ANTI-BACTERIAL AND ANTI-INFLAMMATORY PROPERTY EVALUATION OF PARAMERIA LAEVIGATA (LUPIIT) FOR THE FORMULATION OF AN OINTMENT.

This study evaluated the antibacterial and anti-inflammatory property of Parameria laevigata in the form of topical ointment. Chloroform and ethanolic solvents were used in the plant stem extraction. The extracts were subjected to phytochemical testing in which both extracts were positive with steroids and phenolic compounds. The antibacterial property was evaluated using paper disc diffusion method. The ethanolic and chloroform extracts with different concentration 2.5mg/ml; 5mg/ml; and 7.5mg/ml were evaluated against Escherichia coli, Staphylococcus aureus and Bacillus subtilis . The study showed that ethanolic crude extract of lupiit with the different concentrations have negative results while the chloroform extract was found to be partially active (10-13mm) on Staphylococcus aureus but not active to E. coli and B. subtilis . The chloroform crude extract was used in preparing the topical ointment. The anti-inflammatory activity was studied using albino rats through wound excision. Different concentrations were made 100 mg/kg BW of rat; 300 mg/kg BW of rat and 500 mg/kg BW of rat. The positive control used in the study was Betamethasone Mupirocin (Foskina-B) while the negative was distilled water. Based from the result of the study, all ointments made with different concentrations can be comparable to positive control. There was no significance difference in time duration among the different concentrations of ointment and the positive control. The best formulation was the 500mg ointment and was made as the final product. Therefore, the study showed that Parameria laevigata can be an antibacterial and anti-inflammatory ointment and it can be made available in the market.

This study evaluated the antibacterial and anti-inflammatory property of Parameria laevigata in the form of topical ointment. Chloroform and ethanolic solvents were used in the plant stem extraction. The extracts were subjected to phytochemical testing in which both extracts were positive with steroids and phenolic compounds. The antibacterial property was evaluated using paper disc diffusion method. The ethanolic and chloroform extracts with different concentration 2.5mg/ml; 5mg/ml; and 7.5mg/ml were evaluated against Escherichia coli, Staphylococcus aureus and Bacillus subtilis. The study showed that ethanolic crude extract of lupiit with the different concentrations have negative results while the chloroform extract was found to be partially active (10-13mm) on Staphylococcus aureus but not active to E. coli and B. subtilis. The chloroform crude extract was used in preparing the topical ointment. The anti-inflammatory activity was studied using albino rats through wound excision. Different concentrations were made 100 mg/kg BW of rat; 300 mg/kg BW of rat and 500 mg/kg BW of rat. The positive control used in the study was Betamethasone Mupirocin (Foskina-B) while the negative was distilled water. Based from the result of the study, all ointments made with different concentrations can be comparable to positive control. There was no significance difference in time duration among the different concentrations of ointment and the positive control. The best formulation was the 500mg ointment and was made as the final product. Therefore, the study showed that Parameria laevigata can be an antibacterial and anti-inflammatory ointment and it can be made available in the market.

…………………………………………………………………………………………………….... Introduction:-
The world's population has insufficient access on having life-saving medicines. Further, a large percentage of the world's population has a limited or no access in attaining the proper medical treatment needed for their condition according to UN Millennium Project (2005). In addition, majority of the world's population cannot manage to pay for the cost of western style drugs. In many cases, the Third World countries are experiencing and the reason why 489 traditional medicines are far distributed on the globe. Such that, herbal medicines are much easier to offer without the rules necessary to register drugs nowadays. And with this, the significance of the medicinal plant's safety and efficacy had been challenged by western academic medicine. Hence, the progress of evidence-based traditional medicines appears to be one of the options to satisfy skeptic western physician according to Tabish (2008).
As defined by World Health Organization (2000), traditional medicine is the sum total of the knowledge, skills, and practices based on the theories, beliefs, and experiences indigenous to different cultures, whether explicable or not, used in the maintenance of health as well as in the prevention, diagnosis, improvement or treatment of physical and mental illness. The use of traditional medicines has prolonged globally and gained popularity not only in the use of primary health care of the poor in developing countries but also in countries where conventional medicine is leading in the national health care system.
In the Philippines, Republic Act No. 8423, also known as the Traditional and Alternative Medicine Act (TAMA) of 1997, Article 1, Section 3 promotes and advocates the use of traditional, alternative, preventive and curative health care modalities that have been proven safe, effective, cost effective and consistent with government standards on medical practice. Moreover, Ehrlich (2015) mentioned that traditional medicines are useful resources that is essential to collect, profile and document in order to widen knowledges on traditional medicine for treating diseases. In relation to this, bacterial exacerbations are associated with systemic inflammation according to Murphy (2006). The purpose of the immune system is to prevent or eradicate bacterial infections recognized in the body and it varies to which site has been invaded and the nature of the invader.
In the study conducted by Cuellar, Giner, Recio, Manez, & Rios (2001) on the topical anti-inflammatory activity of extracts of Cassia angustifolia, Rheum palmatum, Coptis chinensis, Phellodendron amurense and Scutellaria baicalensis plants in East Asia as medicine against different skin disorders found out that all the extracts significantly inhibited the edema induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), in single or multiple application, oxazolone, and arachidonic acid (AA). Another study also demonstrated the antibacterial and antiinflammatory property of capric acid and lauric acid and found out that it exerts anti-bacterial and anti-inflammatory effect (Huang, 2014).
Published studies also reported that P. laevigata leaves and bark are found to have embryoprotective and antiteratogenic role (Cajuday & Hererra, 2011). According to Vital (2011), the ethanolic extracts of leaves of P. laevigata A. L. Juss Moldenke can inhibit parasites. Thus, the plant ethanolic extracts can possibly be used to produce alternative forms of antimicrobials. And also, in the study conducted by Tolentino (2014), P. laevigata was evaluated in terms of its anti-inflammatory activity using paw edema method and it was found to treat inflammation. Though in the study, it revealed that P. laevigata ethanoic extract have no antimicrobial activity to establish its safety when used as a medication whether topical or internal. Henceforth, this study was conducted to determine the antibacterial property of the P. laevigata plant crude ethanolic and chloroform extract. Furthermore, the researchers deemed to evaluate the anti-inflammatory activity of the P. laevigata stem using the chloroform extract and to formulate an anti-inflammatory topical ointment. Specifically, the study sough to determine the phytochemical components of P. laevigata (Lupiit) stem; determine the antibacterial property of the crude ethanolic and chloroform extract of P. laevigata (Lupiit) stem; formulate different concentrations of anti-inflammatory ointment from the chloroform extract of P. laevigata (Lupiit) stem; and to determine significant difference between the control group and the formulated concentration of the lupiit ointment in terms of time duration.

Research Design
Experimental research design was used in determining anti-bacterial and anti-inflammatory property of P. laevigata (Lupiit) ethanolic and chloroform extract.

Preparation of lupiit stem extract
Four (4) kilograms of P. laevigata was freshly powdered using a blender. The powdered lupiit stems were soaked in 80% ethanol and in chloroform for 72 hours. The ethanol and chloroform extracts were placed in steam bath at 40-45 0 C until crude extracts was produced. The crude ethanolic and chloroform extracts were weighed and stored in the laboratory refrigerator until phytochemical screening, anti-microbial assay and anti-inflammatory was established.

Laboratory Analyses Preparation of the Plant Extraction for Phytochemical Screening
The crude extract was filled into the Thin Layer Chromatography plates using a capillary tube and placed into a developing chamber with ethanol, toluene-acetic acid (8:1:1). Chromatograms were sprayed with the different reagents in order to have an idea on the bioactive compounds of the extract responsible for its anti-inflammatory property. Table 1 summarizes the spray reagents used and the corresponding compounds that they tested.

Anti-microbial screening
Test for the anti-microbial activity of the extracts on Escherichia coli, Staphylococcus aureus, and Bacillus subtilis was done using the protocol of Guevara (2005).

Formulation of Ointment Preparation of ointment
Incorporation method was used in the study. The components were mixed until a uniform preparation is attained. Extemporaneous compounding was used, components were mixed using mortar and pestle, or a spatula was used to rub the ingredients together on an ointment slab (a large glass or porcelain plate or pill tile). Non-absorbable bent parchment paper was used to cover the working surface. The ointment was prepared by thoroughly rubbing and working the components together on the hard surface and the powdered components, previously reduced to fine powder is mixed with a portion of the base until uniform. Geometric dilution was used and continued until all portions of the powder and base were combined thoroughly and uniformly blended.

Wound Excision
Excision wound model was based from the study of Mulisa, Asres, & Engidawork (2015). On wounding day, animals were anesthetized using subcutaneous injection of lidocaine (1 ml/kg) and the back hair of the animals was depilated by shaving. about 1 cm 2 circular area was marked and the full thickness of the marked area was carefully excised by using sharp sterilized scissors. After 24h of wound creation, the ointment was applied gently once daily, according to the respective grouping as described under grouping and dosing section, to cover the wounded area until complete healing was achieved. Wound contraction, epithelization period was monitored. Wound contraction was measured every 2 days until complete wound closure was achieved.

Application of Ointment and Animal Disposal
A sufficient quantity of ointment was applied on the inflamed area of rats. The Albino rats that were used in the experiment were killed painlessly using chloroform and was buried at the mini forest of Saint Mary's University.

Anti-inflammatory Assay
Albino rats weighing 110-120g were used in the study and were acclimatized at the CNS greenhouse with free access to food and water ad libitum. The animals were divided into five groups, each group containing three animals.

Treatments
Group 1: negative control: white petrolatum and white wax; Group 2: positive control: Bethametasone + Mupirocin; Group 3: 100 mg/kg body weight of the rat; Group 4 : 300 mg/kg body weight of the rat; and Group 5: 500 mg/kg body weight of the rat

Scratch Test
The protocol for the sensitivity test to local and topical applications were done using the protocol of Guevara (2005). The researchers only conducted the scratch test. Using the scratch test, the absence, presence and severity of edema was evaluated based on the parameters listed on Table 2. 492 Severe edema For the scratch test in sensitivity test, albino rats were used. The skin lateral to the spinal groove was shaved and was cleaned. The left side of groove in the animal was utilized as the negative (-) control site and the right side as the test drug site. The negative control refers to the vehicle of the drug. The site was cleaned with 70% alcohol and is immediately followed by the application of the test drug. Both sites are covered with sterilized gauze (1x1 cm in size). Surgical tapes were used to keep the gauze in place, and left undistributed for about 24 to 27 hours during which in time all the test animals are rendered immobile, in scratch test, the application of the test drug, the skin was abraded, lateral to the spinal groove of the test animal, by slightly scratching the skin five (5) to seven (7) times with scalpel.

Treatment of Data
Values were expressed as mean ± standard error of the mean. Statistical significance was calculated using analysis of variance (ANOVA). The values were considered statistically significant when the p-value is less than 0.05 (<0.05).  Table 3, 21% of crude can be extracted from 2kg of P. laevigatathat were soaked in 3250 ml of ethanol and 19% of crude extract can be extracted from 2 kg powdered stem that were soaked in 3,750 ml of chloroform. This shows that in the ethanolic extraction, there is more % yield compared with the chloroform extraction.

Determination of the Phytochemical Components of P. laevigata (Lupiit) Stem.
Phytochemical screening of the crude solvents extract afforded several metabolites summarized in Table 4. Ethanolic and Chloroform stem extract of P. laevigata contains flavanoids, triterpenes, sterols, essential oils, fatty acid, steroids, antraquinones, coumarines, and anthrones. The bioactive compounds provide semi-qualitative information on the active constituents of the extract. . In aromatherapy, essential oils are believed to improve the mood and mental function. They can be used for skin issues, respiratory illnesses and as antiseptics. This is because phenols and phenolic compounds have been widely used in disinfection and remain the standard with which added bactericides are compared. Phenolic compounds are able to inhibit either the production or the action of pro-inflammatory mediators, resulting in anti-inflammatory capacity (Perez, 2015). Phenolic compounds are primarily recognized for their activity as anti-microbial agents in clinical dentistry, and their anti-inflammatory activity is thought to be due to their action as non-specific counter-irritants. Fatty acid may also be responsible for antibacterial and antiinflammatory effect. One study showed the anti-bacterial and anti-inflammatory property of capric acid and lauric acid and was found to exert anti-bacterial and anti-inflammatory effect (Huang, 2014). In connection with this, P. laevigata is positive for phenolic compounds like the flavonoids; hence, it can be said to have potential in suppressing inflammation.
Apparently, in the study, steroids have been detected through phytochemical screening implying that the plant has potential in suppressing inflammation. It is responsible in suppressing the body's immune system, decreasing inflammation as well as blocking the chemical called histamine (causes allergic reaction). The compound is also known as corticosteroids which are used to treat diseases (Tidy, 2015). Corticosteroids are also known as glucocorticosteroids, glucocorticoids or just steroids which are among the most widely used drugs in the world and are effective in many inflammatory and immune diseases (Barnes, 2006). Topical steroids (corticosteroids) are extremely effective anti-inflammatory agents. They suppress various parts of the inflammatory reaction. Topical steroids are available as ointments, creams, gels, lotions, solutions, aerosols and in other forms that can be applied locally on the skin. Moreover, different topical steroids have different potency ranging from mild to very potent topical steroids.

Determination of the Antibacterial Property of the Crude Ethanolic and Chloroform Extract of P. laevigata (Lupiit) Stem Antibacterial Assay: Paper disc diffusion method
The antibacterial assay was done as presented in Guevara (2005) and Benson (2002). The ethanolic and chloroform extracts were tested for their activities against the following microorganism; Bacillus subtilis, Staphylococcus aureus and Escherichia coli. Bacterial cell suspension was prepared from cultures, and swabbed on petri plates prefilled with Nutrient Agar (NA). Control disks (Streptomycin for positive control; distilled water for negative control)). The moistened filter paper discs containing different concentrations (A-2.5mg/ml, B-5mg/ml and 7.5mg/ml) of the crude extract and the positive control drug are laid on the solidified agar using sterile wooden sticks and immediately the petri dishes were covered. The petri dishes were incubated within 24 hours at 37 degree Celsius. After 24 hours of incubation, the petri dishes were observed for any zone of inhibition. The diameters of the zone of inhibition were measured using Vernier caliper then the measurements were recorded. The measured diameters of the zones were expressed in millimeter (mm). Assays were in 3 replicates. Table 5 summarizes the observed zones of inhibition. The zones of inhibition were measured and means were computed. These measured zones of inhibition among the Lupiit ethanolic and chloroform extracts and with the standard (Streptomycin) were observed and compared with the positive control. And based from Table 5, all of the concentrations showed no antibacterial property against  494 Bascillus subtilis and E. coli. Only the chloroform crude extracts exhibit antibacterial property on Staphylococcus aureus at 7.5mg/ml of the Lupiit extract.

Formulation of the Anti-inflammatory Ointment from the Different Concentrations of P. laevigata (Lupiit)
Stem.  Table 6 shows the anti-inflammatory effect of the chloroform extract administered in every after 2-day intervals. Inflammation has four phases. The first phase called inflammatory phase of inflammation occurred at time 0-1. At time 2, phase 2 occurred, in which there was observed epithelialization. The sign and symptoms observed in rats were pain, palpable tenderness, swelling and muscle range in motion. There was a violent reaction of rats so there was tendency of scratching their back. Third phase which is maturation phase occurred at time 3 to time 7. In this phase there was formation of collagen characterized by less warmth, swelling palpable tenderness decreases, pain felt with tissue resistance. Phase 4 occurred at time 8 in which there was removal of epithelialization and the surrounding skin pigmentation.
Initially, around 1cm 2 was inflicted to the rats as to determine the anti-inflammatory property of the Lupiit chloroform extract. And based from table 3, the topical administration of chloroform extract in the form of ointment markedly raised the size of the incurred wound 2 days after wound excision (Time 0). However, across time, it can be gleaned from the table that the sizes of inflammation decreases indicating that the 100mg/kg BW of rat, 300mg/kg BW and 500mg/kg BW of rat concentrations are relatively effective of reducing the skin inflammation.  Tolentino (2015) in her study on anti-inflammatory property of the ethanolic extract of P. laevegata using paw edema method (oral gavage). The results of the study revealed that there is no significant difference in the different concentrations when compared to the control group wherein p value was greater than 0.05. Similarly, as stated by Nordqvist (2015), the inflammation can cause unlikable result since it can become self-perpetuating because more inflammation is created in response to the prevailing inflammation. From the initial incurred wound of 1cm 2 , 2 days after excision, the wounds have increased in sizes. The sizes of inflammation become wider across the concentrations. On the other hand, Wassung (n.d.) stated that the inflammatory response is a natural defense mechanism that is triggered whenever body tissues are damaged in any way. Most of the body defense elements are located in the blood and inflammation is the means by which body defense cells and defense chemicals leave the blood and enter the tissue around the injured or infected site. Moreover, inflammation occurs in response to physical trauma, intense heat and irritating chemicals, as well as to infection by viruses and bacteria. In the conducted study, the topical ointment method was used to determine the anti-inflammatory property of the chloroform extract of P. laevigata. It resulted to no significant difference across concentrations in terms of time excluding day 6. To determine the particular group with significant difference, further test was performed through Bonferroni multiple comparison. Using multiple comparisons, there is no significant difference between the concentrations of ointment across time intervals. This implies that the concentrations have relative effect on the wound of the experimental rats. The concentrations 100mg/kg, 300mg/kg, 500mg/kg, and the positive control group concentration have p values greater than 0.05. This indicates that the different concentrations of chloroform extract of P. laevigata (Lupiit) are relatively comparable with the positive control Bethametasone + Mupirocin. Further, the data suggest that the different concentrations can reduce size of the inflammation and treat the inflammation of the experimental rats. Based on the result conducted in scratch testing, it did not show any edema; hence, the experimental animal has no sensitivity with the 500mg/kg concentration.

Conclusions and Recommendations:-
Based on the results, there was no significance difference among the concentrations and positive control in terms of time duration. The researchers also concluded that P. laevigata ointment is effective as anti-inflammatory agent and 496 it was partially active against Staphylococcus aureus. The best formulation of the ointment was made from 500mg concentration. Hence, the researchers recommended that addition of perfume and colorants can be mixed with the ointment to enhance the appearance. The researchers also recommend that the next researchers may focus on the isolation of the compound responsible in the anti-inflammatory effect.