compared with water. According to these tests, the peel of rambutan is a very rich source of phytoconstituents. The resulting data are shown in Table 3.2 and Figure 3.2

Table 3.2: Estimation of Phytoconstituents of the Ethanol and Water Extracts of Peel of Rambutan.

                              Figure 3.2: Phytoconstituents of the ethanol and watery extracts of the peel of rambutan.

                   Table 3.2: Antimicrobial Activities of Ethanol and Watery Extracts of Peel of Rambutan.

Agar well –     10 mm; 10 mm – 14 mm (+); 15 mm – 19 mm (++); above 20 mm (+++); Absence (-)

Activity-          10 mm – 14 mm (low); 15 mm – 19 mm (medium); above 20 mm (strong)

Figure 3.3: Antimicrobial activities of ethanol and extracts of peel of rambutan.

Table 3.3: Radical Scavenging Activity and IC50 of Ethanol and Peel of Rambutan.

Figure 3.4: Radical scavenging activity of ethanol and water extracts ofpeel of rambutan.

Table 3.4: Radical Scavenging Activity of Standard Gallic Acid.

Figure 3.5: Radical scavenging activity of standard gallic acid.

Table 3.5: Reducing Power Activity and IC50 of Ethanol and Watery Extracts of Peel of Rambutan.