Traditional utilization of bamboo in the Central Siwalik region, Nepal.

Bamboo are the fastest growing perennial woody grasses that have versatile applications. Most of the local people inhabiting the riverine area of the Siwalik region of Nepal rely on bamboo products for economic benefits and medicinal uses. Our objective was to identify the diversity of bamboo species, their ethnomedicinal practices, and economic and ecological importance. Data were collected by direct observation, key informant interviews, participatory rural appraisal, inventory technique, focus group discussions, and a household survey using semi-structured and structured questionnaires. We recorded four genera and nine species of bamboo, of which eight species have been used for agriculture, five for medicine, four for construction, food, fodder, artifacts and religious purpose, three for river embankment, and two for ornamental purpose. As the local people in the study area were deprived of medical facilities, using traditional herbal medicine to cure various diseases was a common practice. The inhabitants responded that they use bamboo-based primary ethnomedicinal care even against snake and scorpion bites. Similarly, they use bamboo young culm for reducing body weight and control diabetes. The value of the informant consensus factor was found to be maximum for the bamboo against snake and scorpion bites (1.0) and minimum for weight loss (0.81). This study concludes that the traditional utilization of all kinds of bamboo in the region is vast despite their less diversity. The recorded bamboo species are used not only for food and fodder but also in preparing artifacts, soil nutrients restoration in the fallow land, construction materials for the rural people, river embankments, and religious and spiritual purposes. Therefore, if grown on a large scale, bamboo can provide sustainable benefits for the local users and ecological aspects. Bambusa tulda and Dendrocalamus strictus have a broad spectrum of pharmacological agents. Considering the multifaceted application of bamboo in the Siwalik area, it is worthwhile to encourage the local people to bamboo plantation, which would contribute to supplement their household requirements and be one of the alternative livelihood options.

Taxol and ß-tubulins from endophytic fungi isolated from the Himalayan Yew, Taxus wallichiana Zucc.

Paclitaxel, better known as the anticancer drug Taxol®, has been isolated from several plant species and has been shown to be produced by fungi, actinomycetes, and even bacteria isolated from marine macroalgae. Given its cytostatic effect, studies conducted in the 1990's showed that paclitaxel was toxic to many pathogenic fungi and oomycetes. Further studies led to the idea that the differences in paclitaxel sensitivity exhibited by different fungi were due to differences in the ß-tubulin protein sequence. With the recent isolation of endophytic fungi from the leaves and bark of the Himalayan Yew, Taxus wallichiana Zucc., and the availability of genomes from paclitaxel-producing fungi, we decided to further explore the idea that endophytic fungi isolated from Yews should be well-adapted to their environment by encoding ß-tubulin proteins that are insensitive to paclitaxel. Our results found evidence of episodic positive/diversifying selection at 10 sites (default p-value threshold of 0.1) in the ß-tubulin sequences, corresponding to codon positions 33, 55, 172, 218, 279, 335, 359, 362, 379, and 406. Four of these positions (i.e., 172, 279, 359, and 362) have been implicated in the binding of paclitaxel by ß-tubulin or formed part of the binding pocket. As expected, all the fungal endophytes grew in different media regardless of the paclitaxel concentration tested. Furthermore, our results also showed that Taxomyces andreanae CBS 279.92, the first fungus shown to produce paclitaxel, is a Basidiomycete fungus as the two beta tubulins encoded by the fungus clustered together with other Basidiomycete fungi.

Annulohypoxylon sp. strain MUS1, an endophytic fungus isolated from Taxus wallichiana Zucc., produces taxol and other bioactive metabolites.

The current study focuses on the isolation and in vitro characterization of bioactive metabolites produced by endophytic fungi isolated from the Himalayan yew (Taxus wallichiana Zucc.). The endophytic fungi were isolated on artificial media from inner tissues of bark and needles. Antimicrobial and antioxidant activity, along with total phenolic- and flavonoid-content assays were used in the evaluation of bioactivity of the fermented crude extracts. The ability of the endophytes to produce the anticancer compound Taxol was also analyzed using thin-layer chromatography (TLC) and reverse-phase high-performance liquid chromatography (RP-HPLC). A total of 16 fungal morphotypes were obtained from asymptomatic inner tissues of the bark and needles of T. wallichiana. Among the 16 isolates, the ethyl acetate (EA) fraction of isolate MUS1, showed antibacterial and antifungal activity against all test-pathogens used (Streptococcus faecalis ATCC 19433, Staphylococcus aureus ATCC 12600, Bacillus subtilis ATCC 6633, Escherichia coli ATCC 25922, Salmonella enterica ATCC 13076, Pseudomonas aeruginosa ATCC 27853, and Candida albicans). MUS1 showed significant inhibition against Pseudomonas aeruginosa ATCC 27853 (minimum inhibitory concentration (MIC): 250 µg/ml) and the pathogenic yeast, Candida albicans (MIC: 125 µg/ml). Antioxidant activity, total phenolic, and total flavonoid content as well as in vitro Taxol production were evaluated for EA fraction of isolate MUS1. Antioxidant activity was evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. At a concentration of 100 µg/ml, the % DPPH radical scavenging activity was 83.15 ± 0.40, 81.62 ± 0.11, and 62.36 ± 0.29, for ascorbic acid, butylated hydroxytoluene (BHT), and the EA fraction of MUS1, respectively. The DPPH-Half maximal inhibitory concentration (DPPH-IC50) value for the EA fraction was 81.52 ± 0.23 µg/ml, compared to BHT (62.87 ± 0.08 µg/ml) and ascorbic acid (56.15 ± 0.19 µg/ml). The total phenolic and flavonoid content in the EA fraction were 16.90 ± 0.075 µg gallic acid equivalent (GAE) and 11.59 ± 0.148 µg rutin equivalent (RE), per mg of dry crude extract, respectively. TLC and RP-HPLC analysis showed that the isolate MUS1 also produces Taxol (282.05 µg/l of fermentation broth). Isolate MUS1 was identified as Annulohypoxylon sp. by internal transcribed spacer (ITS) sequencing. Having the ability to produce antimicrobial and antioxidant metabolites, as well as the anticancer compound Taxol, makes Annulohypoxylon sp. strain MUS1, a promising candidate for further study of naturally occurring bioactive metabolites. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02693-z.

Evaluation of antimicrobial, antioxidant and cytotoxic properties of bioactive compounds produced from endophytic fungi of Himalayan yew ( Taxus wallichiana) in Nepal.

Background: Endophytic fungi are largely underexplored in the discovery of natural bioactive products though being rich sources of novel compounds with promising pharmaceutical potential. In this study, Taxus wallichiana, which has huge medicinal value, was investigated for its endophytic diversity and capability to produce bioactive secondary metabolites by analyzing antioxidant, antimicrobial and cytotoxic properties. Methods: The endophytes were identified by ITS-PCR using genomic DNA samples. The secondary metabolites were extracted by solvent extraction method using ethyl acetate. The antioxidant activity was analyzed by Thin Layer Chromatography, Total Phenol Content (TPC), Total Flavonoid Content (TFC) and DPPH assay, and the antimicrobial activity was analyzed by agar-well diffusion method. Brine shrimp lethality assay was used to analyze the cytotoxicity of the fungal extracts. Results: Out of 16 different Taxus trees sampled from different locations of Dhorpatan, 13 distinctive endophytic fungi were isolated and grouped into 9 different genera: Bjerkandera, Trichoderma, Preussia, Botrytis, Arthrinium, Alternaria, Cladosporium, Sporormiella and Daldinia. The ethyl acetate extracts isolated from three endophytic fungi: Alternaria alternata, Cladosporium cladosporioides and Alternaria brassicae showed significant TPC values of 204±6.144, 312.3±2.147 and 152.7±4.958µg GAE/mg of dry extract, respectively, and TFC values of 177.9±2.911, 644.1±4.202 and 96.38±3.851µg RE/mg of dry extract, respectively. Furthermore, these three extracts showed a dose dependent radical scavenging activity with IC 50 concentration of 22.85, 22.15 and 23.001 µg/ml, respectively. The extracts of C. cladosporioides and A. brassicae also showed promising antimicrobial activity against Escherichia coli, Staphylococcus aureus and Bacillus subtilis with a minimum inhibitory concentration of 250µg/ml for all bacteria. Both the samples showed cytotoxic property against shrimp nauplii with LC 50 of 104.2 and 125.9µg/ml, respectively. Conclusions: The crude fungal extracts obtained from endophytes: A. alternata, C. cladosporioides and A. brassicae upon purification and further identification of the bioactive compounds can be a fascinating source for novel pharmaceutical agents.