Immunomodulator Activity of Effervescent Granule of Wualae Fruit (Etlingera elatior (Jack) R.M. Smith) Based on Specific Phagocytic Activity

Indonesia is a megabiodiversity country where almost all of its natural resources can be utilized as traditional medicines. Etlingera elatior or wualae, in local name, is a family of Zingiberaceae which can be found in Indonesia. Wualae can be utilized as source food and traditional medicine, which almost all of part of wualae from the rhizome to flower can be used (Chan et al., 2011). Active compounds can be found in wualae are tannins, terpenoids, saponins, flavonoids, and carbohydrates. The essential oil also detected in wualae (Lachumy et al., 2010; Jaafar et al., 2007). The results from previous study showed that wualae has activity as immunostimulant with dose at 300 mg/kg BW and 400 mg/kg BW consecutively with higher dose gives higher effect. Phytochemical screening indicated that wualae contains flavonoids which potentially have activity as an immunomodulator (Wahyuni et al., 2017). An immunomodulator is a compound that can enhance the immune system of humans that classify into three groups, consisting of immunostimulatory, immunoregulator, and immunosuppressor. Immunostimulator can enhance the function and activity of the immune system, immunoregulator can regulate Immunomodulator Activity of Effervescent Granule of Wualae Fruit (Etlingera elatior (Jack) R.M. Smith) Based on Specific Phagocytic Activity


INTRODUCTION
Indonesia is a megabiodiversity country where almost all of its natural resources can be utilized as traditional medicines. Etlingera elatior or wualae, in local name, is a family of Zingiberaceae which can be found in Indonesia.
Wualae can be utilized as source food and traditional medicine, which almost all of part of wualae from the rhizome to flower can be used (Chan et al., 2011). Active compounds can be found in wualae are tannins, terpenoids, saponins, flavonoids, and carbohydrates. The essential oil also detected in wualae (Lachumy et al., 2010;Jaafar et al., 2007).
The results from previous study showed that wualae has activity as immunostimulant with dose at 300 mg/kg BW and 400 mg/kg BW consecutively with higher dose gives higher effect. Phytochemical screening indicated that wualae contains flavonoids which potentially have activity as an immunomodulator (Wahyuni et al., 2017).
An immunomodulator is a compound that can enhance the immune system of humans that classify into three groups, consisting of immunostimulatory, immunoregulator, and immunosuppressor.
Immunostimulator can enhance the function and activity of the immune system, immunoregulator can regulate

Abstract
The previous study reported that Etlingera elatior (Wualae) has activity as immunostimulant with dose at 300 mg/kg BW and 400 mg/kg BW. Formulating natural product into effervescent granule (EG) can increase practicality and interest to consume traditional medicine. This study aims to investigate the immunostimulant activity of Wualae ethanolic extract in the effervescent granule. Wualae was macerated with ethanol then formulated into effervescent granule at dose 300 mg/kgBW and 400 mg/kgBW. Formulations were evaluated. Animals were divided into four groups and treated with: group I (positive control); group II (negative control); group III (EG dose at 300mg/kgBW); and group IV (EG at dose 400mg/kgBW) for seven days and on the eighth day, groups were infected by Staphylococcus aureus intraperitoneally. Immunostimulant activity was measured by calculating the Specific Phagocytic Activity (SPA) of macrophage. Data collected from the evaluation and measurement of the SPA then analyzed using SPSS. Results from the evaluation were considered good except the moisture content of effervescent granule. Results from SPA was found that effervescent granule at dose 300 mg/kgBW and at dose 400 mg/kgBW have 26.33% and 32.5% consecutively with significance 0.02 (p<0.05). Thus, effervescent granule has comparable phagocytosis activity with positive control.
the immune system, and immunosuppressor can hamper and ease the immune system if the function is overworked (Murphy, 2012

Wualae fruit collection
Wualae fruit samples were collected from Kalu-kaluku Village of Kodeoha Sub-District, North Kolaka District, Southeast Sulawesi, Indonesia. Collected samples were sorted from their stalks then washed by running water.
The samples then dried in under sun exposure covered by a black cloth.

Wualae fruit extraction
The dried wualae fruit samples (2.1 kg) were macerated with ethanol 96% (Merck®) for three times each for 24 hours and filtered using Whatman filter papers. Their filtrates were concentrated under reduced pressure, and 124.8 g ethanolic extract of wualae fruit yielded.  Table I.

Organoleptic test
The organoleptic evaluation was performed by observing the taste, color, and odor of granule.

Moisture content test
The moisture content evaluation was performed by weighting the wet and dried granule.

Dissolve time evaluation
The dissolution time evaluation was performed by observing how much time is needed by the effervescent granule disperse in water.

pH test
The pH evaluation was performed by using a pH meter.

Flow test
The flow test was performed by measuring the flow time and angle of repose of granule formed.

Animals and ethics
Male albino mice (20-30 g) used in this study were obtained from the animal farm in Surabaya, Indonesia.

Experimental design
After seven days of acclimatization, 30 male mice were divided into four groups (group I -IV) and treated orally for the next seven days with the following treatment:

Measurement of phagocytic activity
Immunostimulant activity was measured by calculating the Specific Phagocytic Activity (SPA) of macrophage under the light microscope (Olympus ® ) with magnification from 10x to 1000x. The formula of SPA = (active macrophages : total macrophages) x 100% (Marusin & Chairul, 2012).

Statistical analysis
Results were statistically analyzed using the Statistical   Results obtained from the angle of repose (α) were shown in Table III, which values 21.42° (F I), 21.37° (F II), and 21.03° (F III). Moisture content has a critical role in effervescent granule since it affects the chemical reaction of acid and base source, and it affects the stability of granule (Swarbick, 2013 (Swarbick, 2013).

RESULTS AND DISCUSSION
The flow test was performed with flow rate and angle of repose of effervescent granule. Results obtained from the angle of repose (α) indicate that formulations are freeflowing granule. The flow rate was affected by particle size and its distribution, shape, density, and moisture content (Patel & Siddaiah, 2018).
The flavonoid in ethanolic extract of wualae fruit is responsible for the phagocytic activity, which is describing its activity as immunostimulator (Wahyuni et al., 2017). The possible mechanism of flavonoids is  Results presented in Table IV show that effervescent granule of wualae fruit ethanolic extracts enhanced phagocytosis activity compared with the negative control.  (Zalizar, 2013).