Jurnal Ilmiah Ibnu Sina, 9.
Oktober 2024, 337-346 p-ISSN: 2502-647X.
e-ISSN: 2503-1902 TOXICITY TEST OF LIME PEEL EXTRACT (Citrus aurantifoli.
USING THE BRINE SHRIMP LETHALITY TEST (BSLT) METHOD FOR DETECTION OF ANTI CANCER ACTIVITY Indah Khairunnisa*.
Endang Linirin Widiastuti.
Nuning Nurcahyani Department of Biology.
Faculty of Mathematics and Natural Sciences.
University of Lampung.
Lampung *Email: indahkhairunnisa44@gmail.
Artikel diterima: 1 Juli 2024.
Disetujui: 27 Oktober 2024 DOI: https://doi.
org/10.
36387/jiis.
ABSTRAK
Kanker termasuk salah satu dari berbagai masalah utama yang terjadi pada masyarakat Indonesia dan juga dunia.
Kurangnya kesadaran masyarakat untuk deteksi dini menyebabkan terjadinya keterlambatan diagnosis.
Salah satu tanaman obat yang banyak dijumpai di Indonesia yaitu jeruk nipis (C.
Pada ekstrak kulit jeruk nipis, terdapat minyak atsiri yang tersusun dari D-Limonene .
94%), -pinen .
66%), -terpineol .
29%), dan terpinen-4-ol .
29%).
Senyawa-senyawa tersebut diketahui memiliki efek yang kuat sebagai anti kanker.
Pada penelitian ini digunakan Rancangan Acak Lengkap (RAL) dengan 5 perlakuan dan 5 kali pengulangan, yaitu 0, 125, 150, 250, dan 1000 ppm.
Metode penelitian yang digunakan yaitu uji fitokimia, analisis FTIR (Fourier-Transform Infrared Spectroscop.
, dan uji BSLT.
Nilai probit dianalisis dengan menggunakan SPSS untuk mendapatkan nilai LC50.
Ekstrak kulit jeruk nipis (C.
memiliki nilai LC50 331,533 ppm termasuk kategori toksik dan berpotensi sebagai agen anti Berdasarkan pada uji fitokimia dan FTIR kulit jeruk nipis (C.
mengandung senyawa alkaloid, flavonoid, dan tanin yang diketahui memiliki sifat antioksidan dan dapat mengganggu keseimbangan Reactive Oxygen Species (ROS) sehingga dapat memicu apoptosis sel kanker.
Kata kunci: Anti kanker.
Artemia salina.
Brine Shrimp Lethality Test (BSLT).
FTIR
ABSTRACT
Cancer is one of the major problems affecting the people of Indonesia and the Lack of public awareness for early detection causes a delay in diagnosis.
One of the medicinal plants that are commonly found in Indonesia is lime (C.
In lime peel extract, there is essential oil composed of D-Limonene .
94%), -pinen .
66%), -terpineol .
29%), and terpinen-4-ol .
32%).
These compounds are known to have strong effects as anti-cancer.
This study used a completely randomized design (CRD) with 5 treatments and 5 repetitions, namely 0, 125, 150, 250, and 1000 ppm.
The research methods used were phytochemical Indah Khairunnisa, dkk | 337 Jurnal Ilmiah Ibnu Sina, 9.
Oktober 2024, 337-346 p-ISSN: 2502-647X.
e-ISSN: 2503-1902 test.
FTIR (Fourier-Transform Infrared Spectroscop.
analysis, and BSLT test.
The probit value was analyzed using SPSS to obtain the LC50 value.
Lime peel extract (C.
has an LC50 value of 331.
533 ppm including the toxic category and has potential as an anti-cancer agent.
Based on phytochemical and FTIR tests, lime peel (C.
contains alkaloid, flavonoid, and tannin compounds which are known to have antioxidant properties and can disrupt the balance of Reactive Oxygen Species (ROS) so as to trigger apoptosis of cancer cells.
Keywords: Anti-cancer.
Artemia salina.
Brine Shrimp Lethality Test (BSLT).
FTIR
INTRODUCTION
According to GLOBOCAN, the potential as anti-cancer agents (Elisya International Agency for Research on plants that are commonly found is Cancer (IARC) there were 14,067,894 Lime contains saponin and new cases of cancer and 8,201,575 alkaloid compounds and has an IC50 deaths in 2012.
According to the 589 ug/ml using DPPH assay.
Total Health Research and Development phenol content of lime is 116.
5 mg Agency of the Indonesian Ministry of GAE/100ml (Permata et al, 2.
et al, 2.
One of the medicinal Health in 2018 through the Basic In lime peel extract, there are Health Research questionnaire that has been conducted, it was known that the Limonene prevalence of cancer in Indonesia is .
66%), -terpineol .
29%), and 8% at all age levels.
Generally, this terpinen-4-ol .
32%).
(Wahyudi et occurs due to a lack of public awareness for early detection, causing flavonoids, alkaloids, saponins, and delays in diagnosis (Hidayati and essential oils from phytochemical Akrom, 2.
Indonesia is known as an .
94%),
Lime -pinen (Rahmatullah et al, 2.
agricultural country that has various Based on research by Sari et al types of plants that are abundant.
, the ethanolic extract of lime Various types of plants can be used as peel has antioxidant activity of IC50 medicine because they contain active 52 ppm with the ethanolic fraction compounds and some types have the Indah Khairunnisa, dkk | 338 Jurnal Ilmiah Ibnu Sina, 9.
Oktober 2024, 337-346 p-ISSN: 2502-647X.
e-ISSN: 2503-1902 Meanwhile, conducted by Novriyanti et al .
ethanol extract (C.
to Artemia salina using BSLT method.
lime peel extract has an IC50 5.
antioxidant activity using the DPPH
RESEARCH METHOD
Place and Time of Research This research was conducted -pinen from December 2023 - February 2024 affects the apoptosis process through at the Botany Laboratory.
Department the Caspase-8 pathway that binds to of Biology.
Faculty of Mathematics death receptors on the surface of and Natural Sciences.
University of cancer cells based on in silico Lampung.
molecular docking assay (Hairunisa et Research Design AAg/ml D-Limonene al, 2.
This research was conducted Brine Shrimp Lethality Test using a completely randomized design (BSLT) is one method that can be used (CRD) with 5 treatments and 5 to determine the toxicity of a material repetitions consisting of negative or compound of natural origin.
The control (K-) 0 ppm, 125 ppm, 250 results of the toxicity test carried out ppm, 500 ppm, and 1000 ppm.
The with the BSLT method are determined based on the Lethal Concentration concentration of 0 ppm only uses (LC.
of the number of Artemia sterile seawater without the use of any salina that die due to the effect of natural substances (Sepvina et al.
Sample Preparation This method is widely used Lime was obtained from Gading because it is relatively easy, relatively Rejo sub-district.
Pringsewu district.
cheap, fast, and also reliable.
The lime peel was cleaned, air dried.
This study was conducted to then oven at 60oC for A 2 days (Sari determine the level of toxicity of lime and Asri, 2.
Then, pulverized with peel (C.
using 96% a blender to make simplisia.
500 g of ethanol solvent so this research will be simplisia was macerated in 96% conducted toxicity test of lime peel ethanol solvent of 3.
75 L for 5 days, then filtered and the filtrate was re- Indah Khairunnisa, dkk | 339 Jurnal Ilmiah Ibnu Sina, 9.
Oktober 2024, 337-346 p-ISSN: 2502-647X.
e-ISSN: 2503-1902 soaked with 96% ethanol of 1.
25 L for transparent thin plate.
Next, the concentrated with a rotary evaporator sample was read using an FTIR tool.
at 50oC (Sapitri et al.
, 2.
The resulting chromatogram was Phytochemical Assay compared with the IR table.
The Phytochemical assay refers to Flavonoid Triterpenoid and Steroid Tannin Treatments Method 1: 2 ml of extract 2 drops of Dragendorff Method 2: 2 ml extract 2 drops HCl 2 drops Mayer 2 ml extract 0.
1 g Mg powder 5 drops HCl 2 ml extract 2 drops HCl 2 drops H2SO4 2 ml of extract 3 drops FeCl3 The aquarium was divided into two parts, the dark part and the light Table 1.
Phytochemical Assay Procedure Type of Test Alkaloid Artemia salina Egg Hatching Okvianingsih et al .
which is shown in Table 1.
Indicators Forms Dragendorff reagent and on Mayer The light part was lighted with a lamp equipped with an aerator to dissolve oxygen.
Then, the aquarium was filled with 500 ml of sterile Artemia salina eggs were weighed first for 100 mg.
Next.
Artemia eggs were put into the dark part of the aquarium and left for 48 Forms a reddishorange color Forms a red or purple color in the test and bluish green color in the steroid test.
Forms a greenishbrown or blue-black Identification of Compounds with FTIR Method Stock Solution Preparation The stock solution was prepared by taking 100 mg of ethanol extract of (C.
and dissolved in 100 ml of sterile seawater to obtain a stock solution with a concentration of 1000 ppm.
Then, the stock solution was re-diluted with concentrations of 125 ppm, 250 ppm, and 500 ppm by diluting sterile seawater to 5 ml (Widyasari et al.
2 mg of lime peel extract and 198 mg of KBr were weighed and Indah Khairunnisa, dkk | 340 Jurnal Ilmiah Ibnu Sina, 9.
Oktober 2024, 337-346 p-ISSN: 2502-647X.
e-ISSN: 2503-1902 Brine Shrimp Lethality Test (BSLT) Test tubes were prepared for each concentration, which are 0 ppm, 125 ppm, 250 ppm, 500 ppm, and 1000 ppm and each was repeated five For negative control, the Table 2.
Phytochemical Assay Results Type of Test Alkaloid Dragendorff Mayer Flavonoid Triterpenoid Steroid Tanin Result concentration of 0 ppm without the Based on Table 2, it is known addition of ethanol extract of lime that the ethanol extract of lime peel (C.
A total of 10 48-hour-old Artemia salina larvae were put into flavonoid, and tannin compounds.
each test tube.
Then, 3 mg of yeast was This is indicated by the formation of dissolved in 5 ml of seawater and added as much as 1 drop into the test Dragendorff reagent and a white tube as a food source for Artemia precipitate on the Mayer reagent salina larvae.
After that, the larvae which indicates that the lime peel (C.
were left to stand for 24 hours.
The aurantifoli.
contains alkaloids.
Then, number of dead larvae was calculated there is a change in color to reddish by subtracting the total number of orange which indicates the presence of larvae at each concentration from the flavonoid compounds and in the number of surviving larvae.
tannin test there is a change in color to greenish brown.
RESULT AND DISCUSSION
Analysis of FTIR Result The FTIR results of lime peel Phytochemical Assay Phytochemical assay conducted extract (C.
with 96% to determine the content of secondary ethanol solvent can be seen in Figure extract of lime peel (C.
can be seen in Table 2.
Indah Khairunnisa, dkk | 341 Jurnal Ilmiah Ibnu Sina, 9.
Oktober 2024, 337-346 p-ISSN: 2502-647X.
e-ISSN: 2503-1902 wave numbers 3000-3500 cm-1 there is a peak with a wide and sharp absorption at wave number 3265.
1 cm1 derived from O-H and N-H groups.
The peak at wave number 2124.
6 cm-1 interprets the stretching vibrations of the C-H bond.
The presence of a peak Figure 1.
FTIR results of lime peel extract (C.
at wave number 1640.
0 cm-1 indicates Based on Figure 1, lime peel carbonyl and ester compounds.
This extract has several peaks, each of compound gives a distinctive aroma to which has a wavelength frequency the lime peel (C.
The value described in Table 3.
bending vibration at wave number Table 3.
FTIR results of lime peel extract (C.
Wave Numbers .
Reference Mustapha Results et al Functional Groups the presence of C=H groups which are 5 cm-1 comes from the secondary alcohol group.
the peak at wave 8 cm-1 Compound stretching vibration of the C-O group.
Flavonoids Stretching O-H Stretching N-H Stretching C-H Stretching C=H Bending C-H Stretching C-O shows the groups composed of O-H.
C-H.
C=C.
Flavonoid.
Alkaloid and C=O groups, alkaloids have N-H functional groups, and tannins have CO functional groups (Berghuis et al.
Flavonoid Carbonyl.
Ester Based on this, lime peel extract (C.
contains flavonoid, alkaloid, and tannin compounds based Flavonoid on the FTIR.
The results obtained are in accordance with the phytochemical Tannin Functional group analysis was carried out using the FTIR method.
assay presented in Table 2.
Brine Shrimp Lethality Test (BSLT) Toxicity tests were conducted to determine the effect of lime peel Indah Khairunnisa, dkk | 342 Jurnal Ilmiah Ibnu Sina, 9.
Oktober 2024, 337-346 p-ISSN: 2502-647X.
e-ISSN: 2503-1902 extract (C.
on Artemia compound has the potential as an anti- salina larvae using the BSLT method.
cancer agent.
This indicates that the The average mortality of Artemia ethanol extract of lime peel (C.
salina larvae can be seen in Table 4.
is toxic to Artemia salina Table 4.
Number of deaths of Artemia salina larvae after 24 hours larvae and has potential as an anti- Concen Number of LC50 Deaths .
0,0A0,00a 3,4A1,14b 5,8A0,84c 331,533 8,0A0,71d 10,0A0,00e Notes: letters a,b,c,d, and e indicate significant differences by LSD .
<0.
cancer agent.
Artemia salina has been widely used in many toxicity tests, such as nanoparticles, bioactive molecules, natural extracts, and metal materials (Banti and Hadjikakou, 2.
This is Based on Table 4, the results show that the concentration of 1000 ppm is the highest average larval mortality, while in the control there is no death of Artemia salina larvae.
The calculation results using SPSS showed that the ethanol extract of lime peel (C.
has an LC50 value of The concentration of the extract tested, the higher the death of Artemia salina larvae which can be seen in Table 4.
Based on research conducted by Setianingsih et al .
that the level categorized into highly toxic (LC50 < 30 pp.
, toxic (LC50 < 1000 pp.
, and non-toxic (LC50 > 1000 pp.
the LC50 value < 1000 ppm, the because Artemia salina has the ability to survive in unfavorable conditions by forming dormancy cysts (Ntungwe et al.
, 2.
In addition.
Artemia salina is known to have the Heat Shock Proteins 70 (Hsp.
gene or a group of proteins that respond to stress conditions, such as high temperature, infection, and toxins (Junprung et al.
Because it has the same type of genes as cancer cells that can be expressed under the same conditions.
Artemia salina can be used as a toxicity test animal (Ntungwe et al.
Compounds found in lime peel (C.
, such as alkaloids, flavonoids, and tannins act as stomach Indah Khairunnisa, dkk | 343 Jurnal Ilmiah Ibnu Sina, 9.
Oktober 2024, 337-346 p-ISSN: 2502-647X.
e-ISSN: 2503-1902 digestion of Artemia salina larvae.
CONCLUSION
Ethanol extract of lime peel (C.
Flavonoids work by inducing an increase in the amount of oxidative flavonoid, and tannin compounds.
The stress in cancer cells so as to inhibit LC50 value of lime peel extract (C.
which is 331.
533 ppm is classified as toxic and can act as an cancer cell apoptosis, necrosis, and anti-cancer agent.
The higher the activate autophagy (Kopustinskiene et concentration of lime peel extract (C.
, 2.
Alkaloid compounds can aurantifoli.
tested, the higher the encourage DNA damage and trigger percentage of death of Artemia salina apoptosis because they work by We would like to express our sincere gratitude to Department of Meanwhile, tannin compounds are Biology.
Faculty of Mathematics and known to activate intrinsic and Natural Lampung for the resources and several signaling pathways (Rajasekar facilities that have been provided so et al.
, 2.
that this research can run successfully.
The (Habli ACKNOWLEDGEMENT Science.
University metabolite compounds contained in REFERENCES