Antiemetic Activity of Trigona spp . Propolis from Three Provinces of Indonesia with Two Methods of Extraction

Introduction: Propolis is a resinous substance collected by bees to protect their hives. One of the least explored function of propolis is its antiemetic activity. The present study aimed to investigate the antiemetic activity of Trigona spp. propolis from three provinces of Indonesia. Methods: Ultrasound-assisted extraction using two kinds of solvent, water and ethanol was conducted to extract propolis from South Sulawesi, South Kalimantan and Banten. Antiemetic activity was observed on copper sulfate–induced emesis in young chicks. Result: All propolis indicated antiemetic activity, in which water extract had higher activity than ethanol extract. Regardless the type of solvents, propolis from South Sulawesi showed higher antiemetic activity than others. Conclusion: This study demonstrated that propolis has antiemetic activity, in which propolis from South Sulawesi showed the highest activity and water extraction is recommended.


INTRODUCTION
Propolis is a resinous substance collected by bees from various plants for its adaptation and honeycomb construction. 1It is the generic name of substance that protects the bees from unexpected environmental threats.Propolis acts as sealer and excluder for the honeycomb. 2,3Propolis also ensures smooth, moist and pleasant hives. 4ropolis is a potential substance that could improve our health.It is considered by many researchers as a new drug. 5Various functions of propolis have been widely investigated either by in vitro or in vivo assay.Some functions confirmed are; as antibacterial, antiviral, antifungal, anticancer, immunomodulatory, antiinflammatory, and antioxidant agents. 6eside of those functions, there is also a less explored biological activity of propolis, which is as antiemetic.Study by Eda and colleagues is the first article to confirm the antiemetic effect from Brazilian propolis.They found 50.9% of retching inhibition in young chick emesis model treated with Brazilian Propolis. 7They also investigated several compounds (dehydrohautriwaic acid, propenoic acid, aromadandrane and dihydrocinamic acid) which are the basis of antiemetic activity of Brazilian propolis.However, propolis properties tend to be inconsistent.It depends on its origins, seasons and plant sources. 8,9he previous study confirmed the diversity of Indonesian propolis compounds. 10The huge diversity of Indonesian propolis has made it generally considered as miscellaneous.In addition, solvent is also a crucial factor that influences propolis biological activity.However, there are conflicting result on types of solvent used to generate highest biological activity.Some studies found ethanol extract had higher antioxidant activity and total phenol compared to water extract.While, some other found water is a better solvent to generate high antioxidant activity and total phenol. 11,12,13This study aimed to investigate the antiemetic activity of propolis from three provinces of Indonesia, using two types of solvent (ethanol and water).We applied ultrasound-assisted extraction to yield more efficient and effective extract. 14

Materials
Raw propolis were collected by bee-keeper in South Sulawesi, South Kalimantan and Banten.The species of bees were all stingless bees (Trigona spp.) namely Tetragonula biroi(South Kalimantan), HeteroTrigona itama (South Kalimantan) and Tetragonula laevicep (Banten).We used copper sulfate (CuSO 4 ) as emetogenic substance and metoclopramide as positive control.Dimethyl sulfoxide (DMSO) and Tween 80 were used as solvent for injected propolis.

Animals
The young chicks (aged 4 days) and weighing from 50-80 g were purchased from Cibadak Indah Sari Farm, Jakarta, Indonesia.All animal experiments were carried out in accordance with the acts of The found water extract of Brazilian propolis demonstrated higher antiemetic activity than hydro alcohol extract.The reason is still unclear.However, it is suspected that there are other water soluble compounds, such as glucoside, polysaccharides, saponins that work as inhibitor of some emesis receptors. 22ome experimental animals have been widely developed as an emetic model such as: ferret, mink, monkey, pig, rat, frog, dog and chick. 23Chick as emesis animal model has been used by many researchers. 7,17,24,25,26hese model is better than frog emesis model because it has some advantages such as, easy to handle and able to visually observed and required shorter time. 26uSO 4 -induced chicks emesis model are suitable for screening for the potency of product as antiemesis. 17CuSO 4 induces emesis through direct stimulation of the stomach wall (gastric irritation) and/or through 5-HT4 stimulation. 22Those are the part of mechanisms that participate in nausea and vomiting action.In addition, this model is also suitable for evaluating brain participation in emerging emesis. 23

CONCLUSION
Our results demonstrated that Indonesian propolis has antiemetic activity, in which propolis from South Sulawesi has indicated the highest activity.Water extracts have shown higher activity compared to the ethanol extract thus we recommend water extraction for generating higher antiemetic activity.

Preparation of extract
The extracts were prepared using ethanol and water extraction.It was carried out in a sonicator bath (Branson 2000).The wave increases cell permeability thus result in optimum solubility of the active component. 15he procedures refered to Trusheva method with slight modification. 16aw propolis was added to solvents (1:10) then treated with ultrasound for 4h.Subsequently, propolis was filtered by what man No.41 and evaporated using vacuum evaporator.

Antiemetic activity assay
The young chicks were divided into 8 groups of treatments, 4 chicks in each group.They were set aside for 10 min for stabilization before any treatment was given.The groups of treatments were; water extract of propolis from South Sulawesi (WS), South Kalimantan (WK), Banten (WB), ethanol extract of propolis from South Sulawesi (ES), South Kalimantan (EK) and Banten (EB), negative control (0.9% saline) and positive control (metoclopramide).The extracted propolis sample were dissolved in 0.9% saline (containing 5% DMSO and 1% Tween 80) to create injection solution.Subsequently, 300 mg/kg of propolis was injected abdominally.10 minutes post injection, CuSO 4 was then administered orally (50 mg/kg).The number of retching was calculated for 10 min. 7,17The results were compared to control group.The percent of inhibition was calculated by the following formula: A: Frequency of retching in negative control group B: Frequency of retching in tested group

Statistical analysis
Number of retching was expressed as mean ± SEM.The difference was determined by univariate analysis of variance and considered to be significant when p< 0.05.

RESULT
Our present study showed that all propolis possess antiemetic activity.Propolis ES, WS, WB and WK were not significantly different in the number of retching to control (+).The inhibition ranged from 44.96 up to 98.51 % (Table 1).For ethanol extract, propolis from South Sulawesi had the highest retching inhibition.While for water extract, all kinds of propolis were not significantly different to control (+).According to univariate analysis of variance, both the solvents and origins of propolis had significant effect on antiemetic activity (p=0.001,respectively).Regardless the type of solvents, propolis from South Sulawesi had the highest inhibition power compared to the others and water extract had higher inhibition power than ethanol extract for all propolis origins.

DISCUSSION
Emesis, nausea and/or vomiting, is an unpleasant state which is a manifestation of several conditions including pregnancy, toxicants ingestion, and side effect of medication, surgery and cancer chemotheraphy. 18Emesis could lead to loss of appetite, fluid and electrolyte disturbance, and decreased nutritional status. 19,20ropolis works as antiemetic through several mechanisms.Hydroxycinamic acids of propolis act as δ (enkephalinergic)-receptor antagonist and inhibit dopamine receptor.Propolis also contains tritepens which are 5-HT 3 , 5-HT 4 and/or NK 1 receptors antagonist.These receptors are involved in emesis stimulation. 21Our research supports the previous study about the antiemetic activity of Brazilian propolis. 7The study also

Table 1 : Antiemetic activity of Trigona spp propolis.
Values in the same column followed by the same uppercased letter are not significantly significant (p<0.05)EB : Ethanol extract of propolis from Banten WB : Water extract of propolis from Banten ES : Ethanol extract of propolis from South Sulawesi WS : Water extract of propolis from South Sulawesi EK : Ethanol extract of propolis from South Kalimantan WK : Water extract of propolis from South Kalimantan *