The Extract of Kincung Flower (Etlingera elatior (Jack) R.M.Sm.) Activity to Decrease IL-4 and IgE Levels in Type I Hypersensitivity White Male Mice

World Allergy Organization (WAO) estimated that the prevalence of allergic diseases from the entire country's population ranges from 10 40%.1 The incidence of allergic diseases was high enough in various parts of the world, both in developed and developing countries. United Kingdom (UK) had the highest prevalence of allergic diseases in the world; more 20% of the population had one or more allergic disorders. In asthma, Scotland had the highest prevalence with 18.4%, the United Kingdom with 15.3%, Canada with 14.1%, United States with 10.9%, while Indonesia was 1.1%.2,3 In rhinitis, The International Study on Asthma and Allergies in Childhood (ISAAC), the prevalence of rhinitis symptoms in children varied between 0.8% to 14.9% at 6 -7 years old and between 1.4% to 39.7% at 13 14 years old. Indonesia was one of the countries with low prevalence, besides Albania, Romania, Georgia and Greece. Whereas the countries with high prevalence were Australia, New Zealand and the United Kingdom.4

The allergic reaction or type I hypersensitivity is one of the dangerous immune system responses because it could damage tissues, so it became a severe disease and ultimately can cause death. 5 IgE antibodies are responsible for type I hypersensitivity reactions. IgE antibodies mediate mast cell degranulation reactions, so that mast cells release allergic mediators such as histamine, serotonin, prostaglandins, and others. If IgE production suppressed, then the antigen reaction with IgE will decrease so that the number of mediators released will decrease. In the production of IgE by plasma cells, the cytokine responsible is IL-4. Plasma cells will produce IL-4 if these cytokine levels are high in the blood and will dominate the binding of B cells. Furthermore, B cells proliferation to plasma cells which produce higher IgE. 6,7 During the second exposure, the antigen is immediately bound by IgE, which is on the surface of basophil cells and mast cells. 8 The cell undergoes a degranulation process and releases allergic mediators such as histamine, serotonin, prostaglandins, and others. Mediators are responsible for the emergence of allergic reactions such as itching, redness, oedema, and impaired tissue function. 9,10 Recent research of kincung flower (Etlingera elatior (Jack) R.M.Sm.) showed it could be used as an alternative drug for anti-allergies because it had activity in inhibiting the active cutaneous anaphylactic reaction in mice. [11][12][13] It could inhibit degranulation of mice mast cells 12,13, which decreased basophils, eosinophils and lymphocytes. It increased neutrophils and the total number of leukocytes. 13 Kincung flower contains many secondary metabolites such as alkaloids, flavonoids, saponins, tannins, terpenoid. 13,[24][25][26] Identification with GC-MS, kincung flowers had compounds such as 1-dodecanol, β-pinene 27 , 17-pentatriacontane 13,28 cyclododecane dan 1,1-dodecanediol diacetate .13,29,30 Last research showed the activities of extract of kincung flower inhibit hypersensitivity of active cutaneous, inhibited mast cell degranulation, and decreased eosinophils and basophils. Based on that explanation, the researchers are interested in continuing research related to the activity of kincung on IgE and IL-4 levels in allergic white male mice.

Place and time
The research conducted in four months in April-September 2019. Preparation and extraction of Etlingera elatior (Jack) R.M.Sm. Also determinating the characterisation from kincung flower's extract were conducted in three weeks at Central Laboratory in Faculty of Pharmacy Universitas Andalas. 13

Tools and materials
The tools in this study were the Evaporator (Buchi® R-210 Rotavapor), UV-vis spectrophotometer (Thermo Scientific GENESYS 10S UV-Vis), Bio-rad spectrophotometer, beaker glass (Pyrex), Erlenmeyer (Pyrex), digital analytical balance (Ohaus), Silica gel 60 F254 (Merck), desiccator, spatula, dark bottle, TLC vessel, sonde instrument, surgical instrument, filter paper, animal cage, centrifuge. Kincung flowers as much as 2 kg was dried until it became dry Simplicia, then it made powder and sifted with sieve number 60. The powder (250 grams) macerated using 70% ethanol solvent (1:10) for 24 hours. Twice repetition using the same type and amount of solvent. It filtered with filter paper, collected the filtrate and evaporated in a rotary evaporator until it became a semi-solid extract (35.276 grams). 31 The TLC and total flavonoid test TLC test: The extract dissolved in methanol P and the comparison (rutin) dissolved in ethanol P. Spot them on TCL plate (Silica gel 60 F25). The chromatography solvent (ethyl acetate P, formic acid P and water (100: 15:17) put into a chromatography vessel. Dry the TLC plate and Place it under an ultraviolet light. Moreover, Calculate the retardation factor (Rf). 13,32 Total flavonoid: The extract and rutin dissolved in ethanol 80%. Then pipette 0.5 mL supernatant and rutin solution, add each of them 1.5 mL of ethanol, 0.1 mL of AlCl 3 10%, 0.1 mL of Na acetate 1 M and 2.8 mL aqua dest. Measure the absorption at the maximum absorption in wavelength 418nm. 13,32 Prepare the mice serum for calculation Of IgE and IL-4 levels After 24 hours of the last extracts are given, mice blood taken by the guillotine method. The blood centrifuged for 30 minutes and 3000rpm until obtained serum. Then the serum was used for testing IgE and IL-4 levels by the ELISA method. 33

Ethical test
In the research used the mice, the subject of study, so it required an ethical test that qualifies to mitigate the treatment gave an adverse impact on a human subject in this research. The ethical test conducted by the Commission of Ethics Faculty of Medicine Universitas Andalas, Padang, Sumatera Barat. 13

RESULTS AND DISCUSSION
Kincung flower extract was a semi-solid extract with characteristic characterisation odour, brown-black colour, and sour taste. The yield percentage was 14.11%, and according to Indonesian Pharmacopoeia Herbal, the yield percentage of kincung extract was not less than 9.86%. 13 The shrinkage of dried kincung flower extract was 6.65%, and according to Indonesian Pharmacopoeia Herbal, it was not more than 10%. 13 The total ash content of kincung flower extract was 4.57%, and according to Indonesian Herbal Pharmacopoeia, it was not more than 7.5%.
In comparison, the ash insoluble in the acid of kincung flower extract was 0.02%, and according to Indonesian Pharmacopoeia Herbal, it was not more than 0.1%. 13 The Determination of total flavonoid used rutin as a standard which tested in UV-vis Spectro, and it obtained wavelength was 418nm. The linear regression of the rutin calibration curve for the calculation of total flavonoid levels was y = 0.0046x -0.0562 with R2 = 0.998. The result of the total flavonoid kincung flower extract was 1.564%, and according to Indonesian Pharmacopoeia Herbal, the total flavonoid content of kincung flower extract was not less than 0.58% which calculated as rutin. 13,32 The UV spectrum of rutin wavelength shown in Figure 1.
The results of Rf values extract obtained as can be seen in Figure 2.
On the first day, the mice sensitized by injecting albumin 20% 0.2 mL/20gBB intraperitoneally. Then the seventh day, the allergic was boosted by injected them subcutaneously. It carried out to increase the formation of IgE antibodies, so the allergic reactions of mice will get worse. Allergic mice used for further treatment, except for the negative control group used healthy mice. The allergic mice will be shown redness to red spots or bumps around the body and injection site. Kincung flower extracts given to mice for seven days, and then the mice take the serum. In several studies showed that the flavonoids could inhibit IL-4 and IL-13 by activation of basophil cells. 36 Rutin had an anti-allergic activity because there was an effect on mast cell activity mediated by IgE. 37 It had an anti-allergic inflammatory effect and protected against allergic rhinitis by reducing levels of inflammatory cytokines. 38 Kincung flowers are rich in flavonoids that could inhibit IL-4 and IL-13 by activation of basophil cells. 36 It had an anti-allergic inflammatory effect and protected against allergic rhinitis by reducing levels of inflammatory cytokines. 38 It suspected that the main compound flavonoids on kincung flower is rutin; it can suppress IL-4 production by macrophage cells. IL-4 level was reducing, the proliferation and differentiation of b cells to become plasma cells which production IgE also decreased. IL-4 has receptors in plasma cells in producing IgE, the more IL-4 that is bound to plasma cells, the higher the production of IgE. 6,35 Thus the decrease in IL-4 will have a direct impact on the decline in IgE production. With the decrease in IgE or the cessation of IgE production, the degranulation process of mast cells and basophils will decrease or not occur at all. 7,35 IgE levels in serum white male mice showed in Table 2. After one-way Anova analysis on the IgE levels after kincung flower extract given at doses of 100, 300, and 1000 mg/Kg BW in type I hypersensitivity mice showed that there was a decrease significantly (<0.05). Duncan test results showed that the effects produced by doses of 1000 mg/kg BW could reduce IgE levels such as healthy mice without allergies.
Flavonoids contained in kincung flowers is rutin, which known had an anti-allergic activity because there was an effect on mast cell activity mediated by IgE. 37 In the type 1 hypersensitivity reactions, IgE was antibodies which played an important role, IgE would be produced more in response to allergies. Furthermore, specific IgE bound to mast cells and basophil cells, and they would degranulate and release oxidative products. The reduction levels of IgE, it reduced IgE bound to mast cells or basophils thereby reduced the process of degranulation and reduced to release of mediators, one of them was the histamine which was a cause of symptoms of type I hypersensitivity reactions. 35 Rutin Figure 2: TLC kincung flower extract with eluent ethyl acetate P: formic acid P: water (100: [15][16][17]   IL-4 level male mice type 1 hypersensitivity showed in Table 1. Oneway Anova analysis of IL-4 level after kincung flowers extract at doses of 100, 300, and 1000 mg/kg BW in type I hypersensitivity mice showed a significant decreased (p<0.05). The result of decreased IL-4 level showed in Table 1. Duncan test results showed that extracts with doses of 1000 mg/kg BW, doses of 300 mg/kg BW and 100 mg/kg BW differed in reduced IL-4 levels and the effects produced by doses of 1000 mg/kg BW and 300 mg/kg BW could reduce IL-4 levels such as healthy mice without allergies.
In type 1 hypersensitivity reactions, the role of cytokines is enormous, especially the process of IgE formation. Interleukins played a role in this regulation was cytokines produced by