Kalanduyung Leaf Bioactives and Secondary Metabolites (Trema tomentosa

(Roxb.)Hara)

Intan Wulan Sari1,* , Nur Masyithah Zamruddin2, Laode Rijai3

Pharmaceutical Research and Development Laboratory “TROPIS FARMAKA”,Faculty of
Pharmacy, Mulawarman University, Samarinda
East Kalimantan, Indonesia
*E-mail correspondence: intanwulan789@gmail.com

Abstract

Kalanduyung leaf (Trema tomentosa (Roxb.) Hara) is a type of wild plant that grows in
the garden or around the forest, based on empirical research from the Buton people,
kalanduyung leaves are used as cough medicine. The purpose of this study was to determine
the % yield of kalanduyung leaf extract, as well as to determine secondary metabolite
compounds and toxicity activity using the BSLT (Brine Shrimp Lethality Test) method. This
laboratory experimental study used 210 larvae of Artemia Salina Leach shrimp which has 7
groups, where 1 group is a negative control and 6 groups is a test solution consisting of 5
ppm, 125 ppm, 250 ppm, 500 ppm, and 1000 ppm. Each group made 3 replications. Based on
the results obtained, the % yield obtained was 12.2% and kalanduyung leaves contained the
following secondary metabolites, namely alkaloids, phenolics, flavonoids, tannins, and
saponins. The results of the toxicity test obtained LC50 values of 120,723 ppm with probit
analysis and 168,8107 ppm with reed & muench analysis. The LC50 results obtained have the
potential for toxicity to Artemia Salina Leach larvae as indicated by the LC50 value < 1000
ppm.

Keywords: Toxicity test, BSLT, kalanduyung leaf (Trema tomentosa (Roxb.)Hara)

1. Introduction
Indonesia has many islands, where there are many forests scattered from Sabang to
Merauke, which contain many wild plants that grow in these forests. However, the
community's focus is not on these wild plants that have no efficacy according to the
community. Most people only have a focus on plants that are nutritious as vegetables,
ornamental plants, and others.
Kalanduyung (Trema tomentosa (Roxb.)Hara) is one of the most common plants found in
the planting area, which acts as a wild plant on farmers' land. Based on empirical studies of the
Buton people, Southeast Sulawesi, this kalanduyung plant is used as cough medicine.
However, for this plant has not been studied by many researchers.
Cytotoxicity test method BSLT (Brine Shrimp Lethality is a preliminary test to determine
the bioactivity of a sample. This test is useful for determining various biological activities in
plants such as cytotoxic activity, phototoxicity, pesticides, enzyme inhibition, and ion
regulation[10].
 BSLT (Brine Shrimp Lethality Test) is one method that working method to search for
new anticancer compounds derived from plants. The BSLT method has been shown to have a
correlation with anticancer activity. In addition, this method is easy to work with, cheap, fast,
and quite accurate

2. Research Method
2.1 Tools and materials
The tools used are analytical scales, oven, blender, glass and non-glass equipment, rotary
evaporator, dry cabinet, aerator, lamp, sonicator, UV-VIS spectrophotometry.

The material used is kalanduyung leaf (Trema tomentosa (Roxb.) Hara) which is taken
from the peaceful alley plantation area, Sidopeace Sub-district, Samarinda. Plant identification
was determined at the Laboratory of Ecology and Conservation of Tropical Forest
Biodiversity (Be-Force) Faculty of Forestry, Mulawarman University. Seawater, yeast, larvae
eggs of Artemia salina L, Methanol (CH3OH), Dimethyl Sulfoxide (DMSO), hydrochloric
acid (HCl), sulfuric acid (H2SO4), DPPH (2,2-diphenyl-1-picryl-hydrazyl), chloroform
(CHCl3), Wegner's reagent, Dragendroff's reagent, Meyer's reagent, Lieberman-Burchard
reagent, Mg powder.

2.2 Procedure
2.2.1 Extraction
A total of 2.5 kg of fresh samples of kalanduyung (Trema tomentosa (Roxb.)Hara) leaves
were sorted wet, washed with running water, then air-dried and dried in an oven for 3 hours at
50°C. The dry sample was then mashed using a blender and put into a glass jar and added 3 L
of methanol. Extraction was carried out using the maceration method where the sample was
soaked for 3x24 hours using methanol as solvent.

2.2.2 Phytochemical Test

2.2.2.1 Identification of Alkaloids
Identification of the alkaloid compound was taken as much as 1 mL of kalanduyung leaf
extract (Trema tomentosa (Roxb.)Hara) added with 3 drops of 10% ammonia and 1.5 mL of
chloroform, then shaken. The chloroform layer was taken and then dissolved in 1 mL of 2 N
sulfuric acid, then shaken. After that, the extract was added with the reagent. The formation of
a white precipitate using Meyer's reagent indicates the presence of alkaloid compounds [4].
The formation of a positive brown precipitate with Wagner's reagent indicates the presence of
alkaloids. Dragendorff's reagent, there is a reddish-purple brown/white precipitate which is
positive for the presence of alkaloids. The formation of an orange precipitate in the second
tube and a white to yellowish precipitate in the third tube indicated the presence of alkaloid
compounds in the sample [6].
2.2.2.2 Identification of flavonoids
The flavonoid test was carried out by means of extract of kalanduyung (Trema tomentosa
(Roxb.)Hara) leaves taken 0.05 g and dropped into 2 test tubes. One part of the test tube as a
control, then the rest can be identified by adding 0.1 mg to 1-2 grains of magnesium metal
powder and a few drops of concentrated chloride (HCl). The formation of orange, purple,
yellow, pink to red colors indicates the presence of flavonoid compounds [7].

2.2.2.3 Identification of tannins/polyphenols

Testing of phenolic compounds was carried out by taking 1 mL of kalanduyung (Trema
tomentosa (Roxb.)Hara) leaf extract and placing it in 2 test tubes. One part of the test tube was
used as a control and the rest was dripped with FeCl3 solution. Phenol is positive when a blue
to black color appears. Furthermore, for the tannin test, 2 mL of sponge sample extract was
added and 100 mL of hot water was added, boiled for 15 minutes and then filtered into 5 mL
of 1% FeCl3 and gelatin solution. A positive result (identified tannin) indicates the formation
of a white or pink precipitate [3].

2.2.2.4 Identification of steroids and terpenoids

Identification of steroid and terpenoid compounds as much as 1 mL of kalanduyung leaf
extract (Trema tomentosa (Roxb.)Hara) added with 2 mL of chloroform in a test tube, then
dropped into a drip plate, and allowed to dry. After that, 1 drop of Lieberman-Burchard
reagent was added. The formation of brown color indicates the presence of terpenoid
compounds and the formation of blue or purple color indicates the presence of steroid
compounds [4].

2.2.2.5 Identification of Saponins

Identification of saponin compounds, namely 1 mL of kalanduyung leaf extract (Trema
tomentosa (Roxb.)Hara) added with 20 mL of distilled water, then heated for 5 minutes.
Transfer the solution to a hot test tube. A total of 10 mL of the solution then shake vigorously
vertically for 10 seconds. The presence of saponins is indicated by forming a stable foam as
high as 1-10 cm for 10 minutes and does not disappear when added with one drop of 2 N HCl
[4].

2.2.3 Testing cytotoxic activity by BSLT (Brine Shrimp Lethality Test) method
2.2.3.1 Hatching of Artemia salina L . shrimp larvae
Add 3 L of seawater then add yeast and dissolve it in seawater. Then add the weighed
Artemia salina L larvae eggs. Install the aerator and turn on the lights to light the eggs of
Artemia salina L larvae.
2.2.3.2 Preparation of stock solution 1000 ppm
The stock solution was made by weighing 10 mg of kalanduyung (Trema tomentosa
(Roxb.)Hara) leaf extract, then 1 mL of DMSO was added, stirred until dissolved, then 9 mL
of methanol was added and homogenized.
2.2.3.3 Preparation of test solution
Making a 500 ppm test solution by taking 2 mL of the stock solution then adding 2 mL
of seawater and then homogenizing it, making a 250 ppm test solution by taking 2 mL of a
500 ppm test solution then adding 2 mL of seawater and then homogenizing it, and so on until
a concentration of 31.25 ppm.
2.2.3.4 Cytotoxic test using BSLT (Brine Shrimp Lethality Test) method
In the toxicity test, each concentration was carried out 3 repetitions with each group
having 10 Artemia salina larvae. Prepared containers for Furthermore, each concentration of
solution should have 10 larvae of Artemia salina. Observation for 24 hours on the mortality of
Artemia salina larvae, ie each concentration was carried out 3 repetitions and compared with
the control. The standard criterion for assessing the mortality of Artemia salina larvae is if the
larvae do not show movement for several seconds of observation[9].

3 . Results and Discussion

3.1 Yield
The results of simplicia as much as 500 grams were then extracted and concentrated using
a rotary evaporator and produced a dry extract of 61 grams. After that the yield is calculated
using the yield formula:

The yield obtained in the methanol extract of kalanduyug (Trema tomentosa (Roxb.)Hara)
leaves was 12.2%. The greater the yield obtained, the more secondary metabolites contained
therein.

3.2 Phytochemical Screening Results

The results of phytochemical screening showed that the kalanduyung leaf extract
contained some of the secondary metabolites. because most of the alkaloid compounds
contained are semipolar and nonpolar, while the flavonoid compounds contained are polar and
semipolar. Results in the table 1
Table. 1 phytochemical test results

Compound Methanol Positive Reaction

Group Extract
Alkaloids + An orange precipitate is formed
in Dragendorph's reagent

Flavonoids +++ Color change to orange

Tannins +++ Color change to dark blue

Steroids - No color change

Terpenoids - No color change
Saponins +++ Foam formed
 3.2.1 Alkaloids
Alkaloids tested using Dragendorff's reagent will produce an orange precipitate, whereas
Mayer's reagent will produce a yellowish-white precipitate, the addition of hydrochloric acid
aims to extract alkaline alkaloids using an acid solution [2,6]. The results of phytochemical
screening showed the presence of sediment, which means that the methanolic extract of the
leaves of Kalanduyung (Trema tomentosa (Roxb.)Hara) contained alkaloid compounds.
3.2.2 Flavonoids
We can determine the presence of flavonoids using concentrated Mg and HCl. Flavonoid
compounds can produce red, yellow or orange colors when reduced with Mg and HCl [8]. The
results of phytochemical screening showed that the methanolic extract of kalanduyung (Trema
tomentosa (Roxb.)Hara) leaves was orange in color and positive for flavonoid compounds.
3.2.3 Tannins
Identification of tannin compounds was carried out by adding FeCl3. Tannin compounds
are polar compounds due to the presence of an OH group, when 10% FeCl3 is added there will
be a change in colors such as dark blue or blackish green which indicates the presence of
tannincompounds [6].
Hydrolyzed tannins will show a blue-black color while condensed tannins will show a
green-black color when added with FeCl3. From the results of phytochemical screening on the
methanol extract of the leaves of kalanduyung (Trema tomentosa (Roxb.) Hara), the results
obtained a green-black color which means that it is positive for condensed tannins [8].
3.2.4 Steroids and terpenoids
Testing of steroids and terpenoids in glacial CH3COOH with concentrated H2SO4 was
based on the ability of steroid and terpenoid compounds to form blue or green colors for
steroids, and red or purple for triterpenoids. Steroids and triterpenoids are compounds that can
be extracted with non-polar or semi-polar solvents [5]. The results of phytochemical screening
showed that the methanol extract did not give a color change and was negative for steroids.
Nothing was formed because the methanol extract of kalanduyung (Trema tomentosa
(Roxb.)Hara) leaves did not contain steroid and triterpenoid compounds.
3.2.5 Saponins
Saponins are surface-active compounds that are easily detected through their ability to
form foam. The components of glycoside bonds contained in saponins cause these compounds
to tend to be polar [5,8]. The presence of saponins was positive if the tested extract formed
foam as high as 1-10cm with an interval of ±10 minutes [1]. Results of phytochemical
screening showed that the methanolic extract of kalanduyung (Trema tomentosa (Roxb.)Hara)
leaves was positive for saponins with a positive reaction to form foam..

3.3 Toxicity Test by BSLT (Brine Shirmp Lethality Test) method

Brine Shrimp Lethaly Test (BSLT) was conducted to determine the cytotoxic activity of a
sample of natural materials. This method is an initial test used to determine anticancer activity
based on the ability of a sample to kill shrimp larvae (Artemia salina).
Toxicity test was carried out using the BSLT (Brine Shrimp Lethality Test) method on
Artemia salina L larvae, the BSLT method was chosen because the process does not require a
lot of cost, and the processing time is short. Artemia salina L larvae were used in this test
because of their small size and easy handling.
 The toxicity test of the methanolic extract of kalanduyung (Trema tomentosa (Roxb.)Hara)
leaves was carried out using the BSLT (Brine Shrimp Lethality Test) method on 48-hour-old
Artemia salina L larvae. The concentrations used in this test were 31.25 ppm, 62.5 ppm, 125
ppm, 250 ppm, 500 ppm, and 1000 ppm. Toxicity test results in tables 2 and 3

Table 2 Results of Toxicity Test Data Using Probit Analysis

Replication Number of dead at sample concentration (ppm)

Control 31,2 62,5 125 250 500 1000

1 0 2 3 4 4 7 10
2 0 3 4 3 5 8 10
3 0 2 3 4 5 7 10
Total - 7 10 11 14 22 30
Inhibition

%inhibition - 23,33 33,33 36,66 46,66 73,33 100

% % % % % %
Probit price 0 4,269 4,569 4,659 4,913 5,619 8,09
LC50 = 120,7 ppm

Table 3 Results of Toxicity Test Data With Reed & Muench Analysis

Ratio
C LogC Amount Accumulated Morality
(ppm) X %
(x+y)
Mati Hidup X Y Jumlah

31,2 1,49 7 23 7 86 93 0,07 7%

62,5 1,79 10 20 17 63 80 0,21 21%
125 2,10 11 19 28 43 71 0,39 39%
250 2,40 14 16 42 24 66 0,63 63%
500 2,70 22 8 64 8 72 0,88 88%
1000 3 10 0 74 0 74 1 100%
LC50 =168,81 ppm
 Based on table 2, it can be seen that the LC50 value of 120.7 ppm was analyzed using
probit analysis. The extract was declared toxic because it was in the range <1000 ppm
including the toxic category. Thus, it can be said that in a concentration of 94,263 ppm,
methanol extract of kalanduyung (Trema tomentosa (Roxb.)Hara) leaves can kill 50% of
Artemia salina L.
Based on table 3, it can be seen that the LC50 value of 168.81 ppm was analyzed using
probit analysis. The extract was declared toxic because it was included in <1000 ppm
including the toxic category. So it can be said that the test results with reed & muench analysis
have activity in the toxic category

4. Conclusion
Based on the results obtained, it can a conclusion that the methanol extract of
kalanduyung (Trema tomentosa (Roxb.)Hara) leaves has a yield weight of 12.2% and contains
secondary metabolites, including alkaloids, flavonoids, tannins, and saponins which means no
contains steroid and terpenoid compounds and has cytotoxic activity with a value of 120, ppm
with probit analysis and 168,81 ppm with reed & muench analysis which means that
kalanduyung leaf extract has cytotoxic activity

5. Acknowledgment
The author would like to thank Allah SWT because of his blessings and guidance, the
author was given health to do activities, and thanks to Mrs. Nur Masyithah as a supervisor
who helped the writer during the research period, and to Mr. Laode Rijai as a supervisor who
always gave directions to the author so that he could arrive at this point. All parties who have
helped the smooth running of the research that the writer cannot mention one by one.

6. Conflict of Interest
There is no conflict of interest in this study.

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