Read more about plaque assay

Dear Abdul Samad!

Here are some valuable information about:

1.Plant extraction and isolation of maesasaponin mixture A

Plant powder (10 g) was extracted exhaustively with aq. EtOH (80%). The solvent was removed in vacuo (T°B40°C). The residue was moistened with 5 ml DMSO and then suspended in 45 ml of sterile phosphate buffer saline, under stirring. The stock-suspension obtained was divided into smaller volumes (2.5±5 ml) and kept at 30°C; it contained the equivalent of 200 mg: ml of plant powder. Prior to the antimicrobial or antiviral screening, the extract stock-suspension was diluted ten-fold in a tryptic soy broth or in an appropriate cell culture medium, respectively. On the other hand, a virucidal 1-BuOHfraction was obtained from the MeOH extract of the dried and ground leaves (1 kg) of M. lanceolata (Myrsinaceae), as described earlier. From a bioassay-guided fractionation procedure using herpes simplex virus type 1 as the target model, the 1-BuOH extract was repeatedly subjected to column chromatography on silica gel (on elution with 1-BuOH: HOAc: H2O (4:1:5) and 1-BuOH: EtOH: H2O (4:1:2.2)) and on Sephadex LH-20 (on elution with MeOH). Two saponin mixtures, made sasaponin mixtures A (2 g) and B (10 g), showing the reddish-purple reaction in the Liebermann±Bur- chard test and forming a stable foam when shaken with water, was obtained. Chromatographed on a silica gel thin layer (precoated plates, Merck), on elution with 1-BuOH: HOAc: H2O (4:1:5), the maesasaponin mixtures A and B migrated to rf-values of 0.53 and 0.35, respectively; they gave both whitish spots with FeCl3. The chemical structure of the maesasaponin mixture B and its biological activities have been re- ported already (Sindambiwe et al., 1996, 1998). The precise chemical structure of the masalaponin mixture A has not been elucidated yet; however, its NMR and mass spectral data suggest similar structural pro®le to that of the maesasaponin mixture B with possible discrepancies in their glycosidic moieties.

.Antibacterial screening

The antibacterial battery was representative of all the important groups of pathogenic bacteria according to their physical and chemical composition, and resistance pattern. The diluted extract suspension was homogenized and the antimicrobial screening was then performed according to the liquid dilution method (Vanden Berghe and Vlietinck, 1991). Minimum inhibitory and minimal bactericidal concentrations were determined. Only bactericidal extracts were considered as active.

3.Antifungal assay

The EtOH extracts of seven Rwandan plants were screened against the yeast Candida albicans ATCC 10231, according to the dilution method previously described for bacteria (Vanden Berghe and Vlietinck, 1991). In addition to C. albicans, isolates of contaminant ®lamentous fungi (Aspergillus fumigatus RV 67686 and Aspergillus niger RV 67644) and Dermatophytes (Epidermophyton ¯occosum RV 71625, Microsporum canis RV 66973, Microsporum langeroni RV 71268, Mi- croides interdigitalis RV 66466 (Trichophyton mentagrophytes var. interdigitale) and Trichophy- ton rubrum RV 58125), provided by Dr Ch. De Vroey, Laboratory for Mycology, Institute of Tropical Medicine (Antwerp, Belgium), were used to screen the maesasaponin mixture A. The anti- fungal assay procedure was recently fully described (Sindambiwe et al., 1998).

4.Antiviral screening and cytotoxicity assay

The battery of antiviral screens (Sindambiwe et al., 1998) was designed to cover as many as possible diverse virus groups in their morphology, replication mechanism, and pathological impor tance. The antiviral activity and the cytotoxicity of the EtOH extracts and the maesasaponin mix- ture A were evaluated as described previously (Vanden Berghe et al., 1993; Sindambiwe et al., 1994). Saponin cytotoxicity was further evaluated by an optimized MTT-assay (Carmichael et al., 1987). The virucidal activity of the plant extracts was also determined as described earlier (Vanden Berghe et al., 1993; Sindambiwe et al., 1994). The virucidal test was incubated at 25°C for both EtOH extracts and maesasaponin mixture A, for 5 min with the former and for 5, 10, 15, 30 and 60 min with the latter. In the virucidal assay, the cytotoxic saponin mixture was separated from the residual virus using ultracentrifugation technique.

 I am also adding some detailed references.

If you need more detailed informations, you should contact me!

Sincerely Yours!

Dr. Bratko Filipič

E-mail: [email protected]

Thank you so much Filipic, for the valuable information

Dear Abdul Samad

Pls. find the attached files

Hoping this will be helpful to your project, good luck

Best regards

Prof. Houda Kawas

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC88925/

https://www.ncbi.nlm.nih.gov/pubmed/25134897

https://www.ncbi.nlm.nih.gov/pubmed/12741619

http://pubmedcentralcanada.ca/pmcc/articles/PMC4663710/

https://www.hindawi.com/journals/ecam/2013/504563/

https://www.omicsonline.org/open-access/plants-as-antiviral-agents-2157-7471-1000254.php?aid=54282&view=mobile

Thank you so much Houda for the valuable information

You are welcome

I want t believe the valuable suggestion above should be sufficient to carry out your project. Regards

Dear Abdul Samad!

Can you include me as coworker to your new project ?

Dr. Bratko Filipič

Email: [email protected]