INTRODUCTION:

Ixora coccinea Linn (Rubiaceae) is known as Jungle of Geranium (or) Flame of the woods or vetchi in Ayurveda. It is a common flowering shrub native to Asia. It is name derived from an Indian deity. It is traditionally used as hepatoprotective, Chemoprotective, antimicrobial, anti-oxidant, anti-nociceptive, anti–mitotic and anti-inflammatory activities. [1] Free radicals or oxidative injury now appears the fundamental mechanism underlying a number of human neurologic and other disorders. For instance in diabetes, increased oxidative stress which co-exist with reduction in the anti-oxidant status has been postulated. [2]

In view of the above, we designed the formulation and evaluate the antioxidant potential and antibacterial effect of Ixora coccinea formulation.

MATERIALS AND METHODS:

Materials:

Clove oil was extracted at own Laboratories. Emulsifying wax, white soft paraffin, liquid paraffin and all other chemicals were of analytical grade and used without further purification. Bacterial culture of Staphylococcus aureus and Escherichia coli were obtained from Department of Pharmacognosy MET’s Institute of Pharmacy.

PREPARATION OF CRUDE EXTRACT:

The dried leaves were exhaustively extracted with 250ml of methanol in a Soxhlet apparatus for 6 Hours. The extract was then concentrated by evaporation methanol and air-dried.

Pic: soxhlet extraction and crude extract obtained

Method for preparation of ointment [3]

MixedIxora cocconiea extract and clove oil. Add this mixture in ointment base with continuous stirring andheated to 70-75°C to melt it completely. Then clove oil and / or Ixora coccinea was / were dissolved in it under stirring and then cooled at room temperature. Disperse acacia and tragacanth in distilled water in a beaker. Add the dispersed phase in the mixture by Doubling Method. Add Preservative. The composition of emulsifying ointment base is given in Table 1 and composition of different ointment formulations is given in Table 2.

Table 1. Composition of emulsifying ointment base

Sr. No.	Ingredients	Quantity
1	Ixora Coccinea Extract	7%
2	Clove oil	0.7%
3	Accacia	1gm
4	Tragacanth	2gm
5	Benzoic Acid	0.5gm
6	Ointment Base	20gm

Spreadability:

Spreadability was determined by modified wooden block and glass slide apparatus. The apparatus consisted of a wooden block with fixed glass slide and a pulley. A pan was attached to another glass slide (movable) with the help of a string. For the determination of spreadability measured amount of ointment was placed in the fixed glass slide, the movable glass slide with a pan attached toit, was placed over the fixed glass slide, such that the ointment was sandwiched between the two slides for 5 min. The weight was continuously removed. Now about 50 g of weight was added to the pan. Time taken for the slides to separate was noted. Spreadability was determined using the following formula:

S = M/T

Where S is the spreadability in g/s, M is the mass in grams and T is the time in seconds.

Extrudability:

A closed collapsible tube containing ointment was pressed firmly at the crimped end. When the cap was removed, ointment extruded until pressure dissipated. Weight in grams required to extrude 0.7 cm ribbon of ointment in 10 seconds was determined.

Viscosity:

Brookfield digital viscometer (model/make-LU-DVE/Brookfield Eng Lab.) was used to measure the viscosity (in cps) of the prepared ointment formulations as such that is in semisolid state.

Phytochemical screening:

Phytochemical screening of the extract was performed using the following reagents and chemicals: Alkaloids with Dragendorffs reagent, flavonoids with the use of Mg and HCl; tannins with ferric chloride and potassium dichromate solutions and saponins with ability to produce suds.

Table 3. Phytochemical screening

Phytochemical screening of Ixora coccinea methanolextracts
Methanol extract
Alkaloids	_{+ +}
Steroids	++
Flavonoids	++
Tannins	+++
Saponins	++
Glycosides	-

+: Present in low range; ++: Present in moderate range; +++: Present in high range;-: Absent

PHARMACOLOGICAL EVALUATION:

Antibacterial activity:

The compounds were tested in-vitro for their antibacterial activity against two microorganisms viz. Escherichia coli (NCTC 10418), and Staphylococcus aureus (NCTC 6571) which are pathogenic in human beings.

a) Method: Cup-plate agar diffusion method using Nutrient agar.

b) Preparation of test solutions:

Each test compound (5 mg) was dissolved in dimethylformamide (5 mL) to give stock solution of concentration 100 mcg/mL. Then 0.1 mL of this solution was used for testing.

c) Preparation of standard solution:

Standard drug Norfloxacin was used. The concentration was 100 mcg/mL.

d) Method of testing:

Nutrient agar plates were prepared by pouring 15-20 mL of the medium into each sterilized petridish and were allowed to set at room temperature. The cell suspension was standardized to the density of 530 nm using spectrophotometer and was inoculated over the surface of agar medium using sterile cotton swab. The three cups were scooped in each plate using a sterile cork borer of 6 mm diameter. Then the solutions of test compounds (0.10 mL) were added in cups by using micropipettes and these plates were incubated at 37°C for 48 hrs. The zone of inhibition was measured in mm for each organism.

Table 4. Inhibition zone diameters of different formulations

Sr.No.	Compd.	Zone of inhibition
Sr.No.	Compd.	*E.coli*	*S. aureus*
1	F-1	22.3	22.9
2	F-2	24.0	24.2
3	F-3	28.5	30.4
4	F-4	31.3	35.8
5	F-5	15.2	16.6
6	F-6	17.7	18.1
7	F-7	18.4	19.0
Standard	Norfloxacin	31	32.2

* ZoI- Zone of inhibition. Values are average of three determinations.

Ferric reducing power assay:

The reducing antioxidant power of the plant extracts were determined by the method of Oyaizu [6]. Different concentrations of plant extracts in 1 ml of distilled water were mixed with phosphate buffer (2.5 ml, 0.2 M, pH 6.6) and potassium ferricyanide [K₃Fe (CN)₆] (2.5 ml, 1%). The mixture was incubated at 50^oC for 20 min. Then, 2.5 ml of trichloroacetic acid (10%) was added to mixture, which was then centrifuged for 10 min at 3000 rpm. The upper layer of solution (2.5 ml) was mixed with distilled water (2.5 ml) and FeCl3 (0.5 ml, 0.1%). The absorbance was measured at 700 nm against a blank using UV-Vis spectrophotometer after 30 mins. Increased absorbance of the reaction mixture indicates increase in reducing power. The redcuing power of the plant material was expressed in terms mg of gallic acid equivalents/g of plant material.Table no.5represents the ferric reducing ability of formulation of IC.[4]

Table 5. Ferric reducing power assay

*Ixora coccinea* parts	Solvents	Amount (mg of gallic acid equivalents/g of plant material)
Leaves	Methanol	11.9

Fig. 1.1: Leaves and flowers of I. Coccinea

Hydrogen peroxide radical scavenging activity:

In this method, when a scavenger is incubated with hydrogen peroxide, the decay or loss of hydrogen peroxide can be measured spectrophotometrically at 230 nm. Different concentrations of Ixora coccinea were prepared in methanol in the concentration range 1-7%. Standard solution of ascorbic acid was prepared in the same concentration range. 1ml of various concentrations (%) of sample and standard were mixed with 2ml of 20 mM hydrogen peroxide in phosphate buffer saline (pH7.5). After 10 minutes absorbance was measured at 230 nm against phosphate buffer saline (pH 7.5) as blank. The experiment was performed in triplicate. Scavenging activity was determined by comparing % inhibition with that of control (100%) containing only H₂O₂ and solvent. Shown in table no.6 [5] The data was expressed as % inhibition. Hydrogen peroxide scavenging activity (%) = [A0– A1/ A1] × 100

Table 6. Hydrogen peroxide radical scavenging activity

Sr.No.	*Formulation*	*Standard (Ascorbic acid)*	IC
1	F-1	19.90 ± 0.13	15.32 ± 0.13
2	F-2	36.21 ± 0.23	24.12 ± 0.52
3	F-3	48.64 ± 0.13	41.22 ± 0.24
4	F-4	70.44 ± 0.15	60.31 ± 0.11
5	F-5	46.21 ± 0.23	24.12 ± 0.52
6	F-6	58.64 ± 0.13	51.22 ± 0.24
7	F-7	89.27 ± 0.12	45.41 ± 0.32

CONCLUSION:

The present study concludes that formulation of Ixora coccinea contain high antioxidant and antibacterial property. The presence of phytochemicals such as alkaloids, tannins, saponins, glycosides, flavonoids, and steroids.

The formulations were evaluated for anti-bacterial activity and antioxidant activity, spreadability, viscosity, extrudability. From the results, it is clearly evident that all formulations showed good extrudability, viscosity and spreadability. From the data, it is evident that formulation F-4 containing both clove oil (0.7%) and Ixora coccinea (7%) showed larger zone of inhibition in comparison to other formulations.So antibacterial and antioxidant activity of formulation F-4 found to be superior to otherformulations. Results of all other evaluation parameters of F-4 were also satisfactory among all the formulations.

REFERENCES:

1. A.Elumalai, ChinnaEswaraiah, Yetcharla Venkatesh, Burle Shiva kumar and ChavaNarendar Phytochemical and pharmacological profile of Ixora coccinea Linn.International Journal of Pharmacy and Life Sciences 2012, 3, (3), 1563-1567

2. Moni Rani Saha, Md. Ashraful Alam,RaushanaraAkter and Rumana Jahangir. In-vitro free radical scavenging activity of Ixora coccinea L. Bangladesh J Pharmacol, 2008,3, 90-96.

3. G. Sandhyarani and K. Praveen Kumar Formulation and Evaluation of Neomycin Sulphate Ointment Containing Natural Wound Healing Agent CouroupitaGuianensis International Journal of Pharmacology Research 2014 , 4, (3),124-127.

4. S. Rohini, M. Shalini, Nithya Narayanaswamy and K. P. Balakrishnan Application of Natural Products In Cosmetics: A Study of Ixora Coccinea Extracts For Their Antityrosinase And Antioxidant Activities International Journal of Research in Cosmetic Science 2012; 2 (1) , 1-7

5. Manimegalai S., Sridharan T. B., Rameshpathy M. and Devi Rajeswari V. Antioxidant, phytochemical screening and antimicrobial activity of CouroupitaGuianensis flower extract Der Pharmacia Lettre, 2014, 6 (6),251-256

Received on 04.05.2018 Accepted on 26.05.2018

Asian J. Pharm. Tech. 2018; 8 (2):88-91.

DOI: 10.5958/2231-5713.2018.00014.4

Item	Material name	Quantity (%)
Item	Material name	F1	F2	F3	F4	F5	F6	F7
1	Clove oil	0.7	0.7	0.7	0.7	--	--	--
2	Ixora coccinea	1	2	4	7	3	5	6
3	Benzoic acid,Accacia and Tragacanth	q.	q.	q.	q.	q.	q.	q.
4	ointment base	s.	s.	s.	s.	s.	s.	s.