Antibacterial and COX-2 Inhibitory Tetrahydrobisbenzylisoquinoline Alkaloids from the Philippine Medicinal Plant Phaeanthus ophthalmicus.

Phaeanthus ophthalmicus (Roxb. ex G.Don) J.Sinclair (previously known as P. ebracteolatus (Presl) Merr) is a Philippine medicinal plant occurring as evergreen shrub in the lowland forests of Luzon islands. It is used traditionally by Filipinos to treat bacterial conjunctivitis, ulcer and wound infections. Based on previous investigations where cyclooxygenase-2 (COX-2) functions as immune-linked factor in infectious sensitivities to bacterial pathogens by triggering pro-inflammatory immune-associated reactions, we investigated the antimicrobial and COX inhibitory activities of the extracts and tetrahydrobisbenzylisoquinoline alkaloids of P. ophthalmicus in vitro and in silico to validate its ethnomedicinal uses. Thus, the dichloromethane-methanol (DCM-MeOH) crude extract and alkaloid extracts exhibiting antibacterial activities against drug-resistant bacterial strains such as methicillin-resistance Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), Klebsiella pneumoniae + CRE and Pseudomonas aeruginosa + MBL afforded (+)-tetrandrine (1) and (+)-limacusine (2) as the major biologically active tetrahydrobisbenzylisoquinoline alkaloidal constituents after purification. Both tetrahydrobisbenzylisoquinoline alkaloids 1 and 2 showed broad spectrum antibacterial activity with strongest inhibition against the Gram-negative bacteria MßL-Pseudomonas aeruginosa Klebsiella pneumoniae + CRE. Interestingly, the alkaloid limacusine (2) showed selective inhibition against ovine COX-2 in vitro. These results were ascertained by molecular docking and molecular dynamics simulation experiments where alkaloid 2 showed strong affinity in the catalytic sites of Gram-negative bacterial enzymes P. aeruginosa elastase and K. pneumoniae KPC-2 carbapenemase (enzymes involved in infectivity mechanisms), and of ovine COX-2. Overall, our study provides credence on the ethnomedicinal use of the Philippine medicinal plant P. ophthalmicus as traditional plant-based adjuvant to treat bacterial conjunctivitis and other related infections. The antibacterial activities and selective COX-2 inhibition observed for limacusine (2) point to its role as the biologically active constituent of P. ophthalmicus. A limited number of drugs with COX-2 inhibitory properties like celecoxib also confer antibacterial activity. Thus, tetrahydrobisbenzyl alkaloids, especially 2, are promising pharmaceutical inspirations for developing treatments of bacterial/inflammation-related infections.

Quantitative ethnopharmacological documentation and molecular confirmation of medicinal plants used by the Manobo tribe of Agusan del Sur, Philippines.

BACKGROUND: The Philippines is renowned as one of the species-rich countries and culturally megadiverse in ethnicity around the globe. However, ethnopharmacological studies in the Philippines are still limited especially in the most numerous ethnic tribal populations in the southern part of the archipelago. This present study aims to document the traditional practices, medicinal plant use, and knowledge; to determine the relative importance, consensus, and the extent of all medicinal plants used; and to integrate molecular confirmation of uncertain species used by the Agusan Manobo in Mindanao, Philippines. METHODS: Quantitative ethnopharmacological data were obtained using semi-structured interviews, group discussions, field observations, and guided field walks with a total of 335 key informants comprising of tribal chieftains, traditional healers, community elders, and Manobo members of the community with their medicinal plant knowledge. The use-report (UR), use categories (UC), use value (UV), cultural importance value (CIV), and use diversity (UD) were quantified and correlated. Other indices using fidelity level (FL), informant consensus factors (ICF), and Jaccard's similarity index (JI) were also calculated. The key informants' medicinal plant use knowledge and practices were statistically analyzed using descriptive and inferential statistics. RESULTS: This study enumerated the ethnopharmacological use of 122 medicinal plant species, distributed among 108 genera and belonging to 51 families classified in 16 use categories. Integrative molecular approach confirmed 24 species with confusing species identity using multiple universal markers (ITS, matK, psbA-trnH, and trnL-F). There was strong agreement among the key informants regarding ethnopharmacological uses of plants, with ICF values ranging from 0.97 to 0.99, with the highest number of species (88) being used for the treatment of abnormal signs and symptoms (ASS). Seven species were reported with maximum fidelity level (100%) in seven use categories. The correlations of the five variables (UR, UC, UV, CIV, and UD) were significant (rs ≥ 0.69, p < 0.001), some being stronger than others. The degree of similarity of the three studied localities had JI ranged from 0.38 to 0.42, indicating species likeness among the tribal communities. Statistically, the medicinal plant knowledge among respondents was significantly different (p < 0.001) when grouped according to education, gender, social position, occupation, civil status, and age but not (p = 0.379) when grouped according to location. This study recorded the first quantitative ethnopharmacological documentation coupled with molecular confirmation of medicinal plants in Mindanao, Philippines, of which one medicinal plant species has never been studied pharmacologically to date. CONCLUSION: Documenting such traditional knowledge of medicinal plants and practices is highly essential for future management and conservation strategies of these plant genetic resources. This ethnopharmacological study will serve as a future reference not only for more systematic ethnopharmacological documentation but also for further pharmacological studies and drug discovery to improve public healthcare worldwide.

Anti-Amyloidogenic and Cyclooxygenase Inhibitory Activity of Guettarda speciosa.

Guettarda speciosa is known in traditional folk medicine for treating cough, cold, sore throat, fever, wounds, epilepsy, and headaches. To discover the scientific pharmacological potential of G. speciosa, we explore its anti-inflammatory, cytotoxicity, and inhibition of amyloid-beta (Aß) aggregation effects. Cyclooxygenase assay of the G. speciosa CHCl3 (GSC) extract and G. speciosa MeOH (GSM) extract are more selective to COX-1 inhibition with a 50% inhibitory concentration (IC50) of 3.56 µg/mL for the GSC extract and 4.98 µg/mL for the GSM extract. Neuroblastoma SH-SY5Y inhibition and thioflavin T assay amyloid-beta (Aß) aggregate inhibition of the GSM and GSC extracts showed their potential therapeutic effects against Alzheimer's disease. The putative compounds from the LC-MS analysis could be responsible for the observed activities. The results suggest that G. speciosa possesses anti-inflammatory and anti-neurodegenerative properties and a promising lead as a source of pharmacologically active compounds.

Standard reference material (SRM) DNA barcode library approach for authenticating Antidesma bunius (L.) Spreng. (bignay) derived herbal medicinal products.

In the Philippines, the herbal medicinal product (HMP) market is flourishing due to the abundance of pharmacologically important species, and the high level of ethnomedicinal knowledge still widely accepted by the public. As such, herbal products from Antidesma bunius (L.) Spreng., locally known as bignay, are popular as medicine for various ailments of the circulatory and digestive systems. Though efficacy is guaranteed, the authenticity of the marketed products is still in question as several other herbal plants can provide the said benefits. Similar morphology between wild species also hinders species identification and contributes confusion especially to the general consumer. The authenticity of the marketed HMPs was established by means of DNA barcoding techniques which offers quick and reliable species identification by means of (1) the Basic Local Alignment Search Tool (BLASTn) and (2) the establishment of the first Standard Reference Material (SRM) Herbal barcode library for Antidesma spp. A total of 56 gene accessions from matK-psbA-trnH-rbcL sequences of 9 wild Antidesma spp. comprised the SRM which was then used to confirm the identity of 11 randomly sampled bignay-derived HMPs. Following the BLASTn and the SRM (maximum likelihood tree reconstruction) criterion, the subjected sequences revealed that only three of the 11 HMPs were authentic A. bunius-derived products. The other eight HMPs contained substitutes that were either fillers or different herbal medicinal plant not indicated in the product labels. These results indicate that product safety should be reinforced with complete HMP authentication using traditional methods supported by molecular data.

Establishment of a standard reference material (SRM) herbal DNA barcode library of Vitex negundo L. (lagundi) for quality control measures.

The majority of the population in the Philippines relies on herbal products as their primary source for their healthcare needs. After the recognition of Vitex negundo L. (lagundi) as an important and effective alternative medicine for cough, sore throat, asthma and fever by the Philippine Department of Health (DOH), there was an increase in the production of lagundi-based herbal products in the form of teas, capsules and syrups. The efficiency of these products is greatly reliant on the use of authentic plant material, and to this day no standard protocol has been established to authenticate plant materials. DNA barcoding offers a quick and reliable species authentication tool, but its application to plant material has been less successful due to (1) lack of a standard DNA barcoding loci in plants and (2) poor DNA yield from powderised plant products. This study reports the successful application of DNA barcoding in the authentication of five V. negundo herbal products sold in the Philippines. Also, the first standard reference material (SRM) herbal library for the recognition of authentic V. negundo samples was established using 42 gene accessions of ITS, psbA-trnH and matK barcoding loci. Authentication of the herbal products utilised the SRM following the BLASTn and maximum-likelihood (ML) tree construction criterion. Barcode sequences were retrieved for ITS and psbA-trnH of all products tested and the results of the study revealed that only one out of five herbal products satisfied both BLASTn and ML criterion and was considered to contain authentic V. negundo. The results prompt the urgent need to utilise DNA barcoding in authenticating herbal products available in the Philippine market. Authentication of these products will secure consumer health by preventing the negative effects of adulteration, substitution and contamination.

Villarinol, a new alkenoyloxyalkenol derivative from the endemic Philippine Rubiaceae species Villaria odorata.

Villarinol (1), a new alkenoyloxy alkenol metabolite, has been isolated from the dichloromethane extract of Villaria odorata, an endemic Rubiaceae Philippine plant, along with the known compounds stigmasterol and 4-hydroxybenzaldehyde. The structure of 1 was elucidated on the basis of spectroscopic and spectrometric studies. The extracts of V. odorata exhibited moderate inhibition against Mycobacterium tuberculosis H37Rv, based on the colorimetric microplate alamar blue assay.