INTRODUCTION:

Phytochemicals which are present abundantly in plants show many biological and pharmacological properties. Hence, they can be utilized for the development and synthesis of drugs which offer profound benefits for the treatment of inflammatory and infectious diseases without many side effects. They also offer affordable treatment compared to allopathic medicines having deleterious side effects and potential toxicities^1,2,3. Phytochemicals not only protect plants from stress conditions but also act as natural drugs for humans^4,5. Secondary metabolites are phytochemicals that can be used as herbal drugs as they offer protection against pathogens and harmful stress conditions. They also act as natural antioxidants and even prevent the risk of Cancer development^6,7.

Unscientific usage of broad spectrum of antibiotics is not only associated with many deleterious side effects but also, it has lead to the development, emergence and global spread of multi-drug resistant strains of pathogenic bacteria^8,9,10. This has caused concern towards public health in preventing the burden of infectious diseases^11,12,13. It is therefore necessary to find out other strategies (naturally occurring compounds) to control and reduce microbial infections^14,15,16. Research findings have elucidated and demonstrated that the active phytometabolites also protect humans against diseases by boosting immune system functions. It is important to note that around 23% of the prescription drugs contain bioactive compounds that are either derived from plants or they are the modified derivatives of plant metabolites^17,18.

Free radicals are atoms with unpaired electrons that can hinder cellular functions causing damage, illness and aging by interacting with different compounds by electron transfer or proton transfer or by addition or abstraction of H atom. Some examples of free radicals that emerge rapidly, spontaneously and recurrently encountered are nitric oxide radical (NO^.), hydroxyl radical (HO^.), alkyl radical (R^.), superoxide anion radical (O₂^.-) and alkoxy radical (RO^.)^21,22,23. Phytochemicals act as natural antioxidants and neutralize the attacking mechanism of free radicals. Antioxidants are of three types namely: Primary antioxidants acting as free radical terminators or scavengers, secondary antioxidants or preventive antioxidants having stabilizing effects on free radicals and tertiary antioxidants aid in repair of the damaged biomolecules. Additionally, there are some endogenous antioxidants like metal binding proteins, uric acid, glutathione, melatonin etc; having the ability to inactivate free radicals, oxidants and their derivatives ^24,25,26.

The body has an adaptive response called inflammation to external harmful stimuli involving certain physiological and pathological processes, characterized by protein denaturation, membrane alteration, increased vascular permeability, release of kinins, prostaglandins and histamines. All these events will in turn lead to edema, heat, swelling and redness at the injured or inflamed site. Inflammation related diseases can be managed and cured by the consumption of phytomedicines as they act as therapeutics and offer various advantages like high potency, less adverse effects and less toxicity when compared to synthetic drugs having deleterious and cytotoxic effects^27,28.

Angiogenesis is the formation, growth and proliferation of new blood vessels from preexisting blood vasculature involving growth and differentiation on epithelial cells that will further lead to physiological processes like embryogenesis, wound healing, ovulation, lactating breast development, development of Corpus luteum etc. Certain pathological conditions like inflammation, arthritis, spondyloarthropathis, Systemic lupus erythematosus and tumous growth also involve angiogenesis²⁹. Tumor growth is completely dependent on angiogenesis that will establish their own blood supply. This formation of blood vessels is brought about by certain proangiogenic factors like FGF, TGF-β, TNF-α and β, angiopoietins, PDGF, angeonins, HGF and VEGF. Among all these factors VEGF is considered as a prime regulator for both normal and tumour angiogenesis^30,31.

Urochloa ramosa also known as Braharia ramosa or Brown top millet is beneficial in controlling root-knot nematodes in herbaceous crops like tomatoes, cucumbers, melons and pepper. The plant is beneficial as it is fast-growing and covers the soil surface thereby controlling soil erosion. It is also used as a nurse crop for maintaining humidity and water content in soil along with the main crop. U.ramosa can be a potent source of bioremediation of contaminated soil, as it has the capacity to accumulate heavy metals like lead, zinc, cadmium and chromium. In the present study phytochemical analysis, antimicrobial, antioxidant, anti-inflammatory and antiangiogenic activities of methanol extract of Urochloa ramosa was determined to check its biological potency.

MATERIALS AND METHODS:

All reagents and solvents were of analytical grade. Resazurin (sigma), Nutrient agar, nitro blue tetrazoleum, 1,1-diphenyl 2-picrylhydrazl, phenazine methosulphate, hydrogen peroxide, EDTA, ferric chloride (Fecl₃), sulphanilamide, hydrogen ortho phosphate, napthylene diamine dihydrochloride. Fertilized poultry eggs were procured from Indian Veterinary Research Institute, Bangalore. Microorganisms were procured from Dept. of Microbiology, University of Mysore.

Urochloa ramosa was identified from its natural habitat and authenticated by an Angiosperm taxonomist from the University of Mysore. The leaves were thoroughly washed, dried under shade, finely powdered and preserved in air tight containers. 100gm of powdered sample was used and subjected to sequential extractions in Soxhlet apparatus using solvents like hexane, chloroform, ethyl acetate, acetone and methanol (non-polar to polar end). Methanol extract was filtered and used in the present study.

Phytochemical Analysis:

The methanol extract of the plant was subjected to preliminary phytochemical analysis to trace the presence of phytochemicals like Carbohydrates, Tannins, Saponins, Alkaloids, Flavonoids, Glycosides, Quinones, Phenols, Terpenoids, Cardiac glycosides, Amino acids, Coumarins, Anthraquinones, Steroids, Lignin, Phlobatannins, Anthocyanin, Balsams, Volatile oils and Fatty acids as per the standard methods^32,33.

Table 1: Phytochemical tests

Phytochemicals	Test	Observation
Carbohydrates	1ml extract + 1ml Molish’s reagent+ 2 drops of sulphuric acid	Purple colour
Tannins	1ml extract + 2ml 5% ferric chloride	Dark blue colour
Saponins	1ml extract + 1ml distilled water+ shaken well	Foam formation
Alkaloids	1ml extract + 1ml HCl + 2 drops of Mayer’s reagent	Green colour
Flavanoids	1ml extract + 1ml 2N sodium hydroxide	Yellow colour
Glycosides	1ml extract + 2ml chloroform + 1ml 10% ammonia solution	Pink colour
Quinones	1ml extract + 1 ml Sulphuric acid	Red colour
Phenols	1ml extract + 1 ml distilled water + 4 drops 10% ferric chloride	Blue/green colour
Terpenoids	1ml extract + 1 ml chloroform + 1ml sulphuric acid	Reddish brown colour
Cardiac glycosides	1ml extract + 1 ml glacial acetic acid + 4 drops of 10% ferric chloride + 1 ml sulphuric acid	Brown colour
Amino acids	1ml extract + 2 drops of 0.2% ninhydrin+ heat	Pink/purple colour
Coumarins	1ml extract + 1 ml 10% sodium hydroxide	Yellow colour
Anthraquinones	1ml extract + 4 drops 10% ammonia solution	Pink colour
Steroids	1ml extract + 1ml chloroform+ 4 drops sulphuric acid	Brown ring formation
Lignin	1ml extract + 1ml Phloroglucinol-HCl	Red/violet colour
Phlobatanins	1ml extract + 4 drops 2% HCl	Red colour precipitate
Anthocyanins	1ml extract + 1ml 2N sodium hydroxide + heat	Bluish green colour
Balsams	1ml extract + 1ml 90% ethanol + alcoholic ferric chloride	Dark green colour
Volatile oils	1ml extract + 4 drops dilute HCl	White precipitate
Fatty acids	1ml extract + 2ml ether + mixed, evaporated on filter paper	Transparency on filter paper

Anti-microbial activity:

Antimicrobial activity of the methanol extract of U.ramosa was assessed by disc diffusion method against Gram-positive bacteria (B.subtilis, S.epidermidis and S.aureus), Gram-negative bacteria (Proteus, Salmonella and Escherichia coli) and yeast type fungi- Candida albicans. Various concentrations of methanol extract (50µg, 100µg, 200µg and 400µg/disc) were loaded on both the sides of the sterile disc (10mm diameter-HiMedia), dried and were placed in the petriplates containing microbial cultures. Ampicillin and Fluconazole (10µg/ml each) served as positive control. To determine the zone of inhibition, petriplates were incubated at 37˚C for 24h. Minimum inhibitory concentration – MIC assay was also performed by Resazurin method³⁴.

MIC Assay:

Minimum inhibitory concentration was calculated by resazurin method. Resazurin is a crystalline dye used in certain microbiological and enzymatic assays. It is also used to study chemical cytotoxicity in cellular assays. The dye is an indicator for oxidation reduction reactions in which non fluorescent blue colour will be converted to fluorescent pink colour.

Preparation of inoculum:

15µl of inoculum was cultured in nutrient broth for 16 hours at 37^oC. Optical density (OD) was checked at 600nm, after blanking the OD. The absorbance was found to be 0.08 which corresponds to 1-2×10⁸CFU/ml. 2ml of 1:100 diluted culture was prepared using OD adjusted culture to get 1-2×10⁵, from this working culture further dilution was prepared to get a concentration of 1-2×10⁵ CFU/ml.

Dilution of Ampicillin at 8 concentrations:

Ten different eppendorf tubes were taken in a row. To the first tube 20µl of 8µg/ml of Ampicillin was added. The adjacent wells were filled with 5µl of water. From the first well, 5µl of 8µg/ml concentration of ampicillin added to the next well and mixed properly to get 4µg/mL concentration of ampicillin. These steps were repeated till the least being 0.06µg/ml concentration. The same exercise was repeated with the methanol extract of U.ramosa.

Working plate

96 well sterile plates was taken, using multi-channel pipette, 99µl of working culture was added to the wells corresponding to the Ampicillin, as per the plate design. 1µl of serially diluted Ampicillin using multi-channel pipette was carefully added to the designated wells as per the plate design and mixed gently to ensure uniformity

Using multichannel pipette, 90µl of working culture was pipetted into the corresponding wells. 10µl of serially diluted different concentrations of ampicillin and methanol extract containing phenolic compounds were added. The tests were conducted in duplicates in working 96 well plates. After setting up the reaction, plate was closed with a lid and incubated overnight for 16 hours at 37^oC. 10µl of 0.002% resazurin was added to all the wells and mixed gently using multi-channel pipette and incubated for 2hours for development of color, the plate was read at 560nm.

In vitro Antioxidant assays:

DPPH radical scavenging assay:

The reaction mixture consisted of different aliquots of 4-20μg of methanol extract, to this 1mM DPPH solution was added and mixed thoroughly for the assessment of DPPH radical scavenging activity. The aliquots were shaken well and incubated at room temperature for 20 minutes. The decrease in absorbance of DPPH at 517nm with that of a blank was measured³⁵.

DPPH radical scavenging activity was calculated using the following equation.

Scavenging effect (%) = 1- sample absorption / control absorption X100 at 517nm

Superoxide radical scavenging assay:

For the assessment of superoxide radical scavenging activity different aliquots of 10-50μg of methanol extract were mixed with 1mM NADH, 0.1mM Phenazinemethosulphate and 1mM Nitrobluetetrazolium chloride dissolved in 0.1M phosphate buffer. The pH of the reaction mixture was set to 7.4. The reaction mixture was incubated for 5 minutes at room temperature and absorbance was read against the blank at 560nm³⁶.

The superoxide scavenging effect was calculated by

Scavenging effect (%) = [1- sample absorption/control absorption] X100 at 560nm

Nitric oxide radical scavenging activity:

Sodium nitroprusside impetuously create nitric oxide which will interact with oxygen forming nitrate ions that can be estimated by Griess reagent (1% sulphanilamide, 2% H₃PO₄ and 0.1% napthylene diamine dihydro chloride). Phenolics in methanol extract of U.ramosa compete with NO^.radical and utilize oxygen thereby preventing the formation of nitrate ions. Different aliquots of 5-25μg extract were mixed with Sodium nitroprusside (5mM). It was incubated at room temperature for 30 minutes followed by the addition of Griess reagent for the formation of chromophore by nitrite undergoing diazotization with sulphanilamide and successive coupling with napthylethylenediamine. The absorbance was recorded at 546 nm³⁷.

No radical scavenging activity was calculated by:

Scavenging effect (%) = [1- sample absorption / control absorption] X100 at 546nm

DNA Protection Assay:

Calf thymus DNA 20µg/20µL was treated with 10µl of methanol extract of U. ramosa (25μg/ml and 100μg/ml). Fenton reaction was performed. Fenton reaction mixture containing 30% H₂O₂ (4μl) and 2 mM FeSO4 (3μl). The reaction mixture was subjected to incubated for 30 minutes at 37°C. After incubation Bromophenol blue dye (3μl) was added to the reaction mixture and agarose gel electrophoresis was performed using 1% agarose gel, ethidium bromide and TAE buffer. Untreated DNA and DNA mixed with FeSO₄ (2 mM) and H₂O₂ was also run as a control simultaneously. Later the gel was observed under UV trans-illuminator³⁸.

In vitro anti-inflammatory activity:

Albumin denaturation inhibition assay:

Different concentrations (100 - 500µg/ml) of methanol extract were mixed with 1% of bovine albumin and the aliquots were incubated at 37°C for 20 minutes. The samples were heated to 51°C for 20 minutes and cooled. Turbidity of the sample was read at 660nm using UV-Vis Spectrophotometer. 100µg/ml Aspirin was used as standard drug³⁹.

Inhibition of denaturation of albumin protein in terms of percentage was evaluated by

Percentage inhibition = (Abs Control – Abs Sample) X 100/ Abs control

Anti-proteinase activity:

Aliquots having 100 - 500µg/ml concentrations of methanol extract of U. ramosa were mixed with 0.05 mg Trypsin. To this mixture 20mM Tris HCL buffer (1ml) with pH- 7.4 was added. The sample reaction mixture was then incubated at 37°C for 5 minutes, followed by the addition of 1ml 8% casein. The aliquots were again incubated for 20 minutes. 70% perchloric acid (2ml) was added to stop the reaction. There was appearance of cloudy white precipitate. The contents were centrifuged and at 210nm the absorbance of supernatant was recorded against the buffer. Aspirin was used as standard drug for comparison⁴⁰.

The percentage of proteinase inhibitory activity was calculated as

Percentage inhibition = (Abs control –Abs sample) X 100/ Abs control

Stabilization of RBC membrane:

Preparation of Red Blood cells (RBCs) suspension:

Non-steroidal anti-inflammatory drug free blood (5ml) was obtained from healthy volunteer. It was centrifuged at 3000rpm for 5 minutes. Later, the blood was washed with (0.9% NaCl) saline. After centrifugation blood was reconstituted as 10% v/ v suspension using isotonic sodium phosphate buffer (10mM, pH- 7.4)⁴¹.

Heat induced haemolysis:

Various concentrations (100-500µg/ml) of methanol extract of U.ramosa were mixed with 10% erythrocyte suspension (1ml). Saline was used as control and standard drug Aspirin was used for comparative studies. All the aliquots were incubated at high temperature of 56°C in water bath for 30 minutes. The reaction mixture was cooled, centrifuged at 2500rpm for 5 minutes and the absorbance of supernatant was read at 560nm⁴². Percentage inhibition of haemolysis was calculated by

Percentage inhibition = (Abs control – Abs sample) X 100/ Abs control

Haemolysis induced by hypotonic solution:

0.5ml of 10 % erythrocyte suspension, phosphate buffer (1ml) and 0.26% hyposaline (2ml) were mixed with different concentrations (100-500µg/ml) of methanol extract. Standard drug Diclofenac sodium was used for comparison. The assay mixtures were incubated at 37°C for 30 minutes to induce breakage of RBC membrane. The reaction mixtures were centrifuged at 3000rpm for 5 minutes. Supernatant was decanted and the absorbance was recorded at 560nm to estimate percentage of inhibition of hypotonicity induced haemolysis. This can be calculated by the following equation

Percentage inhibition = (Abs control – Abs sample) X 100/ Abs control

Chick Chorioallantoic Membrane assay:

The eggs that were procured from IVRI were surface sterilized with 70% ethanol before transferring it to the fan assisted humidifier incubator at 37^oC. After 3 days of incubation, with the help of needle and scalpel a small window was made at the narrow end of the egg. The window was covered with sterile plastic sheet and eggs were returned to the incubator. On day 6, 100μg of methanol extract was added on to the sterile filter paper disc and the disc was then layered over the blood vessels. Eggs were again placed in the incubator. Antiangiogenic activity involving decrease in blood vessel formation was observed after two days^44,45.

RESULTS AND DISCUSSION:

Qualitative analysis of phytochemicals:

The phytochemicals found in methanol extract is presented in table-2. It was found that the extract is rich in phenolic compounds.

Table 2: Showing the presence and absence of some phytochemicals

Phytocompounds	Methanol extract	Functions
Carbohydrates	+	Dietary supplement
Tannins	+	Healing of wounds
Saponins	+	Coagulation of blood
Alkaloids	-	Diuretic
Flavonoids	+	Free radical scavenging
Glycosides	+	Antiinflamatory
Quinones	-	Anticancerous
Phenols	+	Antioxidant
Terpenoids	+	Antiallergenic
Cardiac glycosides	+	Vasodialators
Amino acids	+	Dietary supplement
Coumarins	-	Antiproliferative
Anthraquinones	-	Estrogenic
Steroids	-	Antiinflamatory
Lignin	+	Laxative
Phlobatannins	-	Astringent
Anthocyanin	+	Antiangiogenic
Balsams	-	Antiulcerative
Volatile oils	-	Antiviral
Fatty acids	-	Membrane stabilization

Note: + indicates presence and - indicates absence of phytoconstituents.

Identification of bioactive compound from the leaves of Urochloa ramosa:

Thin Layer Chromatography [TLC]:

Dry weight of methanol extract was found to be 6.74 gm/100gm of dry powder. Using activated preparative TLC plate measuring 2mm (20cm×20cm), methanol extract was loaded and subjected to development using solvent system - methanol: ethyl acetate: chloroform in the ratio of 1.5:1.5:2. This yielded three separate bands. The middle band was found to be chlorophyll and the top band was scraped into beaker, acetone was added and left for a day. The supernatant was decanted and air dried. The purified molecule thus obtained was found to be phenolics as it was confirmed by biochemical test such as appearance of blue color on treating with Folin- Ciocalteu reagent.

Fig. 1: TLC profile of methanol extract of Urochloa ramosa

Table 3: Antimicrobial activity of methanol extract of U.ramosa - zone of inhibition (mm) induced by plant extract by disc diffusion method

Sl. No.	Name of the organism	Zone of inhibition (mm)
Sl. No.	Name of the organism	50μg	100μg	200μg	400μg	Ampicillin 10μg	Flucanazole 10μg
1	Bacillus subtilis	0.8	1.2	1.6	2.6	18	-
2	Staphulococcus aureus	-	0.5	1.0	1.5	19	-
3	Staphylococcus epidermidis	-	0.8	1.0	1.8	17	-
4	Proteus sp.	-	1.3	1.6	2.0	18	-
5	Salmonella sp.	-	0.8	1.3	1.7	16	-
6	E. coli	0.2	0.8	1.3	2.1	19	-
7	Candida albicans	0.8	1.3	1.5	1.8	-	17

Table 4: Antimicrobial activity of different microorganisms with their MIC values.

*U. ramosa*	*B. subtilis* Gram +ve	*S. aureus* Gram +ve	*S. epidermidis* Gram +ve	*Proteus* Gram –ve	*Salmonella* Gram -ve	*E. coli* Gram –ve
*U. ramosa*	MIC values in µg/ml
Methanol extract	300μg/ml	145 μg/ml	378μg/ml	300 μg/ml	245μg/ml	245 µg/ml
Ampicillin	15 µg/ml	10 µg/ml	10 µg/ml	15 µg/ml	10 µg/ml	10 µg/ml

Methanol extract of U. ramosa proved antimicrobial potential in opposition to the test microorganisms used. The highest antibacterial activity is recorded at 400μg concentration in B. subtilis (2.6mm) followed by E. coli (2.1mm) and Proteus sp. (2.0mm). It is also observed that the extract at 50μg concentration was ineffective against S.aureus, S. epidermidis, Salmonella and Proteus sp.. Zone of inhibition was observed in C. albicans at all the concentrations proving potent antifungal activity.

Fig 2: Microtitre plate showing MIC values for various microorganisms

By looking at the table we can conclude that Staphylococcus aureus is highly susceptible to methanol extract with the MIC value of 145μg and Staphylococcus epidermidis is the most resistant with the MIC value of 378μg. Proteus, Bacillus subtilis are having same MIC value with requirement of 300μg/ml. E. coli and Salmonella are having same MIC value with requirement of 245μg/ml.

Table 5: Antioxidant activity of bioactive molecule from UR

Antioxidant activity	IC₅₀ value of standard Butylated hydroxyl toluene	IC₅₀ value of phenolics from methanol extract of UR
DPPH radical scavenging assay	7.8 µg	10.10µg
Superoxide radical scavenging assay	19.00 µg	25.12 µg
Nitric oxide radical scavenging assay	22.98 µg	17.32µg

Ta represented in the table 5 shows IC₅₀ value of DPPH radical scavenging, superoxide anion radical scavenging activity and Nitric oxide radical scavenging of standard butylated hydroxyl toluene and methanol extract of U.ramosa. Methanol extract showed better Nitric oxide radical scavenging activity when compared to BHT.

DPPH radical scavenging activity:

Natural antioxidants have the capacity to scavenge free radicals. Methanol extract of U.ramosa showed less DPPH radical scavenging activity with an IC₅₀ value of 10.10µg when compared with BHT with an IC₅₀ value of 7.8µg. Protonated radical DPPH has maximum absorbance at 517nm. The absorbance may decrease due to the scavenging actions of antioxidants especially phenolics of methanol extract which has hydrogen donating ability¹⁸.

DNA protection assay:

Fig 3: Effective action of methanol extract of U.ramosa on Calf thymus DNA.

Lane 1: DNA+ FeSO₄+ H₂O₂; Lane 2: DNA+ FeSO₄+ H₂O₂+ Methanol extract 25μg/mL; Lane 3: DNA+ FeSO₄+ H₂O₂+ Methanol extract 100μg/mL;

Lane 4: Untreated Calf thymus DNA

When hydrogen peroxide reacts with iron, there will be generation of hydroxyl radicals (Fenton reaction) which in turn react with DNA resulting in strand breaks and degradation. Methanol extract of U. ramosa protected the calf thymus DNA against the deleterious effects of hydroxyl radicals on DNA by scavenging them. Radical scavenging was found to be dose dependent as seen in (fig 3).

Superoxide anion radical scavenging activity:

Our results with methanol extract from U.ramosa exhibited moderate superoxide radical scavenging activity. During normal metabolism superoxide anion is produced in minute quantity. Even though it is a weak oxidant, it can lead to the formation of powerful hydroxyl radicals that contribute to oxidative stress. Hence it is mandate to scavange and terminate the harmful effects of superoxide radicals. The methanol extract of U.ramosa showed scavenging activity of super oxide anion radicals with an IC₅₀ value of 25.12µg/ml when compared to that of BHT with an IC₅₀ value of 19.00µg.

Nitric oxide radical scavenging activity:

Under normal physiological conditions, nitric oxide play significant and noteworthy roles by acting as a neurotransmitter and vasodilator. It also fights against tumour metastasis and infective microbes. Certain signaling molecules like lipo-polysaccharide (LPS), tumor necrosis factor (TNF-α), and interleukin (IL-1) elicit the production of NO·. NO· will inturn reacts with superoxide anion radicals and gets converted to strong oxidant called peroxynitrite that cause high level of oxidative stress. It is noteworthy to record that the methanol extract of U.ramosa possess high nitric oxide radical scavenging activity with an IC₅₀value of 17.32 µg/ml when compared to IC₅₀value 22.98µg/ml of BHT.

Anti-inflammatory activity:

Inhibition of albumin denaturation:

Table 6 : Effect of methanol extract of U. ramosa on heat induced protein denaturation

Treatment (s)	Concentration (µg/ml)	Absorbance at 660 nm	% inhibition of protein denaturation
Treatment (s)	Concentration (µg/ml)	Absorbance at 660 nm	% inhibition of protein denaturation
Control	-	0.55	-
Methanol extract of U. ramosa	100	0.53	04
	200	0.49	11
	300	0.43	22
	400	0.34	38
	500	0.25	55
Aspirin	100	0.10	80

During inflammatory mechanism proteins lose their molecular structure and become non-functional (denaturation). It is evident that maximum inhibition was recorded at 500 µg/ml of extract with 55% in comparison to standard aspirin with inhibitory action at 100 µg/ml.

Proteinase Inhibitory Activity:

Table 7: Effect of methanol extract of U. ramosa on proteinase inhibitory action

Treatment(s)	Concentration (µg/ml)	Absorbance at 210 nm	% inhibition of proteinase action
Control	-	0.46	-
Methanol extract of U. ramosa	100	0.35	23
	200	0.31	33
	300	0.29	37
	400	0.20	57
	500	0.16	63
Aspirin	100	0.09	81

Leukocyte proteinase are present abundantly in lysosomes of neutrophils which are responsible for tissue damage during inflammatory reactions and their action can be prevented by supplying significant level of proteinase enzyme inhibitors. Methanol extract of U.ramosa exhibited sufficient anti-proteinase activity at different concentrations as represented in table 6. Maximum proteinase action inhibition was recorded at 500µg/ml concentration and it was 63%. Aspirin being potent synthetic drug showed 81% of proteinase inhibitory action.

Membrane stabilization:

To prevent elicitation of inflammatory response, stabilization of lysosomal membrane is very much needed during the tissue injury. In-vitro anti-inflammatory activity proved to inhibit distortion of RBC membrane which implies that the extract will also stabilize lysosomal membrane, as RBC membrane is analogous to lysosomal membrane. Extracellular release of constituents of lysosomes from neutrophils (certain lytic substances and proteases) bring about tissue damage indicating development of acute or chronic inflammation¹⁹.

Heat Induced Haemolysis:

Table 8: Effect of methanol extract of U. ramosa on heat induced haemolysis

Treatment (s)	Concentration (µg/ml)	Absorbance at 560 nm	% inhibition of haemolysis
Treatment (s)	Concentration (µg/ml)	Absorbance at 560 nm	% inhibition of haemolysis
Control	-	0.47	-
Methanol extract of U. ramosa	100	0.33	22
	200	0.29	31
	300	0.27	36
	400	0.23	48
	500	0.18	57
Aspirin	100	0.11	74

At the concentration of 500µg/ml the extract recorded inhibition of 57% of heat induced haemolysis. But aspirin, a standard synthetic drug proved to be more effective offering 74% inhibition at 100µg/ml concentration when compared to that of the plant extracts.

Haemolysis induced by hypotonic solution

Table 9: Effect of methanol extract of U. ramosa on hypotonic solution induced haemolysis

Treatment (s)	Concentration (µg/ml)	Absorbance at 560 nm	% Inhibition of haemolysis
Treatment (s)	Concentration (µg/ml)	Absorbance at 560 nm	% Inhibition of haemolysis
Control	-	0.43	-
Methanol extract of U. ramosa	100	0.34	23
	200	0.27	37
	300	0.24	44
	400	0.22	49
	500	0.18	58
Diclofenac sodium	100	0.09	79

There was significant inhibition even in haemolysis induced by hypotonic solution, maximum protection was observed at 500µg/ml with 58% whereas Diclofenac sodium at a concentration of 100µg/ml offered 79% protection.

Antiangiogenesis Tumor growth can be related to angiogenesis as demonstrated by various in-vivo experiments which also include chick chorioallantoic membrane (CAM) assay. Currently, the most convenient approach for limiting tumor angiogenesis is by blocking vascular endothelial growth factor (VGEF). Increased angiogenesis not only lead to tumor growth, but also leads to endothelial cell proliferation. Thus, we can decrease metastatic potential of tumour by inhibiting angiogenesis which can stop endothelial cell proliferation. Present study reveals that methanol extract of Urochloa ramosa showed better antiangiogenic results compared to control as there was reduction of formation of number of blood vessels, dilation, vasoconstriction and disintegration of blood vessels (Fig. 4).

Fig 4: CAM assay -1. Suppression of angiogenesis by the methanol extract of U. ramosa. 2. Number of blood vessels formed were less in (decreased vasculature) treated compared to the control CAM.

CONCLUSION:

Phytochemicals in plants are backbone of traditional medicine systems dating back to hundreds of years. Due to the advent of synthetic chemical compounds there is decline in the use of plant based preparations in contemporary medicine over years⁴⁶. Methanol extract U.ramosa showed zone of inhibition in all the concentrations for B. subtilis, E.coli and C. albicans and further it proved that methanol extract is showing more of antifungal activity than antibacterial potential. By resurine method we could find out that Staphylococcus aureus is highly susceptible to methanol extract with the MIC value of 145μg and Staphylococcus epidermidis is the most resistant with the MIC value of 378μg. IC₅₀ value of DPPH radical scavenging, superoxide anion radical scavenging activity and Nitric oxide radical scavenging of standard butylated hydroxyl toluene and methanol extract of UR were assessed and it was found that methanol extract of U.ramosa showed better Nitric oxide radical scavenging activity when compared to BHT. Methanol extract from U.ramosa protected the calf thymus DNA against the deleterious effects of hydroxyl radicals on DNA by scavenging them in DNA protection assay. The plant extract was also found promising to have anti-inflammatory activities by various assays. Present study reveals that methanol extract of Urochloa ramosa also showed better antiangiogenic results compared to control as there was reduction of formation of number of blood vessels, dilation, vasoconstriction and also disintegration of blood vessels. Antimicrobial, antioxidant, anti-inflammatory and anti-angiogenic activities of methanol extract of leaf of Urochloa ramosa is mainly because of the presence of phenolics in the methanol extract. In the present study we could able to partially purify the phenolics from the methanol extract, further purification and elucidation of the structure is under progress.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors would like to thank JSS Academy of Higher Education and Research, Sri Shivarathreeshwara Nagara, Mysuru-570017 for their kind support for carrying out this research work.

REFERENCE:

1. Doss A. Preliminary phytochemical screening of some Indian Medicinal plants. Anc.Sci.Life.2009; 29:12-16.

2. Ranjitha Dhevi V. Sundar, Mythili Sathiavelu. A Comparative Study on Phytochemical Screening, Antioxidant and Antimicrobial Capacities of Leaf Extracts from Medicinal plants. Research J. Pharm. and Tech 2019; 12(1): 361-366.

3. Arunava Das, M. Bharath, M. Jeevanantham, S. Manoj Kumar, R. P. Thanarithanika J.Bindhu. Phytochemical Screening and Antimicrobial activity of Syzygium cumini (Jamun) seed Extract. Research J. Pharm. and Tech 2018; 11(9): 4096-4100.

4. C. Das, A. Mohanty, S. Dash, D.C. Sahoo, N.S.K. Choudhury, V.J. Patro, S.K. Kanungo. Phytochemical Screening of Crude Bark extracts of Tecoma stans Linn. (Bignoniaceae). Research J. Pharm. and Tech.2 (4): Oct.-Dec. 2009; Page 816-818.

5. Geetha S, Rajeswari S. A Preliminary Study on Phytochemical Screening, Proximate Analysis and Anti-Bacterial Activities of Andrographis paniculata Seed Extract. Research J. Pharm. and Tech. 2019; 12(5):2083-2088.

6. Arunakumar S and Muthuselvam. Analysis of phytochemical constituents and antimicrobial activities of Aloe vera L. against clinical pathogen: World. J. Agril. Sec.2009; 5(5): 572-576.

7. Kumar A, Mahajan A and Begum Z. Phytochemical Screening and In vitro Study of Free Radical Scavenging Activity of Flavonoids of Aloe vera. Research J. Pharm. and Tech 2020; 13(2):593-598.

8. R. Bhatia and J. P. Narain, “The growing challenge of antimicrobial resistance in the South-East Asia Region - are we losing the battle?”. Indian Journal of Medical Research, vol. 132, no. 5, pp. 482–486, 2010.

9. H. W. Boucher, G. H. Talbot and J. S. Bradley et al., “Bad bugs, no drugs: no ESKAPE! An update from the Infectious Diseases Society of America,” Clinical Infectious Diseases, vol. 48, no. 1, pp. 1–12, 2009.

10. Dahikar S. B. In Vitro Antimicrobial Activity of Fruit Extracts of Lagenaria Siceraria (Mol.). Res. J. Pharmacognosy and Phytochem. 2018; 10(2): 183-186.

11. H. Giamarellou, “Multidrug-resistant Gram-negative bacteria: how to treat and for how long,” International Journal of Antimicrobial Agents, vol. 36, Supplement 2, pp. S50–S54.

12. Gonzalez, C. E, Venzon D, Lee S, Mueller B. U, Pizzo P. A and Walsh T. J (1996). Risk factors for fungemia in children infected with human immunodeficiency virus: a case-control study. Clin Infect Dis. 23: 515-521.

13. Basanti Majhi, Kunja Bihari Satapathy, Sagar Kumar Mishra. Antimicrobial activity of Averrhoa carambola L. leaf extract and its Phytochemical Analysis. Research J. Pharm. and Tech. 2019; 12(3): 1219-1224.

14. Sieradzki K, Wu SW and Tomasz A. (1999). Inactivation of the methicillin resistance gene mecA in vancomycin-resistant Staphylococcus aureus. Micro. Drug Resist. 5(4): 253-257.

15. L. S. Patel, R. S. Patel. Antimicrobial Activity of Asparagus racemosus Willd from Root Extracts – A Medicinal Plant. Research J. Pharm. and Tech. 6(10): October 2013; Page 1141-1143.

16. Preeti Tiwari. Antimicrobial Activity of Balarishta Prepared by Traditional and Modern Methods. Research J. Pharm. and Tech. 7(7): July 2014 Page 789-791.

17. Jency George. Bioactive Screening and Antimicrobial Activity of Selected Three Medicinal Plants on Chosen Microbes. Research J. Pharm. and Tech. 7(11): Nov. 2014 Page 1264-1269.

18. Farnsworth NR. The roleof medicinal plants in drug development. In: Krogsgaard-Larsen, editor. Natural products and drug development. Balliere, Tindalland Cox, London 1984; 8-98.

19. Pradeep Kumar Samal. Investigation of Antioxidant activity of Butea monosperma barks. Research J. Pharm. and Tech 6(6): June 2013; Page 610-613.

20. Rekha Rajendran, R Hemachander, T Ezhilarasan, C Keerthana, DL Saroja, KV Saichand, Mohamed Gasim Abdullah. Phytochemical Analysis and In-Vitro Antioxidant Activity of Mimosa pudica Lin., Leaves. Research J. Pharm. and Tech. 3(2): April- June 2010; Page 551-555.

21. Halliwell B, Aeschbach R, Loliger J, Aruoma O. I. Food Chem. Toxic: Chemical characterization of antioxidants,1995; 33:601.

22. Cotelle N, Bernier J.L, Catteau J.P , Pommery J, Wallet J.C and Gaydou E.M. Free Radic. Biol. Med Antioxidant properties of hydroxy-flavones.1996; 20:35.

23. J.S. Vaghela and S.S. Sisodia. In Vitro Antioxidant Activity of Terminalia chebula Fruit Extracts. Research J. Pharm. and Tech. 4(12): Dec. 2011; Page 1835-1843.

24. Aruoma,O.I. Food Chem. Toxic., Nutrition and health aspects of free radicals and antioxidants. 1994; 32: 671.

25. R. Manikandan, A. Vijaya Anand. A Review on Antioxidant activity of Psidium guajava. Research J. Pharm. and Tech. 8(3): Mar., 2015; Page 339-342.

26. U.S MahadevaRao, Khamsah Suryati Mohd, Siti Zulaikha Bt Abd Halim, Masitah Bt Khamis. Screening of Phytochemicals and Comparative Antioxidant activity of Leaf and Fruit of Malaysian Mengkudu Using Aqueous and Organic Solvent Extracts. Research J. Pharm. and Tech. 6(9): September 2013; Page 1064-1072.

27. Mizushima Y, Kobayashi M. Interaction of anti-inflammatory drugs with serum proteins, especially with some biologically active proteins. J of Pharma Pharmacol. 1968; 20:169- 173.

28. B. Meher, T. Satapathy, A.K .Sahu, K .K Ahirwar, P D Kashinath, N P Jain. Screening of Methanolic Extract of Euphorbia hirta linn for Antiinflammatory Activity in Experimental Animals. Research J. Pharm. and Tech. 5(1): Jan. 2012; Page 38-40.

29. Folkman, J. Angiogenesis in cancer, vascular, rheumatoid and other disease, Nat. Med. 1995; 1: 27–31.

30. Folkman, J. Tumor angiogenesis, in: J.F. Holland, E. Frei III, R.C. Bast Jr., D.W. Kufe, R.E. Pollock, R.R. Weichselbaum (Eds.), Cancer medicine, 5th ed, B.C. Decker Inc, Ontario, Canada, 2000; 132–152.

31. G. V. N. Kiranmayi, L. Anil Ricky, L. Sandeep Kumar, M. Lalitha Kala, M. Krishna Vamsi, M. Sai Sureshma, M. Vishnu, M. Kiran Sai. Assessment of Antioxidant and Antiangiogenic Activities of Ethanolic Root extract of Cassia occidentalis. Research J. Pharm. and Tech. 2019; 12(3): 1230-1234.

32. Amrit pal singh. Promising phytochemicals from Indian Medicinal plants. Ethnobotonicals Leaflets vol: 2005 Issue, Article 18.

33. Shabi Ruskin R. S. Ajina. Qualitative Phytochemical Screening and In-vitro Anthelmintic Activity of Adhatoda vasica (Acanthaceae). Research J. Pharm. and Tech. 2017; 10(2): 414-420.

34. Nascimento GGF, Locatelli J, Freitas PC, Silva GL. Antibacterial activity of plant extracts and phytochemicals on antibiotic-resistant bacteria. Braz J Microbiol. 2000;31(1):247-56.

35. Scherer R and Godoy HT. Antioxidant activity index (AAI) by 2,2- diphenyl-1-picrylhydrazyl method. Food Chem 2009; 112: 654-658.

36. Nishikimi M, Rao NA, Yagi K. The occurrence of superoxide anion in the reaction of reduced phenazine methosulphate and molecular oxygen. Biochem Biophys. Res.commun 1972; 46: 849-864

37. Marcocci L, MaguireJJ, Droy-Lefaix MT. The nitric oxide scavenging properties of Ginkgo biloba extract EGb, Biochem, Biophys. Res. Commun 1994; 15: 748-755

38. Tian B. and Hua Y. Concentration – dependence of prooxidant and antioxidant effects of aloin and aloe- emodin on DNA. Food chemistry, 2005: 91; 413-418, 2005.

39. Sakat S, Juvekar AR, Gambhire MN. In vitro antioxidant and anti-inflammatory activity of methanol extract of Oxalis corniculata Linn. International Journal of Pharma and Pharmacological Sciences. 2010; 2(1):146-155.

40. Oyedepo OO and Femurewa AJ. Anti-protinase and membrane stabilizing activities of extracts of Fagra zanthoxiloides, Olax subscorpioides and Tetrapleura tetraptera. Int J of Pharmacong.1995; 33: 65-69.

41. Sadique J, Al-Rqobahs WA, Bughaith, EIGindi AR. The bioactivity of certain medicinal plants on the stabilization of RBS membrane system. Fitoterapia.1989; 60:525-532.

42. Shenoy S, Shwetha K, Prabhu K, Maradi R, Bairy KL, Shanbhag T. Evaluation of antiinflammatory activity of Tephrosia purpurea in rats. Asian Pac J Trop Med. 2010; 3(3): 193-5.

43. Gandhidasan R, Thamaraichelvan A, Baburaj S. Anti inflammatory action of Lannea coromandelica by HRBC membrane stabilization. Fitoterapia 1991; Voll LXII; No1; 81- 83.

44. Shobana S, Vidhya R. Evaluation of in vitro hemolytic activity of different parts of Abutilon indicum (linn.). World journal of pharmacy and pharmaceutical sciences. 2006; 5(5),1182-1196

45. Rekha ND, Aradhya SM and Jayashree K: The Antiangiogenic, Antioxidant and Proapoptotic Chemopreventive Properties of Tannins from Memecylon malabaricum (Cl.). Int J Pharm Sci Res 2015; 6(1): 259-66.

46. Udayashankar AC, Nandhini M, Rajini SB and Prakash HS: Pharmacological significance of medicinal herb Eclipta alba L. - a review. Int J Pharm Sci and Res 2019; 10(8): 3592-06.

47. Udayashankar AC, Rajini SB, Nandhini M, Suhas YS, Niranjana SR, Lund OS, Prakash HS. Acute oral toxicity, dermal irritation and eye irritation study of Eclipta alba aqueous extract in sprague dawley rats and newzealand white rabbits. Int. Res. J. Pharm. 2016; 103-109.

48. Rajini SB, Nandini M, Udayashankar AC, Niranjana SR, Lund OS, Prakash HS. Antifungal Activity of Eclipta alba Metabolites against Sorghum Pathogens. Plants 2019;8(3):72.

49. Konappa N, Arakere U C, Krishnamurthy S, Gangadharaiah K C, Gubbiveeranna V, Shivaiah N, Chowdappa S, Ramachandrappa N S. Evaluation of in vitro antioxidant and antidiabetic activities from Amomum nilgiricum leaf extract. Plant Science Today. 2020; 7(4):638-644.

50. Ty Viet Pham, Hanh Nhu Thi Hoang, Hoai Thi Nguyen, Hien Minh Nguyen, Cong Thang Huynh, Thien Y Vu, Anh Thu Do, Nguyen Hoai Nguyen, and Bich Hang Do. Anti-Inflammatory and Antimicrobial Activities of Compounds Isolated from Distichochlamys benenica. BioMed Research International / 2021

51. Jitendra Pandey, Sushma Bhusal, Laxman Nepali, Maya Khatri, Rasmita Ramdam, Himal Barakoti, Paras Mani Giri, Dhakaraj Pant, Pramod Aryal, Rabindra Kumar Rokaya, and Ravin Bhandari. Anti-Inflammatory Activity of Artemisia vulgaris leaves, originating from Three Different Altitudes of Nepal. The Scientific World Journal / 2021.

52. Taye KebedeID, Eshetu Gadisa , Abreham Tufa. Antimicrobial activities evaluation and phytochemical screening of some selected medicinal plants: A possible alternative in the treatment of multidrug-resistant microbes. PLOS ONE, March 26, 2021.

53. Raquel Costa, Daniela Azevedo, Pedro Barata, Raquel Soares, Luís F. Guido and Daniel O. Carvalho. Antiangiogenic and Antioxidant In Vitro Properties of Hydroethanolic Extract from Euterpe oleracea Dietary Powder Supplement. Molecules,April 2021.

54. Jundi Ismael , Engeda Dessalegn and Workineh Mengesha Fereja. In Vitro antioxidant and antibacterial activity of leaf extracts of Measa lanceolata. International Journal of Food Properties , Volume 24, April 2021.

Received on 13.08.2021 Modified on 23.10.2021

Research J. Pharm. and Tech. 2022; 15(8):3571-3579.

DOI: 10.52711/0974-360X.2022.00599