Cytotoxicity Activities of Ethanol Extract of Hooks Uncaria tomentosa West Kalimantan

Received: May 20, 2020 Accepted: June 19, 2020 Available online: June 30, 2020 Uncaria tomentosa is a member of the plant family Rubiaceae. It has been used as medicinal plants in West Kalimantan. The cytotoxic of ethanol extract from the hooks of U. Tomentosa was determined. This study used Brine Shrimp Lethality Test (BSLT) method with solution concentration 1,000; 5,000 and 10,000 ppm. The extract has LC50 values of 21,754 ppm. It is indicated the extract not toxic. This extract is potent to be used as drugs.


INTRODUCTION
Plants of the genus Uncaria (Rubiaceae) contains approximately 34 species. The genus is distributed mainly in tropical regions, such as Southeast Asia, Africa, and Southeast America. Uncaria is liana or scandent shrub that climb by hooks (Turner, 2018). Uncaria tomentosa is one of the species in West Kalimantan (Iskandar, 2020). It is known as the cat's claw because of its claw-shaped thorns (Honório et al., 2016).
U. tomentosa has been known as medicinal plants. Medicinal plants have been used for various therapeutic purposes. U. tomentosa has been traditionally used to treat asthma, abscesses, fever, urinary tract infections, viral infections, and wounds (Batiha et al., 2020). It also has been used to treating diabetes, cancer, intestinal affections inflammation, cancer, menstrual disorders (de Paula et al., 2015;Zhang et al., 2015). The plants also have a potent for treating most parasites (Santos et al., 2016).
U. tomentosa has several pharmacological activities such as antitumor, anticancer, antioxidant, anti-neoplastic, antiinflammation, antimicrobial, antiprotozoal, and antiviral activities (Batiha et al., 2020). Cytotoxic activity of ethanol extract from hooks U. tomentosa from West Kalimantan is not known. The extract was tested for the cytotoxic against Artemia salina Leach using Brine Shrimp Lethality Test (BSLT) methods. The methods were used to see the toxicity of plant extracts.

Collection and Identification of Plants
Hooks of U. tomentosa were collected from Kapuas Hulu, West Kalimantan. The plants were identified in the Biology Laboratorium, Department of Biology, FMIPA, Tanjungpura University. The cytotoxic of the plant extracts have been carried out in Chemistry Laboratorium, Department of Chemistry, FMIPA, Tanjungpura University.

Preparation of Plants and Plant Extract
The hooks of U. tomentosa was prepared by air-dried at room temperature. Dried samples powdered and macerated with ethanol. Whatman no.1 filter paper was used to filter the mixture. The filtrate was concentrated by rotary evaporation to evaporate the residue of the solvent. Then, ethanol extract of hooks U. tomentosa stored in the refrigerator for further use.

Hatching of Brine Shrimp Nauplii
Brine shrimp eggs were hatched in sterilized seawater. It soaked for 24 hours with aeration in a transparent container. After 48 hours, the nauplii (larvae) were collected by pipette as many five larvae for each replication. The larvae were used for the brine shrimp lethality test (Pisutthanan et al., 2004).

Plant Extracts Solution Preparation
Small amounts of ethanol extract of hooks U. tomentosa were evaporated. Then, the extract was dissolved in DMSO to prepare a stock solution. A serial dilution was prepared by diluting stock extract in several concentrations 1,000; 5,000; and 10,000 ppm.

BSLT Test
The procedure for BSLT was modified from the assay described previously (Meyer et al., 1982). The stock solution pipetted and put in vials with varied concentrations. Each concentration has five replications. Then, the vials were added up to10 mL of seawater containing ten nauplii. The control had no extract in it. The test vials were incubated at room temperature for 24 hours. The numbers of dead nauplii in each vial were counted, and LC 50 values were estimated.

RESULTS AND DISCUSSION
Maceration is an extraction method by soaking samples with solvent. The technique does not damage the chemical entities of the plant. Hooks of U. tomentosa were macerated using ethanol. It is a semipolar solvent that can dissolve polar and non-polar compounds. The solvent is nor associated with toxic effects (Marzuki et al., 2019). The solvent penetrated the cell wall of plants and disrupting the cell membrane. It caused secondary metabolites to come out of the cell and dissolved into the solvent.
Cytotoxic activities were conducted using the BSLT method. This study was a preliminary test before the plants extract applied in vivo. The result of larvae mortality percentage for each concentration of ethanol extract of U. tomentosa showed in Table 1.  tomentosa using Artemia salina Leach showed the LC50 value of 21,754 ppm. The value of LC50 > 1,000. It means that ethanol extract of hooks U. tomentosa was not toxic. It is indicated the extract not toxic to humans and safely used as a drug candidate. In bioactivity evaluation of plant extracts by BSLT, LC50 value greater than 1,000 μg/mL is considered non-toxic. The cytotoxic activity was considered toxic when the LC 50 31 mg/L ≤ LC 50 ≤ 1000 mg/L, as strong when LC 50 ≤ 30 mg/L (Gaikwad et al., 2017). Cytotoxic of other species of Uncaria was reported. The Uc7, terpenoid compound from ethyl acetate extract of U. cordata (Lour.) Merr has a powerful cytotoxic activity with LC50 2,75 µg/mL (Rahmawati et al., 2016). The methanol extract of bark and wood of the roots U. nervosa Elmer was very toxic with LC50 values of 1.76 dan 2.66 ppm, respectively (Maulina and Pratiwi, 2019).
Artemia salina Leach has highly sensitive to the changes in environmental conditions and chemical agents in the environment. Brine shrimp eggs hatched after 48hr incubation. The whole body of nauplii (larvae) was formed completely (Muaja et al., 2013). The nauplii death caused by the presence of toxic secondary metabolites. The compounds of the extracts entered through the mouth of nauplii and absorbed into the alimentary tract. The absorption process occurs through the cell membrane.
After the absorption process, the toxic compounds distributed into the body of nauplii. Finally, the metabolism of nauplii was damaged.

CONCLUSIONS
The ethanol extract of hooks U. tomentosa from West Kalimantan was determined the cytotoxic activities. The LC50 values of the extract are 21,754 ppm. It showed the extract not toxic to the human.