Abstract. Pikulthong V, Bhirompan S, Dechkla M, Mokkapan K, Boonman N, Wanna C, Machana K, Phakpaknam S. 2022. Local herbs for pain relief in the area of Tumbon Khao Hin Son, Chachoengsao, Thailand. Biodiversitas 23: 5012-5019. Local herbs have been used in Thailand for years. However, data collection on the use of local herbs in particular areas has not been thorough. This study aimed to survey local herbs treating pain relief in the area of Tumbon Khao Hin Son, Amphoe Phanom Sarakham, Chachoengsao, Thailand. Semi-structured interviews were used. Purposive sampling was used to determine the sample group. In the survey and study on botanical characteristics, from November 2019 - March 2020, 68 plants were identified and classified into 29 families. The local herbs which were mostly used for pain relief included 7 species from the Acanthaceae family, 6 types from Fabaceae, Lamiaceae, and 5 types from Zingiberaceae. The leaves of herbaceous plants had the highest frequency of use. A remedy, Ko-Klan, was found and prepared using a boiling method that could relieve pain. The surveyed herbs were used to treat 4 main symptoms which were body pain, stomachache, headache, and toothache. It was found that 36 species of herbs, or a majority of them, were used to directly relieve the pain. The interviews showed that 27% of the respondents had some home-grown herbs which were used for cooking and basic illness treatment. The age of the respondents was relevant to the use of herbs. The findings in this study will be the foundation for local herb studies which support and are aware of Thai local wisdom and knowledge as guidance to alter herbs for pharmaceutical purposes in the future.

The three-domain Cry4Aa toxin produced from Bacillus thuringiensis subsp. israelensis was previously shown to be much more toxic to Culexmosquito larvae than its closely related toxin—Cry4Ba. The interaction of these two individual toxins with target receptors on susceptible larval midgut cells is likely to be the critical determinant in their differential toxicity. Here, two full-length membrane-bound alkaline phosphatase (mALP) isoforms from Culex quinquefasciatus larvae, Cq-mALP1263and Cq-mALP1264, predicted to be GPI-linked was cloned and functionally expressed inSpodoptera frugiperda(Sf9) cells as 57- and 61-kDa membrane-bound proteins, respectively. Bioinformatics analysis disclosed that bothCq-mALP isoforms share significant sequence similarity to Aedes aegypti-mALP—a Cry4Ba toxin receptor.In cytotoxicity assays, Sf9 cells expressing Cq-mALP1264, but not Cq-mALP1263, showed remarkably greater susceptibility to Cry4Aa than Cry4Ba, while immunolocalization studies revealed that both toxins were capable of binding to each Cq-mALPexpressed on the cell membrane surface. Molecular docking of the Cq-mALP1264-modeled structure with individual Cry4 toxins revealed that Cry4Aa could bind to Cq-mALP1264 primarily through particular residues on three surface-exposed loops in the receptor-binding domain—DII, including Thr512, Tyr513 and Lys514 in the b10-b11loop. Dissimilarly, Cry4Ba appeared to utilize only certain residues in its C-terminal domain—DIII to interact with such a Culex counterpart receptor. Ala-substitutions of selected b10-b11loop residues (T512A, Y513A and K514A) revealed that only the K514A mutant displayed a drastic decrease in biotoxicity against C. quinquefasciatuslarvae. Further substitution of Lys514 with Asp (K514D) revealed a further decrease in larval toxicity. Furthermore, in silico calculation of the binding affinity change (ΔΔGbind) in Cry4Aa-Cq-mALP1264 interactions upon these single-substitutions revealed that the K514D mutation displayed the largest ΔΔGbind value as compared to three other mutations, signifying an adverse impact of a negative charge at this critical receptor-binding position.Altogether, our present study has disclosed that these two related-Cry4 mosquito-active toxins conceivably exploited different domains in functional binding to the same Culex membrane-bound ALP isoform—Cq-mALP1264 for mediating differential toxicity against Culex target larvae.

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