Plant in vitro Culture Technologies; A Promise Into Factories of Secondary Metabolites Against COVID-19.

The current pandemic has caused chaos throughout the world. While there are few vaccines available now, there is the need for better treatment alternatives in line with preventive measures against COVID-19. Along with synthetic chemical compounds, phytochemicals cannot be overlooked as candidates for drugs against severe respiratory coronavirus 2 (SARS-CoV-2). The important role of secondary metabolites or phytochemical compounds against coronaviruses has been confirmed by studies that reported the anti-coronavirus role of glycyrrhizin from the roots of Glycyrrhiza glabra. The study demonstrated that glycyrrhizin is a very promising phytochemical against SARS-CoV, which caused an outbreak in 2002-2003. Similarly, many phytochemical compounds (apigenin, betulonic acid, reserpine, emodin, etc.) were isolated from different plants such as Isatis indigotica, Lindera aggregate, and Artemisia annua and were employed against SARS-CoV. However, owing to the geographical and seasonal variation, the quality of standard medicinal compounds isolated from plants varies. Furthermore, many of the important medicinal plants are either threatened or on the verge of endangerment because of overharvesting for medicinal purposes. Therefore, plant biotechnology provides a better alternative in the form of in vitro culture technology, including plant cell cultures, adventitious roots cultures, and organ and tissue cultures. In vitro cultures can serve as factories of secondary metabolites/phytochemicals that can be produced in bulk and of uniform quality in the fight against COVID-19, once tested. Similarly, environmental and molecular manipulation of these in vitro cultures could provide engineered drug candidates for testing against COVID-19. The in vitro culture-based phytochemicals have an additional benefit of consistency in terms of yield as well as quality. Nonetheless, as the traditional plant-based compounds might prove toxic in some cases, engineered production of promising phytochemicals can bypass this barrier. Our article focuses on reviewing the potential of the different in vitro plant cultures to produce medicinally important secondary metabolites that could ultimately be helpful in the fight against COVID-19.

Therapeutic potential of medicinal plants against COVID-19: The role of antiviral medicinal metabolites.

There are numerous trials underway to find treatment for the COVID-19 through testing vaccines as well as existing drugs. Apart from the many synthetic chemical compounds, plant-based compounds could provide an array of \suitable candidates for testing against the virus. Studies have confirmed the role of many plants against respiratory viruses when employed either as crude extracts or their active ingredients in pure form. The purpose of this review article is to highlight the importance of phytomedicine against COVID-19. The main aim is to review the mechanistic aspects of most important phytochemical compounds that have showed potential against coronaviruses. Glycyrrhizin from the roots of Glycyrrhiza glabra has shown promising potential against the previously epidemic coronavirus, SARS-CoV. Other important plants such as Artemisia annua, Isatis indigotica, Lindera aggregate, Pelargonium sidoides, and Glychirrhiza spp. have been employed against SARS-CoV. Active ingredients (e.g. emodin, reserpine, aescin, myricetin, scutellarin, apigenin, luteolin, and betulonic acid) have shown promising results against the coronaviruses. Phytochemicals have demonstrated activity against the coronaviruses through mechanisms such as viral entry inhibition, inhibition of replication enzymes and virus release blockage. However, compared to synthetic drugs, phytomedicine are mechanistically less understood and should be properly evaluated before application. Nonetheless, phytochemicals reduce the tedious job of drug discovery and provide a less time-consuming alternative for drug testing. Therefore, along with other drugs currently tested against COVID-19, plant-based drugs should be included for speedy development of COVID-19 treatment.

Comprehensive investigations on anti-leishmanial potentials of Euphorbia wallichii root extract and its effects on membrane permeability and apoptosis.

Clinically available synthetic chemotherapeutics to treat the vector-borne protozoan infection, leishmaniasis, are associated with serious complications such as toxicity and emergence of resistance. Natural products from plants consist of interesting biomolecules that may interfere with DNA or membrane integrity of the parasite and can possibly minimise the associated side effects. In the present study, various fractions of Euphorbia wallichii (EW) root extracts including n-hexane (EWNX), ethyl acetate (EWEA), chloroform (EWCH) and aqueous (EWAQ), were evaluated for their antileishmanial potential against Leishmania tropica followed by investigation of the possible mechanism of action via reactive oxygen species (ROS) quantification, membrane permeability (via sytox green dye) and apoptotic assay (via AO/EB method) using fluorescent microscopy. Two of the fractions i.e. EWEA and EWAQ inhibited the growth of promastigotes (IC50 7.8 and 10.2 µg/mL, respectively) and amastigotes (IC50 9.9 and 13.3 µg/mL, respectively) forms almost at similar concentrations as found for the standard antileishmanial drugs, tartar emetic (TA) and Glucantime (IC50 9.4 and 21.5 µg/mL, respectively). Both the active fractions remained non-toxic towards human blood erythrocytes and were able to cause membrane permeability and apoptotic induction (using Triton X-100 as a positive control) leading to death of Leishmania parasites. However, both the fractions could not triger significant and persistent ROS generation, compared to hydrogen peroxide used as a positive control. Antilesihmanial activity of the two active fractions might be attributed to the presence of high quantity of tannins and saponins.

Biosynthesis of anti-leishmanial natural products in callus cultures of Artemisia scoparia.

Clinically, available synthetic chemotherapeutics in the treatment for leishmaniasis are associated with serious complications, such as toxicity and emergence of resistance. Natural products from plants can provide better remedies against the Leishmania parasite and can possibly minimize the associated side effects. In this study, various extracts of the callus cultures of Artimisia scoparia established in response to different plant growth regulators (PGRs) were evaluated for their anti-leishmanial effects against Leishmania tropica promastigotes, followed by an investigation of the possible mechanism of action through reactive apoptosis assay using fluorescent microscopy. Amongst the different callus extracts, higher anti-leishmanial activity (IC50:19.13 µg/mL) was observed in the callus raised in-vitro in the presence of 6-Benzylaminopurine (BA) plus 2,4-Dichlorophenoxyacetic Acid (2,4-D) at the concentration of 1.5 mg/L, each. Further, the results of apoptosis assay showed a large number of early-stage apoptotic (EA) and late-stage apoptotic (LA) cells in the Leishmania under the effect of callus extract grown in-vitro at BA plus 2,4-D. For the determination of the potent natural products in the callus extracts responsible for the anti-leishmanial activity, extracts were subjected to Gas chromatography-mass spectrometry (GC-MS) for the metabolite analysis. Nonetheless, higher levels of the metabolites, such as nerolidol (22%), pelletierine (18%), aspidin (15%) and ascaridole (11%) were detected in the callus grown in vitro at BA plus 2,4-D (1.5 mg/L, each). This protocol determines a novel method of production of anti-leishmanial natural products through callus cultures of A. scoparia, a medicinal plant.

Five Indigenous Plants of Pakistan with Antinociceptive, Anti-Inflammatory, Antidepressant, and Anticoagulant Properties in Sprague Dawley Rats.

Five medicinal plants of Pakistan were investigated for their antinociceptive, anti-inflammatory, antidepressant, and anticoagulant potential. Antinociceptive activity was estimated by hot plate and writhing assay. In hot plate assay, Quercus dilatata (52.2%) and Hedera nepalensis (59.1%) showed moderate while Withania coagulans (65.3%) displayed a significant reduction in pain. On the other hand, in writhing assay, Quercus dilatata (49.6%), Hedera nepalensis (52.7%), and Withania coagulans (62.0%) showed comparative less activity. In anti-inflammatory assays crude extracts showed significant edema inhibition in a dose dependent manner. In carrageenan assay, the highest activity was observed for Withania coagulans (70.0%) followed by Quercus dilatata (66.7%) and Hedera nepalensis (63.3%). Similar behavior was observed in histamine assay with percentage inhibitions of 74.3%, 60.4%, and 63.5%, respectively. Antidepressant activity was estimated by forced swim test and the most potent activity was revealed by Withania coagulans with immobility time 2.2s (95.9%) followed by Hedera nepalensis with immobility time 25.3s (53.4%). Moreover, the crude extracts of Fagonia cretica (74.6%), Hedera nepalensis (73.8%), and Phytolacca latbenia (67.3%) showed good anticoagulant activity with coagulation times 86.9s, 84.3s, and 67.5s, respectively. Collectively, the results demonstrate that these five plants have rich medicinal constituents which can be further explored.

Plants as Antileishmanial Agents: Current Scenario.

Leishmaniasis is a clinical manifestation caused by the parasites of the genus Leishmania. Plants are reservoirs of bioactive compounds, which are known to be chemically balanced, effective and least injurious as compared with synthetic medicines. The current resistance and the toxic effects of the available drugs have brought the trend to assess the antileishmanial effect of various plant extracts and their purified compound/s, which are summarized in this review. Moreover, it also highlights various traditional remedies used by local healers against leishmaniasis. A systematic cross-sectional study for antileishmanial activity of natural products was carried out using multiple literature databases. The records retrieved since 2000 till year 2016 were analysed and summarized in the form of comprehensive tables and graphs. Natural products are potential source of new and selective agents that can significantly contribute to primary healthcare and probably are promising substitutes of chemicals for the treatment of protozoan diseases like leishmaniasis. Where the researchers prefer to use alcoholic solvents for the extraction of antileishmanial agents from plants, most of the studies are limited to in vitro conditions majorly on using promastigote forms of Leishmania. Thus, there is a need to carry out such activities in vivo and in host macrophages. Further, there is a need of mechanistic studies that can help taking few of the promising pure compounds to clinical level. Copyright © 2016 John Wiley & Sons, Ltd.

Hedera nepalensis K. Koch: A Novel Source of Natural Cancer Chemopreventive and Anticancerous Compounds.

Traditional medicinal plants are often used for both the prevention and the treatment of local diseases. Taking into consideration the medicinal importance of Hedera nepalensis within local Pakistani traditions, the present study was undertaken to analyze the in vitro cancer chemopreventive and cytotoxic properties of the plant. The in vitro cancer chemopreventive testing was performed using nitrite assay, NFκB assay, aromatase assay, and quinone reductase 1 (QR1) assay. The cytotoxic potential was evaluated on three cancer-cell lines: MCF-7, MDA-MB-231, and HeLa using sulforhodamine B (SRB) assay. The results of cancer chemopreventive assays show that n-hexane and ethyl acetate fractions of tested plant have promising cancer chemopreventive potential. Lupeol isolated from n-hexane as well as ethyl acetate fraction showed lowest IC50 (0.20 ± 1.9 µM) in NFκB assay. Crude extract and its fractions inhibited the growth of three cancer cell lines by more than 60%, IC50 value of lupeol varied from 2.32 to 10.2 µM. HPLC-DAD-based quantification of lupeol in different plant tissues demonstrated that leaves of H. nepalensis are a rich source of lupeol (0.196 mg/100 mg dry weight). Our data have shown that H. nepalensis harbors cancer chemopreventive and cytotoxic agents.

Physiological and biochemical mechanisms of allelopathy mediated by the allelochemical extracts of Phytolacca latbenia (Moq.) H. Walter.

In allelopathy, one plant suppresses the growth and development of other plant/plants by negatively affecting a variety of physiological and biochemical reactions. We checked the effects of methanolic extracts (allelochemical extracts) of Phytolacca latbenia (Moq.) H. Walter on antioxidant enzyme activities such as peroxidases (PODs), super oxide dismutases (SODs) and catalase (CAT) and on total protein contents (TPC), cellular injury (CI), and malondialdehyde (MDA) in the germinating seeds of Brassica napus L. (dicot) and Triticum aestivum L. (monocot). Both the crude methanolic extract root (CMER) and crude methanolic extract aerial (CMEA) of P. latbenia at 10000 ppm significantly reduced the POD activity in both the test seeds. The activity of SODs was significantly decreased by both CMER and CMEA in B. napus germinating seeds. A linear increase in the activity of CAT, CI, and MDA contents was found in both the test seeds with the increasing concentrations of CMEA and CMER, while TPC of the germinating seeds was found decreased. It is inferred that both the CMEA and CMER inhibited/delayed the seed germination, reduced the seedling growth by affecting a variety of biochemical and physiological attributes, and also caused cellular membrane injury in the germinating seeds of both the monocot and dicot seeds.

Phytotoxicity evaluation and phytochemical analysis of three medicinally important plants from Pakistan.

This work examines the crude methanolic extracts of three medicinally important plants native to Pakistan for potent phytotoxic activities and important phytochemicals. These plants include Euphorbia wallichii, Bergenia ciliata and Phytolacca latbenia. The phytotoxic effects were checked at 10,000, 1000, and 100 µg/ml against two economically important standard target species, Triticum aestivum (monocot representative) and Brassica napus (dicot representative). The phytotoxicity effects on seed germination, seedling growth and seedling weight were checked. A simple, cost-effective in vitro phytotoxicity assay (that uses petri plates) was used to evaluate the allelopathic properties of crude extracts. At highest concentration, extracts from all the three plants showed phytotoxic activities such that P. latbenia > E. wallichii > B. ciliata. In seedling growth, root length was affected more than shoot length, whereas among the target species B. napus was found to be more sensitive towards extracts when compared with T. aestivum. Phytochemical analysis showed that P. latbenia is rich in saponins and terpenoids, while E. wallichii and B. ciliata are rich in tannins, terpenoids and cardiac glycoside. P. latbenia also carries a moderate amount of cardiac glycosides.

Antioxidant and Cytotoxic Activities and Phytochemical Analysis of Euphorbia wallichii Root Extract and its Fractions.

Euphorbia wallichii a perennial herb growing mainly in Himalayas has been widely used in folk medicines for its medicinal properties. In the present study, the crude methanolic root extract (CME) and its fractions; n-Hexane Fraction (NHF), n-Butanol Fraction (NBF), Chloroform Fraction (CHF), Ethyl acetate Fraction (EAF) and Aqueous Fraction (AQF) of this plant specie were investigated for antioxidant and cytotoxic activities and phytochemical analysis. Antioxidant activity was determined by using 2,2-diphenyl-1-picryl-hydrazyl free radical (DPPH) and DNA protection assay performed on pBR322 plasmid DNA. In both these assays, promising results were obtained for CME as well as other fractions. The IC50 values for DPPH assay were in a range of 7.89 to 63.35 µg/ml in which EAF showed the best anti-oxidant potential and almost all the tested samples showed certain level of DNA protection. The cytotoxic activity was assessed by using Sulforhodamine B (SRB) assay on human cell lines; H157 (Lung Carcinoma) and HT144 (Malignant Melanoma). The IC50 values of the tested samples ranged from 0.18 to 1.4 mg/mL against H157 cell line whereas against HT144 cell line the IC50 values ranged from 0.46 to 17.88 mg/mL with NBF fraction showing maximum potential for both. Furthermore, the phytochemical analysis of CME and its fractions showed the presences of flavonoids, saponins, tannins, terpenoides and cardiac glycosides with varying concentrations.