Approach to Study the Efficacy and Safety of New Complementary and Alternative Medicine Formulations: Lesson during COVID-19 from Sri Lanka.

COVID-19 affected Sri Lanka from early 2020, a time of considerable ignorance accompanied by wide media coverage of a devastating epidemic in Italy and Europe. Many were attracted to complementary and alternative medicine (CAM) or traditional medicine (TM) in this desperate situation. Several preparations were claimed to be effective against COVID-19 globally. Dammika Bandara Syrup© was one such preparation promoted for preventing and treating SARS-CoV-2 infection. It was based on bees' honey, pericarp and mace of Myristica fragrans (nutmeg), the seed of Foeniculum vulgare and fresh rhizome of Zingiber officinale, all believed to have anti-viral properties. Following an unpublished clinical study claiming efficacy, Dammika Bandara Syrup© gained wide media publicity and political patronage. The producer claimed of Goddess Kali revealing the formula added an anthropological, cultural, and religious complexity to the issue. The demand for the product increased rapidly as a debate raged both in public and in the parliament on utilizing such products in combating COVID-19. The Department of Ayurveda, which is statutorily responsible for regulating CAM/TM had to respond to the situation. The legislation to regulate such indigenous medicinal products was weak, and the crisis deepened as thousands converged to the production facility, defying mobility restrictions introduced to control COVID-19. This led to the Ministry of Health requesting academics to form a team and conduct a clinical trial to prove its efficacy. This paper outlines the process and issues faced during the regulatory approval for the trial in a polarized political environment. Some health professionals accused the researchers of bowing to political pressure and questioned the scientific justification for the trial. However, the team considered this as an opportunity to streamline a path for research into CAM/TM therapies in situations such as COVID-19. Several processes were identified and addressed, such as the provisional registration of CAM preparations, assessing the potential efficacy of a CAM product, confirmation of authenticity and safety, standardization and supervision of production respecting cultural identities, obtaining approval for human use, choice of comparators, and ethical issues. We believe the study has helped set standards and a benchmark for CAM and TM research in Sri Lanka.

Wood decay fungi show enhanced biodeterioration of low-density polyethylene in the absence of wood in culture media.

The involvement of microorganisms in low-density polyethylene (LDPE) degradation is widely studied across the globe. Even though soil, landfills, and garbage dumps are reported to be promising niches for such organisms, recently the involvement of wood decay fungi in polyethylene degradation is highlighted. In light of this, 50 fungal samples isolated from decaying hardwoods were assessed for their wood degradation ability and for their depolymerization enzymatic activities. For the LDPE deterioration assay, 22 fungal isolates having wood decay ability and de-polymerization enzymatic activities were selected. Fungal cultures with LDPE sheets (2 cm x 10 cm x 37.5 µm) were incubated in the presence and in the absence of wood as the carbon source (C) for 45 days. Degradation was measured by weight loss, changes in tensile properties, reduction in contact angle, changes of functional groups in Fourier-transform infrared spectroscopy, Scanning electron microscopic imaging, and CO2 evolution by strum test. Among the isolates incubated in the absence of wood, Phlebiopsis flavidoalba out-performed the other fungal species showing the highest percentage of weight reduction (23.68 ± 0.34%), and the lowest contact angle (64.28° ± 5.01). Biodegradation of LDPE by P. flavidoalba was further supported by 46.79 ± 0.67% of the mass loss, and 3.07 ± 0.13% of CO2 emission (mg/L) in the strum test. The most striking feature of the experiment was that all the isolates showed elevated degradation of LDPE in the absence of wood than that in the presence of wood. It is clear that in the absence of a preferred C source, wood decay fungi thrive to utilize any available C source (LDPE in this case) showing the metabolic adaptability of fungi to survive under stressful conditions. A potential mechanism for LDPE degradation is also proposed.

Cytotoxic Bioxanthracene and Macrocyclic Polyester from Endolichenic Fungus Talaromyces pinophilus: In-Vitro and In-Silico Analysis.

Lichens are used in folklore medicines across the globe for wound healing and to treat skin disorders and respiratory diseases. They are an intricate symbiosis between fungi and algae with the domination of fungal counterparts. Recent research studies pointed out that yeast is a third major partner in lichens. Endolichenic fungi (ELF) are also a part of this complex miniature ecosystem. The highly competitive environment of lichens compels ELF to produce toxic metabolites which are comparatively less explored for their chemical diversity and use. Here, we investigated 31 ELF isolated from 32 lichens found on mangrove plants at Puttalam Lagoon of Sri Lanka to find cytotoxic molecules by applying LC-UV-HRMS analysis and in vitro bioassays. The studies resulted in the identification of three potent cytotoxic molecules from endolichenic fungi Talaromyces pinophilus isolated from host lichen Porina tetracerae. The ethyl acetate extract of this fungus showed moderate cytotoxicity against the breast cancer cell line. Chemical characterization of ethyl acetate extract of T. pinophilus produced peniazaphilin B, 152G256α-1, and ES-242-3. The structures of these molecules were confirmed by NMR and MS data. We are reporting ES-242-3 for the first time from the genus Talaromyces and peniazaphilin B and 152G256α-1 from T. pinophilus. The isolated compounds were evaluated for their anticancer potential against breast, oral and cervical cancer cell lines. Compound 152G256α-1 showed potent cytotoxicity against oral cancer (CAL-27 cell line) with an IC50 value of 2.96 ± 0.17 µM while ES-242-3 showed the best activity against breast cancer (MCF-7 cell line) and cervical cancer (HeLa cell line) with IC50 value 14.08 ± 0.2 µM and 4.46 ± 0.05 µM respectively. An in-silico analysis was carried out to predict the mechanism of in-vitro activity, drug likeliness, and pharmacokinetic profile of the isolated compounds. The study confirms the potential of ELF T. pinophilus to produce diverse bioactive scaffolds and encourages the researchers to further explore the fungus and its metabolites with newer technologies to produce potent anticancer leads. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-021-00994-8.

Exploration of Potent Cytotoxic Molecules from Fungi in Recent Past to Discover Plausible Anticancer Scaffolds.

Fungi are known to produce diverse scaffolds possessing unique biological activities, however, to date, no molecule discovered from a fungal source has reached the market as an anti-cancer drug. Every year number of cytotoxic molecules of fungal origin are getting published and critical analysis of those compounds is necessary to identify the potent ones. A review mentioning the best cytotoxic fungal metabolites and their status in the drug development was published in 2014. In this report, we have included 176 cytotoxic molecules isolated from fungi after 2014 and categorized them according to their potencies such as IC50 values below 1 µM, 1-5 µM, and 5-10 µM. The emphasis was given to those 42 molecules which have shown IC50 less than 1 µM and discussed to a great extent. This review shall provide potent scaffolds of fungal origin which can be given priority in the development as a drug candidate for cancer therapeutics.

Apoferritin and Dps as drug delivery vehicles: Some selected examples in oncology.

BACKGROUND: The ideal nanoparticle should be able to encapsulate either pharmaceutical agents or imaging probes so that it could treat or image clinical tumours by targeting the cancer site efficiently. Further, it would be an added advantage if it demonstrates: small size, built in targeting, biocompatibility and biodegradability. Ferritin, which is an endogenous self-assembling protein, stores iron and plays a role in iron homeostasis. When iron atoms are removed apoferritin (AFt) is formed which consists of a hollow shell where it can be used to load guest molecules. Due to its unique architecture, AFt has been investigated as a versatile carrier for tumour theranostic applications. DNA-binding protein from starved cells (Dps), which also belongs to the ferritin family, is a protein found only in prokaryotes. It is used to store iron and protect chromosomes from oxidative damage; because of its architecture, Dps could also be used as a delivery vehicle. CONCLUSIONS: Both these nano particles are promising in the field of oncology, especially due to their stability, solubility and biocompatibility features. Further their exterior surface can be modified for better tumour-targeting ability. More studies, are warranted to determine the immunogenicity, biodistribution, and clearance from the body. GENERAL PERSPECTIVE: This review discusses a few selected examples of the remarkable in vitro and in vivo studies that have been carried out in the recent past with the use of AFt and Dps in targeting and delivery of various pharmaceutical agents, natural products and imaging probes in the field of oncology.

Antimicrobial Compounds Isolated from Endolichenic Fungi: A Review.

A lichen is a symbiotic relationship between a fungus and a photosynthetic organism, which is algae or cyanobacteria. Endolichenic fungi are a group of microfungi that resides asymptomatically within the thalli of lichens. Endolichenic fungi can be recognized as luxuriant metabolic artists that produce propitious bioactive secondary metabolites. More than any other time, there is a worldwide search for new antibiotics due to the alarming increase in microbial resistance against the currently available therapeutics. Even though a few antimicrobial compounds have been isolated from endolichenic fungi, most of them have moderate activities, implying the need for further structural optimizations. Recognizing this timely need and the significance of endolichenic fungi as a promising source of antimicrobial compounds, the activity, sources and the structures of 31 antibacterial compounds, 58 antifungal compounds, two antiviral compounds and one antiplasmodial (antimalarial) compound are summarized in this review. In addition, an overview of the common scaffolds and structural features leading to the corresponding antimicrobial properties is provided as an aid for future studies. The current challenges and major drawbacks of research related to endolichenic fungi and the remedies for them have been suggested.

Pheromone baited biopesticide for control of Leucinodes orbonalis Guenee in brinjal plant.

Leucinodes orbonalis Guenee, Brinjal fruit and shoot borer (BFSB), is the major pest on brinjal world wide. Larvae of this pest cause the damage; which at initial stages adversely affect the shoot growth, and in later stages diminish fruit quality. Spraying of insecticides is the main pest control method. This has been absolutely ineffective due to concealed habit of the larvae. Such a phenomenon apprehends to the development of insecticides resistance which demands continuously increasing doses. Manipulating the insect behavior using semiochemicals could be an opportunity for better management of insect pest. Application of BFSB sex pheromone components (E)-11-hexadecenyl acetate (E11-16: Ac) alone or in combination with (E)-11-hexadecen-1-ol (E11-16: OH) in traps can be used to suppress the populating growth. Goal-oriented interdisciplinary research on semiochemicals for sound administration of BFSB will depend on a better understanding of the key chemical ecology stimuli of relevance to the pest.

Medicinal plants commonly used against cancer in traditional medicine formulae in Sri Lanka.

Cancer is a global burden. In low- and middle-income countries around 70% of deaths are due to cancer. For a number of years natural products have been a good source of agents for combatting cancer and plants have played a huge role in anti-cancer product development. For many centuries, indigenous cultures around the world have used traditional herbal medicine to treat a myriad of diseases including cancer. In Sri Lanka, a number of plants have been reported to have anti-cancer properties and some of the commonly used plants are described in this review with an account of their compounds and modes of action. Only a small number of the plants in Sri Lanka have been tested for their bioactivity and more research is required to determine their medicinal activity with the aim of developing novel drugs to fight this disease.

Anticancer potential of natural products: a review focusing on Sri Lankan plants.

In the pharmaceutical industry, the expected surge in production of new therapeutic entities promised by technological advances, such as high-throughput screening, synthetic libraries and advances in molecular biology and genomics, has not materialized. The unique structural diversity of natural products continues to provide opportunities to discover novel compounds. Secondary metabolites, active components of natural products such as marine organisms, microbial organisms and terrestrial plants, are particularly exciting untapped resources for exploration as medicines. Sri Lanka is home to around 3700 plant species, half of which are considered as medicinal plants. Seventy per cent of the Sri Lankan population relies on this plant-based traditional medicine system for treating various illnesses such as tumors. As such these medicinal plant sources should be used to conquer terminal diseases and for prevention of diseases. Sri Lankan researchers have found several plant species that possess cytotoxic activity. This review summarizes the current information regarding the Sri Lankan plant materials that possess anticancer properties.

Molecular phylogeny and bioprospecting of Endolichenic Fungi (ELF) inhabiting in the lichens collected from a mangrove ecosystem in Sri Lanka.

Endolichenic fungi (ELF) are unexplored group of organisms as a source for the production of bioactive secondary metabolites with radical scavenging activity, antilipase and amylase inhibitory activities. Endolichenic fungi in lichens collected from mangrove or mangrove associated plants are least known for their fungal diversity and potential to produce bioactive compounds. A total of 171 ELF strains were isolated from the lichens collected from mangrove and mangrove associated plants in Puttalam lagoon. Out of this collection, 70 isolates were identified using rDNA-ITS region sequence homology to the GenBank accessions and a phylogenetic analysis was performed. Commonly isolated genera of ELF from lichens were Aspergillus, Byssochlamys, Talaromyces, Diaporthe, Phomopsis, Endomelanconiopsis, Schizophyllum, Cerrena, Trichoderma, Xylaria, Hypoxylon, Daldinia, Preussia, Sordaria, Neurospora, and Lasiodiplodia. In the present study, the effectiveness of ethyl acetate extracts of the ELF isolates were investigated against antioxidant activity, antilipase activity and α-amylase inhibition activity in in-vitro conditions. The results revealed that the extracts of Daldinia eschscholtzii, Diaporthe musigena and Sordaria sp. had the highest radical scavenging activity with smaller IC50 values (25 µg/mL to 31 µg/mL) compared to the IC50 values of BHT (76.50±1.47 µg/mL). Antilipase assay revealed that 13 extracts from ELF showed promising antiobesity activity ranged between 25% to 40%. Amylase inhibitory assay indicated that the test extracts do not contain antidiabetic secondary metabolites.

Phosphate fertilizer is a main source of arsenic in areas affected with chronic kidney disease of unknown etiology in Sri Lanka.

Chronic Kidney Disease of unknown etiology (CKDu) has escalated into an epidemic in North Central Province (NCP) and adjacent farming areas in the dry zone of Sri Lanka. Studies have shown that this special type of CKD is a toxic nephropathy and arsenic may play a causative role along with a number of other heavy metals. We investigated the hypothesis that chemical fertilizers and pesticide could be a source of arsenic. 226 samples of Fertilizers and 273 samples of pesticides were collected and analyzed using atomic absorption spectrometry and inductively coupled plasma mass spectrometry for arsenic and other heavy metals in two university laboratories. Almost all the agrochemicals available to the farmers in the study area are contaminated with arsenic. The highest amount was in triple super phosphate (TSP) with a mean value of 31 mg/kg. Also TSP is a rich source of other nephrotoxic metals including Cr, Co, Ni, Pb and V. Annually more than 0.1 million tons of TSP is imported to Sri Lanka containing approximately 2100 kg of arsenic. The next highest concentration was seen in the rock phosphate obtained from an open pit mine in NCP (8.56 mg/kg). Organic fertilizer contained very low amounts of arsenic. Arsenic contamination in pesticides varied from 0.18 mg/kg to 2.53 mg/kg although arsenic containing pesticides are banned in Sri Lanka. Glyphosate the most widely used pesticide in Sri Lanka contains average of 1.9 mg/kg arsenic. Findings suggest that agrochemicals especially phosphate fertilizers are a major source of inorganic arsenic in CKDu endemic areas. Organic fertilizer available in Sri Lanka is comparatively very low in arsenic and hence the farmers in CKDu endemic areas in Sri Lanka should be encouraged to minimize the use of imported chemical fertilizer and use organic fertilizers instead.

Drinking well water and occupational exposure to Herbicides is associated with chronic kidney disease, in Padavi-Sripura, Sri Lanka.

BACKGROUND: The chronic kidney disease of unknown etiology (CKDu) among paddy farmers in was first reported in 1994 and has now become most important public health issue in dry zone of Sri Lanka. The objective was to identify risk factors associated with the epidemic in an area with high prevalence. METHODS: A case control study was carried out in Padavi-Sripura hospital in Trincomalee district. CKDu patients were defined using health ministry criteria. All confirmed cases (N = 125) fulfilling the entry criteria were recruited to the study. Control selection (N = 180) was done from people visiting the hospital for CKDu screening. Socio-demographic and data related to usage of applying pesticides and fertilizers were studied. Drinking water was also analyzed using ICP-MS and ELISA to determine the levels of metals and glyphosate. RESULTS: Majority of patients were farmers (N = 107, 85.6%) and were educated up to 'Ordinary Level' (N = 92, 73.6%). We specifically analyzed for the effect modification of, farming by sex, which showed a significantly higher risk for male farmers with OR 4.69 (95% CI 1.06-20.69) in comparison to their female counterparts. In the multivariable analysis the highest risk for CKDu was observed among participants who drank well water (OR 2.52, 95% CI 1.12-5.70) and had history of drinking water from an abandoned well (OR 5.43, 95% CI 2.88-10.26) and spray glyphosate (OR 5.12, 95% CI 2.33-11.26) as a pesticide. Water analysis showed significantly higher amount of hardness, electrical conductivity and glyphosate levels in abandoned wells. In addition Ca, Mg, Ba, Sr, Fe, Ti, V and Sr were high in abandoned wells. Surface water from reservoirs in the endemic area also showed contamination with glyphosate but at a much lower level. Glyphosate was not seen in water samples in the Colombo district. CONCLUSION: The current study strongly favors the hypothesis that CKDu epidemic among farmers in dry zone of Sri Lanka is associated with, history of drinking water from a well that was abandoned. In addition, it is associated with spraying glyphosate and other pesticides in paddy fields. Farmers do not use personnel protective equipments and wears scanty clothing due to heat when spraying pesticides.

Sesquiterpene quinones and related metabolites from Phyllosticta spinarum, a fungal strain endophytic in Platycladus orientalis of the Sonoran Desert.

Five new metabolites, (+)-(5 S,10 S)-4'-hydroxymethylcyclozonarone ( 1), 3-ketotauranin ( 3), 3alpha-hydroxytauranin ( 4), 12-hydroxytauranin ( 5), and phyllospinarone ( 6), together with tauranin ( 2), were isolated from Phyllosticta spinarum, a fungal strain endophytic in Platycladus orientalis. The structures of the new compounds were determined on the basis of their 1D and 2D NMR spectroscopic data and chemical interconversions. All compounds were evaluated for inhibition of cell proliferation in a panel of five cancer cell lines, and only tauranin ( 2) showed activity. When tested in a flow cytometry-based assay, tauranin induced apoptosis in PC-3M and NIH 3T3 cell lines.

Uncovering biosynthetic potential of plant-associated fungi: effect of culture conditions on metabolite production by Paraphaeosphaeria quadriseptata and Chaetomium chiversii.

In an attempt to uncover the biosynthetic potential of plant-associated fungi, the effect of culture conditions on metabolite production by Paraphaeosphaeria quadriseptata and Chaetomium chiversii was investigated. These studies indicated that the production of the major metabolites by P. quadriseptata differ when the water used to make the media was changed from tap water to distilled water. It resulted in the isolation of six new secondary metabolites, cytosporones F-I ( 1- 4), quadriseptin A ( 5), and 5'-hydroxymonocillin III ( 6) together with monocillin III ( 7), a metabolite new to P. quadriseptata, in addition to monocillin I ( 8), a previously known metabolite from this organism. Aposphaerin B ( 9) encountered was suspected to be an artifact originating from cytosporone F ( 1). Incorporation of heavy metal ions to P. quadriseptata culture medium induced production of monocillin I ( 8) by this fungus. Cultivation of C. chiversii in liquid medium resulted in the isolation of chaetochromin A ( 12) as the major metabolite instead of radicicol ( 10), the major constituent of this organism when grown in a solid medium. Compounds 1- 7 and 12 were evaluated for their potential to inhibit Hsp90 and antiproliferative activity toward the cancer cell lines NCI-H460, MCF-7, and SF-268. Only compounds 6, 7, and 8 exhibited significant activity in both assays.

Heptaketides from Corynespora sp. inhabiting the cavern beard lichen, Usnea cavernosa: first report of metabolites of an endolichenic fungus.

Two new heptaketides, corynesporol (1) and 1-hydroxydehydroherbarin (2), along with herbarin (3) were isolated from an endolichenic fungal strain, Corynespora sp. BA-10763, occurring in the cavern beard lichen Usnea cavernosa. The structures of 1-3 were elucidated from their spectroscopic data. Aerial oxidation of corynesporol (1) yielded herbarin (3). Acetylation of 1 afforded the naphthalene derivative 4, whereas acetylation of 3 gave the corresponding naphthoquinone 6 and dehydroherbarin (5). All compounds were evaluated for their cytotoxicity and ability to inhibit migration of human metastatic breast and prostate cancer cell lines MDA-MB-231 and PC-3M, respectively. Dehydroherbarin (5) inhibited migration of both cell lines at concentrations not toxic to these cell lines. This is the first report of metabolites from an endolichenic fungus.