Sep 24, 2014 - of secondary metabolites from plants cranberry (Vaccinium macrocarpon). The procedure based on standard procedure. [14] . a) Alkaloids.
WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES
Resmi et al.
World Journal of Pharmacy and Pharmaceutical Sciences
SJIF Impact Factor 2.786
Volume 3, Issue 10, 194-210.
Research Article
ISSN 2278 – 4357
ANALYSIS TOTAL FLAVONOID CALCULATED AS GENISTEIN, ANTIOXIDANT ACTIVITY AND ANTI-INFLAMMATORY PROPERTIES OF CRANBERRY PLANT ETHANOL EXTRACT Resmi Mustarichie*, Moelyono Moektiwardoyo, Yoga Windu Wardhana, Danni Ramdhani Faculty of Pharmacy, Universitas Padjadjaran, Jatinangor, Indonesia 45363 ABSTRACT Article Received on 13 August 2014, Revised on 04 Septembe 2014, Accepted on 24 September 2014
Raw cranberries and cranberry fruit are abundant food sources of flavonoids such as proanthocyanidins, flavonols and quersetin. Reviews These compounds have demonstrated activity as a possible anti-cancer agents in vitro. In previous research has been done proving the interaction of test compounds quersetin computing that is nutritious
*Correspondence for
content of the stems and leaves of cranberries with an anti-
Author Dr Resmi Mustarichie
inflammatory receptor H4R. Cytotoxicity properties of cranberries for
Faculty of Pharmacy,
this plant..This study reports on the phytochemical screening, total
Universitas Padjadjaran,
flavonoids calculated as quercetin, antioxidant activity as well as anti-
Jatinangor, Indonesia 45363
inflammatory of ethanol extract of cranberry. Antioxidant activity was carried out by DPPH method. Testing antiinflammatory activity
performed by the method of induction of carrageenan and total flavonoids calculated as quercetin done by spectrophotometry. It was found that the herbs that we checked positive compounds containing alkaloids, flavonoids, tannins and polifenolat, triterpenoids and steroids, as well as monoterpenoid and sesquiterpenoid quinones, and negative for saponins. From the results of this experiment, the percentage content of total flavonoids in cranberry herbal at 0.099% w/w. IC50 of antioxidant activity of cranberry ethanol extract was found lower than its vitamin C as control positive. The antioxidant activy might be due to flavonoid content.The test results indicated that the anti-inflammatory activity of extracts of cranberry plants had inflammation inhibition percentage of 36.47%, 40.07% and 51.27%. It was found that extracts of a dose of 1000 mg/kg was the most good in the inhibition of inflammation. The results of this study support previous research that the cranberry extract can be used as an adjuvant therapy in the treatment of inflammation caused by cancer. www.wjpps.com
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Keywords: Cranberry, anti-inflammatory activity, quercetin, antioxidant, cancer. INTRODUCTION Mantovani et al.
[1]
describes the relationship between inflammation and cancer where the
cancer followed by acute inflammation. Inflammation can be accompanied by symptoms of fever, redness, swelling, tenderness/pain, impaired function. Inflammatory process include microvascular damage, increased vascular permeability and leukocyte migration into inflamed tissue, with symptoms of fever, redness, swelling, tenderness/ pain, impaired function. Mediators released include histamine, bradykinin, leukotrienes, prostaglandins and PAF. Inflammation is a process fisiopatologi common in some human diseases. Principal signs of acute inflammation include swelling/edema, redness, heat, pain, and change in function. The things that happen in the process of acute inflammation largely made possible by the release of a wide variety of chemical mediators, including vasoactive amines, plasma proteases, arachidonic acid metabolites and leukocyte products. Cranberry fruit and cranberry juice are abundant food sources of flavonoids such as proanthocyanidins, flavonols [2, 3, 4] and quersetin [5, 6]. This compound has shown activity as a possible anti-cancer agents in vitro
[7, 8, 9, 10]
. In a previous study
[11]
we have verified
computing interactions of quercetin which is nutritious content of the stems and leaves of cranberries with an anti-inflammatory receptor H4R. Antioxidant properties and cytotoxicity of methanol extracts of this plant have also been reported [12, 13]. This paper reports on the invivo anti-inflammatory test from ethanol extracts of cranberry plants, phytochemical screening and total flavonoid content calculated as genistein. METHODS Chemical methods Extraction Extraction of cranberry plants in this study was performed using Soxhlet method using 96% ethanol. Soxhletation was carried out till a clear droplets. Liquid extract was then evaporated with a rotary evaporator at low pressure-temperature 50oC and then poured into the vaporizer cup evaporated on a water bath for so obtained extract thick with a constant weight. Furthermore determined percent yield using the formula: % yield = (extract mass/simplisia mass) x 100 %
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Phytochemical screening: Phytochemical screening was conducted to determine the content of secondary metabolites from plants cranberry (Vaccinium macrocarpon). The procedure based on standard procedure [14]. a) Alkaloids Materials basified with ammonia, and then added a mixture of chloroform and shaken. Pipetted liquid chloroform, divided into three sections and placed on a drip plate to evaporate the chloroform was then added a solution of hydrochloric acid 2 N. In each plate drops added to a solution of Dragendorff, Mayer, and Bouchardad. The presence of alkaloids with the formation of precipitate or orange brown turbidity. b) Flavonoids Material dissolved in a little water, then transferred to a test tube containing powdered magnesium and hydrochloric acid solution 2 N. The whole mixture was heated for five minutes. After the hot-filtered, the filtrate was allowed to cool, the filtrate was added amyl alcohol and then shaken vigorously. The presence of color in the amyl alcohol layer showed positive for flavonoids. c) Tannins and Polyphenols Dissolved material in a test tube with water and boiled for a few minutes. Once filtered, the filtrate was dripped into the gelatin solution, and then observed the occurrence of precipitation or clotting. The presence of clumping showed that the filtrate contained tannin class of compounds, the precipitate was filtered gelatin. The filtrate spilled ferric chloride reagent solution. If the black color meaned formed in the crude drug class of compounds contained tannins and polyphenols. d) Saponins The extract was heated for 15 minutes in a water bath and then filtered. The filtrate was placed in a test tube and then shaken vigorously for 20 minutes. Formation of foam at least one centimeter consistent for five minutes and did not disappear with the addition of one drop of dilute hydrochloric acid showed that the extract contained saponins. e) Triterpenoids and Steroids Crushed material with ether and then ether phase in the pipette by filtration through cotton and evaporated in the vaporizer cup to dry. Against residue dripped Lieberman-Bourchardad
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reagent solution. If the green color was formed by means of blue material tested contained steroids. f) Monoterpenoids and Sesquiterpenoids Material dissolved with ether, then filtered with a pipette. The filtrate was placed in the vaporizer cup, then allowed to evaporate to dryness. Results of drying the filtrate was added a solution of 10% vanillin in concentrated sulfuric acid. The emergence of the colors indicated the presence of monoterpenoid compounds and seskuiterpenoid. g) Quinone The extract was heated for 15 minutes over a water bath and then filtered. The filtrate was sprinkled with a solution of potassium hydroxide. Yellow to pink color indicated the presence of quinone compounds. Analysis of Total Flavonoids Ethanolic extract was aanalyzed using modification of Chang et al.
[15]
method.
Quantification of total flavonoids was done using standard addition method with the help of shifting-AlCl3 reagent. Total flavonoids calculated as quercetin equivalent (QE) w/w sample. Phamacological methods Antioxidant activity assay: Antioxidant activity test was carried out by DPPH method [16,17]
in which Vitamin C was used as
positive control. DPPH Operating time was
determined, and IC50 was calculated. Test solution and the positive control were incubated with several concentrations in waterbath at 37ºC for 30 minutes, absorbance was measured at a wavelength of 517 nm using a maximum in the ultraviolet-visible light spectrophotometer. The percentage of inhibition was calculated by the following formula: % Inhibitor = (Ablank – Asample) × 100% Ablank Anti-inflammatory Activity Tests carried out by the method of induction of anti-inflammatory carrageenan
[18]
. The
extract was tested for 5 Wistar rats of the test group. Determination of the number of mice used was determined by using the Federer formula [19].
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with: t = number of groups n = number of rats Because used 5 test groups, then the calculation was as follows: (n-1) (5-1) ≥ 15 4 (n-1) ≥ 15 4n ≥ 19 n ≥ 4.75 rounded to 5 mice. Testing antiinflammatory Ethanol extract of cranberry plants inflammatory activity was tested using the method of induction of edema in the feet of mice. There were three variations of the doses tested, ie 250 mg/kg (low dose), 500 mg/kg body weight (medium dose), and 1000 mg/kg (high dose). Edema induced by carrageenan performed after administration of a cranberry plant extract to test animals. The amount of edema formed was measured by means of pletismometer. Activity of ethanol extract of cranberry crop production indicated by the percent given to the formation of edema was calculated by the formula:
% inhibition =
× 100%
with Vt = average volume feet rats Vo = volume of foot carrageenan induced rat before.
after
carrageenan-induced
The inflammation inhibition percentage was calculated showing the anti-inflammatory activity of the test material. % Anti-inflammatory = % inhibition control - % inhibition test x 100 % % inhibition test The volume of foot edema in rats was measured every hour for five hours using pletismometer. The testing procedure was carried out as follows:
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There were five groups of mice positive control group, negative control, the group a dose of 250 mg/kg (low dose), the dose group of 500 mg/kg body weight (medium dose), and group dose of 1000 mg/kg (high dose). Body weight of each rat was weighed and measured leg volume (expressed as the initial volume, Vo). Each test group was given a different treatment. positive control group was given acetosal suspension 10 mg/kg BW. negative control group was given a suspension PGA 2%. The dose of 250 mg/kg (low dose) were given ethanol extract of cranberry plants as much as 250 mg /kg rat. The dose of 500 mg/kg body weight (medium dose) were given ethanol plant cranberry extract 500 mg/kg rat. The dose of 1000 mg/kg (high dose) were given ethanol extract of cranberry plants
as
much as 1000 mg / kg rat. One hour later the animals edema induced by carrageenan test were injected subcutaneously in the soles of the feet. Volume 4 feet measured one hour after administration of oral dosage. The soles of the feet of mice dipped in pletismometer tool. This procedure was done every hour for five hours. The difference in foot volume was recorded each time the rat was measured. Volume measurement data were calculated percent rat foot edema and inflammation inhibitory percentage results were then compared between the positive control group, negative control group, and the dose groups. Data processing with statistical calculations were also performed to prove the results of scientific research. RESULTS AND DISCUSSION Extraction Results Plants obtained fresh cranberries, wet sorted first stage which was necessary because the raw materials should be true and pure botanicals, meaning that was derived from plant raw material crude drug in question, instead of other crops. For that, one needs to separate and disposal of foreign organic matter or plant or other plant parts were shipped. Raw maerilas must also be clean, meaning that should not be mixed with soil, gravel, or other impurities, for example, insects or any portion thereof.
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Washing was done by using tap water, then drained after washing so that excess water flows up first. The plant material that had been washed further dried in the open air under the sun, the goal was to stop the enzymatic reaction or hydrolysis that occurs in cells or tissues of plants, and to evaporate the water contained in the plant tissue. Once dried, the plants then smoothed by using a blender, with the aim to increase the surface area of touch with current solvent extraction process, in order to obtain results which extracts more. Once the sorting was done dry, to separate impurities, foreign organic matter, and crude drugs damaged by the previous process. The process of extraction was conducted using soxhlet with 96% ethanol. 96% ethanol solvent chosen because it can dissolve almost all secondary metabolites contained in crude drugs. From the extraction of as much as 145 grams of plant material, extracts obtained thick weighing 22.08 grams. So we got a value of 15.227% yield. Phytochemical screening Results of phytochemical screening of ethanol extract of cranberry plants, can be seen in Table 1. From this table, it can be seen that testing positive for alkaloid compounds, flavonoids, tannins and polifenolat, triterpenoids and steroids, as well as monoterpenoid and sesquiterpenoid quinones, and negative for saponins. Table 1. Results of phytochemical screening of ethanol extract of cranberry plants Compounds Alkaloids Flavonoids
Results Chocolate precipitate Yellow colour of amyl alcohol
Tannins & Polyphenols
Black colour
Saponins
No foam
Triterpenoids & Steroids Monoterpenoids & sesquiterpenoid Quinone
Notes + + + -
Green colour
+
Colour formed
+
Brownish yellow
+
Total flavonoids calculated as quercetin Analysis of the levels of total flavonoids in cranberry plants performed using ultravioletvisible spectrophotometer instrument. Flavonoids itself is a compound that has a basic structure that is typical, the two aromatic rings form a chromophore which is called ring A and ring B. When the wave ultraviolet or passed on flavonoids appear, there will be a
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transition of an electron from the π to π * on ring A and ring B. This is why flavonoids in general may issue a peak at a specific wavelength. Generally be detected two maxima for the flavonoid, which is in the range 240-285 nm band called II and in the 300-400 nm region is referred to as band I
[20]
. The wavelength range will be different for each type of flavonoids
depends on auxochromes attached to the chromophore group in the flavonoid structure. Auxochrome attached to the chromophore will give bathochromic shift causing different wavelengths. Quantitative analysis for total flavonoids done by standard addition method with the help of sliding AlCl3 reagent. Because the sample to be analyzed in the form of extracts, the content of the compound in the sample not only flavonoids alone. Therefore, the standard addition method was chosen to equalize the conditions of the sample solution and matrix standard solution, so that the calculation was obtained more accurate levels. The use of sliding AlCl3 reagent aimeds to confirm the flavonoids in the sample. Flavonoids form a complex between hydroxyl acid resistant and neighboring ketones and acids form complexes with the group not stand orthodihidroksil. This causes changes in the structure of flavonoids auxochrome group which in turn affects the resulting wavelength shift. Estimation of total flavonoid content in this study was calculated as quercetin, because standard used is quercetin. Sebenrnya determination of total flavonoids quercetin as standard using current could be due to flavonoids including quercetin and flavonol class of flavonoids identifier is a compound that has been commonly used to analyze flavonoids [15, 21]. Absorbance values were used to create a standard addition curve was the value of absorbance at the maximum wavelength, ie 365 nm. At the maximum wavelength, the maximum sensitivity was also due to the wavelength of the maximum absorbance change for each unit of concentration is the greatest. In addition, the wavelength of maximum absorbance curve flat shape so on the condition of the Lambert-Beer law be fulfilled [22]. After making measurements using instruments, different absorbance values obtained at the same wavelength of each sample were measured. Absorbance values were obtained based on raw concentration added to the sample can be seen in Table 2.
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Table 2 Absorbance obtained based on standard addition added Amoun of sample in solution (mL) 5 5 5 5 5
Amoun of standard in solution (mL) 0.0 1.0 1.5 2.0 2.5
Concentration o f standard in solution (ppm) 0.0 2.5 3.75 5.0 6,25
Absorbance average at 375 nm 0,0010 0,2139 0,3462 0,4215 0,5400
From the absorbance values obtained in accordance with standard concentrations were added to the analyte, the standard addition curve can be made to see the effect of concentration on the absorbance of the resulting sample. Standard addition curve obtained had line equation y = 0.0821x - 0.0128 with R2 = 0.998, which indicated that the concentration could explain the diversity of the absorbance at 99.8%, and only about 0.2% was explained by other factors. Based on the standard curve adducts that have been created, it was able to determine the total flavonoid content calculated as quercetin. Moment where it crosses the x axis with a value of y = 0 can be considered as the maximum concentration of flavonoids in the sample solution. When performing measurements with a calibration curve method, when there was no value of absorbance at the maximum wavelength of raw concentration values are no or zero. Meanwhile, if using a calibration curve method, when there was no value of absorbance at the maximum wavelength at that moment there was a difference between the raw value of the analyte concentration in the sample solution. From the results of this experiment, the percentage content of total flavonoids in cranberry herbal at 0.099% w/w. Antioxidant activity The time chosen for the DPPH reaction time was 30 minutes. Absorbances was measured at λ max. 517 nm (see Fig. 1)
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Fig.1 λ max of DPPH DPPH (1,1-diphenyl-2-pycrilhydrazil) is a stable free radical which when reacts with a substance that can donate hydrogen (antioxidants) would be reduced to 1,1-diphenyl-2pycilhydrazin. Based on the reaction between DPPH and antioxidants derived from the extract cranberry, can be obtained IC50 value which is the concentration that can provide a certain 50% inhibitory effect of free radical. Inhibition against DPPH free radicals was measured by spectrophotometry of visible light by the color change. DPPH (1,1-diphenyl-2-pikrilhidrazil) when dissolved with methanol will be colored purple, and after reacting with cranberry extract that act as antioxidants, will be transformed into 1,1-diphenyl-2-pycrilhydrazin yellow. IC50 (Inhibition Concentration 50) is the antioxidant concentration (mg/mL) were able to reduce free radicals by 50% compared to the control via a linear line equation. IC50 values obtained from the intersection line between the power constraints and concentration axis, then put in the equation y = a + bx, with y = 50 and x indicate IC50 values. Extracts confirmed active as an antioxidant when IC50 values less than 200 mg / mL [23]. The test results of water-phase antioxidant activity can be seen in Table 3. Antioxidant effect in shown by ethanol extract cranberry might be caused by the presence of total flavonoids contained in the ethanol extract of cranberry such as quercetin. The possibility of these antioxidant properties due to the presence of phenol compounds have been also reported [11].
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Table 3 Test results of antioxidant activity in the calculation of the IC 50 against cranberry ethanol extract and vitamin C. C (µg/mL) (x)
Sample
Ab
As
%Inhibition (y)
Cranberry
5
0,732
15,0766
extracts
10
0,673
21,0836
0,644 0,551
24,3816 35,3357
0,424 0,836 0,644 0,423 0,2165 0,051
50,2945 14,6366 34,3910 57,1136 77,9939 94,9846
25 50 100 2 4 6 8 10
Vitamin C
0,855
0,979
IC50 (µg/mL)
96,62
5,45
Notes: C
= Conc. test solution
Ab
= Absorbance blank (control negative) As = Absorbance test solution
Anti-inflammatory Activity Testing the anti-inflammatory activity in this study was conducted using the carrageenan induced rat foot. Anti-inflammatory activity can be seen from the test material's ability to inhibit the swelling of the feet of mice. Carrageenan edema chosen as the inducer compound because it can cause swelling without causing tissue damage, not cause scars, and can lead to a response that is sensitive to anti-inflammatory compounds. Edema produced by carrageenan, can survive in a time of 6 hours and then gradually decreased and eventually disappeared within 24 hours. Swelling that occurs in the legs of mice caused by induction of carrageenan was measured using a pletismometer. This tool works by Archimedes law, which is an object that is partially or completely submerged in a liquid will experience upward force in proportion to the weight or volume of liquid displaced. The results of the calculations can be seen in the bar chart below.
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dose1 250 mg/kg bw
dose2 500 mg/kg bw
dose 3 1000 mg/kg bw
control (+) acetosal 10 mg/kg bw
Figure 2. Rod diagram of average percentage inhibition of inflammation of each group From Figure 2 it could be concluded that the positive control (acetosal 10 mg/kg BW) inhibition had the best ability that was equal to 59.33% followed by ethanol extract of cranberry plants test dose 3 (1000 mg/kg BW) of 47.16%; test dose 2 (500 mg/kg BW) of 38.07%, and the next test dose 1 (250 mg/kg BW) of 26.58%. The data then analyzed statistically. Data analysis was one statistical method to determine the effect of variables contained in the study [24]. Requirements of data analysis consisted of a normal distribution of data, the same data variance, and the data derived from independent samples. Below is a Table 4 of test results to determine the normality of data distribution. Table 4. Results of Testing Normality using the PASW Statistics 18 software
Inhibition
Treatmen t + D1 D2 D3
Kolmogorov-Smirnova Statistic df Sig. ,177 5 ,200* ,135 5 ,200* ,270 5 ,200* ,177 5 ,200*
Shapiro-Wilk Statistic df Sig. ,937 5 ,643 ,995 5 ,995 ,913 5 ,488 ,937 5 ,643
Normality test is divided into two types, namely the Kolmogorov-Smirnov and Shapiro-Wilk. Its use depends on the number of samples used in the study. Kolmogorov-Smirnov test is used if the sample is more than 50 and the Shapiro-Wilk used if the sample amounted to less than 50 Because this study used a sample of less than 50, the significance value of the Shapiro-Wilk seen. From the results of this test for normality can be seen that the data are
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normally distributed. This is indicated by the significant value of more than 0.05. Furthermore, homogeneity testing, which aims to find common variance in the data. Here's a Table 5 of the results of homogeneity testing. Table 5. Results of Testing Homogeneity of Variance Data using the PASW Statistics 18 software Levene Statistik 3,776
df1 4
df2 20
Sig. ,019
In the homogeneity test, the data is said to have the same variance if the significance value of data is more than 0.05. From the table above it can be seen that the significance value is 0.019 (
