Determination of phenylethyl alcohol by reversed-phase high-performance liquid chromatography (RP-HPLC) in Budesonide nasal spray

Phenylethyl alcohol is used as an antimicrobial preservative in many pharmaceutical products, especially nasal sprays. A simple and accurate reverse phase high performance liquid chromatographic method was developed to assay of phenylethyl alcohol in budesonide nasal spray preparations. A waters C18 symmetry column chromatographic system (150 × 4.6 mm, 5 µm particle size) was performed with a 50:50 (%V/V) mixture of water and acetonitrile as a mobile phase. The detection of the phenylethyl alcohol was carried out at 220 nm and flow rate was employed 1.0 ml/min. The retention time of phenylethyl alcohol was about 2.8 min. Linearity was established in the concentration range of 173.28 to 259.92 mg/ml (80 to 120% of the target concentration), with a regression coefficient of 0.9991. Specificity was tested in the presence of placebo; no interference was detected at the retention time of phenylethyl alcohol. The results of the analysis were validated statistically and recovery percentage studies confirmed the accuracy and precision of the proposed method. 
 
   
 
 Key words: Phenylethyl alcohol, budesonide, nasal spray, reversed-phase high-performance liquid chromatography (RP-HPLC), preservative.


INTRODUCTION
Allergic rhinitis is a common disease and refers to inflammation of the nasal passages including sneezing, itching, nasal congestion and runny nose.Intranasal corticosteroids are among the most effective treatments for permanent allergic rhinitis.Some individuals unable to tolerate aerosols may prefer an aqueous nasal spray (Mygind, 1993).
Budesonide is a white to off-white, tasteless, odorless powder that is practically insoluble in water and in heptanes, sparingly soluble in ethanol, and freely soluble in chloroform.Its partition coefficient between octanol and water at pH 7.4 is 1.6×10 3 .Budesonide is an antiinflammatory corticosteroid that exhibits potent glucocorticoid activity and weak mineralocorticoid activity.In standard in vitro and animal models, budesonide has approximately a 200-fold higher affinity to the glucocorticoid receptor and a 1000-fold higher topical anti-inflammatory potency than cortisol.As a measure of systemic activity, budesonide is 40 times more potent than cortisol when administered subcutaneously and 25 times more potent when administered orally in the rat thymus involution assay (Rice- Thomas et al., 2009).
Antimicrobial preservatives are included in preparations to kill or inhibit the growth of microorganisms inadvertently introduced during manufacture or use.They are used in sterile preparations such as eye-drops and multidose injections to maintain sterility during use and in cosmetics, foods, and non-sterile pharmaceutical products such as oral liquids, creams, inhalations and nasal sprays to prevent microbial spoilage.The choice of a suitable preservative for a preparation depends on pH, compatibility with other ingredients, the route, dose and frequency of administration, partition coefficients with ingredients and containers or closures, degree and type of contamination, concentration required, and rate of antimicrobial effect (Thomas et al., 1989).
Phenylethyl alcohol is an excipient of budesonide nasal spray.It is an antimicrobial preservative designated chemically as 2-Phenylethanol.The empirical formula of phenylethyl alcohol is C 8 H 10 O (Figure 2) and its molecular weight is 122.17.Phenylethyl alcohol is a clear, colorless liquid with an odor of rose oil.It has a burning taste that irritates and then anesthetizes mucous membranes (Rowe et al., 2009;O'Neil et al., 2001).Phenylethyl alcohol is very soluble in alcohol, in fixed oils, in glycerin, and in propylene glycol, and sparingly soluble in water and slightly soluble in mineral oil Franson et al., 2012).
Phenylethyl alcohol in relatively low concentrations (1:400) exerts an effective inhibitory action on Gramnegative bacteria and may thus be used for differential inhibition (Lilley and Brewer, 1953;Hodges et al., 1996).Assay and detection of phenylethyl alcohol in nasal sprays is one of the important experiments during manufacturing process in quality control laboratory.
Therefore the aim of this study was finding a fast and valid measurement method to assess phenylethyl alcohol in budesonide nasal sprays.High performance liquid chromatography (HPLC) is one of the most powerful analysis methods.In recent years the use of reversed-phase high-performance liquid chromatography (RP-HPLC) method for determination of drug substances is very common and HPLC instruments and RP-HPLC solvents are available in most pharmaceutical laboratories.For this reasons, we developed a RP-HPLC method for determination of phenylethyl alcohol in Budesonide nasal spray and similar formulations which this analysis method is simple, fast, short response time, cheap price, with high accuracy and high precision.This analysis method was fully validated and can be done easily in any laboratories.

MATERIALS AND METHODS
HPLC grade acetonitrile was procured from Merck Company (Germany), pure standard of phenylethyl alcohol (99.9 % w/w) was obtained from LGC Company (England) and HPLC grade water was prepared by using Millipore Milli Q plus purification system (USA).The 0.45 µm nylon filter was obtained from Millipore Company (USA) and L1 columns were procured from both Waters Company (USA) and Agilent Company (USA).All other chemicals were analytical grade and commercially available.
Chromatography (Waters HPLC system, USA) was performed with a 1525 separation module through inherent manual injector jointed to 2487 UV detector and also a 2695 separation module with inbuilt auto injector and 2996 photodiode array detector.Waters C18 symmetry column (150×4.6 mm, 5 µm particle size) and Agilent C18 column were used for chromatographic separation under isocratic elution.Detection was carried out using an UVspectrophotometric detector at 220 nm and Waters Breez and Empower software was used.The mobile phase was a 50:50 (% v/v) mixture of prepared water and acetonitrile.Mobile phase was sonicated and degassed before use.The flow rate of mobile phase was adjusted at 1.0 ml/min.The column temperature was maintained at ambient conditions.The injection volume was 20 µl and total run time was 5 min.The phenylethyl alcohol was identified by retention time of the standard phenylethyl alcohol peak.Also in specificity test, phenylethyl alcohol peak was identified against standard compound peak in the presence of placebo.

Validation of the method
Based on previous and similar chromatographic methods, the best system for determining of phenylethyl alcohol was selected (Harris, 1991;Skoog et al., 1991;Moffat et al., 2011).The method is intended to assay phenylethyl alcohol in budesonide nasal spray during analytical method validation.The method was validated, in accordance with ICH guidelines (Authors Group, 2005) and other similar works (Rao et al., 2010;Blanco et al., 1999).All validation factors such as linearity, specificity, accuracy, precision, repeatability, reproducibility and robustness were assessed.

Linearity
Linearity was obtained with the concentration range of 173.28 to 259.92 mg/L for phenylethyl alcohol.Linearity was performed with different dilutions.Calibration solutions were 80 to 120% of the target concentration.
Calibration graph was plotted on the basis of analysis of calibration solutions.The coefficient of regression was obtained 0.9991 and the slop of 10283 was achieved (Figure 3).Standard stock solution of phenylethyl alcohol (2.166 g/L) was prepared by dissolving it in water.From this stock, concentrations of 173.28, 194.94, 216.60, 243.67, 259.92 mg/L were prepared in acetonitrile.Each solution was injected three times except that the target solution (216.60 mg/L) was injected six times.The results are shown in Table 1 and Figure 3.

Specificity
Specificity was tested against standard compound and against potential interferences in the presence of placebo.As Figures 4, 5 and 6 depict, no interference was detected at the retention time of phenylethyl alcohol in placebo solution.

Accuracy
Accuracy was determined by the two methods including: 1) Spiking the phenylethyl alcohol standard in placebo.2) Using linearity curve.Standard stock solution of phenylethyl alcohol (2.1508 g/L) was prepared in water.1.0 ml of this solution was transferred into 10 ml volumetric flask and diluted with acetonitrile to achieve a   final concentration of 215.08 mg/L.Concentrations of 187.5, 206.25, 176.25 mg/L with acetonitrile were prepared from budesonide nasal spray.

Spiking the phenylethyl alcohol standard in placebo
Accuracy was evaluated by spiking the phenylethyl alcohol standard in placebo at three different concentrations level and were calculated the recovery percentages with external standard method.Results are presented in Tables 2 and 3. Recovery percentages were in the range of 98.0 to 102.0% that show this method has suitable accuracy.

Using linearity curve
Accuracy was estimated by this method at three different concentration levels and recovery percentages were calculated with use linearity curve.In this method concentrations were obtained by a linear equation and recovery percentages were in the range of 98.0 to 102.0% that demonstrate this method has suitable accuracy.These results are represented in Table 4. Spiking in placebo and using linearity curve overall confirm which accuracy of this method is in the series of very high-quality.

Precision
Precision was studied in the three levels including: Repeatability (Intra-assay precision), ruggedness and solution stability (Intermediate precision) and reproducibility.Standard stock solution of phenylethyl alcohol (2.1508 g/L) was prepared in water.1.0 ml of this solution was moved to 10 ml volumetric flask and diluted with acetonitrile to achieve a final concentration of 215.08 mg/L.Concentrations of 200, 250 and 220 mg/L with acetonitrile were prepared from budesonide nasal spray.

Repeatability
Repeatability was studied at three different concentration      5 and 6 were found less than 2.0%.

Ruggedness and solution stability
The ruggedness of the method was studied on three different days with different analysts.The relative standard deviations of results were found less than 2.0%.
To demonstrate the stability of both standard and sample solutions during analysis, both solutions were analyzed over a period of 48 h at room temperature.The results showed that for all solutions, the retention times and peak areas of phenylethyl alcohol remained almost unchanged (RSD<2.0%)which indicating that no significant degradation occurred within this period.Both solutions were stable for at least 48 h.These results are presented in Tables 7 and 8.

Reproducibility
The reproducibility of method was studied in the two   9 and 10.

Robustness
The robustness of the method was determined by making slight changes in the chromatographic conditions that is, mobile phase ±5%, flow rate ±0.1 ml/min (Woolfson et al., 2014).Also this method was done with another C18 column (150×4.6 mm, 5 µm, Agilent Company), and finally similar results were obtained.

DISCUSSION
The purpose of this study was development a method to determination of phenylethyl alcohol in budesonide nasal spray and other similar nasal anti allergic formulations.The mixture of water and acetonitrile in different ratios was examined as a mobile phase and lastly a mixture of water and acetonitrile in the ratio of 50:50 (V/V) and flow rate of 1.0 ml/min was selected.The optimum wavelength for detection was considered at 220 nm (because of no interfering and suitable shape).After obtaining these final conditions of the chromatographic system, validation of the method was performed.
(i) Linearity was recognized in the concentration range of 173.28 to 259.92 mg/ml (80 to 120% of the target concentration) with a regression coefficient of 0.9991.(ii) Specificity was experienced in the presence of placebo; no interference was detected at the retention time of phenylethyl alcohol.
(iii) Accuracy was determined by the two technique of spiking and linearity curve.Recovery percentages were calculated and results were in the range of 98.0 to 102.0%.
(iv) Precision was studied in the three levels including; repeatability, ruggedness and reproducibility.
Percentage relative standard deviations of the results were calculated that were less than 2.0%.The results of the analysis were validated statistically and confirmed the accuracy and precision of the proposed method.
We concluded that proposed RP-HPLC method for determination of phenylethyl alcohol in budesonide nasal spray is simple, precise, specific, and highly accurate and this method is very less time consuming in quality control laboratories.So, this method can definitely be used in phenylethyl alcohol drug substance analysis and determination of phenylethyl alcohol in budesonide nasal spray and other similar nasal anti-allergic formulations such as Fluticasone nasal spray, Beclometasone nasal spray, Mometasone nasal spray and etc.The advantages of this method over other old methods are short retention time for determination of phenylethyl alcohol (about 2.7 min), simple mobile phase, economical and practical procedure to assay phenylethyl alcohol in other similar pharmaceutical products.

INTRODUCTION
Plants have been used for medicinal purposes over the years, which have provided mankind with a source of essentials of life such as food, medicine and raw materials for clothing and shelter (Midawa et al., 2010).et al., 2003).Reactive oxygen species (ROS) such as superoxide radical, hydroxyl radical, singlet oxygen, and hydrogen peroxide are produced in the body during normal metabolism or on exposure to exogenous factors.These reactive species can initiate deterioration of biomolecules such as proteins, lipids, carbohydrates and nucleic acids and are implicated in several diseases such as ageing, atherosclerosis, inflammatory injury, cancer, cardiovascular disease, neurological disorders etc. Oxidative stress results, when the balance between the generation of ROS and antioxidant defense system of the body is disturbed.Cells have innate defense system which protects against the adverse effects caused by these ROS and includes enzymatic and non-enzymatic defense.However, during pathophysiological conditions, there is an extra need for antioxidants from exogenous sources.Synthetic antioxidants have been suspected to cause or promote negative health effects.Hence, there is a need for development of safer antioxidants particularly from natural sources.Many studies have demonstrated the efficacy of plant derived products as antioxidants against various diseases induced by these free radicals (Koleva et al., 2000).It has been shown that the antioxidant nature of plants is mainly attributed to phenolic compounds, such as flavonoids and phenolic acids (Pietta, 2000).Senna alata L. has been ethnobotanically used extensively in traditional medicines for the treatment of a variety of diseases such as skin problems, arthritis, high blood pressure (HBP) and laxative using its organs.Therefore, there is need to investigate these plant organs for the bioactive compounds to be used for therapeutic purposes.

Collection, identification and preparation of plant material
Leafy plant of S. alata was obtained from Edokota forest along Bida-Zungeru road, Bida, Niger State, Nigeria.The identity was confirmed by plant taxonomist from the National Institute of Pharmaceutical Research Development, Idu-Abuja where a voucher specimen was deposited with Herbarium No.1369.The samples (leaf and root bark) collected from the experimental sites were washed with distilled water to remove impurities and dried at room temperature.These were then grind into uniform powder manually.It was then sieved, weighed, bottled, labelled and used for laboratory analysis.

Extraction of the plant extracts
Powdered flower and seed of S. alata were extracted with 70% aqueous methanol at room temperature.The extract solution of each sample was filtered, and the solvent was evaporated under reduced pressure at 35°C (Figure 1).

Qualitative phytochemical screening of the samples
Phytochemical screening procedures carried out were adopted from Mann ( 2014), where tannins, saponins, steroids, alkaloids, cardiac glycoside, terpenoids and flavonoids were determined.

Quantitative phytochemical analysis of the samples
Standard analytical methods were used for the quantitative phytochemical analysis of these samples (Edeoga et al., 2005).Tannins and saponins were determined using standard method of Onwuka (2005), while flavonoids and alkaloids were determined using standard method as described by Harborne (1989) and the total phenolic content was estimated using the modified Folin-Ciocalteu photometric method by Schuler (1990).

Anti-nutritional properties of the samples
Oxalate and cyanide contents were determined using the method of Day and Underwood (1986).Phytate content was determined by the method described by Wheeler and Ferrel (1971).

Antioxidants activities of the samples
The ferric reducing antioxidant power (FRAP) assay was done according to Benzie and Strain (1996) with some modifications while total phenolics of various fractions of plant were determined by reported method of Valentao et al. (2002).

GC/MS analysis of the samples
GC-MS analysis was carried out on a Shimadzu (Kyoto, Japan) GC-MS model QP 2010 at National Research Institute for Chemical Technology, Zaria, according to the EN 14103 standard method (Adams, 2007;Orishadipe et al., 2010).The GC column oven temperature (70°C), injecting temperature (250°C), flow control mode (linear velocity), total flow (40.8 ml/min) column flow (1.80 ml/min), pressure (116.9 kpa), linear velocity (49.2 cm/s) and purge flow (3.0 ml/min) were employed for this analysis.A sample volume of 8.0 µl was injected using split mode (split ratio of 20:0).The peak area, that is, the % amount of every component was calculated by comparing its average peak area to the total areas.Software was used to handle mass spectra and chromatogram.

Identification of components from the samples
Interpretation of mass spectrum GC-MS was conducted by  comparing the database peaks of National Institute of Standard and Technology (NIST) library with those reported in literature, the mass spectra of the peaks with literature data (Stein et al., 2002).The spectrum of the unknown component was compared with the spectrum of the known components stored in the NIST library.Component relative percentages were calculated based on GC peak areas without using correction factors.The name, molecular weight and structure of the components of the test materials were ascertained.

Statistical analysis
All the experiments were conducted in triplicate unless stated otherwise and statistical analysis of the data was performed by analysis of variance (ANOVA), using SPSS 11.0 for Windows software.A probability value of difference p ≤ 0.05 was considered to denote a statistically significance.All data were expressed as mean values ± standard deviation (SD).

RESULTS AND DISCUSSION
The results of qualitative analysis of the crude methanolic extract of two parts of S. alata shown in Table 1 revealed the presence of tannins, flavonoids, terpenoids, saponins, alkaloid, glycosides, anthraquinone, which are the basis Values are means ±SD of three determinations, different superscripts along the same row are significantly different (p≤0.05).
of therapeutic potentials of medicinal plants.Similar results were reported for Senna obtusifolia by Essiett and Bassey (2013) where the presence of saponins, tannins, alkaloids, terpenoids, anthraquinone and a trace of steroids were reported.The presence of tannins as reported in this work may be the cause of lowering of available protein by antagonistic competition and can therefore elicit protein deficiency syndrome "Kwashiokor" (Maynard, 1997).Saponin may be responsible for its antiyeast, anti-fungal, antidote, antimicrobial and antiinflammatory activities.It is also believed that the role of saponin is to protect plant against attack by potential pathogens (Sparg et al., 2004).Flavonoids which are also known as vitamin p or plan modifier, elicit a wide range of therapeutic activities as antihypertensive, antirheumatism as well as antimicrobial as identified with flavonoids (Veerachari and Bopaiah, 2011).Essiett et al. (2010) reported that many plants containing flavonoids have diuretic and antioxidant properties.The leaf and root bark of this plant can equally be used accordingly; glycosides were detected in the extracts and this class of compound has been found useful in the treatment of asthma (Trease and Evans, 1989;Evans, 2002).Steroids were also found and their pharmaceutical importance might hinge on their relationship with such compounds as sex hormones (Bell, 2007).Glycosides were detected in the leaf and root bark of S. alata.Glycoside has been used for over two centuries as stimulant in cases of cardiac failure and diseases (Taiwo et al., 2009).This perhaps justifies the already locally established function of the plant in the treatment and management of hypertension (Duke, 1985).Alkaloids have been found to have microbiocidal effect and their antidiarrheal effect is probably due to their action on small intestine.In addition, they effect antihypertensive antifungal, anti-inflammatory, and anti fibrogenic effect (Awoyinka et al., 2007).However, the results of this work are similar to the findings of McDevitt et al. (1996) who reported the presence of alkaloid in Cnidoscolusa conitifolius.Some alkaloids are useful against HIV infection as well as intestinal infection associated with AIDS (Scalbert, 1991).The results of quantitative analysis of the parts of S. alata as presented in Table 2 showed saponin contents of 40.57±0.57and 33.02±0.07mg/100 g for leaf and root bark respectively.It was observed that saponin concentrations were higher in leaf than root bark.These results were high compared to 12.1 mg/100 g of M. utilis reported by Siddhuraju and Becker (2005).Saponins are naturally occurring surfaceactive glycosides.They are mainly produced by plants, but also by lower marine animals and some bacteria (Riguera, 1997).The results of quantitative analysis of alkaloid content obtained from this plant organ were 14.09±0.50and 15.89±0.72 mg/100 g for leaf and root bark respectively.The alkaloid contents were higher in root bark than leaf.This is similar to the values reported for S. alata flower (8.50±0.01mg/100 g) by Abdulwaliyu et al. (2013).Alkaloids are more or less toxic substances which act primarily on the central nervous system (Hegnuauer, 1963).The tannin contents analyzed in this work were 59.48±0.50 and 44.38±0.72 mg/100 g for leaf and root bark respectively.The concentration was high with S. alata leaf while root bark had the least.The contents of tannin obtained in these were similar to 46.08 mg/100 g of M. utilis reported by Siddhuraju and Becker (2005).The values of flavonoids analyzed from the two samples were 42.28±0.90and 36.52±0.38 mg/100 g for leaf and root bark respectively.The flavonoid contents were higher in S. alata leaf than root bark.The phenol contents analyzed from this work were 7.84±0.49and 9.91±0.68mg/100g for leaf and root bark respectively.The concentration of the root bark was found to be high while leaf had the least value.These values were high compared to 2.00±0.21mg/100 g for S. alata leaf reported by Abdulwaliyu et al. (2013).
Anti-nutritional factors affect the availability of nutrients required by the body and interfere with metabolic process so that growth and development of the body is negatively influenced (Richard et al., 2006).The results of antinutritional factors obtained for this work were presented in Table 3.The phytate content in the samples analyzed was 12.44±0.31and 15.07±0.58mg/100 g for root bark and leaf respectively.The content of phytate was higher in S. alata leaf.Phytate helps in adequate iron bioavailability.The result obtained in this study was high when compared to the 3.55 mg/100 g of S. alata leaf reported by Abdulwaliyu et al. (2013).The contents of oxalates obtained from this work were 7.84±0.74and    et al. (2013).The presence of oxalate in food causes irritation in the mouth and interferes with absorption of divalent minerals particularly calcium by forming insoluble salts (Ola and Oboh, 2000).The cyanide content in the samples analyzed ranged from 13.04±0.09and 21.69±0.11mg/100 g for root bark and leaf respectively.The contents of cyanide were higher with S. alata leaf.These values are low when compared to the toxic level of 26.05±0.45mg/100 g, reported for S. alata leaf by Abdulwaliyu et al. (2013).
Table 4 shows the antioxidants properties obtained from this study using phenolic and ferric reducing properties.The antioxidant values of leaf and root bark were 1.47±0.66and 3.72±0.35mg/g respectively for phenolic properties with root bark exhibiting higher value.These values were similar to 3.58 mg/g reported for S. hirsute by Essiett and Bassey (2013).The ferric reducing properties obtained from this work were 0.62±0.13and 0.32±0.24µmol/mg for leaf and root bark respectively.From these results, high ferric reducing properties was recorded for leaf (0.62±0.13 µmol/mg) over root bark (0.32±0.24 µmol/mg).These values were higher when compared to 0.17±0.04µmol/mg reported for Pueraria mirifica by Buran and Supak (2007).
Tables 5 to 6 show the analytical parameters for GC-MS for the two organs of S. alata.It was observed that the organ of this plant contains all important fatty acid needed in the body for proper functioning.The fatty acids  5).The content of S. alata leaf was similar to Cassia alata reported by Isiaka et al. (2010).

Conclusion
There is need for more research on the activity of the extracts in this plant against a wider range of bacteria and fungi and on the toxicology and further purification of the extracts for isolation of the pure active constituents.However, the two parts of the plant studied can contribute to human medication.It can be concluded that the plant contains various phytochemical constituents such as tannins, flavonoids, terpenoids, saponins, alkaloids, glycosides, steroids, phenol and anthraquinone.The presences of these secondary metabolites can inhibit the growth of micro-organisms and also have potentials of being developed for pharmaceuticals.
several diseases, and therefore its prevention can play an important role in the cure of those diseases (Kanwal et al., 2011;Kulisica et al., 2004;Sharma and Trivedi, 2002;Smith et al., 2007).For example, oxidative stress has been widely postulated to be involved in the development and progression of some chronic diseases such as cardiovascular disease, neuronal disease, cataracts, and several types of cancer (Gua et al., 2009).There is increasing search for antioxidants that remove occurring naturally in vegetables, fruits and functional herbs to replace synthetic antioxidants.It has been found out that some synthetic antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) have been revealed to be potentially toxic and carcinogenic, they have been found to induce DNA damage (Helle et al., 2004).
T. minuta is a wild shrub in Uganda that thrives mostly in the rainy season (Tabuti et al., 2003).Tagetes species, commonly known as marigold are also grown as ornamental plants and thrive in varied agro-climates (Vasudevan et al., 1997b).Bioactive extracts of different Tagetes parts exhibit nematocidal, fungicidal and insecticidal activity (Vasudevan et al., 1997b).T. minuta has been used by the local people in Uganda to relieve a number of ailments (Hamil et al., 2000;Paul and Kasenene, 2007).
Previous work on Tagetes species, Tagetes maxima reavealed strong antioxidant properties of its ethylacetate extracts (Parejo et al., 2005).T. maxima was found to exhibit strong radical scavanging and antioxidant activities (Parejo et al., 2005) .There is a great possibility of similar activity in other Tagetes species.Antioxidant activity of T. minuta from Uganda has not been determined according to literature, but since it belongs to the same family as other Tagetes species with strong antioxidant properties, it was necessary to determine its potential as an antioxidant.
In this research, antioxidative compounds of T. minuta were isolated both from the Ethyl acetate extract and the essential oil.It was done by determining the scavenging Christine et al. 99 activity using 2,2-diphenyl-1-picrylhaydrazyl free radical (DPPH).The active compounds were tested quantitatively for their radical scavenging activity.

EXPERIMENTAL Plant material
Fresh aerial parts of T. minuta were collected from Mabira Forest in the morning hours in the month of November 2012.The sample was transported to Makerere University, Department of Chemistry Laboratory.Essential oils from T. minuta were extracted on arrival in the Laboratory.The remaining plant material was dried under shade for 3 weeks, ground in a mortal to obtain fine powder.A voucher specimen (CK001) was deposited at Makerere University Herbarium.

Hydrodistillation
Essential oil from fresh T. minuta was extracted by hydro-distillation in a Clevenger type apparatus for 3 h with a separated extraction chamber.The resulting essential oils were dried over anhydroussodium sulphate to extract the water.The oil was kept in refrigerated conditions at 8°C prior to the antioxidant activity determination and GC-MS analysis (Conti et al., 2010;Polatoglu et al., 2012).

Chemicals
All chemicals and reagents used in extraction, isolation and analysis of the active compounds were obtained from Sigma-Aldrich (Germany).These chemicals and reagents were of analytical grade.The standards were also purchased from sigma-Aldrich.

Solvent extraction-Cold extraction
T. minuta dry powder (1000 g) was extracted four times with 2000 ml of ethyl acetate at 40 to 45°C.The supernatant (extract) was separated from the residue by paper filtration (Whatman No. 1 filter, whatman paper Ltd., UK).It was dried in vaccum using a rotary evaporator at 40°C to remove all the ethyl acetate to give a residue.The powder was dried and re-extracted three times with 2000 ml methanol.The extract was combined and evaporated at 40°C to dryness.Both methanolic and ethyl acetate extracts were kept in a dry place for further testing (Gua et al., 2009).

DPPH assay
The capacities to donate hydrogen atoms/electrons by the essential oil and solvent extracts from the test samples were preliminarily detected using thin layer chromatography (TLC) and further measured spectrophotometrically.

DPPH spectrophotometric assay
This assay uses DPPH as a reagent (Argolo et al., 2004;Brand-Williams et al., 1995;Burits et al., 2001;Helle et al., 2004).50 µl of various concentrations of the volatile oils were added to 5 ml of 0.004% methanolic solution of DPPH.After 30 min of incubation period at room temperature, the absorbance was read against the blank at 517 nm using a U-1100 UV-VIS Spectrophotometer (Hitachi Ltd; Tokyo Japan).The tests were carried out in duplicate.DPPH solution (1.0 ml; 0.3 mM) plus methanol (2.5 ml) was used as a negative control.After 30 min the absorbance values were measured at 517 nm and converted into the percentage antioxidant activity (AA) using the following formula: %AA, which was, %aa=((AC(0) -AA(t))/AC(0))*100 Where AC(0) is the AA for the control solution at t=o minutes, and AA(ti) is the AA after the given time intervals, for I = 5, 10, 15, 20, 25, and 30 min (Kulisica et al., 2004).

GC-MS analysis
GC-MS analysis was used to identify the compounds in the essential oil and solvent extracts that had antioxidant activity.The GC-MS results of T. minuta was already determined in a previous research (Kyarimpa et al., 2014).

High performance liquid chromatography
The active fractions were purified with HPLC.A Dionex Ultimate 3000 HPLC (Dionex) equipped with a diode array detector and operated by Chromeleon Version 6.80 SR9 software.2.0 ml each of the active fractions was injected onto a 150 x 2.1 mm, 100 A, 2.6 µm Phenomenex Kinetex C18-column at 35°C.N-Hexane was used as a mobile phase.The flow rate was 237 µl/min.To selectively detect antioxidants, the detector recorded the signal at 520 nm (Application Note 281, Dionex Corporation, Sunnyvale, CA, USA).HPLC was carried out only samples with antioxidant activity and the solvent system was chosen based on Rf values of the TLC experiments.

Nuclear magnetic resonance (NMR) spectroscopy
Nuclear magnetic resonance (NMR) spectroscopy is (arguably) the most powerful tool available for determining the structure of organic compounds.It is used to identify and/or elucidate detailed structural information about chemical compounds.In this case it was used to determine the structure of the pure active compound in the sample.
All NMR spectra were recorded on a Bruker Avance II 400 (resonance frequencies 400.13 MHz for 1 H and 100.63 MHz for 13C) equipped with a 5 mm broadband observe probe head (BBFO) with z-gradients at room temperature with standard Bruker pulse programmes.The sample was dissolved in 0.6 ml of CDCl3 (99.8% D).Chemical shifts are given in ppm, referenced to residual solvent signals (7.26 ppm for 1 H, 77.0 ppm for 13 C). 1 H NMR data were collected with 32k complex data points and apodized with a Gaussian window function (lb = −0.3Hz and gb = 0.3 Hz) prior to Fourier transformation. 13C-jmod spectra with WALTZ16 1 H decoupling was acquired using 64k data points.Signal-to-noise enhancement was achieved by multiplication of the FID with an exponential window function (lb = 1Hz).All two-dimensional experiments were performed with 1k × 256 data points, while the number of transients (2-8 scans) and the sweep widths were optimized individually.The resulting FIDs were zero-filled to a 2k × 1k data matrix and apodized with a sine function for COSY in both the ω1 and ω2 dimensions prior to Fourier transformation.Heteronuclear spectra were zero-filled only in F1 to a 1k × 512 data matrix, and apodized in both dimensions with a shifted sine function.The heteronuclear single quantum coherence (HSQC) experiment was acquired using adiabatic pulse for inversion of 13 C and GARP-sequence for broadband 13 C-decoupling, optimized for 1J(CH) = 145 Hz.

RESULTS AND DISCUSSION
The essential oil from tagets minuta exhibited strong antioxidant activity on TLC.Some components of the crude extract were also found to be active (Figure 2).Track 1 on the first TLC plate from the left (Figure 2) was the essential oil, the other tracks were different extracts from T. minuta and Tephrosia Vogelli.It was noted that some of the components of the two plants had strong antioxidant activity.These fractions were isolated using Column Chromatography, HPLC.The pure fractions were analysed using NMR spectroscopy.Track 1 had very strong antioxidant activity as shown by the DPPH reaction on the TLC plate (Figure 2).This oil was later used for quantitative measurement again using DPPH and Ultra Violet Spectrophotometer (Table 1).The total antioxidant capacity revealed that the essential oil from T. minuta had a high antioxidant activity.Free radical scavenging activity of the extracts was assesed using the stable free radical DPPH.Plant extracts which reduce DPPH by donating hydrogen ions are considered as antioxidants having free radical scavenging activity.The results from Table 1, were used to calculate the amount of DPPH scavenged over a period of time according to the formula {%AA, which was , %aa=((AC(0) -AA(t))/AC(0))*100}, and the LC 50 was determined (Appendix 1B).DPPH solution alone served as control (A0).It is evident from the study, that the investigated extracts and essential oil have the ability to quench free radicals.This indicates that T. minuta is a potential source of natural antioxidants.

Nuclear magnetic resonance (NMR) Spectroscopy
One active pure compound was identified using both the proton NMR and the carbon NMR as shown in Figure 1 and Table 2.The 1 H NMR spectra of this compound revealed the presence of two isolated olefinic spin systems without any further coupling partners: on the one hand a vinyl group, indicated by its characteristic ABXsystem at α 5.05 (d, J = 10.8Hz), α 5.23 (d, J = 17.6 Hz) and α 6.37 (dd, J = 17.6 , 10.8 Hz), and on the other hand an olefinic methylene group with broad singuletts at α 4.99 and α 5.00 ppm, respectively.Besides a triplett at   α 2.18 ppm, a bulk of aliphatic methylene and methine protons in the region between 1.60 to 1.00 ppm and signals of different methyl groups at around 0.90 ppm no more signals were found in the 1H NMR spectra.In combination with the hsqc experiment the j-modulated 13C nmr spectra showed signals of 4 olefinic carbonsone quaternary, one methine, and two methylenes -4 methyl, 3 methine and 9 methylene carbons.In addition to the molecular mass peak at m/z = 278 these results indicated that compound is a noncyclic, nonoxidized diterpene with a molecular formula of C 20 H 38 .Extensive analysis of heteronuclear 2D NMR led to the elucidation of that structure which turned out to be neophytadiene, a widespread component of essential oils from different plant sources.Whereas Burkhardt et al. published only NMR data of the olefinic part of the molecule, we present here to our best knowledge for the first time the fully assigned nmr resonances (Table 2).Neophytadiene, is a fatty acid-related compound which plays an important part in competitive inhibition of cyclooxygenase or lipoxygenase in an inflammation reduction, resulting in decreased production of prostaglandins and leukotriene (Pillai and Nair, 2013).

Conclusion
DPPH is a free radical, stable at room temperature, which produces a violet solution in methanol.It is reduced in the presence of an antioxidant molecule, giving rise to uncoloured methanol solutions.The use of DPPH provides an easy and rapid way to evaluate antioxidants.According to the results obtained from data in Tables 1  and 2 as well as those of statistical analysis, we can say that extracts from T. minuta as well as its essential oil possess strong antioxidant properties with an LC 50 of 1.49 g/l -1 after 30 min as compared to other antioxidants reported in Parejo et al. (2005)

INTRODUCTION
Despite the requirement for healthy environment, the environment has been found to be contaminated with various pollutants.This has now posed a great challenge to human wellbeing.Such pollutants may be found in air, water soil, coastal erosion, overfishing and deforestation as well as disposal of waste, which constitute several heavy metals.Contamination of water by heavy metals is one of the most challenging environmental issues currently.Cadmium is one of the most toxic metals apart from lead and mercury.It has been reported to cause renal dysfunction, hypertension, lung insufficiency, bone lesions and cancer (Feng et al., 2010) which is a leading cause of death.The cadmium drinking water guidelines value recommended by WHO is 0.003 mg L -1 (WHO, 2008).Cadmium accumulates both in the environment and the body causing long term damage to life (Nida et al., 2012).Cadmium is one of the heavy metals with a greatest potential hazard to humans and environment *Corresponding author.E-mail: santurakiah65@gmail.com Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License (Fouad et al., 2012).
The principal sources of Cd into the environment are electroplating, smelting, alloy manufacturing, pigments, plastic, battery, mining and refining processes (Gupta and Nyaka, 2012).Once released into the environment cadmium is toxic to plants animals and microorganisms (Bailey et al., 1999).The metal is non-biodegradable, persistent and bioaccumulate mainly in the kidneys and liver of vertebrates, invertebrates and also in algae (Ajay et al., 2005).A number of methods have been employed to remove Cd (II) ions from the environment including, ion exchange, reverse osmosis (Gupta and Nyaka, 2012) membrane filtration, electrochemical treatment, and adsorption.etc where each of them have limitations such as high cost and production of hazardous by-products are found expensive and sometimes ineffective, especially when metals are present in solution at very low concentration within the range 1 to 100 mg/cm 3 (Santhi and Manonmani, 2012).
Consequently, it is essential to find new methods for effective removal of cadmium from water and wastewater.Compared with other traditional methods, adsorption is quite popular due to its simplicity and high efficiency, as well as the availability of a wide range of adsorbents (Orhan and Buyukgungor, 1993;Babel and Kurniawan, 2003).Activated carbon is the best useful adsorbent of heavy metals from waste water.However, the cost of activated carbon is high; its regeneration also requires additional expense.Therefore there is need to come up with other alternative that provide an easy, feasible, reliable, low cost adsorbents especially those of biological origin commonly referred to as biosorbents to improve the water quality.Biosorption has gained a lot of credibility currently because of its eco-friendly nature, excellent performance and cost-effectiveness (Davis et al., 2003).Equilibrium isotherm models and kinetic models were applied to the data obtained for a better understanding of the adsorption process.Thus the objective of the present study is to investigate the binding of metal ion cadmium (II) by Lonchocarpus laxiflorus roots (LLR) in its immobilized form from aqueous solutions and to study the effect of various factors affecting the efficiency of the process.

Plant collection and treatment
The roots of L. laxiflorus plant were collected from a tree behind Modibbo Adama Federal University of Technology Yola, Nigeria.The plant was wash thoroughly under running water to remove dust and any adhering particle and then rise with distilled water.The sample was air dry for 2 weeks and the dry roots was grinded in analytical mill and sieve to obtain adsorbent of known particle size range.The biomass powder was kept in an air tight bottle for further study (Igwe and Abia, 2006).Our studies indicated that though there was no big difference in the adsorption rates of the various parts of plant, the roots were better.Therefore roots were used in this adsorption experiments.

Chemicals
All chemicals used in the present work were of analytical grade.The stock solution of Cd 2+ ions was prepared in 1.0 g L -1 concentration.Cadmium solution of 1000 mg/cm 3 concentration was prepared by dissolving 2.103 g of Cd(NO3)2 in 250 cm 3 of distilled water and make up to 1000 cm 3 in a volumetric flask.The pH of the solutions was adjusted using 0.1 mol L -1 HCl and NaOH solutions.

Analysis of metal ions
Atomic absorption spectrophotometer equipped with an air acetylene flame, controlled with computer was used to investigate the concentration of cadmium metal ion.The hallow cathode lamp of Cd, was used at 283.3 nm wavelength, while slit is 0.2 nm, operated at 8 mA (Suleiman et al., 2007).

Reparation of sodium alginate and calcium chloride stock solution
Sodium alginate was prepared by weighing 4.00 g and making it up to 100 cm 3 mark with distilled water in a volumetric flask and left overnight for complete dissolution.0.12 M of calcium chloride was prepared by weighing 26.28 g in to 1000 cm 3 volumetric flask and making up to mark with distilled water according to a standard procedure described by Osemeahon and Esenowo, (2012).

Immobilization of the roots of L. laxiflorus plant
Sodium alginate was used for immobilization of the roots of L. laxiflorus plant.50 cm 3 of sample solution prepared by dissolving 4 g of each test sample in 100 cm 3 of distilled water and mix with 50 cm 3 of 4% stock solution of sodium alginate and stir vigorously in 250 cm 3 beaker, to obtain a homogenous mixture.After mixing, the solution the solution was drawn through hyperdemic needles and was added drop wise to a stirred solution of 1 M CaCl2.A retention time of 1 h was allowed for the reaction to obtain complete precipitation of the immobilized leave powder of L. laxiflorus plant.The beads thus formed that is, sodium alginates were kept in fresh CaCl2 solution.Before sorption studies, the beads were removed and allowed to dry at room temperature.The dried solid mass was stored in a polythene bag for further use (Mishra, 2013).

Biosorption experiments
The experiments were carried out in the batch mode for the measurement of adsorption capacities.From 100 ppm of cadmium metal ion solution, 50 ml was taken into a 250 ml conical flask and 0.2 g of the LLR was added corked with a rubber bung and shaken with a flask shaker for 2 h at room temperature (30°C) at 180 rpm.The separation of the adsorbents and solutions were carried out by filtration with whatman filter paper No 42 and the filtrates were stored in sample cans for use.The residual metal ions concentrations were determined using atomic absorption spectrophotometer (AAS).Pyeunicam Model SP.For studies on effect of temperature the adsorption studies were carried out at 25, 30, 40, 50, 60 and 70°C.The percentage adsorption was calculated using the following equation: (1) Where Ci= Initial metal ion concentration and Cf = Equilibrium metal ion concentration (mg/L).
The amount of metal absorbed by the biosorbent was also calculated as Where, q is the amount of metal ion adsorbed in mg/g; Co is the initial metal ion concentration in mg/cm 3 ; Ce is the final concentration in mg/cm 3 , V is the volume of metal ion solution in liters; M is the mass of the root of L. laxiflorus powder used in gram.

RESULTS AND DISCUSSION
The FTIR is measured in the range of 400 to 4000 cm -1 wave number.The FTIR of the adsorbent displays a number of adsorption peaks, indicating the complex nature of the studied adsorbent.The IR bands consisted of four regions, the broad OH band (3200-3600 cm -1 ), C-H stretching region (2800-3000 cm -1 ), carbonyl group stretching region (1550 -1750 cm -1 ), and finger print bands (below 1550 cm -1 ) (Shin et al., 2007).From Figure 1, the absorption of peak at approximately 3400 were due to stretching vibrations of hydroxyl groups which are one of the main components of cell wall polysaccharides of the plants (Suantak et al., 2011).The adsorption peak at 2930 cm -1 is likely due to the presence of C-H asymmetric stretching vibration in -COOH group (Kumar et al., 2012) of methylene groups on the surface.The absorption band at 1630 cm -1 may be assigned to Amide I and II (protein) respectively (Pradhan et al., 2007).While the peaks in the range of 1427 to 1328 cm -1 could be attributed to carboxylate group (Pradhan et al., 2007).The broad peak at 1240 could be due to (C-C) or (C-H) or (C-O) stretching of the carboxyl groups (Singh et al., 2010).The peak in the region 1051 cm -1 is due to the presence of C-C Stretching of the polysaccharides (Singh et al., 2010).In conclusion, the FTIR spectroscopic analysis of the plant biomasses indicated the presence of hydroxyl, amide and carboxylate groups as the main functional groups involved in the complexation of metal ions for biosorption processes.The adsorption capacity of the adsorbent depends upon porosity as well as chemical reactivity of the functional groups at the adsorbent surfaces.It seems that these functional groups participate in metal binding process.Scanning electron microscope (SEM) analysis is another important tool used in the determination of the surface morphology of an adsorbent.The SEM image and micro-analysis of LLR plant is shown in Figure 2. The external surface of LLR is full of cavities with well developed porous structure.The external surfaces show a rough area having different pore diameters distributed over the surface of the biomass which may be responsible for metal removal.This analysis reveals a highly porous structure for LLR biomass.It could also be seen that more uneven and rough surface morphology exist in all the adsorbent.X-ray diffraction (XRD) analysis was carried out by using XRD system with Cu-Kα radiation.The XRD patterns for LLR in raw powdered form is shown in Figure 3.These gives information about the changes in the crystalline and amorphous nature of the adsorbents.Sharp intensity XRD peaks have been observed at typical scanning angles of 2θ = 20.The sharp peaks present in the figures indicated the crystalline nature of the material.In addition, the presence of other weak intensity peaks in the spectra indicates the amorphous nature of the three adsorbents.The amorphous nature of the adsorbents suggests that metal ions can easily penetrate the surface which is desirable for an effective removal.These results are in good agreement with those reported Kugbe et al. (2009).

Effect of pH on metal biosorption
Hydrogen ion concentration is one of the important factors that influence the adsorption behavior of metal ions in aqueous solutions.It affects the solubility of metal ions in solution, replaces some of the positive ions found in active sites and affects the degree of ionization of the adsorbate during the process of biosorption.This is because it affects solution chemistry and also the  speciation of the metal ions.The effect of initial pH on biosorption of Cd (II) ions onto L. laxiflorus was evaluated in the pH range of 3.0 to 7.0.Studies in pH range above 7.0 were not attempted as there is precipitation of cadmium (II) hydroxides.
From Figure 4 it could be seen that Cd (II) ions adsorption increased as the pH increased.At low pH values, protons occupy the biosorption sites on the biosorbent surface and therefore less Cd (II) ions can be adsorbed because of electrostatic repulsion between the metal cations and the protons occupying the binding sites.When the pH was increased, the biosorbent surface became more negatively charged and the biosorption of the metal cations increased drastically until equilibrium was reached at pH 6.0 to 7.0.At pH of >7.0 there is formation of hydroxylated complexes of the metal ions and these complexes compete with the metal cations for the adsorption sites hence a reduction in the effective metal cations removal.Therefore adsorption experiments at pH above this were not considered.

Effect of biosorbent dosage
The effect of biomass dosage on adsorption of Cd (II) ions is indicated in Figure 5.The number of available binding sites and exchanging ions for the biosorption depends upon the amount of biosorbent in the biosorption system.This is attributed to the fact that it determines the number of binding sites available to remove the metal ions at a given concentration.The dosage also determines the adsorption capacity of the biosorbent with an increase in mass reducing the biosorption capacity as the mass increase from 0.1 to 2.5 g per 20 ml of adsorbate.An increase in the % adsorption is attributable to an increase in the number of binding sites for the metal cations.Similar results were recorded in the literature for other adsorbents.However, the mass could not be increased infinitely as at some point all the solution is sequestered leaving no residual solution for concentration determination.Similar trend have been found by Mahajan and Sud (2011).

Effect of initial metal concentration
The initial concentration remarkably affected the uptake of Cd (II) ions in solution.The efficiency of Cd (II) ions adsorption by LLR at different initial concentrations (10 to 80 mg L -1 ) was investigated as shown in Figure 6.At a lower concentration, the adsorption sites take up the available metal ions much quickly due to less competition among the metal ions for the available binding sites which are fixed in this case.However, as the concentration increases the competition for the limited  binding sites sets in as the binding sites become saturated (Mahajan et al., 2013).

Effect of contact time
Contact time is an important parameter for any successful use of the biosorbents for practical purposes.Effect of contact time on adsorption of Cd (II) ions was investigated keeping the biomass in contact with the metal ion solution for different time periods between 0 to 60 min.It was noted that as adsorption proceeds, the sorbent reaches saturation state, at this point the sobbed solute tends to desorbs back into solution (Figure 7).Eventually, the rate of adsorption and desorption are equal at equilibrium.When the system attains equilibrium, no further net adsorption occurs.The time taken to attain equilibrium is very important for process optimization.The rate of adsorption is very fast at first and over 95% of total biosorption of Cd (II) ions occurs in the first 5 min and thereafter it proceeds at a slower rate Santuraki and Muazu 109 and finally no further significant adsorption is noted beyond 20 min of contact time.The very fast adsorption makes the material suitable for continuous flow water treatment systems (Sarin and Paint, 2006).

Effect of temperature
Temperature of the medium affects the removal efficiency of pollutants in aqueous solutions.This is because a change in temperature in turn affects the solubility of pollutants and also the kinetic energy of the adsorbing ions.Therefore the effect of temperature on adsorption of Cd (II) ions was investigated and the data is shown in Figure 8.The results indicate that the percentage adsorption increases with increase in temperature up to 40°C, after that any increase in temperature is accompanied by a reduction in % adsorption.This can be attributed to the fact that with increase in temperature of the solution, the attractive forces between the biomass surface and Cd (II) ions are weakened thus decreasing the sorption efficiency.This could be due to increase in the tendency for the Cd (II) ions to escape from the solid phase of the biosorbent to the liquid phase with increase in temperature.Finally increased temperature beyond 40°C could have destroyed some of the binding sites on the biosorbent surface due to bond rupture (Meena et al., 2005).

Biosorption kinetics
Kinetic study provides useful information about the mechanism of adsorption and subsequently investigation of the controlling mechanism of biosorption as either mass transfer or chemisorption.This helps in obtaining the optimum operating conditions for industrial-scale batch processes.A good correlation of the kinetic data explains the biosorption mechanism of the metal ion on the solid phase (Garima and Dhiraj, 2013).In order to evaluate the kinetic mechanism that controls the biosorption process, the pseudo -first-order models (Lagergren, 1898) were applied for biosorption of Cd (II) ions on the biosorbent.The Lagergren pseudo-firstorder rate model is represented by the equation: = Where q e and q t are the amounts of metal adsorbed (mg g -1 ) at equilibrium and at time t respect ively, and K 1 is the rate constant of pseudo-first-order biosorption (min -1 ).
The q e and rate constant were calculated from the slope and intercept of plot of log (q e -q t ) against time t.
The pseudo-second-order equation (Ho and Mckay, 1999) assumes that the rate limiting step might be due to chemical adsorption.According to this model metal cations can bind to two binding sites on the adsorbent surface.The equation can be expressed as shown below: = + (4) Where k 2 is the rate constant of the pseudo-secondorder adsorption (g/mg/min ).If the adsorption kinetics obeys the pseudo-second-order model, a linear plot of t/q t versus t can be observed as shown in Figure 9.

Biosorption isotherms
For optimization of the biosorption process design, it is imperative to obtain the appropriate correlation for the equilibrium data.Biosorption isotherms describe how adsorbate interacts with the biosorbent and the residual metal ions in solution during the surface biosorption.The isotherms also help in determination of adsorption capacity of the biosorbent for the metal ions.The data on Cd (II) ions biosorption was fitted with the Langmuir (1918) and Freundlich (1906) isotherms (Figures 10 and 11).The Langmuir isotherm assumes monolayer coverage of the adsorbate onto a homogeneous adsorbent surface and the biosorption of each cation onto the surface has equal activation energy.The Langmuir isotherm can be exppressed as: = + (5) Where qmax is the monolayer capacity of the biosorbent (mg g -1 ), and b is the biosorption constant (L mg -1 ).The plot of ce/qe versus ce should be a straight line with a slope of 1/qmax and intercept of 1/q max b when the biosorption follows Langmuir equation.The Freundlich equation can be expressed as: = + (6) Where K f and 1/n are the Freundlich isotherm constants related to biosorption capacity and biosorption intensity respectively.If the equation applies then a plot of log qe versus log Ce will give a straight line of slope 1/n and intercept as K f LLR has a higher value for n suggesting multilayer sorption on the surface of the plant biomass, which is due to its various chemical functional groups.The values of b and K obtained for LLR also confirms a higher sorption capacity and superior performance of LLR adsorbents for Cd (II) ions adsorption from aqueous solution.Similar results are reported by Mahajan et al. (2013), Iqwe et al. (2005) and Kurniawan and Thiam (2010).

Conclusion
Adsorption of cadmium ions from aqueous solution using the Lonchocarpus laxiflorus plant roots material was investigated.Various contributing parameters such as contact time, initial metal ion concentration, solution pH, and adsorbent dose was optimized for maximum removal efficiency.The sorption data fitted well with Langmuir isotherm with high R 2 values.The kinetic studies indicated that the pseudo second order model was the best one in describing the kinetics of cadmium (II) adsorbed onto roots powder.A large number of carbonyl and hydroxyl groups were observed in the FTIR analysis, XRD studies reveal the crystalline structure of the biosorbent and SEM studies showed the presence of various moieties that enhances the adsorption phenomenon.Excellent removal efficiency in its encapsulated form explores the utilization of the biomass at the commercial scale for small scale industries, making it of potential commercial use.

Figure 1 .
Figure 1.The structural formula of budesonide.

Figure 2 .
Figure 2. The structural formula of phenylethyl alcohol.

Figure 3 .
Figure 3. Linearity curve of phenylethyl alcohol in budesonide nasal spray preparation.

Figure 4 .
Figure 4. Chromatograms of Standard solution, Test Solution and Placebo solution (with PDA detector).

Figure 5 .
Figure 5. Three-dimensional chromatogram of phenylethyl alcohol peak in Standard solution (with PDA detector).

Figure 6 .
Figure 6.Three-dimensional chromatogram of phenylethyl alcohol peak in Sample solution of budesonide nasal spray preparation (with PDA detector).

Figure 1 .
Figure 1.The structure of Neophytadiene

Figure 2 .
Figure 2. TLC plate before (left) and after applying DPPH (right).The yellow spots on a purple background indicate antioxidant activity of the compounds at that particular spot.

Figure 2 .
Figure 2. SEM of LLR powder in raw form.

Figure 4 .
Figure 4. Effect of pH on % adsorption of Cd (II) ions on LLR biomass.

Figure 5 .
Figure 5.Effect of dosage on % adsorption of Cd (II) ions on LLR biomass.

Figure 6 .
Figure 6.Effect of initial cadmium concentration on adsorption of LLR biomass.

Figure 7 .
Figure 7. Effect of contact time on adsorption of Cd (II) ions by LLR.

Figure 8 .
Figure 8.Effect of temperature on % adsorption of Cd (II) ions by LLR.

Table 1 .
Linearity results of phenylethyl alcohol in budesonide nasal spray preparation.

Table 2 .
Chromatographic results of accuracy test.

Table 3 .
Accuracy results of phenylethyl alcohol in budesonide nasal spray preparation by external standard method.

Table 4 .
Accuracy results of phenylethyl alcohol in budesonide nasal spray preparation by linearity curve.

Table 5 .
Chromatographic results of repeatability test.

Table 6 .
Repeatability results of phenylethyl alcohol in budesonide nasal spray preparation by external standard method.

Table 8 .
Ruggedness results of phenylethyl alcohol in budesonide nasal spray preparation by external standard method.

Table 9 .
Chromatographic results of reproducibility test.

Table 1 .
Qualitative phytochemical evaluation of methanol extracts of S. alata leaf and root bark.

Table 2 .
Quantitative evaluation of methanol extracts of S. alata leaf and root bark (mg/100 g).

Table 3 .
Anti-nutrient factors of leaf and root bark of S. alata (mg/100 g).

Table 4 .
Determination of Antioxidant contents of leaf and root bark of S. alata.
bValues are means ±SD of three determinations, different superscripts along the same row are significantly different (p≤0.05).

Table 5 .
Chemical compounds deduced from GC-MS spectrum of S. alata leaf.

Table 6 .
Chemical compounds deduced from GC-MS spectrum of S. alata root bark.

Table 1 .
Ultraviolet Spectroscopic results showing the effect of sample concentration on the absorbance of DPPH.

Table 2 .
NMR resonance of the fully assigned Neophytadiene.
Percentage absorbance of DPPH at different concentrations of the Essential oil of Tagetes Minuta vs time in minutesWe notice that radical scavenging or absorbance is inversely proportional to time.
APPENDIX A: