INTRODUCTION

Herbal products obtained from natural sources including plants, animals, and minerals have been employed by humans as a medicine for treatment, prevention, mitigation, and cure of human ailments, disorders, and diseases [1, 2]. Decades ago, a raw crude drug from natural origin and semi-pure extracts of natural and herbal sources of medicine are the only available medications for the prevention, mitigation, and restoration of health. In the 20th century, a revolution occurs, which develops the idea that the effect of any drug in the human body is mediated by a specific interaction of the administered drug entity with macromolecule which is abundantly present in the human body. This idea gives a leads to scientists that the individual chemical entity present in the extract is only responsible for the desired effect or biological and pharmacological activity of the drug. This was the starting of a new epoch in the field of pharmacology and pharmacognosy. Isolated chemical compounds have become a standard treatment option for various diseases [3].

Malaria is an infectious disease that occurs due to protozoa parasites that belong to the genus plasmodium which are transmitted to human beings after the female anopheles bites the human. Male anopheles feed only on plants juices and nectar, cannot transmit malaria. This disease is a serious global public health issue. Approximately 100 species of parasite plasmodium exist but only four of them can spread the infection to human beings [4].

As available in ayurvedic literature, leaves of N. arbor (Harsingar) treat malaria effectively and reduce symptoms like increased body temperature, chills immediately after administration [5-8]. Whereas Tinospora cordiflia (Guduchi), which is termed as Amrita means nectar, which gives strength to the human body by modulating immunity. Guduchi is also known as Indian quinine [9-12]. Ocimum sanctum (Tulsi) is termed as a divine herb in Ayurveda that averts and treats various bacterial infections. It is used to cure malaria, as stated in literature; infusion of leaves is administered to the patient [13-16].

Modern dosage form tablet is a modified form of Ayurvedic dosage form vati. Vati Kalpana plays a prominent role in the pharmaceutics of Ayurveda, has many advantages like easy administration, palatability, ease for dispensing, and transportation. Vati Kalpana is a pharmaceutical manufacturing procedure in which the powder of crude drugs is triturated together with certain liquids or even honey and the medicines are prepared in the form of pills or tablets [17, 18].

The main aim of this research work deals with better as well as more effective eradication of malaria parasite from the human body with lesser side effects and strengthening of the human immune system.

MATERIALS AND METHODS

Collection and identification of crude material

Leaves of N. arbor stems of T. cordifolia & leaves of O. sanctum were collected from village Barlai Jageer, Indore, Madhya Pradesh. The crude plant materials were identified by Dr. Chitralekha Soni Kadel, Department of Botany, Vikram University, Ujjain (MP), and voucher specimens O. sanctum voucher no. MIPS/O/01/2017, T. cordifolia voucher no. MIPS/T/02/2017, and N. arbor voucher no. MIPS/N/03/2017 was deposited in the herbarium of the Department of Pharmacognosy, MIPS, Ujjain (MP).

Processing of collected crude drug

All the collected crude materials were dried separately in shade at normal, room temperature by spreading them uniformly. Dried crude materials were pulverized individually in the grinder to obtain a powder. They were then subjected to extraction using soxhlet apparatus, ethanol was used as solvent [19].

Phytochemical screening

Phytochemical screening of all three extracts was performed as per the procedure prescribed [20, 21].

Qualitative analysis

Qualitative analysis of all three crude drugs was performed as per the procedures prescribed [22-25].

Formulation of vati

Fine crude powder of N. arbor, extract of T. cordifolia, and extract of O. sanctum were mixed well together by blending according to formula (Table 1). Jaggery was taken and a little amount of water was added to it to make paka. Paka was added to the above mixture to make vati. Manually formulated vati was then allowed to dry [26-28].

Table 1. Formula for vati

In-vitro antimalarial evaluation

The designed formulation was screened for anti-malarial activity in the Micro-care laboratory & TRC, Surat, Gujarat. The in-vitro antimalarial assay was carried out in 96 well micro-titre plates according to the microassay protocol of Rieckmann and co-workers with minor modifications. The P.falciparum strain cultures were maintained in medium RPMI        1640 supplemented with 25 mMHEPES, 10% heat-inactivated human serum, 0.23% sodium bicarbonate, and 1%D-glucose. To obtain only the ring stage parasitized cells, the asynchronous parasites of P.falciparum were synchronized after 5% D-sorbitol treatment. For carrying out the assay, an initial ring stage parasitemia of 0.8 to 1.5% at 3% haematocritina total volume of 200 µl of medium RPMI-1640 was determined by Jaswant Singh Bhattacharya staining to assess the percent parasitemia (rings) and uniformly maintained with 50% RBCs (O⁺). A stock solution of 5mg/ml of each of the test samples was prepared in DMSO and subsequent dilutions were prepared with a culture medium. The diluted samples in 20 µl volume were added to the test wells to obtain final concentrations (at five-fold dilutions) ranging between 0.4µg/mlto100 µg/ml in duplicate well containing parasitized cell preparation. The culture plates were incubated at 37˚C in a candle jar. Thin blood smears from each well were prepared and stained with Jaswant Singh Bhattacharya stain after 36 to 40 h incubation. To record the ring-stage parasites maturation into schizonts and trophozoites when different concentrations of the test agents present, the slides were microscopically observed. The concentration of test that inhibited the complete maturation into schizonts was recorded as the minimum inhibitory concentrations (MIC) [29-35]. The reference drug that was used was Chloroquine.

RESULTS AND DISCUSSION

In this research work, three medicinal plants were selected for formulation development for malaria infection. The basic motive of this formulation development is to provide a treatment option to the patient with better results in terms of cure with minimal side effects. N. arbor tristis, T. cordifolia, and O. sanctum were selected and analyzed for this purpose.

Extraction of crude drugs was done individually by using soxhlet apparatus and ethanol as solvent, percent yield was calculated and was found to be 32% for N. arbor, 21% for T. cordifolia, and 17% for O. sanctum.

Phytochemical screening

Qualitative phytochemical analysis was performed and results show that N. arbor contains phytoconstituents such as carbohydrates, alkaloids, phenolics, tannins, flavonoids, fixed oils, and glycosides. T. cordifolia contains alkaloids, glycosides, phenolics, tannins, and steroids, whereas O. sanctum contains alkaloids, flavonoids, glycosides, phenolics, tannins, steroids, and volatile oils (Table 2).

Table 2. Phytochemical screening of extracts of N. arbor, T. cordifolia, and O. sanctum

+ present, ++ moderately present, +++ strongly present, - absent