INTRODUCTION:

The world is rich in medicinal plants. From ancient times herbs are in use as medicine throughout the world. Many of the drugs currently in use have been derived from herb, faunae or mineral deposits. Around the globe, 80% of inhabitants apparently use herbs for remedies¹. The remedial property of the plants is endorsed to the chemicals as secondary metabolites available in the plant.

The leaves of Putranjiva roxburghii Wall plant was used for the study, which belongs to family Euphorbiaceae. They are refrigerant and used in catarrh and to improve fertility. Seeds are sweet, anti-inflammatory and are used to treat vata and pitta, hyperdipsia, opthalmopathy, azoospermia, habitual abortion and sterility². While the fruits are anodyne, anti dysentric, stimulant and tonic. Leaf decoction sometimes prescribed for washing infected eyes³. The fruit stones are believed to protect from evil eye and the lives of children. The leaves, the fruits and the fruit stones are used in fever and cold⁴. The nuts of Putranjiva roxburghii, wall; in Sanskrit putra-jiva or putram-jiva, “that which makes the child live”, are hung round the necks of children to keep them in good health. It is mentioned in the nighantus as being also Garbha-kara, “productive of impregnation” and medicinal properties are attributed to them. The band wrinkled nuts are generally worn only as a charm, but are sometimes given internally in colds on account of their supposed heating properties⁵. The beads prepared from the roots of Putranjva tree is used to prevent miscarriage by the locals of Gandaiarea of Chhattisgarh, India⁶. Phytochemical analysis, antimicrobial and antioxidant study on different leaf extracts of Putranjiva was undertaken to substantiate the above facts.

MATERIALS AND METHODS:

Materials:

Streptomycin (CSC Pharmaceuticals, Mumbai), Nutrient agar, mycological peptone (Mitushibiopharma, Ahmedabad), Laboratory grade solvents was procured locally. Strains of organisms (ATCC), Bacillus subtilis (6633), Staphylococcus aureus (29737), Salmonella typhi (19430), Klebsiella pnemoniae (10031), Escherichia coli (10536) and Pseudomonas aeurginosa (9027) were provided by microbiology laboratory, Bharat Institute of Technology, Hyderabad, India.

Collection of plant materials:

Putranjiva roxburghii leaves and fruits of were collected from the forests of Phulbani in Kandhamala district of Odisha, India. Certification of the plant specimen was done by Department of Botany, Bharat Institute of Technology, Hyderabad. Leaves were shade dried and in room temperature.

Pharmacognostical evaluation:

The leaf powder’s organoleptic properties (taste, colour and odor) were documented. Leaf powder was observed under microscope for the presence of cell fragments, fibers, epidermal cells, vessels, crystals of calcium oxalate and starch grains. The ash and extractive values, moisture level, other parameters were found using standard methods.

Extraction:

The leaf extract of Putranjiva were obtained by successive Soxhlet extraction using different solvents. The leaf powder was packed in a pouch of filter paper and put in the Soxhlet extractor. The extraction was done successively starting with petroleum ether (60-80°C) followed by chloroform, benzene, acetone, ethanol. Aqueous extract was obtained by 24h maceration of dried marc in chloroform-water. The solvents in the extracts were removed by evaporation on the water-bath. The consistency, color and produce of the extracts were charted.

Screening of the phytochemicals:

Chemical analysis (Qualitative and Quantitative) was done on crude extracts and the isolated constituents. Chemical components of the extracts were detected by standard phytochemical tests.

Chromatography (TLC):

Sample extracts were prepared by dissolving in their solvent. Samples were and applied to the silica gel G and GF TLC plates followed by development in suitable solvent system to detect chemical components^7,8. Developed plates were dried, the spots on the chromatogram were located to observe the separation of the components of extracts. The locations of steroidal glycosides were observed visually and under UV lamp. Carbohydrates were located by using Anisaldehyde and Sulphuric acid. Saponins by vanillin and sulphuric acid, flavonoids by UV lamp and natural product reagent.

Isolation of chemical components:

The method of Harborne along some changes was followed for the separation of chemical compounds. The leaf powder was mixed with 2M HCl and boiled for one hour in a beaker, cooled and filtered followed by successive extraction with petroleum ether and ethyl acetate. Phytochemical test was done on the crystalline compounds collected in the parts of ethyl acetate and petroleum ether⁹. TLC of the isolated compounds were done on silica gel GF and solvent system benzene: ethyl acetate: glacial acetic acid (9:12:0.5). Spots on the TLC were located using UV lamp.

High performance thin layer chromatography studies:

HPTLC (Camag2016) was used to analyze the ethanol extract and isolated compound.

Visible Spectrophotometric estimation of total Polyphenolic constituent:

The total polyphenolic constituent in the ethanol, water extracts and the separated compound were determined with reference to gallic acid. One ml of 5, 10, 15, 20, 25 µg/ml concentration of the extract was added separately to 1ml of 95% ethanol, 5ml distilled water and 50% folin-ciocalteu reagent (0.5ml). The mixtures were kept away from light for 1 hr followed by immediate recording of absorbance at lmax i.e., 725nm.

Assay of antimicrobial activity:

Petroleum-ether, chloroform, alcoholic and water extracts of Putranjiva was taken to determine their activity against gram +ve and –ve test bacteria using streptomycin as standard and DMSO as control. Cup plate method was followed for the study. All bacteria used were American Type Culture collection.

Preparation of test solutions:

Extracts having 500, 1000 and 1500mg/0.1ml concentration in DMSO, Streptomycin (100mg/ml) was prepared as standard.

Procedure:

The antibacterial activity of the extracts were done following the procedure reported by Kothari and Shrivasthava^10,11, 3-4mm thickness layer of nutrient agar media in 9cm diameter petri dish containing 25ml of media was used. Using sterile borer, cups of 8mm diameter (5 nos) were made after solidification of the medium. Using micropipette 0.1ml solution of extracts and standard were put in the cups. Diffusion of the extracts and standard was facilitated by keeping the plates in room temperature, followed by incubation for one day at 37±1°C. The above steps were followed simultaneously for control and DMSO vehicle for the extract to assess the activity. The study was done in triplicate. Zone of inhibition (diameter) was measured and recorded.

Free radical scavenging activity using DPPH:

Okada & Okada method¹² technique was followed to assess the DPPH free radical for aging action. Fresh ethanolic solution of DPPH (0.05mM) was prepared by adding 96% ethanol with vigorous shaking then kept in dark at 4⁰C. 300μl of the prepared DPPH was mixed with 40μl each from 0.025- 2mg/ml concentration of extract solution. The absorbance (Abs) of the mixture was checked at 517nm using spectrophotometer after 5 minutes. Ethanolic solution of DPPH was used as blank. The determinations were repeated three times. Following equation was used to determine percentage of inhibition, which is the radical scavenging activity^13-17.

Percent of DPPH inhibition= [(Abs blank – Abs test)/Abs blank] × 100

Where Abs blank and Abs test are the absorbance values of the blank and test sample respectively.

Assay of reducing power:

2.5ml phosphate buffer (2 M, pH 6.6), 2.5ml potassium ferricyanide (10g/l) and1 ml of 20-100µg/ml plant extract solution was mixed and kept for 20min at 50°C, added 2.5ml 10% Tri-chloroacetic acid and centrifuged at 1500rpm for 10 min. 2.5ml of the supernatant solution was diluted with equal volume of distilled water and to this 0.5ml FeCl₃ (0.1%) was added. The absorbance was measured at 700nm in UV-Visible Spectrophotometer (Elico UV visible Spectrophotometer 196, INDIA) taking phosphate buffer as reference and ascorbic acid as standard. The absorbance was presented as mean ± standard deviation of the end mixture of two corresponding experiments. Higher absorbance of the mixture indicates higher reducing power¹⁸.

Assessment of peroxide radical scavenging activity:

20-100µg/ml of extracts were prepared in phosphate buffer saline (PBS), mixed with 0.6ml of 4mM H₂O₂ solution prepared in PBS and incubated for 10min. The absorbance at 230nm using ascorbic acid as standard was measured¹⁹.

Assessment of total antioxidant activity:

0.2ml of the extracts (100-500µg/ml) was diluted with 1.8ml of distilled water, to this 2ml of phosphomolybdium reagent solution containing 0.6M Sulphuric acid, 28mM sodium phosphate and 4mM ammonium molybdate was added, stored at 95°C for 90min, allowed for cooling to room temperature and absorbance was measured at 695nm^20-25. Ascorbic acid equivalent was used to represent the antioxidant activity.

Table 1: Rf values for different components by thin layer chromatography of different extracts of p.roxburghii

Compound	Rf value
	Pet Eth Ext	Benz Ext	Ace Ext	Chlor. Ext	Eth. Ext	Wat. Ext
Steroidal glycosides	0.44	0.35	-	-	-	-
Carbohydrate	-	-	-	-	0.22 0.53 0.71	0.80 0.71 -
Saponins	-	-	-	0.53 0.94	0.93 -	0.82 -
Flavonoids	-	-	-	0.26 0.44 0.86	0.44 0.62 0.86	0.26 0.82 0.93

Pet Eth E: Petroleum ether extract; AE: Acetone extract; Eth Ext: Ethanol extract; Benz Ext: benzene extract; Ace Ext: Aqueous extract; Chlorof Ext: Chloroform extract.

Table 2: Assessment of total phenolic compound in p.roxburghii leaf extract.

Concentration	Absorbance at 725nm
	Ethanolic extract	Water extract	Isolated compound	Gallic acid (std)
10	0.003±0.001	0.004±0.001	0.004±0.007	0.17±0.014
20	0.005±0.003	0.071±0.007	0.062±0.002	0.28±0.002
30	0.22±0.002	0.26±0.001	0.26±0.015	0.37±0.006
40	0.26±0.002	0.44±0.001	0.35±0.02	0.53±0.002
50	0.51±0.002	0.69±0.003	0.62±0.15	0.87±0.003

Fig. 1: HPTLC Chromatogram of isolated compound (?).

Fig. 2: HPTLC Chromatograms of Ethanol extract.

Fig. 3: 3D photographs of the isolated compound and ethanol extract.

Fig. 4: Radical Scavenging activity DPPH, of leaves extracts of P. roxburghii and Vitamin- C

Fig. 5: Reduction power of leaves extracts of P. roxburghii and Vitamin-C

Fig. 6: H₂O₂ Scavenging activity, of leaves extracts of P.roxburghii and Vitamin-C

Fig.7: Sum of Antioxidant activity of leaves extracts of P.roxburghii and Vitamin-C

RESULTS AND DISCUSSION:

Study on Pharmacognosy:

Shade dried Putranjivaleaves were coarsely powdered. The organoleptic, microscopic and physical characteristics of the powdered drug were evaluated. The color of the powders was greenish red with distinct odor without taste. The leaves were coriaceous dark green, shining, 6.3–10cm by 2.2–3.8cm, elliptic oblong, obtuse or acuminate, distantly serrulate, main nerves slender, with reticulate venation between; petioles 6mm long; stipules small triangular acute. Microscopic evaluation of the leaf powder was done. The result showed presence of rectangular to square shaped thick walled epidermal cells with and prominent cuticle, wide angular thick walled short radial multiples of xylem elements, stomata is anomocytic and presence of sporadic Calcium oxalate (Spherocrystals).The powdered leaves found to have 8%, 3.6%, 5.8% of total, acid insoluble and water soluble ash with water soluble extractive 6.2%, 3.9% of alcohol soluble extractive and 8.6% total moisture content. These differences in values are due to the variation in the chemicals, specifically the polar plant constituents.

Phytochemical analysis:

Powdered drug was extracted by successive soxhlation with different solvents like petroleum ether, chloroform, benzene, acetone, ethanol and water. Finally, maceration of the marc was done with chloroform water I.P. Primary phytochemical analysis of the extracts was done. The P.roxburghii leaf extracts contain carbohydrates, flavonoids, saponins and phytosterols. Carbohydrate was found the successive ethanol and aqueous extract. Saponins are present in ethanol, acetone and aqueous extract. Flavonoids were found in all extraction solvent except petroleum ether and benzene extracts, whereas phytosterols were found in Petroleum ether extracts.

Thin layer chromatography findings:

R_f values were calculated for all the spots presented in Table 1. Benzene and petroleum ether extracts do not contain Carbohydrates, saponins or flavonoids. Saponins and flavonoids were found in acetone, chloroform and ethanol extract. Carbohydrates were found in acetone and ethanolic extract. Extracts of petroleum ether and benzene found to have steroidal glycosides. The difference in R_f value may be resulting due to impurity in the solvent, composition of solvent system, used adsorbent, solvent polarity, absorbent substance etc.

Isolation of chemical compounds:

The ethyl acetate, petroleum ether fraction and isolated crystalline compounds were tested for presence of phyto chemicals.The results revealed the presence of carbohydrates and tannins, ethyl acetate fraction contained flavonoids. Petroleum ether fraction contained steroids and steroidal glycosides. Isolated compounds were only positive to phenolic compounds test. TLC of ethanolic extract was developed. The R_f value for the spot of the isolated compound was 0.29matching to a spot found in ethanolic extract. Five spots were seen in the HPTLC profile of ethanolic extract. The R_f values for the 5 spots were 0.11, 0.29, 0.41, 0.56, 0.71 and the % area under curve of the were 30.7, 19.13, 16.81, 11.31 and 23.03, respectively. The isolated compound has R_f value 0.27 and % area under curve 33.11, matching with a spot of Rf value 0.29 seen in the ethanolic extract. The concordance spot found in both TLC and HPTLC reveals the co-presence of isolated compound with others. Presence of phenolic content in the isolated compound from the ethanol extract was confirmed by the chemical analysis. Figures 1 and 2 represents the HPTLC chromatograms of isolated compound and ethanol extract and figure 3 shows the 3D photographs of the isolated compound and ethanol extract.

Estimation of total polyphenolic content:

Sum of the polyphenolic content for the aqueous, ethanolic extract and separated compound was assessed by UV-Visible Spectrophotometric method.The findings are given in Table 2. The water extract showed presence of significant amounts of phenolic compounds in comparison to ethanolic extract and the standard.

Antimicrobial activity:

The antimicrobial activity was assessed by cup plate method against various strains of bacteria. Chloroform, petroleum ether, ethanolic and aqueous extracts of the leaves of P. roxburghii was used for the study. The zone of inhibition in mm (Mean ± standard deviation, n = 3) for the standard streptomycin was found to be. 6.0±1.9, 5.4±1.9, 6.2±1.2, 6.0±0.2, 9.0±0.1 and 6.0±1.5 for Klebsiella pneumoniae, Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeurginosa and Salmonella typhi, respectively. In this study, the extracts did shown activity against mentioned microorganism (table 3), which was in concordance with the report^26,27 that stated that P. roxburghii methanolic extract showed a significant antimicrobial activity²⁸ and this justifies its use to wash infected eyes.

In vitro antioxidant activity:

Leaf extracts (Petroleum ether, Chloroform, aqueous and alcoholic) and isolated compound of P. roxburghii were evaluated for antioxidant action using Vitamin-C as standard. Radical scavenging activity DPPH, reduction power, H₂O₂ scavenging activity and sum of antioxidant activity of leaves extracts of P. roxburghii and Vitamin-C are expressed in Figures 4, 5, 6 and 7. There are reports about the antioxidant or free radical scavenging activity of flavonoids, tannins or phenolic compounds of plants²⁹. The DPPH test gives insight about the scavenging activity of the compounds against steady free radicals. DPPH shows high absorption in the visible range at 517nm. DPPH upon reaction with open radical, the single electrons gets paired, which causes change in color from deep violet to light yellow results in reduced absorption. The reduction in absorbance is the indicator of the radical scavenging potential. The aqueous extract of P. roxburghii leaves seems to be equipotent as Vitamin-C with a highest inhibition of 59.16% at 100µg/ml whereas for Vitamin-C it was 66.4% for the same concentration. Extracts by petroleum ether had less inhibition in comparison to isolated compound, other extracts and Vitamin-C. Reductants are responsible for the antioxidant activity, and they cause breaking of the chain of free radicals by giving a hydrogen atom. The reductants in P.roxburghii leaves extracts reduces Fe³⁺ ferricyanide complex to the ferrous (Fe²⁺) which can be measured spectrophotometrically at 700nm due to formation of Perl’s Prussian blue. Reductive potential of the extracts and ascorbic acid has been represented in Figure 5. P.roxburghii leaves extracts have strong reducing power, which is dose dependent. The aqueous leaf extract (100µg/ml) of P. roxburghii has maximum absorbance of 0.35 where as Vitamin-C has 1.5 at the same concentration. Water extracts found to have better hydrogen peroxide scavenging activity than Vitamin-C in terms of the absorbance value. Petroleum ether and chloroform extracts has poor antioxidant activity, so other extracts and isolated compounds have been considered for estimating antioxidant in total and compared with Vitamin-C taking five concentrations ranging from 100-500µg/ml with a difference of 100µg/ml. The water extract was found to have better total antioxidant activity in terms of absorbance than Vitamin-C. This investigation revealed the antioxidant potential of the plant against the oxidative damage. In the recent past there several reports about the usage of plant having antioxidant activity for the green synthesis of nanoparticles^27,30,31.

CONCLUSION:

The plant P. roxburghii has phenolic compounds and other compounds contributing significantly to its antimicrobial and antioxidant property favoring its use as traditional medicine. However structural elucidation of the isolated compound using IR, NMR and mass spectroscopy is highly recommended as a part of future study.

ABBREVIATION USED:

TLC-Thin Layer Chromatography, HPTLC- High Performance Thin Layer Chromatography, DPPH-α, α-diphenyl-β-picryl hydrazyl, DMSO-Dimethyl Sulfoxide\

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Received on 04.10.2020 Modified on 29.11.2020

Research J. Pharm. and Tech 2021; 14(12):6216-6222.

DOI: 10.52711/0974-360X.2021.01076

Extract	Bacillus subtilis	Staphylococcus aureus	Klebsiellaphuemoniae	Escherichia coli	Pseudomonas aeurginosa	Salmonella typhi
streptomycin	5.4±1.9	6.2±1.2	6.0±1.9	6.0±0.2	9.0±0.1	6.0±1.5
petroleum ether	1.4±1.3	1.1±1.6	2.0±1.4	1.8.0±0.8	2.0±0.13	1.6.0±1.7
chloroform	3.3±1.7	4.5±1.8	5.0±1.2	5.2±0.2	7.7±0.6	5.1±1.3
ethanolic	6.3±1.5	6.7±1.4	5.6±1.8	5.7±0.8	8.0±0.5	7.0±1.6
aqueous extracts	2.2±1.4	3.2±1.8	4.0±1.2	3.0±0.8	5.4±0.4	3.0±1.7