Open Access Original Research Article

Phytochemical Screening, In-vitro Evaluation of Antioxidant and Free Radical Scavenging Activity of Leaves, Stems and Roots of Xanthium strumarium L., (Compositae)

Anjoo Kamboj, Pooja Atri, Ajay Kumar Saluja

Journal of Pharmaceutical Research International, Page 1-22
DOI: 10.9734/BJPR/2014/3667

Introduction: Many diseases are associated with oxidative stress caused by free radicals. Current research is directed towards finding naturally occurring antioxidants of plant origin. Xanthium strumarium L. a cocklebur or bur weed is a reputed medicine in Europe, China, Indo-china, Malaysia and America. It is used in treatment of common disease such as hemicrania, leucoderma, biliousness, poisonous bites of insects, epilepsy, long standing malaria, relieving constipation, diarrhoea, vomiting etc. The present research deals with phytochemical screening and in-vitro evaluation of antioxidant activities of the various extracts of leaves, stems and roots of X. strumarium L.
Method: Successive extracts of leaves, stems and roots was subjected for phytochemical screening. Various extracts of leaves, stems and roots were screened in-vitro for total antioxidant potential. Inhibition of oxygen derived free radicals, viz., assay for free radical scavenging of nitric oxide, hydrogen peroxide, the antioxidant capacity by phosphomolybdenum, reducing power ability and determination of phenolic and flavonoids content in the extracts of leaves, stems and roots were performed. DPPH scavenging activity or the Hydrogen donating capacity was quantified in presence of stable DPPH radical on the basis of Blois method. NO scavenging activity was performed in the presence of nitric oxide generated from sodium nitroprusside using ascorbic acid as standard in both methods. The phenolic content was determined by using Folin-Ciocalteu reagent and flavonoid content was determined by aluminium chloride.
Result: The preliminary phytochemical screening revealed the presence of saponins, sterols, flavonoids, alkaloids and phenolic compounds in the extracts. The scavenging activity was found to be dose dependent. The reducing capacity serves as significant indicator of antioxidant activity. The reducing power increased with the increasing concentration of sample. The 100mg powder of leaves yielded 0.069, 0.523, 1.620 mg/g phenolic content and 0.17, 0.45, 0.95 mg/g flavonoid content with solvents such as petroleum ether (60º-80ºc), chloroform, and ethanol respectively. Similarly, in case of stems and roots the phenolic content yielded 0.063, 0.324, 1.324 mg/g and 0.040, 0.159, 0.41 mg/g and flavonoids content 0.00, 0.11, 0.23 mg/g and 0.00, 0.05, 0.18 mg/g respectively, using quercetin as standard.
Conclusion: The present study provides evidence that X. strumarium L., is a potential source of antioxidants and the extracts have constituents which were capable of showing antioxidant activity and the said in-vitro antioxidant activity may also be due to the presence of antioxidant principles present in the extracts like flavonoid and phenolic compounds. So the folklore use of X. strumarium L. has been proved in present research work. These findings confirm the great interest of the herb whose phytochemistry and phytopharmacology should be investigated further in order to detect possible phytotherapeutic uses in the prevention of ageing related diseases, cardiovascular disorders and Alzheimer disease.

Open Access Original Research Article

Phytochemical and Biological Activities of the Wild Grape Fruit Extracts Using Different Solvents

Boonsod Yardpiroon, Sangdee Aphidech, Srihanam Prasong

Journal of Pharmaceutical Research International, Page 23-36
DOI: 10.9734/BJPR/2014/5626

Aims: To investigate the phytochemical, antioxidant and antibacterial activities of the solvent extracts of wild grape fruits in different colors.
Study Design: The solvent extracts of wild grape (Ampelocissus martinii Planch.) fruits were prepared by mixing grinded fruit in each solvent. The filtrates were evaporated using a rotary evaporation at 45ºC until the weight of evaporated filtrate were less than 10% of the original weight.
Place and Duration of Study: Department of Chemistry, Faculty of Science, Mahasarakham University, Thailand, between August 2012 and May 2013.
Methodology: All extracts were investigated for their total phenolic (TPC) and flavonoid contents (TFC) by Folin-Ciocalteu and colorimetric aluminum chloride assays, respectively as well as antioxidant activity using 2,2΄-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) assays. They also tested for their antibacterial activity against infective bacteria using agar well diffusion method.
Results: Methanolic extracts showed the highest of TPC comparison to other. The methanolic extract from green wild grape has the highest of TPC, followed by ethanolic extract. The ethanolic extracts of red and black wild grape fruits found the highest of TFC whereas the green wild grape using methanol showed the highest TFC. All methanolic extracts showed the lowest of IC50 values when compared to other solvents in the same color. Among them, the methanolic extract from green wild grape has the lowest of IC50 values which considered to be the highest powerful of antioxidant activity. The obtained results were directly trend with the FRAB values. However, the ethanolic extract showed antioxidant activity similar as the methanolic extract. The methanolic extract from green wild grape showed good antibacterial activity. All ethanolic extracts showed widely and similarly inhibition of selected bacteria, but no activity in all water extracts. The MIC and MBC of all extracts were arranged of 500-250 µg/mL.
Conclusion: Methanol and ethanol should be used as good solvent extraction of wild grape fruits to obtain high TPC and TFC and good biological activities.

Open Access Original Research Article

Optimized Artemether-loaded Anhydrous Emulsion

N. C. Obitte, L. C. Rohan, C. M. Adeyeye, C. O. Esimone

Journal of Pharmaceutical Research International, Page 37-59
DOI: 10.9734/BJPR/2014/5279

Objective: The objectives of this study were to identify stable anhydrous emulsions via pseudo ternary phase diagram, optimize artemether-loaded batches using factorial design and subsequently evaluate the antimalarial activity.
Methodology: Using labrasol®, triacetin® and lauroglycol 90® as the surfactant, oil and co-surfactant respectively, pseudo ternary phase diagram was generated from the quantitative titration of water with the anhydrous emulsion. Stable combinations from the phase diagram were subjected to a 23 full factorial experimental design. The 22 software-generated formulations were experimentally formulated and characterized for droplet size, polydispersity index, viscosity and thermodynamic stability. Droplet size was chosen and subsequently fitted into the Response column of the software, thus prompting the generation of graphs and Desirability table of 125 predicted formulations. Out of the 125 predictions, three with the least droplet sizes (less than 100 nm) were adjudged as optimized batches. Subsequently, they were formulated, converted to powder by adsorption on magnesium aluminum metasilicate and evaluated. Antimalarial effectiveness of the drug-loaded formulation was also investigated.
Results: Triacetin® most significantly (P<0.05) contributed to droplet size variation. The droplet size of the experimental formulations approximated that of the statistical predictions. The anhydrous emulsions (AEs) were powderable and the granulations rated fair and passable according to Carr’s scale. Artemether-loaded anhydrous emulsion (AE) demonstrated highest antimalarial activity.
Conclusion: We therefore conclude that optimization proved a useful tool for the identification of excipient proportions with optimal effects.

Open Access Original Research Article

Formulation and Evaluation of Oxcarbazepine Suspension: In vitro/In vivo Correlation

A. Prameela Rani, V. Hema

Journal of Pharmaceutical Research International, Page 60-69
DOI: 10.9734/BJPR/2014/4763

Aims: Oxcarbazepine was formulated as suspension in order to perk up the palatability, augment bioavailability of the product, furthermore extended to estimate good degree of in vitro in vivo correlation (IVIVC).
Study Design: Formulation and Evaluation of Oxcarbazepine Suspension: In Vitro/In Vivo Correlation
Place and Duration of Study: Birla Institute of Technological Sciences- Hyderabad, India between July 2011 to January 2012
Methodology: Oxcarbazepine had poor aqueous solubility, thus the solubility was increased by hydrotropes then formulated into suspension using taragum as viscosity enhancer. Further validity of dissolution study was extended by In vitro/In vivo correlation using level A method.
Results: These suspensions were observed for in vitro dissolution profile and studied for in vivo pharmacokinetic profile, from the obtained values, a level A IVIVC modeling was observed, interestingly from the results attained suspension showed almost 98% drug release (FDR) and 85% drug absorbed (FDA) in 90min. Fascinatingly, from the correlation between FDR and FDA, slope and regression co-efficient obtained was near to 1.0 indicated good linearity.
Conclusion: In conclusion, a point-to-point link from the level A which was a keystone of acceptable and reliable correlation was achieved.

Open Access Original Research Article

Economic Burden of Drug Therapy in Hypertension Management in a Private Teaching Hospital in Nigeria

Kehinde A. Ganiyu, Ismail A. Suleiman

Journal of Pharmaceutical Research International, Page 70-78
DOI: 10.9734/BJPR/2014/3983

Background: Information on economic burden of hypertension is needed for relevant decisions and policies due to escalating cost of disease management.
Aims: The study assessed economic burden of pharmacotherapy in hypertension management on the National Health Insurance Scheme (NHIS) of Nigeria and the economies of antihypertensives selection.
Study Design: Cross-sectional study.
Place and Duration of Study: Out-patient-department of a private teaching hospital located in Lagos, Nigeria over four-month duration in 2011.
Methodology: Two hundred and fifty case notes of hypertensive patients were randomly selected. These were assessed for costs of pharmacotherapeutic management of hypertension. Patients’ details such as demographic data, drug regimens and funding status were extracted from the case notes. Drugs’ prices were obtained from the hospital billing guide. Data presentation was by using descriptive statistics.
Results: Two hundred and eight (83.2%) of the selected case notes met the study criteria. Diuretics were the most economical at an average monthly cost per prescription of NGN858.6 ($5.51) followed by the beta-blockers at NGN1,101.1 ($7.07) while fixed dose combinations were the costliest at NGN10,425.0 ($66.93). Health Maintenance Organizations (HMOs) having 104 (50.0%) of the cohort as enrollees incurred most of the cost at NGN446, 325.0 ($2,865.47) followed by NHIS 75 (36.0%) at NGN321, 354.0 ($2,063.14). An average monthly cost of antihypertensives per patient was highest for private patients NGN4, 314.47 ($27.69) and least for NHIS NGN4, 284.72 ($27.50). The national cost implication using the least average monthly antihypertensive cost per patient of NGN4,284 .72 ($27.50) for NHIS implies an average of NGN51,416.64 ($330.10) per annum for each patient and a whooping sum in excess of NGN1.054 trillion (over $6.76billion) for over 20 million affected hypertensive patients in Nigeria.
Conclusion: Cost burden of hypertension management is high, incurred mostly by HMOs and NHIS. Diuretics were the most economical of all prescribed regimens.

Open Access Original Research Article

Development and Validation of Densitometry TLC Stability Indicating Method for Quantitative Determination of Azelastine Hydrochloride and Emedastine Difumarate in Their Drug Products

Nahla N. Salama, Sawsan A. Abdel-Razeq, Shimaa Abdel-Atty, Naglaa El-Kosy

Journal of Pharmaceutical Research International, Page 79-92
DOI: 10.9734/BJPR/2014/3693

Aims: Stability indicating densitometry-TLC assay was established and validated for determination of azelastine hydrochloride (AZT) and emedastine difumarate (ETD) in the presence of their acid and oxidative degradants.
Methodology: Forced degradation was performed using 30% H2O2 and 5 M HCl. The method was based on thin-layer chromatographic separation of the two drugs from their degradants, using methanol- 10% ammonia (9.5:0.5, v/v) as developing system, followed by densitometric measurements of the intact drug spots at 292 and 283 nm, for azelastine hydrochloride and emedastine difumarate respectively.
Results: The linear range was 0.5 - 10.0 μg/spot, with mean recoveries of 100.09 ± 0.53% and 100.36 ± 0.40% for azelastine hydrochloride and emedastine difumarate respectively.
Conclusion: The proposed method was successfully applied for the routine quality control analysis of both drugs in laboratory prepared mixtures and commercially available preparations. The degradation products were identified by IR and MS and the pathways were illustrated. The method was validated according to ICH.

Open Access Original Research Article

In vitro Synergistic Action of Honey and Essential Oils against Two Species of Aspergillus: A Preliminary Study

Hasan A. Alzahrani, Laïd Boukraa, Fatiha Abdellah, Balkees Bakhotmah, Si Mohamed Hammoudi

Journal of Pharmaceutical Research International, Page 93-100
DOI: 10.9734/BJPR/2014/5332

In order to evaluate the synergistic action of essential oils (EO) on the antifungal activity of honey, a comparative method of adding honey with and without EO to culture media was used. One variety of honey and five EO types were used to determine the minimum inhibitory concentration (MIC) against two clinical isolates of microfungi; namely Aspergillus niger and Aspergillus flavus. In a second step, lower concentrations of honey than the MIC were mixed with a set of sub-MIC of EO and then added to media to determine the minimum synergistic inhibitory concentration (MSIC). The MIC of honey without EO was 47% (v ⁄ v) against A. niger and 50% (v ⁄ v) against A. flavus. The MIC of EO varied strongly from one variety to another one with that of Eugenia caryophylata being the most effective against the tested Aspergillus species. When the EO was mixed with honey, a MIC drop was observed with each variety of EO. Isobolographic representation shows a synergistic action between honey and all EO types against the tested fungi. Further research studies are needed to elucidate and optimize the effective combination of these natural products in clinical practice.

Open Access Original Research Article

Use of Antimicrobial Medicines among University Students in Sierra Leone

M. O. Afolabi, L. Macarthy, K. P. Osemene

Journal of Pharmaceutical Research International, Page 101-112
DOI: 10.9734/BJPR/2014/5972

Aims: Misuse of antimicrobial medicines is a major contributory factor to development of resistant strains of micro-organisms, therapeutic failure and increased healthcare costs in many countries. To identify pattern of antimicrobial use among undergraduate students of the University of Sierra Leone and to determine possible gaps in their understanding of appropriate use of these therapeutic agents.
Study Design: A cross-sectional survey of the students using a structured questionnaire and a stratified random sampling method to obtain the respective number of students from each college.
Place and Duration of Study: Registered undergraduates in the three Colleges of the University of Sierra Leone, between March and June 2012.
Methodology: A 25-item structured questionnaire was administered on a random sample of four hundred and eighteen (418) undergraduates of the University. The instrument explored respondents’ pattern of self-medication with antimicrobials, knowledge of the indications for use and sources of supply.
Results: Most students reported having self-medicated with antimicrobials at various times and there were gaps in their understanding of the medicines; with about 67% having some knowledge of correct indications for use. Majority of them (70%) obtained the medicines on demand from open drug markets without prescriptions and the medicines were used for such ailments as common colds and diarrhea. Previous experiences of treating similar symptoms ranked highest as the factor affecting demand and penicillins topped the list of commonly used antimicrobials. Most of the students did not complete full regimen of the medication for reasons of cost, long duration of treatment and side effects.
Conclusion: There were knowledge gaps in the proper use of antimicrobial medicines and unrestricted access to prescription drugs was a major factor of misuse. The existing drug laws in the country should be strengthened to control indiscriminate sale and distribution. Basic courses on rational medicine use may need to be incorporated in the general studies programme of the University; with emphasis on the consequences of indiscriminate use of antimicrobial medicines.

Open Access Original Research Article

Comparative Effect of Antidepressants (Duloxetine) and NSAID (Dexibuprofen) in a New Rat Model of Chronic Pain Induced Depression Associated with Monosodium Iodo Acetate (MIA) Induced Osteoarthritis in Rats

V. S. Saravanan, V. Krishnaraju

Journal of Pharmaceutical Research International, Page 113-124
DOI: 10.9734/BJPR/2014/4040

Objective: To evaluate the role of anti-depressants (Duloxetine) and NSAID (Dexibuprofen) in a new rat model of chronic induced depression.
Methods: Twenty four male wistar rats were divided into 4 groups of 6 animals each. Group I to IV served as vehicle control, osteoarthritis (OA) control, duloxetine and dexibuprofen treated groups respectively. Group I received intra-articular Injection of 50 µl of 0.9% normal saline, and Group II to IV received 50 µl MIA, and the treatment of drugs started on the same day. The animals will be monitored for OA parameters and/or depression on pre-dose day (day 0) and on day 1, 3, 5, 7, 11, 14, 18, 21 and 28 th day.
Results and Discussion: In MIA treated group, the rise in knee inflammation is maximum on day 3 (12.31±0.85 mm; p<0.001) and reduced near to normal on day 7 (9.26±0.57 mm; p<0.001). Dexibuprofen and duloxetine decreased the inflammation from day 3, and the decrease is comparatively better in dexibuprofen group. Also, dexibuprofen increased vocalization threshold of knee compression force for 7 days and decreased thereafter, whereas duloxetine has no effect for first 7 days and increased thereafter. Duloxetine was significantly (p<0.001) effective on neuropathic pain (Punctate allodynia, mechanical gripstrength, threshold angle of knee extension) and depression (forced swim test and locomotor activity) compared to dexibuprofen.
Conclusion: The present study has shown that dexibuprofen has the potential in the initial phase of chronic OA and duloxetine in the later stage, where neuropathic and depressive component dominates.

Open Access Original Research Article

Antioxidant and Cytotoxic Activities of Gmelina arborea ROXB. Leaves

Hussein A. Shoeb, Hassan M. F. Madkour, Laila A. Refahy, Mona A. Mohamed, Amal M. Saad, Mosad A. Ghareeb

Journal of Pharmaceutical Research International, Page 125-144
DOI: 10.9734/BJPR/2014/6018

Aims: The leaves of Gmelina arborea (ROXB.) (Family Verbenaceae) are widely used in the folklore to treat various types of diseases. In this study, the antioxidant and cytotoxic activities of different methanolic extracts and the derived subfractions of 90% methanolic extract of this plant were evaluated.
Methodology: The antioxidant activity was carried out via three different quantitative assays as well as qualitative one. Total phenolic was determined via Folin-Ciocalteu and total flavonoid via AlCl3 assays. The cytotoxic activity was carried out via brine shrimp test and toward human cancer cell line; HepG2 using Sulphorhodamine-B assay. The 90% methanolic extract was fractionated using pet. ether then the 90% defatted methanol undergoes fractionation using (CHCl3, EtOAc and n-BuOH).
Results: The antioxidant results showed that the; DPPH antioxidant activity was (19.20, 14.10 and 28.94 µg/ml); total antioxidant capacity was (412.69, 518.45 and 390.41; mg AAE /g extract); reducing power was (0.649, 0.715 and 0.396; 200 mg/ml) and total phenolic was (330.22, 400.66 and 244.76; mg GAE/g extract), respectively for 90% methanol, n-BuOH and EtOAc. The cytotoxic results showed that the; mortality of brine shrimp larvae (LC50) against different dosages of defatted 90% methanol, n-BuOH and EtOAc respectively was (158.48, 39.81 and 199.52; µg/ml) and the results of HepG2 assay showed that n-BuOH fractions have cytotoxic activity with IC50 ≤ 20 µg/ml (IC50 = 17.3 µg/ml) which falls within the American Cancer Institute criteria followed by 90% methanol and EtOAc (IC50 = 22.1 µg/ml).
Conclusion: It was concluded that Gmelina arborea extracts possess a powerful antioxidant and cytotoxic activities.

Open Access Original Research Article

Simple Reversed-Phase High Performance Liquid Chromatographic Estimation of the Antiretroviral Agent Efavirenz from Human Plasma

Benjamin U. Ebeshi, Oluseye O. Bolaji, Margaret Oluka, Vaikosen N. Edebi, Julius O. Soyinka, Anastasia Guantai

Journal of Pharmaceutical Research International, Page 145-157
DOI: 10.9734/BJPR/2014/4123

Aims: Sequel to the resurgence of TB co-infection in HIV/AIDS patients in sub-Saharan Africa, efavirenz has become an important component of the highly active antiretroviral treatment (HAART). The objective of this study therefore is to provide a simple reversed-phase high performance liquid chromatographic (HPLC) method for the determination of efavirenz in human plasma.
Study Design: Method development and experimental study.
Place and Duration of Study: School of Pharmacy, University of Nairobi, Nairobi, Kenya, between October 2009 and September 2010.
Methodology: A 500µl drug-free plasma sample was each placed in six different centrifuge tubes (2ml) and varying aliquots of the stock solution (100μg/ml) of efavirenz were spiked and vortexed for 60sec to give concentrations of 0.5, 1.0, 2.0, 4.0, 8.0 and 16µg/ml for calibration standards and 2.0, 4.0, 8.0 and 16.0µg/ml for quality control samples. The off-column sample pretreatment was carried out by protein precipitation using ice-cold acetonitrile. The samples were chromatographed in a phenomenex (C18) 5µm particle size column with 250x4.6mm I.D and UV detection at 254nm using a mobile phase, which was made up of a mixture of solutions A and B. Both consisted of acetonitrile, 25mM ammonium acetate buffer and glacial acetic acid in proportions of 90:10:0.1 and 10:90:0.1(v/v), respectively. The analytical technique was validated for precision, accuracy and analyte recovery.
Results: The calibration plot for efavirenz was found to be linear over the concentration range of 0.5 to 16.0µg/ml with the regression line equation obtained as y=26842x–409.4 and the regression coefficient (R2=0.999), which allows for accurate reading of the concentrations of the test samples. The RSD (%) in intraday and interday assays ranged from 0.44 to 0.78%. Accuracy ranged from 92 to 110% and the recovery was >97%.
Conclusion: This new HPLC method is simple, reproducible and cost-effective and can be used for therapeutic drug monitoring of efavirenz in HIV/AIDS patients on HAART as demonstrated in this study.

Open Access Original Research Article

The Effects of Extracts of Christmas Bush (Alchornea cordefolia) on Sickle Cell Hemoglobin

R. N. Nwaoguikpe, W. Braide, G. N. Okwu, B. O. Anyanwu, C. O. Ujowundu

Journal of Pharmaceutical Research International, Page 158-171
DOI: 10.9734/BJPR/2014/4467

The study aims at determining the antisicking effectiveness of the extracts of the leaves of a herbal medicinal plant Alchornea cordefolia. The plant is widely distributed in the South-eastern region of Nigeria. The study is designed to involve phytochemical exploration, the nutrient and mineral compositions, the free amino acid, ascorbic acid and the amino acid constituents. Apart from these, the rates of sickle cell hemoglobin polymerization were assayed with different fractions of extracts and compared with the control to ascertain their antisickling effectiveness. Sickle cell blood samples were donated by a total of forty patients (25 males and 15 females) of ages (17-32 years) whose sickle cell status were confirmed by electrophoresis of sickle cell blood by staff of the hematology unit of the centre . The donors were co-opted into the study by the personnel of the sickle cell unit of the Federal Medical Centre, Owerri, Nigeria. The determination of the Fe2+/Fe3+ ratio was to assess the oxygen affinity of sickle cells or drepanocytes. The antisickling properties of leaf extracts of Christmas bush (Alchornea cordefolia) were investigated. Results from phytochemical analysis of crude aqueous extract (CAE) revealed the presence of flavonoids (4.2±0.1%), alkaloids (5.7±0.12%), saponins (4.60±0.10%), tannins (6.50±0.1%), phenol (3.0±0.10%) and oxalate (5.47±0.1%). Proximate composition showed the following results: moisture (11.05±0.0%), ash (6.8±0.1%), crude fat (6.03±0.0%), protein (6.10±0.0%), fiber (24.5±0.2%) and carbohydrates (44.50±0.2%). Assay of mineral composition, revealed a preponderance of such, which include: Potassium (150.30 mg/100g), Sodium (228.20 mg/100g), Calcium (1.60 mg/100g), Magnesium (2.40 mg/100g) and Phosphorus (1.00 mg/100g) of dry weight of sample. The determination of the antisickling effects of the extracts of Alchornea cordifolia was assessed based on the inhibition of sickle cell hemoglobin polymerization and the improvement of Fe2+/Fe3+ ratio. Fifty grams (50 g) of the powdered sample was used for the batch extraction procedures with chloroform, methanol, butanol and distilled water to obtain the fat soluble fraction (FAS), the butanol soluble(BUS) and water soluble fractions (WAS) respectively. The FAS, BUS and WAS fractions exhibited profound antisickling effectiveness by inhibiting the HbSS polymerization to varying degrees from (47.50% for the BUS to 98.12% for the WAS fractions respectively in 20 min. The WAS and FAS fractions improved the Fe2+/Fe3+ ratio remarkably, except the BUS fraction . Thin layer chromatographic (TLC) analysis revealed the following amino acids - Phenylalanine, Alanine, Glutamate, Histidine, etc. The total free amino acid concentration of the fractions were as follows: the FAS (526.8 mg/100g); the WAS (79.33mg/100 g and the BUS (15.65 mg/100 g). The total vitamin C concentration was found to be 1929.18 mg/100 g of sample. Alchornea cordefolia leaf extracts, with the preponderance of micro and macro-nutrients, vitamins, amino acids and others, may be very beneficial for the management of sickle cell disease.