Isolation and Characterization of Novel Huperzine-Producing Endophytic Fungi from Lycopodiaceae Species.

Huperzine A (HupA) is an important drug for treating Alzheimer's disease (AD) and is primarily extracted from the Huperzia serrata (Lycopodiaceae). Failures in the chemical synthesis of Hup and in vitro culture have put H. serrata in danger of extinction, and there is a need for an extensive investigation of Hup from alternative perspectives. The aim of this study is to identify endophytic fungi that produce high Hup or simultaneously produce many types of Hup and have high genetic stability derived from other Lycopodiaceae species as a source of materials for natural Hup production. In this work, Hup-producing endophytic fungi were isolated from three species: Lycopodium clavatum, Phlegmariurus squarrosus, and P. phlegmaria. Of these, L. clavatum and P. squarrosus were confirmed as novel sources of Hup-producing fungi. Based on morphological characteristics and nuclear ribosomal DNA ITS sequences, four endophytic fungi Colletotrichum siamense THG1-17, Epicoccum sorghinum THG01-18, Phoma sp. TKH3-2, and Phyllosticta sp. THG2-27 were firstly isolated from these Lycopodiaceae plants, which were capable of simultaneously producing both HupA and HupB, as evidenced by high-performance liquid chromatography analysis. The four strains showed stability in Hup yield over 50 generations of culture with an in vitro storage period of 3 months. These isolated fungi will provide a new source of materials for further research to develop drugs containing HupA as well as HupB for AD treatment in the future.

The apoptotic effect of the Lycopodium clavatum extracts on MCF-7 human breast cancer cells.

Breast cancer is a significant health problem worldwide, and the search for effective treatments is critical. Side effects of cancer treatments such as surgery, radiotherapy, and chemotherapy reduce the patient's standard of living. Recently, natural compounds from plants have gained attention as potential anticancer agents due to their safety, low toxicity, and potential efficacy. Lycopodium Clavatum (LC) is an herb abundant in tropical regions and Europe and is known for its various medicinal properties. In this study, we investigated the cytotoxic and apoptotic effects of LC Water Extract (LC-WE) and LC Ethanol Extract (LC-EE) plant extracts on MCF-7 human breast cancer cells. Our results showed that LC treatment led to a dose and time-dependent cytotoxic effect on MCF-7 cells, indicating its potential as an anticancer agent against human breast cancer. Additionally, we observed that LC treatment activated apoptosis-related proteins, including BAX, Caspase-3, and Caspase-9. These results suggest that LC may induce apoptosis as a mechanism underlying its cytotoxic effect on MCF-7 human breast cancer cells. Previous studies have shown the anti-cancer potential of LC against different types of cancer. However, the anti-cancer effect of LC on human breast cancer cells has not been investigated to date. Therefore, our study provides novel insights into the potential of LC as an anti-cancer agent against breast cancer. Overall, our results highlight the potential of LC as a promising natural compound for breast cancer treatment.

Alergias alimentares na infância: uma abordagem homeopática / Food allergies in childhood: a homeopathic approach

As alergias alimentares são condições muito frequentes dentre as queixas pediátricas, sendo a alergia às proteínas do leite de vaca (APLV) o tipo mais comum na infância. Apesar de sua prevalência, há controvérsias na literatura sobre o diagnóstico e o tratamento convencional. A Homeopatia oferece uma opção valiosa para a abordagem dessas patologias, uma vez que age conforme a totalidade sintomática característica de cada caso, isto é, de forma individualizada. O presente relato é sobre uma criança, do sexo masculino, com diagnóstico prévio de APLV, apresentando crises respiratórias frequentes e uso recorrente de antibióticos, além de estar submetido a uma dieta restrita. A mãe buscou tratamento homeopático como alternativa à terapêutica vigente, uma vez que os resultados obtidos até então eram pouco satisfatórios. O caso foi conduzido segundo os preceitos da homeopatia clássica hahnemanniana, com obtenção de um medicamento único segundo o método de repertorização. Optou-se pelo tratamento com o medicamento Lycopodium clavatum, considerado o mais semelhante segundo a individualidade do paciente. A criança foi acompanhada por pouco mais de um ano, observando-se melhora clínica importante da queixa inicial, além de mais qualidade de vida à criança e à família.

Food allergies are very common conditions amongst pediatric complaints, and cow's milk protein allergy (CMPA) is the most common kind in childhood. Despite its prevalence, there are controversies in the literature about diagnosis and conventional treatments. Homeopathy offers a valuable option for approaching these pathologies, since it acts according to the characteristic symptomatic totality of each case, that is, in an individualized way. This report is about a child, male, with a previous diagnosis of CMPA, with frequent respiratory crises and recurrent use of antibiotics, in addition to being submitted to a restricted diet. His mother looked for homeopathic treatment as an alternative to current therapy, since the results obtained so far were unsatisfactory. The case was conducted according to the precepts of classical Hahnemannian homeopathy, obtaining a single medicine according to the repertorization method. We opted for treatment with Lycopodium clavatum, considered the most similar according to the individuality of the patient. The child was followed up for a little over a year, and significant clinical improvement of the initial complaint was observed, in addition to better quality of life for the child and family.

Cytotoxic and nitric oxide inhibitory activities of triterpenoids from Lycopodium clavatum L.

Using combined chromatographic separation techniques, three new triterpenoids named lycomclavatols A-C (1-3), a new natural product, methyl lycernuate-A (4), as well as seven known compounds (5-11), were isolated from the methanol extract of the whole plants of Lycopodium clavatum. Their chemical structures were established based on 1 D/2D NMR and HR-ESI-MS spectroscopic analyses. Among the isolates, compound 1 exhibited inhibitory activity on NO production in LPS-stimulated BV2 cells (IC50 = 36.0 µM). In addition, 1 was cytotoxic against both HepG2 and A549 cancer cell lines, with IC50 values of 40.7 and 87.0 µM, respectively. Compounds 10 and 11 showed cytotoxicity on only HepG2 and A549 cells, with IC50 values of 91.2 and 57.6 µM, respectively. Our results contribute to understanding more the secondary metabolites produced by L. clavatum and provide a scientific rationale for further investigations of anti-inflammatory and anticancer effects for this valuable medicinal plant.

Fractionation of Lycopodiaceae Alkaloids and Evaluation of Their Anticholinesterase and Cytotoxic Activities.

In view of the abundant evidence that Lycopodiaceae alkaloids, including the well-known huperzine A (HupA), are among the potent acetylcholinesterase (AChE) inhibitors, an attempt was made to search for new compounds responsible for this property. For this purpose, three plant species belonging to the Lycopodiaceae family, commonly found in the Euro-Asia region, were subjected to the isolation of bioactive compounds, their identification and subsequent evaluation of their anticholinesterase and cytotoxic activities. Methanolic extracts of two Lycopodium and one Hupezia species were obtained via optimized pressurized liquid extraction (PLE) and then pre-purified using innovative gradient vacuum liquid chromatography (gVLC). For the first time, three sorbents of different porosity packed in polypropylene cartridges and mobile phase systems of different polarity were used to elute the target compounds. This technique proved to be a rapid tool for the obtainment of alkaloid fractions and allowed one to select the appropriate process conditions to yield potent AChE inhibitors in each of the species studied. More than 100 collected fractions were analyzed via HPLC/ESI-QTOF-MS, which enabled one to detect more than 50 compounds, including several new ones previously unreported. Some of them were present in high purity fractions (60-90% of the established purity). TLC bioautography assays proved that the analyzed species are rich sources of AChE inhibitors, but H. selago showed the highest anti-AChE activity. Additionally, the modified silanized silica gel sorbent used allowed one to isolate L. clavatum alkaloids more efficiently using an aqueous reversed-phase solvent system. Furthermore, the tested extracts from the three plant extracts were found to be safe, as they did not exhibit cytotoxicity to skin fibroblasts.

Optimization of Pressurized Liquid Extraction of Lycopodiaceae Alkaloids Obtained from Two Lycopodium Species.

Alkaloids of the Lycopodiaceae family are of great interest to researchers due to their numerous properties and wide applications in medicine. They play a very important role mainly due to their potent antioxidant, antidepressant effects and a reversible ability to inhibit acetylcholinesterase (AChE) enzyme activity. This property is of immense importance due to the growing problem of an increasing number of patients with neurodegenerative diseases in developed countries and a lack of effective and efficient treatment for them. Numerous studies have shown that Lycopodiaceae alkaloids are a rich source of AChE inhibitors. In the obtaining of new therapeutic phytochemicals from plant material, the extraction process and its efficiency is crucial. Therefore, the aim of this work was to optimize the conditions of modern PLE to obtain bioactive alkaloids from two Lycopodium species: L. clavatum L. and L. annotinum L. Five different solvents of different polarity were used for prepared plant extracts in order to compare the alkaloid content in and thereby effectiveness of the entire extraction. PLE parameters were used based on multiple studies conducted that gave the highest alkaloids recovery. Crude extracts were purified using solid-phase extraction (SPE) on Oasis HLB cartridge and examined by HPLC/ESI-QTOF-MS of the highly abundant alkaloids. To the best of our knowledge, this is the first time such high recoveries have been obtained for known Lycopodiaceae alkaloids. The best extraction results of alkaloid-lycopodine were detected in the dichloromethane extract from L. clavatum, where the yield exceeded 45%. The high recovery of annotinine above 40% presented in L. annotinum was noticed in dichloromethane and ethyl acetate extracts. Moreover, chromatograms were obtained with all isolated alkaloids and the best separation and quality of the bands in methanolic extracts. Interestingly, no alkaloid amounts were detected in cyclohexane extracts belonging to the non-polar solvent. These results could be helpful for understanding and optimizing the best conditions for isolating potent AChE inhibitors.

Serratane triterpenoids isolated from Lycopodium clavatum by bioactivity-guided fractionation attenuate the production of inflammatory mediators.

Lycopodium clavatum has been used in traditional medicine for the treatment of kidney disorders, rheumatic arthritis, cystitis, and gastritis. We isolated a new serratane triterpenoid (2), and five known triterpenoids (1, 3-6) from the ethyl acetate fraction of L. clavatum by bioactivity-guided fractionation based on their suppression of inflammatory cytokines. Two different cell lines, RAW 264.7 and HT-29 were used to determine the anti-inflammatory activity of the isolated compounds. Among them, compounds 1, 2, 4, and 5 significantly inhibited the production of lipopolysaccharide (LPS)-induced NO in macrophages. Compounds 1, 2, 4, and 5 reduced inducible nitric oxide (iNOS) expression in RAW 264.7 cells and compounds 1 and 6 downregulated COX-2, which correlated with the reduced expression of PGE2. Compounds 1, 2, 4, and 5 downregulated pro-inflammatory cytokines, such as interleukin-1ß (IL-1ß) in macrophages, and additionally suppressed the levels of IL-8 in HT-29 cells. To determine the signaling pathways involved in the suppression of NO production by these compounds, we investigated ERK1/2 and nuclear factor-kappa B (NF-κB) expression by western blot analysis. We observed that these compounds downregulated the expression of LPS-induced NF-κB and pERK 1/2 in RAW 264.7 cells. Our results demonstrate that serratane triterpenoids isolated from L. clavatum may be used as potential candidates for treating inflammatory bowel disease (IBD) due to their anti-inflammatory effects.

Relato de caso de rinossinusite em criança tratado com lycopodium clavatum / Case report of rhinosinusitis in a child treated with lycopodium clavatum

As rinites e sinusites são processos inflamatórios da mucosa nasal e sinusal. Nas crianças é comum denominarmos rinossinusites devido à frequência com que ocorrem associados os dois processos inflamatórios. O caso em descrição é de uma criança de três anos, portadora de rinossinusite crônica, e histórico de insuficiência respiratória noturna, com obstrução nasal, respiraçao bucal com ruído acentuado, levando a criança a acordar frequentemente. Fez vários tratamentos da medicina hegemônica alopática, sem bons resultados, e com indicação para adenoamigdalectomia. Após consulta homeopática, tendo sido feito repertorização, utilizando a técnica simples, sem sintoma diretor, usando o repertório digital Dr. Ariovaldo; foram selecionados sete sintomas. Chegou-se ao medicamento Lycopodium clavatum. Após o início do tratamento homeopático houve melhora surpreendente no quadro clínico. (AU)

Antibacterial Activity of Homeopathic Medications Lycopodium clavatum and Arsenicum album Against Periodontal Bacteria / Evaluación antibacteriana de los medicamentos homeopáticos sobre bacterias periodontales

Abstract There are several controversies regarding the efficacy of homeopathic substances; however, these remedies are used in many countries for the treatment of various pathological conditions. The purpose of this study was to evaluate the in vitro antibacterial activity of two homeopathic tinctures Arsenicum album (mineral extract) and Lycopodium clavatum (plant extract) on the periodontal bacteria Actinomyces israelii, Streptococcus sanguinis, Prevotella intermedia, Aggregatibacter actinomycetemcomitans and Phorphyromonas gingivalis (P. gingivalis). Materials and methods: Equal numbers of bacteria were seeded on agar plates containing enriched media with the homeopathic solutions at 1dH and 1cH dilutions. After 7 days of incubation under anaerobic conditions, colony forming units (CFUs) were counted. The antibacterial effect was calculated based on the total number of CFUs observed on non-tincture containing agar, and on the tincture containing plates. Results: No visible growth of any of the strains was observed on the plates containing Arsenicum album at any of the dilutions tested. In contrast, when Lycopodium clavatum at 1cH dilution was tested, only P. gingivalis was susceptible to this compound. Conclusions: The results suggest that the mineral extract tincture had a greater antibacterial activity than the plant extract tincture, also Lycopodium clavatum preparation could be an effective inhibitor of periodontal pathogens bacteria such as P. gingivalis.

Resumen Se necesita un mayor número de estudios in vitro e in vivo para validar estos resultados.

Increased of the hepatocytes and splenocytes apoptosis accompanies clinical improvement and higher survival in mice infected with Trypanosoma cruzi and treated with highly diluted Lycopodium clavatum.

Recent evidence includes apoptosis as a defense against Trypanosoma cruzi infection, which promotes an immune response in the host induced by T cells, type 1, 2 and 17. Currently, there is no medicine completely preventing the progression of this disease. We investigated the immunological and apoptotic effects, morbidity and survival of mice infected with T. cruzi and treated with dynamized homeopathic compounds 13c: Kalium causticum (GCaus), Conium maculatum, (GCon), Lycopodium clavatum (GLy) and 7% alcohol solution (control, vehicle compounds, GCI). There was significant difference in the increase of apoptosis in the treated groups, compared with GCI, which might indicate action of the compounds in these cells. Infected animals treated with Lycopodium clavatum presented better performance compared with other groups. GLy showed a higher amount of hepatocytes and splenocytes undergoing apoptosis, higher number of apoptotic bodies in the liver, predominance of Th1 response, increased TNF-α and decreased IL-6, higher survival, lower morbidity, higher water consumption, body temperature, tendency to higher feed intake and weight gain compared with GCI. Conium maculatum had worse results with increased Th2 response with increased IL-4, worsening of the infection with early mortality of the animals. Together, these data suggest that highly diluted medicines modulate the immune response and apoptosis, affecting the morbidity of animals infected with a highly virulent strain of T. cruzi, being able to minimize the course of infection, providing more alternative approaches in the treatment of Chagas disease.

Predominance of Th1 response, increase of megakaryocytes and Kupffer cells are related to survival in Trypanosoma cruzi infected mice treated with Lycopodium clavatum.

We investigated the number of megakaryocytes, Kupffer cells and ratios of Th1/Th2 and Th1/Th17 cytokines in survival of mice infected with Y strain of Trypanosoma cruzi and treated with Lycopodium clavatum. In a blind, randomized and controlled assay, Swiss male mice, 8weeks-old, infected with 1400 trypomastigotes (Y strain) were divided into groups and treated with: GLy - Lycopodium clavatum dynamization13c and GCI - alcohol solution 7° GL (vehicle medicine). The treatment was offered two days before infection and on the 2nd, 4th and 6th days after infection, overnight (1mL/100mL) and ad libitum. Parameters assessed were: survival rate, number of megakaryocytes and Kupffer cells, cytokines dosage (TNF-α, IFN-γ, IL-2, IL-4, IL-6, IL-10, IL-17), Th1/Th2 and Th1/Th17 ratios. The increase in megakaryocytes, Kupffer cells, predominance of Th1 response, with increased TNF-α, IL-10, TNF-α/IL-4, TNF-α/IL-17 and decreased IL-6 IL-6/IL-4, are related to increased survival in mice infected with T. cruzi and treated with Lycopodium clavatum 13c. This result demonstrates the possibility of an alternative approach for the treatment of Chagas disease with dynamized drugs.

Prevalence and likelihood ratio of symptoms in patients with good therapeutic response to Lycopodium clavatum. A retrospective study.

BACKGROUND: Assessment of the likelihood ratio (LR) of symptoms has been proposed as a rational means for detecting indicators to homeopathic medicines. AIMS: To investigate the prevalence and LR of symptoms commonly attributed to the homeopathic medicine Lycopodium clavatum (Lyc). Secondarily, to answer the question if experienced homeopaths could intuitively infer which the main symptoms of this medicine are. METHODS: The presence of 35 selected symptoms, prescribed medicines and therapeutic response were assessed retrospectively. The symptoms' prevalence in the Lyc responding population and the LR of the symptoms compared to their prevalence in the remainder of the population were calculated. RESULTS: Two hundred and two Lyc and 550 non Lyc cases (total 752) were included for analysis. Twenty-two symptoms were confirmed as pertaining to Lyc's semiology (prevalence %; LR): contemptuous (3.3; 6.7), urinary stones history (2.7; 5.4), egotism (5.6; 3.6), dictatorial (33.3; 3.4), haughty (8.7; 3.3), sleeps on abdomen (3.3; 3.3), intolerance to clothing in abdomen (12.0; 3.1), reproaches (4.0; 3.0), helplessness (24.0; 2.7), fear of failure (10.7; 2.6), irritability on waking in the morning (16.7; 2.5), constipation alternating with diarrhea (8.7; 2.5), intolerant to contradiction (59.3; 2.3), want of self confidence (30.0; 2.4), abdominal distension after eating (23.3; 2.1); ailments from anticipation (32.0; 1.9), irritability before menses (23.3; 1.8), conscientious (26.0; 1.6), desire of sweets (52.0; 1.6), desire of chocolate (16.7; 1.6), lack of vital heat (41.3; 1.3), and flatterer (1.3; ∞). Surveyed homeopaths' intuitive inferences correlated well with symptoms' prevalence but not with their LR. CONCLUSIONS: Lycopodium's main symptoms are well known by homeopaths, but their knowledge correlates well with the symptoms' prevalence and not with their LR. Retrospective assessment of prevalence and LR of symptoms in good responders might be a means for better selection of symptoms for prospective studies.

Effect of homeopathic Lycopodium clavatum on memory functions and cerebral blood flow in memory-impaired rats.

BACKGROUND: Lycopodium clavatum (Lyc) is a widely used homeopathic medicine for the liver, urinary and digestive disorders. Recently, acetyl cholinesterase (AchE) inhibitory activity has been found in Lyc alkaloid extract, which could be beneficial in dementia disorder. However, the effect of Lyc has not yet been explored in animal model of memory impairment and on cerebral blood flow. AIM: The present study was planned to explore the effect of Lyc on learning and memory function and cerebral blood flow (CBF) in intracerebroventricularly (ICV) administered streptozotocin (STZ) induced memory impairment in rats. MATERIALS AND METHODS: Memory deficit was induced by ICV administration of STZ (3 mg/kg) in rats on 1st and 3rd day. Male SD rats were treated with Lyc Mother Tincture (MT) 30, 200 and 1000 for 17 days. Learning and memory was evaluated by Morris water maze test on 14th, 15th and 16th day. CBF was measured by Laser Doppler flow meter on 17th day. RESULTS: STZ (ICV) treated rats showed impairment in learning and memory along with reduced CBF. Lyc MT and 200 showed improvement in learning and memory. There was increased CBF in STZ (ICV) treated rats at all the potencies of Lyc studied. CONCLUSION: The above study suggests that Lyc may be used as a drug of choice in condition of memory impairment due to its beneficial effect on CBF.