Identification and Characterization of Bioactive Compounds of Leaves of Justicia Gendarussa Burm. F

The present investigation has been carried out to find the phytochemicals present in Justicia gendarussa leaves extract. The leaves of Justicia gendarussa were collected from Kadukaval in January 2018 Thanjavur, Tamil Nadu, India. The powder leaf was extracted with aqueous and 70% methanol for 24 hours and studied for FTIR, HPLC and NMR. A preliminary phytochemical testing of the leaves extract where than to identify the phytoconstiuents, which reveals that presence of tannin, saponin, flavonoids, steroids, terpenoids, triterpenoid, carbohydrate, anthroquinone, polyphenol, glycoside bioactive compound was isolated by column chromatography technique. The collected flavonoid fractions was purified by thin layer chromatography. FTIR, HPLC and NMR studies were carried out to find the structure of bioactive compound Quercetin. 1 HNMR and 13 C – NMR that reveals the structure of flavonoids. The compound was identified as 3, 3’, 4’, 5, 7 – pentahydroxyflavanone by 1 HNMR and 13 C – NMR. All these data obtained in the present investigation supported the rich source of phytochemicals preent in J.gendarussa leaves extract and therby traditional claim associated with J. gendarussa literature. KeywordsJusticia gendarussa, phytochemical, FTIR, HPLC and NMR


I. INTRODUCTION
World Health Organization estimate over 80% of the people in developing countries depend on traditional medicines for their primary health care 1 . India is one of the largest producers of medicinal herbs and is rightly called the botanical garden of the world as it is sitting on a gold mine of well-recorded and traditionally well practiced knowledge of herbal medicine. Nearly 17,000 species of Indian flora, and 7500 species of higher plants are reported to possess medicinal value and in other countries it is projected about 7% and 13%. There are estimated to be around 25,000 effective plant-based formulations, used in folk medicine and known to rural communities in India 2 .
The search for new molecules, nowadays, has taken a slightly different route where the science of ethno botany and ethno pharmacognosy are being used as guide to lead the chemistry towards different sources and classes of compounds 3 . Plant derived natural products hold great promise for discovery and development of new pharmaceuticals 4 .
The search for biologically active compounds from natural sources has always been of great interests to researchers looking for new sources of drugs useful in various diseases. The indigenous population has developed vast knowledge on the uses of plant as traditional medicines to protect themselves and their crops, plants are known to contain numerous biologically active compounds which possess curative properties. Within a decade, there were a number of dramatic advances in analytical techniques including TLC, UV, NMR, FTIR, HPLC, HPTLC and GC-MS that were powerful tools for separation, identification and structure determination of phytochemicals 5 . Biological screening is necessary to provide a scientific basis for validating the traditional utilization of medicinal plants. A great number of screening programs are going on worldwide for new plant based bioactive molecules. NMR, HPLC and FTIR can be used to study traditional medicines and characterize the compound of interest. In the present study to investigate the bioactive compound in J. gendarussa leaves.

Collection of Plant materials:
The leaves of Justicia gendarussa were collected from Kadukaval in January 2018 Thanjavur, Tamil Nadu, India. Identified with the help of Flora in Carnatic 6 .

Preparation of alcoholic extract:
The leaves of J. gendarussa were first washed and dust was removed. The leaves were washed several times with distilled water to remove the traces of impurities from the leaves. The leaves were dried at room temperature and coarsely powdered. The powder was extracted with aqueous and 70% methanol for 24 hours. The extract was stored in refrigerator until used.

Phytochemical screening:
Chemical tests were carried out on the alcoholic extract and on the powdered specimens using standard procedures to identify the constituents as described by Sofowara 7 , Trease and Evans 8 and Harborne 9,10 .
Quantitative assay: Determination of total phenols by spectrophotometric method. Flavonoid determination by the method 11 .

HPLC Analysis:
Flavonoids were analyzed by using HPLC method 12 .

Column Chromatography:
Separation of flavonoid compound using in column chromatography adopted by the method 13 .

Thin layer chromatography :
Thin layer Chromatography is based upon the principles of column and partition Chromatography. A thin layer of the stationary phase is formed on a suitable flat surface, such as glass. Separation of a mixture in this case is achieved over a thin layer of silica gel to which they are absorbed by different physical forces 9,10 .
Fourier Transform Infrared (FTIR) spectroscopic analysis FTIR spectrophotometer (Perkin Elmer Spectrophotometer system, USA) used to investigation of spectrum. A small amount of plant extract was respectively placed directly on sample holder of the infrared spectrometer with constant pressure applied and data of infrared absorbance, collected over the wave number ranged from 4000 cm -1 to 400 cm -1 and computerized for analyses by using the 21 CFR part 11 software. The reference spectra were acquired from the cleaned blank crystal prior to the presentation of each sample replicate. The peak values of FTIR were recorded.

NMR Spectroscopy
After the separation of plant extract to fractions using Column chromatography, Thin Layer chromatography was used for further purification of collected fraction. The NMR experiment was carried out in BRUKER-AMX400 MHz instrument with 5mg of purified compound in DMSO were used for 1 H NMR and 13 C NMR spectra recorded. Tetra Methyl Silane is used as the internal standard and chemical shifts are expressed in ppm.

Qualitative analysis
The phytochemical characters of the leaves of J. gendarussa investigated and summarized in Table 1

Column chromatography
Column chromatography of J. gendarussa leaves extract afforded 3 fractions. The result of chromatographic separation is given in Table 2

Thin Layer Chromatography
The presences of secondary metabolites in the extracts were detected by TLC using suitable reagents. The presence of flavonoid was detected by information of pale yellow colour spot in the positive reaction by exposure of ammonia. In the present study flavonoid was in the leaves detected of J. gendarussa (Table 3).

HPLC
HPLC study reveals the presence of quercetin in the leaves of J. gendarussa (Table 4 and Fig 6).

NMR spectrum leaves of J. gendarussa extract
Nuclear magnetic resonance (NMR) spectroscopy has evolved as one of the most powerful analytical techniques. It allows the visualization of single atoms and molecules in various media in solution as well as in solid state. NMR is nondestructive and gives molar response that allows structural elucidation and quantification simultaneously. Magnetic interactions between NMR active nuclei along covalent bonds result in spin-spin couplings. Through space interactions can be detected using the Nuclear Overhauser Effect (NOE). Both these interactions facilitate the three dimensional structure elucidations. One dimensional and two dimensional NMR data can be collected. The 1D NMR experiments are 1H, 13C, 31P, 19F, etc. The 1D NMR techniques will give information regarding the chemical shifts, spin-spin couplings and intensities. The chemical shifts will give the information regarding environment of the protons. Nuclei which are close to one another exert an influence on each other's effective magnetic field. This effect shows up in the NMR spectrum when the nuclei are non-equivalent. If the distance between non-equivalent nuclei is less than or equal to three bond lengths, this effect is observable. This effect is called indirect spin-spin coupling. The intensities will give the relative number of protons under the peak. The 2D NMR experiments are COSY, TOCSY, HSQC, HMBC, NOESY, ROESY, etc. These 2D experiments provide information regarding through bond or through space interactions.
The intensity or the integral of a signal is considered to be the area under that signal. The comparison of the signal intensities in a spectrum will give the ratios of the protons in the molecule. If there are multiples in a spectrum, the whole group of peaks should be integrated. Just like the chemical shifts and indirect spin-spin couplings, the signal intensities are also important for the structure determination. The signal intensities will help in the quantification of mixtures. In principle, the intensities of carbon-13 signals can also be used to infer the number of carbons responsible for the signal. Practically, the low abundance and sensitivity of the carbon-13 isotope will affect the quantification of number of carbons in a molecule. Due to this reason the carbon signals are generally not integrated in 13C NMR spectrum. The quantification of carbon-13 signal can be made possible with high digital resolution, suppression of NOE, a pulse repetition rate that is not too fast and small spectral width and high pulse power. The one dimensional NMR spectra have two dimensions, the abscissa and the ordinate. The abscissa corresponds to the frequency axis and the ordinate gives the signal intensities. But, in two dimensional (2D) NMR spectra, both the abscissa and the ordinate represent frequency axes; the third dimension gives the intensities. In the 2D J-resolved NMR spectrum, the chemical shifts will be plotted along one of the axes and the coupling constants along the other dimension. If both axes are chemical shifts, then it is called 2D (shift) correlated NMR spectrum. Most often the shift correlated 2D NMR data is used in structure elucidation. The correlations could be homo nuclear (H1-1H) or hetero nuclear (1H/13C) ( Table 5).

IV. DISCUSSION
Phytochemicals in plant material have raised interest among scientists, food manufacturing and pharmaceutical industry, as well as consumers for their roles in the maintenance of human health. Phytochemicals are the bioactive, non-nutrient, and naturally occurring plant compounds found in fruits, vegetables, and whole grains. They can be categorized into various groups, i.e., polyphenols, organo sulfur compounds, carotenoids, alkaloids, and nitrogen-containing compounds. Many phytochemicals are potent effectors of biologic processes and have the capacity to influence disease risk via several complementary and overlapping mechanisms 14 . The phytochemical screening of aqueous extract of J. gendarussa leaves showed that the presence of flavonoids, terepenoids, steroids, saponins, triterpenoids, phenolics, alkaloids, carbohydrate, anthriquinone and glycosides while phlobatannins, tannin and protein were absent.
Abuzar et al. 15 reported the phytochemical analysis of Heliotropium dasycarpum and evaluating the presence of secondary metabolites like alkaloids and cardiac glycosides while the saponins, anthroquinone, glycoside and tannins were absent in the plant extract.
A simple, accurate, and reproducible high-performance liquid chromatography (HPLC) method has been developed and validated for the quantification of flavonoids 16 . Quercetin were confirmed in Justicia gendarussa using HPLC 17 were carried out to characterize the phenolic acids and flavonoids in methanolic extracts of Withania somnifera leaves by HPLC. Five phenolics (gallic, syringic, benzoic, p-coumaric and vanillic acids) and three flavonoids (catechin, kaempferol and naringenin) have been identified in Withania somnifera leaves. Similarly catechol, gallic acid, ellagic acid, and catechin of compounds were identified in Cissus vitiginea leaves among the four compounds of this present study.
Paranthaman et al. 18  Fourier Transform Infrared (FTIR) Spectroscopy is a rapid, noninvasive, high resolution analytical tool for identifying types of chemical bonds in a molecule by producing an infrared absorption spectrum that is like a molecular fingerprint 19 . FTIR has been shown to be a valuable tool for differentiating, classifying and discriminating closely related microbial strains, plants and other organisms 20,21 . It is one of the most widely used methods to identify the chemical constituents and elucidate the structural compounds and has been used as a requisite method to identify medicines in pharmacopoeia of many countries.
It is well known that the medicinal materials comprise hundreds of components and produce their curative effects through mutual effects of many ingredients, so the limited numbers of specific components cannot availably reflect the real qualities of the herbal medicines. Therefore, an effective and inexpensive analysis method to entirely monitor the whole constituents of the medicinal materials and their corresponding extract products is required 22 .
FTIR has played a vital role in pharmaceutical analysis in recent years 23 . FTIR spectroscopy is a physicochemical analytical technique that does not determine concentrations of individual metabolites but provides a snapshot of the metabolic composition of a tissue at a given time 19 . The FTIR method measures predominantly the vibrations of bonds within chemical functional groups and generates a spectrum that can be regarded as a biochemical or metabolic "fingerprint "of the sample 24 .
FTIR has proven to be a valuable tool for the characterization and identification of compounds or functional groups (chemical bonds) present in an unknown mixture of plants extract 25,26 . FTIR spectrum of the J. gendarussa leaf extract was pronounced absorbance was recorded in the region between 4000 and 400 cmˉ¹. The peak indicates alcoholic and phenolic groups, alkenes (C-H strech stretch), alkenes (C-H stretch), carboxylic acids (O-H strech), alkenes (-C=C-stretch), alkynes (C=C-stretch), aromatics and alkenes (C-C stretch (in-ring) and C-H bend), aromatics (C-C stretch (in-ring), aromatic amines (C-N stretch), alkynes C-O stretch and C-N stretch), 1049.33 indicates aliphatic amines C-N stretch).
Karpagasundari and Kulothungan 27 screened the bioactive components of Physalisminima leaves have been evaluated using UV-visible and FTIR. The UV-visible profile showed the peaks at 315.09 nm, 408.09 nm and 676.50 nm with the absorption 0.247, 0.106 and 0.003 respectively. The results of FTIR analysis confirmed the presence of phenol, alkanes, aldehyde, secondary alcohol, amino acid, aromatic amines and halogen compound. The results of this study offer a platform of using Physalisminima leaves as herbal alternative for various diseases.
Nuclear magnetic resonance (NMR) spectroscopy is usually the method of choice for natural product structure determination and it is not surprising that this powerful technique has come to the fore in plant metabolomics. The data requirements for metabolomics are the qualitative and quantitative analyses of the maximum number of metabolites in the highest achievable throughput. Most metabolomics laboratories deploy a range of spectroscopic technologies but use of NMR spectroscopy, particularly as a first pass screen, has a number of advantages over other analytical platforms currently being used. Sample preparation is relatively simple when compared to other analytical methods and a high sample throughput with little instrument drift is readily achieved. NMR is not discriminatory unlike certain mass spectrometry methods that rely on the prior derivatization of metabolites or the ability of them to ionize. Metabolite screening requires maximum sensitivity with a broad compound coverage. Based on 1 H and 13 C data has been characterized as Quercetin (Molecular Formula: C 15 H 10 O 7 ). Sumit Arora and Prakash Itankar 28 showed the extraction, isolation and identification of flavonoid from Chenopodium album aerial parts. The flavonoids contained in C. album aerial parts were extracted, identified and characterized. Sequential soxhlet extraction was subjected to preliminary screening and flavonoid quantification. The results showed that maximum yield of the flavonoid (7.335 mg/g) were obtained from acetone extract. This acetone extract was subjected to flash chromatography for isolation of flavonoid. Characterization of isolated flavonoid was done by UV, IR, 1H & 13C NMR and MS. On the basis of chemical and spectral analysis structure was elucidated as 2-(3, 4-dihydroxyphenyl)-3, 5, 7-trihydroxy-4H-chromen-4one, a flavonoid.
Ahmadu et al. 29 examined flavonoid glycosides from Byrsocarpus coccineus leaves. The bioactive ethyl acetate and N-butanol soluble parts of an ethanolic extract of Byrsocarpus coccineus leaves was subjected to column chromatography over silica gel G (60 -120µ) and repeated purification of the flavonoid rich fraction over sephadex LH-20 eluted with methanol led to the isolation of three flavonoid glycosides identified as quercetin 3-O-αarabinoside (I), quercetin(II) and quercetin 3-β-D-glucoside.
Present study concluded that FTIR, HPLC and NMR studies were carried out to find the structure of bioactive compound Quercetin. 1 H-NMR and 13 C -NMR that reveals the structure of flavonoids. The compound was identified as 3, 3', 4', 5, 7pentahydroxyflavanone by 1 H-NMR and 13 C -NMR.
All these data obtained in the present investigation supported by traditional claim associated with J. gendarussa literature. Further biological studies along with chemical characterization of the separated compounds will have the way for a better understanding of the bioactive product and its subsequent application in disease.