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