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Bar-cas12a, a novel and rapid method for plant species authentication in case of Phyllanthus amarus Schumach. & Thonn.

ABSTRACT: Rapid and accurate species diagnosis accelerates performance in numerous biological fields and associated areas. However, morphology-based species taxonomy/identification might hinder study and lead to ambiguous results. DNA barcodes (Bar) has been employed extensively for plant species identification. Recently, CRISPR-cas system can be applied for diagnostic tool to detect pathogen's DNA based on the collateral activity of cas12a or cas13. Here, we developed barcode-coupled with cas12a assay, "Bar-cas12a" for species authentication using Phyllanthus amarus as a model. The gRNAs were designed from trnL region, namely gRNA-A and gRNA-B. As a result, gRNA-A was highly specific to P. amarus amplified by RPA in contrast to gRNA-B even in contaminated condition. Apart from the large variation of gRNA-A binding in DNA target, cas12a- specific PAM's gRNA-A as TTTN can be found only in P. amarus. PAM site may be recognized one of the potential regions for increasing specificity to authenticate species. In addition, the sensitivity of Bar-cas12a using both gRNAs gave the same detection limit at 0.8 fg and it was 1,000 times more sensitive compared to agarose gel electrophoresis. This approach displayed the accuracy degree of 90% for species authentication. Overall, Bar-cas12a using trnL-designed gRNA offer a highly specific, sensitive, speed, and simple approach for plant species authentication. Therefore, the current method serves as a promising tool for species determination which is likely to be implemented for onsite testing.

SUBMITTER: Buddhachat K

PROVIDER: S-EPMC8536675 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundPhyllanthus amarus has been used widely in various traditional medicines to treat swelling, sores, jaundice, inflammatory diseases, kidney disorders, diabetes and viral hepatitis, while its pharmacological and biochemical mechanisms underlying its anti-inflammatory properties have not been well investigated. The present study was carried out to investigate the effects of 80% ethanolic extract of P. amarus on pro-inflammatory mediators release in nuclear factor-kappa B (NF-?B), mitogen activated protein kinase (MAPK) and phosphatidylinositol 3-kinase/Akt (PI3K-Akt) signaling activation in lipopolysaccharide (LPS)-induced U937 human macrophages.MethodsThe release of prostaglandin E2 (PGE2) and pro-inflammatory cytokines, tumor necrosis factor (TNF)-? and interleukin (IL)-1? in a culture supernatant was determined by ELISA. Determination of cyclooxygenase-2 (COX-2) protein and the activation of MAPKs molecules (JNK, ERK and p38 MAPK), NF-?B and Akt in LPS-induced U937 human macrophages were investigated by immunoblot technique. The relative gene expression levels of COX-2 and pro-inflammatory cytokines were measured by using qRT-PCR. The major metabolites of P. amarus were qualitatively and quantitatively analyzed in the extract by using validated reversed-phase high performance liquid chromatography (HPLC) methods.ResultsP. amarus extract significantly inhibited the production of pro-inflammatory mediators (TNF-?, IL-1?, PGE2) and COX-2 protein expression in LPS-induced U937 human macrophages. P. amarus-pretreatment also significantly downregulated the increased mRNA transcription of pro-inflammatory markers (TNF-?, IL-1?, and COX-2) in respective LPS-induced U937 macrophages. It downregulated the phosphorylation of NF-?B (p65), I?B?, and IKK?/? and restored the degradation of I?B?, and attenuated the expression of Akt, JNK, ERK, and p38 MAPKs phosphorylation in a dose-dependent manner. P. amarus extract also downregulated the expression of upstream signaling molecules, TLR4 and MyD88, which play major role in activation of NF-?B, MAPK and PI3K-Akt signaling pathways. The quantitative amounts of lignans, phyllanthin, hypophyllahtin and niranthin, and polyphenols, gallic acid, geraniin, corilagin, and ellagic acid in the extract were determined by HPLC analysis.ConclusionThe study revealed that P. amarus targeted the NF-?B, MAPK and PI3K-Akt signaling pathways to exert its anti- inflammatory effects by downregulating the prospective inflammatory signaling mediators.

Project description:Despite the growing interest in the miracle plant worldwide due to its numerous applications, the threats and the wild harvest of the species hamper its sustainable utilisation. Moreover, traditional knowledge so far documented on the species is limited to a narrow geographical coverage of its natural distribution range, which is West and Central Africa. This study analysed the use variation and knowledge acquisition pattern of the miracle plant among West African sociolinguistic groups and deciphered the drivers of populations' willingness and readiness to engage in cultivating the species. Semi-structured interviews were conducted with 510 respondents purposively selected from nine sociolinguistic groups in Benin and Ghana using the snowball sampling approach. Information was collected on respondents' socio-demographic profile, miracle plant ownership, plant parts used and preparation methods, knowledge of the species bioecology, perceived threats on the species, willingness to cultivate, maximum acreage to allocate to the species and maximum price to pay for a seedling. Descriptive statistics, generalized linear models, classification and regression tree models were used for data analysis. The miracle plant ownership mode depended on the age category. Sociolinguistic affiliation, level of schooling, migratory status and religion significantly affected the number of trees owned. We recorded 76 uses belonging to six use categories. The overall use-value of the miracle plant significantly varied according to the respondent sociolinguistic affiliation, main activity and religion. Men were the main source of knowledge and knowledge is mainly acquired along the family line. Knowledge related to food and social uses was mostly acquired from parents and people of the same generation, while magico-therapeutic and medicinal use-related knowledge were inherited from parents and grandparents. Sociolinguistic affiliation, awareness of taboos and market availability were the most important drivers of respondent willingness to cultivate the miracle plant. While the respondent's level of schooling and perception of plant growth rate determined the maximum acreage they were willing to allocate to the species in cultivation schemes, their main activity, sociolinguistic affiliation and knowledge of the species time to fruiting drove the maximum purchase price they were willing to offer for a seedling of the species. Our findings provide key information for the promotion of miracle plant cultivation in the study area.

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Bar-cas12a, a novel and rapid method for plant species authentication in case of Phyllanthus amarus Schumach. & Thonn.

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