The toxicity of brown algae (Sargassum sp) extract to mice (Mus musculus)

  ugust 2016, Volume 1, Number 2: 109-115 P-ISSN.2503-0817, E-ISSN.2503-0825

  

  A bstract O OObbbjjjectivvve: The purpose of this study is to know the toxicity of brown algae extract sargassum sp given orally to mice. The research performed is an experimental laboratory research type with experimental post-test- only control group design. The research samples used are females white mice (mus musculus).

  M M Maaateriaaal aaannnd M M Methods: The demand of seaweed in the world increases due to increasing use of seaweed for various purpose in the field of industry, food, textile, paper, paints, cosmetics, medical and pharmaceutical field. Alginate is commonly used in the field of dentistry as printed materials to create study models. Materials in the field of dentistry must be biocompatible to the oral cavity tissues. The materials should be stable, safe, comfortable and certainly nontoxic to the oral cavity tissues and other tissues in human body.

  Treatment group 1 was given 500 mg/kg b.w. dose of sargassum sp, group 2 was given 1000 mg/Kg b.w. dose of sargassum sp, group 3 was given 1500 mg/Kg b.w. dose of sargassum sp, group 4 was given 2000 mg/Kg b.w. dose of sargassum sp and a control group was given only dose of Sodium carboxymethyl cellulose.

  Resuuults: This study is a dose in humans that is converted into 2000 mg/Kg b.w. in mice, is a dose that does not cause the death of whole animals.

  Connncluuusionnn: Based on the acute toxicity category, the extracts of sargassum sp obtained from Punaga Takalar Regency, South Sulawesi contains mild toxicity.

  Keywords: Sargassum sp, Toxicity, Mice, Mus musculus Cite this Article: Wariz R, Asfar NW, Fauzi A. 2016. The Toxicity of Brown Algae (Sargassum sp) Extract to Mice (Mus musculus). Journal of Dentomaxillofacial Science Introduction

  The potential of Indonesia to develop and exploit its marine biological resources is expected to esca- late development rate and descend dependence on mainland. One of the marine biological resources is seaweed. The application of seaweed in Indonesia has extensively been

  Developed, and has become one of income sources for Indonesians living in coastal areas, which has high potency of seaweed. Nevertheless, the seaweed application has not significantly increased in develop countries. It becomes very pathetic circumstance, considering that Indonesia is one of the countries that has the largest seaweed area in the world Brown alga is one of seaweeds, which spread out in territorial waters of Indonesia. There are several genera of Brown algae. One of the brown algae gener- ally founded in South Sulawesi, Indonesia is the genus Sargassum sp. The seaweed is pretty much spread out in several areas in South Sulawesi. Although Sargassum sp is not widely cultivated, it is still consumed by local community for industrial need Brown seaweed contains active compound with various bioactivities so that it has potencies to be developed as a nutraceutical materials. Its metabo- lite source such as carotenoids, laminaria, alginate, fucoidan, mannitol and phlorotannin has economic value. Brown alga is better known as alginate and is a source of iodine. Pigment content in thali (Brown alga) is dominated by chlorophyll a, c, β-carotene, violaxanthin, and fucoxant Alginate is one of common materials adminis- tered in the field of dentistry as printed material to create study model. A material administered in the field of dentistry obviously must have good biocom- patibility against oral cavity tissue and its surround- ings. Materials administered have to be stable, safe, pleasurable, and obviously not have toxic character, both the oral cavity tissue and surroundings as well as other tissues in human’s body.

  Based on the research by Nursid et al., on administering six kinds of brown seaweeds, such as: Sargassum ilicifolium (S. ilicifolium), Sargassum binderi (S. binderi), Turbinaria decur- rens (T. decurrens), Turbinaria ornata (T. ornata), Padina australis (P. australis), and Hormophysa triquetra (H. triquetra). The results of cytotoxic test conducted reveals that H. triquetra extract stands at the first rank in hampering the HeLa and T47D cells and T. decurrens stands at the second place. The highest fucoxanthin level is produced by H. triquetra extract, followed by T. ^1,2

  Department of Prosthodontics, Faculty of Dentistry, Hasanuddin University ₃ Departement of Oral and Maxillofacial Surgery, Faculty of Dentistry, Hasanuddin University, Makassar, Indonesia

• * Correspondence to: Rahmat Wariz,

  Department of Prosthodontics, Faculty of Dentistry, Hasanuddin University Received: 29 July 2016 Revised: 15 August 2016 Accepted: 17 August 2016 Available Online: 31 August 2016

  The toxicity of brown algae (Sargassum sp) extract to mice (Mus musculus) Rahmat War Nur W.Asfar, ² Abul ³ decurrens and P. australis, whereas the fucoxan- thin level of S. Ilicifolium, T. ornata and S. binderi extract is relatively much low (<20 ppm). The fukosantin specifically works against particular tumor cell and obstructs their proliferation through several mechanisms. Astawan et al. reveals that methanol extract from brown seaweed (Sargassum echinocar- pum) contains tannins, polyphenol, saponins, ately toxic and safe for consuming at a dose of 1250 mg/kg b.w. On consuming the methanol extract of Sargassum echinocarpum at approx- imately ≥1250 mg/kg b.w. may obstruct the enhancement of mice weight. However, a dose up to 5000/kg b.w. does not induce death. The research conducted by Ayyad et al ^{nalyzed four pure compounds reveals that fucoxanthin level in Sargassum sp has high potency as antioxidant and cytotoxic against breast cancer (MCF-7) with}

  IC50=11.5 mg/ml. The fucoxanthin is naturally safe and it can be consumed as antioxidant and antitumor supplement.

  15

  30 60 120 180 240

Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD

  15

  10

  5

  

Time (minute)

p-value

  Na-CMC 0.40 ± 0.54 0.60 ± 0.54 1.40 ± 1.51 2.40 ± 0.89 1.60 ± 1.14 1.60 ± 1.14 1.40 ± 0.89 1.20 ± 0.83 0.010 500 2.20 ± 0.83 1.00 ± 0.00 1.00 ± 0.00 0.40 ± 0.89 0.40 ± 0.89 1.00 ± 0.70 1.00 ± 1.22 0.80 ± 1.09 0.024 1.000 0.40 ± 0.54 0.20 ± 0.44 0.60 ± 1.34 1.60 ± 1.34 1.00 ± 0.70 0.20 ± 0.44 0.80 ± 0.44 0.00 ± 0.00 0.097 1.500 0.20 ± 0.44 0.40 ± 0.54 0.60 ± 0.54 0.60 ± 0.54 0.80 ± 044 1.80 ± 0.44 3.00 ± 0.00 3.00 ± 0.00 0.000 2.000 1.00 ± 0.00 0.40 ± 0.54 0.20 ± 0.44 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.003 Table 2 The result of catalepsy test based on dose and time Dose (mg/

  30 60 120 180 240

Mean ± SD mean±SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD

  10

  The Sargassum polycystum extract positively contains alkaloid in extract using hexane solvent and steroid in methanol, ethyl acetate, and hexane. The results of toxicity test of Sargassum polycystum extract show that it has toxic effect against Artemia salina with chronic category at hour 24 hours of observation, in other words Sargassum polycystum extract tends to be toxic against living organism Material and Methods

  5

  Table 1 The result of activity test based on dose and time Dose (mg/

Time (minute)

p-value

  Based on the test results suggested , the highest p-value is 0.09 in 1.000 mg/kg b.w. dose group that the value (0.9 or > 0.005) indicates that

  Based on the research conducted to observe the toxic effect of brown seaweed Sargassum sp against mice, we obtain the results and that can be referred to

  Results

  The observation of toxicity effect of brown seaweed extract Sargassum sp are performed by three means, i.e. physical symptom test, increase of respiration rate, descent of moving activities, diarrhea, urine and excessive saliva, test of daily weight change and observation of mice death.

  The extraction method administered in this research is maceration method. This research was begun with the preparation of test animals. Before the animals were provided the treatment, they obtain acclimatization for 7 days to adapt with their circumstance and have to fast for 3–4 hours.

  The type of the research performed is laboratory experiment research, with post-test only with control group design of the research contrivance. This research was conducted in Phytochemicals and Biopharmaceuticals Laboratory, Faculty of Pharmacy, Hasanuddin University. The research samples are classified into five groups that consisted of five female mice for each treatment group.

  Na-CMC 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 - 500 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 - 1.000 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 - 1.500 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.20 ± 0.44 0.00 ± 0.00 0.374 2.000 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.20 ± 0.44 0.00 ± 0.00 0.374

  Table 3 The result of urination test based on dose and time Time (minute)

  5

  10

  15

  30 60 120 180 240 p- Dose (mg/ Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD value

Na-CMC 0.00 ± 0.00 0.20 ± 0.44 0.00 ± 0.00 0.40 ± 0.54 0.80 ± 0.83 2.00 ± 1.00 0.60 ± 0.54 0.00 ± 0.00 0.007

500 0.00 ± 0.00 0.20 ± 0.44 0.00 ± 0.00 0.60 ± 1.34 1.40 ± 0.54 0.80 ± 0.83 0.80 ± 0.83 0.80 ± 0.44 0.097

1.000 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.40 ± 0.89 1.40 ± 0.89 2.20 ± 0.44 2.00 ± 0.83 0.80 ± 1.30 0.001

2.000 0.00 ± 0.00 0.40 ± 0.54 0.20 ± 0.44 1.40 ± 0.54 0.60 ± 1.34 3.00 ± 0.00 1.60 ± 0.89 0.20 ± 0.44 0.000

Table 4 The result of defecation test based on dose and time Time (minute)

  5

  10

  15

  30 60 120 180 240 p- Dose (mg/ Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD value

Na-CMC : 0.20 ± 0.44 0.00 ± 0.00 0.20 ± 0.44 0.80 ± 1.09 0.80 ± 0.83 0.00 ± 0.00 0.60 ± 0.54 0.140

500 0.00 ± 0.00 0.20 ± 0.44 0.00 ± 0.00 0.60 ± 0.89 0.20 ± 0.44 1.40 ± 1.14 1.20 ± 0.83 0.80 ± 0.83 0.023

1.000 0.20 ± 0.44 0.00 ± 0.00 0.80 ± 1.30 1.40 ± 1.14 1.00 ± 1.00 1.00 ± 1.22 1.40 ± 0.54 0.80 ± 0.83 0.043

1.500 0.00 ± 0.00 0.40 ± 0.54 0.60 ± 0.89 0.80 ± 0.83 1.40 ± 0.54 1.20 ± 1.30 1.40 ± 0.54 0.80 ± 1.09 0.016

2.000 0.20 ± 0.44 0.60 ± 0.89 0.80 ± 1.30 1.20 ± 1.30 1.60 ± 1.14 1.60 ± 1.51 0.80 ± 0.44 0.00±0.00 0.199

Table 5 The result of salivation test based on dose and time Time (minute)

  5

  10

  15

  30 60 120 180 240 p- Dose (mg/ mean ± SD mean ± SD mean ± SD mean ± SD mean ± SD mean ± SD mean ± SD mean ± SD value

• Na-CMC 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00

  500 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.40 ± 0.54 1.80 ± 1.30 1.60 ± 1.34 1.40 ± 1.14 0.80 ± 1.09 0.004

  1.000 0.00 ± 0.00 0.00 ± 0.00 0.40 ± 0.54 0.40 ± 0.54 1.00 ± 1.22 1.20 ± 1.09 0.80 ± 0.83 0.00 ± 0.00 0.065

  1.500 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.20 ± 0.44 0.00 ± 0.00 0.374

  2.000 0.00 ± 0.00 0.40 ± 0.54 2.20 ± 0.83 0.60 ± 0.54 0.80 ± 0.83 2.00 ± 1.00 0.80 ± 0.83 0.40 ± 0.54 0.000

  Table 6 The result of vascular test based on dose and time Time (minute)

  5

  10

  15

  30 60 120 180 240 p- Dose (mg/ mean ± SD mean ± SD mean ± SD mean ± SD mean ± SD mean ± SD mean ± SD mean ± SD value

• Na-CMC 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00

  500 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.40 ± 0.54 0.20 ± 0.44 0.00 ± 0.00 0.20 ± 0.44 0.00 ± 0.00 0.263

  1.000 0.20 ± 0.44 0.20 ± 0.44 0.40 ± 0.89 0.40 ± 0.89 0.60 ± 1.34 0.60 ± 1.34 0.60 ± 1.34 0.60 ± 1.34 0.374

  1.500 0.20 ± 0.44 0.20 ± 0.44 0.40 ± 0.54 0.20 ± 0.44 0.40 ± 0.54 0.40 ± 0.54 0.20 ± 0.44 0.00 ± 0.00 0.638

  2.000 2.00 ± 0.83 2.00 ± 0.70 2.20 ± 0.44 2.40 ± 0.89 2.20 ± 0.83 2.00 ± 0.70 1.40 ± 0.89 1.20 ± 1.09 0.050

  there is no frequency differences of activation inci- dose group at minute 30, 60, 120, 180 and 240 as dent in every minute of observation, meanwhile the well as in 1000 mg/kg BB dose group at 240 minute. lowest p-value is in 2000 mg/kg b.w. dose group. The In catalepsy test, the highest p-value is 0.375 in significant data are shown in 1.500 mg/kg b.w. and 1.000 mg/kg b.w. and 2.000 mg/kg b.w. dose group, 2000 mg/kg b.w. dose group with (p<0.005). The meanwhile the lowest mean is p (-) or <0.005 in highest mean for activation test is in 1.500 mg/kg Na-CMC, 500 mg/kg BB, and 1.000 mg/kg b.w. b.w. dose group at the 180 and 240 minute, whereas dose group, indicating that there is no difference at the lowest mean is 0.00 mostly in 2000 mg/kg b.w. all from the first until the last minute. Whereas the

  Table 7 The result of mice weight for 7 days after providing Sargassum sp extract

Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7

Dose mean ± SD mean ± SD mean ± SD mean ± SD mean ± SD mean ± SD mean ± SD p-value (mg/kg b.w.)

  Na-CMC 26.89 ± 0.95 26.21 ± 5.04 26.53 ± 5.09 26.66 ± 5.06 25.91 ± 3.84 26.17 ± 3.87 26.34 ± 3.72 0.709 500 mg 28.98 ± 3.13 29.59 ± 3.30 29.98 ± 3.44 29.98 ± 3.60 27.98 ± 1.18 29.97 ± 3.27 28.44 ± 2.81 0.335 1.000 mg 22.15 ± 4.81 22.05 ± 4.76 21.70 ± 4.57 21.46 ± 4.56 21.70 ± 4.57 21.20 ± 4.47 20.74 ± 4.35 0.026 1.500 mg 24.67 ± 2.21 23.63 ± 1.84 23.59 ± 1.42 23.33 ± 0.53 22.41 ± 1.41 22.30 ± 1.41 21.20 ± 1.40 0.028

  In salivation test, the highest mean is 2.20 in 2.000 mg/kg BB dose group at minute 15 and the lowest mean is 0.00 in Na-CMC group. The highest p-value is 0.374 in 1.500 mg/kg b.w. dose group and the lowest p-value is (-) or <0.005 in Na-CMC group which means that there is no differences at all from the first until the last minute.

  In vascular test, the highest mean is 2.40 in 2.000 mg/kg b.w. dose group at minute 30 and the lowest mean is 0.00 in Na-CMC group. The highest p-value is 0.638 in 1.500 mg/kg b.w. dose group and the lowest p-value is (-) in Na-CMC dose group, which means that there is no significant differences at all from minute 5 until 240. After performing toxicity examination by testing the six variables it was continued to scale the mice weight conducted for 7 days, starting from the first day after giving the Sargassum sp extract until day 7. The table of mice weight observation may be monitored in the t

  The lowest mean is (21.10), (20.75), (20.48), (20.38), (20.16), (19.92), and (17.81) successively suggested by 2.000 mg/kg b.w. dose group from the first until the seventh days.

   reveals that the Na-CMC group in Figure 1 The mice weight after giving the Sargassum sp extract

  the first day does not encounter dominant weight highest mean is in Na-CMC and 2.000 mg/kg b.w. difference counting from the first day (26.89) until dose group at minute 180. the seventh day (26.34), however on day 5, the mice

  The highest p-value in 500 mg/kg b.w. dose weight encounters a descent (25.91). In 500 mg/Kg group based on the data 3 is 0.09 or b.w. dose group, the mice weight does not encounter (ρ>0.005) and the p-value of Na-CMC dose group modification of dominant weight descent. The mice is 0.007 atau (ρ>0.005). Whereas, the lowest p-value weight runs into escalation and descends periodically, is 0.000 in 2.000 mg/kg b.w. dose group. Meanwhile, and at day 7, it runs into escalation again the highest mean is 3.00 in 2.000 mg/kg b.w. dose (26.34). group at the minute 120 and the lowest mean is From data analyzed in 500 mg/kg b.w. dose 0.000 in every dose group at minute 5 and 15 except group, we obtain that mean is in 1.000 mg/kg b.w. 2.000 mg/kg b.w. dose group and 1.000 mg/kg b.w. dose group, the mice weight encounters descent dose group at minute 10. from day 1 until day 4 (21.46), and encounters

  For the results of defecation test, the highest escalation again at day 5 (21.70). The difference is mean is 1.60 in 2.000 mg/kg b.w. dose groups at suggested by 1500mg/kg b.w. dose group, the mice minute 60 and 120. Meanwhile the lowest mean weight gradually encounters descent from day 1 is 0.00 in Na-CMC, 500 mg/kg b.w., and 1500 mg/ with mean value (24.67) until day 7 (21.20). It is the kg b.w. dose group. The highest p-value is 0.199 in same as 1500 mg/kg b.w. dose group, the 2.000 mg/ 2.000 mg/kg b.w. dose group and the lowest p-value kg b.w. dose group also reveals that there are grad- is 0.016 in 1.500 mg/kg b.w. dose groups. ual descents of mice weights where the means from day 1 until day 7 are successively (21.10), (20.75), (20.48), (20.16), (19.92) and (17.81).

  Discussion

  This research is administering five treatment groups consisting of the first group as control group, which only provided Na-CMC; the second group provided 500 mg/kg b.w. Sargassum sp extract; the third group provided 1.000 mg/kg BB Sargassum sp extract; the fourth group provided 1.500 mg/kg b.w. Sargassum sp extract and the fifth group provided 2000 mg/kg b.w. Sargassum sp extract. Each treat- ment group consists of five mice. The observation of toxic effect is monitored at the minute 5, 10, 15, 30, 60, 120, 180, and 240. The toxic effect may be monitored when the mice run into descent of motion activity, escalation of catalepsy (imbalance test), salivation (spending saliva), urine (spending urine) and vascular (vasoconstriction). After moni- toring the toxic effect, the mice weight is scaled after performed intervention (giving extract) for 7 days to observe the change of mice weight.

  Based on the result of analyzed data we can recognize that p-value (0.000) < 0.005 is in the 1.500 mg/kg b.w. and 2.000 mg/kg b.w. dose groups. It reveals that there is significant difference on the descent of motion activity because of the toxic from Sargassum sp extract among the minute observations. The highest mean is 3.00 in 1.500 mg/kg b.w. dose group and ρ-value is 0.000 atau <0.005, the lowest mean is in 2.000 mg/kg b.w. dose group. It also indicates that the descent of motion activity is connected to depression of the s system (CNS). ³⁰ Based on the Seomardji et al. ³¹ research, he clarifies that the reduction of motor activity can be manifestation of tranquilizer activity, depression of central nerves, relaxation of muscle, paralysis, or anesthesia.

  The catalepsy test revealed that there is no imbalance of dominant toxic effect against mice, because the imbalance effect only occurs at minute180 in the highest dose that is 1.500 mg/ kg b.w. and 2.000 mg/kg b.w., and the mean is 0.20 and the p-value is 0.375. Meanwhile, the results of analysis in other mice groups do not exhibit that there is f toxic effect at all. It can be observed in table 2 that the p-value is (-) or < 0.005 in Na-CMC, 500 mg/kg b.w., and 1.000 mg/ kg b.w. dose group which means that change does not occur from the first until the last minutes. Based on the data it is indicated that the imbalance in mice occurs because of the Sargassum sp extract that is given although it is relatively minor and only occurs in 1.500 mg/kg b.w. and 2.000 mg/kg b.w. dose group. Occurrence of the imbalance is connected to depression of central nervous system compo- sition and muscle relaxation in mice. ³⁰ Catalepsy test or imbalance test is also called as righting reflex test that is the one restoring body position returning to normal position. Loss of reflex can indicate the existence of restriction of sry nervous, spinal synapses, or efferent pathways. ² Based on the resultum analysis of uri- that there is significant difference in 1.000mg/kg

  BB, 1.500 mg/kg b.w., and 2.000 mg/kg b.w. dose groups. It is referred to the p-value (<0.005) which is different from Na- CMC and 500mg/kg b.w. dose group not indicated significant difference (ρ > 0.005). The highest mean is 3.00 in 2.000 mg/kg b.w. dose pointed that the frequency of occurrence of the highest e excretion is in 2.000 mg/kg b.w.. Rasyid et al. ³⁰ clarifies that there is exaggerated urine excretion connected to cholinergic effect in mice.

  Differed from the results of urination test, the defecation test reveals that there are no signif- icant differences in all of dose groups, which is (ρ However, the results of defecation test in table 4 reveal that the lowest p-value is 0.016 atau (p > 0.005) in Na-CMC group indicated that the lowest frequency of occurrence of feces excretion during each minute of observation is in Na-CMC group and the highest is in 2.000mg/kg b.w. dose group. Rasyid et al. ³⁰ assumes that the occur- rence of exaggerated excretion of feces (diarrhea) is connected to cholinergic effect in mice.

  The results of salivation test clarified in table 5 reveal that there are significant differences among doses in Na-CMC, 500 mg/kg b.w., and 2.000 mg/ kg b.w. groups which is (p < 0.005) meaning the data is significant. Meanwhile the data in Na-CMC group in which the p-value is (-) or <0.005 also indicate that there is no occurrence of toxic effect of salivation at all, in other words we can assume that the mice in Na-CMC grot encounter exaggerated saliva excretion. Table 5 also indicates that there is high saliva excretion in 2.000 mg/kg b.w. dose group suggested with the highest average of mean, wis in 2.000 mg/kg b.w. dose group. Rasyid et al. ³⁰ clarifies that the exaggerated saliva excretion is connected to cholinergic in mice. The results of vascular test performed indicate that the Na-CMC dose group represents significant datificant differences. It can be referred to the table 6 , in which p-value is (-) or <0.005 also meaning that there is no escalation at all connected to Na-CMC group. Meanwhile the highest mean on the average is shown in 2.000 mg/kg b.w. dose group. The occurrence of vascular escalation connected to vasoconstriction inducing sympathetic nervous system will support potencies to escalate blood- pressure. The occurrence of sel toxic effects that is parallel to Riyanto et al., ⁶ stating that the

  Sargassum polycystum extract is able to induce the toxic effect in experimental animal, Artemia salina. It is assumed that Sargassum polycystum extract, which is found in Artemia salina medium, diffuses into the body and interrupts the metabolic and enzymatic process such as individual cell respira-

  Based on the results of data analysis performed, we can assume that the higher dose can provide highly dominant toxic effect in mice. The observation results indicate that the extract with higher concentration has more effect. It can be witnessed from the frequency of toxic symptom presenting from the descent of motion activities, occurrence of imbalance, escalation of diarrhea symptom, urination, and salivation as well as escalation of vaso-constriction. The results of datum analysis reveal that treatment with provided with 500 mg/kg b.w. dose of methanol extracts of Sargassum sp does not indicate the descent of dominant weight in mice. It indicates that the extracts of Sargassum sp do not inflict metabolism disorder in experiml animal where in this circumstance, refers to mice (Mus musculus). Nagayama et al. and Loomis ³⁵ stated that mice provided with Ecklonia kurome extracts do not indicate growth disorder. The growth of organism depends on absorption and bioavailability of nutrient, including protein, in the body. The absence of obstruction of the weight escalation reveals that the process of protein absorption in digestive canal is not by the presence of phlorotannin.

  The observation results of mice for 7 days after providing the intervention, shown in table 7 , in 1.000 mg/kg b.w., 1.500mg/kg b.w., and 2.000 mg/ kg b.w. dose group causes descent of mice weight but the dominant descent mice weight occurs in 2.000mg/kg b.w. dose group until day 7 after the extract was provided. The same case was also reported by Firdaus et al. ³ who stated that the

  Sargassum echinocarpum extract indicates the descent of mice weight as experimental animal except in control treatment and in the lowest dose group, which is 625 mg/kg b.w.

  Based on the results of data analysis performed the toxic effect can be observed from the decrease of mice weight dominantly occurs in 2.000 mg/kg b.w. dose group from day 1 until day 7. It is also because of the assumption that the higher concen- tration of extract, the higher doses, and more active compound is ntained in extract suspension. as we know that the dose is the main problem in determining whether the chemical substance is categorized as toxic. ^{the higher concentra- tion of extract, the possibilities of metabolism disorder occurring in experimental animals are also high. It can be observed in table 7 where Stern et al. and Loomis 35 reported that the brown seaweed extract could obstruct the growth of the organism because of the presence of phlorotannin ability to form hydrogen bond with protein including enzyme in digestion canal. tum extracts contain phytochemical compound such as, alkalosteroids and triterpenoids. 6 Sargassum cinereum generally contains chemical compounds, which are alkaloids, steroids, sapo- nins and tannins. 32 Methanol extract of Sargassum duplicatum contains alkaloids, saponins, quinone, phenolic steroids, and avonoid compound. Meanwhile, ethyl acetate extract of Sargassum duplicatum contains alkaloids, saponins, steroids, and flavonoid compound. 14 It was Estimated that one of the several active compouncontained in}

  Sargassum sp extract is an active compound, which is responsible for occurrence of toxicity effect in mice. According to Riyanto et al. ⁶ the alkaloids in Sargassum polycystum extract is toxic, because the compound indicates extenely physiological activity, almost all without exception, which tends to be alkali.

  The results of Firdaus et al. ³ research revealed that consumption of methanol extract of Sargassum echinocarpum obtained from water of Talango Island, East Java orally once up to 5.000 mg/kg BW of dose does not inflict death in experimental mice.

  It is indict or compound contained in methanol extract of Sargassum echi- nocarpum is categorized as relatively non-toxic compound. ³ Derelanko et al. ³³ categorizes that if a material or compound is consumed orally in experimental animals as many as 5.000 mg/ kg b.w. and does not induce death, it includes the moderate-toxin. In addition, when consumed more than 15.000 mg/kg b.w., it includes as non-toxic compound.

  The research result obtained reveals that no mouse was dead during treatment. Based on the results, the data cannot be processed by using SPSS 22 for Windows. According to the agreement acceptey authorities, if the maximal dose does not evoke death of experimental animals, the LD50 is called as ‘pseudo’ LD50 with taking the maximal dose ⁴ so that in this research, LD50 known as pseudo LD50 is 2.000 mg/Kg b.w. The result cannot be placed in classification of acute toxicity category because LD50 obtained is not the real LD50, but the dose 2.000 mg/Kg b.w. is the conversion of maximal dose of human against mice based on the ratio of body surface area. Based on the authorities, when the maximal dose of a compound does not create death in experimental animals, the compound is categorized as mildly toxic.

  Conclusion

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  Conflict of Interest

  The authors report no conflict of interest

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