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Anti-Inflammatory and Anti-Bacterial Efficacy of Aratiles Leaves (Muntingia Calabura Linn) Against Staphylococcus Aureaus

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Submitted By pudine
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Pages 30
Abstract

Plant-based antimicrobials represent a vast untapped source of medicines and further exploration of plant antimicrobials needs to occur. Antimicrobials of plant origin have enormous therapeutic potential. . Antimicrobials of plant origin have enormous therapeutic potential. They are effective un treatment of infectious disease while simultaneously mitigating many of the side effects that are often associated with synthetic antimicrobials.
Present study was executed to mainly investigate the anti-inflammatory and antibacterial efficacy of Muntingia calabura Linn against Staphylococcus aureaus. This study is done to provide people with an inexpensive, natural and safe anti inflammatory agent. In addition, it may ease the pain of arthritis. The output of this study will help to make anti inflammatory products out of aratilis (Muntingia calabura linn) flowers and leaves. This study might also provide new knowledge about the properties of aratilis (Muntingia calabura Linn).
Extracts of aratilis leaves are subjected to several solutions of oil, water and ethanol at 1:1 ratio. Antibacterial efficacy is done using cup cylinder assay by measuring the diameter (mm) of the clear zone around the cup. The results showed that the oil solution showed an average value of 9.03mm, the ethanol solution showed an average value of 18.27 mm and the water solution with an average of 20.10 mm. The negative controls – oil, ethanol and water – exhibited an average of inhibition with values, 8.87 mm, 14.67 mm, and 12.10 mm, respectively. The positive control, Streptomycin sulfate, exhibited a wide range of inhibition with a 24.53 mm clearing. The ointment has no range of inhibition that may due to dilution to the petroleum jelly. When concentrated with the Aratilis leaves extract, different active compounds may exhibit a synergistic effect to effectively control bacterial strains. The eucalyptus oil of Vics improves the quality of Aratilis Ointment. Patients feel some reducing of swelling and they confirm that they have the strength to do their work. As well as the Aratilis tea, it improves and continuous reducing the pain and swelling of Artritis. These medicinal plants can be further subjected to further research and antimicrobial evaluation to examine their full spectrum of efficacy.

Introduction

Herbal medicine is still the mainstay of about 75-80% of the whole population, mainly in developing countries, for primary health care because of better cultural acceptability with human body and fewer side effects. Because of the side effects and the resistance that pathogenic microorganisms build against antibiotics, recently much attention has been paid to extracts and biologically active compounds isolated from plant species used in herbal medicine. Antimicrobials of plant origin have enormous therapeutic potential. They are effective in the treatment of infectious diseases while simultaneously mitigating many of the side effects that are often associated with synthetic antimicrobials.The constituents and chemical properties of Muntingia Calabura Linn are the root that has 12 new flavonoids were isolated (7 flavans, 3 flavones, two biflavans). Most of the isolates demonstrated cytotoxic activity and some exhibited selective activities when evaluated with a number of human cancer cell lines and the leaves and stems yielded cytotoxic flavonoids: chrysin, 2',4'-dihydroxychalcone and galangin 3, 7-dimethyl ether. The compounds were active against one or more panels of human and murine cell lines. The ability of tannins to form chelates with metal ions, particularly iron, which lead to the disruption of the S. aureus membrane, could be one of the possible factors that contribute to its antimicrobial activity (Akiyama et al., 2001). This study will evaluate the leaves extracts of Muntingia Calabura Linn against Staphylococcus aureus.

Methodology

Extraction of the active compound. The leaves of Aratiles were extracted with water by boiling the leaves for 30mins and filtered. The filtrated extracts of aratiles leaves is set aside for 10 mins. The extracts serve as the sample for the assay.

Antimicrobial assay. The extracts from leaves were assayed against Staphylococcus aureus 25923, common skin infections by cup cylinder method. Ten (10) ml of sterile Mhueller Hinton Agar(MHA) was placed in the sterile plates ad solidify. Five(5)ml of tap agar seeded with test organism was placed onto base agar and solidify. All of petri dishes contain tap agar with test organisms, label the dishes from 1-3 correspondingly. After a while, using a laboratory tweezers, place 7 cap cylinders in the circular manner following the label indicated. Place in the incubator, incubated at 37 centigrade overnight

Table 1 Zone of Inhibition exhibited by Aratilis leaves against Staphylococcus aureaus 25923 on Petri dish 1 | Sample | Zone of Inhibition (diameter in mm) | Oil 1 | 11.5 | Ethanol 1 | 15.5 | Water 1 | 19.8 | Oil (Negative control) | 10.9 | Ethanol (Negative control) | 12.7 | Water (Negative control) | 12.9 | Streptomycin sulfate(Positive control) | 23.4 | Ointment (product) | 0 |

Individual assay of the Aratilis leaves by cup cylinder revealed that all samples exhibited zone of inhibition against S. aureaus. The Oil 1, Ethanol 1 and Water 1 samples contains the Aratilis leaves extract with a 1:1 ratio. Results showed that the different solutions exhibited zone of inhibition with values 11.5 mm, 15.5mm, and 19.8 mm, respectively.

The negative control which is the oil, ethanol and water alone showed a considerable zone of inhibition with values 10.9 mm, 12.7 mm and 12.9 mm respectively.

The positive control, Streptomycin sulfate, showed the highest zone of inhibition with a value of 23.4 mm compared to the Aratilis leaves ointment with only a value of 0 mm. The low value of the ointment sample could be due to dilution to the petroleum jelly. When concentrated with the Aratilis leaves extract, different active compounds may exhibit a synergistic effect to effectively control bacterial strains.

Table 2 Zone of Inhibition exhibited by Aratilis leaves against Staphylococcus aureaus 25923 on Petri dish 2 | Sample | Zone of Inhibition (diameter in mm) | Oil 1 | 7.3 | Ethanol 1 | 21.3 | Water 1 | 18.6 | Oil (Negative control) | 7.5 | Ethanol (Negative control) | 12.1 | Water (Negative control) | 12.5 | Streptomycin sulfate(Positive control) | 22.7 | Ointment (product) | 0 |
Individual assay of the Aratilis leaves by cup cylinder revealed that all samples exhibited zone of inhibition against S. aureaus. The Oil 1, Ethanol 1 and Water 1 samples contains the Aratilis leaves extract with a 1:1 ratio. Results showed that the different solutions exhibited zone of inhibition with values 7.3 mm, 21.3 mm, and 18.6 mm, respectively.

The negative control which is the oil, ethanol and water alone showed a considerable zone of inhibition with values 7.5 mm, 12.1 mm and 12.5 mm respectively.

The positive control, Streptomycin sulfate, showed the highest zone of inhibition with a value of 22.7 mm compared to the Aratilis leaves ointment with only a value of 0 mm. The low value of the ointment sample could be due to dilution to the petroleum jelly. When concentrated with the Aratilis leaves extract, different active compounds may exhibit a synergistic effect to effectively control bacterial strains.

Table 3 Zone of Inhibition exhibited by Aratilis leaves againstStaphylococcus aureaus 25923 on Petri dish 3 | Sample | Zone of Inhibition (diameter in mm) | Oil 1 | 8.3 | Ethanol 1 | 18.0 | Water 1 | 21.9 | Oil (Negative control) | 8.2 | Ethanol (Negative control) | 19.2 | Water (Negative control) | 10.9 | Streptomycin sulfate(Positive control) | 27.5 | Ointment (product) | 0 |

Individual assay of the Aratilis leaves by cup cylinder revealed that all samples exhibited zone of inhibition against S. aureaus. The Oil 1, Ethanol 1 and Water 1 samples contains the Aratilis leaves extract with a 1:1 ratio. Results showed that the different solutions exhibited zone of inhibition with values 8.3 mm, 18.0 mm, and 21.9 mm, respectively. The negative control which is the oil, ethanol and water alone showed a considerable zone of inhibition with values 8.2 mm, 19.2 mm and 10.9 mm respectively.

The positive control, Streptomycin sulfate, showed the highest zone of inhibition with a value of 27.5 mm compared to the Aratilis leaves ointment with only a value of 0 mm. The low value of the ointment sample could be due to dilution to the petroleum jelly. When concentrated with the Aratilis leaves extract, different active compounds may exhibit a synergistic effect to effectively control bacterial strains.

Results and Discussion

Extracts of aratilis leaves are subjected to several solutions of oil, water and ethanol at 1:1 ratio. Antibacterial efficacy is done using cup cylinder assay by measuring the diameter (mm) of the clear zone around the cup. The results showed that the oil solution showed an average value of 9.03mm, the ethanol solution showed an average value of 18.27 mm and the water solution with an average of 20.10 mm. The negative controls – oil, ethanol and water – exhibited an average of inhibition with values, 8.87 mm, 14.67 mm, and 12.10 mm, respectively. The positive control, Streptomycin sulfate, exhibited a wide range of inhibition with a 24.53 mm clearing. The ointment has no range of inhibition that may due to dilution to the petroleum jelly. When concentrated with the Aratilis leaves extract, different active compounds may exhibit a synergistic effect to effectively control bacterial strains.

Based on the results, the ointment having different solutions of the plant extract, aratilis leaves showed inhibition against Staphylococcus aureaus. Since the extracts of aratilis leaves(Muntingia Calabura Linn) showed a few or having no sign of inhibition, it is recommended to use different concentrations. It is also suggested to use another substance for extraction like organic solvents.

Conclusion
It is anticipated that phytochemicals with adequate antibacterial efficacy will be used for the treatment of bacterial infections. Secondary plant metabolites (phytochemicals compounds have been extensively investigated as a source of medicinal agents. Among those antimicrobial compounds, phenolic compounds, terpenoids, and alkaloids’ are very important compounds in antimicrobial or antioxidant effects. Further research is necessary to determine the different antibacterial compounds from these herbs and their full spectrum of efficacy. However, the present study of in vitro antimicrobial evaluation of these herbs forms a primary platform for further phytochemical and pharmacological studies. These promissory extracts open the possibility of finding new clinically effective antimicrobial compounds.

Introduction

A. Background of the study

Herb is an immeasurable wealth of nature not only from the global environmental perspective but also from the medicinal point of view. It plays a significant role ameliorating the disease resistant ability and combating against various unfavorable metabolic activities within the living system. Numerous infectious diseases have been known to be controlled by herbal remedies that have been proved variously since primitive to present history of the mankind. Since time immemorial, man has used various parts of plants in treatment and prevention of various ailments. Unimaginably unrevealed and unmatched varieties of compounds are present in the diversified herbs on earth. From these points of view, it is obvious that natural products, either in the form of pure compounds or as standardized plant extracts, provide unlimited opportunities to develop a variety of new drugs.

Muntingia calabura, the sole species in the genus Muntingia, is a flowering plant native to southern Mexico, the Caribbean, Central America, and western South America south to Peru and Bolivia. Common names include (English) Jamaican cherry, Panama berry, Singapore cherry, Strawberry tree; (Spanish) bolaina, yamanaza, cacaniqua, capulín blanco, nigua, niguito, memizo or memiso; (Indonesia) kersen; and (Filipino) aratilis, aratiles, manzanitas and sarisa.
It is a small tree 7-12 meters tall with tiered and slightly drooping branches. It has serrated leaves 2.5-15 cm long and 1-6.5 cm wide. The flowers are small, white and slightly malodorous. It gives rise to 1-1.5 cm light red fruit. The fruit is edible, sweet and juicy, and contains a large number of tiny (0.5 mm) yellow seeds.

It is a pioneer species that thrives in poor soil, able to tolerate acidic and alkaline conditions and drought. Its seeds are dispersed by birds and fruit bats. It is cultivated for its edible fruit, and has become naturalised in some other parts of the tropics, including southeastern Asia. As a pioneer plant, it could help condition the soil and make it habitable to other plants. However, it might also be considered as an invasive species since it might out-compete indigenous plants.

Septic, or infectious, arthritis is infection of one or more joints by microorganisms. Normally, the joint is lubricated with a small amount of fluid that is referred to as synovial fluid or joint fluid. The normal joint fluid is sterile and, if removed and cultured in the laboratory, no microbes will be found. With septic arthritis, microbes are identifiable in an affected joint fluid.
Most commonly, septic arthritis affects a single joint, but occasionally more joints are involved. The joints affected vary somewhat depending on the microbe causing the infection and the predisposing risk factors of the person affected. Septic arthritis is also called infectious arthritis.
Septic arthritis can be caused by bacteria, viruses, and fungi. The most common causes of septic arthritis are bacteria, including Staphylococcus aureus and Haemophilus influenzae. In certain "high-risk" individuals, other bacteria may cause septic arthritis, such as E. coli and Pseudomonas spp. in intravenous drug abusers and the elderly, Neisseria gonorrhoeae in sexually active young adults, and Salmonella spp. in young children or in people with sickle cell disease. Other bacteria that can cause septic arthritis include Mycobacterium tuberculosis and the spirochete bacterium that causes Lyme disease.
Viruses that can cause septic arthritis include hepatitis A, B, and C, parvovirus B19, herpes viruses, HIV (AIDS virus), HTLV-1, adenovirus, coxsackie viruses, mumps, and ebola. Fungi that can cause septic arthritis include histoplasma, coccidiomyces, and blastomyces.

Antibiotic resistance has become a global concern. The clinical efficacy of many existing antibiotics is being threatened by the emergence of multidrug-resistant pathogens. There is a continuous and urgent need to discover new antimicrobial compounds with diverse chemical structures and novel mechanisms of action for new and re-emerging infectious diseases. Therefore, researchers are increasingly turning their attention to folk medicine, looking for new leads to develop better drugs against microbial infections . The increasing failure of chemotherapeutics and antibiotic resistance exhibited by pathogenic microbial infectious agents has led to the screening of several medicinal plants for their potential antimicrobial activity. Recent, studies have suggested that several plants species exhibit promising antimicrobial effects.

Plant-based antimicrobials have enormous therapeutic potential as they can serve the purpose with lesser side effects that are often associated with synthetic antimicrobials. In recent years, it has been proposed that the herbal extracts may be used as natural antifungal agents to inhibit the growth of food-borne pathogen and as a source of various medicinal agents.

B. Statement of the problem

Staphylococcus aureus can cause a range of illnesses from minor skin infections, such as pimples, impetigo, boils (furuncles), cellulitis folliculitis, carbuncles, scalded skin syndrome, and abscesses, to life-threatening diseases such as pneumonia, meningitis, osteomyelitis, endocarditis, toxic shock syndrome (TSS), chest pain, bacteremia, and sepsis. Its incidence is from skin, soft tissue, respiratory, bone, joint, endovascular to wound infections. It is still one of the five most common causes of nosocomial infections, often causing postsurgical wound infections.

Sadly, the cost of imported synthetic antibiotics in the country is fast rising. Nowadays multiple drug resistance has developed due to the indiscriminate use of commercial antimicrobial drugs commonly used in the treatment of infectious disease. In addition to this problem, antibiotics are sometimes associated with adverse effects on the host including hypersensitivity, immune-suppression and allergic reactions. This situation forced scientists to search for new antimicrobial substances. Given the alarming incidence of antibiotic resistance in bacteria of medical importance, there is a constant need for new and effective therapeutic agents. Therefore, there is a need to develop alternative antimicrobial drugs for the treatment of infectious diseases from medicinal plants.

Several screening studies have been carried out in different parts of the world. There are several reports on the antimicrobial activity of different herbal extracts in different regions of the world.

Because of the side effects and the resistance that pathogenic microorganisms build against antibiotics, recently much attention has been paid to extracts and biologically active compounds isolated from plant species used in herbal medicine. Plant-based antimicrobials represent a vast untapped source of medicines and further exploration of plant antimicrobials needs to occur. Antimicrobials of plant origin have enormous therapeutic potential. They are effective in the treatment of infectious diseases while simultaneously mitigating many of the side effects that are often associated with synthetic antimicrobials. Since Philippine is blessed with vast natural resources, it has a wide range of medicinal plants that may able to use and study. This can provide us an answer to this dilemma.

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C. Significance of the Study

Its been a sad reality that Aratiles tree has been forgotten and the young generation today doesn’t even know it or have any familiarization of its features using the antimicrobial and anti inflammatory property of the leaves of this wonderful tree. This could provide an inexpensive, natural and safe kind of ointment or oil extracts and tea. Not only the adult suffered from arthritis or muscle pain but even teenagers. The antimicrobial property will help even the below average member of the society to purchase and to cure their muscles pain. This study might also provide new knowledge about the properties of Aratiles(muntingia calabura linn) as medicinal plants.

D. Scope and Limitations

The study focuses on the anti-inflammatory and anti-bacterial activity of Aratiles (Muntingia Calabura linn). The extract of the leaves was tested on S. aureaus in the form of coconut oil extract and ointment. The leaves was processed into tea and tested on several patients with Arthritis. The study was conducted in one of the researchers house, Chemistry Laboratory at Christian School International and microbial assay at BIOTECH, UPLB from June 2011 to January 2012.

Review of Related Literature

Herbal medicine is still the mainstay of about 75-80% of the whole population, mainly in developing countries, for primary health care because of better cultural acceptability with human body and fewer side effects. Because of the side effects and resistance that pathogenic microorganisms build against antibiotics, recently much attention has been paid to extracts and biologically active compounds isolated from plant species used in herbal medicine.

Plant-based antimicrobials represent a vast untapped source of medicines and further exploration of plant antimicrobials needs to occur. Antimicrobials of plant origin have enormous therapeutic potential. They are effective un treatment of infectious disease while simultaneously mitigating many of the side effects that are often associated with synthetic antimicrobials.

All plants containing active compounds are important. The beneficial medicinal effects of plant materials typically result from the combinations of secondary products present in the plant. In plants, these compounds are mostly secondary metabolites such as alkaloids, steroids, tannins, and phenol compounds, which are synthesized and deposited in specific parts or in all parts of the plant. These compounds are more complex and specific and are found in certain taxa such as family, genus and species, but heterogeneity of secondary compounds is found in wild species.

The medicinal actions of plants are unique to a particular plant species or group, consistent with the concept that the combination of secondary products in a particular plant is taxonomically distinct. The plants secondary products may exert their action by resembling endogenous metabolites, ligands, hormones, signal transduction molecules or neurotransmitters and thus have beneficial medicinal effects on humans due to similarities in their potential target sites. Therefore, random screening of plants for active chemicals is as important as the screening of ethnobotanically targeted species.

Muntingia Calabura Linn
Aratiles/Cherry tree

Botanical Information: Muntingia Calabura Linn is a small tree 7-12 meters tall with tiered and slightly drooping branches. It has serrated leaves 2.5-15 cm long and 1-6.5 cm wide. The flowers are small, white and slightly malodorous. It gives rise to 1-1.5 cm light red fruit. The fruit is edible, sweet and juicy, and contains a large number of tiny (0.5 mm) yellow seeds.

Taxonomy of Muntingia Calabura Linn

Kingdom: Plantae
(unranked): Angiosperms
(unranked): Eudicots
(unranked): Rosids

Order: Malvales
Family: Muntingiaceae
Genus: Muntingia
L.
Species: M. calabura
Constituents and Chemical Properties: The constituents and chemical properties of Muntingia Calabura Linn are the root that has 12 new flavonoids were isolated (7 flavans, 3 flavones, two biflavans). Most of the isolates demonstrated cytotoxic activity and some exhibited selective activities when evaluated with a number of human cancer cell lines and the leaves and stems yielded cytotoxic flavonoids: chrysin, 2',4'-dihydroxychalcone and galangin 3, 7-dimethyl ether. The compounds were active against one or more panels of human and murine cell lines.

Folkloric Medicinal Uses: There were also different folkloric uses of this plant. Flowers are antispasmodic. The decoction of flowers is used for abdominal cramps. The decoction is used as emollient. The flowers used as antiseptic and to treat spasms and also used to relieve colds and headaches. In the Antiles, it is used as antispasmodic. The bark decoction is mucilaginous and used as emollient.

Recent studies: There are also recent studies done about Muntingia Calabura Linn. Last January 2008, the leaves have been previously identified by a botanist at University Putra Malaysia (UPM) Serdang Selangor, and a voucher specimen (SK 964/04) has been preserved at the Herbarium of the laboratory of Natural product, Institute of Bioscience, UPM, Selangor, Malaysia. Scientifically, several types of flavonoids and flavones have been isolated and identified from this plant (Keneda et al., 1991; Su et al., 2003; Chen et al., 2005) with the first two authors also reported on their anti-tumor activity. The aqueous extract of this plant also possesses opiodmediated antinociception (Zakaria et al., 2007, 2005a).

In addition, the M. calabura leaves extracts also possesses anti-inflammatory and anti-pyretic properties (Zakaria et al., 2007a, b, 2008), antibacterial activity (Zakaria et al., 2006) and antistaphyloccocal activity (Zakaria et al., 2007d). Therefore, the main objective of this study is to search for the active fraction with strong antimicrobial activity which could serve as a good candidate for the development of new antimicrobial agents. There has been an alarming increase in nosocomial staphylococcal infections by multiple drug resistance strains of S. aureus in recent years (Al-Masaudi et al., 1991; Hiramatsu et al., 1997). Therefore, there is a need to find new antimicrobial compounds, particularly antistaphylococcal agents. The lower MIC/MBC conferred by fraction for the two strains of S. aureus compared to extracts and partitions (2500 to 78 ìg/ml) could be due to the fact that the fractions consist of pure compounds while the extracts as well as the partitions contained various types of compounds, which included the ineffective fractions A1 until A8.

Findings by Zakaria et al. (2006) have demonstrated the presence of flavonoids, triterpenes, saponins steroids and tannins in M. calabura leaves. Several mechanisms of action could be suggested with regards to those groups of chemical compounds, particularly flavonoids and tannins, which are presence in the M. calabura extracts. Some of the flavonoids that favor polar solutes entry, like rutin and quercetin, bind to the bacteria’s structural membrane proteins called porines, causing changes in the tridimentional conformation exposuring the hydrophilic character of the pore, which lead to an easier passage of other polar bioactive compounds via diffusion (Alvarez et al., 2006).

The ability of tannins to form chelates with metal ions, particularly iron, which lead to the disruption of the S. aureus membrane, could be one of the possible factors that contribute to its antimicrobial activity (Akiyama et al., 2001). In addition, tannins, like tannic acid, has a greater relative binding efficiency to iron and may act with iron from the medium to form chelates and deplete iron from microorganisms. It is well known that aerobic microorganisms require iron to perform a variety of functions, like reduction of ribonucleotide and formation of haem (Chung et al., 1998). Besides, tannins of the catechin group have also been shown to exhibit antimicrobial activity via mechanisms that involved damage to the membrane, for example the leakage of 5,6-carboxyfluorescin from phosphatidyl choline liposomes (Ikigai et al., 1993). In conclusion, the present study successfully isolated fractions with antistaphylococcal activity from the methanol extract of M. calabura.

Staphylococcus aureus

Staphylococcus aureus literally the "golden cluster seed" or "the seed gold" and also known as golden staph and Oro staphira, is a facultatively anaerobic, Gram-positive coccus and is the most common cause of staph infections. It is frequently part of the skin flora found in the nose and on skin. About 20% of the human population are long-term carriers of S. aureus.

S. aureus can cause a range of illnesses from minor skin infections, such as pimples, impetigo, boils (furuncles), cellulitis folliculitis, carbuncles, scalded skin syndrome, and abscesses, to life-threatening diseases such as pneumonia, meningitis, osteomyelitis, endocarditis, toxic shock syndrome (TSS), chest pain, bacteremia, and sepsis. Its incidence is from skin, soft tissue, respiratory, bone, joint, endovascular to wound infections. It is still one of the five most common causes of nosocomial infections, often causing postsurgical wound infections. S. aureus was discovered in 1880 by the surgeon Sir Alexander Ogston in Aberdeen, Scotland.
Recent studies show that S. aureus developed resistance to methicillin. Methicillin is one antibiotic commonly used to treat staph infections. This bacterium is called the methicillin-resistant staphylococcus aureaus or MRSA. Symptoms in serious cases of MRSA may include fever, lethargy and headache. It can cause urinary tract infections, pneumonia, toxic shock syndrome and even death. People are susceptible to MRSA infection are those elderly and very ill person. Intravenous drug users and people with long term illnesses are also increased at risk. Fortunately, there are still antibiotics that can get rid of MRSA infections.
Methodology

I. Materials
Sterilized petri dishes, MHA (Mueller Hinton Agar), bacterial strain (Staphylococcus aureaus), Plant(Aratilis leaves:Muntingia Calabura Linn) Pipette, rubber aspirator, aratilis extracts, coconut oil extracts, ethanol extracts and water extracts, positive control(Streptomycin sulfate)

II. Methods

A. Preparation of plant extracts
Chop the aratilis leaves and place it in a clean bowl. Prepare the pan and boil 900ml water. While waiting, get 250 ml of beaker and measure the coconut oil to 50ml. Get the chopped aratils leaves and place them (8 grams each) inside the beaker in order to get the extract. Place the beaker with raw materials to the pan and wait for 30 minutes to let it cook.

B. Preperation for Ointment and Tea
Oinment: Get the stainless steel cooking pan and boil 900ml of tap water. While waiting, get the beaker and pour 50ml of coconut oil. Then get the aratilis leaves (8grams each) and place it in a substance. Place the beaker with the raw material and wait until it cooks. After that, get the beaker and collect the extract. Mix it well with the petroleum jelly.

Note: We measured using the graduated cylinder the extracted substance. In this case, 70 ml extracted substance. We got a measurement to proportion the amount of extract per ml. We got 1:2 ratios which mean that for every 1ml of extracted substance, we have to mix with 2ml petroleum jelly.

C. Antibacterial Assay
To start the antimicrobial assay, prepare 3 sterilized petri dishes. While waiting for it to be sterilized, preheat the tap and base agar. When the base agar (MHA) is ready, using a pipette and rubber aspirator, pour 10 ml to the sterilized petri dishes. Set aside for some minutes. After that, when the tap agar with the test organism is ready, using pipette and rubber aspirator, pour 5ml to the sterilized petri dishes with the base agar. After all of petri dishes contain tap agar with test organisms, label the dishes from 1-3 correspondingly. After a while, using a laboratory tweezers, place 7 cap cylinders in the circular manner following the label indicated. Place in the incubator, incubated at 37 centigrade overnight.

Notes: * The base agar used is MHA (Mueller Hinton Agar). It is a microbiological growth medium that is commonly used for antibiotic susceptibility testing. MHA is also used to isolate and maintain Neisseria and Moraxella species. It typically contains (w/v): 30.0% beef infusion 1.75% casein hydrolysate 0.15% starch 1.7% agar pH adjusted to neutral at 25 °C. * The positive control is Streptomycin sulfate. It prevents infections that are proven or strongly suspected to be caused by bacteria. Streptomycin is a water-soluble aminoglycoside derived from Streptomyces griseus. The molecular formula for Streptomycin Sulfate is (C21H39N7O12)2-3H2SO4. * There are three Petri dishes per test organism. It was labeled as follows: O1

+C

E1

W

W1

E

O

* The extracts are placed in this manner, numbered according to the arrangement as follows: * O1- aratilis coconut oil extract * E1- aratilis ethanol extract * W1- aratilis water extract * O- oil * E- ethanol * W- water * +C- positive control(Streptomycin sulfate) * SA- ointment
Materials used in making the Assay:
Hotplate
Autoclave Autoclave Mixing Machine

Balance Beam Vernier Caliper

Making Samples and Agar for the Assay:

Measure 3.8grams of Mueller Hinton for Tap agar and 1.9grams of Mueller Hinton for base agar using the balance beam.

For tap agar (3.8g) mix it with 100ml of distilled water. For base agar (1.9g), mix it with 50ml of distilled water.

Pictures of the all agars mix with water

Collecting the extracts and preparing for samples:

Anti Micro-bacterial Assay

(Preparing the cup cylinder inside the petri dish which contain Staphylococcus aureaus)

(Putting the plant extract inside the cup cylinder)

(Ethanol extract (e1), water extract (w1) and positive control shows better results.

Results of the assay:

Vernier Caliper is a device used to measure the distance between two opposing sides of an object. A caliper can be as simple as a compass with inward or outward-facing points. The tips of the caliper are adjusted to fit across the points to be measured, the caliper is then removed and the distance read by measuring between the tips with a measuring tool, such as a ruler.

After we used the laboratory, we cleaned it up before we leave.

Methodology: Tea Making

Washing the fresh leaves of Aratiles leaves and prepare it for getting the extracts for the tea.

Collect the extract of aratiles leaves and mix it with honey and lemon to have some taste.

Filter the collected extracts with a strainer and put in a container. Cool it for a while then place it on the bottle.

Methodology: Ointment Making

Extracting the Aratiles leaves using coconut oil that is placed in beaker.

Beaker and graduated cylinder is used to collect and measure the extracts.

Mixing the collected extracts of
Aratiles in petroleum jelly using stirring rod.

Placing the mixed extracts in a container and this is the final product:

Pictures of patients and the part where their Arthritis occur:

PATIENT #1

Elsa Reyes

PATIENT #2

Cresencia Ballon

PATIENT #3

Arnel Lopez

PATIENT #4

Erlinda Cupon

PATIENT #4

Ruben Pagado

Trials 1, 2 & 3: Aratiles Tea

Trial#1
Patient’s Response to the Effectiveness of the Aratilis Leaves Tea

Patients’ Rating to the Effectiveness of Aratilis Leaves Tea(5 highest – 1 lowest) | Patient’s Name | Taste | Odor | Appearance | Over-all Effectiveness of the Tea | Elsa Reyes | 3 | 3 | 3 | 3 | Cresencia Ballon | 3 | 3 | 3 | 3 | Arnel Lopez | 3 | 2 | 2 | 2.3 | Erlinda Cupon | 2 | 2 | 3 | 2.3 | Ruben Pagado | 3 | 2 | 3 | 2.67 | | | | | | Mean | 2.8 | 2.4 | 2.8 | 2.65 | Standard Deviation | 0.447214 | 0.547723 | 0.4472136 | 0.350114 |

Mr. Pagado told that there is no effect yet it needs a long time to determine its effectiveness. Mr. Lopez told that it has a bitter taste. He suggested that more filtration must occur. Other patients have the same response.

Trial#2
Patient’s Response to the Effectiveness of the Aratilis Leaves Tea

Patients’ Rating to the Effectiveness of Aratilis Leaves Tea(5 highest – 1 lowest) | Patient’s Name | Taste | Odor | Appearance | Over-all Effectiveness of the Tea | Elsa Reyes | 3 | 4 | 3 | 3.3 | Cresencia Ballon | 4 | 3 | 3 | 3.3 | Arnel Lopez | 4 | 2 | 3 | 3 | Erlinda Cupon | 3 | 4 | 4 | 3.7 | Ruben Pagado | 3 | 2 | 3 | 3 | | | | | | Mean | 3.4 | 3 | 5.3 | 3.26 | Standard Deviation | 0.547723 | 1 | 0.447214 | 0.288097 | Erlinda Cupon told that there is slight effect. Elsa Reyes told that it has a slight bitter taste. She suggested that more filtration must occur. Other patients have the same response.

Trial#3
Patient’s Response to the Effectiveness of the Aratilis Leaves Tea

Patients’ Rating to the Effectiveness of Aratilis Leaves Tea(5 highest – 1 lowest) | Patient’s Name | Taste | Odor | Appearance | Over-all Effectiveness of the Tea | Elsa Reyes | 4 | 4 | 3 | 4 | Cresencia Ballon | 4 | 4 | 4 | 4 | Arnel Lopez | 5 | 5 | 3 | 4 | Erlinda Cupon | 4 | 3 | 4 | 4 | Ruben Pagado | 4 | 4 | 3 | 4 | | | | | | Mean | 4.2 | 4 | 3.4 | 4 | Standard Deviation | 0.45 | 0.71 | 0.55 | 0 |

Mr. Pagado told there is effectiveness in drinking that kind of tea and it swelling. Mr. Lopez told that it has a bitter after taste. He suggested filtration machine can filter it fully because precipitate appear after a long time. He also suggested blood chemistry to check uric acid level due to taking of the tea. Other patients have the same response.

Patient’s Response to the Effectiveness of the Aratilis Leaves Ointment
Trial# 1:
Patient # 1: Clarita Banasihan
These data shows that the ointment of aratilis leaves(Muntingia calabura linn) is effective based on our patient #1. Date | Results | August 20,2011(3:00pm) | It lessens the pain after applying, but at evening the arthritis attack again and gives much pain. | September 3,2011(2:30 pm) | It continues to lessen the pain and gout. At evening, the arthritis rarely attacks. | September 17,2011(3:00pm) | The inflammation lessened. | October 1,2011(2:30pm) | The inflammation lessened. |

Patient’s Name | Texture | Odor | Appearance | Over-all Effectiveness of the Ointment | Clarita Banasihan | 3 | 2 | 3 | 3 |

Patients’ Rating to the Effectiveness of Aratilis Leaves Ointment(5 highest – 1 lowest) | Patient’s Name | Texture | Odor | Appearance | Over-all Effectiveness of the Ointment | Elsa Reyes | 4 | 5 | 4 | 4 | Cresencia Ballon | 5 | 3 | 4 | 1 | Arnel Lopez | 5 | 3 | 5 | 3 | Mean | 4.7 | 3.7 | 4.7 | 2.7 | Standard Deviation | 0.58 | 1.15 | 0.58 | 1.53 |

Ms. Reyes told that the ointment is effective but improve the oily texture of the ointment. Ms. Ballon experienced no effect yet and skipped one morning treatment. Mr. Lopez noticed change in swelling and the pain is removed.
Trial #3:
Patient’s Response to the Effectiveness of the Aratilis Leaves Ointment

Patients’ Rating to the Effectiveness of Aratilis Leaves Ointment(5 highest – 1 lowest) | Patient’s Name | Texture | Odor | Appearance | Over-all Effectiveness of the Ointment | Elsa Reyes | 4 | 4 | 4 | 5 | Cresencia Ballon | 5 | 5 | 4 | 5 | Arnel Lopez | 4 | 5 | 5 | 4 | Erlinda Cupon | 3 | 4 | 4 | 4 | Ruben Pagado | 4 | 5 | 3 | 5 | | | | | | Mean | 4 | 4.6 | 4 | 4.6 | Standard Deviation | 0.71 | 0.55 | 0.71 | 0.55 |

Mrs. Cupon told that there is less pain and less swelling. Mr. Lopez told that pain and tenderness relieved. Mr. Pagado told that itchiness and pain is gone and there is less pain and swelling. Both of the other patients have the same response.

Results and Discussion
Antibacterial Assay Results Table 1 Zone of Inhibition exhibited by Aratilis leaves against Staphylococcus aureaus 25923 on Petri dish 1 | Sample | Zone of Inhibition (diameter in mm) | Oil 1 | 11.5 | Ethanol 1 | 15.5 | Water 1 | 19.8 | Oil (Negative control) | 10.9 | Ethanol (Negative control) | 12.7 | Water (Negative control) | 12.9 | Streptomycin sulfate(Positive control) | 23.4 | Ointment (product) | 0 |

Individual assay of the Aratilis leaves by cup cylinder revealed that all samples exhibited zone of inhibition against S. aureaus. The Oil 1, Ethanol 1 and Water 1 samples contains the Aratilis leaves extract with a 1:1 ratio. Results showed that the different solutions exhibited zone of inhibition with values 11.5 mm, 15.5mm, and 19.8 mm, respectively.

The negative control which is the oil, ethanol and water alone showed a considerable zone of inhibition with values 10.9 mm, 12.7 mm and 12.9 mm respectively.

The positive control, Streptomycin sulfate, showed the highest zone of inhibition with a value of 23.4 mm compared to the Aratilis leaves ointment with only a value of 0 mm. The low value of the ointment sample could be due to dilution to the petroleum jelly. When concentrated with the Aratilis leaves extract, different active compounds may exhibit a synergistic effect to effectively control bacterial strains.

Table 2 Zone of Inhibition exhibited by Aratilis leaves against Staphylococcus aureaus 25923 on Petri dish 2 | Sample | Zone of Inhibition (diameter in mm) | Oil 1 | 7.3 | Ethanol 1 | 21.3 | Water 1 | 18.6 | Oil (Negative control) | 7.5 | Ethanol (Negative control) | 12.1 | Water (Negative control) | 12.5 | Streptomycin sulfate(Positive control) | 22.7 | Ointment (product) | 0 |

Individual assay of the Aratilis leaves by cup cylinder revealed that all samples exhibited zone of inhibition against S. aureaus. The Oil 1, Ethanol 1 and Water 1 samples contains the Aratilis leaves extract with a 1:1 ratio. Results showed that the different solutions exhibited zone of inhibition with values 7.3 mm, 21.3 mm, and 18.6 mm, respectively.

The negative control which is the oil, ethanol and water alone showed a considerable zone of inhibition with values 7.5 mm, 12.1 mm and 12.5 mm respectively.

The positive control, Streptomycin sulfate, showed the highest zone of inhibition with a value of 22.7 mm compared to the Aratilis leaves ointment with only a value of 0 mm. The low value of the ointment sample could be due to dilution to the petroleum jelly. When concentrated with the Aratilis leaves extract, different active compounds may exhibit a synergistic effect to effectively control bacterial strains.

Table 3 Zone of Inhibition exhibited by Aratilis leaves against Staphylococcus aureaus 25923 on Petri dish 3 | Sample | Zone of Inhibition (diameter in mm) | Oil 1 | 8.3 | Ethanol 1 | 18.0 | Water 1 | 21.9 | Oil (Negative control) | 8.2 | Ethanol (Negative control) | 19.2 | Water (Negative control) | 10.9 | Streptomycin sulfate(Positive control) | 27.5 | Ointment (product) | 0 |

Individual assay of the Aratilis leaves by cup cylinder revealed that all samples exhibited zone of inhibition against S. aureaus. The Oil 1, Ethanol 1 and Water 1 samples contains the Aratilis leaves extract with a 1:1 ratio. Results showed that the different solutions exhibited zone of inhibition with values 8.3 mm, 18.0 mm, and 21.9 mm, respectively. The negative control which is the oil, ethanol and water alone showed a considerable zone of inhibition with values 8.2 mm, 19.2 mm and 10.9 mm respectively.

The positive control, Streptomycin sulfate, showed the highest zone of inhibition with a value of 27.5 mm compared to the Aratilis leaves ointment with only a value of 0 mm. The low value of the ointment sample could be due to dilution to the petroleum jelly. When concentrated with the Aratilis leaves extract, different active compounds may exhibit a synergistic effect to effectively control bacterial strains.

Summary, Conclusion, and Recommendations

Based on the results, the ointment having different solutions of the plant extract, aratilis leaves showed inhibition against Staphylococcus aureaus. Since the extracts of aratilis leaves(Muntingia Calabura Linn) showed a few or having no sign of inhibition, it is recommended to use different concentrations. It is also suggested to use another substance for extraction like organic solvents.

It is anticipated that phytochemicals with adequate antibacterial efficacy will be used for the treatment of bacterial infections. Secondary plant metabolites (phytochemicals compounds have been extensively investigated as a source of medicinal agents. Among those antimicrobial compounds, phenolic compounds, terpenoids, and alkaloids’ are very important compounds in antimicrobial or antioxidant effects.

Further research is necessary to determine the different antibacterial compounds from this small tree and their full spectrum of efficacy. However, the present study of in vitro antimicrobial evaluation of these tree forms a primary platform for further phytochemical and pharmacological studies. These promissory extracts open the possibility of finding new clinically effective antimicrobial compounds.

Literature Cited

Philippine Medicinal Plants, Tilacea: Aratilies Muntingia Calabura Linn Cherry Tree

http://www.medicinet.com/septic Arthritis page3.htm

http://www.medicinenet.com/septic_arthritis/article.htm

In vitro Antibacterial Activity of Muntingia calabura extracts / Z A Zakaria, C A Fatimah, A M Mat Jais et al / Internation Journal of Pharmacology, 2 (45): 439-442, 2006

Activity-guided isolation of the chemical constituents of Muntingia calabura using a quinone reductase induction assay

Antinociceptive, anti-inflammatory and antipyretic effects of Muntingia calabura aqueous extract in animal models / Z. A. Zakaria et al

Plant anticancer agents, XLVIII. New cytotoxic flavonoids from Muntingia calabura roots / Kaneda N, Pezzuto JM et al /

Cytotoxic Constituents of Muntingia calabura Leaves and Stems Collected in Thailand / C M Nshimo et al / Summary Pharmaceutical Biology • 1993, Vol. 31, No. 1, Pages 77-81 / DOI 10.3109/13880209309082922

Effects of Muntingia calabura L. on isoproterenol-induced myocardial infarction / Nivethetha M et al / Singapore Med J 2009; 50 (3) : 300

The Antinociceptive Action of Aqueous Extract from Muntingia calabura Leaves: The Role of Opioid Receptors / Zainul Amiruddin Zakaria et al / Med Princ Pract 2007;16:130–136 / DOI: 10.1159/000098366

Appendix

A. Costing

Quantity | Item | Unit prize | | Total prize | 1 | Coconut oil (1st trial) | 100.00 | | 100.00 | 1 | Coconut oil (2nd and 3rd trial) | 74.00 | | 74.00 | 2 | Petroleum jelly(1st trial) | 42.00 | | 84.00 | 5 | Petroleum jelly (2nd trial) | 37.25 | | 186.25 | 5 | Eucalyptus Vics (3rd trial) | 105.00 | | 535.00 | 2 | Honey | 150.00 | | 300.00 | 8 | Lemon | 22.00 | | 176.00 | 3 | Sticker printing and research handout | 30.00 | | 250.00 | | BIOTECH | | 1,700.00 | Total: | 3, 405.25 |

B. Calculations

Aratiles Ointment

Trial # 1

Patient’s Name | Texture | Odor | Appearance | Over-all Effectiveness of the Ointment | Clarita Banasihan | 3 | 2 | 3 | 3 |

For getting the mean:

* = = 3 * = = 2 * = = 3 * = = 3
For getting the standard deviation:

Trial#2

Patient’s Name | Texture | Odor | Appearance | Over-all Effectiveness of the Ointment | Elsa Reyes | 4 | 5 | 4 | 4 | Cresencia Ballon | 5 | 3 | 4 | 1 | Arnel Lopez | 5 | 3 | 5 | 3 | Mean | 4.7 | 3.7 | 4.7 | 2.7 | Standard Deviation | 0.58 | 1.15 | 0.58 | 1.53 |

For getting the mean:

For getting the standard deviation:

Trial#3

Patient’s Name | Texture | Odor | Appearance | Over-all Effectiveness of the Ointment | Elsa Reyes | 4 | 4 | 4 | 5 | Cresencia Ballon | 5 | 5 | 4 | 5 | Arnel Lopez | 4 | 5 | 5 | 4 | Erlinda Cupon | 3 | 4 | 4 | 4 | Ruben Pagado | 4 | 5 | 3 | 5 | | | | | | Mean | 4 | 4.6 | 4 | 4.6 | Standard Deviation | 0.71 | 0.55 | 0.71 | 0.55 |

For getting the mean:

For getting the standard deviation:

Aratiles Tea

Trial#1

Patient’s Name | Taste | Odor | Appearance | Over-all Effectiveness of the Tea | Elsa Reyes | 3 | 3 | 3 | 3 | Cresencia Ballon | 3 | 3 | 3 | 3 | Arnel Lopez | 3 | 2 | 2 | 2.3 | Erlinda Cupon | 2 | 2 | 3 | 2.3 | Ruben Pagado | 3 | 2 | 3 | 2.67 | | | | | | Mean | 2.8 | 2.4 | 2.8 | 2.65 | Standard Deviation | 0.45 | 0.55 | 0.45 | 0.35 |

For getting the mean:

For getting the standard deviation:

Trial#2

Patient’s Name | Taste | Odor | Appearance | Over-all Effectiveness of the Tea | Elsa Reyes | 3 | 4 | 3 | 3.3 | Cresencia Ballon | 4 | 3 | 3 | 3.3 | Arnel Lopez | 4 | 3 | 3 | 3 | Erlinda Cupon | 3 | 4 | 4 | 3.7 | Ruben Pagado | 3 | 2 | 3 | 3 | | | | | | Mean | 3.4 | 3.2 | 3.2 | 3.3 | Standard Deviation | 0.55 | 0.84 | 0.45 | 0.29 |

For getting the mean:

For getting the standard deviation:

Trial #3

Patient’s Name | Taste | Odor | Appearance | Over-all Effectiveness of the Tea | Elsa Reyes | 4 | 4 | 3 | 4 | Cresencia Ballon | 4 | 4 | 4 | 4 | Arnel Lopez | 5 | 5 | 3 | 4 | Erlinda Cupon | 4 | 3 | 4 | 4 | Ruben Pagado | 4 | 4 | 3 | 4 | | | | | | Mean | 4.2 | 4 | 3.4 | 4 | Standard Deviation | 0.45 | 0.71 | 0.55 | 0 |

For getting the mean:

For getting the standard deviation:

Excess Pictures:

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