RETRACTED ARTICLE: Improved treatment of Asthma by using natural sources of antioxidants

Asthma is a chronic disease characterized by inflammation of the airways (Buse and Lemanske 2001), a complex disorder characterized by variable and recurring symptoms, airflow obstruction, bronchial hyperresponsiveness, and an underlying inflammation. The interaction of these features determines the clinical manifestations and severity of asthma, and it has been reported as a disease of increasing prevalence (Expert Panel Report 3: Guidelines for the diagnosis and management of asthma. NIH Publication No. 07–4051. 2007).

The pathogenesis of asthma is unknown but imbalances between oxidants and antioxidants are believed to play a fundamental role. One key component of the oxidant-antioxidant hypothesis centers on the huge burden of oxidants derived from inflammatory cell infiltration into the lung. The eosinophil, in particular, is implicated as a major source of oxidative injury, including protein nitration (MacPherson et al. 2011). Dysfunctional mitochondria in lung cells are another potential source of oxidants. Mitochondrial injury to airway epithelium occurs in murine models of allergic asthma (Aguilera-Aguirre et al. 2009; Mabalirajan et al. 2008). There is evidence to support its role in human asthma as well, including increased oxidative injury to mitochondrial epithelial cell superoxide dismutase (SOD)( Comhair et al. 2005), enhanced mitochondrial proliferation in bronchial smooth muscle (Trian et al. 2007), and mutations in mitochondrial DNA (Reddy 2011). Overall, this oxidative burden, generated by both inflammatory and lung cells, can overwhelm antioxidant defense to cause oxidant stress during asthma. Thisk stress can alter or inactivate the function of essential proteins, lipids and nucleic acids culminating in severe cell injury, dysfunction and death.

Among many unknown and complicated mechanisms, involvement of airways inflammation with an oxidant/antioxidant imbalance such as reactive oxygen species (ROS) can lead to lung injury as a result of direct oxidative damage to epithelial cells and cells shedding. As inflammation is often associated with an increased generation of reactive oxygen species (ROS), it is rational to surmise that an oxidant stress could be mechanistically important in asthma. ROS have been shown to be associated with the pathogenesis of asthma by inducing bronchial hyperreactivity as well as directly stimulating histamine release from mast cells and mucus secretion from airway epithelial cells (Ryszard 2000).

The great external surface area (1–2) m² of the human airway epithelium plus its direct contact with the environment, makes the respiratory tract a major target for oxidative injury from inhaled oxidants such as cigarette smoke, ozone, hyperoxia, nitrogen and sulphur oxides and other airborne pollutants. It has been well recognized that biological systems are capable of forming highly reactive moieties, both free radicals and non-radicals named reactive oxygen species (ROS) and reactive nitrogen species (RNS). Free radicals can especially be generated in a wide variety of chemical and biological systems, including the formation of plastics, the ageing of paints, the combustion of fuels and in the human body. In living organisms, the levels of free radicals and other ‘reactive species’ are controlled by a complex web of antioxidant defences, which minimize (but do not completely prevent) oxidative damage to biomolecules (Roberfroid and Calderon 1995; Gaston et al. 1994; Halliwell (2005).

These biologically active species serve in cell signaling as messenger molecules of the autocrine or paracrine system (Saran and Bors 1989; Suzuki et al. 1997 ) and also in host defense, (biocidal effects against microbial and tumor cells) (Babior 1978) but their excessive production may result in tissue injury and inflammation (Halliwell et al. 1992; Gutteridge and Halliwell 1994). Reportedly, any excessive production of oxidants is kept to a minimum by a well coordinated and efficient endogenous antioxidant defense mechanism. It has been proposed that a deficit in the precise balance between exposure to oxidants and endogenous antioxidants results in oxidative stress which appears to be involved in the pathogenesis of a growing number of diseases, including lung pathologies such as respiratory distress syndrome, asthma, idiopathic and iatrogenic pulmonary fibrosis, cystic fibrosis, HIV-associated lung disease, lung cancer and other pulmonary diseases and conditions (Clement and Housset 1996; Barnes 1995) . As excessive ROS levels damage lipids, proteins and nucleic acids through oxidation and thus are associated with various diseases, such as atherosclerosis, arthritis, neurodegenerative disorders, and cancers , a regular supplement of antioxidants can assist the endogenous defense systems to counterbalance the harmful effects of excessive ROS (Balsano and Alisi 2009; Kaur and Geetha 2006).

There has recently been a remarkable increment in scientific articles dealing with oxidative stress (Urguiaga and Leighton 2000). Consequently, knowledge about reactive oxygen and nitrogen species metabolism; definition of markers for oxidative damage; evidence linking chronic diseases and oxidative stress; identification of flavonoids and other dietary polyphenol antioxidants present in plant foods as bioactive molecules; and data supporting the idea that health benefits associated with fruits, vegetables in the diet are probably linked to the polyphenol antioxidants they contain. In addition, more than 8,000 polyphenolic compounds have been identified in various plant species and reported to possess many useful properties including antiallergic, antiinflammatory, antimicrobial, antiviral, antioxidant, oestrogenic, enzyme inhibition, vascular and cytotoxic anti- tumor activity (Pandey and Rizvi 2009; Asha et al. 2012).

According to Ji-Xiao Zhu et al., among many other contained phenolic compounds plants, Rhizoma Homalomenae has been shown to be positive linear correlations between total phenolic content and antioxidant activity of the extracts in the DPPH (R = 0.9817), ABTS (R = 0.9873), b-carotene/linoleic acid (R = 0.8347) and reducing power (R = 0.9876) tests, respectively.

Another source of natural antioxidants is naturally occurring from traditional Chinese medicines sources, have been identified as free radical or active oxygen scavengers (Duh 1998; Pan et al. 2007). Natural antioxidants can protect the human body from free radicals and retard the progress of many chronic diseases as well as retard lipid oxidative rancidity in foods or medicinal materials (Kang et al. 2008). Among the many mentioned sources of naturally occurring antioxidant, Seahorse (Hippocampus kuda Bleeker) has been well known for its special medicinal composition. According to Zhong-Ji Qian et al., the methanol extracts of seahorse contained high amount of phenolic compounds and those extracts exhibited good antioxidant activity by effectively scavenging various free radicals such as DPPH radicals, hydroxyl radicals, superoxide anion radicals, alkyl radicals, and reducing the ferric to ferrous ion in different antioxidant systems.

In a search for treatment that might stop the recurrent attacks of breathlessness and wheezing to make it more susceptible to at least, providing relief for asthmatic patients, and if possible to treat the asthmatic disease, a method has emerged that seems to be extremely useful for application of natural sources of antioxidants for treatment of asthma. The method was using finely extracted powders from the seahorse (Hippocampus kuda) and Rhizoma Homalomenae (with a ratio of 1: 1 w/w) in honey to form into pill of 500mg. All the hand-rolled pills were dried in an oven at 55°C until the moisture content of the pill was consistent.

In this paper, successful application of extracted powders from the seahorse (Hippocampus kuda) and Rhizoma Homalomenae together with honey in the form of medical pill for treatment of asthma is reported.

Data on total antioxidant activity of BRONAS were shown in the Table 1 and Figure 1. The values obtained were also shown that the DPPH scavenging effect in percentage of BRONAS were three times higher than those extracted from green tea using the same method of extraction (Yen and Chen 1995)

The Forced Volume Capacity (FVC), Forced Expiratory Volume in First Second (FEV1), Peek Expiratory Flow Rate (PEFR), and Maximal Mid-expiratory Flow Rate (MMEFR) were extensively applied for determining the response to the therapy and monitoring the rate of progression. The results have been summarized and shown in the Table 2 and Figure 2.

Drug	FVC %		FEV1 %		MMEFR %		PEFR
	Before	After	Before	After	Before	After	Before	After
PREDISONE	63.45 ± 11.94	65.55 ± 12. 50	51.14 ± 11.63	*54.78 ± 12.20	50.28 ± 22.45	51.72 ± 11.60	2.96 ± 1.34	3.57 ± 2.32
BRONAS	61.38 ± 15.60	*69.35 ± 5. 32	55.39 ± 15.63	*65.32 ± 5.16	53.28 ± 22.45	*62.05 ± 3.60	3.26 ± 1.54	*5.94 ± 2.50

*Results were significant at (p<0.05)

The ratio of FEV₁ to FVC (v/v) after 21 days of treatment with PREDNISONE was 84.50%.

The ratio of FEV₁ to FVC (v/v) after treatment with BRONAS was 94%.

As the ratio of FEV₁ to FVC (v/v) after 21 days of treatment with PREDNISONE was improved but lower (84.50%) than the one obtained from the treatment with BRONAS (94%) in the same period of treatment process, the asthmatic patients in Group A was then continuously given BRONAS with a single dose of 1200 mg/kg daily for another 21 days of treatment. The collected data in this stage of treatment has been summarized and shown in the Table 3 and Figure 3.

Drug	FVC %		FEV1%		MMEFR %		PEFR
	Before	After	Before	After	Before	After	Before	After
BRONAS	65.63 ± 12.44	* 73.80 ± 9.67	62.34 ± 14.21	* 71.95 ± 10.18	52.34 ± 19.16	* 60. 92 ± 11.06	3.15 ± 1.75	* 5.86 ± 2.40

*Results were significant at (p<0.05)

After all the all ninety two patients having had treatment with BRONAS as given dose in the treating process mentioned above, sets of pulmonary functions test were conducted and found that till the 83^th day of treatment, all the asthmatic patients were fully or almost fully recovered. The result of pulmonary functions test has been summarized and shown in Figure 4.

This statement has been consolidated by the fact that no clinical symptoms of the severe asthmatic disease such as Spasm of the bronchial muscles and shortness of breathing, cough, expectoration, and prolonged expiratory phase with wheezing were seen again.

From the Table 2, it could be reasoned that there was no significant increase in FVC%, MMEFR% and PEFR but only FEV₁% by using single PREDNISONE in group A. In addition, the ratio of FEV₁ to FVC (v/v) after 21 days of treatment was 84.50%.

In group B, a significant improvement in Pulmonary Function Tests associated with improvement in patients’ clinical conditions was observed. After taking the BRONAS for the same period of treatment, the ratio of FEV₁ to FVC (v/v) was pretty high (94%).

Data collected from the oral administration of BRONAS of patients in group A, of which shown in Table 3 and Figure 4 would again support and explain the beneficial effect of taking BRONAS in reducing the incidence of asthma when the ratio of FEV₁ to FVC (v/v) was around 97.50%.

FEV₁ has been found to be a better physiologic index than PEFR in the measurement of airflow obstruction (Christophe et al. 1998). So in this study, the values of PEFR were used as a reference to possibly support and/or explain the beneficial effect of orally administering the prescribed and prepared medical tablet/ pill in reducing, even treating the incidence of asthma.

The strong antioxidant properties of the BRONAS may be attributed to the presence of the combined bioactive antioxidant compounds from the extract of Hippocampus kuda and those of Rhizoma Homalomenae. Based on the report of Zhong et al. (2008), the extracted antioxidants from Hippocampus kuda contained effective reducing power, DPPH radical scavenging, hydroxyl radical scavenging, superoxide radical scavenging, alkyl radical scavenging and inhibitory intracellular ROS. Zheng and Wang (2001) reported that the strong antioxidant properties of the Rhizoma Homalomenae extracts may be due to the presence of phenolics compounds such as protocatechuic acid, vanillic acid, syringic acid, caffeic acid, p-coumaric acid, ferulic acid and apigenin. Phenolic acids are the main phenols consumed by humans (Ghasemzadeh and Ghasemzadeh 2011). The six phenolics mentioned above are all prominent and naturally occurring, and all of them individually possess potent antioxidant activity (3 Joskova et al. 2013; Gao et al. 2012). The honey was intentionally used with the dried extract powder of Rhizoma Homalomenae and that of Hippocampus kuda since it has been scientifically considered as an antimicrobial agent and chelating in traditional medicine. In addition, there is a great variety of minor components, including phenolic acids and flavonoids (Natella et al. 1999; Joskova et al. 2013), the enzyme glucose oxidase, ascorbic acid, carotenoids, organic acids, amino acids, proteins and α-tocopherol (Kesic et al. 2009).

By and large, the results obtained from this study showed that BRONAS can protect cellular components from many types of oxidative stress by both free radical scavenging mechanism and stabilizing the cell membranes and this may explain the improvement that occurred in both dynamic performance of the lung in moving air and the elastic recoil force of the lung, especially when the ratio of FEV₁ to FVC reached at least 94%.

The study demonstrates that the combined antioxidants from extracted powders of Hippocampus kuda and Rhizoma Homalomenae together with honey in a form of medical pill (BRONAS) can help improve the treatment of asthma. BRONAS can be used to replace synthetic antioxidants, which are being restricted due to their side effects such as carcinogenicity. In addition, BRONAS can be prepared at a lower cost and in a more friendly way.

The ACTRN12612000766819  was the Retrospectively registered and approved by Australian New Zealand Clinical Registry.