FLUORESCENCE ANALYSIS, PHYTOCHEMICAL AND ANTIOXIDANT ACTIVITIES IN LEAVES AND STEM OF EMBELIA RIBES BURM. F

Objective: The objective of the present study is to carry out fluorescence analysis, phytochemical, and antioxidant activities using different solvent extract in leaves and stem of Embelia ribes. Methods: In the present study, fluorescence analysis, phytochemical, and antioxidant activities of leaves and stem of E. ribes are carried out using standard procedures. Result: The fluorescence analysis under visible and ultraviolet light for leaves and stem powder of E. ribes treated with various chemical reagents shown different fluorescence effect. In the phytochemical analysis, the methanolic extract of leaves has shown the highest total alkaloid content (19.05±0.42 mg CE/g DW), followed by the stem. The methanolic extract of stem exhibited highest total phenolic content (59.82±2.98 mg GAE/g DW), and ethanolic extract showed highest total flavonoid content (10.05±0.36 mg RE/g DW), followed by leaves. The ethanolic extract of stem possesses highest antioxidant activity toward 1,1-diphenyl-2-picrylhydrazyl (DPPH) (84.86±0.11%), whereas methanolic extract of stem reported highest Ferric reducing antioxidant power (FRAP) activity (72.22±0.31 mg Fe+2E/g DW), followed by leaves. Antioxidant activities (DPPH and FRAP) were significantly correlated with TPC. Conclusion: The results of the present study revealed that the fluorescence analysis of leaves and stem of E. ribes can be helpful for the standardization and quality control of indigenous drug. Both leaves and stem serve as the good source of secondary metabolite and antioxidant agents.


INTRODUCTION
Medicinal plants have been used as resourceful medium in traditional medicinal system from primitive period. The potentiality of plant depends on the presence of phytochemicals such as amino acids, proteins, alkaloids, phenols, flavonoids, and tannins. At present, about 75% of population depends on the traditional system of medicine for the treatment of various diseases [1]. Plant-based chemical substances show very minor side effect and serve as the best curative materials. Medicinal plants, during development, produce a variety of secondary metabolites, in which, phenolic compounds play a key role as antioxidants [2].
Alkaloids and their synthetic derivatives can be utilized for their analgesic and antispasmodic effects [3]. Natural antioxidants act as free-radical scavengers, chain breakers, complexes of pro-oxidant metal ions, and quenchers of singlet oxygen formation such as reactive oxygen species (ROS), which may damage cellular components of DNA, proteins, and lipids [4]. Flavonoid is reported to act as an antioxidants, which are scavengers of a wide range of ROS and also have inhibitors of lipid peroxidation [5].
Embelia ribes Burm F., belongs to family Primulaceae, is a woody liana found in the semi-evergreen to evergreen forest of India and is reported to be red-listed species [6]. The fruits are used as an anthelmintic, diuretic, carminative, contraceptive, antibacterial, antiinflammatory, and anti-astringent as reported in various literatures [7]. Conventionally, the seeds are employed as a remedy for toothache, headache, and snakebite. The seeds are mainly used for maintaining healthy skin and to support the digestive function. It is also effective in the treatment of fevers and for the diseases of chest [8]. Embelin is the principle chemical compound present in the berries of E. ribes. The other chemical constituents isolated from the seeds are quercitol, tannin, christembine, vilangin, resinoid, and volatile oil [9,10]. Hence, taking into consideration the importance of E. ribes, the study was carried out to investigate the fluorescence analysis, along with phytochemicals and antioxidant potential of aqueous and various solvent extracts of leaves and stem using the spectrophotometric method.

Materials
All the chemicals used in the present study including standards were purchased from the Sigma-Aldrich; Himedia.

Sample preparation for fluorescence analysis
Powdered drug of different parts of plant gave different fluorescence under ultraviolet (UV) radiation. Each fluorescence characteristic of the treated sample was observed under visible light and then under UV light of both long and short wavelengths [11]. Therefore, fluorescence evaluation is used for the identification of plant and powdered drug [12]. Some crude drugs are often assessed qualitatively in this way, and it is an important parameter of pharmacognostic evaluation [13,14].
The leaves and stem of E. ribes were dried in the shade to prevent decomposition of active principle and make fine powder for the fluorescence study. A small quantity (1 gm) of dried and finely powdered leaves and stem of E. ribes was treated with freshly prepared acids, alkaline solutions, and different solvents. The drug powders were treated with acids (10% HCl, Conc. HCl, Conc. H 2 SO 4 , and Conc. HNO 3 ), alkaline solutions (1 N aqueous NaOH, 1 N alcoholic NaOH, and 5%

Preparation of extract for phytochemical studies
Leaves and stem powder of E. ribes were extracted with 80% of methanol, ethanol, petroleum ether, and distilled water. Each sample (1 g) was extracted in 10 ml of each solvent separately by continuous shaking on an orbital shaker for 9 h at 110±2 rpm at controlled temperature (30±1°C). The extracts were then filtered through Whatman filter paper No. 1 and stored in air tight vials in a refrigerator.

Determination of total alkaloid content (TAC)
TAC of leaves and stem extracts of E. ribes was measured using 1, 10-phenanthroline method [15]. A volume of 100 µl of plant extract was mixed with 1 ml of 0.05 M of 1, 10-phenanthroline in ethanol and 1 ml of 0.025 M FeCl 3 in 0.5 M HCL, and volume was adjusted to 10 ml by adding distilled water. The reaction mixture was incubated for 30 min in water bath maintained at 70°C. Above reaction mixture excluding plant extract, substituted by distilled water served as a blank. The absorbance was measured at 510 nm against reagent blank. The concentration of TAC was determined by milligrams colchicine equivalent per gram of dry weight by using calibration curve obtained from colchicine (mg/ml) standard solution [ Fig. 1c]. All the samples measurements were performed in triplicates.

Determination of total phenolic content (TPC)
The determination of TPC of the various solvent extracts of leaves and stem of E. ribes was performed using Folin-Ciocalteu assay with some modification [16]. In brief, 125 µl of extract was mixed with 1.8 ml of Folin-Ciocalteu reagent and kept for 5 min at 25°C. After this, 1.2 ml of 15% Na 2 CO 3 was added to the reaction mixture and kept for 90 min at room temperature. The absorbance of the reaction was measured at 765 nm. The concentration of the TPC was determined as milligrams of gallic acid equivalents per gram dry weight (mg GAE/g DW) using an equation obtained from gallic acid calibration curve [ Fig. 1a]. All the samples estimation was performed in triplicates.

Determination of total flavonoid content (TFC)
TFC of various plant extracts of leaves and stem of E. ribes was quantified using the aluminum chloride colorimetric method [17]. The each solvent extract (0.5 ml) was mixed with 1.5 ml methanol, 0.1 ml of 10% aluminium chloride, 0.1 ml of 1 M potassium acetate, and 2.8 ml of distilled water. The mixture was vortexed, and the reaction was kept at the room temperature for 30 min and absorbance of reaction mixture was measured at 415 nm. The concentration of the TFC was determined as milligram of rutin equivalent per gram dry weight (mg RE/g DW) using the calibration curve obtained from rutin (mg/ml) standard solution [ Fig. 1b]. Estimation of all the samples was carried out in triplicate.

Free-radical scavenging assay (DPPH)
The DPPH radical scavenging activity of the various extracts of leaves and stem of E. ribes was estimated using modified and stable DPPH assay [18]. Stock reagent solution was prepared by dissolving 2.5 mg of DPPH in 100 ml of chilled methanol. A volume of 100 μl extract was allowed to react with 3 ml of DPPH solution. The reaction mixture was vortex and kept in the dark at room temperature for 30 min, and absorbance was recorded at 517 nm. A control sample with no added extract was also analyzed, and the results were expressed as percent inhibition/radical scavenging activity (%RSA) and calculated using the following formula: where, A = absorbance at 517 nm.

Ferric reducing antioxidant power (FRAP) activity
The FRAP assay of various solvent extract of leaves and stem of E. ribes was carried out according to the described method by Benzie and Strain, 1996 with some modifications [19]. FRAP reagent formed by assimilation of the acetate buffer (0.3M -pH-3.6), 2, 4, 6-tripyridyl-s-triazine (TPTZ, 10 mM)) in 40 mM HCL, and FeCl 3 . 6H 2 O (20 mM) in 10:1:1 ratio former to use and heated to 37 o C in water bath for 10 min. The plant extracts of various concentrations were allowed to react with 2.7 ml of the FRAP reagent, and the final volume of the reaction was adjusted to 3 ml with distilled water; the reaction mixture was kept in dark for 30 min, and the absorbance was recorded at 593 nm. The results were expressed as FeSO 4 equivalent antioxidant capacity [ Fig. 1d].

Statistical analysis
The experiments were done in triplicates. Results were expressed as mean±standard deviation using Excel.

Fluorescence analysis
The fluorescence study of leaves and stem powder of E. ribes was treated with different chemical reagents and was observed under visible light and UV light (254 nm and 366 nm), which shown predominantly fluorescence effect in both leaves as well as stem. Among the various chemical treatments, the leaves and stem powder of E. ribes showed the characteristic fluorescent green color when treated with 1 N NaOH, 10% HCL, Conc. HCl, Conc. HNO 3 , Conc. H 2 SO 4 , 5% iodine, 5% KOH, 5% FeCl 3, acetone, and distilled water under short UV light [Tables 1 and 2].

Total alkaloid content (TAC)
The amount of TAC determined in different solvent extracts of leaves and stem of E. ribes [ Table 3] showed the methanolic extract of leaves has highest TAC, that is, 19.05±0.42 mg CE/g DW, followed by ethanolic extract with 14.59±0.11 mg CE/g DW.
The petroleum ether extract of leaves has shown 13.38±1.10 mg CE/g DW of TAC, and water extract has shown 9.94±2.86 mg CE/g DW of TAC. The methanolic extract of stem has shown 11.60±2.86 mg CE/g DW of TAC, followed by 10.88±0.41 mg CE/g DW of TAC in ethanolic extract. The petroleum ether extract has shown 8.10±0.58 mg CE/g DW of TAC, and water extract has shown 7.37±4.97 mg CE/g DW of TAC. From the present study, it is found that the TAC is higher in methanolic extract of leaves as compared to the stem of E. ribes.

TPC
The amount of TPC determined in different solvent extracts of leaves and stem of E. ribes [ Table 3] reveals that the methanolic extract of stem shown the highest TPC, that is, 59.82±2.98 mg GAE/g DW, followed by ethanolic extract with 44.65±3.35 mg GAE/g DW.
The water extract of stem has shown the 30.44±1.43 mg GAE/g DW of TPC, and petroleum ether extract reported least 14.39±0.52 mg GAE/g DW of TPC. The methanolic extract of leaves has shown the 55.27±1.29 mg GAE/g DW of TPC, followed by ethanol with 32.23±1.29 mg GAE/g

Ferric reducing antioxidant power activity (FRAP)
FRAP free-radical scavenging activities of different solvent extracts of E. ribes leaves and stem [ Table 4] reported highest amount of FRAP activity in the methanolic extract of stem with 72.22±0.31 mg Fe +2 /g, followed by ethanolic extract with 65.05±0.26 mg Fe +2 /g activity. The aqueous extract of stem has shown 36.89±0.62 mg Fe +2 /g FRAP activity and the lowest FRAP activity, that is, 17.50±0.10 mg Fe +2 /g is exhibited by petroleum ether extract of stem. The methanolic extract of leaves has shown 66.66±0.27 mg Fe +2 /g of FRAP activity, followed by ethanolic extract with 64.91±0.36 mg Fe +2 /g. The aqueous extract of leaves has shown 39.27±1.26 mg Fe +2 /g of FRAP activity. The lowest FRAP activity with 14.95±0.69 mg Fe +2 /g is exhibited by petroleum ether extract of leaves. From the present study, it is found that the FRAP free-radical scavenging activity is higher in methanolic extract of stem as compared to the leaves of E. ribes.

DISCUSSION
Fluorescence analysis is an efficient, responsive, and precise method for the determination of various components in short period of time compared to several time-consuming dilutions steps involved in the analysis of pharmaceutical samples [20,21]. The fluorescence color shows specificity for each compound. The various plant materials show different coloration when subjected to different chemical reagents. It acts as a preliminary pharmacognostic parameter for identification and standardization of a particular drug from its adulterants. At varied wavelength, crude powder of plants shows diverse fluorescence due to the presence of different chemical constituents in the drug [14,22].
A correlation is found between a compound present in the drugs and their fluorescent behavior under different conditions. Under short UV radiation coumarin, especially hydroxyl amino acid derivative such as o-Coumaric acid appears yellowish green in alkaline condition.
Flavonones which appear to be light yellow in aqueous condition, but under UV light, it appears to be bright yellow under alkaline conditions. Phytosterols when treated with 50% H 2 SO 4 appear to be green under UV light. Sapogenin and terpenoids show yellow green fluorescence under short UV light [23]. Quinine, aconitin, berberin, and emetin show specific colors of fluorescence. Least fluorescence is shown by fixed oils and fats [11]. In the present study, the major bioactive compounds present in the crude drug of E. ribes leaves and stem were found to be phenols, flavonoids, tannins, alkaloids, steroids, and quinine.
Alkaloids are utilized as stimulants, narcotics, pharmaceuticals, and poisons. At present, they are used in clinical treatments such as antimalarial quinine, the anesthetic cocaine, the stimulant caffeine and nicotine, the analgesics morphine and codeine, the gout suppressant colchicine, the antibiotic sanguinarine, the antiarrhythmic ajmaline, the anticancer vinblastine, taxol, and certain sedative [24,25]. Here, we are reporting the TAC from leaves and stem of E. ribes using different solvent extracts. Similar result for TAC in different plant parts of Chinchona officinalis has been reported [26].
Phenolic compound enhances the quality of plant by helping in plant growth and reproduction; they are produced in the plant as a response to environmental factors such as pollution, light, and irradiation [27]. From the leaves exact of E. ribes, 57.89±2.94 mg GAE/g dry basis TPC was reported [28], whereas bark reported 26.59 mg GAE/100g of TPC [29].
In the present study, it was noticed that TPC content was higher in stem as compared to leaves in E. ribes. Flavonoid is a polyphenolic secondary metabolite which helps in pollination, as a repellent to pest, responsible for pigmentation of various parts of plant and in nodule formation [30][31][32]. It has potential antioxidant property [33]. From the bark extract of E. ribes, 01.35 mg QE/100g was reported [29]. Since no data are available for TFC in leaves and stem of E. ribes, from the present study, we are reporting the TPC and TFC contents in stem are found to be higher than leaves. Similar kind of work done using different solvent extraction is carried out in P. minima Linn and Eclipta prostrata (L.) L [34,35] Antioxidant, molecules act as protector against free-radical damage. They have shown a major contribution to the prevention of many chronic diseases such as cancer, cardiovascular disease, hepatitis, asthma, atherosclerosis, arthritis, and diabetes [36][37][38]. Since some plant extracts show complication, use of different methods is recommended for the evaluation of antioxidant activity [32]. In the present study, we have used DPPH and FRAP antioxidant assays for leaves and stem of E. ribes. FRAP activity of 60.75±0.16 mg Asc AE/g dry basis was reported in the leaves of E. ribes [28]. It seems no data are available for the DPPH and FRAP free-radical scavenging activities on the stem of E. ribes. It was observed that antioxidant activity in stem is higher than that of leaves. There is influence of different solvents on the extraction of plant material. Similar kind of results are reported from wheat and Salacia chinensis L [38,39]. Antioxidant activity was observed in the phenolic extract from Russula laurocerasi with the similar kind of result [40]. DPPH free-radical scavenging assay was used to observed antioxidant activity in various solvent extract of sweet orange peel and makrut lime [41,42].

CONCLUSION
From the present study, it can be concluded that leaves and stem of E. ribes show the presence of different bioactive compounds in the fluorescence analysis, which might be ideal for various pharmaceutical applications. It is clearly indicated from the present study that polarity of solvent plays a major role in extraction of phytoconstituents. Among the four solvents used for extraction, methanolic extract of stem was found to be the superior compared to ethanolic, water, and petroleum ether extracts with respect to TAC, TPC, and FRAP. Ethanolic extract of stem found to be superior for TFC and DPPH activities. The leaves and stem of E. ribes are good source of antioxidants and secondary metabolite, which can be used for the various pharmaceutical applications.