Formulation and Evaluation of Herbal Gel of Basella alba for wound healing activity

Mohammed Haneefa K.P1*, Anu Abraham2, Saraswathi.R3, Guru Prasad Mohanta4, Chandini Nayar5

1, 2, 3 Al shifa College of Pharmacy, Poonthavanam Post, Kerala, India *Research Scholar, School of Chemical & Biotechnology, SASTRA University, Tamil Nadu,

India 4Department of Pharmacy, Annamalai University, Tamil Nadu, India

5School of Chemical & Biotechnology, SASTRA University, Tamil Nadu, India

Abstract: Basella alba is used in many parts of the world for its medicinal use as laxative, anti-inflammatory, rubefacient, soothing as well as its cooling effects when applied to burns and scalds. It’s also used for haemorrhages, skin diseases, sexual weakness, ulcers and as laxative in children and pregnant women. In Nigeria, used for fertility enhancement in women, burn wounds etc. Hence the present study was chosen to evaluate its scientific validity. In the present study aqueous extract of leaves of Basella alba were formulated as a gel and investigated for its physicochemical as well as for its burn wound healing activity. Aqueous extract of dried leaves of Basella alba were subjected to preliminary phytochemical evaluation and wound healing activity studies. Different gel formulations of aqueous extract of Basella alba (2% w/v) were prepared using polymers carbopol 934 and carbopol 940 by varying their concentration. These formulations were evaluated for the following parameters: drug content, pH, viscosity, extrudability, spreadability, primary skin irritation, pharmacological activity and stability. Wound healing studies of aqueous extract revealed that Basella alba treated animals were found to epithelise in 23 days while the solvent control and untreated rats epithelise within 35days and 39 days respectively. The formulation 1.5% w/w carbopol 934 was found to be more promising as it shows better physicochemical characteristics, higher pharmacological activity and better stability compared to other formulations. So it was concluded that Basella alba aqueous extract shows significant improvement in burn wound contraction and hence this is a spromising candidate in burn wound healing. Keywords: Basella alba, Burn wound healing, Herbal gel, Carbopol 934, Carbopol 940

INTRODUCTION Gels are defined as semi rigid systems in which movement of the dispersing medium is restricted by an interlacing three dimensional network of particles or solvated macromolecules of the dispersed phase [1]. Gels have better potential as a vehicle to administer drug topically in comparison to ointment, because they are non-sticky, require low energy during formulation, are stable and have aesthetic value. The increased viscosity caused by the interlacing and consequential internal friction is responsible for the semisolid state. Basella alba is a perennial fast-growing, soft-stemmed vine. Leaves are thick, semi-succulent, heart-shaped with mild flavour and mucilaginous texture. It is reported to possess laxative, anti-inflammatory, rubefacient, soothing as well as its cooling effects when applied to burns and scalds. The present study was conducted to formulate an herbal gel of Basella alba using gelling agents like Carbopol 934, Carbopol 940. The prepared gels were evaluated for

physicochemical properties as well as for their pharmacological activity. MATERIALS AND METHODS Materials Plant material Basella alba were collected from Calicut and were authenticated by Mr.A.K.Pradeep, Curator, Department of Botany, University of Calicut. Chemicals Carbopol 934 and Carbopol 940 were obtained from Loba Chem.Pvt.Ltd, Mumbai. Methyl Paraben obtained from HiMedia Laboratories Pvt. Ltd. Glycerine and Triethanolamine were obtained from Nice Chemicals Pvt. Ltd, Mumbai Methods Preparation of extract The foreign, earthy matter and residual materials were removed carefully from the leaves and then cleaned and dried in the shade. It was then powdered and used for extraction.

Mohamman Haneefa K P et al /J. Pharm. Sci. & Res. Vol.4(1), 2012, 1642-1648

1642

The powdered material was macerated for a period of one week, filtered and concentrated under reduced pressure. It was then refrigerated. Animals A total of 56 rats, healthy male albino rats weighing 150-200g were used for wound healing studies and the same were used for primary skin irritation test. They were bred locally in animal house of Al shifa College of Pharmacy. They were housed under controlled conditions of temperature (25±20C), humidity (50±50C) and 10-14hrs of light and dark cycles. The study was conducted after obtaining approval from Institutional Animal Ethical Committee (Reg no: 1195/ac/08/CPCSEA) STUDY DESIGN Wound healing studies of extract [2] [3] Rats were anaesthetized with ketamine + xylazine(50 mg/kg+5mg/kg ) and the hair on the back was clipped with electric clippers. Burn wounds were created by using a device with a iron piece and a wooden handle placed on the back of the rat. It is heated to red hot over flame and was placed in contact with the back of anaesthetized rat up to 10 seconds without any pressure. After this each animal was placed in a separate cage for full recovery from anaesthesia before being returned to holding rooms. The wound of test animal was treated with extract in glycerol and the control group 1 was treated with glycerine and control group 2 were left untreated. The application was repeated daily for the next 20 post operative days and the following parameters were studied Epethelization period: It was monitored by noting the number of days required for scar to fall away, leaving no raw wound behind. Wound contraction: To monitor this, progressive changes in wound area were

followed planimetrically. Leaving the wounding day, wounds were traced on a transparent paper on an alternate day. The animal was restrained in proper position during tracing. The tracings were then transferred to 1 mm2 graph sheet. From this, wound areas were read and the percent of wound contraction was calculated taking the initial size of wound (250 mm2) as 100% %

100

Formulation of gel The gel was prepared using varying concentrations of Carbopol 934 and Carbopol 940 (0.5-2%). Accurately weighed quantity of Carbopol 934 and Carbopol 940 was dispersed in distilled water with constant stirring using a mechanical stirrer for 30 min at 1200rpm. After all the Carbopol was dispersed, the extract dissolved in water and the preservatives were added and mixed well. The pH was adjusted to neutral using triethanolamine until a clear consistent gel was obtained. Evaluation of gel The prepared gels were evaluated for appearance, pH, drug content, spreadability, extrudability, permeability studies and stability studies. All the gels were visually evaluated for presence of fibres and particles. Appearance: All the gels were brown and translucent in nature. pH: The pH values of prepared gels were checked by using a digital pH meter (Systronics, Model Apx 175 E, India) Drug content [4]: 1g each formulation containing approximately 20 mg of drug was taken in a 50 ml volumetric flask and diluted with water and shaken to dissolve the drug in water. The solution was filtered through whatmann filter paper.1 ml of the filtrate was pipette out and diluted to 10 ml with water.

Mohamman Haneefa K P et al /J. Pharm. Sci. & Res. Vol.4(1), 2012, 1642-1648

1643

The content of the drug was estimated spectrophotometrically by using standard curve plotted at 302 nm. Spreadability [5]: Two sets of glass slides of standard dimensions were taken. The herbal gel formulation was placed over one of the slides. The other slide was placed on the top of the gel, such that the gel was sandwiched between the two slides in an area occupied by a distance of 7.5 cm along the slide. 100g weight was placed upon the upper slides so that the gel between the two slides was pressed uniformly to form a thin layer. The weight was removed and the excess of gel adhering to the slides were scrapped off. The two slides in position were fixed to a stand without slightest disturbance and in such a way that only the upper slide to slip off freely by the force of weight tied to it. A 20g weight was tied to the upper slide carefully. The time taken for the upper slide to travel the distance of 7.5 cm and separated away from the lower slide under the influence of the weight was noted. The experiment was repeated by three times and the mean time taken for calculation. Spreadability was calculated by using the following formula.

S – Spreadability, m – Weight tied to the upper slide (20g) l - Length of the glass (7.5 cm) t - Time taken in seconds Extrudability [6]: A closed collapsible tube containing above 20g of gel was pressed firmly at the crimped end and a clamp was applied to prevent any rollback. The cap was removed and the gel extrudes until the pressure was dissipated. pH: pH measurements of the gel were carried out using a digital pH meter by dipping the glass electrode completely in to the gel system to cover the electrode.

Viscosity [7]: Viscosity of the gels was determined using Brookfield viscometer. Spindle type, model LVDV-E at 10 rpm. 100 g of the gel was taken in a beaker and the spindle was dipped in it for about 5 minutes and then the reading was taken. Permeability studies [8]: The in vitro diffusion studies of the gels were performed using dialysis membrane. The membrane was soaked in phosphate buffer pH 6.8 for 6-8 hr & was clamped carefully to one end of the hollow glass tube. Phosphate buffer of pH 6.8 was used for in vitro release as a receptor medium. The gel sample was applied on the membrane and then fixed in between donor and receptor compartment of glass tube. The receptor compartment contained phosphate buffer (100ml) of pH 6.8. The temperature of diffusion medium was thermostatically controlled at 37º ± 1º by surrounding water in jacket and the medium was stirred by magnetic stirrer at 500rpm. The samples were withdrawn at predetermined intervals and were replaced by equal volume of fresh fluid. The samples withdrawn were spectrophotometrically estimated at 302nm using phosphate buffer as blank. Pharmacological studies of formulation Primary skin irritation test: The primary skin irritation test was performed on albino rats weighing about 150-200g. The animals were maintained on standard animal feed and had free access to water ad libitum. The animals were kept under standard laboratory condition. The total mass was divided into four batches, each batch containing seven animals. Two batches of each were used for control and test. Dorsal hairs at the back of the rats were clipped off one day prior to the commencement of the study. Animals showing normal skin texture were housed individually in cages. 50mg of the each formulation of different concentrations were applied over one square centimetre area of

Mohamman Haneefa K P et al /J. Pharm. Sci. & Res. Vol.4(1), 2012, 1642-1648

1644

Table No.1: BURN WOUND HEALING STUDIES OF AQUEOUS EXTRACT SHOWING

PERCENTAGE REDUCTION OF WOUND SIZE IN RATS (% CLOSURE)

Conc: of alcoholic extract (%w/v) 

Day 4  Day 8  Day 12 Day 16 Day 20  Epithelization time

(in days) 

Sol.control  3.12±0.15  21.87±0.22  30.07±0.42 46.87±0.63 62.57 ±0.91  35±0.612%  9.03±0.17  27.08±0.24  57.62±1.1 80.20±0.09 93.74 ±1.45  23±0.354%  7.02±0.12  22.91±0.15  53.64±1.05 73.95±1.12 87.76 ±1.2  25±0.216%  4.68±0.08  29.22±0.28  45.83±0.71 66.14±0.81 78.64 ±1.13  29±0.488%  5.02±0.95  26.01±0.32  43.25±0.65 53.3±0.63 71.66 ±0.95  32±0.5110%  5.98±0.85  21.87±0.12  37.5±0.45 52.05±0.91 69.76 ±1.15  33±0.61Untreated control 

1.87±0.02  9.03±0.15  21.87±0.13 37.5±0.35 52.5 ±0.85  39±0.71

intact and abraded skin to different animals. Aqueous solution of 0.8% formalin was applied as standard irritant. The animals were observed for seven days for any signs of oedema and erythema. Wound healing studies of formulation: Wound healing studies of formulation were carried out similar to wound healing studies of extract in which the test animals were treated with gel formulation and control groups with dummy gel. The application was repeated daily for the next 20 post operative days and the following parameters were studied, epithelisation period and wound contraction. Table No.2: Drug content of Basella alba gel

FORMULATION  ABSORBANCE CONCENTRATION*

(µg/ml) DRUG CONTENT

(%w/w)

F1  0.042  31.70588  79.26471F2  0.04  30.52941  76.32353F3  0.052  37.58824  93.97059F4  0.035  27.58824  68.97059G1  0.047  34.64706  86.61765G2  0.041  31.11765  77.79412G3  0.036  28.17647  70.44118G4  0.035  27.58824  68.97059

* n=3

Stability studies [9]: The selected formulations were subjected to stability studies. The selected formulations were filled in to aluminium collapsible tubes and stored at room temperature (250C to 300C), 370C ± 0.50C, 40C to 50C. All the gel formulations

were stored for a period of six weeks. Samples were withdrawn at weekly interval for a period of six weeks and assessed for drug content. At the end of six week they were evaluated for physical parameters and integrity of the product. Results and Discussion: The preliminary step involved was the extraction of leaves of Basella alba using different solvents. Then these extracts were subjected to phytochemical screening for identification of constituents. Since the aqueous extract contained maximum number of constituents it was selected for further study. Different concentrations of aqueous extract was prepared and subjected to pharmacological studies in order to fix the dose of the extract. The pharmacological studies showed that 2% concentration of extract showed max: wound healing activity with a percentage wound closure of 93.74 after 20 days. The results are shown in table no: 1. Eight different formulations of herbal gel was prepared using carbopol polymer and were evaluated. The drug content of various formulations were determined and shown in table 2.Various parameters such as drug content , physical evaluation pH, viscosity, spreadability, extrudability were determined and the results are shown in tables: 2-7.The in vitro release studies were conducted and

Mohamman Haneefa K P et al /J. Pharm. Sci. & Res. Vol.4(1), 2012, 1642-1648

1645

Table No: 3 Physical Observations TYPE  FORMULATION NATURE OF GEL

 CARBOPOL 934 GEL  F1  Brown, slightly translucent with less gel consistency

F2  Brown, translucent , with less consistency F3  Brown, translucent , non greasy gel F4  Brown translucent , greasy gel

CARBOPOL 940 GEL  G1  Brown, slightly translucent with less gel consistencyG2  Brown, translucent, with less consistency G3  Brown, translucent, greasy gelG4  Brown, translucent, greasy gel

Table No: 4 pH OF GEL FORMULATION TYPE  FORMULATION pH OF GEL

CARBOPOL 934 GEL F1 6.8

F2 7.2

F3 6.8

F4 6.9

CARBOPOL 940 GEL G1 6.8

G2 6.8

G3 7.1

G4 6.8

*Average of three readings

Table No: 5 Viscosity of Gel Formulation

TYPE  FORMULATION VISCOSITY OF GEL FORMULATION ( cps) CARBOPOL 934 GEL  F1 31260 ± 0.31 

F2 49251 ± 0.41 F3 55580 ± 0.32 F4 63149 ±0.18 

CARBOPOL 940 GEL  G1 57318 ± 0.09 G2 69241 ± 0.04 G3 80145 ± 0.28 G4 89240 ± 0.21 

*Average of three readings

Table No: 6 Spreadability of Gel Formulation

TYPE  FORMULATION SPREADABILITY OF GEL

FORMULATION (g cm/sec)

CARBOPOL 934 GEL 

F1 32.45 ± 0.61 F2 24.96 ± 0.45 F3 21.15 ± 0.38 F4 16.66 ± 0.31 

CARBOPOL 940 GEL 

G1 29.82 ± 0.55 G2 20.07 ± 0.4 G3 15.48 ± 0.29 G4 17.81 ± 0..31 

*Average of three readings

Mohamman Haneefa K P et al /J. Pharm. Sci. & Res. Vol.4(1), 2012, 1642-1648

1646

Table No: 7 EXTRUDABILITY OF GEL FORMULATION

TYPE  FORMULATION EXTRUDABILITY OF GEL FORMULATIONCARBOPOL 934 GEL  F1 + +

F2 + + +F3 +++F4 ++

CARBOPOL 940 GEL  G1 ++G2 +++G3 ++G4 ++

+++ Excellent ++ Good + Not satisfactory

Table No:8 IN VITRO DRUG RELEASE OF F3 FORMULATION

SL.NO  TIME(hrs)  ABSORBANCE AMOUNT

RELEASED (µg)

CUMULATIVE PERCENTAGE RELEASE 

1  0.5  0.096  6347.059 40.03711± 2  1  0.119  8017.353 50.57329± 3  2  0.133  9225.882 58.19666± 4  4  0.157  11063.82 69.79036± 5  6  0.175  12619.41 79.60297± 6  8  0.196  14404.41 90.86272± 

Table No: 9 BURN WOUND HEALING STUDIES OF CARBOPOL 934 GEL SHOWING PERCENTAGE REDUCTION IN WOUND SIZE IN RATS (% CLOSURE)

Formulation  Day 4  Day 8  Day 12  Day16  Day 20 Epithelization

timeControl  4.65±0.08  25.07±0.41  46.87±0.85  56.32±1.11  62.46±0.95  32±0.41 

F1  7.62±0.13  30.11±0.5  56.46±0.95  78.46±1.3  85.06±1.60  24±0.46 

F2  5.68±0.09  29.44±0.51  49.84±0.87  62.56±1.23  79.86±1.39  25±0.48 

F3  8.57±0.11  32.87±0.61  57.46±0.85  79.63±1.35  90.16±1.68  22±0.48 

F4  4.68±0.07  27.08±0.48  45.83±0.75  67.97±1.15  70.18±1.31  30±0.54 

Table No:10 Physical Evaluation of F3 Formulation

PARAMETERS  ROOM TEMP 370C ± 0.50C 40C to 50C VISUAL APPEARANCE  No change  No change No change pH  6.8 6.8 6.8 EXTRUDABILITY  +++  +++ +++ PHASE SEPARATION  -  - - LEAKAGE  -  - - NATURE  Smooth  Smooth Smooth 

Mohamman Haneefa K P et al /J. Pharm. Sci. & Res. Vol.4(1), 2012, 1642-1648

1647

Table No:11 Chemical Evaluation of F3 Formulation

FORMULATION (F3) DRUG CONTENT (%) INITIAL  93.97

FIRST WEEK  93.96SECOND WEEK  93.88THIRD WEEK  93.79

FOURTH WEEK  93.71FIFTH WEEK  93.55SIXTH WEEK  93.57

results are shown in table:8. Pharmacological evaluation of gels revealed that all formulations are non sensitizing and safe for use. The wound healing activity studies were carried out and the results are shown in table no:9. The selected formulation was subjected to stability studies and the results are shown in table no: 10-11. No significant difference in physical and chemical parameters was observed and the formulation was found to be stable for six weeks.

CONCLUSION Eight gel formulations of the Basella alba were prepared using Carbopol 934 and Carbopol 940. The formulations were then subjected to different evaluations. All the formulations were found to be neutral (pH 6.8-7.2) and the drug content was found to be in the range of 68-93%w/w. On physical evaluation of all the formulations F3 (1.5% carbopol 934) was found to be optimum in terms of consistency, spreadability and extrudability. sPharmacological studies of the formulations showed that F3 (1.5% carbopol 934) has greatest pharmacological activity. Based on the evaluations F3 was selected as the best formulation and was subjected to accelerated stability studies over a period of six weeks. Stability studies revealed that there was no significant difference in physical and chemical parameters. It was finally concluded that the formulation F3( 1.5% Carbopol 934) was found to be more promising formulation as it shows better physicochemical characteristics and higher pharmacological activity compared to other

formulations. From the results it can be concluded that Basella alba alcoholic extract when formulated as gel shows significant improvement in burn wound contraction and hence this is a promising candidate in burn wound healing

References: [1] Peter Goldman;Herbal Medicines Today and the Roots of Modern Pharmacology. Annals of internal medicine: 2001, Page no: 594–600 [2] Nayak B S; Investigation of wound healing activity of Lantana camara L. in Sprague dawley rats using a burn wound model; International Journal of Applied Research in Natural Products; Vol. 1(1), March/April 2008), pp. 15-19 [3] Farnood Shokuhi; The efficacy of alcoholic extract of garlic on the healing process of experimental burn wound in the rabbit, Journal of Animal and Veterinary advances 8 (4), 2009, Page no: 655-659 [4] Benoy Brata Bhowmik; Formulation Development and Characterization of Metronidazole Microencapsulated Bioadhesive Vaginal gel; International Journal of Pharmacy and Pharmaceutical sciences;Vol 1, Issue 1, 2009 [5] Mônica V. da silva; Development of Gel with Matricaria recutita L. extract for Topic Application and Evaluation of Physical-Chemical Stability and Toxicity; Latin American Journal of Pharmacy; 28 (4), 2009, Page no: 574-9 [6] Gowda Bhaskar; Formulation and Evaluation of Topical Polyherbal Antiacne Gels containing Garcinia mangostana and Aloe vera; Pharmacognosy magazine; Vol 5, 2009, Page no: 93-99 [8] Panigrahi L; “Effect of Permeation Enhancers on the Release and Permeation Kinetics of Lincomycin Hydrochloride Gel Formulations through Mouse Skin”; Indian J Pharm Sci. 68: (2006), Page no:205-11 [9] U. D. Shivhare; Formulation Development and Evaluation of Diclofenac Sodium gel using water soluble polyacrylamide polymer; Digest Journal of Nanomaterials and Biostructures; Vol. 4, No.2, June 2009, Page no: 285 – 290

Mohamman Haneefa K P et al /J. Pharm. Sci. & Res. Vol.4(1), 2012, 1642-1648

1648