Open Access Original Research Article

Different Approaches for Quantification of the Bioactive Metastable Thiosulfinates in Garlic Using a Validated High Performance Liquid Chromatography Method

Florence A. Opondo, Were L. L. Munyendo, Patrick M. Shem, Raphael O. Nyayieka

International Journal of Biochemistry Research & Review, Page 1-9
DOI: 10.9734/IJBCRR/2017/35180

Aims: In this study Allium sativum extracts were evaluated for antibacterial activity, High Performance Liquid Chromatography method was developed and validated for direct quantification of alliin and indirect quantification of metastable bioactive thiosulfinates.

Study Design: The experimental research design composed of bioassay using Disc-diffusion method and chemical assay which involved differential quantification of bioactive metastable thiosulfinates in Allium sativum using a validated HPLC method.

Place and Duration of Study: Chemical Engineering Laboratory, School of Engineering, Moi University, Eldoret, Kenya. The study duration was from June 2015 to August 2016.

Methodology: Antibacterial activity conducted for gram-positive Staphylococcus aureus and Pseudomonas aeroginosa while Escherichia coli was bioassayed as the gram-negative bacteria. The bioactive metastable thiosulfinates were differentially quantified using HPLC method through direct quantification of alliin.

Results: The highest percent yield of 0.32% was realized from a garlic cloves blend marc of 25 g. The activity of the extracts was noted to be dependent on the duration of time after reconstitution but before assaying. This was revealed from evaluation of antibacterial activity constancy which indicated that the garlic extract exhibited a pattern of decreasing zone of inhibition from above 14 mm at 3 hours reducing to 6 mm after 24 hours. The HPLC method developed enabled elution of alliin at 2.5 minutes illustrating high levels of accuracy from the calculated mean percent recovery ± SD that ranged from 99.06 ± 0.08 to 99.56 ± 0.11 and 99.08 ± 0.12 to 99.34 ± 0.03 for the inter-day and intra-day respectively which is regarded optimum for the method application. The data obtained for quantification is in agreement with the results of bioactivity constancy evaluation in that as the bioactivity of the garlic extracts diminishes with time after extraction, so the % bioactive thiosulfinates falls along the time intervals from 22.9% at 0 hour to 10.0% after 24 hours.

Conclusion: The devised method for differential quantification of bioactive thiosulfinates proved to be valid and accurate hence applicable for evaluating the metastable bioactive constituents of garlic extracts.

Open Access Original Research Article

Ethanolic Extract of Solanum melongena Linn Fruit Mitigated Monosodium Glutamate-Induced Oxidative Stress

Uchendu O. Mbah, Anthony Cemaluk C. Egbuonu

International Journal of Biochemistry Research & Review, Page 1-8
DOI: 10.9734/IJBCRR/2017/35055

Background/Aim: Monosodium glutamate (MSG), a flavour enhancing food additive generally regarded as safe by food regulatory bodies at low concentration can induce oxidative stress at high concentration. The heightened search for underutilized plant-sourced food necessitated this study aimed at determining some phytochemicals and vitamins in Solanum melongena Linn fruit and the influence of the ethanol extract of the sample on MSG–induced oxidative stress.

Study Design/Methodology: Standard protocols were employed in the determination of the studied phytochemicals and vitamins of the sample. In the animal study, twenty four Wistar rats with average weight of 105.00 ± 7.00 g were assigned into six groups and fed thus: Group 1 (control, feed and distilled water only), Group 2 (8000_mg/kg body weight MSG), Group 3 (300_mg/kg body weight the sample extract), Group 4(8000_mg/kg body weight MSG+ 100_mg/kg body weight the sample extract), Group 5(8000_mg/kg body weight MSG+ 300_mg/kg body weight the sample extract) and Group 6(8000_mg/kg body weight MSG+ 500_mg/kg body weight the sample extract). Exposure was oral and daily for 14 days.

Results: The determined vitamins in the sample  were vitamin A (0.36 ± 0.02IU), vitamin B3 (9.03 ± 0.07 mg/100 g) and vitamin C (369.67± 7.54 mg/100 g) while the phytochemicals were alkaloids (1.13 ± 0.10 mg/100 g), saponins (5.54 ± 0.37 mg/100 g), tannins (11.87 ± 1.87 mg/100 g), cyanogenic glycogenic (6.21 ± 0.22 mg/100 g) and phytates (30.62 ± 1.54 mg/100 g). The MSG only fed group significantly (P =.05) increased malondialdehyde (MDA) concentration  but decreased (P =.05) glutathione (GSH), catalase (CAT) and superoxide dismutase (SOD) activity compared to the control, suggesting induction and enhanced generation of reactive oxygen species (ROS) which compromised the antioxidant defense of the MSG-fed rats. Co-administration of MSG with ethanol extract of Solanum melongena Linn fruit significantly (P=.05) reduced the MDA concentration to a value non-significant (P=.05) compared to that of the control rats, and conversely, significantly (P=.05) increased GSH, CAT and SOD activities compared to the group 2 rats.

Conclusion: The Solanum melongena Linn fruit contains the studied phytochemicals and vitamins while the ethanol extract of the fruit could significantly mitigate MSG-induced oxidative stress in the rats.

Open Access Original Research Article

Assessment of Some Biochemical Oxidative Stress Markers in Type II Diabetics and Non-diabetics with Chronic Periodontitis

Abdul Samad Aziz, Madhav Govind Kalekar, Adinath Narayan Suryakar, Rahul Kale, Tabita Benjamin, Madhurima Dikshit

International Journal of Biochemistry Research & Review, Page 1-9
DOI: 10.9734/IJBCRR/2017/35143

Aims: Comparative assessment of some biochemical oxidative stress markers in Type II diabetics and non-diabetics with chronic periodontitis.

Study Design: The cross sectional study groups were clinically evaluated and their biochemical parameters were assessed and statistically compared.

Place and Duration of Study: Departments of Biochemistry and Dentistry, Grant Medical College and Sir J J Group of Hospitals, Mumbai, India, between May 2010 and July 2012.

Methodology: 168 individuals with generalized chronic periodontitis (CAL ≥ 3 mm; American Academy of Periodontology criteria) were divided them into non-diabetics (F Glucose ≤ 5.5 mmol/L; CP group, n = 86) and diabetics (F Glucose ≥ 7 mmol/L; WHO criteria, CPDM group, n = 82). The diabetic status was ascertained by measuring the Fasting plasma glucose (F Glucose). Apparently healthy individuals (CAL ≤ 3 mm and F Glucose ≤ 5.5 mmol/L; C group, n = 120) were recruited as controls. The periodontal status for the control and the aforementioned study groups was evaluated by measuring gingival index (GI), plaque index (PI), papillary bleeding index (PBI), probing depth (PD) and clinical attachment level (CAL). The biochemical oxidative stress markers namely total antioxidant capacity (TAC), superoxide dismutase (SOD), glutathione peroxidase (GPx), vitamin C, malondialdehyde (MDA) were estimated.

Results: The clinical periodontal parameters were significantly (p≤0.05) higher in CPDM than CP, and both the diseased group v/s controls. The biochemical markers also showed similar trend as that of clinical parameters. TAC, GPx, vitamin C got significantly reduced and SOD, whereas, MDA got significantly increased.

Conclusion: The individuals with diabetes and chronic periodontitis may be at a higher risk of oral and systemic oxidative stress damage compared to non-diabetic with chronic periodontitis.

Open Access Original Research Article

A Comparison of Parasitological Techniques (Microscopy) and Loop-mediated Isothermal Amplification (LAMP) of DNA in Diagnosis and Monitoring Treatment of Trypanosoma brucei rhodesiense Infection in Vervet Monkeys (Chlorocebus aethiops)

L. M. Chimbevo, J. B. Malala, P. S. Oshule, W. F. Muchiri, D. O. Otundo, S. Essuman, F. O. Oginga, M. K. Webale, G. O. Munyekenye, J. M. Ndambuki

International Journal of Biochemistry Research & Review, Page 1-14
DOI: 10.9734/IJBCRR/2017/32393

Microscopy and LAMP were compared in diagnosis and treatment follow-up of HAT with T. b. rhodesiense infected vervet monkeys (Chlorocebus aethiops) treated with diminazene aceturate (Berenil®) and Melarsoprol (Mel-B®). Saponin method and heat treatment were used to extract pure and crude DNA, amplified on thermocycler and water bath. SYBR-green and UV-illumination in agarose gel were used to assess results. Parasitaemia, CSF-parasitosis, PCV, WBC and CSF-protein concentration were determined. DNA was detected 7dpi in blood and serum and 21dpi in CSF. It cleared 56dpi in blood and serum of both monkeys and re-appeared 77dpi and 129dpi in blood and serum of monkey A and B respectively after Berenil® treatment. DNA cleared 40 and 90 days and 90 and 150 days in blood and serum and CSF of A and B respectively after Mel-B® treatment. Using blood and CSF, detection of microscopy was 28.21% and 21.18% while LAMP was 60.26%, 55.13% and 79.49% using blood, serum and CSF respectively. X2 was 16.734 (p=0.000) and 38.023 (p=0.000) between LAMP and microscopy in blood and CSF, pure DNA using thermocycler it was 60.27%, 55.13% and 78.12%, and 46.15%, 48.72% and 75.64% on water bath in blood, serum and CSF respectively. Crude DNA had %detection of 56.41%, 56.41% and 76.92% using thermocycler and 48.72%, 44.87% and 64.10% on water bath using blood, serum and CSF respectively. Crude DNA using thermocycler, LAMP and microscopy had Kappa (k) of 0.397 and 0.602 and X2 13.141 (p=0.000) and 35.247 (p=0000) using blood and CSF respectively. In late stages, k and X2 of 0.600 and 15.000 (p=0.000) were obtained using CSF. For treatment follow-up, k and X2 were 0.472 and 19.429 (p=0.000) and 0.527 and 21.346 (p=0.000) in blood and CSF respectively. Heat treatment and amplification using water bath may be used for sample preparation and amplification respectively.

Open Access Original Research Article

Antimicrobial Activity and Isolation of Methylated Flavanone from Methanol Stem-bark Extract of Nauclea diderrichii

M. E. Khan, J. O. Amupitan, I. Y. Sudi

International Journal of Biochemistry Research & Review, Page 1-10
DOI: 10.9734/IJBCRR/2017/28200

The antimicrobial activities of the crude methanol fraction of the stem bark extract of Nauclea diderrichii was investigated using agar diffusion and broth dilution techniques. Clinical isolates obtained from the Department of Medical Microbiology, Ahmadu Bello University Teaching Hospital, Zaria, Nigeria were used for the studies. The crude fraction showed activity against Staphylococcus aureus, Streptococcus pyrogenes, Pseudomonas aeruginosa, Klebsiella pneumonia and Samonella typhi, but were below detectable limits (BDL) in methicillin resistant S. aureus, Bacillus subtilis, Corynobacterium ulcerans, Escherichia coli and the fungus Candida albicans. Low values for minimum inhibitory concentration (MIC, 1.25 μg/ml), and minimum bactericidal concentration (MBC, 2.25 μg/mL), suggested that the crude extract had a good antimicrobial activity against the susceptible organisms. The minimum fungicidal concentration MFC, was 5.00 μg/mL.Chromatographic separation using a combination of silica and sephadex LH-20 led to the isolation of a methylated flavanone. The structure of this isolated compound was determined using different spectroscopic techniques including both 1 and 2D nuclear magnetic resonance (NMR). All the above validates the ethno medicinal uses of the N. diderichii in the treatment of various bacterial infections.