INTRODUCTION:

Medicinal herbs have long been used by archipelagic indigenes, including the people of modern-day Indonesia, for disease prevention, as well as treatment in improving health.

The biodiversity of Indonesia is rich in natural, as well as traditional medicinal ingredients, and has been exploited from generation to generation¹. In the last decade, medicines produced from natural ingredients have received increasing attention in both developing and developed countries. According to the World Health Organization (WHO), about 65% of the population of developed countries have resorted to the use of traditional medicines².

Efforts are currently being accomplished to develop traditional medicine which will be compatible with modern medicine. Various R&D projects that exploit technological advances are also being conducted to improve both the quality and safety of products, while further increasing confidence in the benefits of traditional medicines. These projects are sanctioned by the Indonesian Ministry of Health Regulation Number 760/MENKES/PER/lX/1992 concerning fitofarmaka. The term “fitofarmaka” refers to traditional medicines which have passed pre-clinical and clinical trials, as well as toxicity tests, proving the drugs are safe and effective. This means simplicia quality control is required for medicinal raw materials or galenic preparations³.

A common method of controlling the quality of simplicia is standardization, a process where a uniform raw material is obtained and analyzed, to ultimately guarantee the pharmacological effect of plants³. This process is also a prerequisite for ensuring the safety, toxicity, and stability of medicinal raw materials. Previous studies have shown the success of drugs derived from plants indigenous to Indonesia, the world’s most bio-diverse country, has brought about a return to the exploitation of natural materials. This development confirms the important role of plants as a source of medicines, and consequently, a potential source of income.

Safflower (Carthamus tinctorius Linn), a member of the Asteraceae family, is a medicinal plant administered for measles treatment, by the people of South Sulawesi by brewing⁴. The plant is also commonly used to reduce high cholesterol levels, thromboangitis, hypertension, cancer, menstrual pain, and postpartum abdominal pain, as well as to treat sarampa and chickenpox⁵. Previous studies by Utami et al., (2016) and Gratitude et al., (2012) reported safflowers to tend to increase the activity of immunoglobulins, including IgG, IgA, and IgM. Immunoglobulins are serum molecules capable of neutralizing a range of pathogenic microorganisms^6,7.

Hamsidi et al., (2015) studied the ethanol extract of Safflower and confirmed the extract’s ability to inhibit the growth of Plasmodium falciparum 3D7⁸. Several other studies have also reported numerous pharmacological activities exhibited by this extract. This study, therefore, aims to characterize the ethanol extract of Safflower by investigating its specific and non-specific parameters.

The onset of degenerative and chronic diseases, including diabetes, cancer, liver damage, arthritis, inflammation, and neurological disorders have been attributed to oxidative stress. Despite the equipment of cells with small antioxidant molecules and significant enzymes stock, studies showed the low potential for these agents to adequately normalize redox conditions. This includes situations of oxidative stress, which result from adverse pathological, environmental, or physicochemical circumstances, where free radical scavenging is hindered, and the generation is improved⁹.

Antioxidants are known to significantly prevent substrate oxidation instigated by the presence of free radicals, both in large and small concentrations. Free radical molecules are highly reactive molecules due to the presence of unpaired electrons in their outer orbitals, which tend to react with body cell molecules by binding to the cell's molecular electrons. The continuous production of free radicals during normal metabolic processes is considered to be the cause of damage to the function of body cells, ultimately, triggering the onset of degenerative diseases^9,10.

Previous studies identified liver damage as a major side effect of synthetic antioxidants, including BHT (butylated hydroxytoluene) and BHA (butylated hydroxy aniline). Nature-based counterparts with relatively safe and effective characteristics were also evaluated. These include beta carotene, vitamin C, flavonoids and others, which endorses further exploration of natural antioxidant sources¹¹.

MATERIAL AND METHODS:

Materials:

Safflower (C. tinctorius L.) flowers were collected from plantations in Amali Sub-district, Bone District, South Sulawesi Province, and identified at the Materia Medica Garden Center Batu. The extract was produced by maceration for 72 hours, with filtering carried out every 24 hours. Subsequently, the filtrate was collected and evaporated, using a rotary vacuum evaporator at a temperature of 50°C. The extract obtained was then standardized using specific and non-specific parameters¹².

Etanol 70% (Merck®), kloroform (Merck®), aquadest, etanol 95% (Merck®), methanol (Merck®), N-heksan (Merck®), etil asetat(Merck®), petroleum eter (Merck®), H2SO4 2M (Merck®), mayer reagent (Merck®), Dragendorf reagent, Mg powder, HCl concentrated (Merck®), FeCl₃ 1% (Merck®), NaOH 1N (Merck®), eter, Lieberman-Buchard reagent, HCl 4N (Merck®), Na asetat 1M (Merck®), H2SO4 (Merck®), HNO₃ concentrated (Merck®), Potato Dextrose Agar (Merck®), Buffered Peptone Agar (Merck®), Plate Count Agar (Merck®), HCl 4N (Merck®).

Equipment:

The identification specific parameters, non-specific parameters was using porcelain cup, petri dish, filter paper, pipette, oven, furnace, crucible, piknometer (Pyrex^®), incubator (Memmert^®), KLT plate (Merck®), desicator, Atomic Absorption Spectrophotometer (Zhimadzu AA-7000), laminar air flow (Esco), fume hood (Esco), Micropipet (Thermo), coloni counter (BZG-30), rotary vacuum evaporator (Rotavapor, Buchi^®), blender (philips), analytical balance (Precisa^®), measuring flask (pyrex^®), electromantel (Stuart^®), erlenmeyer (Pyrex^®), funnel (Pyrex^®), test tube (Pyrex^®), beaker (Pyrex^®), volumetric flask (Pyrex^®).

Method:

Specific Parameters:

Extract Identity:

Nomenclature description, plant’s other names, including the Indonesian name, as well as the plant parts used^10,11,12.

Organoleptic Evaluation of Extracts:

The organoleptic properties evaluated include the extract’s color, smell, and taste^13,14.

Determination of the Content of Dissolved Compounds in Certain Solvents:

The content of water-soluble compounds:

A total of 1 g of the extract was immersed in 20mL of a water-chloroform P (1:1) mixture for 24hours, then filtered. Furthermore, 20mL of the filtrate were evaporated till dry, using an evaporating cup, then heated at 105ºC, until a fixed weight is obtained. The percentage content of water-soluble compounds was then calculated using the formula below^13,14,15.

A₁- A₀

% Water-soluble compound = ------------------ X 100

Explanation:

A₁= Cup weight + residue after heating (g)

A₀ = Empty cup weight (g)

B = Initial sample weight (g)

The content of ethanol-soluble compounds:

A total of 1g of the extract was immersed in 20mL of 80% ethanol for 24 hours and filtered. Subsequently, 20 mL of the filtrate was evaporated till dry, using an evaporating cup, then heated at 105ºC, until a fixed weight was obtained. The percentage content of ethanol-soluble compounds was then calculated using the formula below^13,14,15.

A₁- A₀

% Ethanol-soluble compound = ------------------ X 100

Explanation:

A₁ = Cup weight + residue after heating (g)

A₀ = Empty cup weight (g)

B = Initial sample weight (g)

Evaluation of the Extract’s Chemical Properties:

Phytochemical Screening:

For this screening, the extract was first dissolved in 70% ethanol and the following evaluations were performed.

a. Alkaloid identification was performed by adding 5 mL of HCL 2 N to the extract and heating the mixture in a water bath. Furthermore, the mixture was filtered, and a few drops of Mayer’s reagent are added to the filtrate. The sample was then observed for the formation of cloudy precipitation or sediments¹⁶.

b. Flavonoid identification was accomplished by pouring 0.5g of magnesium powder and 3 drops of concentrated HCL into the extract. The formation of orange to red coloration indicates the presence of flavones, while red to deep red coloration indicates the presence of flavanols, and deep red to purplish-red coloration shows the existence of flavanones¹⁶.

c. Saponin identification was achieved by adding 20 mL of aquabidest to the extract, shaking the mixture, and leaving it to stand for 15 to 20 minutes. The presence of foam indicates a negative result, while the manifestation of foam above 1 cm, 1.2 cm, and 2cm, indicates a weak positive, positive, and strong positive result, respectively¹⁶.

d. Triterpenoid identification was completed by adding 1mL of chloroform and 1 mL of acetic anhydride to the extract and leaving the mixture to cool before adding H₂SO₄is added. The reddish coloration then signifies the presence of triterpenoids¹⁷.

e. Tannin identification was carried out by dissolving 0.5g of the extract in water and heating the mixture for 5 minutes. The mixture was then filtered and 1% FeCl₃ was combined to the filtrate and the bluish-green coloration exhibits the presence of tannins^13,17.

f. Steroid identification was excecuted by adding 2mL of chloroform to the extract and dropping 2mL of concentrated H₂SO₄ slowly into the mixture from the side of the test tube wall. The formation of a red-colored ring exhibits the presence of steroids¹⁷.

g. Quinone identification was conducted by dissolving 0.5g of the extract in water and heating the mixture for 5 minutes. Subsequently, the mixture was filtered and 1N NaOH was included to the filtrate and the red coloration is an indication of quinones presence¹⁸.

Chromatogram Pattern:

For this evaluation, 1mg of extract was dissolved in 1 mL of ethanol to obtain a test solution. Subsequently, the test solution was dropped on a TLC plate and eluted using about 2mL of mobile phase comprising n-hexane and ethyl acetate in a 6:4 ratio, then the Rf-value was calculated¹⁹.

Non-Specific Parameters:

Drying Shrinkage:

1mg of extract was placed in a cup and heated at 105ºC for 30 minutes, weighed, and flattened by shaking until a layer about 5mm to 10mm thick was formed. Subsequently, the extract was dried to a fixed weight at the determination temperature. The cup lid was then opened for a while then closed back and the cup was closed then cooled to room temperature, using a desiccator, and the fixed weight was recorded^13,14,15.

A₁- A₀

% Drying shrinkage = ------------------ X 100

Explanation:

A = Sample weight before heating (g)

B = Sample weight after heating (g)

Relative density:

The relative density of the extract was measured using a pycnometer, which was weighed first, then filled with aquadest and set at 25°C, then weighed again. Subsequently, the aquadest was removed and the pycnometer was dried, then filled with the 5% liquid extract, set at 25°C, and weighed^13,14,15. The relative density was then calculated using the formula below.

W₂- W_o

Relative density = ------------------

W₁-W_o

Where:

W_o = Empty pycnometer weight (g)

W₁ = Pycnometer weight + aquadest (g)

W₂ = Pycnometer weight + liquid extract (g)

Water Content (Gravimetric Method):

For this evaluation, 1 gram of extract was poured into a pre-weighed container. Subsequently, the extract was oven-dried at 105ºC for 5 hours, then weighed, and the percentage water content was determined using the formula below^13,14,15.

A- B

% Water content = --------------- X 100

Explanation:

A = Sample weight before heating (g)

B = Sample weight after heating (g)

Total Ash Content:

1 gram of extract was poured into a crucible and the temperature was gradually increased to 600 ± 25ºC. The extract was annealed until carbon-free, then cooled in a desiccator, and the ash was weighed. Subsequently, the percentage ash content was measured using the formula below^13,14,15.

A₁- A₀

% Total ash content = ------------------ X 100

Explanation:

A₁ = Crucible weight + extract after annealing (g)

A₀= Empty crucible weight (g)

B = Initial sample weight (g)

Acid-Insoluble Ash Content:

The ash content determination involved boiling the sample in 25mL dilute sulfuric acid for 5 minutes. The insoluble part of the medium was then collected, and ash-free filter paper was used to filter the ash, before subjecting the residue to a hot water rinse. Subsequently, the filter paper containing the ash was placed in a silicate crucible and annealed in a furnace, with the temperature gradually increased to 600±25°C. The percentage acid-insoluble ash content was then performed using the formula below^13,14,15.

A₁– (C x 0.0076) – A₀

% Insoluble ash = ------------------------- X 100

Where:

A₁ = Crucible weight + extract after annealing (g)

A₀ = Empty crucible weight (g)

B = Initial sample weight (g)

C = Ash-free filter paper weight (g)

0,0076 = Ash-free filter paper if becoming ash

Microbial Contamination:

About 135mL of BPW (Buffered Peptone Water) media was prepared and mixed with 15g of the extract, to obtain a homogenous mixture. Subsequently, the mixture was left to stand until two layers were formed, and 1mL of each layer was pipetted into a petri dish, then 10-15ml of PCA (Plate Count Agar) media thawed at 45^oC was poured into each petri dish. The Petri dishes were then incubated at 35^oC for 48 hours and the colony growth was recorded^13,20.

Mold/ Yeast Contamination:

About 135mL of Peptone media was prepared and mixed with 15g of the extract, to obtain a homogenous mixture. Subsequently, the mixture was left to stand until two layers were formed, and 1mL of each layer was pipetted into a sterile petri dish and 10-15mL of PDA media thawed at 45^oC was poured into each petri dish. The Petri dishes were then incubated at 25^oC for 5 days and the colony growth of mold/yeast was recorded²⁰.

Antioxidant assay:

This was performed using the DPPH (1,1-diphenyl-2-picrylhydrazyl) method, which measures a sample’s absorbing power against DPPH free radicals. DPPH reacts with hydrogen atoms from free radical scavenging compounds to form more stable or non-radical compounds²¹.

The antioxidant assay was performed using the Burits and Bucar method. For this assay, 1 ml of ethanol extract of C. tinctorius L. flower (1, 2, 3, 4, and 5ppm concentration) was mixed with 1 ml of 0.4 mM DPPH solution. Subsequently, the mixtures were incubated in the dark for 30 minutes, and the absorbance was measured at 528nm, using a UV-visible spectrophotometer. Each measurement was carried out in triplicate and vitamin C was used as a comparison²¹, and the percentage IC₅₀ value was calculated using the formula below.

A _Blanco - A_Extract

% IC₅₀ = ----------------------- x 100

A _Blanco

Explanation:

A Blanco = Absorbance does not contain samples

AExtract = Extract Absorbance

RESULT AND DISCUSSION:

Simplicia powder obtained is as much as 740.5g. Then extraction is carried out using the maceration method. After that, the result of maceration is collected and concentrated using a rotary evaporator to separate the ethanol solvent. Then 153.42g of the viscous extract is obtained, so the extract yield value is 20.718%.

In the development of herbal medicine, it is very important to standardize the raw materials utilized. Standardization in pharmacy constitutes several forms, procedures and parameters of measurement. Consequently, the results usually include elements connected with the pharmaceutical paradigm as well as quality, particularly in the aspect of attaining certain biological and chemical standards. These include the guarantee of meeting the limits in terms of stability, in pharmaceutical products, generally²². Therefore, to ensure that the extract raw material remains stable during the extraction process, the temperature used in this process must not be too high in order to avoid damaging the components in the sample that are not heat resistant²³. The extract obtained must pass the standardization stage to ensure the quality of the drug’s raw materials is stable for every fixed quality requirement²⁴.

Determination of specific and non-specific standard parameters of extracts involves macroscopic and microscopic examination, determination of ash content, determination of drying losses, determination of water content, determination of ash content that is insoluble in acid, and determination of microbial contamination levels. The organoleptic observation of the extract was intended as an objective and simple initial introduction using the five senses by describing shape, color, smell, and taste. Determination of water-soluble extracts and ethanol has no impact related to their pharmacological effect but, rather, as an estimate for polar (water-soluble) compounds and semi-polar-nonpolar (ethanol soluble) active compounds^1,25.

Specific Parameters:

Characterized by specific parameters that includes identity, organoleptic, and content of compounds which dissolve in certain solvents can be seen in table 1.

Table 1: The characterization of specific parameters of extract C. tinctorius L.

Parameters

Characteristics

Identity

Scientific name for plants

Used parts

Indonesian Name Plants

Carthamus tinctorius L.

Flos

Kasumba Turate

Organoleptic

Form

Color

Smell

Taste

Thick

Deep red

Distinctive

Astringent

The content of soluble compounds

Water

Ethanol

0,12 %

0,22 %

The parameter of extract identity needs to be carried out to provide objective identity from the name and the specific identify of the compound. The content of ethanol-soluble compounds is greater than the content of water-soluble compounds. It happens because the solvent used in the extraction process is organic, that is ethanol, so that the absorbed organic compounds are greater than the absorbed inorganic compounds.

Phytochemical Screening:

Phytochemical including non-chromatographic assay that characterized mono and polyherbal of the product. The result form qualitative analysis can showed by phytochemicals²⁶. Phytochemicals are one of the major sources of antioxidants, which are potentially capable to scavenge free radicals to decrease the risk of many disease such as antimicrobial, antiviral, malarial, anti-tumor, cancer, and cardiovascular disorders^27,28. The phytochemical screening of the extract is carried out by testing the groups of alkaloid, flavonoid, saponin, triterpenoid, tannin, quinone, and steroid compounds. The results of phytochemical screening can be seen in table 2.

Table 2: Phytochemical screening

Compound grup

Results

Alkaloids

Flavonoids

Saponins

Triterpenoids

Tannins

Quinones

Steroids

Explanation : - (Negative) / + (Positive)

The identification of the chemical compound groups contained in the ethanol extract of safflower (C. tinctorius L.) is carried out by using a chemical reaction (color and sediment). Based on the results of the identification of chemical groups of the extract, they indicate that the extract contains flavonoids, saponins, triterpenoids, tannins, quinones, and steroids.

Chromatogram Pattern:

The determination of the chromatogram pattern using thin layer chromatography (TLC) is carried out by using a mobile phase ratio of n-hexane: ethyl acetate with a ratio of 6:4 and using UV 364. The chromatogram pattern can be seen in Figure 1.

Figure 1: TLC profile of extract with mobile phase ratio of n-hexane: ethyl acetate 6: 4

Thecalculation of Rf₁produces a value of 0,1, whereas the calculation of Rf₂ produces a value of 0.7. The value of Rf is very characteristic for certain compounds in certain eluents. It can be used to identify differences in compounds in the sample. The purpose of phytochemical screening is to evaluate the secondary metabolites present and also to generate a qualitative description. Furthermore, compounds with higher Rf tend to demonstrate lower polarity, and otherwise because the stationary phase is polar. This involves the firm hold of more polar compounds to the stationary phase and consequently results in lower values. The TLC Rf is considered to be good between the range of 0.2 to 0.8, and higher values indicate a need to reduce the eluent polarity²⁰.

Non-Specific Parameters:

Non-specific parameter testing was carried out by testing drying shrinkage, relative density, water content, total ash content, and acid-insoluble ash content. The results of testing can be seen in table 3.

Table 3: Non-specific parameter test

Parameters

Results

Terms

Drying shrinkage

0.96%

Relative density

Water content

Ash content

Acid-insoluble ash content

1.02%

0.91%

0.23%

0.32%

5-30% (Voigt, 1994)

< 10% (MMI, 1980)

< 4,0% (MMI, 1980)

The determination of drying shrinkage in the extract is a requirement that must be met in the standardization of medicinal plants. The determination of relative density is carried out by using pycnometer to describe the amount of mass per unit volume to provide a boundary between liquid extract, viscous extract, and dry extract. Besides, the relative density related to how to determine the purity of a substance is determined from its relative density^13,14,15.

The measurement of water content in a certain material is vital because high water content can result in the growth of mold that is not good for health.

The ash content is related to the minerals of a certain material that can be organic and inorganic salts. Ash content testing is an important standardization parameter because the ash content can indicate the feasibility of a sample or extract for further processing.

Beside those several tests, non-specific parameters are carried out to discover microbial contamination. The results of tests can be seen in table 4.

Table 4: Microbial contamination test

Parameters

Results

Terms

Microbial contamination

< 1.0 x 10-1

10⁴ colony/mL

(BPOM RI, 2006)

Mold/yeast contamination

< 1.0 x 10-1

1x10³ colony/mL (BPOM RI, 2006)

The determination of non-specific parameters is carried out by microbial contamination testing, namely the total plate number which is one of the extract purity tests. This test includes determining the number of microorganisms that are allowed and to show the absence of certain bacteria in the extract.

At a temperature of 105°C, water will evaporate and compounds that have a lower boiling point than water will also evaporate. Consequently, this test can provide a maximum limit (range) of the amount of compounds lost in the drying process^13,14,15. Determination of specific gravity is completed using a pycnometer with the aim of describing the amount of mass per unit volume to provide a boundary between liquid extract, viscous extract, and dry extract. In addition, specific gravity is related to how to determine the purity of a substance is determined by its specific gravity^13,14,15.

This measurement of water content was determined, in addition to avoiding the rapid growth of fungi in the extract, in order to maintain the quality of the extract during storage²⁶. The presence of ash content that is insoluble in acid indicates the continuing presence of sand or other impurities. Therefore, it can be seen that the mineral content of the extract and that which is not dissolved in acid are within the maximum range related to purity and contaminants. The low growth of bacteria and mold/yeast occurs due to the low water content of the extract. In addition, ethanol, the solvent used in the extraction process, can inhibit the growth of bacteria or microbes present in the extract.

These activities are best controlled by plant products, based on their intrinsic metabolic pathway. In addition, studies showed a significant increase in global interest in antioxidant compound identification, particularly for those with pharmacological potentials and little to no side effects. This implies much attention is attributed to plants as potential antioxidant sources. There has been an increase in requests for dietary, pharmaceutical and cosmetic applications, and is a major challenge for the past decades in scientific and industrial studies²⁷.

One of the leading causes of cellular damage is oxidative stress. The possible effects depend on various factors, including the extent of change, where a cell is capable of overcoming smaller perturbations and subsequently recuperate to an original state. Furthermore, reactive oxygen species production is a particular aspect of oxidative stress, known to include peroxides and free radicals^28,29. The DPPH radical is characterized by an unpaired electron and consequently exhibits a strong violet colour in solution (peak absorbance at 517 nm). This property is exploited in its application as a reagent during the free radical assessment to evaluate the scavenging ability of antioxidants²⁷. Those with positive attributes are of high significance in disease prevention therapies^30,.

The DPPH method has a working principle featuring the presence of hydrogen atoms generated from antioxidant compounds with a strong bond to the free electrons present. This further instigates the modification of a free radical, including diphenylpicrylhydrazyl to non-radical compounds (diphenylpicrylhydrazine). The indicator under these circumstances is a change in colour from purple to yellow (free radical compounds are reduced by the presence of antioxidants)³¹.

While using the DPPH method to determine antioxidant activities, certain IC₅₀ parameters are adopted, including the sample concentration required for the capture of up to 50% DPPH radical. Table 5 and figure 2 are displays the results obtained from antioxidant assessment following the use of the DPPH method. In addition, the ethanol extract C. tinctorius L. showed very strong antioxidant activities with an IC₅₀ of 2.511 ppm, while 0.004 ppm was observed for vitamin C, which served as a comparison.

Figure 2. Antioxidant Activity of ethanol extract C. tinctorius L.

Furthermore, the IC₅₀ value is inversely proportional to the antioxidant activity, meaning an increase in the antioxidant activity implies a reduction in the IC₅₀ value. The antioxidant activity of the ethanol extract of C. tinctorius L flowers is due to the high content of flavonoid compounds. This implies an increase in the total flavonoid content of a plant extract increases the extract’s antioxidant activity. Flavonoids compounds (polyphenols) of plant origin have been reported as scavengers and inhibitors of lipid peroxidation²⁹. Several flavonoids and other phenolic compounds are considered to be antioxidants not only because of their free radical scavenging activity but also because they chelate metals, contributing to increasing antioxidant capacity³².

CONCLUSION:

Based on the results of the Carthamus tinctorius L ethanol extract testing, it can be mentionable that Indigenous extract has strong potential of free radical scavenging activity against the DPPH and the characteristics for the ethanol extract of C. tinctorius L. flowers according to the quality standards of raw materials.

ACKNOWLEDGMENT:

The authors are grateful to the Ministry of Research, Technology, and Higher Education of the Republic of Indonesia for the provision of financial aid towards this study, under the Hibah Penelitian Dasar Scheme.

CONFLICT OF INTEREST:

None declared.

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Received on 19.11.2021 Modified on 22.03.2022

Research J. Pharm. and Tech 2023; 16(2):791-798.

DOI: 10.52711/0974-360X.2023.00136

Sample	Concentration (ppm)	Absorbance (Å)			Average	% Inhibition	IC50 (ppm)
DPPH	0,4 mM	0.183	0.179	0.178	0.18	-
Vitamin C	1	0.077	0.096	0.094	0.089	50.55%	0.004
	2	0.067	0.095	0.072	0.078	56.66%
	3	0.097	0.098	0.078	0.091	49.44%
	4	0.040	0.092	0.083	0.071	60.55%
	5	0.080	0.095	0.083	0.086	52.22%
Ethanol Extract C. tinctorius L	1	0.06	0.010	0.013	0.027	85%	2.511
	2	0.020	0.015	0.015	0.016	91.11%
	3	0.015	0.011	0.016	0.014	92.22%
	4	0.017	0.005	0.038	0.02	88.88%
	5	0.040	0.012	0.035	0.029	83.33%