Anti-biofilm activity of Marula – A study with the standardized bark extract

Ethnopharmacological relevance

Marula (Sclerocarya birrea; family – Anacardiaceae) is an African plant, which enjoys wide socio-economic importance particularly in southern part of Africa. The fruits are consumed as food and also as alcoholic beverage (cream liquor). In different parts of Africa, the decoction of the bark is traditionally used for the treatment of dysentery, diarrhoea, and various other infectious conditions. The aim of the study was to investigate the anti-biofilm properties of the methanol extract of Marula bark (stem bark of Sclerocarya birrea), with a view towards combating the emergence of antimicrobial resistance often associated with bacterial biofilms.

Materials and methods

The standardized methanol extract was initially tested for its antimicrobial property. The crystal violet assay was used for evaluating anti-biofilm (biofilm formation by Pseudomonas aeuginosa) activity. Further in order to study the mechanism of anti-biofilm activity, the same was evaluated for understanding its role on various quorums sensing mediated phenomenon (swarming motility assay, protease and pyoverdin assay) that are known to be associated with the formation of biofilms and pathogenicity.

Results

The methanol extract showed no inhibition of bacterial growth up to a concentration of 200 µg/ml. Interestingly, the sample produced anti-biofilm activity (around 75% decrease; 100 µg/ml) at sub-lethal concentration. Further it also significantly reduced the QS mediated swarming motility. The release of various virulent factors (protease and pyoverdin) was found to be lowered when pre-treated with the extract.

Conclusion

The present study illustrates the anti-biofilm property Sclerocarya birrea. The standardized extract significantly disrupted the quorum sensing mediated production of biofilm formation and also inhibited swarming ability of the cells. The extract displayed a regulatory role on the secretion of protease and pyoverdin, two QS dependent pathogenic factors found in Pseudomonas aeruginosa. This study also validates the ethnobotanical use of Marula.     

Estimation of Apigenin, an Anxiolytic Constituent, in Turnera aphrodisiaca

An HPTLC densitometric method has been developed to estimate apigenin in Turnera aphrodisiaca aerial and its segregated parts (leaves, stems, flowers and fruits) so that plant can be standardized on the basis of its bioactive marker. The apigenin content in methanol extract of T. aphrodisiaca aerial parts was found to be about fourteen times less than acid hydrolyzed methanol extract of the plant indicating the presence of most of apigenin in glycosidic form. Amongst different plant parts, flowers possessed maximum content of apigenin followed by leaves. The apigenin content was also determined in three marketed formulations of T. aphrodisiaca viz., NLK, DWSG and SBL. DWSG contained higher content of apigenin. Aerial parts of the plant were collected at bimonthly intervals over a period of one year in the months of January, March, May, July, September and November. The plant material collected in September showed maximum content of apigenin.     

丹参中水溶性酚酸类成分的薄层扫描测定法

应用高效薄层扫描法对丹参根中的原儿茶醛、咖啡酸、迷迭香酸及其甲酯、丹酚酸A,B和C7种有效成分进行了含量测定。选择了最佳提取分离方法,即生药用热水提取并酸化后用乙酸乙酯萃取,萃取液浓缩后进行薄层色谱分离。展开剂A:氯仿乙酸乙酯——苯—甲酸(2.4:2:1:0.6)分离原儿茶醛,展开剂B:氯仿-乙酸乙酯-苯-甲酸甲醇(1.5:2:1;1:0.1)分离其余6种成分。并用所建立的分析方法分析比较了4种丹参根中7种有效成分的含量。     

Sulfate incorporation into peripheral nerve endoneurial glycolipids after crush and permanent transection injury

The sulfation of peripheral nerve glycolipids was examined at 35 days after both crush injury or permanent transection of the adult rat sciatic nerve by in vitro incorporation of [35S]sulfate into endoneurial slices. These experimental models of neuropathy are characterized by the presence and absence of both axonal regeneration and subsequent myelin assembly. Although the sulfo-glucuronosyl glycosphingolipids (SGGLs) were not detected by alpha-napthol reagent after HPTLC separation of the total acidic lipid extract, fluorographic analysis after sulfate incorporation revealed a 4.7-fold increase in [35S]sulfate in the sulfo-glucuronosyl paragloboside (SGPG) and a 3.5-fold increase in the sulfo-glucuronosyl-lactosaminosyl paragloboside (SGLPG) after the crush injury compared to permanent transection. These [35S]sulfate-labeled lipids were identified by comigration after HPTLC separation by immunostaining with specific IgM monoclonal antibodies from a patient with demyelinating neuropathy and plasma cell dyscrasia. Enhanced incorporation of sulfate in the crushed nerves was also observed in the sulfatides and in several unknown lipids migrating between GM2 and GM3, between GM1, and GM2, slightly above the origin, and at the origin. Since previous studies (Yao and Poduslo: J Neurochem 50:630-638, 1988) have shown [35S]sulfate incorporation, but not [3H]Gal or [3H]Glc, into sulfatides at 35 days after transection, it is possible that the sulfation observed in the present studies does not represent de novo biosynthesis but rather sulfation of an endogenous pool of glycolipids that results from the nerve injury.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacognostic Evaluation of the Root of Berberis asiatica

Berberis asiatica Roxb. ex. DC. (Berberidaceae) is a very common substitute to "Daruharidra", that is, B. aristata DC., which is used in the Ayurvedic system of medicine. Being an important medicinal plant, it is used extensively for treating a variety of ailments, that is, affection of eyes, skin disease, jaundice, and rheumatism. The current study was therefore carried out to provide requisite pharmacognostic details. Morphological, anatomical, and phytochemical aspects were carried out to identify the diagnostic features of B. asiatica root. Some of the diagnostic features of the root drug noted from the anatomical study are patches of pericyclic fiber, pitted sclerieds, and berberine-containing cells and heterocyclic medullary rays. Physicochemical studies revealed the presence of total ash 2.650%; acid insoluble ash 0.266%; alcohol soluble extractive 11.833%; water soluble extractive 15.333%; tannins 1.723%; sugar 0.332%; starch 16.444%; and alkaloidal content (berberine) 2.4%. A comparative high performance thin layer chromatography (HPTLC) analysis with B. aristata showed a similar profile. Berberine was identified as the major constituent, with a slightly lower percentage (2.4%) in the former. The R_f value of other bands was also calculated.     

Anti-inflammatory activity and qualitative analysis of different extracts of Maytenus obscura (A. Rich.) Cuf. by high performance thin layer chromatography method

Objective

To perform aqueous ethanol soluble fraction (AESF) and dichloromethane extract of aerial parts of Maytenus obscura (A. Rich.) Cuf. using high performance thin layer chromatography (HPTLC) and to test anti-inflammatory activity of these extracts.

Methods

HPTLC studies were carried out using CAMAG HPTLC system equipped with Linomat IV applicator, TLC scanner 3, Reprostar 3, CAMAG ADC 2 and WIN CATS-4 software were used. The anti-inflammatory activity was tested by injecting different groups of rats (6 each) with formalin in hind paw and measuring the edema volume before and 1 h later formalin injection. Control group received saline i.p. The extracts treatment was injected i.p. in doses of 100 and 200 mg/kg 1 h before formalin administration. Indomethacin (30 mg/kg) was used as standard.

Results

The results of preliminary phytochemical studies confirmed the presence of protein, lipid, carbohydrate, phenol, flavonoid, saponin, triterpenoid, alkaloid and anthraquinone in both extracts. Chromatography was performed on glass-backed silica gel 60 F254 HPTLC plates with the green solvents toluene: ethyacetate: glacial acetic acid (5:3:0.2, v/v/v) as mobile phase. HPTLC finger printing of AESF revealed major eight peaks with R_f values in the range of 0.28 to 0.80 and the dichloromethane revealed major 11 peaks with R_f values in the range of 0.12 to 0.76. The purity of sample was confirmed by comparing the absorption spectra at start, middle and end position of the band. Treatment of rats (i.p.) with AESF and dichloromethane in doses of 100 and 200 mg/kg inhibited singnificantly (P<0.05, n=6) formalin-induced inflammation by 50%, 55.9%, 45.5%, and 51.4%, respectively.

Conclusions

HPTLC finger printing of AESF and dichloromethane of Maytenus obscura revealed eight major spots for alcoholic extracts and nine major spots for dichloromethane extracts. These HPTLC profiles may be of great usefulness in the quality control of herbal products containing these extracts. The anti-inflammatory activity of both extracts also revealed the medicinal importance of these extracts. The plant can be further explored for the isolation of phytoconstituents having anti-inflammatory activity.     

Antioxidant potential of Rumex vesicarius L.: in vitro approach

ObjectiveTo assess in-vitro antioxidant activity of different fraction and perform high performance thin layer chromatography fingerprint analysis of most active fraction of Rumex vesicarius L. (R. vesicarius).MethodsIn the present study, acetone, ethyl acetate, n-butanol, and methanol extracts of R. vesicarius were evaluated for radical scavenging activity by studying the inhibition of the level of lipid peroxidation induced by Fe(++)/ascorbate, DNA sugar damage, scavenging of hydrogen peroxide, diphenylphosphine DPPH radical scavenging activity, total phenolic content, total flavonoids content and total proanthocyanidin. High performance thin layer chromatography finger print profiling of R. vesicarius L. was also done.ResultsLipid peroxidation induced by the iron/ascorbate system, hydrogen peroxide, diphenylphosphine and DNA sugar damage were inhibited by the addition of different extract of R. vesicarius. Among them, methanolic extract showed maximum efficacy. The methanolic extract showed the highest total phenolic, total flavonoids and total proanthocyanidin contents.ConclusionsThe results suggest that the extracts can be a vital source of phytochemical antioxidants.     

Chromatographic finger print analysis of anti-inflammatory active extract fractions of aerial parts of Tribulus terrestris by HPTLC technique

Objective

To develop HPTLC fingerprint profile of anti-inflammatory active extract fractions of Tribulus terrestris (family Zygophyllaceae).

Methods

The anti-inflammatory activity was tested for the methanol and its fractions (chloroform, ethyl acetate, n-butanol and aqueous) and chloroform extract of Tribulus terrestris (aerial parts) by injecting different groups of rats (6 each) with carrageenan in hind paw and measuring the edema volume before and 1, 2 and 3 h after carrageenan injection. Control group received saline i.p. The extracts treatment was injected i.p. in doses of 200 mg/kg 1 h before carrageenan administration. Indomethacin (30 mg/kg) was used as standard. HPTLC studies were carried out using CAMAG HPTLC system equipped with Linomat IV applicator, TLC scanner 3, Reprostar 3, CAMAG ADC 2 and WIN CATS-4 software for the active fractions of chloroform fraction of methanol extract.

Results

The methanol extract showed good antiedematous effect with percentage of inhibition more than 72%, indicating its ability to inhibit the inflammatory mediators. The methanol extract was re-dissolved in 100 mL of distilled water and fractionated with chloroform, ethyl acetate and n-butanol. The four fractions (chloroform, ethyl acetate, n-butanol and aqueous) were subjected to anti-inflammatory activity. Chloroform fraction showed good anti-inflammatory activity at dose of 200 mg/kg. Chloroform fraction was then subjected to normal phase silica gel column chromatography and eluted with petroleum ether-chloroform, chloroform-ethyl acetate mixtures of increasing polarity which produced 15 fractions (F1-F15). Only fractions F1, F2, F4, F5, F7, F9, F11 and F14 were found to be active, hence these were analyzed with HPTLC to develop their finger print profile. These fractions showed different spots with different R_f values.

Conclusions

The different chloroform fractions F1, F2, F4, F5, F7, F9, F11 and F14 revealed 4, 7, 7, 8, 9, 7, 7 and 6 major spots, respectively. The results obtained in this experiment strongly support and validate the traditional uses of this Sudanese medicinal plant.     

HPTLC-FLD-SERS as a facile and reliable screening tool: Exemplarily shown with tyramine in cheese

The serious cytotoxicity of tyramine attracted marked attention as it induced necrosis of human intestinal cells. This paper presented a novel and facile high performance thin-layer chromatography (HPTLC) method tailored for screening tyramine in cheese. Separation was performed on glass backed silica gel plates, using methanol/ethyl acetate/ammonia (6/4/1 v/v/v) as the mobile phase. Special efforts were focused on optimizing conditions (substrate preparation, laser wavelength, salt types and concentrations) of surface enhanced Raman spectroscopy (SERS) measurements directly on plates after derivatization, which enabled molecule-specific identification of targeted bands. In parallel, fluorescent densitometry (FLD) scanning at 380</400 nm offered satisfactory quantitative performances (LOD 9 ng/zone, LOQ 17 ng/zone, linearity 0.9996 and %RSD 6.7). Including a quick extraction/cleanup step, the established method was successfully validated with different cheese samples, both qualitatively (straightforward confirmation) and quantitatively (recovery rates from 83.7 to 108.5%). Beyond this application, HPTLC-FLD-SERS provided a new horizon in fast and reliable screening of sophisticated samples like food and herb drugs, striking an excellent balance between specificity, sensitivity and simplicity.     

A Rapid Method for the Isolation of Swertiamarin from Enicostemma littorale

We report a simple method for the isolation of swertiamarin, a secoiridoid glycoside, from the whole plant of Enicostemma littorale Blume. Methanol extract of defatted plant material when treated with diethyl ether gave a precipitate containing swertiamarin as one of the major components. Swertiamarin was separated from this precipitate by column chromatography over silica gel. The identity of the compound isolated was confirmed through infrared (IR), ultraviolet (UV), nuclear magnetic resonance (NMR), and mass spectra and melting point and co-chromatography with a reference standard on thin-layer chromatography (TLC). The purity of the compound was confirmed from the UV absorption spectrum, NMR, mass, high performance thin layer chromatography (HPTLC), and differential scanning calorimetry.