Phytochemicals as Antimicrobials: Prospecting Himalayan Medicinal Plants as Source of Alternate Medicine to Combat Antimicrobial Resistance.

Among all available antimicrobials, antibiotics hold a prime position in the treatment of infectious diseases. However, the emergence of antimicrobial resistance (AMR) has posed a serious threat to the effectiveness of antibiotics, resulting in increased morbidity, mortality, and escalation in healthcare costs causing a global health crisis. The overuse and misuse of antibiotics in global healthcare setups have accelerated the development and spread of AMR, leading to the emergence of multidrug-resistant (MDR) pathogens, which further limits treatment options. This creates a critical need to explore alternative approaches to combat bacterial infections. Phytochemicals have gained attention as a potential source of alternative medicine to address the challenge of AMR. Phytochemicals are structurally and functionally diverse and have multitarget antimicrobial effects, disrupting essential cellular activities. Given the promising results of plant-based antimicrobials, coupled with the slow discovery of novel antibiotics, it has become highly imperative to explore the vast repository of phytocompounds to overcome the looming catastrophe of AMR. This review summarizes the emergence of AMR towards existing antibiotics and potent phytochemicals having antimicrobial activities, along with a comprehensive overview of 123 Himalayan medicinal plants reported to possess antimicrobial phytocompounds, thus compiling the existing information that will help researchers in the exploration of phytochemicals to combat AMR.

Clinostomum complanatum: Anthelmintic potential of curcumin on the infective progenetic metacercarial stage.

The emerging resistance against commonly used antiparasitic drugs has driven investigators to explore alternative approaches using plant-derived active ingredients. These compounds have been tested for antiviral, antibacterial, and anthelmintic properties, particularly against adult worms. However, their effects on larval forms have been neglected. Curcumin is a polyphenol that is a significant constituent of the rhizome of Curcuma longa and possesses various biological activities, including antioxidant, anti-inflammatory, anti-infectious, and anti-carcinogenic. In the present study, the anthelmintic potential of curcumin was tested in vitro for its efficacy against the zoonotically important larval form, the progenetic metacercariae of Clinostomum complanatum, which were procured from the forage fish, Trichogaster fasciatus. Curcumin produced time and concentration-dependent inhibition in the motility of treated metacercarial worms, with the maximum inhibition of motility reported at 60 µM along with a significant increase of (36-92%) in ROS and (57-112%) in GSH levels at the end of a period of 6 h. In contrast, curcumin at the highest concentration significantly inhibited the activities of the antioxidant and detoxification enzymes SOD (36%) and GST (16%), respectively, in addition to altering the polypeptide profile and inhibiting cysteine proteases. The tegumental surface appeared to be highly disrupted in curcumin-treated worms, exhibiting severe blebbing, shearing of the tegument, and spine erosion. Such changes would affect the tegumental functions and survival of worms in the hostile microenvironment. This would render worms more susceptible to host-mediated rejection responses. Based on the results of the present study, it is inferred that C. complanatum could serve as an excellent model for screening novel anthelmintic drugs against larval trematodes of great economic significance. Furthermore, we conclude that curcumin could be exploited as an excellent phytotherapeutic agent against the virulent larval form under investigation.

Oxidative status and changes in the adenosine deaminase activity in experimental host infected with tropical liver fluke, Fasciola gigantica.

Fine tuning of the metabolic, physiological and immunological cues along with interplay between the biomolecules of the host and the parasite could be responsible for the successful establishment of parasitic infections. The present investigation was aimed at evaluating the oxidative status and the level of adenosine deaminase (ADA) in the serum and liver of rabbits experimentally infected with Fasciola gigantica. A significant increase in level of ROS, MDA and 4-HNE along with a decline in the SOD, CAT, GR and GST activity was evident in rabbits experimentally infected with Fasciola gigantica. However, there was an increase in the GPX activity in the sera of infected rabbits. The increased GPX activity and decreased GR activity would have resulted in the depletion of GSH, a key non-enzymatic antioxidant, in the infected animals. The level of GSSG was also found to be higher in the sera and liver tissues of the infected rabbits along with a decline in the GSH/GSSG ratio, indicating a high level of oxidative stress in the infected animals, which also showed a significant increase in the activity of the marker enzymes of liver pathology, AST and ALT. Further, a significant inhibition of the adenosine deaminase (ADA) activity in the infected rabbits was accompanied with the reduction in the level of pro-inflammatory cytokine, IL-6 while the anti-inflammatory cytokine, IL-4 level was significantly elevated. In conclusion, the F. gigantica induced significant oxidative stress as evident from the increased levels of ROS and lipid peroxidation along with the disruption of antioxidant and detoxification cascade ultimately lead to pathogenic and inflammatory responses in the experimental host. Whereas, the altered ADA activity could modulate the host's immune responses toward Th-2 type and would facilitate the successful establishment of flukes within their host, thus indicating that ADA could be exploited as a target for the development of novel anthelmintic drugs against fasciolosis.

Glutathione-S-transferase: an important diagnostic antigen of liver amphistome Gigantocotyle explanatum, infecting the Indian water buffalo.

The foodborne trematodiases pose a significant health problem to the animals as well as the human population living in close proximities with the livestock and are still considered as the neglected tropical diseases by the World Health Organisation. The digenetic trematode, Gigantocotyle explanatum infecting the liver of Indian water buffalo, Bubalus bubalis, has been identified as one of the most common helminth parasite responsible for the disease, amphistomosis, in livestock. Despite huge abattoir prevalence, the epidemiological data and the actual economic losses incurred due to this parasite alone are yet to be established probably due to the limitations of routinely used diagnostic tests. The gold standard for the confirmation of such infections under field conditions is still the fecal egg count (FEC). However, the poor sensitivity and cumbersome nature of these tests necessitates the development of a more sensitive, reliable and easy to perform workflow/method. Immunological diagnosis of helminthic infections is still considered as an alternative to the FEC. Therefore, efforts have been made to utilize glutathione-S-transferase (GST), a vitally significant molecule of the adult G. explanatum, for the serodiagnosis of amphistomosis under both laboratory and field conditions. The GST antigen was first affinity purified from the somatic extract of the adult worms since its highest level was recorded in the somatic extracts followed by eggs and the excretory/secretory products. A five-fold affinity purified native GST antigen of about 25 kDa was found to be highly immunogenic as evident from high titre (1:25,600) of the polyclonal antibodies raised in the rabbits. The immunoblotting results revealed differential presence of GST in the adult worms, their eggs and excretory/secretory products. The immunolocalization studies revealed that the vitelline glands are the major source of GST in liver amphistome. Further, we were able to successfully screen animals naturally infected with G. explanatum using anti GST polyclonal antibodies in dot blot assay. High levels of both circulating GST antigen and anti GST antibodies were detected in the serum of the animals naturally infected with G. explanatum, while no cross reactivity was observed with the tropical liver fluke, F. gigantica which often infects the buffalo liver concurrently. The findings of the present study indicate that GST could be used as an important antigen for the diagnosis of G. explanatum infection in Indian water buffaloes.

Generation of oxidative stress and induction of apoptotic like events in curcumin and thymoquinone treated adult Fasciola gigantica worms.

Fasciolosis is a neglected tropical disease caused by the liver fluke Fasciola gigantica. The absence of successful vaccine and emerging resistance in flukes against the drug of choice, triclabendazole, has necessitated the search for alternatives including phyto-therapeutic approaches. Curcumin and thymoquinone, the active ingredients of Curcuma longa and Nigella sativa plants respectively, were first screened for their binding affinity with Glutathione-S-transferase (GST) molecule through in silico molecular docking followed by in vitro treatment of worms with varying concentrations of the test compounds. The in silico molecular docking of curcumin and thymoquinone with sigma GST revealed strong hydrogen bonding as well as hydrophobic interactions with high fitness scores but showing inter-specific differences. The in vitro treatment of F. gigantica worms with both curcumin and thymoquinone resulted in a significant increase in the generation of reactive oxygen species (ROS) whereas the level of reduced glutathione, a primary redox regulator, was found to be significantly decreased (p < 0.05). The two compounds not only inhibited the GST activity, which is an important detoxification enzyme and also a key drug/vaccine target for the control of fasciolosis but also significantly inhibited the activity of antioxidant enzymes glutathione peroxidase and glutathione reductase that are vital in maintenance of redox homeostasis. The immunohistochemistry performed using anti sigma GST polyclonal antibodies revealed that both the compounds used in the present study significantly reduced immunofluorescence in the vitellaria, developing eggs present in the ovary and the intestinal caecae indicating inhibition of GST enzyme in these regions of the worms. Further, following treatment with curcumin and thymoquinone, chromatin condensation and DNA fragmentation was also observed in F. gigantica worms. In conclusion, both curcumin and thymoquinone generated oxidative stress in the worms by production of ROS and significantly inhibiting their antioxidant and detoxification ability. The oxidative stress along with induction of apoptotic like events would compromise the survival ability of worms within the host. However, further studies are required to establish their anthelmintic potential alone and in combination with the commonly used anthelmintic drugs under in vivo conditions.

Low virulence potential and in vivo transformation ability in the honey bee venom treated Clinostomum complanatum.

The helminth parasites possess great capabilities to adapt themselves within their hosts and also develop strategies to render the commonly used anthelmintics ineffective leading to the development of resistance against these drugs. Besides using anthelmintics the natural products have also been tested for their anti-parasitic effects. Therapeutic efficacy of honey bee venom (HBV) has been tested in various ailments including some protozoal infections but very little is known about its anthelmintic properties. To investigate the anthelmintic effect of HBV the excysted progenetic metacercariae of Clinostomum complanatum, a heamophagic, digenetic trematode with zoonotic potential, infecting a wide variety of hosts, were obtained from Trichogaster fasciatus, a forage fish, which serves as the intermediate host. The metacercarial worms were in vitro incubated in RPMI-1640 medium containing HBV along with the controls which were devoid of HBV for the analysis of worm motility, enzyme activity, polypeptide profile and surface topographical changes. The motility of the worms was significantly reduced in a time dependent manner with an increase in the concentration of HBV. Following incubation of worms the release of cysteine proteases was inhibited in the presence of HBV as revealed by gelatine substrate gel zymography. As well as the polypeptide profile was also significantly influenced, particularly intensity/expression of Mr 19.4 kDa, 24 kDa and 34 kDa was significantly reduced upon HBV treatment. The HBV treatment also inhibited antioxidant enzyme, superoxide dismutase (SOD) and Glutathione-S-transferase (GST) significantly (p < 0.05) in the worms. The scanning electron microscopy of the HBV treated worms revealed tegumental disruptions and erosion of papillae as well as spines showing vacuolation in the tegument. The HBV treated worms also showed a marked decline in the transformation rate when introduced into an experimental host which further reflect the anthelmintic potential of HBV.

Immunolocalization and immunodetection of the excretory/secretory (ES) antigens of Fasciola gigantica.

The digenetic trematode Fasciola gigantica is a parasite of great agricultural and economic importance. Along with Fasciola hepatica, F. gigantica incurs huge economic losses to the agricultural sector. Because of unavailability of an effective and commercial vaccine, the earliest diagnosis of the disease is the only way to control the disease. The conventional coprological techniques are able to detect the disease only after the parasites get matured and starts releasing their eggs with the faeces of host, therefore prepatent infection remain undiagnosed. The alternative method is by serological tests that uses circulatory antigens. Despite high sensitivity, their reliability is quite low because of the common antigens shared between different helminth parasites. To overcome this, investigation was shifted to identify the copro-antigens which could be more sensitive and reliable. In the present study, we tried to identify some of the immunodominant proteins from the Excretory Secretory (ES) product of F. gigantica which can be further characterized and used for early detection of infection and also as drug and vaccine candidates. The ES products of F. gigantica were collected and used for raising the polyclonal antibody in rabbit. The polypeptide profile was generated as well as immunogenic polypeptides were identified. The Source of ES antigen was immunolocalized using confocal microscopy and dot blot assay was performed to diagnose field infection. The polypeptide profile of ES products revealed a total of 24 polypeptides out of which 12 immunogenic polypeptides were identified by western blotting. Confocal micrographs showed the immunolocalization of antigens in the intestinal caecae, vitalline glands, gonads as well as in the tegument of the worm. The dot blot assay confirmed the utility of ES products for the detection of field infection. Subsequently, cross reactivity was found negative with Gigantocotyle explanatum; an amphitome parasite of same habitat. However, the cross reactivity with other helminths needs to be worked out.

Genotoxic potential of Fasciolagigantica infection in experimentally infected rabbits.

A number of parasitic platyhelminthes are known to cause genotoxicity in humans and animals. However no such information is available on tropical liver fluke, Fasciola gigantica, which incurs huge economic losses worldwide. In the present study the genotoxic potential of F. gigantica infection in rabbits, experimentally infected with the metacercarial cysts of this parasite, has been investigated using the standard comet assay and micronucleus (MNi) test on the isolated hepatocytes and the whole blood from the infected rabbits. The tail length of the comet in both hepatocytes and reticulocytes from the infected animals was significantly prominent (p < 0.05) as compared to the controls. About 61.17 % of the hepatocytes from the infected rabbits were positive for MNi formation. A number of blood cells also showed cellular deformities, which were recognised as spicule type, schistocytes, tear drop type, acanthocytes and dumbbell type. It is possible that during the establishment of host-parasite relationship the worms might have released some products which could have contributed to the induction of cellular and DNA damage. However, long term studies are required to understand the serious implications of such an effect caused by F. gigantica, though hepatic carcinoma has not been reported so far due to fasciolosis, however, considering the present results the possibility may not be rule out for the disease progression in this direction.

Anthelmintic Potential of Thymoquinone and Curcumin on Fasciola gigantica.

Fasciolosis an economically important global disease of ruminants in the temperate and tropical regions, caused by Fasciola hepatica and F. gigantica, respectively, also poses a potential zoonotic threat. In India alone it causes huge losses to stakeholders. Anthelmintics including triclabendazole have been used to control this menace but the emerging resistance against the available compounds necessitates identification of novel and alternative therapeutic measures involving plant derived natural compounds for their anthelmintic potential. Thymoquinone (T) and curcumin (C), the active ingredients of Nigella sativa and Curcuma longa respectively have been used as antiparasitic agents but the information on their flukicidal effect is very limited. Adult flukes of F. gigantica were in vitro exposed to different concentrations of thymoquinone and curcumin separately for 3h at 37+ 1°C. A significant (p<0.05) reduction in the worm motility at 60 µM concentration of both T and C was observed though all the worms remained alive after 3h exposure, whereas the effect on egg shedding was statistically insignificant. Pronounced tegumental disruptions and erosion of spines in the posterior region and around the acetabulum was evident. A significant (p<0.05) decrease in glutathione-S-transferase and superoxide dismutase activity and reduced glutathione (GSH) level was observed, while protein carbonylation increased differentially. A significant inhibition of CathepsinL (CatL) gene expression in thymoquinone treated worms was also evident. Further, in silico molecular docking of T and C with CatL revealed a stronger interaction of curcumin with the involvement of higher number of amino acids as compared to thymoquinone that could be more effective in inhibiting the antioxidant enzymes of F. gigantica. It is concluded that both the compounds understudy will decrease the detoxification ability of F. gigantica, while inhibition of CatL will significantly affect their virulence potential. Thus, both thymoquinone and curcumin appeared to be promising anthelmintic compounds for further investigations.

Time-dependent tegumental surface changes in juvenile Fasciola gigantica in response to triclabendazole treatment in goat.

Triclabendazole (TCBZ), the anthelmintic drug active against both mature and immature liver flukes, was used to investigate the effect of in vivo treatment on the tegumental surface of juvenile Fasciola gigantica. Five goats were infected with 150 F. gigantica metacercariae each by oral gavage. Four of them were treated with single dose of TCBZ at 10mg/kg at four weeks post-infection. They were euthanized at 0 (untreated), 24, 48, 72 and 96h post treatment. Juvenile flukes were manually retrieved from the goat livers and processed for scanning electron microscopy. In control flukes, the anterior region was adorned with sharply pointed spines projecting away from the surface, while in the posterior region, spines become shorter and narrower, loosing serration and with the appearance of distinct furrows and papillae. The dorsal surface retained the same pattern of surface architecture similar to that of ventral surface. Flukes obtained from 24h post-treatment did not show any apparent change and were still very active. However, there were limited movements and some blebbing, swelling, deposition of tegumental secretions and some flattening displayed by the flukes of 48h post-treatment. All the worms were found dead 72h post-treatment and showed advanced level of tegumental disruptions, consisting of severe distortion of spines, sloughing off the tegument to expose the basal lamina, formation of pores and isolated patches of lesions. By 96h post-treatment, the disruption was extremely severe and the tegument was completely sheared off causing deeper lesions that exposed the underlying musculature. The disruption was more severe at posterior than anterior region and on ventral than dorsal surface. The present study further establishes the time-course of TCBZ action in vivo with 100% efficacy against the juvenile tropical liver fluke.