Asthma is a chronic inflammation of the respiratory tract. This study aimed to prove Moringa oleifera Lam. leaves extract's effect on reducing the eosinophil count and mast cells in asthmatic mice. Twenty BALB/c mice that met the inclusion criteria were divided into four groups of standard control (K1), negative control (K2), dexamethasone (K3), and M. oleifera leaves extract (K4). On days 0 and 7, intraperitoneal ovalbumin (OVA) was sensitized. On days 14, 16, and 18, mice were re-sensitized by inhalation using 1% OVA in sterile 0.9% NaCl. On days 18 to 25, K1 and K2 groups were given Na-CMC, the K3 group was given dexamethasone 1.3 µg day, and the K4 group was given M. oleifera leaves extract 3.9 mg day. On the 25th day, the mice were terminated to analyze the eosinophil count and stable bronchiolar mast cells. In conclusion, M. oleifera leaves extract was proven to decrease the eosinophil count with a p-value 0.05 and could stabilize bronchiolar mast cells with a p-value 0.05.

Asthma is a diseasewith an increasing prevalence rate, and there is a strong interaction betweenairway epithelial cells and the immune system against the pathogenesis ofasthma1. The most commonimmunopathology in asthma is inflammation of type 2 characterized by processesin the airway epithelium involving cytokines2. Symptoms of asthmainclude wheezing, shortness of breath, heavy chest, and coughing at night andearly morning. There is muscle contraction in the bronchial walls, swelling ofthe bronchial mucosa, and increased mucus production in the secretory cells ofthe bronchial mucosa, resulting in increased resistance to airflow through thenarrowed bronchi. This condition occurs in shortness of breath3,4. The increase inthe eosinophil count in asthmatic patients correlated with the severity of therespiratory failure and was predicted to increase the risk of sequentialsub-exacerbations. Immunoglobulin E (IgE) is involved early in the inflammatorycascade and may be considered a cause of allergic asthma5,6.

Moringa oleifera Lam. is considereda miracle plant with many benefits for humans and is credible for providinggood nutrition and healing and preventing many diseases7. Thepharmacologically reported effects include antibacterial, antifungal,antiviral, anti-inflammatory and analgesic, antioxidant, hypotensive,antiulcer, cardioprotective anesthetic, antiurolithic, and wound healingactivity8-12. The M. oleiferaplant has various therapeutic properties. For example, the general nutritionalcontent of M. oleifera to several specific properties, includinganti-inflammatory, antimicrobial, antihyperglycemic, antioxidant and anticancerproperties13. The secondarymetabolites of M. oleifera leaves are alkaloids, saponins, phenolics,tannins, flavonoids, and steroids14. Oraladministration of M. oleifera aqueous extract has been shown to reducesymptoms of type I allergy in ovalbumin-induced rats. Aqueous extract of M.oleifera altered the T helper (Th)1/Th2 balance towards Th1 dominance inthe allergy model in mice, resulting in suppression of mast cell activation,followed by histamine release15.

Treatment decisionsmust consider the biological background in this case of inflammation as theytend to predict the patient's response to asthma treatment16. Agrawal and Mehta17 studied fine powder dryseeds of 3 g on M. oleifera seeds for three weeks showing a significantincrease in forced vital capacity, forced expiratory volume in one second, andpeak expiratory flow rate values in patient subjects. Moringa oleiferaleaves extract was proven to decrease the eosinophil count and could stabilizebronchiolar mast cells18. However, research on theanti-plasma activity of M. oleifera leaves extract reduces theeosinophil count, and the stability of the number of mast cells in asthmaticmice has not been reported. Therefore, this study aims to prove the effect of M.oleifera leaves extract on reducing eosinophils and mast cells in asthmaticmice.

The materials used in this studywere M. oleifera Lam. leaves extract (Deltomed Laboratories, Indonesia),dexamethasone (Cayman Chemical, US), ovalbumin (OVA) (Worthington BiochemicalCorporation, US), and alhydrogel® (InvivoGen, US). The main instruments used inthis study were electronic nebulizer, automated hematology analyzerXS1000i/800i (Sysmex, Japan), and Olympus digital microscope OptiLab Pro 6.1. Thesoftware used for data analysis was IBM SPSS Statistics ®16.0.

Preparation of test animals

The test animals used were femaleBALB/c mice weighing 20-30 g, which has been acclimatized for a week. All test animalswere given drink and standard feed ad libitum. Female BALB/c mice were usedbecause they showed a better response to OVA than males19.

Research design

This study used a laboratoryexperimental quantitative approach with a post-test only control group designin asthmatic mice in vivo. The test group was divided into four groups:standard control (K1), negative control (K2), positive control (K3), and M.oleifera leaves extract group (K4). The treatment dose for M. oleiferaleaves extract produced by Deltomed Laboratories was 1500 mg/day, with the conversiondose for mice was 1500 mg x 0.0026 = 3.9 mg/day. For the standard controlgroup, BALB/c mice were given sterile 0.9% NaCl by intraperitoneal injection ondays 0 and 7. On days 14, 16, and 18, inhalation was carried out with sterile 0.9%NaCl as much as 8 mL per treatment by inhalation using an electronic nebulizerfor 20 minutes with airflow volume and volume nebulization on a scale of 1 onday 1. On days 18, 19, 20, 21, 22, 23, and 24, mice were treated with 0.5%Na-CMC orally 1 mL/day. For K2, K3, and K4 groups, all BALB/c mice havesensitized with 10 µg OVA + 1 mg alhydrogel® suspended in 0.5 mL of 0.9%sterile NaCl by intraperitoneal injection on days 0 and 7. On days 14, 16, and18, inhalation was carried out with sterile 0.9% NaCl as much as 8 mL pertreatment by inhalation using an electronic nebulizer for 20 minutes withairflow volume and volume nebulization on a scale of 1 on day 1. On days 18,19, 20, 21, 22, 23, and 24, mice were treated with 0.5% Na-CMC orally 1 mL/day(K2), 1.3 µg dexamethasone orally 1 mL/day (K3), and 3.9 mg M. oleiferaleaves extract orally 1 mL/day (K4). The research design schematic was shown inFigure 1.

Figure 1. Research design. R: Randomization; O1-O4:Observation of the eosinophil count and bronchial mast cells

Examination of eosinophil count

Blood sampling was performed to theanesthetized mice by an intraperitoneal injection of ketamine 200 μg/g. Theblood collection location was in the retro-orbital sinus/eye of mice using ahematocrit capillary pipette. The application could be made by inserting thepipette at an angle of 45°. This method could produce large volumes of blood,and samples could be obtained in both eyes alternately, accommodated in theEDTA blood tube20. The examination of the bloodeosinophils count was the eosinophils cell count x 109/L of bloodplasma, calculated by an automated hematology analyzer. This variable sizescale was a ratio. Data analysis was performed using one-way ANOVA and analyzedby post hoc test to determine the difference between the two treatment groups.Data were presented as average ± SD. Significance was defined at the p <0.05level.

Examination of bronchiolar mast cells

Mast cell examination was carriedout by histopathology of the mice's lungs with Toluidine blue staining andobserved with a 400x magnification software microscope Image OptiLab Pro 6.121. This variable size scale was aratio. Data analysis was performed using one-way ANOVA and then analyzed bypost hoc test to determine the difference between the two treatment groups.Data were presented as average ± SD. Significance was defined at the p <0.05level.

Ethical considerations

This research was conducted after obtaining approval from the healthresearch biotech commission of the Faculty of Medicine, Universitas IslamSultan Agung Semarang with Ethical Clearance No. 110/IV/2020/BioethicalCommission.

Normality and homogeneity analysis of the eosinophil count and mastcells

The data normality test for theeosinophil count was performed with Shapiro-Wilk, with a p-value >0.05,indicating that the data were normally distributed as presented in Table I. As for the homogeneity of the data, the post hoctest results were obtained with a p-value >0.05, indicating that the dataobtained for the eosinophil count and mast cells were homogeneous, as shown in Table II.

Table I. Normality of data withShapiro-Wilk test

Table II. Datahomogeneity by post hoc test

Examination of eosinophil count

The statistical analysis of the test results for the average, SD, Fvalue, and p-value of the eosinophil count in all groups were presented in Table III. The K2 had the highest average value of all othergroups. This finding indicates that the high average value of eosinophils inthe K2 could be reduced by administering dexamethasone or M. oleiferaleaves extract. The average difference between the two groups analyzed by posthoc test showed that the average eosinophil count between the K2 was 2±0.71 x109/L, and the K3 was 0.4±0.11 x 109/L, with a p-value of0.002 indicate that there was a significant difference between K2 and K3. Theaverage difference in the K3 was 0.4±0.11 x 109/L, and the K4 was0.48±0.15 x 109/L, with a p-value of 0.503, suggesting nostatistically significant difference between the K3 and K4 groups in reducingthe eosinophil count. The analysis results of the average difference aboveresulted in an F value of 6.773 and a p-value of 0.001, suggesting that theeosinophil count was significantly different between each study group.

Eosinophils are promoters of type-2inflammatory environment, which contribute to airway renovation in asthma,wherein eosinophils can increase airway hyperresponsiveness (AHR) and mucushypersecretion22,23. Moringa oleifera seed oilis proven to contain flavonoid compounds that have biological activity asanti-asthma by reducing the thickness of the bronchial epithelium of asthmaticmice24. The subchronic toxicity effect of M.oleifera leaves extract based on liver histopathology shows mild reversibledamage, and kidney histopathology shows that the renal filtration function ofall treatment groups is still expected25. Moringa oleifera leavescontain phenolic compounds that contribute to higher antioxidant activity. Theycan be used as a potential source of natural antioxidants in pharmaceuticals toimprove the function of the endogenous antioxidant system and help reduce freeradical levels in the body26. Eosinophils contribute to asthmaseverity and may persist despite guideline-based treatment27. The characteristic picture ofinflammation is characterized by an increase in the number of activatedeosinophils causing damage to the airway epithelium28.

Table III. Average, SD, F value, and p-valueof eosinophils count

Examination of bronchiolar mast cells

The test results for the average, SD, F value, and p-value of the mastcells in groups K1, K2, K3, and K4 were presented in Table IV. The analysis of the average difference aboveresulted in an F value of 10.062 and a p-value of 0.001, which means that thenumber of mast cells was significantly different between each study group. TheK3 had the highest average number of stable mast cells than the K2 and K4.

The average difference between thetwo groups analyzed by post hoc test showed that the average number of mastcells that were stable between the K2 was 2 ± 0.71 and the K3 was 4 ± 1, with ap-value of 0.003, indicates that there was a significant difference between theK2 and K3 groups. It could be assumed that dexamethasone could stabilize mastcells. The average difference between the K3 was 4 ± 1, while the K4 was 3.2 ±0.84, with a p-value of 0.176, which means that statistically, there was nosignificant difference between the K3 and K4 groups in stabilizing mast cells.

Table IV. Average,SD, F value, and p-value of mast cells

The analysis of the number of stablebronchial mast cells in all groups was carried out by taking pulmonary organs.The histopathological test was carried out with Toluidine Blue staining,observed microscopically with a magnification of 400x. The histopathological pictureof stabilizer mast cells was presented in Figure 2. In the lungs of patients with allergic asthma,mast cells accumulate in smooth muscle, bronchial epithelium, and alveolarparenchyma. This unique location of pulmonary mast cells can be replicatedusing a mouse model of allergic asthma29. Mast cells migrate to inflamedtissue and release pro-inflammatory mediators by degranulation upon activationof cell surface receptors such as high-affinity IgE receptor (FcεRI),prostaglandin E2 receptor (PGE2), or receptors for stem cell factor in humanand mouse mast cells30.

Figure 2. Comparison of stable mast cells in thestandard control group (K1), negative control group (K2), positive controlgroup (K3), and Moringa oleifera leaves extract group (K4), withtoluidine blue staining and 400x magnification. Red arrows indicate stable mastcells

Thisstudy shows that M. oleifera leaves could reduce the eosinophil countand stabilize mast cells; in line with the research of Suresh et al.31, whichreported that the methanol extract of M. oleifera leaves given to guineapigs shows anti-plasma activity such as blocking the release of inflammatorymediators to local lung tissue and bronchospasm, mast cell degranulation,immune reactions, and anaphylactic reactions. This was found to inhibit the inflammatorymediator, histamine. Moringa oleifera extract can inhibit the release ofβ-hexosaminidase, histamine, and tumor necrosis factor (TNF)-α more activelythan ketotifen fumarate. Moringa oleifera leaves has mast cellstabilizing activity and its potential to inhibit the final phase of theallergic response32.

Moringa oleifera Lam. leaves extract has been shown to reduce theeosinophil count and can stabilize the number of mast cells in asthmatic mice.There was no statistically significant difference between M. oleiferaleaves extract. Compared with positive controls given dexamethasone in reducingthe eosinophil count, the p-value was 0.503, and there was no statisticallysignificant difference between M. oleifera leaves extract. Whilecompared with positive controls (dexamethasone) to stabilize mast cells, thep-value was 0.176.

The authors would like to thank the laboratorytechnicians and staff of the department of pharmacology, Universitas IslamSultan Agung, Semarang. We also thank STIKES Cendekia Utama Kudus for thepermission for the research. All authors contribute to financing this work.

Dian Arsanti Palupi: conceptualization, supervision, methodology, datacuration, data analysis, validation, writing-original draft & editing. TriWahyuni Prasetyowati: conceptualization, supervision, data analysis,writing-review & editing. Dwi Murtiningsih: conceptualization,methodology, data curation, data analysis, writing-review & editing. DedeMahdiyah: project administration, data curation, data analysis,writing-review & editing.

None.

The authors declare no conflict of interest.

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