Chemical, Biological and Comparative Clinical Evaluation of Linkus Kids Syrup to Determine the Safety and Efficacy for the Treatment of Cough

Abstract

Introduction more than 102/ ml, Enterobacteria and some other gram- negative bacteria – not more than 102/ g, Salmonella absent in 10 ml and E. coli, Staphylococcus aureus – absent / ml. Linkus lozenges revealed average weight (2.375g to 2.625g), weight uniformity (2.5g ± 5%), thickness (7mm ± 1mm) and diameter (17mm ± 1mm) by Vernier caliper. Linkus cough syrup and lozenges both contains Adhatoda vasica vasicinone and vasicine (helps to reduce cough, common cold and serve as an expectorant),root of Glycyrrhiza glabra contains active glycyrrhizin (helps for upper respiratory tract infections),Piper longum fruit and root contains active piperlonguminine , piperine, piperlongumine (helps for cough and other respiratory tract infections), Hyssopus officinalis, Alpinia galangal, Zingiber officinale and Cordia latifolia (used to control coughing and respiratory tract infections). In current study, Standardize Linkus syrup and lozenges have been prepared in amber color bottle. Various Physiochemical parameters including organoleptic parameters, pH (3.0-5.0) and Density (1.25 - 1.35 g/ml) were calculated for Syrup. On qualitative evaluation tanning agents and ascorbic acid were confirmed however the quantitative analysis showed total alkaloid content as vasicine were not less than 0.035 %.Microbial purity was checked and confirmed that Bacteria – not more than 104/ ml, Fungi – not Cough is the most common symptom in children and causes discomfort in health care utilization .Over the counter (OTC) cold and antitussive medicine is very common in kids under 2 to 5 years of age and almost all children experienced cough and associated symptoms .The usefulness of anti-cough medications remains debatable in spite its wide spread consumption. Different Mata analysis and Clinical trials are investigating their role and claimed lack of evidence. A systematic review of randomized controlled trials on different kinds of these medications concluded they were not superior to placebo. The recognitions and advancements in evidence based herbal treatment have gained popularity along with the allopathic medication. Herbal medicines have gained attraction and captured the market during the last decades .WHO claims that 65-80% population in developing countries prefer plants for the treatment in health care division . For investigating the efficacy and toxicity of Linkus Syrup and lozenges in experimental animal Albino Rats were choosing. It is a first of its kind studies to further authenticate the safety of linkus lozenges. The Acute toxicity test for Linkus dosage foams was performed on Albino rats Strain Haffkine with both Sex ratio 1:1.Weight was between 150 – 250 g and ages were 4 – 6 months. 0.21 g / kg is the therapeutic dose of the preparation. 20 times (4g/kg) the therapeutic dose was tested for acute toxicity. For chronic toxicity testing both gender albino rats with strain Haffkine ,1:1 ratio (weight between 150 – 200 g) were selected for experimentation. The morphological examination of organs and estimation of biochemical parameters were assessed .Recommended adult x The biomarker Vasicine was analyzed qualitative and quantified through HPTLC by UV visible spectrophotometry, which was not less than 0.02 mg per lozenges and 0.080mg respectively. The quantitative estimation of Piperine and Glycyrrhizin biomarkers via HPLC & HPTLC method were used in Linkus Kids syrup. Chromatography included HPTLC and HPLC has been used to determine the biomarkers included Piperine and Glycyrrhizin using Benzene: Ethyl acetate (v/v): Methanol: Water: Formic Acid, 15:5:1:1 (v/v/v/v) as a solvent system with ultra violet light 345 for Piperine and 254 nm for Glycyrrhizin. The HPLC chromatograms of Piperine with standard at an optimum wavelength on 345nm at a retention time of 1-8.09 min however in HPTLC silica gel G60F254 with the use of CAMAG Linomet 5,CAMAG Scanner method the Rf value seemed to be <1 (0.1-0.20). Linkus dosage foam tested for anti-microbial activity against Staphylococcus aureus, Streptococcus pyogenes, Salmonella and Pseudomonas Aeruginosa. McFarland principles were adopted for maintaining the turbidity in bacterial microorganism. Antimicrobial testing was performed on agar and well diffusion method. As per disc diffusion, the disc was 0.007 m and well prepared with the help of a cork-borer were 0.85 cm. The outcomes of the study exhibited that linkus cough shows strong sensitivity against Staphylococcus Aureus, Streptococcus pyogenes and Salmonella in both disc and well diffusion method and showed resistivity against Pseudomonas Aeruginosa. However with the comparison of Amoxicillin and Ampicillin, linkus showed more sensitivity against Salmonella and Streptococcus pyogenes. dose was 15 g/day, Dose /kg of body weight was 0.21 g/kg however the therapeutic dose of the preparation was 20 (4g/kg) and was tested for chronic toxicity. Housed condition was 75ºF or 25˚C ± 1˚C and nourishment at labium. No mortality and no abnormality were seen inlinkus dosage foam and suggested it to be the safest choice The Phase IV randomize control trial was establish to evaluate the safety and efficacy of Linkus with other 2 arms included Aminophylline with Diphenhydramine group and Acefyllin Piperazine with Diphenhydramine cough syrup on children having cough and sleep difficulty associated with cough. Simple randomization pattern were adopted for patient randomization.To determine the effects of Linkus polyherbal syrup (group A) and compared with other parallel allopathic groups (Group B and C) for cough on children and associated sleep quality and improvement. 360 children having cough inducted in 3 different groups randomly selected. Ethnic groups, frequency of cough and diseases illness (<0.05) were determine on every group on the investigational syrup. Cough impact on child and its sleep of three different syrups (every group) were assessed on day1 and day 14(p< 0.001) via a likert scale. For the evaluation of pain assessment Wong baker face scale were used and level of significance in each group (p <0.001).Significant results were observed in the Linkus Group as compared to the other parallel groups including Aminophylline plus Diphenhydramine and Acefyllin Piperazine with Diphenhydramine on day 14 (p<0.001).Side effects on group B and group C (Aminophylline with Diphenhydramine and Acefyllin Piperazine with Diphenhydramine) were almost similar in number however linkus syrup have minimum side effects on study duration. Polyherbal syrup Linkus shows better results in treatment of cough including side effects as compare to the other parallel groups B and C (Aminophylline with Diphenhydramine and Acefyllin Piperazine with Diphenhydramine). The interquartile ranges on the same selected marketed group were established with Linkus. It was open label multi centric simple randomize control trial. The study was conducted on different private schools of East and West Malir ,Karachi Pakistan with the special approval from the school honors .Informed consent and assents were taking before the enrollment of the study subjects . The study enrolled participants were 196 who evaluate on cough and associated problems include 140± females and 56± were male.Participants were divided into 3 interventional group according to the treatment regimen .One group of participant received Linkus Syrup however the 2nd group received Acefyllin Piperazine and 3rd group received Aminophylline Diphenhydramine group. The dosing of Linkus syrup were 1-2 table spoon (15 ml) 3-4 times daily However the 2nd and 3rd group taking dose as per the standard 2.5 to 5ml 3-4 times daily . One way analysis of variance was used to compare the mean across treatment groups and Pearsonchi square test was used to see the association of qualitative variables. Wilcoxon signrank test was applied to see the effect of treatment outcomes at pre and post stages,Median and inter quartile ranges also reported along with p-values. Box plot, bar and pie chart used to display the information in graphical way, all p-values less than 0.05 were considered significant. The frequency of the cough on linkus Syrup was considered to be achieved on the basis of interquartile relationship (1st and 3rd Quartile) and impact has been observed on child and parent sleep and found significant (p <0.01). The frequency of the side effects observed significantly reduced (p<0.01) in Linkus Group and consider to be the effective with minimum side effects however the pharmacological treatments are considered to be more unwanted effects on human subjects.