FT-IR spectra of a lyophilized extract of Z. officinale are shown in Fig. 1. The spectrum of Z. officinale showed the prominent transmittance types of bands. This absorbance peak values can be compared to the standard IR spectrum available online (Table 1).
Maximum non-toxic dose (MNTD) assay
The MNTD of Z. officinale plant extract was calculated with serial dilution in the Vero cell line followed by MTT assay. The microplate reader provides the numerical value against the different concentrations of extract in respective wells. Blank and control values were recorded at 0.036 and 0.92 respectively. MNTD of Z. officinale plant extract in the Vero cell line was 62.5 µg/ml.
Determination of tissue culture infective dose (TCID50)
TCID50 estimates the concentration of CHIKV which induce CPE in 50% of the cells. Under observation, the CHIKV infected cells undergo structural changes, like syncytia formation and blebbing, when seen under an inverted microscope. TCID50 value was calculated as 103 TCID50/mL for anti-viral assay.
Determination of antiviral property of plant extract of Z. officinale
The antiviral assay was performed to determine the anti-viral effect of the aquatic extract of Z. officinale with the TICD50 valve of CHIKV. Anti- CHIKV effects of Z. officinale extract was determined by inhibition of CPE and an increase in cell viability (Fig. 2). However, upon treating the CHIKV infected cells with plant extract of the Z. officinale with MNTD, an increase in the percentage of cell viability was observed and calculated by MTT assay. However, upon treating the CHIKV infected cells with plant extract of Z. officinale MNTD and half of MNTD, an increase in the percentage of cell viability was observed. Our study revealed that in pre-treatment, CHIKV infected Vero cells when treated with plant extract at MNTD and half of MNTD showed 51.05% and 35.1% increase in cell viability. Similarly, in co-treatment, plant extract at MNTD and half of MNTD brought 52.9% and 49.02% increase in cell viability, respectively (Fig. 3).
Suddenly viral outbreaks in India are increasing like pandemic Influenza , Nipah virus , Zika virus , dengue and Chikungunya virus is one of them. Chikungunya virus is a highly infectious viral disease and well notorious since its origin from Tanzania and has the potential to re-emerging the global level [20, 21]. There is no vaccine and antiviral for CHIKV. Chemically synthesized antivirals have a variety of side effects as compared to natural medicinal plant remedies .
According to the Indian Ayurvedic system, Z. officinale (Ginger) is a well-known medicinal plant and very helpful in controlling gastrointestinal problems by reducing nausea. Ginger is already utilized to treat morning sickness, heartburn, colic, stomach upset, gas, flatulence, bloating, diarrhea, loss of appetite and dyspepsia [23,24,25]. Ginger act as an analgesic and helpful for muscle soreness, arthritis, chest pain, stomach pain, low back pain, and menstrual pain. Ginger is a well-known medicinal plant to treat cough, respiratory tract infections, bronchitis. Fresh and dilute juice of ginger is very useful in skin burns treatment. Active components of ginger are utilized as a laxative and antacid medication. As per Ayurveda system, Ginger is well known for the treatment of a variety of cancers including skin, oral, breast, liver, gastric, pancreatic, colon, renal, prostate, brain, ovarian and cervical cancer. Ginger has antioxidant, anti-inflammatory and anti-mutagenic properties .
The present study has been conceptualized to check the anti-chikungunya activity of aquatic plant extract from the rhizome of Z. officinale. The anti-chikungunya effect of Z. officinale plant extract was checked in vitro model by using a Vero cell-line. In- vitro assessment of maximum non-toxic dose of aquatic medicinal of plant extract are crucial before their therapeutic uses to the possibilities of their side-effects. Cytotoxicity of aquatic medicinal plant extract was observed on the Vero cell-line in terms of cytopathic effect. In present study, MNTD of Z. officinale aquatic plant extract was found to be 62.50 μg/mL.
Anti-chikungunya effects of the medicinal plant extract were determined on Vero cell with MNTD and half of MNTD through pre- and co-treating mode of treatment. The results point out that the aquatic plant extract of Z. officinale has anti-chikungunya potential. Antiviral assays were performed to study the effect of plant extract, they may hamper the CHIKV replication in Vero cell-line. Our study revealed that Vero cells when pre-treated with MNTD and half of MNTD of Z. officinale plant extract, their viability increase 51.05% and 35.1% respectively. Vero cell viability increases 52.9% and 49.02% when co-treatment with MNTD and half of MNTD Z. officinale plant extract.
Medicinal plants and their secondary metabolites have therapeutic potential against other similar viruses like dengue . Although C. papaya leaves are generally taken during dengue fever, C. papaya may not interfere with viral replication but responsible for increasing the platelets [28, 29]. In previous other similar studied on andrographolide show anti-chikungunya potential when a study in the HepG2 cell-line . In another study, plant extract and their AgNPs of T. cordifolia, have the potential to interfere with CHIKV and able to increase the 75% and 56% Vero viability when these cells were treated MNTD and half of MNTD, respectively. Medicinal plants are very sensitive, specific and cost-effective against various old and emerging viruses. In another similar study on influenza virus, aqueous Isatidis Radix extract were screening for the anti-influenza property in Madin-Darby Canine Kidney (MDCK) cells, less influenza viruses were observed by scanning electron microscope (SEM) in the Isatidis Radix extract pre-treated cells than the cells without Isatidis Radix extract treatment . Despite the huge immunization and modern medication, many viruses lack preventive vaccines and effective antiviral therapies. In the situation, identification of novel antiviral drugs from medicinal plants are more importance as natural products are an excellent source and safer. Medicinal plants are good source of antiviral and effective herbal medicines .
Medicinal plants compounds have antibacterial [33,34,35] and anticancer  properties. Antiviral activities of medicinal plants have been reported against various medically important viruses including, vaccinia virus, HSV, RSV, FLU, MPU, HIV and HBV [37,38,39,40,41,42,43,44,45]. Medicinal plants could interfere with viral replication through various mechanisms. It is reported that components from certain medicinal plants, interfere with the binding of viron to the cellular receptors, therefore can avoid their entrance in the cell. The exact mechanism of medicinal plants for their antiviral actions has not been fully understood. Therefore, the interaction of medicinal plant extracts with different types of cells is a complex phenomenon that requires further details study.
In conclusion, aquatic medicinal plant extract successfully manufactures the from the fresh rhizome of Z. officinale. The aquatic plant extract of Z. officinale showed excellent anti-chikungunya activity. A large number of compounds may involve in combating the CHIKV. Plant extracts are comparatively simple, capable, economical and an environment-friendly as compared to the chemically synthesized drugs. Medicinal plants could be an alternative source to develop a wide range of antiviral agents which could very helpful the alternative treatment of viruses. Zingiber officinale not only has a therapeutic potential but also combat drug resistance in antivirals against CHIKV. It is advisable, before using aquatic plant extract or their nano-material as therapeutic agents in the form of antiviral, their toxicity or side-effects and molecular mechanism should be studied properly.