STUDY ON THE ANTIBACTERIAL ACTIVITY OF FRUIT EXTRACTS OF KLUTUK BANANA (MUSA BALBISIANA COLLA) AGAINST SHIGELLA DYSENTERIAE ATCC 13313

Objective: The purpose of this study is to determine the antibacterial activity of Klutuk Banana (Musa balbisiana colla) fruit extracts against Shigella dysenteriae ATCC 13313 and the amount of potassium to the discovery of anti-dysentery drug candidates.Methods: The simplisia of Klutuk banana fruit was extracted with ethanol using a maceration method. The phytochemical screening of ethanol extract was performed using standard procedures. Determination thin layer chromatography (TLC) profile of the extract was performed using a thin layer plate. The antibacterial activity was investigated using agar well diffusion technique. The minimum inhibitory concentrations (MIC) were determined by a serial microdilution method, whereas the minimum bactericidal concentration (MBC) was done by subculturing the MIC result onto agar medium. Potassium levels of the extract were carried out quantitatively using atomic absorption spectrophotometry.Results: The phytochemical analysis revealed the presence of flavonoids, polyphenols, tannins, monoterpenoid and sesquiterpenoids, quinones, and saponins. The TLC results prove the existence of flavonoids in the tested extract. The content of secondary metabolites that can act as an antibacterial, strengthen the antibacterial activity of ethanol extract of Klutuk banana against S. dysenteriae 13313 with MBC values in the range of 5-10%w/v. Potassium levels in the ethanol extract of Klutuk banana fruits contain potassium as much as 2.919% (29 190 ppm).Conclusion: It can be concluded that the ethanol extract of Klutuk banana fruits is more potent as antibacterial against S. dysenteriae than as potassium supplier in hypokalemia therapy.

Extraction of Artemisinin, an Active Antimalarial Phytopharmaceutical from Dried Leaves of Artemisia annua L., Using Microwaves and a Validated HPTLC-Visible Method for Its Quantitative Determination

A simple, rapid, precise, and accurate high-performance thin-layer chromatographic method coupled with visible densitometric detection of artemisinin is developed and validated. Samples of the dried Artemisia annua leaves were extracted via microwaves using different solvents. This method shows the advantage of shorter extraction time of artemisinin from leaves under the influence of electromagnetic radiations. Results obtained from microwave-assisted extraction (MAE) were compared with hot soxhlet extraction. Chromatographic separation of artemisinin from plant extract was performed over silica gel 60 F254 HPTLC plate using n-hexane : ethyl acetate as mobile phase in the ratio of 75 : 25, v/v. The plate was developed at room temperature 25 ± 2.0°C. Artemisinin separation over thin-layer plate was visualized after postchromatographic derivatization with anisaldehyde-sulphuric acid reagent. HPTLC plate was scanned in a CAMAG’s TLC scanner 3 at 540 nm. Artemisinin responses were found to be linear over a range of 400–2800 ng spot−1 with a correlation coefficient 0.99754. Limits of detection and quantification were 40 and 80 ng spot−1, respectively. The HPTLC method was validated in terms of system suitability, precision, accuracy, sensitivity (LOD and LOQ), and robustness. Additionally, calculation of plate efficiency and flow constant were included as components of validation. Extracts prepared from different parts of the plant (leaves, branches, main stem, and roots) were analyzed for artemisinin content, in which, artemisinin content was found higher in the leaf extract with respect to branches and main stem extracts; however, no artemisinin was detected in root extract. The developed HPTLC-visible method of artemisinin determination will be very useful for pharmaceutical industries, which are involved in monitoring of artemisinin content during different growth stages (in vitro and in vivo) of A. annua for qualitative and quantitative assessment of final produce prior to commercial-scale processing for assessment of cost-benefit ratio.

High-Performance Thin-Layer Chromatographic Quantification of Rosmarinic Acid and Rutin in Abnormal Savda Munziq

A high-performance thin-layer chromatographic (HPTLC) method has been established for simultaneous analysis of rosmarinic acid and rutin in Abnormal Savda Munziq (ASMq). A methanol extract of ASMq was used for quantification. The compounds were separated on silica gel H thin layer plate with ethyl acetate-formic acid-acetic acid-water 15 : 1 : 1 : 1.5 (v/v) as a developer, trichloroethanol as the color reagent. The plates were scanned at 365 nm. The linear calibration data of rosmarinic acid and rutin were in the range of 0.0508 to 0.2540 μg(r=0.9964), 0.2707 to 1.35354μg(r=0.9981), respectively. The recovery rate of rosmarinic acid was 99.17% (RSD = 2.92%) and rutin was 95.24% (RSD = 2.38%). The method enables rapid screening, precise, selective, and sensitive quantification for pharmaceutical analysis.

In Vitro Inhibitory Activity of Antimicrobial Peptides Cecropin, α-Thionin DB4, and γ-Thionin RsAFP1 Against Several Pathogens of Strawberry and Highbush Blueberry

As part of a program to develop transgenic highbush blueberry (Vaccinium corymbosum L.) and strawberry (Fragaria ×ananassa Duchesne) cultivars with increased levels of disease resistance, we have investigated the feasibility of introducing genes for the antimicrobial peptides cecropin B and MB39, α-thionin DB4 (DB4) and γ-thionin RsAFP1 (RsAFP1) by testing the effects of these peptides on several important pathogens of these two crop species. A thin-layer plate bioassay was conducted with these peptides and the pathogens Botrytis cinerea (Pers.ex. Fr.), Botryosphaeria dothidea (Mouq.ex. Fr.) Ces & de Not., Colletotrichum acutatum Simmonds, C. gloeosporioides (Penz.) Penz.et Sacc., C. fragariae Brooks, Monilinia vaccinii-corymbosi Reade (Honey), Phytophthora fragariae Hickman and Xanthomonas fragariae Kennedy and King. The minimum lethal concentration (μm) for cecropin ranged from 0.02 for X. fragariae strains 10 and 128 to 72.8 for C. gloeosporioides isolate Akp1. For DB4, the minimum inhibitory concentration (μm) ranged from 0.03 for X. fragariae strain 6 to 87.2 for B. cinerea isolate cc. For RsAFP1, the minimum inhibitory concentration (μm) ranged from 0.13 for X. fragariae strain 6 to 61.4 for M. vaccinii-corymbosi isolate 9423-x-45. These results indicate that introducing genes for either cecropin, DB4 or RsAFP1 into strawberry may be useful for controlling bacterial angular leaf spot disease caused by X. fragariae.

Cutting Apple Fruits Induces Cellulase Activity in the Abscission Zone

Preharvest abscission of apple [Malus ×domestica (L.) Borkh.] fruits causes significant crop loss in many years. In this study, fruit cutting was used to induce abscission in August and September. Abscission zones of `Redchief Delicious' Mercier strain fruits were sampled 0, 2, 4, and 6 days after cutting. Thin-layer-plate assays were developed and used to identify hydrolytic enzymes active in the abscission zone (AZ) after induction. Increased activity of cellulase, but not polygalacturonase, was detected in the AZ following cutting. Cellulase activity was consistently high in AZs 4 days after cutting. Both AVG (652 mg·L–1) and NAA (10 mg·L–1) applied 2 or 4 days after cutting delayed drop, but NAA delayed drop 1.6 days longer than did AVG. Fruits treated with AVG dropped over a longer period than did control or NAA-treated fruits. Chemical names used: aminoethoxyvinylglycine (AVG); naphthaleneacetic acid (NAA).

Thin Layer Chromatographic Confirmation of Aflatoxin Mi Extracted from Milk

Aflatoxin M1 can be confirmed directly on a thin layer plate by reacting the toxin with a mixture of reagents containing p-anisaldehyde. This confirmatory procedure requires only 2 elutions in the same direction using 2 different solvents. The mixture containing p-anisaldehyde is overspotted on Mi after the plate has been developed in toluene-ethyl acetate-ethyl ether-formic acid (25 + 35 + 40 + 5). The plate is heated at 110°C for 10 min and then developed in hexanc-acetonechloroform (15 + 50 + 35). The Rf value of the green fluorescent derivative is less than that of the M1 standard. This confirmatory procedure requires only one-dimensional TLC, so several sample extracts and the standard can be run simultaneously. The minimum detectable quantity of aflatoxin M1 on the TLC plate with this test is 0.3 ng. p- Anisaldehyde reagent solution may also be used as a spray reagent for the confirmation of aflatoxin M1. The procedures described were satisfactory for confirming the mycotoxin in spiked samples of powdered and liquid milk.