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A novel nucleic acid extraction method from aromatic herbs and dried herbal powders using cow skim milk.

ABSTRACT: Authenticity of dried aromatic herbs and herbal powders for the ASU (ayurvedic, siddha, unani) drug formulations is a key of their clinical success. The DNA based authentication is an answer; however, extraction of PCR quality DNA from such material is often problematic due to the presence of various co-extracted PCR inhibitors. Here, we report a novel DNA isolation and purification method utilizing cow skim milk that successfully yields PCR quality DNA from the aromatic herbs and dried herbal powders. The improved method presented in this study could be used as an alternative to successfully extract PCR quality DNA from such plant materials. Further, we present a set of robust matK primers which could be used as plant barcoding resource in future studies.

SUBMITTER: Verma SK

PROVIDER: S-EPMC7359320 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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A novel nucleic acid extraction method from aromatic herbs and dried herbal powders using cow skim milk.

Verma Sunil Kumar SK Biswas Nabanita N

Scientific reports 20200713 1

Authenticity of dried aromatic herbs and herbal powders for the ASU (ayurvedic, siddha, unani) drug formulations is a key of their clinical success. The DNA based authentication is an answer; however, extraction of PCR quality DNA from such material is often problematic due to the presence of various co-extracted PCR inhibitors. Here, we report a novel DNA isolation and purification method utilizing cow skim milk that successfully yields PCR quality DNA from the aromatic herbs and dried herbal p ...[more]

PMID: 32661429

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Project description:Localized aerosol delivery of gene therapies is a promising treatment of severe pulmonary diseases including lung cancer, cystic fibrosis, COPD and asthma. The administration of drugs by inhalation features multiple benefits including an enhanced patient acceptability and compliance. The application of a spray dried powder formulation has advantages over solutions due to their increased stability and shelf life. Furthermore, optimal sizes of the powder can be obtained by spray drying to allow a deep lung deposition. The present study optimized the parameters involved with spray drying polyplexes formed by polyethylenimine (PEI) and nucleic acids in inert excipients to generate a nano-embedded microparticle (NEM) powder with appropriate aerodynamic diameter. Furthermore, the effects of the excipient matrix used to generate the NEM powder on the biological activity of the nucleic acid and the ability to recover the embedded nanoparticles was investigated. The study showed that bioactivity and nucleic acid integrity was preserved after spray drying, and that polyplexes could be reconstituted from the dry powders made with trehalose but not mannitol as a stabilizer. Scanning electron microscopy (SEM) showed trehalose formulations that formed fused, lightly corrugated spherical particles in the range between 1 and 5 µm, while mannitol formulations had smooth surfaces and consisted of more defined particles. After redispersion of the microparticles in water, polyplex dispersions are obtained that are comparable to the initial formulations before spray drying. Cellular uptake and transfection studies conducted in lung adenocarcinoma cells show that redispersed trehalose particles performed similar to or better than polyplexes that were not spray dried. A method for quantifying polymer and nucleic acid loss following spray drying was developed in order to ensure that equal nucleic acid amounts were used in all in vitro experiments. The results confirm that spray dried NEM formulations containing nucleic acids can be prepared with characteristics known to be optimal for inhalation therapy.

Project description:Soluplus is a polymer that has been explored to prepare nanocomposites for pulmonary drug delivery and is non-toxic. However, its aerosolization attributes when spray-dried have not been investigated. Hence, this work aimed to investigate the aerosol performance of soluplus-based spray-dried powders. In addition, the potential use of leucine to improve the aerosolization of such particles was also investigated by including leucine at 10 or 20% w/w. 4% w/w salbutamol was used as a model drug in all the formulations primarily to aid quantification during aerosolization evaluation and for assessing the interaction between the drug and soluplus using infrared spectroscopy with the multivariate analysis approach of principal component analysis (PCA). Three formulations (4% salbutamol/96% soluplus, 4% salbutamol/86% soluplus/10% leucine, 4% salbutamol/76% soluplus/20% leucine) were prepared. The formulations were characterized in terms of solid-state, water content, particle size/morphology, and aerosolization. Similarly, two additional formulations (14% salbutamol/86% soluplus and 24% salbutamol/76% soluplus) were prepared to assess potential non-covalent interactions between salbutamol and soluplus. The formulations with only salbutamol and soluplus were amorphous, as evident from X-ray diffraction. Leucine was crystalline in the formulations. All the spray-dried formulations were irregular spheres with surface corrugation. The 96% soluplus powder showed an emitted fraction (EF) and fine particles fraction (FPF) of 91.9 and 49.8%, respectively. The inclusion of leucine at 10% did not increase the EF; however, an increase in FPF (69.7%) was achieved with 20% leucine. PCA of the infrared spectra suggested potential non-covalent interactions between salbutamol and soluplus. It hinted at the potential involvement of ketone groups of the excipient. This study concludes that soluplus-based spray-dried powder with or without leucine can potentially be utilized for pulmonary drug delivery. In addition, PCA can effectively be utilized in assessing interactions and overcoming limitations associated with visual assessment of the spectra of such formulations.

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A novel nucleic acid extraction method from aromatic herbs and dried herbal powders using cow skim milk.

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A novel nucleic acid extraction method from aromatic herbs and dried herbal powders using cow skim milk.

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