FORMULATION AND CHARACTERIZATION OF AN ANTI-BACTERIAL CREAM USING HEMPEDU BUMI (ANDROGRAPHIS PANICULATA)

FORMULATION AND CHARACTERIZATION OF AN ANTI-BACTERIAL CREAM USING HEMPEDU BUMI (ANDROGRAPHIS PANICULATA)

A. Dahlan ^{* 1}, W. S. Yip ¹ and K. Y. Pin ²

Department of Pharmaceutics, Faculty of Pharmacy ¹, Cyberjaya University College of Medical Sciences, Lot 3410 Jalan Teknokrat 3, Cyberjaya 63000, Selangor, Malaysia.

Herbal Technology Centre, Medicinal Plant Division ², Forest Research Institute Malaysia, 52109, Kepong, Selangor, Malaysia.

ABSTRACT: Staphylococcus aureus (S. aureus), a bacterium that causes skin infection as a result of skin colonization, is becoming increasingly resistant to many commonly used antibiotics. Andrographis paniculata (A. paniculata) or hempedu bumi, traditionally used for the treatment of inflammation and infection, in extract form has been shown to exhibited anti-bacterial activity against various bacteria. This study aims to examine the anti-bacterial properties of A. paniculata towards S. aureus and formulate A. paniculata into cream to determine its anti-bacterial properties. Plant extraction using water was performed at Forest Research Institute Malaysia. Different concentrations (5%, 25%, 50%, 75%, and 100%) of aqueous extract of and cream containing A. paniculata were used in the disc diffusion method for determination of antibacterial activity towards S. aureus. The creams were evaluated physically and chemically. Microbiological pre- and post-testing were carried out for both extract and cream formulations to determine their anti-bacterial properties. Both extract and cream containing A. paniculata showed good anti-bacterial properties against S. aureus. The anti-bacterial activities were proportional to the concentration of extract alone and in the cream. All cream formulations showed satisfactory physical properties with a smooth texture, emollient, non-greasy, and easy to remove with water. The creams also showed good stability in various temperatures after prolonged storage. It is concluded that A. paniculata has the potential to be developed as a cream for skin infections caused by S. aureus. Future research is required to confirm the effectiveness of the A. paniculata cream formulation.

Staphylococcus aureus, Skin infection, anti-bacterial, Andrographis paniculata, hempedu bumi, cream, formulation

INTRODUCTION: Plants produce a diverse range of bioactive molecules that make them rich sources of different types of medicine. Many medicinal plant species worldwide are used in traditional medicine as a treatment for skin diseases caused by fungi and bacteria ¹.

Andrographis paniculata (A. paniculata), locally known as hempedu bumi or Bile of Earth, is a plant which can be found in abundance in Malaysia.

It has been previously shown to exhibit a wide range of pharmacological effects in animals such as anti-diabetes, antidiarrhoeal, anti-viral, anti-inflammatory, hepatoprotective, anti-malarial, anti-HIV, anti-cancer, anti-venom and immune stimulatory ². The main chemical constituents of A. paniculata which is diterpenoids and flavonoids are believed to be responsible for most of the biological activities of this plant ³. A. paniculata is considered beneficial to the skin and is used both externally and internally for the treatment of skin diseases ⁴.

Staphylococcus aureus (S. aureus) causes a variety of skin infections.⁵ These infections are characterized by the formation of pus containing blisters, which often begin in hair follicles and spread to adjoining tissues. There are a few types of S. aureus infections, including folliculitis, furuncles, carbuncles, mastitis, impetigo, and cellulitis.⁶ The common treatments for S. aureus skin infection include administration of antibiotics and surgical.

Most research focuses on determining anti-bacterial properties of crude extract of A. paniculata. So far no pharmaceutical cream dosage form that incorporates A. paniculata has been produced or marketed. There is a clear gap between research and product commercialization. Plant-based medicine can be a viable alternative to antibiotics subject to proper investigation and appropriate documentation. Therefore, this study aimed to determine the antibacterial activity of cream formulations containing A. paniculata against S. aureus.

MATERIALS AND METHODS:

Andrographis paniculata:

Selection of Plant: A. paniculata plants with an annual growth of 0.60-0.70 m in height were selected in which the mature leaves were used. The leaves were dark green in color and free of crops and free from insecticides and pesticides.

Collection of Plant: Fresh plant materials leave (75 kilograms) (kg) of A. paniculata were procured from the botanical garden of Forest Research Institute of Malaysia (FRIM), Kuala Lumpur, Malaysia.

Plant Identification: Specimen sample was authenticated by Dr. Richard Chung (Taxonomist, FRIM) and deposited (sample number: PID 180712-12) in the herbarium of FRIM, Malaysia. A. paniculata is from the family of Acanthaceae with the scientific name of A. paniculata (Burm. f.) Wall. Ex Nees. Its local name includes hempedu bumi and akar cerita. The collector for the fresh plant materials leaves was En Mat Asri Ngah Sanah with the collection number of FRI 38708 from Perlis.

Preparation of Plant Sample Material: Fresh plant leaves of A. paniculata were air dried at room temperature for five days to remove excess water in the leaves. After five days, the leaves of A. paniculata were dried in a Protech laboratory dryer (LDD-720) from Germany at 37 °C for seven days to remove water further and prevent the growth of fungus on the surface of the leaves. The final weight for the oven-dried A. paniculata leaves is 20 kg. The preparation was conducted by Ethno Herbs Resources Sdn. Bhd.

Preparation of Aqueous Extract: The preparation of aqueous extract of A. paniculata was conducted at Herbal Technology Centre (HTC), Forest Reserve Institute of Malaysia, Kuala Lumpur. Three major processes were involved, including the extraction, concentration and freeze-drying processes for the preparation of aqueous extract into the powdered form. These processes were carried out using automated equipment on an industrial scale and in compliance with Good Manufacturing Practices (GMP). Briefly, the dried leaves of A. paniculata were first ground into smaller fragments of leaves using a grinder (EXTA, model GETF-1200) to increase the surface of the leaves and to remove their moisture.

Extraction: The extraction process was carried out by using an EXTA extractor (EETF-1600) with aqueous or reverse osmosis (R.O.) water at 60°C. A ratio of 1:20 was used for the determination of R.O. water required for the extraction process. The extraction took three days to be completed, which was then followed by extract concentration.

Concentration: The extract was transferred into an EXTA evaporator (EETF-950) to concentrate the extract from 400 liters to 14 liters. The temperature was maintained at 60 °C under vacuum. 2 liters of the aqueous concentrated extract was then stored at -18°C before using.  The remaining aqueous extract was freeze-dried under vacuum using a Millrock freeze dryer (85XL) for 48 hours. 0.82 kg of powdered A. paniculata was obtained and stored in at room temperature before use.

Antimicrobial Screening of Extract and Cream Formulation: Aqueous extract of A. paniculata leaves was evaluated for in-vitro antibacterial activity against skin diseases, causing positive bacteria strain S. aureus using the disc diffusion method at five different concentrations of the extract.

Preparation of Aqueous Extracts: Various concentrations of aqueous A. paniculata extracts prepared from both concentrated liquid extract and powder. The two different types of aqueous extracts were evaluated for in-vitro antibacterial activity. All procedures involved in this preparation were done under strict aseptic conditions to avoid contamination of the extracts. All glassware including beaker, volumetric flask, dropper, measuring cylinder, pipette, conical flask, laboratory glass bottles were autoclaved at 121°C for 20 min before use.

Preparation of different concentration of extracts from the concentrated liquid extract of A. paniculata: Five different concentrations of aqueous extracts 5% v/v, 25% v/v, 50% v/v, 75% v/v and 100% v/v (percentage of volume over volume) were prepared from concentrated liquid extract of A. paniculata. Normal saline solution was used as a buffer solution.

Agar Disc Diffusion Method: S. aureus stock cultures with ATCC number 25923 (Thermo Fisher Scientific, Malaysia) was initially grown on nutrient agar plates or blood agar at 37 °C and incubated for 24 hours followed by transferring them onto blood agar using the streak-plate technique. The inocula were prepared in Mueller-Hinton agar (100 mm) at a density adjusted to a 0.5 McFarland turbidity standard for the disc diffusion method. Gentamicin 6-mm discs (Becton Dickinson, USA) were used as a positive control in this study.

For negative control, 10% aqueous Dimethyl sulfoxide (DMSO) and normal saline were prepared. The diameter of the zone of inhibition was visually measured using a ruler.

Modified Agar Well Diffusion Method: Modified agar well diffusion method was performed for extract cream formulations, as described previously ⁷. Mueller-Hinton agar and S. aureus were used as mentioned earlier. 8 mm diameter wells were punched into the agar and filled with the cream formulations followed with 24-incubation at 37 °C. Standard gentamicin cream and blank oil-in-water (O/W) cream (without any active ingredient) were used as positive and negative controls, respectively.

Preparation of Dried Filter Paper Discs: A. paniculata extracts prepared from concentrated liquid extracts and powdered were initially passed through a 0.45 mm membrane filter. Whatman no. 1 filter paper was used to prepare discs with 6 mm in diameter using a single hole puncher. The discs were placed in a sterile covered petri dish and sterilized in an oven at 160 °C for an hour.

The sterile filter discs of 6mm were then impregnated with 10μL of the aqueous extracts prepared from both concentrated liquid extract and powder A. paniculata at a various concentration (5, 25, 50, 75 and 100%^v/_v). The cover of the Petri dishes was closed immediately once the impregnation process done was to minimize the risk of contamination. The discs were later allowed to dry at room temperature for an hour before transferring them onto the Mueller-Hinton agar.

Preparation and Evaluation of Andrographis paniculata Cream:

Cream formulation: An O/W cream formulation was used based on the previous study.⁸ The formula for the various creams is shown in Table 1. The oily phase components such as stearic acid, white beeswax, cetyl alcohol, mineral oil, and stearyl alcohol (R&M) was purchased from Dynamic Sains Sdn. Bhd. For the aqueous phase components, propylene glycol, methylparaben, and propylparaben were purchased from Euro-Chemo Pharma Sdn. Bhd. while triethanolamine was sourced from Becton Dickinson, USA.

Briefly, the components of the oily and aqueous phases were weighed and dissolved in the water bath at 75 °C separately. The aqueous phase was gradually added to the oily phase and stirred continuously until the mixture cooled, and the cream was transferred into a plastic container and labeled accordingly.

Physical Characterization: In addition to the antimicrobial tests as described earlier, each of cream was subjected to visual inspection, pH test using a digital pH meter (Terri + William, USA) and also basic rheological studies.

TABLE 1: FORMULATION OF VARIOUS CREAMS

Statistical Analysis: Statistical analysis was performed using the Statistic Package for Social Sciences program version 19 (SPSS 19.0) and Microsoft Excel 2007. ANOVA followed by posthoc ‘Tukey’s test’ were conducted to determine the significant difference between groups. A p-value of less than 0.05 was accepted as statistically significant.

RESULT AND DISCUSSION:

Evaluation of Anti-bacterial Properties of A. paniculata aqueous extract: The polar aqueous extracts prepared from the concentrated liquid extract and powder A. paniculata were investigated at five different concentrations (5%, 25%, 50%, 75%, and 100%) using disc diffusion method against S. aureus. Based on Fig. 1, all polar aqueous extracts displayed good antibacterial activity against gram-positive strain S. aureus (p<0.05). However, S. aureus was less sensitive to the 5% polar aqueous plant extract (2.08 ± 0.67 mm) compared to higher concentrations. 100% polar aqueous extract exhibited the highest zone of inhibition (17.08 ± 0.67 mm) which shows that the Gram-positive S. aureus strain was susceptible to growth inhibition by the plant extracts at high concentration.

Fig. 2 shows the zone of inhibition exhibited by various concentrations of polar extracts prepared from powder A. paniculata. All polar aqueous extracts displayed positive anti-bacterial activity against Gram-positive strain S. aureus (p<0.05). However, in comparison with the results using liquid extracts, polar extracts of A. paniculata prepared from concentrated liquid extract displayed a higher anti-bacterial activity with greater diameter of inhibition zones (2.08 ± 0.67 mm to 17.08 ± 0.67 mm) compared to the polar extracts of powder A. paniculata (1.33 ± 0.49 mm to 15.25 ± 0.62 mm).

Based on this study, all polar aqueous extracts prepared from both concentrated liquid extract and powder A. paniculata displayed antibacterial activity against gram-positive bacteria strain S. aureus (p<0.05). Active plant constituent such as the andrographolide is mostly concentrated in the leaves, which is very bitter and exhibits natural anti-bacterial properties. There are many other phytochemicals which can be isolated from the plant, especially the leaves, roots and stems including sesquiterpenes, lactones, and flavonoids that may contribute to the anti-bacterial properties of the plant.

Although, both polar extracts prepared from concentrated liquid and powder A. paniculata displayed antibacterial activity against Gram-positive strain S. aureus, they still possess a lower anti-bacterial activity compared to gentamicin antibiotic discs, the positive control (p<0.05). However, the anti-bacterial activity for both concentrated liquid and powder extract in this study were lower than the one reported in an earlier study ⁹. This might be due to the differences between the maturity and the origin of the plant used.

Characterization of Cream Formulation: Table 2 shows the 11 cream formulations which have been formulated for this study. The cream formulated with A. paniculata displayed different green intensity depending on the extract concentration (shown in Fig. 3 below). Generally, the higher the concentration of extract, the higher the green intensity of the cream produced.

TABLE 2: VARIOUS CREAM FORMULATIONS AND THEIR NAME

Table 3 shows the pH of the cream formulations. The pH of the cream formulations appears to be more alkaline, going across the concentration gradient.

TABLE 3: MEAN pH OF CREAM FORMULATIONS (n=3)

FIG. 3: DIFFERENT COLOURS OF CREAM EXHIBITED BY DIFFERENT CONCENTRATION OF EXTRACTS

Evaluation of Anti-bacterial Properties of Cream Formulations containing A. paniculata: All of the A. paniculata creams with and without preservatives displayed antibacterial activity against Gram-positive strain S. aureus (p<0.05) (as shown in Fig. 4 and 5. As expected, S. aureus was less sensitive to the 5% A. paniculata cream compared to the higher concentrations. 100% A. paniculata cream exhibited the highest zone of inhibition, which shows that the Gram-positive strain was susceptible to growth inhibition by the plant extracts at high concentration when incorporated into the cream. Both extract alone and cream exhibited good anti-bacterial activity, although it was slightly reduced in the form of cream.

FIG. 4: ZONE OF INHIBITION EXHIBITED BY CREAM FORMULATIONS (WITH PRESERVATIVES) WITH DIFFERENT CONCENTRATION OF EXTRACT (n=3)

FIG. 5: ZONE OF INHIBITION EXHIBITED BY CREAM FORMULATIONS (WITHOUT PRESERVATIVES) WITH DIFFERENT CONCENTRATION OF EXTRACT (n=3)

Effect of Extract Concentration on the Anti-bacterial Activity of A. paniculata Cream: Based on this study, the higher the concentration of the extract incorporated in the cream, the higher the anti-bacterial activity (p<0.05). Based on Fig. 6 below, there is a decrease of anti-bacterial activity when a lower concentration of the extract was incorporated into the cream when compared to liquid extract alone, and this may be due to the dilution effect of the extract.

A lower concentration of extract contains a lower amount of active components, which results in a reduction in the antibacterial activity of the cream ⁸. A study on the formulation and evaluation of an anti-bacterial cream using Oxalis corniculata (common yellow woodsorrel) polar aqueous extract showed there was an increased anti-bacterial activity of the cream when a higher concentration of the extract was used ¹⁰.

In formulating commercial anti-bacterial cream, a high concentration of extract may be incorporated into the preparation of semisolid dosage form to treat adult skin infection caused by S. aureus while a lower concentration of extract may be more suitable for children because both high and low extract concentration have been shown to exhibit anti-bacterial properties when incorporated into cream.

FIG. 6: ZONE OF INHIBITION EXHIBITED BY 100% POLAR AQUEOUS EXTRACT AND DIFFERENT TYPE OF CREAMS (n=3)

FIG. 7: ZONE OF INHIBITION EXHIBITED BY TWO A. PANICULATA CREAM FORMULATIONS (WITH AND WITHOUT PRESERVATIVES) WITH DIFFERENT CONC. OF EXTRACT

Effect of Preservative on the Anti-bacterial Activity of A. paniculata Cream: The zone of inhibition exhibited by cream with preservatives is greater than the zone of inhibition exhibited by the creams without preservatives (p<0.05). As expected, creams containing preservatives have higher antibacterial activity. Figure 8 below shows the zone of inhibition exhibited by both cream formulations with preservatives and without preservatives.

The preservatives methylparaben and propylparaben, both act as an antimicrobial preservative ¹¹. Although the antibacterial activity is statistically different (p<0.05), this result confirmed that A. paniculata on its own still possesses good anti-bacterial properties.

CONCLUSION: To our best knowledge, this is the first study to incorporate A. paniculata into a topical cream. In this study, different concentrations (5%, 25%, 50%, 75% and 100%v/v) of aqueous extracts of A. paniculata, prepared from both concentrated liquid and powder, exhibited anti-bacterial activity towards S. aureus. There seems to be a correlation between the concentration of the aqueous extract and its anti-bacterial properties.

The anti-bacterial activity increases as the concentration of polar aqueous extract increases. Cream formulations using A. paniculata were shown to be a potential candidate for anti-bacterial cream at concentration of 100%v/v. The presence of preservative in the formulation contributes to the anti-bacterial properties. Moving forward, more extensive in-vitro and in-vivo research is required to evaluate the antibacterial activity of the plant to reach commercialization.

ACKNOWLEDGEMENT: The authors wish to acknowledge Cyberjaya University College of Medical Sciences for the research grant provided for this research.

CONFLICT OF INTEREST: Nil

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    How to cite this article:

    Dahlan A, Yip WS and Pin KY: Formulation and characterization of an anti-bacterial cream using Hempedu bumi (Andrographis paniculata). Int J Pharm Sci & Res 2014; 5(9): 3633-40. doi: 10.13040/IJPSR.0975-8232.5(9).3633-40.

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