PHYTOCHEMICAL ANALYSIS OF BULUNG BONI ( Caulerpa cylindracea S.) N-HEXANE EXTRACT WITH GC-MS METHOD AND TOXICITY TEST ON MICE

One of the marine resource commodities owned by Indonesia is seaweed. Bulung boni is one type of seaweed that grown in Indonesia. Bulung boni is the regional name of Caulerpa cylindracea . Bulung boni is usually used as a food and medicine for most people in Bali. The purpose of this research is to study the content of phytochemical compounds in n-hexane extract of bulung boni and determine the toxicity effect on mice ( Mus musculus ). The methods used in this study were maceration, GC-MS (Gas Chromatography-Mass Spectrometry), observation, and calculation with the Thomson and Weil formula. This study used 24 mice as experimental animals. Mice were divided into 6 treatment groups, group 1 as control was given 1% Na-CMC solution, groups 2-6 were given test extracts with doses, 100, 200, 300, 400, 500 mg/30 g BW mice. The results showed the highest compound contained in the n-hexane extract of bulung boni was Hexadecanoic acid, methyl ester with an AUC value of 35.21%. Toxicity tests on mice showed no deaths experienced by mice, so the LD 50 value determined was pseudo at 16.6 g/ kg BW and fell into the practically non-toxic category.


INTRODUCTION
Bulung boni is one type of seaweed that grows in Indonesia.Bulung Boni is the regional name of Caulerpa cylindracea.
This seaweed is usually used as a vegetable by the Balinese people.In the study of Julyasih (2009) stated the ethanol extract of bulung boni contains 37,249,000 µg carotenoids in 100 mg samples.
Carotenoids are antioxidants that are very potential in protecting lipid membranes against peroxidation (Siems et al., 2002).The use of antioxidant compounds has recently grown rapidly along with the increasing knowledge of free radical activity against several degenerative diseases (Hanani et al., 2005).
Epidemiological studies shows that consuming antioxidants derived from natural materials such as vegetables and fruits shows protective effects against various degenerative diseases such as coronary heart disease, stroke, and cancer (Budiana, 2008).Therefore, bulung boni has the potential to be developed into processed food and medicine products.
In processing a raw material, the content of the compounds in the material must be known.Phytochemical analysis is a test conducted to identify bioactive components in a material.Bioactive components are active compounds in functional foods that are responsible for metabolic reactions that benefit health (Subroto, 2008). of bulung boni n-hexane extract is harmful to humans if consumed at doses that are not recommended and in long term use.

Preparation of Bulung Boni Extract
The bulung boni that has been obtained is washed thoroughly then dried in the air for 2 days and then dried using an oven for 5 days at a temperature below 45 o C. Then ground using a blender and sifted with a 60 mesh sieve, then added nhexane solvent and then evaporated using a rotary evaporator to get a condensed extract of bulung boni.

Compounds in Bulung Boni Extracts
The analysis was performed using

Administration of Bulung Boni Extract to Mice
The experimental animals used were male mice aged 60 days.The mice used were healthy as many as 24 mice with a weight 20-30 g.Mice were divided into 6 groups, namely group 1 as control and groups 2-6 as treatment groups.Previously, the mice were acclimatized first for 1 week to adapt the mice's living environment to the laboratory environment.Mice were also fed (can still be given a drink) for 14 hours before treatment.Each group of mice was put into 1 cage.Each group was treated as follows: (K) given 1% Na-CMC (P1) given 100 mg/30 g BW of extract (P2) given 200 mg/30 g BW of extract (P3) given 300 mg/30 g BW of extract (P4) given 400 mg/30 g BW of extract (P5) given 500 mg/30 g BW of extract.
The treatment was given orally

Bulung Boni (Caulerpa cylindracea)
Bulung boni has a green thalus consisting of many erect branches that are about 2.5-6.0 cm tall.The main stem measures between 16-22 cm.There is a flatshaped sphere at the top of the branch, the length of each branch top is about 2.5-10.0cm (Trono and Ganzon, 1988).Bulung boni is mostly found in coastal areas that have coral reef flats, growing on dead substrates, dead coral fragments, mud sand and mud.(PubChem, 2005).Tetradecane that functions as a chemical intermediary and solvent (PubChem, 2004).Tremor may be caused by a disturbance in the neuromuscular system of mice after administration of n-hexane extract of bulung boni at a dose of 500 mg/30 g BW.
Neuromuscular occurs due to a failure of interaction between motor nerves and muscle cells which causes muscle contraction disorders (Lu and Kacew, 2002).Mice that experience tremors then return to normal the following day.

Determination of LD50
Determination of the LD50 value is based on the presence or absence of death in the test animals.The results of observations made during 14 days showed no deaths.The data that has been obtained from each group within 24 hours until day 14, then used to calculate the LD50 value using the Thomson and Weil formula.With no death of test animals, it shows that there is no f factor obtained from the Thomson and Weil table, so the calculation of the LD50 value cannot be continued.This is in accordance with the criteria for acute toxicity tests conducted to assess LD50 that based on the agreement taken by experts, if the maximum dose given does not cause the death of test animals, then the LD50 is expressed with pseudo LD50 or not the real LD50 (Loomis, 1978).According to Armansyah et al., (2016) in the acute toxicity test that has been carried out does not cause the death of test animals after giving a single dose of test substance, so to determine the LD50 value of the extract, the highest dose given to the test animals is used.Based on this statement, the apparent LD50 value of bulung boni n-hexane extract in this study is 16.6 g/ kg BW and bulung boni n-hexane extract is included in a practically non-toxic category.If up to a dose of 5000 mg/ kg BW does not cause death, then the test does not need to be continued using a higher dose of test substance (BPOM, 2014).In general, the smaller the LD50 value the more toxic the compound, and vice versa the greater the LD50 value the lower the toxicity.

CONCLUSION
The n-hexane extract of bulung boni Figure. 1.The Maps of Bulung Boni Site Qualitative data was obtained by observing the clinical symptoms experienced by the mice.Quantitative data was obtained by calculating the LD50, which is the number of mice that died and those that were still alive in each group.LD50 will be calculated using the Thomson and Weil method with the equation (1).log m = log D + d(f+1) ………………………………………………………………….(1) Description: m = LD50 value D = smallest dose used d = log of the multiple of the dose f = a value in Weil's table due to a certain mortality rate (r) Agilent 7890B MSD 5977B and Wakosil ODS/5C18-200 silica column with size 4.6 x 200 mm using N2 as a carrier.The injection temperature used was 290 o C for 27 minutes with constant flow and a flow rate of 1 ml/Min.Identification was conducted by looking at and comparing the retention time on each chromatogram peak with the reference database.

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single dose) to mice using a sonde.The observation parameters in this study were the number of mortality mice after 24 hours of administration n-hexane extract bulung boni and toxicity symptoms experienced by mice for 14 days.Symptoms of toxicity observed were hypersalivation, convulsions, diarrhea, behavioral changes, and tremor.Phytochemical Analysis of Bulung Boni (Caulerpa cylindracea S.) N-Hexane Extract with Gc-Ms Method and Toxicity Test on Mice (Mus musculus L.) Silalahi, M.D.B., Wirawan, I.G.P., Wijaya, I.N., Suada, I.K., Phabiola T.A. & Astaykinta, A.

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Caulerpa cylindracea) mostly contains fatty acid derivative compounds.The most abundant compound contained in the extract is the compound Hexadecanoic acid, methyl ester with an AUC value of 35.21%.The n-hexane extract of bulung boni (Caulerpa cylindracea) at a dose of 500 mg/30 g BW did not result in death in 50% of the test animals.The LD50 value determined was a pseudo of 16.6 g/ kg BW Phytochemical Analysis of Bulung Boni (Caulerpa cylindracea S.) N-Hexane Extract with Gc-Ms Method and Toxicity Test on Mice (Mus musculus L.) Silalahi, M.D.B., Wirawan, I.G.P., Wijaya, I.N., Suada, I.K., Phabiola T.A. & Astaykinta, A.

Table 1 .
Identified Chemicals Compounds in Bulung Boni Chromatogram