Quality Control of Turmeric Rhizome (Curcuma domestica Val) as

Traditional Medicine from Wonogiri, Central Java

Fatimah Nisma, Ema Dewanti, Rini Prastiwi, Alexander, Wanda Puspita Sari and Wido Artanto

Faculty of Pharmacy and Science, Universitas Muhammadiyah Prof. DR. HAMKA

Islamic Centre Jl. Delima II/IV, Klender Jakarta Timur

Keywords: Curcuma domestica Val, Aflatoxin, Endosulfan, Malathion, HPLC, curcumin, pathogenic bacteria

Abstract: Turmeric is one of the plants that can be used as traditional medicine. To improve the quality of turmeric as

a traditional medicine, turmeric must be free from contamination of pesticide residues, aflatoxin, pathogen

bacteria, and curcumin content contained therein. The aim of this research was to investigate the

contamination of endosulfan and malathion pesticides, aflatoxin B1, Escherichia coli microbial

contamination, Salmonella sp., Staphylococcus aureus and Pseudomonas aeruginosa, as well as to know the

content of curcumin contained in turmeric rhizomes. The sample in this research was taken from Wonogiri

region of Central Java, Indonesia by random sampling. The methods used were HPLC for Aflatoxin B1

analysis and curcumin and Gas Chromatography for residual pesticide analysis of Endosulfan and

Malathion pesticides. Microbial testing included the establishment of Total Plate Count, AKK, MPN

Coliform, and analysis of Escherichia coli microbial contamination, Salmonella sp., Staphylococcus aureus

and Pseudomonas aeruginosa. The results showed that the samples were not contaminated by Aflatoxin B1

and Endosulfan pesticides, but contained a residual malathion with levels of 0.014 mg/kg. Microbial test

results showed that the turmeric samples from the Wonogiri market did not meet the quality requirements

due to contamination of Salmonella sp. and the chopped AKK exceeded the specified limits.

1 INTRODUCTION

Turmeric plant (Curcuma longa L.) is a plant of

biopharmaceutical, a plant that is useful in medicine

and consumed as an effort to overcome health

problems. Treatment using traditional medicine of

turmeric rhizome is one of the alternative therapy

which is done from generation to generation.

Turmeric rhizome contains the active compounds

such as curcumin, essential oils capable of inhibiting

the growth of gram-negative and gram-positive

bacteria. Curcumin in turmeric is an active

compound that gives the yellow colour to turmeric

rhizome, curcumin is produced naturally from

turmeric rhizome together with two other curcumin

analogue compounds that is demethoxycurcumin

and bisdemethoxycurcumin (BPOM, 2011).

Traditionally turmeric is used for the treatment of

itching, tingling, swollen gums, abdominal pain,

ulcers, jaundice, and gastrointestinal.

Turmeric rhizome (Curcuma longa L.) is used

extensively for food, beverage, medicine, cosmetics

and textiles. Standard quality of turmeric to be used

for raw materials of the drug should also be

considered. The quality standard of turmeric should

be highly regarded, the standard quality of turmeric

as a traditional medicine is free of pesticide

contamination, aflatoxin, heavy metals, microbial,

and curcumin levels (BPOM, 2016). Turmeric

contamination of pesticides occurs in turmeric plants

attacked by pests then farmers will spray turmeric

with pesticides. The pesticides which commonly

used are the type of malathion and endosulfan. Both

of these insecticides have a broad spectrum and non-

systemic. The content of pesticides allowed in

turmeric is 0.05 mg/kg for the type of

organophosphate. In addition to using pesticides to

control pests, farmers are also use manure pile for

turmeric growth. Manure pile is derived from animal

waste and can contaminate turmeric, because

bacteria or pathogenic microbes like to live on the

faeces (Paramitasari, 2011). Microbes and

pathogenic bacteria can be coliform, Escherichia

coli, Salmonella sp., Staphylococcus aureus and

Pseudomonas aeruginosa.

The wrong technique of post-harvest turmeric

processing, temperature conditions in the tropics,

Nisma, F., Dewanti, E., Prastiwi, R., Alexander, ., Sari, W. and Artanto, W.

Quality Control of Turmeric Rhizome (Curcuma domestica Val) as Traditional Medicine from Wonogiri, Central Java.

DOI: 10.5220/0008241101590168

In Proceedings of the 1st Muhammadiyah International Conference on Health and Pharmaceutical Development (MICH-PhD 2018), pages 159-168

ISBN: 978-989-758-349-0

159

and high humidity cause the turmeric rhizome to be

easily overgrown by Aspergillus flavus toxigenic

strain, A. parasiticus and A. nonius (Wrather and

Sweet, 2016). The product produced by this

toxigenic strain is aflatoxin. Aflatoxin is myotoxic,

as a secondary metabolic outcomes of these

Aspergillus strains, that can affect immunity, acute

necrosis, cirrhosis and liver carcinoma.

According to WHO, countries in Africa, Asia

and Latin America use herbal medicine as a

complement to the primary treatment they receive.

The traditional medicine failure for certain diseases

such as cancer and the extensive information about

herbal medicine around the world (Sukandar, 2006).

Traditional herbal medicine is a mean of traditional

medicine which is very important for the

distribution of public health. It is seen that these

herb has a huge potential and the prospect to be

developed to be an opportunity for herbalists to

develop their business.

Based on the Regulation of BPOM, traditional

medicine used as an internal medicine should be

aware of pesticide content, aflatoxin, heavy metals

and the presence of microbes such as Salmonella,

Escherichia coli, Staphylococcus aureus, and

Pseudomonas aeruginosa. These microbes should

not be contained in traditional medicine (BPOM RI,

2014). Escherichia coli bacteria is used as an

indicator of contamination, its presence in processed

products indicates contamination of human or

animal faeces through the water used.

Staphylococcus aureus bacteria is a normal flora

found in the skin and human lining membrane.

While Salmonella sp. is a bacteria that causes

infection. If swallowed into the body, it will cause

symptoms that called Salmonellosis.

Based on the preface above, it is necessary to

conduct a research about the quality of turmeric

rhizome originating from Wonogiri, Central Java,

because the region became the centre of traditional

herbal medicine industry including turmeric (Sakti,

2009). The method used to determine the pesticides

types, malathion and endosulfan, is by gas

chromatography for analysis of Aflatoxin B1 and

curcumin using HCV microbial testing conducted

include the determination of Total Plate Count,

AKK MPN Coliform as well as analysts.

2 METHODOLOGY

The instruments used in this research were: HPLC,

GC (Variant 450GC), column C18. The materials

used were: Turmeric, Peptone Dilution Fluid (PDF),

Plate Count Agar (PCA), Potato Dextrose Agar

(PDA), Lactose Broth (LB), Brilliant Green Lactose

Bile Agar 2% (BGLB 2%), Eosin Methylene Blue

Agar (EMBA), Mac-Conkey broth (MCB), Nutrient

Agar (NA), Tryptone Broth (TB), methyl red-Voges

proskauer (MR-VP), Simmon’s Citrate Agar,

Trypticase Soy Broth (TSB), Baird Parker Agar

(BPA), Brain-Heart Infusion Broth (BHIB),

Tetrathionate Brilliant Green Broth (TBGB),

Selenite Cystine Broth (SCB), Salmonella-Shigella

Agar (SSA), Triple Sugar Iron Agar (TSIA),

Cetrimide Agar. The turmeric (Curcuma longa L.)

rhizomes used were from Wonogiri, Central Java

and in the form of powder and dry sliced

2.1 Simplicia Characteristic Tests

The macroscopic examination was performed by

observing the morphology of turmeric rhizomes by

considering the colour, shape, size, and texture.

Microscopic examination of the rhizomes was by

putting the simplicia powder on the object glass that

is dripped with distilled water and covered with a

cover glass, and then being observed under a

microscope.

2.2 Pesticide Test

A total of 10 grams of turmeric rhizome powder was

added with 75 ml of acetone mixture: 1: 1 v/v

dichloromethane and left for one night for static

extraction process. The powder is filtered with

Whatman no. 40 filter paper, then concentrated with

a vacuum rotary evaporator until only 1 mL

remaining. The sample was then purified by passing

it to a chromatographic column containing

anhydrous sodium sulfate. Samples were then ready

to be injected into gas chromatography (Deptan,

2006).

2.3 Aflatoxin Test

As total of 10 grams of turmeric rhizome powder

was added with methanol and aqua bikes mixture

(80:20), and left for one night for static extraction

process. The powder was then filtrated with

Whatman no. 40 filter paper, then concentrated by

vacuum rotary evaporator, then rinsed with methanol

gradually, and collected in test tube up to 10 mL.

Samples are then ready to be injected into high

performance liquid chromatography (Deptan, 2006).

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2.4 Identification and Determination of

Curcumin Levels

2.4.1 Determination of Curcumin Levels

with HPLC

A total of 100.0 mg of turmeric extract samples was

added into a 100.0 ml measuring flask of 15.0 ml of

0.01 N H

, then in the ultrasonic and added 96%

ethanol until the limit mark. The solution was

filtered with 0.2 μm membrane into the VCC vial

and injected as much of 50 μl for 12 minutes.

Identification of active substance were done by

comparing between sample retention time and

standard curcumin retention time.

2.4.2 Qualitative Identification of Curcumin

with Thin Layer Chromatography

Method

Qualitative analysis of the curcumin was performed

using thin layer chromatography method using

stationary phase silica gel silicone 60 F254 and the

mobile phase used was chloroform: methanol (95: 5)

v / v., then observed on 366 nm UV rays

2.5 Microbial Contamination on

Turmeric Sample Test

2.5.1 Homogenization and Sample Dilution

A total of 10 grams of turmeric samples were

dissolved in 90 ml of Peptone Dilution Fluid (PDF)

media, resulting from 10

-1

dilution. The resultant

dilution was piped as much as 1 ml and inserted into

the first tube and dilution of the PDF, resulting 10

-2

-3

, 10

-4

, 10

-5

, and 10

-6

(Radji, 2011).

2.5.2 Total Plate Count Determination

Each result of turmeric sample was piped as much

as 1 ml, then inserted into a Petri dish containing

15-20 ml of Plate Count Agar (PCA) medium. After

the media froze, the Petri dish is incubated at 37°C

for 24-48 hours with the position reversed. (Radji,

2011).

2.6 Total Number of Mold and Yeast

Examination

The petri dish which contained 15-20 Potato

Dextrose Agar (PDA) medium was prepared in

advance. As much as 0.5 ml diluted sample of

turmeric was put into the surface of the PDA

medium, then it was incubated at 20-25°C for five

days in a reversed position. The colonies growing on

the media were

observed and counted on the fifth

day (Radji, 2011).

2.7 Coliform Examination

Coliform examination was performed using Most

Probable Number method, 5 tubes system. This

method includes:

2.7.1 Presumptive Test

The samples were taken into tubes containing

Lactose Broth Media (LB). Next, the samples were

inserted into second 5 tubes containing 5 ml of LB

medium of single concentration. The area of

inverted Durham tube. 0.1 ml of the sample was

inserted into a row containing 5 ml of single

nutritional LB medium and inverted Durham tube

(Radji 2011).

Then the tubes were incubated at 37 °C for 24 -

48 hours. The gas-filled tube proceeded with the

assertion test. This estimate test was to detect the

presence or absence of bacteria capable of

fermenting lactose that indicates the presence of

colibacteria (Radji, 2011).

2.7.2 Confirmative Test

A total of 1 culture from the number of tubes which

were forming the gas in the LB media estimator test

was transferred into a tube containing 10 ml of

Brilliant Green Lactose Bile Broth 2% (BGLB 2%),

with an inverted Durham tube in it and 2 tubes of the

same for sample and control (Radji, 2011).

All tubes were incubated at 37 °C for 24 - 48

hours, until the gas was formed in tubes. A tube

with gas was tested with Eosin Methylene Blue Agar

medium. Using an inoculation needle, the gas-

formed tube was inoculated on the EMBA plate by

scraping it and then incubated at 37 °C for 24 hours

(Radji, 2011).

2.7.3 Complete Test

The gas-containing tube in the assertion test was

taken using Ose, then it was scraped onto EMBA

media. The tubes were incubated at 37 °C for 24 - 48

hours (Radji, 2011).

2.7.4 Gram Staining

A small amount of microbial growth was taken

using the Ose tip, then spread onto droplet of water

Quality Control of Turmeric Rhizome (Curcuma domestica Val) as Traditional Medicine from Wonogiri, Central Java

161

over the object glass and dried by fixating on a small

flame. The main paint solution (Crystalline violet)

then washed with running water and dried. Then the

preparation was stained with Lugol solution (I

+ KI

solution) and allowed for 45-60 seconds. The color

disappears by dipping the preparations into 96%

alcohol while being shaken for 30 seconds or until

no more dyestuff flows from the preparation. Then

the safranine was dropped into the preparation and

be left for 1 minute, and rinse with running water

and then dry. The object was then examined under

the microscope (Radji, 2011).

2.8 Microbial Pathogen Contamination

Analysis

2.8.1 Sample Homogenization and Dilution

• Contamination Analysis of Escherichia coli

The total of 10

-1

dilution was taken and inoculated

into three tubes containing the Mac Conkey broth

medium (MCB) and there was a Durham tube in it.

While contamination analysis of Staphylococcus

aureus, homogenized and diluted with a PDF

solution until 10

-1

dilution. Then the sample was

taken and added into a test tube containing 18 ml

Trypticase Soy Broth (TSB).

• Contamination Analysis of Salmonella thypi

This analysis used Lactose Broth (LB) solution, by

transferring 25 ml samples aseptically into a bottle

containing 225 mL of LB media sample.

• Contamination Analysis of Pseudomonas

aeruginosa

This analysis used enrichment medium of 100 ml of

Tetrathionate Brilliant Green Broth (TBGB) medium

and 100 ml of Selenite Cystine Broth (SCB)

medium, each then incubated at 37 °C for 24 hours

(Radji, 2011).

2.8.2 Growing into Selective Use

The tube which was positive of E. coli, the gas on

the Durham, was taken into a selective solid medium

Eosin Methylene Blue Agar (EMBA). The next step

was confirmatory testing, IMVIC test, by

inoculating bacterial culture on NA media into

indole, methyl red, Voges Proskauer, and citrate

(IMVIC).

Staphylococcus aureus was inoculated into

Brain-Heart Infusion Broth (BHIB) media. As much

as 1 ml of each culture in BHIB was piped and

transferred into a sterile test tube. Furthermore, 0.3

ml plasma was added in each tube. Plasma clotting

showed positive Staphylococcus aureus coagulase.

Salmonella sp. bacteria, was identified by

inoculating 1 tangle culture in a Petri dish containing

Salmonella-Shigella Agar (SSA) medium. The

alleged Salmonella sp. colony was characterized by

a colorless colony to pink, clear to opaque (Radji,

2011). The next phase was confirmation test. In this

test, 2-5 specific colonies of SSA selective medium

were selected and inoculated on NA media. Then the

colonies on NA medium were inoculated with

puncture and scratching methods on the Triple Sugar

Iron (TSIA) medium. If on the slant, Salmonella

ferment lactose or sucrose then the color of the

media turns yellow and if Salmonella does not

ferment lactose or sucrose then the color of the

media remains red or unchanged.

While Pseudomonas aeruginosa used the TSB

cultures and inoculated on the surface of Cetrimide

Agar (Cet.A) medium. Observed the growth of

greenish colony (Radji, 2011). Further test on

suspected colonies of Pseudomonas aeruginosa, a

catalase test of the NA culture was tilted by taking

the colony using an aseptic Ose needle. Placed on a

glass object that was previously cleaned with 70%

flattened alcohol, then added a drop of 3% hydrogen

peroxide solution. Positive results are characterized

by gas formation (Radji, 2011).

3 RESULT AND DISCUSSION

3.1 Materials

The materials used were rhizomes and turmeric

powder (Curcuma domestica Val.) which were

obtained from Wonogiri market region of Central

Java, because Wonogiri is a central area of

traditional medicine (herbal medicine), and in that

area there are many small or medium industries that

produce traditional medicine (herbal medicine)

where one of the raw materials is turmeric.

3.2 Sample Identification

3.2.1 The Macroscopic Test

The Macroscopic examination aimed to observe the

colour, shape, size and texture of turmeric rhizomes.

As seen in table 1.

According to Pharmacopoeia Herbal Indonesia

(2008), fresh turmeric colourized by yellow-orange,

reddish orange-yellow to brownish orange-yellow

and has a round shape up to rounded, sometimes

branching, a width of 0.5 cm to 3 cm, length 2 cm to

6 cm. Based on the results of macroscopic tests, it

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162

showed that the inside was yellow in colour, while

the exterior was brownish and elliptical with a width

of ± 2.5 cm, and length ± 4.5 cm.

3.2.2 Microscopic Test

This microscopic test was performed to see the

anatomy of turmeric tissue, by putting the simplicia

powder on the object glass that has been dripped

with distilled water and covered with a cover glass,

then viewed under a microscope. Microscopic test

results from turmeric powder showed that it has

epidermal tissue, stomata cells, bearing files and hair

cover. It has one-layer epidermis, polygonal-shaped

flat, cell wall pouring. Covering hair, conical,

straight, or slightly crooked; length of 20 μm to 890

μm, thick wall. Perindrem consisted of 6 to 9 layers

of cells in the shape of a long facet, the wall

penetrates. Single grain starch is oval end having

bulge or round to almost triangle with one side

rounded (Farmakope Herbal Indonesia, 2008). The

anatomy of tissue in turmeric has a characteristic

that is the presence of parenkim, cell clumps and

hair cover. The observable tissue anatomy included

wood vessels, parenchyma and starch grains. It can

be seen in the Figure 1

The pictures above shows that the microscopic

test using a microscope with 10x18 magnification,

produced the anatomy of turmeric tissue namely

xylem, covering hair, and starch grain periderm.

This result was in accordance with the requirements

of Pharmacopoeia Herbal Indonesia Issue 1 (2008).

3.2.3 Chemical Identification Test

The identification of the turmeric sample was carried

out using chemical reactions. Turmeric that had been

dubbed and added with Materia Medika Indonesia

volumes VI 1977. The resulting chemical

identification test of turmeric rhizomes can be seen

in Table 2.

The table above shows that the sample used is

true turmeric powder because the results obtained

are in accordance with the chemical identification

test according to Materia Medika Indonesia (1977).

3.2.4 Loss on Drying Test

Loss on drying aimed to provide a maximum (range)

of the number of compounds lost in the drying

process (MOH RI, 1995). Loss in drying test results

is seen on Table 3.

The result above showed that the sample meets

the requirements because according to

Pharmacopoeia Herbal Indonesia Edition I (2008),

the limit of loss on drying is less than 12%.

Table 1: Macroscopic test result of turmeric.

Macrosco

ic Turmeric

Colour yellow on the inside, brown

on the outside

Sha

e oval sli

hlt

roun

Table 2: Results of chemical turmeric powder

identification test.

Turmeric

owde

Reagent Result

2 m

5 dro

of sulfuric acid P Blood re

2 m

5 dro

of chloric acid P Brown

2 mg 5 drop of natrium dioxide

solution 5% b/v

Red-

orange

2 mg 5 drop of ammonia (25%)

Red-

orange

2 mg 5 drop of iron (III)

chloride solution P 5% b/v

Brown

2 mg 5 drop of lead (II) acetate

solution P 5% b/v

Pink

Table 3: Loss on drying test result.

Handling Result

Loss on Drying 5,37%

Figure 1: The microscopic test result of turmeric: a)

xylem with thickening of stairs and parenchyma with

secretion cells, b) Starch grain periderm, c) Covering

hair with irregular lumps colourized by yellow to

brown.

(a)

(b)

(c)

Quality Control of Turmeric Rhizome (Curcuma domestica Val) as Traditional Medicine from Wonogiri, Central Java

163

3.3 Pesticide Test

3.3.1 Standard Normative Endosulfan

Curve

Preparation of standard solution of Endosulfan by

making five concentrations in acetone solution.

Standard normative uptake measurements were

performed using gas chromatography with an

electron detector (ECD), an oven temperature of

150ºC. The raw solution was injected into the ECD

for 20 minutes to get the peak. The result of the

linear line equation of the curve is y = 64935x +

19368 with the correlation coefficient (r) = 0.991.

The value of (r) = 0.991 on the endosulfan

calibration curve (figure 2) showed that the line

formed between the endosulfan concentration with

the area are in accordance with line linearity

requirements, because the linearity test is done by

making the calibration curve, which can produce the

equation of the regression line with the correlation

coefficient (r) ≥ 0.9990.

Table 4: Results of pesticide test of endosulfan and

malathion types.

Tested subtance

Content

(pp

)

Average

(ppm)

1 2

Organochorine 0 0 0 0

Lindan

(

ɤ-BHC

)

0 0 0 0

Aldrin 0 0 0 0

taklo

0 0 0 0

Dieldrin 0 0 0 0

DDT 0 0 0 0

Endrin 0 0 0 0

Endosulfan* 0 0 0 0

ano

hos

hate

Diazinon 0 0 0 0

Fenitrotion 0 0 0 0

Metidation 0 0 0 0

Malathion * 0,028 0,0015 0,014 0,0187

Klorfiri

os 0 0 0 0

Parathion 0,041 0,090 0,065 0,035

Profenopos 0 0 0 0

* Tested Substance

3.3.2 Standard Normative Malathion Curve

Standard normative uptake measurements were

performed using gas chromatography with an

electron detecting detector (ECD), an oven

temperature of 150 ºC. The standard solution was

injected into the apparatus for 20 min to get the peak

in appendix 4. The result of the linear line equation

of the curve is y = 12666x + 4449 with the

correlation coefficient (r) = 0.992 (figure 3).

3.3.3 Pesticide Content on Sample

The sample of turmeric was measured using gas

chromatography. In this gas chromatography, the

system was arranged with 1mL/minute flow

velocity, using gas phase N

with 80 psi flow

pressure, stationary phase VFRV 1701 Pesticide

capillary with diameter 0.25 μm, 30 m long, ECD

detector (Electron Capture Detector) and at a

temperature of 300 ° C. The measurement results is

seen on Table 4.

The results of pesticide residue analysis on

turmeric rhizome showed that the sample was not

detected containing pesticide residue organochlorine

especially for endosulfan type, because the use of

pesticides for the group of organochlorines has been

banned by the Minister of Agriculture with Law No.

434.1/kpts/TP.270/7/2001 due to its persistent in the

environment (Isnawati, 2005). The result of the test

was the contamination of organophosphate pesticide

residue of malathion type with concentration of

0.014 ppm. Judging from the resultant content of the

sample, it still fulfilled the requirement because

based on the quality requirement of turmeric

simplistic of the Food and Drug Control Agency

concerning the Maximum Limit of Pesticide Residue

by 0,05ppm (BPOM, 2006). The test results also

detected other types of pesticides group, other

organophosphates Parathion with levels of 0.065

ppm. This is because the organophosphates in the

0 0.2 0.4 0.6

Total area counts

Endosulfan Concentration (ppm)

Figure 2: Results of endosulfan calibration.

0 0.2 0.4 0.6

Total area counts

Malathion Concentration (ppm)

Figure 3: Results of malathion calibration curve.

MICH-PhD 2018 - 1st Muhammadiyah International Conference on Health and Pharmaceutical Development

164

turmeric rhizome can be induced by the use of

pesticides directly and indirectly (due to

contamination of surrounding pesticides). Pesticides

move from agricultural land to rivers and lakes

carried by rain or evaporation left behind or

dissolved in the flow of the surface, located on the

soil layer and dissolved along with the groundwater

flow. (Djojosumarto, 2008).

3.4 Aflatoxin Contamination Analysis

The result of analysis of aflatoxin contamination on

turmeric rhizome by HPLC method. The prepared

sample was then measured using HPLC with a

wavelength of 365 nm.

Analysis of turmeric rhizome aimed to

identify the absence of aflatoxin B1 in the sample.

Aflatoxin B1 analysis on samples showed negative

or undetectable results, this may be due to the

aflatoxin content of the turmeric sample was small,

so the test did not show results, because handing in

pre and post-harvest was good, so that the turmeric

was not contaminated by aflatoxin.

3.5 Curcumin Analysis

3.5.1 Qualitative and Quantitative

Curcuminoid Analysis

The qualitative test of curcumin compound was done

by Thin Layer Chromatography (TLC) method using

chloroform: methanol (95: 5) v/v and the stationary

phase used was silica gel 60 F254 (Pharmacopoeia

Herbal, 2008). The result of curcuminoid

identification by Thin Layer Chromatography can be

seen in Figure 4.

The curcumin identification performed was

observed with UV cabinet on 366 nm UV rays, the

detection did not use spray reagent because

curcuminoid was visible under UV light at 366 nm

wavelength. The curcuminoid identification result

shows that there are 3 rickshaws in which curcumin

compound (X) is a constituent of turmeric, while

desmethoxycurcumin (Y) and

bidesmethoxycurcumin (Z) are the identifying

compounds of curcuminoid. The result of

identification of the spots obtained looks like

standard spotted results with Rf value of 0.68 and

the Rf value of the four samples is also equal to the

standard. The spots are curcumin compounds

because they have the same color and Rf values in

each sample and the comparators used.

3.5.2 Curcumin Content Test with HPLC

After testing curcumin with TLC, High Performance

Liquid Chromatography was performed. 96%

ethanol was used as a solvent in the determination of

curcumin content by using HPLC. Curcumin testing

method with gradient technique using 0.1%

Trifluoroacetic acid (TFA) motion phase and

Acetonitrile at 425 nm wavelength for 12 minutes

with a flow rate of 1.0 ml/min. Tests using a

gradient system aimed to separate samples

containing components with a very diverse polarity

that can provide good results. Determination of the

level of curcumin with this gradient system obtained

3 chromatograms at the standard and the first

chromatogram sample is curcumin ranging from

retention time 8.6 minutes, followed by

chromatogram desmethoxycurcumin at retention

time 9,2 minutes and bidesmethoxycurcumin at

retention time 9.7 minutes

Result of curcumin with HPLC test found that

the turmeric plants a good place to grow turmeric

growth. Land in the Wonogiri area has alluvial soil

type where this type of soil is good for turmeric

growth (Raharjo and Rostiana, 2005). Alluvial soil is

a soil formed from fine deposits. This type of soil is

widely used in agriculture due to its nature which

has a high nutrient content. Turmeric plants can

grow well in rainfall ranging from 2000 to 4000

ml/year and has rainfall ranging from 2,790 mm.

Figure 4: Result of curcumin content identification with

TLC at wavelength 366 nm. (X): Curcumin; (Y):

Demetoxicurcumin; (Z): Bisdemetoxicurcumin; (STD):

Standard of Curcumin.

Quality Control of Turmeric Rhizome (Curcuma domestica Val) as Traditional Medicine from Wonogiri, Central Java

165

3.6 Bacteria Test

3.6.1 Total Plate Count Test Result

The powder and rizhomes turmeric samples were

grown on Seed Plate Count Agar (PCA) hatching

medium calculated bacterial growth. Bacteria that

can be calculated ranges from 30-300 colonies, so

the results obtained as in Table 5.

The total number or amount of aerobic bacteria

from the turmeric sample powder and chopped is

between 1.17 X 10

to 3.45 X 10

bacteria per ml of

sample. According to the provisions stipulated by

the Head of the Food and Drug Supervisory Agency

No. 12 of 2014, on the requirements of traditional

medicine that the total number of chopped plates and

powder ≤ 106 then the turmeric sample from the

eligible Wonogiri Market is set.

3.6.2 Total Number of Mold and Yeast

Result

In this test, samples of turmeric powder and chopped

were grown on Potato Dextrose Agar media (PDA)

and calculated the growth of mold and yeast.

Colonies that can be calculated ranging from 30-300

colonies, resulting in Table 6.

The fungi were a group of eukaryotic

microorganisms that vary widely, so the ability to

take advantage of nutrients from the environment

and metabolic abilities of fungi also vary widely.

The total amount of mould and yeast from the

turmeric sample obtained was 8.85 X 10

to 4.6 X

mould and yeast per ml sample. The total

amount of mould and yeast in the turmeric powder

had fulfilled the requirements set by the Head of the

Food and Drug Administration Law No. 12 of 2014

of traditional medicine that is 10

, but the chopped

sample did not meet it, maybe the case in tttthe

process of storing the simplicia that have not met the

criteria.

3.6.3 Most Probable Number (MPN) of

Coliform Examination Result

Coliform examination results of turmeric (powder

and chopped) showed a positive result of the

presence of Coliform bacteria, thus the examination

was followed by IMVIC test. The complete

Coliform test consists of 3 stages: test of predictor,

test of confirmation and complete test. In the LB

(Lactose Broth) probe test, a positive result is seen

by bubbles forming on the Durham tube. The

formation of gas in Durham tubes as a result of

lactose fermentation does not necessarily indicate

the number of Coliform bacteria, because lactose can

also be fermented by other microbes such as lactic

acid bacteria. Therefore, the positive probable test

should be continued with the assertion test, the

BGLB (Brilliant Green Lactose Bile Broth) test

containing bile salt is a component that can inhibit

bacterial growth besides Coliform, and give

Coliform bacteria chance to grow well.

The result of the research of turmeric samples

in the form of powder and chopped on medium

(BGLB) showed negative result, with MPN value

9/100 small on chopped and <2/100 ml on powder

(table 7). Furthermore, the tube showed a positive in

the assertion test, followed by a test by inoculating a

dispute of the assay test results into a Petri dish

containing the Eosin Methylene Blue Agar (EMBA)

medium and incubated at 37 °C for 24-48 hour

Table 5: The result of tests of total plate count on

powder and chopped.

Turmeric Number of

colonies

(Colony/g)

Maximum

Limit of

Microbial

Contamination

Information

Powder 1,17 X 10

≤ 10

colony/g Qualifie

Choppe

7 X 10

≤ 10

colony/g Qualifie

Table 6: Result of determination of total number of

mold and yeast turmeric powder and chopped.

Turmeric

Sample

Number of

Colonies

(Colony/g)

Maximum

Limit of

Microbial

Contamination

Information

Powder 0,885 X

≤ 10

colony/g Qualified

Chopped 1,13 X 10

≤ 10

colony/g Not

qualifie

Table 7: Results of the Coliform MPN examination

turmeric powder and chopped.

Turmeric

Sample

Lactose Broth

Number

Postive

Tubes

MPN/100

0,1

Chopped 2 3 0 2-3-0 9

Powder 0 0 0 0-0-0 <2

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3.6.4 Pathogen Microbial Contamination

Analysis Result

The presence of pathogenic bacteria in food should

be avoided so that product users are protected from

adverse effects caused by consumed products. One

cause of disease transmission and the cause of

poisoning is contamination of microbes in a food.

Microbes in terms of bacteria, such as Escherichia

coli, Staphylococcus aureus, Salmonella, and

Pseudomonas aeruginosa can contaminate food

consumed by humans. The examination results (

table

8) of pathogenic microbial contamination was

obtained as follows:

Escherichia coli

The formation of gas in Durham tubes as a result of

lactose fermentation does not necessarily indicate

the number of Coliform bacteria, because lactose can

also be fermented by other microbes such as lactic

acid bacteria. The results of the E. coli test showed a

negative on the chopped sample and the positive on

the powder sample in MCB medium. The positive of

the powder samples were scratched onto the EMBA

media. The results of the casting on the EMBA

media showed negative or no gloss, so the negative

E. coli test results on both the turmeric sample were

concluded.

Staphylococcus aureus

Staphylococcus aureus is a normal flora found in

human skin. It is a type of pathogenic bacteria that

can cause infection and abnormalities in the skin

(Radji, 2011). Ecologically, Staphylococcus aureus

is closely related to humans especially in the skin,

nose and throat. Thus, food and drink are mostly

polluted through management by humans. Overall,

these organisms are not strongly competitive with

others and consequently these bacteria have no

important role in uncooked food ingredients.

However, in cooked or salted food, where

existing organisms have been damaged by warming

or growth inhibited by salt concentrations,

Staphylococcus aureus cells may continue to

progress to a dangerous level. Poisoning due to

Staphylococcus aureus contaminated food is mostly

related to food products that have been cooked

especially those managed by humans. The symptoms

of Staphylococcus aureus contaminated food are

intoxicated. The growth of these organisms in food

produces toxic enterotoxins, which when ingested

may result in abrupt on slaught, stomach cramps and

severe vomiting. Diarrhea may also occur (Buckle et

al., 2007).

The examination result of pathogenic microbial

Staphylococcus aureus planted into BPA media

(Baird Parker Agar) showed negative result. This

medium contained lithium chloride and tellurite to

grow the microbes in the sample, as well as pyruvate

and glycine that support the growth of

Staphylococcus aureus bacteria. If the samples

contain Staphylococcus aureus bacteria, colony will

grow glossy black colour, as the results of the

analysis did not show a shiny black result, turmerics

from Wonogiri Market were negative from

Staphylococcus aureus microbial pathogens.

Salmonella sp.

Salmonella test is used to establish the presence of

pathogenic microbial Salmonella sp. These

microbial pathogens are Gram-negative microbes

that are stick-shaped and cause typhoid, paralysis

and foodborne diseases. Salmonella sp. consists of

2500 serotypes which are all pathogenic in both

humans and animals. TSIA is rich in lactose,

sucrose, dextrose, ferrous sulfate. The medium is

used to sort out microorganisms that have the ability

to degrade sulfur and ferment carbohydrates. With

the fermentation of phenol red, if microorganisms

can not ferment the three types of sugar (sucrose,

lactose, glucose) present in the media then the media

will turn yellow. If microorganisms can only

ferment dextrose. The occurrence of dextrose

fermentation by Salmonella will decrease the pH to

acid condition. This condition causes phenol red (red

medium) changes to yellow. That is what happened

on examination of bacterial pathogen powder and

turmeric samples from Wonogiri Market.

Table 8: Result of pathogen microbes.

Turmeric Sample

Bacteria Identification Test

Escherichia coli Staphylococcus

aureus

Salmonella Pseudomonas

aeruginosa

Chopped

Negative Negative Positive Negative

Powder

Negative Negative Positive Negative

Quality Control of Turmeric Rhizome (Curcuma domestica Val) as Traditional Medicine from Wonogiri, Central Java

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Pseudomonas aeruginosa

Medium Cet. A (Cetrimide Agar) is commonly used

for the isolation of Pseudomonas aeruginosa. Cet. A

is a quarternary ammonium compound that can

inhibit the growth of other bacteria, but does not

occur in Pseudomonas aeruginosa. The

examination result of chrysanthemum and turmeric

powder from Wonogiri are negatively contain

Pseudomonas aeruginosa.

4 CONCLUSION AND

SUGGESTION

4.1 Conclusion

Based on the research on turmeric rhizome from

Wonogiri area, it can be concluded that the samples

did not contain endosulfan type organochlorinated

pesticide residues, but it contained pesticidal residue

from organophosphate type that is malathion, with

0,014 ppm concentration level. The curcumin level

met the requirements of Pharmacopoeia Herbs of ≤

6.6% and the curcumin content was 7,8482%.

Contamination of Salmonella sp. bacteria was found,

and thus it did not meet the requirements of the

Regulation of the Head of the Food and Drug

Supervisory Agency No. 12 of 2014 about

traditional medicine

4.2 Suggestion

We recommend to test other quality standards such

as the heavy metal test on turmeric samples from

Wonogiri.

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