Anti - plasmodial potential of selected medicinal plants and a compound Atropine isolated from Eucalyptus obliqua

: The present research study was aimed to inves - tigate the e ﬃ ciency of selected medicinal plants against Plasmodium vivax. Crude methanolic extracts from the seeds of leaves of Datura stramonium , Parthenium hyster - ophorus , Calotropis procera , and Dodonaea viscosa were prepared. In addition, Atropine was also isolated from alka - loid components of Eucalyptus obliqua to evaluate their in vitro anti - plasmodial e ﬀ ects. It was observed that proguanil ( positive control ) and Atropine displayed strong anti - plasmo - dial activity ( 94.04 and 68.02%, respectively ) against P. vivax at 0.1 mg/mL concentration while the leaf extracts of other medicinal plants did not exhibit any notable anti - plasmodial activity. It was concluded that alkaloids of E. obliqua plant ’ s extracts were rich in anti - plasmodial compound Atropine, which exhibit a remarkable anti - plasmodial activity against P. vivax . Anti - plasmodial action of medicinal plants are attributed to these phytochemicals. In vitro studies using medicinal plant ’ s extracts and standardized methods will help to make more powerful and cost - e ﬀ ective anti - plasmo - dial compounds.


Introduction
Medicinal plants are tbeing used as a treatment for several microbial infections for hundreds of years, but they are also being used as health and growth promoters in animal feed [1].Many medicinal plants are extensively used in the preparation of therapeutic drugs to treat a variety of microbial infections [2].Due to improper diagnosis and an elevated exposure of antibiotics and antimalarial compounds, the risk of drug resistance is alarmingly increased [3,4].
Malaria is an erythrocytic blood infection caused by a hemo-parasite of genus Plasmodium [5].About one-half of the world's population is at risk of malaria infection [6].Following the Sustainable Development Goals 2030, World Health Organization has specified the Global Technical Strategy for the eradication of malaria worldwide.This strategy is intended to reduce the death rate caused by malaria infection up to 90%.It is also intended to get 35 countries free of malaria infection and also to prevent malaria comeback in 20 countries that were malaria-free in 2001 [7].Building solid alliances and government commitment, developing evidence-based policy for malaria eradication in various transmission zones, and investing in effective control measures and surveillance systems are all part of this strategy [8].
Multi-drug resistance in pathogens is increasing nowadays, which leads to difficulty in treating life-threatening viral, bacterial, and parasitic infections [9].Alteration in drug binding sites and enzymes involved in drug inactivation is responsible for evolving resistance against commercial drugs.Alterations of enzymatic pathways are also considered to contribute to the drug resistance in microorganisms [10].
For thousands of years, medicinal plants are being used to cure a variety of diseases in humans and animals.Medicinal plants are traditionally being used for various disorders in Arabic herbal studies, such as bacterial infections, conjunctivitis, asthma, GIT dysfunctions, and hypertension.The therapeutic impact of medicinal plants is recently a hot topic.Research is being conducted on medicinal plants to cash its pharmacological properties to treat cancer and gastrointestinal diseases.Medicinal plants are also an active area of research in the recent era [11].It is also reported that the oil of Eucalyptus obliqua decreases the level of free radicals and has a high impact on chemokines such as prostaglandins and cytokines [12].
Atropine is a naturally occurring compound found in most of the medicinal plants.It is listed in the essential medicines list by the WHO due to its dire importance and medical uses.The most common medical effect of Atropine is in gastric, bronchial, and salivary secretions [13].
The leaves and roots of Datura stramonium, Calotropis procera, and Dodonaea viscosa also traditionally used as folk medicines for the treatment of different diseases including skin infections, healing of wounds, a bronchodilator, rheumatic pains, skin ulcers, skin burns, painful menstruation, antispasmodic, and anti-diabetic [14][15][16].There is a massive decrease in options for treating bacterial and parasitic infections worldwide; therefore, there is a dire need to search for new plant-based remedies to cope with such infections effectively.Therefore, keeping in view the importance of medicinal plants, the present study was designed to investigate the efficiency of selected medicinal plants against Plasmodium vivax and Staphylococcus aureus.
Atropine was isolated and purified from alkaloids of E. obliqua.Plant samples were transported to the Molecular Parasitology Laboratory, Kohat University of Science and Technology (KUST), Kohat, Khyber Pakhtunkhwa, Pakistan.

Isolation and purification of Atropine through Gradient Solvent Systems and high performance liquid chromatography (HPLC)
Gradient Solvent Systems were designed to isolate the antiplasmodial components in alkaloids of E. obliqua crude extract through Silica Gel Column Chromatography.Various concentrations of n-hexane and methanol were prepared, and extracts were yielded by the repeated experimentation.Finally, samples were subjected to HPLC and Atropine was isolated (Figure 2).

Plasmodial culture collection
Prepared culture of P. vivax was obtained from Molecular Parasitology Laboratory, KUST, Kohat, Khyber Pakhtunkhwa, Pakistan, while anti-plasmodial activity of crude methanolic extract of selected medicinal plants was evaluated using standard protocols described by Nyandwaro et al. [18].

In vitro anti-plasmodial activity
A total of 32 wells were prepared in microtiter plates containing agar medium.Each well was filled with parasitized RBCs.Furthermore, five concentrations of Proguanil were used as a positive control based on field survey.It was revealed in field survey that proguanil products were in trending use and giving good results against malaria infection in the first five wells, while five concentrations of plant extracts, i.e., Atropine, D. stramonium, P. hysterophorus, P. hysterophorus, and D. viscosa were used as experimental group.In addition, distilled water was used as negative control.After the incubation period of 48 h, the supernatants from each well were removed, and smear were prepared for further anti-plasmodial assay as per protocol of WHO [19].Maturation and inhibition percentages were evaluated using the following formula as described by Kwansa-Bentum et al. [20].However, mean inhibition, standard error, and standard deviation were calculated using Statistix 9 software:

Discussion
The study was aimed to investigate the in vitro anti-plasmodial potential of Atropine, D. stramonium, C. procera, P. hysterophorus, and D. viscosa extracts against P. vivax.
The results revealed that the methanolic extracts of all these medicinal plants except Atropine exhibited a minimal antiplasmodial activity against P. vivax.Anti-plasmodial potential: Atropine and other selected medicinal plants  3 Atropine is an amalgamation of L-hyoscyamine and D-hyoscyamine which is identified as a tropane alkaloid.L-Hyoscyamine is responsible for most of its physiological effects.Pharmacologically it is very important as it binds to muscarinic receptors and induce pharmacological actions as antimuscarinic mediator.Stereoselective metabolism of Atropine is also reported in various studies.Sulfate (monohydrate) is the most common Atropine which is used in the medicines.About 60% of the Atropine, when taken, are excreted from the body unchanged in the urine, rest of the 40% become hydrolyzed.Glands regulated by the parasympathetic nervous system perform their activity in "rest and digest rule."Generally Atropine counters the rest and digest activity [21].
As reported by our previous studies [22] the alkaloid components of E. obliqua exhibit a remarkable anti-plasmodial potential.It was concluded that alkaloids contain some anti-plasmodial compounds which are responsible for the mortalities of the P. vivax in vitro.In addition, several studies have already been conducted to evaluate the anti-microbial activity of medicinal plants [23,24].
Moreover, in vitro studies using comparable extracts and standardized methods are required to make more definitive claims regarding medicinal plants and its derivatives' powerful anti-plasmodial effects in pharmacological formulations.
Medicinal plant-based research is seen as a universal solution to inhibit the pathogenesis of microorganisms.Genetic variations and polymorphism in genes of the Plasmodium lead to the drug resistance in Plasmodium species [25].Leaf extracts of D. stramonium and C. procera exhibited a noteworthy anti-plasmodial activity, i.e., 8.04, and 4.02% inhibition, respectively, against P. vivax.The current findings on a crude extract of leaves also support prior results, demonstrating the widespread usage of medicinal plants as an anti-malarial agent by Western Cameroon's endogenous traditional healers [26].Xu et al. [27] reported alkaloids, flavonoids, saponins, and tannins during preliminary phytochemical screening of leaf extracts of medicinal plants and concluded that the extract's anti-plasmodial action might be attributed to or more groups of these components.
In the current study, in vitro anti-plasmodial activity of medicinal plant's extracts is also in agreement with the study of Satish et al. [26], which revealed that D. stramonium had anti-plasmodial potential against drug-resistant Plasmodium species.The anti-plasmodial activity of E. obliqua was due to the presence of alkaloids which contain Atropine, i.e., an anti-plasmodial compound.Further, in vivo studies are required to understand the detailed mechanism of anti-plasmodial activity of active anti-plasmodial compounds in other selected medicinal plant extracts.In this study, the anti-plasmodial efficacy of P. hysterophorus was not found, which is in quite contradiction to the study of Ahmed et al. [28] who reported the in vitro anti-plasmodial potential of P. hysterophorus on chloroquine-sensitive strains of Plasmodium falciparum.The possible reason might be that Ahmed et al. [28] used whole plant in the preparation of extracts to perform the in vitro activity, while in the current study, only leaves of P. hysterophorus and D. viscosa were used in the preparation of extracts.Similarly, no satisfactory antimalarial activity of C. procera was observed in the current study, which is in contradiction to the study of Adejoh et al. [29], who used phosphate buffer extract from the latex of C. procera and evaluated the in vivo antimalarial activity on mice.Studies suggested that medicinal plants from Eucalyptus species are traditionally being used for the treatment of malaria infection in various parts of Africa [30].One of the species, i.e., Eucalyptus globulus has also traditional popularity in healing malaria infection in Brazil [31].Moreover, C. procera, D. stramonium, D. viscosa, and P. hysterophorus are being used traditionally to repel the malaria vector [32,33].

Conclusions
In the current study it was concluded that Atropine isolated from E. obliqua extract displayed anti-plasmodial activity against P. vivax at all concentrations; however, Atropine is reported for the first time for its anti-plasmodial potential particularly for P. vivax.A strong anti-plasmodial activity was found at higher concentration, i.e., 0.1 mg/mL.On the other hand, it was also concluded that the leaf extracts of D. stramonium, C. procera, P. hysterophorus, and D. viscosa did not reveal any prominent anti-plasmodial activity against P. vivax.Moreover, it was suggested that further in vitro and in vivo studies are needed to explore the medicinal plants and its derivatives' powerful anti-plasmodial and anti-bacterial effects in future.Furthermore, the mode of action of Atropine and the detailed study of the target site of this compound is needed to be investigated.

Figure 2 :
Figure 2: HPLC peak of isolate compound "Atropine." the current research study, it was observed that methanolic extracts of proguanil and Atropine exhibited 94.04 and 68% inhibition, respectively, against P. vivax with the LD 50 0.068.On the other hand, leaf extracts of D.