Antiasthmatic effect of Curcuma aeruginosa extract on isolated organ of the trachea [version 1; peer review: 1 approved with reservations, 2 not approved]

Background: Asthma is a major health problem worldwide. Antiasthma drugs have side effects and can be expensive. It is important to develop antiasthma drugs from medicinal plants that have fewer side effects and are cheaper. One of the medicinal plants used for antiasthma treatment comes from Curcuma aeruginosa ( Zingiberaceae family). The aim of the research is to examine spasmolytic activity of ethanol extract of C. aeruginosa on isolated guinea pig tracheas to determine the antiasthma effects. Methods: The spasmolytic activity of C. aeruginosa extracts was tested in separated organs of guinea pig trachea. Guinea pig was sacrificed and its trachea rings were suspended in L-shaped wire loops in organ baths containing the Krebs solution aerated with carbogen. Isometric contractions of tracheal rings were measured by the transducer coupled to the amplifier. The trachea rings were exposed to DMSO as negative control, aminophylline as positive control and C. aeruginosa extracts. The single concentration-relaxation curve was obtained in every preparation. Results: The result showed that the decrease of the spasmolytic activity in the guinea pig tracheal tone due to C. aeruginosa extract was significantly better (p=0.022) when compared to the negative control. Meanwhile, the EC50 value of aminophylline (0.019 ± 0.05) was not significantly different (p=0.454) with C. aeruginosa (0.024 ± 0.05). Conclusion: It could be concluded that C. aeruginosa extracts have the potency to be further developed as a new natural source of the antiasthma agents. Open Peer Review


Introduction
Asthma is an inflammatory airway disease characterized by the occurrence of an respiratory airway hyper response and reversible narrowing of the airway 1 . Asthma is one of the major non-communicable diseases in the world. About 235 million people worldwide suffer from asthma, particularly children. The strongest risk factors for developing asthma are a combination of genetic susceptibility to certain inhalable allergens and environmental exposure to them 2 . Asthma medications are given to manage asthma sufferers 3 . Herbal preparations are one of the most popular complementary treatments used by asthmatic patients. Many important asthma drugs such as B2-agonists, anticholinergics, methylxanthines, and cromones have herbal origins 4 . Some medicinal plants have the effect of reducing smooth muscle stiffness, similar to the mechanism of asthma drugs, especially the anticholinergic drugs 5 . Research has also shown that some medicinal plants have the anti-inflammatory effects, following the same mechanism of corticosteroid drug used in asthma treatment 6 .
The genus Curcuma (family Zingiberaceae) consisting of more than 100 species is used widely as food a and in traditional medicines 7 . Indonesia is home to many species of Curcuma. The various species of Curcuma often used are C. longa (turmeric), C. xanthorrhiza, C. heyneana, C. aeruginosa, C. mangga, and C. zedoaria. Turmeric is the most frequently used plant for traditional medicine in Indonesia. C. aeruginosa considered as indigenous Curcuma species in Indonesia are currently not extensively studied, yet 8 . Important medicinal plants from the genus Curcuma with antiasthmatic potential include C. longa 9 . Other rhizomes of Curcuma species are traditionally used in the treatment of asthma, i.e. C. aeruginosa 10 , C. mangga 11 , C. caesia 12 , and C. zedoaria 13 . The antiasthma effects of C. aeruginosa are currently known, therefore, the objective of this study was to establish the tracheospasmolytic activity of C. aeruginosa applied on isolated tracheas of guinea pigs.

Plant materials
The sampling of medicinal plants was conducted in Kutai Kartanegara District, East Kalimantan (0°59'51.1"S 116°58'33.1"E). Plants were then identified in the Faculty of Mathematics and Natural Sciences, Mulawarman University by comparing to the university herbarium collection.

Plant extractions
The rhizomes of C. aeruginosa were sliced and dried at room temperature for 3 days, crushed and transferred into a glass container. Approximately 1 kg of crushed rhizomes was soaked in 1 L of absolute ethanol (9401-03 Alcohol, Anhydrous, Reagent, J.T. Baker) for 5 days. The mixture was shaken occasionally with a shaker (3525 Incubator Orbital Shaker, Lab-Line, US). After 5 days, the materials were filtered (Whatman Filter Paper 11µm, Sigma-Aldrich) and evaporated using a rotary evaporator (RV06-ML Rotary Evaporator, IKA, Germany). The dried extracts were obtained and stored at 4°C in a dark bottle until use.

Experimental model
One male guinea pig (Cavia porcellus) (6 months old, 485 g) was obtained from Animal House Faculty of Medicine (Mulawarman University). They were treated in a controlled room temperature of 25°C, with a 12-hour light/dark cycle, and access to food pellets and filtered water ad libitum. The guinea pig was anesthetized intraperitoneally with a ketamine injection (Hameln Pharmaceuticals, Germany) at a dose of 60 mg/kg before the trachea was taken. After anesthetized, animals were euthanized by cervical dislocation. The trachea was quickly dissected by adhering fat and connective tissue of guinea pig.

Spasmolytic activity
The trachea rings were suspended in L-shaped wire loops in 10 ml organ baths (PL3508B6 Panlab Organ Bath System, ADInstruments), containing the Krebs solution (K3753 Krebs-Henseleit Buffer, Sigma-Aldrich) aerated with carbogen by maintaining the temperature at 37°C. Isometric contractions of tracheal rings were measured by the transducer (7004 Isometric Force Transducers, Ugo Basile) coupled to the amplifier (FE 221 BridgeAmp, ADInstruments) connected to PC running LabChart V5 software. An equilibration period of 90 minutes was done in Krebs solution. At the end of the equilibration period, the tracheal rings were stimulated with histamine in order to establish viability. After equilibration, the tracheal rings were exposed to DMSO (W387520 Methyl sulfoxide, Sigma-Aldrich) as the negative control, aminophylline (A1755 Aminophylline, Sigma-Aldrich) as positive control drugs and extract of C. aeruginosa according to the experimental protocol by Janbaz et al. 14 . The dosage for DMSO, aminophylline and plant extract were 0.0001, 0.0003, 0.001, 0.003, 0.01, and 0.03 mg/ml given 700, 750, 800, 850, 900, and 950 seconds after equilibration on the organ baths. The dose-response curve for trachea relaxation activity was obtained in every preparation.

Data analysis
Trachea relaxation activity is tabulated in the mean ± SD curve of the dose-response curve. The value of EC 50 was calculated with Microsoft Excel 2016 as shown in Dataset 1. Data were analyzed using the Mann-Whitney because not normally distributed. All statistical analysis was performed using SPSS version 16.0 for Windows. A p-value of ≤ 0.05 was considered to be significant.

Ethical considerations
All protocols used in this experiment received approval from the Ethical Animal Care from the Medical and Health Research Ethics Commission, Faculty of Medicine, Mulawarman University No. 72/KEPK-FK/V/2018. All efforts were made to ameliorate any suffering of animals used in this research.

Results
The results of trachea relaxation between negative control, aminophylline, and C. aeruginosa extract presented in Figure 1. The result showed that the decrease of spasmolytic activity of C. aeruginosa extract was significantly better (p=0.000) than that in negative control. Meanwhile, the EC 50 value of aminophylline (0.019 ± 0.05) was not significantly different (p=0.454) with C. aeruginosa (0.024 ± 0.05), as shown in Figure 2.   File 1) is known in Indonesia as temu ireng or "pink and blue ginger" in English 15 . The color of fresh the rhizome can be yellows or greenish blue in color and mildly aromatic with a ginger-like aroma 16 . C. aeruginosa has been used as a traditional medicine in South and Southeast Asia 17 . The rhizomes have been used for gastrointestinal and uterine disorders, as well as parasitic and fungal infection 18 .

C. aeruginosa (Supplementary
Other pharmacological activities of C. aeruginosa that have been reported include inhibition of HIV, anti-cancer activity, hepatoprotective, antiandrogenic, estrogenic properties, antimicrobial, antioxidant, antiplatelet-activating factor-like, antipyretic, antinociceptive, and anti-inflammatory 19 . Germacrone, zedoarone, dehydrocurdione, curcumenol, zedoarondiol, and isocurcumenol were chemical constituents from sesquiterpenes isolated from rhizomes of C. aeruginosa 20 . In this study, the examination of antiasthma effects of C. aeruginosa has been reported. Further research is needed to identify the chemical compounds from C. aeruginosa that could convey antiasthma activity.

Conclusion
The results of this study indicate that ethanol extract of C. aeruginosa has an antiasthma effect based on the tracheospasmolytic activity. Therefore, C. aeruginosa can be developed as a possible source of new antiasthma drugs.

Institute of Environmental Medicine (IMM), Karolinska Institute (KI), Stockholm, Sweden
The authors attempt to evaluate if an ethanolic extract of the plant Curcuma Aeruginosa possesses bronchodilatory actions in the guinea pig trachea. Figure 1 shows that the influence of the extract is no different from a presumed negative control (DMSO). The data therefore do not support the author's conclusion that the extract has anti-asthmatic effects. The title of the manuscript is therefore wrong.

Specific major concerns:
Although figure 1 displays data without indications of the variability, the curves for the extract and the negative control are almost superimposed, whereas the curve for aminophylline shows the expected relaxation. 1.
The calculations for Figure 2 are impossible to understand, and as far as I can see incorrect.
EC 50 values are often calculated as 50% of the maximal response to e.g. a supramaximal dose of aminophylline. Here, it seems that the value causing 50% of the response to the highest dose tested (3 mg/mL) has been used for aminophylline, but this dose causes only 35% relaxation of the preparation. It is even more difficult to understand how an EC 50 value can be calculated for the extract because figure 1 indicates it causes no relaxation. In addition, the weight of an extract containing many undefined ingredients cannot be compared for potency with a pure pharmacologic agonist. The only comparison that would be relevant would be the qualitative documentation that the extract has bioactivity. The study has failed to show that.

2.
The method description is unclear. Was the extract and the controls tested in a histamineprecontracted preparation, or only in a preparation kept at baseline tension? 3.
Minor comments: Why was DMSO the negative control? Was really the lyophilized extract dissolved in DMSO for the tests? 1.
The dosage of DMSO is given in mg/mL and seems to be quite high.

2.
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