Improved treatment of Asthma by using natural sources of antioxidants.

ABSTRACT: A combined composition of the extracted powders from Hippocampus kuda and Rhizoma Homalomenae together with honey in a form of medical pill (named as BRONAS) for the treatment of asthma has thoroughly been investigated under this study. BRONAS has shown its high anti-inflammatory effects and strong inhibition upon the pathogenesis of asthma. In comparison with other treatments without using BRONAS, the restoration of patients' health was improved by a factor of 2-3. CLINICAL TRIALS WITH ACTR NUMBER: ACTRN12612000766819.

Introduction

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.

Materials and methods

Preparation of extracts from seahorse (Hippocampus kuda)

The antioxidant extracts were prepared by adopting the method of Zhong-Ji Qian to check the total phenolic content and determine the total antioxidant activity.

Preparation of extracts from Rhizoma Homalomenae

The plant materials of Rhizoma Homalomenae used in this study was donated by DUC- HUNG- a traditional medicine shop in Nhatrang City- a central part of Vietnam. The dried materials were ground to the fine powder and passed through a 20-mesh sieve for the preparation of extracts.The sieved powder was subjected to water distillation for 5 hrs by adopting the method of Zeng et al. 2011.

Preparation of medicinal pills

In this study, a dosage of 500mg pill was prepared and named as BRONAS, and described as below:

BRONAS was prepared from 200 mg of dried extract powder of Hippocampus kuda*, 200 mg of dried extract powder of Rhizoma Homalomenae** and 130 mg of honey***, and then hand – rolled into pills of around 500 mg, each.

Notes

*The moisture content of the dried extract powder of Hippocampus kuda was 3%.

**The moisture content of the dried extract powder of Rhizoma Homalomenaee was 3%.

***The moisture content of the used honey was 17%.

All the hand- rolled pills were dried in an oven at 55°C for 34 to 46 hours. Moisture content of the pill was determined by the standard AOAC method (AOAC 2000)

Determination of total antioxidant activity of BRONAS

The antioxidant activity of BRONAS was determined right after drying the pills to the consistent moisture content. The antioxidant potential of BRONAS was determined on scavenging activity of the DPPH Free- Radical by adopting the method described by Sadhu et al. (2003).

The maximum absorption (λ max) of a stable DPPH in methanol is 520 nm and the results are expressed as IC50 values. The percent inhibition, radical scavenging capacity was calculated using the following equation:

Where: A control = Absorbance of DPPH alone.

A sample = Absorbance of DPPH along with different concentrations of extracted sample.

IC50 was calculated from the slope obtained by plotting a graph of concentration versus % inhibition.

According to Molyneux P (Molyneux 2004) DPPH is the molecule of 1,1-diphenyl-2-picrylhydrazyl (α, α-diphenyl β picrylhydrazyl) characterized as a stable free radical by virtue of the delocalization of the spare electron over the molecule as a whole, so that the molecules do not dimerize, as would be the case with most other free radicals. The delocalization also gives rise to the deep violet colour, characterized by an absorption band in ethanol solution centered at about 520 nm. When a solution of DPPH is mixed with that of a substance that can donate a hydrogen atom, then this gives rise to the reduced form. With the loss of this violet colour (although there would be expected to be a residual pale yellow colour from the picryl group still present). Representing the DPPH radical by Z* and the donor molecule by AH, the primary reaction is Z* + AH = ZH + A*

Where:

ZH is the reduced form

A* is free radical produced in this first step.

The latter radical will then undergo further reactions which control the overall stoichiometry, that is, the number of molecules of DPPH reduced (decolorized) by one molecule of the reductant.

In this study, stock solutions of the BRONAS sample were used for preparing various concentrations of 200, 400, 600, 800 and 1000 (mg/L).

Patients

This study was performed at the Home Clinic, 345 D5 Street, Binh Thanh District of Ho Chi Minh city, Vietnam between September 2011 to early April 2013.

Ninety two asthmatic patients (55% males and 45% females) who come from different parts of Vietnam, were randomly selected and included in the study. Their age range was 12–65 years. All Patients were desperately suffering from disease of asthma and all of them were many times hospitalized and measured value of FEV1/FVC were from 50% to 60%, of which the shortest disease history was 3 years and the longest was 11 years. It is worth noting here that most of the participating patients experienced a set of clinical symptoms which result in the sensation of difficulty breathing such as Spasm of the bronchial muscles and shortness of breathing, cough, expectoration, and prolonged expiratory phase with wheezing. In addition, all of the participating patients were those, who had ever used high doses of inhaled corticosteroids and particularly long acting inhaled β2-adrenergic agonists as the relief of bronchial constriction to reduce the asthmatic episodes.

, adopting the method of Faruk et al. 2010 for checking possible effects of the given medicines on the treated patients. By applying this, a total computerized spirometer (Discom-14 Autospiror, Chest Corporation Tokyo, Japan) that measures Forced Volume Capacity (FVC), Forced Expiratory Volume in First Second (FEV1), Peek Expiratory Flow Rate (PEFR), and Maximal Mid-expiratory Flow Rate (MMEFR) was extensively applied as it would provide predicted values.

The patients were then randomly allocated to 2 groups of A and B. The group A of 40 (18 Males: 22 Females) patients was given PREDISONE with a single dose of 10 mg/kg of body weight, daily for each 21 days and stopped for pulmonary function check. The group B (24 Male: 28 Female) was given BRONAS with a single dose of 1200 mg/kg of body weight, daily for 21 days and stopped for pulmonary function check as well. The group A was then given the same dose of BRONAS as to the group B for 21 days for further comparing the anti-inflammatory effect of BRONAS with the anti-inflammatory effect of PREDISONE.

Data analysis

All data are expressed as means ± standard deviation representative of similar tests carried out in triplicate. Statistical differences were determined by student’s t-test in which, p<0.05 was considered as statistically significant.

Ethics

All participated patients gave their consent prior to participation in the study.

Results

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)

Table 1

OD values after being measured in triplicate

	Blank	200 mg/L	400 mg/L	600 mg/L	800 mg/L	1000 mg/L
	0.53	0.40	0.38	0.28	0.24	0.19
	0.51	0.41	0.33	0.25	0.22	0.10
	0.53	0.42	0.37	0.30	0.23	0.21
Mean	0.52	0.41	0.36	0.28	0.23	0.20
SD	0.01	0.01	0.02	0.03	0.01	0. 01
CV (%)	2.10	1.72	6.30	9.54	5.51	4.30
DPPH (%)	0	22.91	32.21	47.32	56.62	62.70

Figure 1

The DPPH free radical scavenging of the BRONAS sample.

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.

Table 2

The effects of BRONAS and PREDISONE on pulmonary functions test

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)

Figure 2

Percentage increment in FVC, FEV , MMEFR after treatment with PREDNISONE and BRONAS.

The ratio of FEV1 to FVC (v/v) after treatment was calculated using the formula:

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.

Table 3

The effects of BRONAS on pulmonary functions test in the post treatment of PREDISONE

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)

Figure 3

Percentage increment in FVC, FEV , MMEFR in the post treatment of PREDISONE with BRONAS.

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 83th 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.

Figure 4

Percentage increment in FVC, FEV , MMEFR in the 83 treatment with BRONAS.

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.

Discussion

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%.

Conclusion

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.

Ethical approval

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