Formulation development and Evaluation of Cleome gyandra based gel

Nilima A. Thombre¹*, Swati Madhukar Gaikwad¹, Sanjay J. Kshirsagar¹, Daksha Attarde²

¹Department of Pharmaceutics, MET Institute of Pharmacy, Bhujbal Knowledge City, Adgaon, Nasik-422003, Maharashtra, India

²Department of Pharmacognosy, MGV’S College of Pharmacy, Nashik.

*Corresponding Author E-mail: nilimathombre@gmail.com, swatigaikwad092@gmail.com

ABSTRACT:

The plant Cleome gyandra family Cleomaceae has analgesic activity. Extraction of whole plant was done using by drying and grinding whole plant in water and ethanol solvent by using soxhlet apparatus. flavonoids and tannins present in the plant are responsible for the analgesic activity. Herbal gel was developed using laboratory collected water and ethanol extract whereas water extract contains tannin and ethanol extract contains flavonoids. Herbal gel formulation was prepared by using different concentration of gelling agent like carbapol940, different concentration of tannin and flavonoids obtained from water and ethanol extract. Herbal gel was evaluated using parameter such as physical appearance like color, Odor, Consistency, pH, Viscosity, Spreadability, Drug content etc. Topical gel formulations had pH in the range of 6.8-7.2 i.e. in the neutral range. Viscosity of gel was determined by Brookfield viscometer, viscosity were determined, Batches F2, F6 and F8 had high viscosity as compared to other batches of gel. The spreadability of the gel formulations was determined, by measuring 1gm of gel between horizontal plates after 1 min. the standardized weight tied on the upper plate. Spreadability values of the formulation (F1-F9) were determined. Batches F1-F4 has higher spreadability value in comparison with others batches of gel. The analgesic activity of plant extract was detected by in vivo study like skin irritation method and acetic acid induced writing test. The prepared herbal gel was evaluated for its skin irritant effect, where no erythema or edema was observed for all the formulations F1 to F9 after 24 hours.

KEYWORDS: Cleome gyandra, analgesic activity, Gel Evaluation, Skin irritation, acetic acid induced writhing test.

INTRODUCTION:

India has rich tradition of plant-based knowledge of healthcare. Herbal product has negligible side effects. The use of the plant-based medication is gradually becoming popular throughout the world. Cleome gyandra Linn belonging to family Cleomaceae, have been used in Indian medicines, used as analgesic used in pain condition. Gel formulation for several routes of administrations they are useful as liquird formulation in oral, topical, vaginal Gel. Gel can be clear formulations when all of the particles completely dissolved in the dispersed medium. The classification of Gel is single phase and two-phase Gels, and types of Gels is hydrogels, xerogels, porganogels. Gelling agent used as carbapol, acacia, pectin, xanthum gum etc.

Gels are easily penetrate through skin, improve patient compliance due to easy of application. Herbal gels are less toxic as compared to conventional formulation due to bilayer composition and structure⁽¹⁾. So the aim of the present study was to formulate and evaluate of herbal gel containing Cleome gyandra linn plant extract.

MATERIALS AND METHODS:

Plant Materials:

The plant Cleome gyandra Linn was collected (Palkhed Dam, Dindori, Nashik, Maharashtra, India.) The authentication of the plant was done by Botanical Survey of India Western Regional Centre, 7-Koregoan Road, Pune-411001, and Maharashtra, India.

Chemicals:

Carbopol 940, Methyl Paraban, Propyl Paraben, Propylene glycol-400, Triethanolamine.

Extraction of tannic acid from Cleome gyandra:

20gm Powder of Cleome gyandra was added in 300mL of water, extraction was done using soxhlet extraction apparatus. Boil it for 24hrs after that filtered it and dried.

Isolation of tannic acid from Cleome gyandra:

700mg extract of Cleome gyandra was added in 50mL of water and this mixture was taken in separating funnel, in same mixture 10mL 6% of petroleum ether was added and it was slightly shaken then petroleum ether layer was removed. Chloroform was added in aqueous layer, removed chloroform layer. Ethyl acetate was added in aqueous layer, slightly shaken it and separated the water layer. Water layered was dried and collected tannin. Phytochemical screening was performed for tannin.⁽¹⁵⁾

Extraction of flavonoids from Cleome gyandra:

20gm Powder of Cleome gyandra was added in 300mL of ethanol, extraction was done using soxhlet extraction apparatus. Boil it for 24hrs after that filtered it and dried.

Method for Preparation of Gel:

In first beaker 0.25gm, 0.50gm and 1g extract of Cleome gyandra was taken and added in the mixture of Carbopol 940 was dispersed in 50ml of distilled water with continuous stirring. 5ml of distilled water was taken in another beaker and required quantity of methyl paraben and propyl paraben were dissolved by heating on water bath. Cool the solution, then to that added Propylene glycol 400 mixed both beakers solution, Volume make up to 100ml by adding remaining distilled water. triethanolamine was added drop wise to the formulation for adjustment of required skin pH (6.8-7) and to obtain the gel at required consistency. The same method was followed for preparation of control sample without adding any Cleome gyandra extract⁽²⁾.

Determination of total Flavonoids:

Total flavonoids content of Cleome gyandra was determined by aluminium chloride colorimetric method. Quercetin was used as standard and the flavonoid content of the extract was expressed as mg of quercetin equivalent/gm of dried extract.⁽¹³⁾

Method for detection of Flavonoids:

Preparation of 1M potassium acetate solution:

9.8gm of potassium acetate was taken in a 100mL of volumetric flask and small amount of distilled water was added and dissolved in it. Then the final volume was made up to the mark by adding required amount of distilled water.⁽¹³⁾

Preparation of standard quercetin acid solution:

1mg quercetin was dissolved into 1mL distilled water, so the concentration of the solution is 1mg/mL or 1000µg/mL which was applied as stock solution then serial dilution was performed in order to prepare different concentrate solution (10µg/mL, 50µg/mL, 100 µg/mL and 200µg/mL) ⁽¹³⁾

Preparation of blank:

Blank consist of all the reagents, except for the extract.

Preparation of extract solution:

200µg/mL concentrated extract solution was prepared as follows:

Method for detection of flavonoids:

1mL of plant extract or standard of different concentration solution was taken in a test tube. 3mL of ethanol was added, 200µl of 10% aluminium chloride solution was added into the test tube, 200µl of 1M potassium acetate solution was added into the test tube, and 5.6mL of distilled water was added into the test tube. The test tube was then incubated at room temperature for 30 min. to complete the reaction. Then the absorbance was measured at 420nm using a spectrophotometer against blank. The total content of flavonoid compounds in plant extract in quercetin equivalent was calculated by the following formula equition:

C= (e*v) / m …………………………equation no.1

Where,

C= Total content of flavonoid compounds, mg/g plant extract, in quercetin equivalent (QE);

e= The concentration of quercetin established from the calibration curve, mg/mL;

V= The volume of extract mL.

M= The weight of pure plant extracts, gm.⁽¹³⁾

Tannin contents determination:

Tannins concentration in gel was measured by UV spectrophotometer. 1g of the prepared gel was dissolved in 100mL of phosphate buffer (pH7.4) the solution was sonicated to dissolve the tannins in phosphate buffer. About 1 mL of solution was withdrawn and further diluted to 10mL. Then absorbance was measured at 255nm in UV/VIS spectrophotometer. The % tannins content was calculated using the equation, which was obtained by linear regression analysis calibration curve of gallic acid in phosphate buffer (pH70.4)⁽⁵⁾

UV- Spectrophotometry:

Gallic acid used s marker compound for the examination of tannins in the range 200-400nm. A 100ppm solution of gallic acid in phosphate buffer shows absorption maxima at about 255nm⁽⁴⁾.

Preparation of stock solution:

A stock solution of gallic acid (100μg/mL). Was prepared. Accurately 10mg of gallic acid was weighed and dissolved in 100mL of phosphate buffer pH 7.4.⁽⁴⁾

Selection of analytical concentration range:

The aliquots of stock solution were transferred to 10mL volumetric flasks and volume was made up with 10 mL of methanol and phosphate buffer pH 7.4. Respectively, the absorbance of solutions was measured at 255nm for phosphate buffer pH 7.4. and plotted against concentration. The concentration range over which the drug obeyed beer’s law was chosen. Beers law was obeyed in the concentration range of 5-25μg/mL for gallic acid⁽¹⁰⁾.

Phytochemical screening of extract of Cleome gyandra:

Phytochemical screening and fractionation of phytoconstituent from aqueous. Extract of gyndropsisgyandra phytochemical screening was performed as mentioned below^.(8).

I. Anthraquinones:

Powdered plant material was boiled with 10% HCl for a few minutes, filtered and allowed to cool. This was then partitioned against equal volumes of chloroform. Formation of a rose-pink colour in the aqueous layer on addition of 10% ammonia solution indicated the presence of combined anthraquinones.⁽¹⁰⁾

II. Cardiac glycosides:

Sample was extracted with 10mL of 80% methanol for 5 minutes on a steam bath, filtered and diluted with an equal volume of distilled water. A few drops of lead acatate solution were added, shaken and filtered after a while. Filtrate was then extracted with methylene chloride (two times) and was evaporated to dryness on a steam bath. Then, about 1mL of 2% 3, 5-dinitro benzoic acid in ethanol was added to the residue and the solution was made alkaline with 5% NaOH. The formation of a brownish purple colour is indicative of the presence of unsaturated lactones.⁽¹²⁾

III. Cyanogenetic glycosides:

About 1 g of powdered sample was boiled with distilled water and moist sodium picrate paper held inside the tube with a cork. A colour change from yellow to brick-red of the picrate paper was positive for cyanogenetic glycosides.

IV. Steroidal nucleus:

A few grams of the powdered material were heated over a Bunsen flame with 10% HCl and FeCl3 for 15 mins, after which it was filtered and cooled. Then extracted with 2 volumes of CHCl3. The organic phase was concentrated to a small volume and acidified with 5mL acetic anhydride. The formation of a reddish-brown ring at the interface on carefully pouring conc. H2SO4 is indicative of the presence of a steroidal nucleus⁽¹²⁾

V. Tannins:

Ferric chloride test:

Plant material was boiled with water for a few minutes; this was filtered and diluted with more water. Bluish-black color formation on addition of a few drops of ferric chloride is indicative of the presence of tannins.

1% solution of gelatin containing 10% Nacl – precipitate indicates a positive test for tannins.

Lead acetate solution (10%) – gives a buff colored precipitate for phenolic compounds.

VI. Test for saponins:

A. About 1mL of extract was dilute with water to 20mL and shaken in a graduated cylinder for 10min. a 1 cm layer of foam indicates the presence of saponins.

VII. Test for gums and mucilage:

B. About 1mL of extract was added slowly to about 25mL of alcohol with constant stirring precipitate indicates the presence of gums and mucilage⁽¹⁶⁾.

Determination of total ash:

About 2 to 3g accurately weighed powdered was incinerated, of the ground drug in tared platinum or silica dish at a temperature not exceeding 450ºc until free from carbon, cool and weighed.⁽¹⁴⁾ If a carbon free ash cannot be obtained in this way, exhaust the charred mass with hot water, collect the residue on an ash less filter paper, incinerate the residue and filter paper, the filtrate was, evaporate to dryness and ignite at a temperature not exceeding 450ºC ⁽¹⁵⁾

Determination of moisture content:

Procedure set forth here determines the amount of volatile matter (i.e. water drying off from the drug). About 1.5g of drug (without preliminary drying) after accurately weighing (accurately weighed to within 0.01g) was placed in tarred evaporating dish. After placing the above said amount of the drug in the tared evaporating dish was dried at 105ºc for 5 hours, and weighed. Drying and weighing at one hr. interval was done until difference between two successive weights corresponds was found not more than 0.25 percent. When constant weight was reached two consecutive weights after drying for 30 min. and cooling for 30 min. in a desiccators, showed not more than 0.01g difference.⁽¹⁵⁾

Thin layer chromatography:

For identification of active constituents, Thin layer chromatography was done on silica coated plates. The plates were developed in chloroform-methanol-water (65:35:10) as mobile phase. After development the plates were visualized using iodine chamber, Gallic acid was used as maker, then the Rf value of marker and sample was calculated.⁽⁸⁾

Formulation development:

From the trial batches, carobopol 940 as a gelling agent in concentration of 1% and 1.5% contains of 1% and 1.5% of tannins and flavonoids was selected for formulation of herbal gel.^(3).

3² Full Factorial Experimental Design Layout:

Variables for Experimental Designs

Independent variable :

X1 = Concentration of carbapol 940

X2 = Concentration extract

Dependent variable:

Y1 = Viscosity

Y2 = Spreadability

Y3 = pH

X1 = Carbapol 940 concentration :

0.50gm, 1gm, and 1.5gm was used

X2 = Concentration extract :

0.25gm, 0.50gm and 1gm was use.

Evaluations:

Preliminary evaluation of formulations was carried out as follows:

Physical evaluation:

Physical appearance and homogeneity of the prepared gels were evaluated visually,

pH:

The pH of various formulations was determined by using digital pH meter. To determine pH, 1g of each gel formulation was transferred into 10mL beaker and pH was measured by using the digital pH meter. (Raghupstruni, J.L.et al, 2013)

Viscosity:

Viscosity had been measured using Brookfield Viscometer (model LV-DV- II, Helipath- spindle type S-96). As the system is Non- Newtonian spindle no.S-96 were used.

Spreadability:

Spreadability was determined by apparatus, which is suitably modified in the laboratory and used for the study. It consists of a wooden block, which is provided by pulley at one end. By this method, spread ability was measured on the basis of ‘slip’ and ‘drag’ characteristics of gels. A ground glass slide was fixed on this block. An excess of gel (about 1gm) under study was placed on this ground slide. The gel was then sandwiched between this slide and another glass slide having the dimension of fixed ground slide and provided with the hook. The top plate was then subjected to pull off with the help of string attached to the hook and the time (in seconds) required by the top slide to cover a distance of 7.5 cm be noted and weight in gram was recorded. Spreadability was calculated using following equation:

S = M*L /T …………………………….Equation No.2

Where M= Weight tide to upper slide,

L= Length proceeded onward glass,

T= time taken (Raghupstruni.J.L. etal, 2013)

Table.1

Formulation code	Variable level
Formulation code	Extract (water extract: ethanol extract)	X2 carbapol 940
F1	0.25:0.25	0.50
F2	0.25:0.25	1
F3	0.25:0.25	1.5
F4	0.50:0.50	0.50
F5	0.50:0.50	1
F6	0.50:0.50	1.5
F7	1:1	0.50
F8	1:1	1
F9	1:1	1.5

Table.2

Ingredient	Batch A	Batch B	Batch C	Batch D	Batch E	Batch F	Batch G	Batch H	Batch I
Extract (gm)	0.25:0.25	0.25:0.25	0.25:0.25	0.50:0.50	0.50:0.50	0.50:0.50	1:1	1:1	1:1
Carbopol940(gm)	0.50	1	1.5	0.50	1	1.5	0.50	1	1.5
Propylene glycol(mL)	10	10	10	10	10	10	10	10	10
Water(mL)	100	100	100	100	100	100	100	100	100
Triethanolamie	q.s	q.s	q.s	q.s	q.s	q.s	q.s	q.s	q.s
Preservatives	0.012	0.012	0.012	0.012	0.012	0.012	0.012	0.012	0.012

In-vitro drug release:

The in vitro diffusion studies of the gel were performed by using cellophane membrane. The membrane was soaked in phosphate buffer (pH7.4) for 6-8 hr. was clamped carefully to one end of the hollow glass tube of diffusion cell (2.3cm diameter, 4.16cm²area). 100mL of phosphate buffer was taken in a beaker, which was used as receptor compartment for the study. 1gm of each formulation was spreaded uniformly on the receptor compartment and the temperature was kept in contact with the receptor compartment and the temperature was maintained 37.5⁰c ± 0.5⁰c the solution on the receptor side were stirred by magnetic beads. At pre-determined time intervals. 5mL of solution from the receptor compartment was pipette out and immediately replaced with 5mL fresh phosphate buffer solution. The tannins were determined spectrophotometrically at 255nm against appropriate blank. The mount of tannin permeated of all formulation was calculated.⁽⁶⁾

Equation 3

Cn = drug concentration of the receptor medium at each sampling time,

Ci = drug concentration of the each sample,

V₀and Vi = The volumes of the receiver solution and the sample respectively

S = effective diffusion area

Stability Studies:

The stability study was evaluated as per ICH guidelines, in the below conditions. (Nataranjan R, et al., 2014).

In this study, effect of temperature and humidity was studied by analyzing the optimized batch kept in environmental chamber maintained at 40⁰C± 2⁰C/ 75% ± 5% RH for 1 months. The formulation was evaluated for changes in appearance, pH, and drug content.

Analgesic activity study:

Skin irritation study:

For the skin irritation test, healthy male mice were divided into three groups of six rats area of dorsal portion of all the mice were shaved and wiped with surgical spirit. A constant measure quantity of the formulation was applied over the test sites. The test sites were observed for any toxic effects, erythema and edema on skin surface for 48hrs after application.⁽⁷⁾

Table.3

Group (n=6)	Treatment
Test	Herbal gel formulation
Control	Aqueous gel base

Acetic acid induced writhing in mice:

(1% v/v) was administered intraperitoneally, to all the groups at the dose of 1mL/kg body after the application of formulated gels. A writhed was indicated by abdominal constriction and full extension of hind limb. The number of abdominal constrictions (writhed) and stretching with a jerk of the hind limb were counted for 30 minutes after administering acetic acid. Percent protection against writhed movement was taken as index of analgesic effects. Analgesic activity was expressed as the percentage inhibition of abdominal constrictions between control animals and mice pre-treated (n=6) with the herbal gel formulations using the formula (Control mean – Treated mean) x 100 / Control mean.⁽⁹⁾

Table.4

Sr. No.	Group (n=6)	Treatment, Dose and Route
1	Control	Aqueous gel base
3	Test 1 (Herbal gel formulation)	Topical application
4	Test 2 (Herbal gel formulation)	Topical application.

B) RESULTS AND DISCUSSION:

Selection and authentication of plant:

The authentication of Cleome gyandra L. was done by Botanical Survey of India,

Western Regional Centre, 7-Koregoan Road, Pune-411001, Maharashtra India.

Table.5

Specimen No.	Plant Name	Family
PG 01	*Cleome gyandra L.*	Cleomaceae.

Extraction and Isolation from Cleome gyandra L.

The percent yield of obtained tannins from Cleome gyandra L. Was 10.34%.±0.32.

*±SD (n=3

Determination of total flavonoids:

The total flavonoids content was to be 0.40mg/mL of extract.

Phytochemical screening of extract of Cleome gyandra L.

Table.6

SR. No	TEST	OBSERVATION	REFERENCE
A	For alkaloids
1.	Dragondrof reagent (potassium bismuth iodide)	No orange brown precipitate	Alkaloids might be absent
2.	Mayers reagent (mercury potasium iodide)	No cream precipitate	Alkaloids might be absent
3.	Hagers reagent (saturated picric acid)	No yellow precipitate	Alkaloids might be absent
4.	Wagners reagent (iodine reagent)	No reddish brown precipitate	Alkaloids might be absent
B	For Tannins
1.	Dilute ferric chloride (5%)	Intense blue black	Tannins might be present
2.	gelatin solution (1%) +Nacl solution (10%)	White precipitate	Tannins might be present
3.	Lead acetate (10%)	Buff precipitate	Tannins might be present
4.	Dilute HNO₃	Red colouration	Tannins might be present
c.	For carbohydrate
1.	Molisch's test	Violet colour ring absent.	Carbohydrate might be absent.
D.	For Saponins
1.	Water was added to the extract	No foam was observed	Saponins might be absent
E.	For flavonoids
1.	Shinoda Test	Red coloration	Flavonoids might be present

Figure No. 1 Test for alkaloids

A. Dragondrof test B. Mayers reagent

C Hagers reagent D Wagners reagent

Fig. 2. Test for tannin

A. Gelatin solution test B. Lead acetic test

C. Ferric chloride test D. HNO3 test

Fig. 3. Test for flavonoids (Shinoda test)

Fig 3. Test for carbohydrate (Molisch’s test)

From the Table 5 we were said that the extract obtained after soxhlet crude powder of gyandropsis gyandra in water contained Tannins and ethanol contained flavonoids.

Determination of Total Ash:

The total ash value of extract was 9.10%±0.10 which was within the Indian Pharmacopeial Limit (NMT 27%).

Determination of Moisture content:

The moisture content of extract was found to be 5.11±0.55 which was within the Indian Pharmacopeial limit

Thin layer chromatography

Mobile Phase: Toluene-Ethyl actetate-formic acid-methnol (3:3:0.8:0.2)

1. Rf value of Extracted Tannins =

Distance moved by the analyte from origin

––––––––––––––––––––––––––––––––––– Equation 4

Distance moved by the front from origin

3.5

––– = 0.63

5.5

2. Rf value of tannic acid (MARKER)

Distance moved by the analyte from origin

––––––––––––––––––––––––––––––––––– Equation 5

Distance moved by the front from origin

3.6

––– = 0.65

5.5

TLC of Extract

Table.7

Sample	Calculated Rf value	Reported Rf value
Extract	0.63	0.65

Figure no.1

The Calculated Rf value of extract and marker were 0.63 and 0.65 respectively given in table7. The calculated Rf value was near to observed value. From thin layer chromatography concluded that tannin might be present in extract.⁽¹¹⁾

Table.8

Batches	pH	Viscosity(cps)	Spreadability (g cm/sec)	Tannins content
F1	6.82±0.09	2561±31.34	71.16±16.23	82%±0.85
F2	6.17±0.06	4125±30.60	72.16±12.25	89%±0.62
F3	6.20±0.08	2165±92.34	64.8±20.85	83%±0.45
F4	6.51±0.15	2361±92.30	68.8±45.12	91%±0.95
F5	7.07±0.02	2135±40.88	56.9±23.14	85%±0.46
F6	6.23±0.12	4558±51.28	58.5±65.14	88%±0.35
F7	6.02±0.08	3612±16.24	19.21±32.12	92%±0.28
F8	6.93±0.07	4850±20.76	21.02±84.25	94%±0.60
F9	6.7±0.06	3584±25.30	26.25±50.23	92%±0.25

*±SD (n=3)

The formulated gel was evaluated for pH and found that all formulations had pH in the range of 6.8-7.2 i.e. in the neutral range. The pH of different formulation batches are shown in Table.7

The topical preparation ideally should have neutral pH. If the pH of the formulation is acidic skin irritation, skin hyper pigmentation is occurred and if it is on alkaline side, the formulation upon application may causes disruption of stratum corneum and damage to the barrier function of skin

Viscosity:

The viscosity of different gel formulation was evaluated

From the above viscosities value observed that formulation containing carbopl940 possess viscosity. Gel formulation majorly depends upon its viscosity. Viscosity of the formulation affects the drug release as well as applicability and spreadability of the gel. If a gel consists of more viscosity then the drug release from the formulation is decreased and if the same gel possess less viscosity the drug diffuses immediately into the diffusion medium. Hence, for the gel formulation optimum viscosity is necessary to get the maximum drug release.⁽¹⁷⁾

Spreadability:

Spreadability value of gel

The above table indicates the spreadability values of the formulation (F1-F9). Batches F1-F4 has higher spreadability value in comparison with others. The higher spreadability value indicates less time required by the slide to drag. Formulation point of view, higher spreadability value is required for application of the semisolid on the affected area. Hence, it can be concluded that the developed formulation had good spreadability which was due to carbopol 940 as a gelling agent.

Tannins content in formulation

The tannins content of all the formulation F1-F9 were in the range of 82%-94%.

In vitro diffusion study:

Table 9 In-vitro release

Time

(min)

2512.23±

0.35

2746.97±

0.56

912.123±

0.85

1514.516±

0.25

1254.177±

0.14

1069.35±

0.25

1254.48±

0.19

1797.95±

0.45

1525.14±

0.25

3067.67±

0.56

3212.94±

0.42

1530.424±

0.82

2131.123±

0.43

2337.202±

0.53

1825.64±

0.16

1346.43±

0.36

2522.21±

0.58

2563.21±

0.31

3126.075±

0.19

3331.855±

0.84

2113.287±

0.72

3131.39±

0.34

2096.586±

0.65

2254.24±

0.84

2351.63±

0.56

2251.21±

0.65

3541.14±

0.51

4520.658±

0.31

4656.861±

0.26

3089.877±

0.82

3964.933±

0.12

3995.349±

0.26

3424.5±

0.15

2585.13±

0.59

3882.15±

0.62

2145.15±

0.74

5027.343±

0.53

5168.596±

0.32

4229.964±

0.84

4125.06±

0.24

3397.936±

0.62

3648.32±

0.61

3551.49±

0.18

2571.75±

0.85

3145.15±

0.25

5886.33±

0.81

6366.891±

0.31

5321.24±

0.64

5477.289±

0.16

4022.158±

0.26

3543.15±

0.83

3145.10±

0.35

4656.78±

0.91

4521.15±

0.65

*±SD (n=3)

The cumulative drug release and the flux after six hours of formulation batches (F1-F9) are given in Table 10. From the cumulative release it was observed that the formulation containing Carbopol 940 as a gelling agent had higher value of flux after the period of six hours. Also the cumulative release of Carbopol 940 gel was observed. is TT.

Table 10: Cumulative release of tannins from gel.

Batches	Cum release µg/sqcm	Flux
F1	5696.33±0.32	949.38±0.52
F2	6426.89±0.26	1071.14±0.63
F3	5137.24±0.56	856.31±0.35
F4	5631.28±0.45	938.54±0.34
F5	46352.15±0.95	725.52±0.42
F6	4972.15±0.61	828.87±0.14
F7	3503.26±0.35	583.20±0.61
F8	4352.15±0.16	725.6±0.16
F9	4215.32±0.65	695.5±0.25

*±SD (n=3)

Figure 2: Graphical representation of tannins release

Stability Study:

Effect of temperature and humidity on formulation was carried out by analyzing the optimized formulation keeping at 40ºC±2ºC and 75%±5% relative humidity in stability chamber for one month. The formulation was analysed for appearance, pH, and Tannins content formulation.

Table.11

Evaluation Parameters	Appearance	PH	Drug content (%)	Viscosity (cps)	SPREADABILITY (g cm/sec )
Batch1	Brown colour	6.8±0.16	81%±32.16	5258cps±0.16	79.34±15.28
Batch 2	Dark brown colour	6.7±0.82	90%±23.02	1544cps±0.23	72.26±16.23
Batch 3	Dark brown colour	7.2±0.52	82%±12.34	2154cps±0.61	62.66±12.25
Batch 4	Brown colour	7.1±0.32	92%±92.31	5164cps±0.12	62.82±20.85
Batch 5	Dark brown colour	6.8±0.61	84%±15.36	1232cps±0.34	65.8±40.12
Batch 6	Brown colour	7±0.63	87%±64.23	13160cps±0.31	52.9±21.10
Batch 7	Brown colour	7.2±0.64	92%±34.12	12000cps±0.34	55.5±62.14
Batch 8	Dark brown colour	6.9±0.31	94%±61.35	28300cps±0.92	29.01±52.12
Batch 9	Dark brown colour	6.5±0.52	85±54.58	45125cps±0.85	24.03±84.15

*±SD (n=3)

Table.11

formulation	Dose (mg/kg.I.P.) Acetic acid (1% v/v)	No. of writhes in 5-15 min after treatment	% Inhibition
F1	10	42.2±2.541	70.25
F2	8.10	15.5±1.58	28.31
F3	11.86	32.25±3.85	31.18
F4	14.38	28.51±3.51	33.54
F5	14.82	21±2.512	50.42
F6	14.83	24.2±1.548	68.42
F7	15.65	28.6±3.581	32.51
F8	15.65	24.4±3.851	33.54
F9	16.07	28.1±3.712	32.55

Analgesic activity study:

Skin irritation test:

The prepared herbal gel was evaluated for its skin irritant effect, where no erythema or edema was observed for all the formulations F1 to F9 after 24 hours. Thus the study indicated that the prepared herbal gel formulation was found to be safe.

Acetic acid (1% v/v) was administered intraperitoneally, to all the groups at the dose of 1mL/kg body weight 15 min after the application of formulated gels.

Aqueous and ethanol extract produced significant inhibition of writhing reaction induced by acetic acid compared to control group. % inhibition of writhed reaction induced by extracts was in the order as aqueous > ethanol. These extracts delayed the onset of the writhed response compared to control group.

Intraperitoneally injection of acetic acid produced pained through activation of chemo sensitive nociceptors or irritation of the visceral surfaced, which leaded to liberation of histamines, bradykinins, prostaglandins and serotonins. Thus analgesic activity of opioid partial agonist and non- steroidal anti-inflammatory drug delivery systems can be determined by the writhed test, the mechanism of action of the analgesic effects of extracts of Cleome gyandra plant could probably due to the blocked of effect or release of endogenous substances that excited pained nerved.

Overall we could say that ethanol and aqueous extract showed analgesic activity, preliminary phytochemical tests showed the presence of flavonoids in ethanol and tannin in aqueous extract. Flavonoids and tannins are reported to inhibit prostaglandin synthesis. From this, we can conclude that the antinociceptive activity observed may be due to flavonoids and tannins.

ACKNOWLEDGEMENTS:

Authors would like to acknowledge to Trustees, Bhujbal Knowledge City, MET’s institute of pharmacy, Adgaon, Nasik, Maharashtra, India, for providing the necessary facilities to carry out this work.

CONCLUSION:

Thus, it was concluded that the tannins was obtained from aqueous extract and flavonoids was obtained from ethanol extract whereas tanninsand flavonoids showed analgesic activity. From the evaluation of gel prepared by tannins and flavonoids we concluded that prepared gel was safe.

CONFLICT OF INTEREST:

Nil.

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Received on 10.02.2020 Modified on 14.03.2020

Asian J. Pharm. Tech. 2020; 10(2):71-80.

DOI: 10.5958/2231-5713.2020.00014.8