Validating the properties of Ranunculus sceleratus Linn . by performing spectroscopic techniques andmodern chromatography

Ranunculus sceleratus Linn. is one of the well-known medicinal plants, being used from the ancient time in India and commonly called as “Jal dhaniya”. It belongs to Ranunculaceae family derived from two LatinWords “Rana”means frog and “unculus” means little and referred together as “little frog” and also species sceleratus for cursed. It is an aquatic perennial herb. It consists of a herbaceous hollow stem, irm tap root, branched rhizome and leaves having a smooth upper side. The green plant is toxic for livestock and uncomfortable to human skin. This plant can grow up to 0.60 m tall, and also used as a food, medicine and possess other uses. Ranunculus sceleratus Linn. is commonly spread in the temperate and cold region in Global distribution (Indonesia, Malaysia, Nepal, Sri Lanka and India). According to Bentham and Hooker classi ication “Genera Plantarum”, this plant belongs to the division Polypetale of Dicotyledones which processes more than 600 species. The Phytochemical screening was performed according to API norms, in addition to this UV VIS, FTIR, TLC and HPLC test were also carried out for further validation. The spectroscopy and chromatography examination revealed the presence of lavonoids, phenols and various other Phytoconstituents in Ranunculus sceleratus Linn. The spectroscopy and chromatography validation can help us it for medicinal and commercial purposes.


INTRODUCTION
Ranunculus sceleratus Linn. is also known as Kandira (Lucas, 2008), it is grown in ponds, lakes and water bodies, having modi ied Root, Stem and coriander leaf type leaves, branched rhizome, striated seed and globular fruit (Das, 2012). The whole plant is used as a medicine in Plague disease (Sharma, 2004).
Rananculus sceleratus such as it is a virulent poisonous plant, producing violent effect if taken internally and the bruised leaves applied to the skin act very ef icaciously as a vesicant, it is used by beggers to keep open sores once caused by vesication or other means. In traditional medicine, Ranunculus sceleratus is used in malaria, scorpion bite, blood stasis, acute icteric hepatitis and internal abscess, Ranunculus sceleratus Linn. showed pharmacological effect such as antibiosis and relief of articular effusion.
Although it is one of the primitive types of species a very brief illustration is found in different botanical and Ayurvedic treatises. The scarcity over the information regarding the characteristics and its uses motivates the author to review it extensively in various research journals and other related literature. And to standardized the parameter after performing phytochemical, physiochemical, TLC, FTIR studies. Such exploration and validation help the society in the treatment of various disease and for medicinal and commercial purpose.

Collection
Ranunculus sceleratus Linn. was collected from its natural habitat near Banaras Hindu University, Varanasi, in February 2018.

Authentication of plants
Sample (Voucher specimen no. Ranunculus.2018/3) was authenticated by the experts from the Department of Botany, Institute of Science, Banaras Hindu University, Varanasi. Plant specimen was deposited in the museum of Department of Dravyaguna, Faculty of Ayurveda, for future reference.

Chemicals
All analytical grade chemicals used in the study were purchased through Advanced Quality traders, E. Merk, Germany.

Phytochemical screening
Plant was extracted in seven solvents (Petroleum ether, chloroform, Acetone, Benzene, Ethanol, Methanol and Distil water) are determined by their relevant chemical test with appropriate testing agents or reagents.

UV-VIS (ultraviolet, visible spectroscopy)
One gram of plant extracts was added in 10 ml of distilled water then iltered with the help of cartilage (0.2µm). Afterwards, it was scanned under ultraviolet, visible spectrophotometer (λ 25 Perkin Elmer) at a range of 200-900nm to measure the size of biomolecules and uncertainty source that may arise from nature of the compound of plant extract.

FTIR (Fourier-transform infrared spectroscopy)
A pinch of powder drug was taken and placed over the crystal present on stage. The IR spectrum (Perkin Elmer, Spectrum-2) was scanned between 4000 to 400-1 and transmittance was recorded. Before scanning the sample, the background signal was also recorded. The peaks thus obtained were matched against IR interpretation chart, and the functional groups were noted.

Modern chromatography TLC (Thin layer Chromatography)
The extract was applied 2 cm on the lower edge of the plate by the help of a microcapillary tube. And then extracts were loaded in small-volume spot on each plate, the plate was taken out, the solvent front was marked, and the plate was dried at room temperature. Thin-layer chromatography was detected by observation of spots for identical Rf value and to determine the purity of a sample.

RESULTS AND DISCUSSION
Directly or indirectly, herbal plants are used upon their characteristics, and their character features are detected through different parameters like spectroscopy and chromatography.

Phytochemical screening
Phytochemical Screening of Ranunculus sceleratus Linn. in a different solvent.

UV-VIS (Ultraviolet, visible spectroscopy)
The Ultraviolet-visible spectroscopy pro ile of Figure 1 of the extract was observed at 200-900 nm wavelength range and 265 nm recorded band. In the previous study, Phenolic and lavonoids components generally absorb at 230-290 nm (Mishra et al., 2015). Hence, it con irms the presence of pheno and ϑlavonoids in the extract of Ranunculus sceleratus.

FTIR (Fourier-transform infrared spectroscopy)
FTIR is a characterisation method which gives the vibration energy based on peak value (Kumar and Ramaswamy, 2014;Mishra et al., 2015). The compressing act of the functional group that are available on the extract of Ranunculus sceleratus. The major bands were observed at 3288,2920,1594,1403,1314,1237 and 1026 cm −1 in Figure 2.
According to Table 2, The peak indicates OH stretching might be alcohol, carboxylic acid. OH bending show phenol. CH stretching is alkyne and alkane. NH stretching is amine salt, and NH bending is an amine. C=C Stretching is Cyclic alkene. S=O Stretching is Sulfone, Sulfate and Sulfonyl Chloride. C-F Stretching is Fluro compound. C-N Stretching is Amine and Aromatic Amine. C-O Stretching is Alkyle arile ether, and C-X (X=Cl or Br) is halo compound.

Modern chromatography TLC (Thin layer Chromatography)
According to the Table 3, Chloroform extract showed maximum mean R f value which is 0.641 and Distil Water could not detect any peak that's, why the mean R f value is 0. In the previous study, Maximum R f value shows highly pure compound and less R f value indicates impurity of the compound (Kanoujiya et al., 2016).

HPLC (High-performance liquid chromatography)
A typical HPLC chromatogram of all standard recorded at 368 nm is present in different igure (Figure 3), and a brief summary of HPLC instrument working is shown in analytical condition (Table 4).
According to In the previous study, Rutin and Caffeic acid both were present in another species of R. arvensis (Bhatti et al., 2015).

CONCLUSION
The present study evaluates phytochemical, spectroscopy and chromatography of the whole plant of Ranunculus sceleratus Linn. for correct identi ication and standardisation and also indirect developing for further research. In Phytochemical study,

ACKNOWLEDGEMENT
The corresponding author expresses her sincere gratitude to her supervisor and co-supervisor for their guidance.
Thanks, to the Head of the department of zoology and interdisciplinary school of life science for providing basic facilities during this study, Dr Jasmeet Singh, Arun Srivastava, Shalvi Agrawal, Suraj and to providing support. Also, thankful to Dr Anurag Mishra for the valuable advice and Veena for the required editing in the manuscript.
I acknowledge sincere thanks to DST for providing support to my studies done in Department of Kriya Sharir and Dravyaguna Laboratory.

Funding support
None.

Con lict of Interest
The corresponding author declare no con lict of interest.