TOXICITY, PHYTOCHEMICAL CONTENTS AND STARCH CHARACTERS OF GADUNG (Dioscorea hispida Dennst.) TUBER ACCESSION TIMOR LESTE, KALIMANTAN TENGAH AND DAERAH ISTIMEWA YOGYAKARTA Toksisitas, Kandungan Fitokimia dan Karakter Pati Aksesi Gadung (Dioscorea hispida D
TOXICITY, PHYTOCHEMICAL CONTENTS AND STARCH CHARACTERS OF
GADUNG (Dioscorea hispida Dennst.) TUBER ACCESSION TIMOR LESTE,
KALIMANTAN TENGAH AND DAERAH ISTIMEWA YOGYAKARTA
Toksisitas, Kandungan Fitokimia dan Karakter Pati Aksesi Gadung
(Dioscorea hispida Dennst.) Timor Leste, Kalimantan Tengah dan
Daerah Istimewa Yogyakarta
L. Hartanto Nugroho and Anna Estyaniyana
Plant Structure and Development Laboratory,
Faculty of Biology, Universitas Gadjah Mada, Yogyakarta, Indonesia.
E-mail: [email protected].
ABSTRAK
Gadung (Dioscorea hispida Dennst.) dikenal sebagai tanaman obat. Di Timor Islan, gadung digunakan sebagai re-
pellant kutu beras (Leptocorisa oratorius F.). Selain itu, di beberapa tempat gadung digunakan sebagai makanan
alternatif. Tujuan dari penelitian ini adalah untuk mengetahui toksisitas, kandungan fitokimia dan karakter pati
umbi gadung yang dikoleksi dari Timor Leste, Kalimantan Tengah, dan Daerah Istimewa Yogyakarta (DIY). Umbi
kering digunakan untuk uji toksisitas dan analisis kandungan fitokimia, sedangkan umbi segar digunakan untuk
mengamati karakter pati gadung. Karakter pati meliputi bentuk, diameter, dan jumlah butir pati. Umbi diekstraksi
menggunakan alat Sokletasi menggunakan pelarut kloroform dan metanol. Uji toksisitas umbi gadung dilakukan
dengan menggunakan Brine Shrimp Lethality Test (BSLT). Pengaruh toksisitas dari masing-masing ekstrak diiden-
tifikasi dengan persentase kematian larva dan dihitung dengan analisis probit (LC50). Deteksi kandungan alkaloid,
flavonoid, terpenoid ekstrak umbi gadung menggunakan kromatografi lapis tipis. Karakter pati diamati dengan
menusuk umbi segar berulang kali dengan jarum, ekstraksi cairan putih kental dan diletakkan pada kaca benda
serta pembuatan penampang umbi korteks kemudian diamati di bawah mikroskop cahaya. Hasil penelitian menun-
jukkan bahwa toksisitas tertinggi ditunjukkan oleh ekstrak kloroform aksesi gadung dari Timor Leste dengan LC50
adalah 10 µg/mL. Ekstrak dari tiga aksesi mengandung alkaloid, flavonoid, dan terpenoid. Tiga aksesi memiliki
karakter pati yang sama yaitu berbentuk bulat dan agregat setiap butir. Aksesi DIY aksesi mempunyai ukuran di-
ameter pati terbesar dan jumlah agregat terbanyak di setiap sel parekim.Kata kunci: Gadung (Dioscorea hispida Dennst), umbi, pati, fitokimia, toksisitas.
ABSTRACT
Gadung (Dioscorea hispida Dennst.) known as a yam internationally is a medicinal plant. In Timor Islan, gadung is
used as repellant of Rice bug (Leptocorisa oratorius F.). Moreover, in same places, gadung is used as an alternative
food. The aims of the research were to know the toxicity, phytochemical contents and starch characters of gadung
tuber which were collected from Timor Leste, Kalimantan Tengah, and Daerah Istimewa Yogyakarta (DIY). Dry
tubers were prepared for toxicity and phtyochemical contents analyses and fresh tuber were used to observe the
character of gadung starch. The starch characters were shape, diameters, and amounts of starch grains. Tubers
were extracted by Soxhletation apparatus using chloroform and methanol. The toxicity of gadung tuber was done
using Brine Shrimp Lethality Test (BSLT). Effect of toxicity from each extract was identified with the percentage
of death larva and counted by probit analysis (LC50). Futhermore, the content of alkaloid, flavonoid, terpenoid of
gadung tuber extract were detected using Thin Layer Chromatography. The starch characters were observed by
stabbing the fresh tuber repeatedly with needles, was extracted viscous white fluid and attached to the glass slide
and making cross section of tuber cortex, following by observation under light microscope. The results revealed
that the highest toxicity was shown by chloroform extract of gadung accession from Timor Leste with the LC50
was 10 µg/mL. Extract of three accessions contained alkaloids, flavonoids and terpenoids.
Three accessions starch grain had same characters. The characters were round shape and aggregate of each grain.
Among the three accessions, DIY accession presented the bigest size of starch diameters and much number of
aggregate in each parencyma cells.Keywords: Gadung (Dioscorea hispida Dennst.), tuber, starch, phytochemical, toxicity.
INTRODUCTION
The development of science and tech- nology which encourage the development of advanced technology could not remove the use of herbal medicine. Public interest on natural or herbal medicine has grown in tandem with the slogan “back to nature”. From herbal medi- cine researches showed that it had lower side effects compared to chemical drugs. The use of herbal medicine is an effort in the utilization of biological resources which are abundant in Indonesia.
Gadung (Dioscorea hispida Dennst.) known as a yum (internationally) is the poten- tial biological resources in Indonesia, which need to be developed as an alternative me- dicinal plant as well as an alternative food in several regions in Indonesia. Some research states that yam tubers can lower blood glucose levels (Sunarsih et al., 2007) and is capable of preventing atherosclerosis disease (Maligan, 2006) while the ethanolic extract of leaves of yam had antitumor activity as well as antiox- idant (Punith et al. 2010). The effect of vari- ous yam tuber extract in medicinal aspect is due to its secondary metabolite content. The content is characteristic for each plant spe- cies, although environment factor also play an important rule in plant secondary metabolite content.
As the first step to find out bioactiv- ity and chemical content in yam tuber, it can be done by toxicity test and phytochemical screening. Toxicity test can be performed using Brine Shrimp Lethality Test (BSLT). The test- ing is based on the ability of toxic compound to kill the shrimp (Artemia salina Leach) larvae (Meyer et al. 1982).
Moreover, according to Anderson (1991) in Hertiani et al
. (2002) the test is also for early screening of cytotoxic compounds. Phytochemical screening is used to de- tect secondary metabolites contained in yam tubers. Various secondary metabolites such as alkaloids, flavonoids, and terpenoids have biological effects in inhibiting cancer growth, antioxidant, lowering blood cholesterol levels, lowering blood glucose levels, antibiotic, and immune enhancement effects (Sumastuti & Soelimar, 2002).
Yam spreaded all over Indonesia with diverse name in different areas, such as gad- ung ribo (Minangkabau), gadung (Sundanese and Javanese), sikapa (Makasar), Salapa (Bu- gis), bituke (Gorontalo), and gadu (Bima). Character yam tuber starch is important to be investigated for further utilization of the tuber, especially in relation to food alternatives. Yam tubers contain carbohydrates that are stored in the form of starch. Starch reserves are usu- ally stored in grain form in the cell vacuole. Ac- cording to Youngken (1951), the type of starch grains have different shapes depending on the type of plant where the starch is obtained. Therefore, it can be used as the basis of the characterization of the relevant plants. The differences in anatomical characters are also strongly influenced by adaptation the plant to the environment (Jones & Luchsinger, 1986). Therefore, region differences where yam growing will influence the nature of toxicity, phytochemical and starch of yam.
This study was conducted to deter- mine the toxicity of yam tuber extracts ac- cession Timor Leste, Kalimantan Tengah, and D.I.Yogyakarta L. Hartanto Nugroho and Anna Estyaniyana
KALIMANTAN TENGAH AND DAERAH ISTIMEWA YOGYAKARTA
Between the two compartments was placed holed septum as a divider. Container was filled with sea water to above the existing divider hole. The eggs which would be hatched were first soaked in distilled water for ± 1 hour. The floated eggs were discarded and the settles eggs were moved into the dark section of hatch- ery compartment. After a day (± 24 hours) the eggs hatch into larvae that will move toward the light compartment. Shrimp larvae were used for toxicity test was 48 hours old larvae after hatching (Hertiani et al., 2002).
Toksisitas, Kandungan Fitokimia dan Karakter Pati Aksesi Gadung(Dioscorea hispida Dennst.) Timor Leste, Kalimantan Tengah dan Daerah Istimewa Yogyakarta
TOXICITY, PHYTOCHEMICAL CONTENTS AND STARCH CHARACTERS OF GADUNG (Dioscorea hispida Dennst.) TUBER ACCESSION TIMOR LESTE,
TLC process was started by preparing the mobile phase. Mobile phase for the detection of alkaloids, flavonoids, and terpenoids were the mixture of toluene:ethyl acetate:diethyl amine in the ratio 7:2:1 (v/v); ethyl acetate:formic acid:glacial acetic acid:water in the ratio 100:11:11:27 (v/v); hexane:ethyl acetate in the ratio 93:7 (v/v) respectively.
Phytochemical Screening by Thin Layer Chro- matography (TLC)
E). Furthermore, from a solution of B, C, D, and E were pipetted into other vials in amount of 100 mL. Into vial containing the extract solu- tion were added 10 larvae of Artemia salina leach aged 48 hours. Each concentration was made 5 times replication. As a control, ten lar- vae were grown in 200 mL seawater without any extract. The mixture was left for 24 hours under a 18 watt fluorescent light. After 24 hours, dead shrimp larvae from each container were calculated. Data were analyzed by probit analysis in order to obtain LC50 values for each treatment (Jamal, 2001, with modifications).
A total of 4 mg of chloroform or metha- nol extract samples were dissolved in 10 mL dimethyl sulfoxide (DMSO) and 10 µg/mL of solvent added to 2 mL of sea water (2.000 µg/ mL) as a solution A (stock solution). Stock so- lution was diluted with sea water to 1.000 µg/ mL (solution B), 500 µg/mL (solution C), 100 µg/mL (solution D) and 10 µg/mL (Solution
Toxicity Test with Brine Shrimp Lethality Test
against shrimp larvae Artemia salina Leach, profiles of secondary metabolites and its starch character.
MATERIALS AND METHOD Materials
Hatching Eggs Artemia salina Leach
Material collected from three differ- ent areas were divided in two. The first was fresh material for anatomical analyses and the second was dry material for toxicity test and phytochemical screening. Dry material was grounded and extracted with chloroform and methanol using soxhlet apparatus. The extract was then dried for the next step of experiment.
Methods Extract preparation
The material and tools for anatomical observation were methanol 70%, 1% safranin, glycerin, sliding microtome, hotplate, glass slide, microscope and canon digital camera.
The material and tools for phytochemi- cal screening were methanol and chloroform p.a. grade, TLC plate (silica gel GF254), Dragen- dorffs reagent, Citroburat reagent, Anisaldehid reagent, TLC chamber, hairdryer, UV light 254 and 365 nm, and canon digital camera.
The material and tools for toxicity test were shrimp Artemia salina Leach. eggs col- lected form Samas Bantul, sea water, dimethyl sulfoxide (DMSO), tray, microtube, and fluo- rescen light 18 watt.
The materials and tools for tuber extrac- tion were chloroform and methanol p.a. grade, soxhlet, analytical balance, and petridisc.
The main material for the research was yam (Dioscorea hispida Dennst.) tuber collect- ed from Timor Leste, Kalimantan Tengah, and D.I.Yogyakarta.
Container to incubate the eggs is a shal- low rectangular plastic box. The container was divided into two compartments, dark and light compartment. L. Hartanto Nugroho and Anna Estyaniyana
The mobile phases were then poured into TLC RESULTS AND DISCUSSION chamber and saturated for ± 15 minutes in order to achieve optimum elution conditions Toxicity Test with Brine Shrimp Lethality
Test (BSLT)
(Harborne, 1987). Each accession of yam tu- ber dry extract was diluted with the same sol- This research was carried out in various vent. With a capillary pipette, extract was spot- stages i.e. egg hatching Artemia salina Leach., ted on the silica gel GF with a distance of 1 preparation of the test sample concentration,
254 cm between the spot. Then, the spotted plate and toxicity testing using the method of Meyer.
was put in the TLC chamber. The chamber is The results of toxicity tests are listed in the fol- closed and left until the mobile phase up to the lowing table. upper limit of 0.5 cm from the top edge of the plate. After the plate was dried with a hairdry- er, the next process was the staining. To detect the presence of alkaloids, flavonoids and ter- penoids plate were sprayed with Dragendorf, Sitroborat and Anisaldehid sulfate reagent re- spectively. Furthermore the compound spots were recorded using a canon digital camera under visible light, UV light 254 nm and 366 nm, also before and after spraying with the re- agent.
Characterization of yam tuber starch
Slides were made directly from fresh yam tuber. Yam tubers were stabbed repeat- edly with needles to come out viscous white fluid. White viscous fluid was the spotted on glass objects, diluted with distilled water and then covered with a glass cover. Slide observa- tions were performed using a nikon light mi- croscope with various objective lens magni- fication. The observed anatomical characters were: the form of starch grains, starch aggre- gates form, the existence of the hilum, and the existence of lamellae. To observe the starch ag- gregate amount of each cell in the cortex and pith parenchyma cells, the amount of starch grains in each aggregate, the aggregate size of the starch and starch grain size were prepared by making fresh transverse yam tubers. Cal- culation of aggregate size and grain starch is done with Corel DRAW Graphics Suite X5 soft- ware for Windows. Each measurement was made with 10 replications.
KALIMANTAN TENGAH AND DAERAH ISTIMEWA YOGYAKARTA
33
Toksisitas, Kandungan Fitokimia dan Karakter Pati Aksesi Gadung(Dioscorea hispida Dennst.) Timor Leste, Kalimantan Tengah dan Daerah Istimewa Yogyakarta
TOXICITY, PHYTOCHEMICAL CONTENTS AND STARCH CHARACTERS OF GADUNG (Dioscorea hispida Dennst.) TUBER ACCESSION TIMOR LESTE,
26 Control Non toxic DMSO + see water
10
23
26 D.I.Yogyakarta 1000 20 >1000 Non toxic 500 13 100
10
23
20 Kalimantan Tengah 1000 20 >1000 Non Toxic 500 10 100
10
20
20 Methanol Timor Leste 1000 26 >1000 Non toxic 500 26 100
10
Toxicity tests with Brine Shrimp Lethal- ity Test (BSLT) is early screening for the active compounds contained in plant extracts using shrimp Artemia salina larvae as bio-indicators. Based on Table 1., the chloroform extract of yam tuber accession Timor Leste at a concen- tration of 10 µg/mL was known could kill half (50%) of the amount of shrimp larvae tested.
Therefore, chloroform extract of yam tuber ac- cession Timor Leste could be said to have LC
10
50
50 Kalimantan Tengah 1000 63 100 Toxic 500 40 100
10
53
10 Toxic 500 56 100
65
Ectract Yum Extract Mortality LC50 Note accession concentration (%) (µg/mL) (ppm) Chloroform Timor Leste 1000
50 Table 1. Toxicity Test of chloroform and methanol extract of yam tuber with Brine Shrimp
Lethality Test (BSLT)
Shrimp Lethality Test (BSLT)val- ues of less than 1.000 µg/mL, while the pure compound said to be toxic if it had LC
50
Chloroform extract of yam tuber accession Kalimantan Tengah is able to kill half of the tested shrimp larvae at a concentration of 100 µg/mL. While the chloroform extract of the tu- ber accession DIYogyakarta able to kill half of the tested shrimp larvae at a concentration of 1.000 µg/mL. According to Meyer et al. (1982), a toxic extract is an extract wich have LC
50 of 10 µg/mL.
23 D.I.Yogyakarta 1000 50 1000 Non toxic 500 33 100 L. Hartanto Nugroho and Anna Estyaniyana
in Thin Layer Chromatography method. In cur- values of less than 200 µg/mL. In conclusion, the chloroform extract of yam tuber accession rent experiment, mobile phase used to sepa- Timor Leste and Kalimantan Tengah toxic to rate and detect the presence of alkaloids in the larval shrimp Artemia salina Leach as having extracts of yam tuber was a combination of tol- uene, ethyl acetate, and diethyl amine. Mobile
LC50 values below 1.000 µg/mL, while the phase used is a combination of non-polar and yam tuber accession D.I. Yogyakarta wasnot polar compounds. It is expected that the non- toxic to shrimp larvae. Furthermore, different concentration levels of the tuber methanol ex- polar, semi-polar, polar compounds would be able to separate through the TLC plate opti- tract were not able to kill half (50%) of the total number of shrimp larvae tested. In fact, at the mally. The stationary phase used was silica gel . highest concentration level (1.000 µg/mL) of
60 F
254
tuber methanol extract of the three accessions With the presence of chromophore group, plates were observed under UV light only able to kill about 21% of the total sample of shrimp larvae as a test animal. In conclusion, with a wavelength of 254 nm and 366 nm and the methanol extract of three accessions yam would give a certain color response. The plate tubers tested by similar methods showed have was then sprayed using Dragendorf reagent to no toxic properties. The negative control con- detect the presence of alkaloids. Results of al- kaloids detection in the chloroform extract of tained blank solution (dimethyl sulfoxide, and sea water) were also applied. Control results yam tubers can be seen in Figure 1. showed mortality of 0%, which means no dead shrimp larvae in the control solution. Death of shrimp larvae revealed by observation using a magnifying glass and is indicated by the move- ment absence of shrimp larvae. According to Croghan (1958), the shrimp larvae mortality due to the presence of bioactive compounds into the body. The die of shrimp larvae in the sample solution thought to be affected by the toxic compounds contained in the extract, it was not caused of other factors. In addition, the body’s defense system in shrimp larvae have not been able to prevent and tolerate the active compounds that pollute the media of its
Figure 1. Chromatogram profile: (A) under UV light life.
λ 254 nm, (B) λ 366 nm, (C) under visible light after sprayed with Dragendorff Detection of Alkaloids, Flavonoids and Ter- reagent,(D) Monograf of chloroform yam extract. penoids using Thin Layer Chromatography
Note: a.) Accession D.I.Y., b.) Accession Kali (TLC) mantan Tengah, c.) Accession Timor Leste,
Detection of Alkaloids in Root Extract yam tu- d.) Quinin sulfat as a standard. ber
Right combination of organic solvent used for mobile phase is required to accommo- date the separation of secondary metabolites
From the TLC results, chromatogram profile under UV light 254 nm showed blue color fluorescence but under UV light 366 nm showed an invisible color fluoresce. The pro- file of the chromatogram after spraying with Dragendorffs reagents appeared orange stain.
From the results, it can be concluded that chlo- roform extract of three yam tuber accession positive for alkaloid compounds. This is in ac- cordance with the orange stain revealed qui- nine sulfate as a alkaloids standard.
Rf value (retention factor) is a value that indicates the location of the compound spots in the chromatogram. Rf value calculation is based on the distance traveled of dissolved compounds divided by distance of solvent traveled (Kealey & Haines, 2002). In the chlo- roform extract of yam tuber accession Timor Leste, Kalimantan Tengah and D.I.Yogyakarta contains two orange alkaloids spots with Rf value of 0.312 and 0.637. While quinine sulfate as the alkaloids standard has Rf value of 0.412
Based on Fig 2., the methanol extract of yam tuber accession of Timor Leste contains two alkaloids components with a Rf value of 0.312 and 0.65. In the methanol extract of yam tuber accession of Kalimantan Tengah contain one alkaloids components with the Rf value of 0.312, while the methanol extract of yam tuber accession D.I.Yogyakarta contains two alkaloid components with the Rf values of 0.312 and 0.65.
According to Kealey & Haines (2002), spot which had the same Rf values likely to contain the same components of secondary metabolites. So the difference of Rf value from each other can be interpreted as different com- ponents. Based on the results, the chloroform extract of yam tuber accession Timor Leste, Kalimantan Tengah and D.I.Yogyakarta contain similiar alkaloid componen with the Rf value of 0.312 and 0.637. It is not include the compo- nent of quinine sulfate.
Detection of Flavonoid in Root Extract yam tu- ber
The mobile phase used to detect the presence of flavonoids in the extract of yam tuber was a combination of ethyl acetate, for- mic acid, glacial acetic acid, and water while the stationary phase used was silica gel 60 F
254
. Upon elution ends, the plate was observed under UV light with a wavelength 254 nm and 366 nm then the plate was sprayed using a specific color reagent manely Sitroborat. From the TLC results, the profile chromatogram af- ter spraying using Sitroborat reagents showed the spot of flavonoid identified as a yellow spot which was the same color with spot of rutin, since the flavonoids standard used was rutin.
Figure 2. Chromatogram profile: (A) under UV light λ 254 nm, (B) λ 366 nm, (C) under fisible light after sprayed with Dragendorffs re agent, (D) Monograph of methanol yam extract. Note: a.) Accession DIY, b.) Accession Kalim antan Tengah, c.) Accession Timor Leste, d.) Quinin sulfat as a standard.
TOXICITY, PHYTOCHEMICAL CONTENTS AND STARCH CHARACTERS OF GADUNG (Dioscorea hispida Dennst.) TUBER ACCESSION TIMOR LESTE,
Toksisitas, Kandungan Fitokimia dan Karakter Pati Aksesi Gadung(Dioscorea hispida Dennst.) Timor Leste, Kalimantan Tengah dan Daerah Istimewa Yogyakarta
KALIMANTAN TENGAH AND DAERAH ISTIMEWA YOGYAKARTA
L. Hartanto Nugroho and Anna Estyaniyana
Based on Figure 4., the methanol extract of yam tuber accession Timor Leste, Kaliman- tan tengah and D.I.Yogyakarta contained two flavonoids components with Rf values of 0.225 and 0.963.
Detection of Terpenoid in Root Extract yam tu- ber
The mobile phase used to detect the presence of terpenoids on yam tuber extract was a combination of hexane and ethyl acetate. Both organic solvent are low polarity, thus it
Figure 3. Chromatogram profile: (A) under UV light
will minimize the absorption of the component
λ 254 nm, (B) λ 366 nm, (C) under visiblelight
solvent mixture. Additionally, mobile phase
after spraying with Sitroborat reagent, (D)
combination also increases the speed of mi- monograph of chloroform extract of yamtuber. gration of solvent (Wagner and Bladt, 1996).
Note: a.) Accession D.I.Yogyakarta, b.)Acces- sion Kalimantan Tengah, c.) Accession Timor
After developing ends, the plate was observed Leste, d.) Rutin as a standard. under UV light with a wavelength of 254 nm and 366 nm. Then the plate was sprayed using
Based on Figure 3., the chloroform ex- a Anisaldehid sulfuric acid reagent as a spe- tract of yam tuber accession Timor Leste, Ka- cific color reagents for terpenoids. Terpenoid limantan Tengan and D.I.Yogyakarta contained detection results on tuber yam extracts can be one flavonoid components with Rf value of seen in the following figure. 0.937, while rutin has Rf value of 0.52.
Figure 5. Chromatogram profile: (A) under UV light
Figure 4. Chromatogram profile: (A) under UV light λ 254 nm, (B) λ 366 nm, (C) under visible light
after spraying with Sitroborat reagent, λ 254 nm, (B) λ 366 nm, (C) under visible light after spraying with reagent Sitroborat, (D) monograph of chloroform extract of yam- tuber. (D)monograph of methanol extract of yamtuber.Note: a.) Accession D.I.Yogyakarta, b.) Ac- cession Kalimantan Tengah, c.) Accession Note : a.) Accession D.I.Yogyakarta, b.)
Accession Kalimantan Tengah, c.) Acces Timor Leste, d.) Tymol as a standard. sion Timor Leste, d.)Rutin as a standard. From the TLC results, profile chromato- gram after spraying using Anisaldehid sulfu- ric acid reagent showed positive results in the form of purple stain. This is in accordance with the purple color of tymol used as a standard. Based on Figure 5., the chloroform extract of yam tuber accession Timor Leste and Kalim- antan Tengah contains 3 terpenoids compo- nents with Rf value of 0.187; 0.25 and 0.375, while the accession D.I.Yogyakarta contain two terpenoids component with Rf value of 0.187 and 0.25. however, tymol Rf value was Rf 0.56.
Figure 6. Chromatogram profile: (A) under UV light λ 254 nm, (B) λ 366 nm, (C) under visible light after spraying with Sitrobo rat reagent, (D) monograph of methanol extract of yam tuber. Note: a.) Accession D.I.Yogyakarta, b.) Ac cession Kalimantan Tengah, c.) Accession Timor Leste, d.) Tymol as a standard.
Based on Figure 6., methanol extract of three accessions tubers did not contain terpe- noid components (non-detected). This occur because of the possibility at the time of extrac- tion using methanol solvent, the solvent is not able to bind existing terpenoid compounds in yam tuber.
Overall, the chloroform extract of three accessions yam tuber contained alkaloids, fla- vonoids and terpenoids. While the methanol ex- tract containing only alkaloids and flavonoids. Among the classes of these compounds, one is predicted to be toxic was terpenoid.
This is supported by the results of the phy- tochemical screening of the methanol extract of the three accessions yam tuber that con- tain only alkaloid and flavonoid compounds. It did not contain terpenoids. Therefore, when methanol extract of three accessions yam tu- bers were tested with BSLT, the extract did not toxic to the larvae of shrimp Artemia salina Leach. Research of Indriyani et al
. (2006) re- ported that the leaf extract of pecut kuda toxic to larvae of Artemia salina Leach. The analyses showed that the leaf contained terpenoid. Ter- penoid is also known as one class of chemical compounds in the plant extracts which have anticancer and antioxidant activity (Lisda- wati, 2002). In conclusion, extracts of which are toxic and are assumed to have potential as anti-cancer was yam tuber chloroform extract accession Timor Leste and Kalimantan Ten- gah because they contain characteristic terpe- noids.
Starch Character of yam tuber AccessionTimor Leste, Kalimantan Tengah and D.I. Yogyakarta
This study has been carried out to ob- serve the character of three accession of yam tuber starch. Results obtained were qualitative and quantitative characters. Qualitative char- acters include starch grain structure, the pres- ence of lamella and hilum, and color of grain starch. Quantitative characters were starch grain size, starch aggregate size, and the num- ber of aggregates in each tuber parenchyma cells. The following data were characteristics of tuber starch in the three yam accessions.
TOXICITY, PHYTOCHEMICAL CONTENTS AND STARCH CHARACTERS OF GADUNG (Dioscorea hispida Dennst.) TUBER ACCESSION TIMOR LESTE,
Toksisitas, Kandungan Fitokimia dan Karakter Pati Aksesi Gadung(Dioscorea hispida Dennst.) Timor Leste, Kalimantan Tengah dan Daerah Istimewa Yogyakarta
KALIMANTAN TENGAH AND DAERAH ISTIMEWA YOGYAKARTA
L. Hartanto Nugroho and Anna Estyaniyana No. Character
Dioscorea hispida Dennst. Accession: Timor Leste Kalimantan Tengah D.I.Yogyakarta
1.Length of starch aggregate (µm) 2,14 ± 0,339 3,11±0,155 4,97 ± 0,486
2.Number of starch aggre-gate in each parencym cell 15 ± 0,447 20 ± 2,549 11 ± 1,643
3.Structure of starch polyhedral polyhedral polyhedral
4.Length of starch grain (µm) 0,55 ± 0,096 0,42 ± 0,061 1 ± 0,105
5.Lamela position consentris consentris consentris
6.Hilum position eccentris eccentris eccentris
Starch is a carbohydrate or polysaccha- ride in the plastids. Forming plastids called leu- koamiloplas. Leukoamiloplas most commonly found in the cortex and pith parenchyma tis- sue of tubers. According to Wallis (1957), ag- gregation of starch can be a single starch grains (simple grains), grain starch compound (com- pound grains), and compound starch grains which have gathered lamella to form a clump (semi-compound grains).
A B C
Figure 7. . Starch types: simple grain (A.), semi-compound grain (B.), and compound grain (C).
Wallis (1957).
Based on the observation, the grains of starch in tubers of yam joined to form a single entity called the aggregate structure. Aggregate starch is a structure composed of a collection of small starch grains. In aggregate yam tuber starch grains are believed to form clumps com- pound wrapped with lamella. Santosa (1989) stated that semi-compound starch had more than one hilus and each hilus was wrapped by its own lamella followed by outer lamella wrapped all of hilum.
Figure 8. Starch agregate structure of yam (Dioscorea hispida Dennst. ) tuber Accession Timor Leste.
KALIMANTAN TENGAH AND DAERAH ISTIMEWA YOGYAKARTA
The authors would like to thank to Elisio Da Costa Netto (Alumni of Biology Postgradu- ate School Program, Universitas Gadjah Mada) for donating yam tuber accession Timor Les- te.
Toksisitas, Kandungan Fitokimia dan Karakter Pati Aksesi Gadung(Dioscorea hispida Dennst.) Timor Leste, Kalimantan Tengah dan Daerah Istimewa Yogyakarta
Daya Air Tanah dan Geologi Lingkungan (PAG)Kabupaten Sleman Daerah TOXICITY, PHYTOCHEMICAL CONTENTS AND STARCH CHARACTERS OF GADUNG (Dioscorea hispida Dennst.) TUBER ACCESSION TIMOR LESTE,
Anonymous 2 , 2013. Database Pusat Sumber
org/wp-content/2012/12/Geology- and-Soils-in-Timor-Leste. html. Ac- cessed 20 th February 2013
Timor Leste . http://seedsoflifetimor.
Anonymous 1 , 2013. Geology and Soils in
REFERENCES
From Table 2., it can be seen that quali- tative characters of three accession of yam tuber showed similar each other. However, quantitative data showed that starch of yam tuber accession D.I.Yogyakarta was the biggest compared to others. According to Salisbury & Ross (1995), the amount of starch in various tissues depends on genetic factors (species) and the environmental conditions.
Aggregate starch tuber accession Timor- Leste has smallest number and diameter of the aggregate starch compared to two other ac- cessions. Based on the data, the average yield of dried sample weight against fresh tuber weight of Timor Leste accession was 31.03%.
Based on the results and discussion, it can be concluded that highest toxicity derived from the chloroform extract of yam tuber ac- cession Timor Leste with LC50 of 10 ppm. Methanol extract of three accessions yam tu- bers did not toxic to Artemia salina Leach. The three accessions contain alkaloids, flovonoids and terpenoids. Active compounds are predict- ed of causing toxic was terpenoid. The tuber starch grains of tree accessions had a common polyhedral shape. The shape and size of starch aggregates have differences in their respective accession yam.
CONCLUSION
From the observations using fresh sam- ple, the type of yam tuber starch was eccentri- cally because the position of the hilum alleg- edly being on the edge, although not clearly observed (indistinct). This is supported by Seidemann (1964) in Brunnschweiler (2004) which examines the characteristics of starch tuber Dioscorea spp., showed that several spe- cies of Dioscorea had starch granule-type ec- centrically because the hilus was on the edge.
alata.
According Tetchi et al . (2012), factors such as cultivars or varieties, storage time and differences parts tubers sampled indirectly af- fect the size of the starch grains in Dioscorea
Tuber accession D.I. Yogyakarta has the bigest number and diameter of the aggregate starch compared to the two other accessions. Based on the data, the average yield of dried sample weight against fresh tuber weight of D.I.Yogyakarta accession was 10.58%. This is because the environmental conditions of the area where the sample grown is volcanic soil type. The volcanic soil is soil material that is formed from the volcanic eruptions that have undergone weathering (Anonymous 2 , 2013). The land is very fertile because it contains a lot of nutrients and soil water nutrients needed by plants. Yam that grows in moist environments, enough water, and enough light, will produce photosynthetic product higher than yam that grows in poor neighborhoods the water.
This is because the land of Buibau village is vertisol soil type. Vertisol soil is kind of a dark- colored clay containing minerals (smectite) in bulk (Anonymous 1 , 2013) During the dry sea- son the soil is cracked and become very sticky in the rainy season. Therefore, the type of soil drainage is impeded so that the possibility of soil water availability is less.
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