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The Salicylates Obtained from the Bark of a Weeping Willow Tree

THE SALICYLATES OBTAINED FROM THE BARK OF WEEPING WILLOW (Salix sepulcralis, Salicaceae) AS A STIMULANT Undergraduate Thesis In Partial Fulfillment of the Requirements for the Degree Bachelor of Science in Pharmacy GLAIZALINE A. AGUSTIN JOANNA MARIE S. BAUTISTA WINNIE G. ERORITA Mariano Marcos State University College of Health Sciences Department of Pharmacy 2906 City of Batac, Ilocos Norte March 2010 APPROVAL SHEET This thesis manuscript entitled THE SALICYLATES OBTAINED FROM THE BARK OF WEEPING WILLOW (Salix sepulcralis, Salicaceae) AS A STIMULANT prepared and submitted by Agustin, Glaizaline A. Bautista, Joanna Marie S. , Erorita, Winnie G. , in partial fulfillment of the requirements for the degree of Bachelor of Science in Pharmacy, is hereby endorsed. Imelda B. Corpuz, RPh, MS Adviser Augustus Caesar C. Pati, RPh Janelyn V. Rojas, RPh, MST Member Member Artemio Seatriz, Ed. D. Dexter A. Adriatico StatisticianEnglish Critic Accepted in partial fulfillment of the requirements for the degree of Bachelor of Science in Pharmacy. Aileen O. Camangeg, RPh, MA, MS Lailani S. Galutira, RN, MHPEd Chairman, Dept. of Pharmacy Dean, College of Health Sciences ACKNOWLEDGEMENT

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Amidst all the challenges that appeared over the horizon which brought unclear visions of tomorrow, some people were considerate enough to become the researchers’ lanterns in the uncertain path. They are the people who believed in their unique capabilities, trusted their strengths and helped mold themselves in order to fill their visible weaknesses. Thus, they would like to extend the greatest appreciation to all the people who contributed in the completion of this manuscript: To their dearest families and friends for their outmost support which served as their greatest foundation of inspiration and determination;

To Ms. Imelda B. Corpuz and Ms. Janelyn V. Rojas, Thesis adviser and committee member, for the patience, understanding, pieces of advices, and encouragements which initiated the to work as hard as possible the for completion of this manuscript; To the faculty and staff of the Department of Pharmacy for the unending support and guiding hands in completing certain tasks; To Mr. Dexter A. Adriatico, English Critic, for dedicating his expertise in editing and polishing the manuscript; To Dr. Artemio Seatriz, statistician, for his expertise in analyzing and interpreting the gathered data;

To all the persons whose names were not mentioned, for all the hands lent; and Above all to the Almighty God for His bestowed knowledge and wisdom for the realization of this challenging yet achievable work. The Researchers CONTENTS PAGE Approval Sheetii Acknowledgementiii Contentsv List of Tablesvii List of Figurexi Abstractxii CHAPTER IINTRODUCTION Background of the Study1 Statement of the Problem3 Significance of the Study4 Scope and Limitations4 Operational Definition of Terms5 IIREVIEW OF LITERATURE Literature7 Coffee13 Studies16 Conceptual Framework19 Research Hypothesis21 IIIMETHODOLOGY Research Design22

Data Gathering Procedure23 Statistical Treatment of Data27 IV PRESENTATION, ANALYSIS AND INTERPRETATION OF DATA Identification Test31Pharmacological Screening32 v VSUMMARY, CONCLUSIONS AND RECOMMENDATIONS Summary of Findings40 Conclusions41 Recommendations41 BIBLIOGRAPHY43 APPENDICES45 APreparation of Reagents Used in the Confirmatory Test Of Salicylates46 BWeight of the Test Animals and Dose Computation47 CData on the Motor Coordination of the Swiss Mice50 DData on the Respiratory Rate of the Swiss Mice51 EData on the Swimming Endurance of the Swiss Mice52 FDescriptive Statistics of Motor Coordination53

GDescriptive Statistics of the Respiratory Rate60 H Descriptive Statistics of Swimming Endurance66 IDocumentation73 CURRICULUM VITAE80 List of Tables TABLESPAGES 1Analysis of Variance28 2Identification Test for Salicylates31 3 Concentration and Treatment Means of Motor Coordination32 4 Results of the Two-way ANOVA on the Motor Coordination32 5Least Significant Difference (LSD) Test for Motor Coordination33 6Concentration and Treatment Means of Respiratory Rate34 7Results of the two-way ANOVA on the Respiratory Rate 34 8Least Significant Difference (LSD) Test for Respiratory Rate35 9Concentration and Treatment Means of Swimming

Endurance36 10Results of the Two-way ANOVA on the Time after Exhaustion37 11Least Significant Difference (LSD) Test for Swimming Endurance38 12 Weight and Dose of the Sample Control Group used for The Oral Administration of Salicylates. 47 13 Weight and Dose of the Negative Control Group Used for The Oral Administration of Distilled Water. 48 14 Weight and Dose of the Positive Control Group used for The Oral Administration of Caffeine49 15 Time Until the Mice Stops Running on the Rota-Rod Before The Oral Administration of the Different Treatments50 16Time Until the Mice Stops Running on the Rota-Rod After

The Oral Administration of the Different Treatments. 50 17Time of 50 Breaths Before Oral Administration of the Different treatments in Sec51 18Time of 50 Breaths After Oral Administration of the Different treatments51 19Time of total exhaustion of swimming52 20Dependent Variable for Motor Coordination53 21Tests of Between-Subjects Effects on the Dependent Variable Motor Coordination53 22Grand Mean of Motor Coordination54 23Concentration Estimates of Motor Coordination54 24Concentration Pairwise Comparisons on Motor Coordination54 25Concentration Univariate test on Motor Coordination55 6Group Estimates on Motor Coordination56 27Group Pairwise Comparison on Motor Coordination56 28Group Univariate Test on Motor Coordination56 29Post Hoc Test on the Concentration Multiple Comparison on Motor Coordination57 30Concentration of Homogeneous Subsets on Motor Coordination58 31 Group Multiple Comparisons on Motor Coordination58 32Homogenous Subsets on Motor Coordination59 33Univariate Analysis of Variance Between-Subjects Factors On Motor Coordination59 34Descriptive Statistics on the Respiratory Rate60 35Test of Between-Subjects Effects on the Respiratory Rate60 36Grand Mean on the Respiratory Rate61 7 Estimates on the Respiratory Rate61 38Pairwise Comparison on the Respiratory Rate61 39Univariate Test on the Respiratory Rate62 40Group Estimates on the Respiratory Rate62 41Group Pairwise Comparisons on the Respiratory Rate63 42Group Univariate Tests on the Respiratory Rate63 43Post Hoc Test Concentration Multiple Comparisons On the Respiratory Rate 64 44Homogeneous subsets on the Respiratory Rate64 45Univariate Analysis Of variance Between-Subject factors65 On the Respiratory Rate 46Descriptive Statistics of Swimming Endurance66 47Tests Between-Subject Effects on Swimming Endurance67 8Estimated Marginal Means of Grand Mean on Swimming Endurance67 49Concentration Estimates on Swimming Endurance67 50Concentration Pairwise comparison on Swimming Endurance68 51Concentration Univariate Tests on Swimming Endurance68 52Group Estimates on Swimming Endurance68 53Group Pairwise Comparison on Swimming Endurance69 54Group Univariate Test on Swimming Endurance69 55Post Hoc Tests on Concentration for Comparisons on Swimming Endurance69 56Concentration Homogenous Subsets of Swimming Endurance70 57Concentration Multiple Comparison on Swimming Endurance70 58Homogenous Subsets of Swimming Endurance71 9Univariate Analysis of Variance between-Subject Factors on Swimming Endurance72 LIST OF FIGURES FIGURE PAGES 1Weeping Willow Tree6 2Research Paradigm 20 3Flowchart of Methods30 4Coffee Beans (Kapeng Barako)73 5Representative of Test Animals (Swiss Mice) Used74 6Weighing of Finely Divided Weeping Willow Bark74 7Decoction Process75 8Filtration of Weeping Willow bark Extract75 9Chemicals Used for determination of Salicylates76 10Determination of Salicylates76 11Isolation of Salicylates77 12Isolation of Caffeine78 13Swimming Endurance78 ABSTRACT AGUSTIN, GLAIZALINE A. , BAUTISTA, JOANNA MARIE S. ERORITA, WINNIE G. Undergraduate Thesis. Mariano Marcos State University, College of Health Sciences, Department of Pharmacy, City of Batac, 2906 Ilocos Norte, March 2010. THE SALICYLATES OBTAINED FROM THE BARK OF WEEPING WILLOW (Salix sepulcralis, Salicaceae) AS A STIMULANT. Adviser: Imelda B. Corpuz This study was conducted to investigate the stimulant effect of the salicylates obtained from the bark of the Weeping Willow (Salix sepulcralis). It utilized the experimental method in the conduct of the study. Twenty-eight healthy male Swiss mice were divided into three groups.

The mice were treated by an oral administration with four (4) different doses starting with an initial dose of 1mg/kg and the increasing dose were computed using the 0. 6 log intervals with a maximum of four log intervals. The treatments were, T1=Distilled; T2=Caffeine, T3=Salicylates obtained from the Weeping Willow Bark. The Analysis of Variance (ANOVA) was used to determine if there is a significant difference between Caffeine and Salicylates obtained from Weeping Willow bark. Similarly, this is utilized to determine which among the doses of salicylates is the most effective.

The study came out with the following findings: First, the crude extract from the Weeping Willow (Salix sepulcralis) bark showed a positive result in the confirmatory test for salicylates; Second, the salicylates obtained from the Weeping Willow (Salix sepulcralis) bark posses stimulant activity in varying doses. The greater the dose, the greater the stimulant activity it has. Thus, it is comparable to the positive control since there is no significant difference between the two. The salicylates obtained from the Weeping Willow (Salix sepulcralis) bark, however, are not comparable with the negative control.

Finally, based on the findings, the salicylates obtained from the Weeping Willow (Salix sepulcralis) bark serves as good stimulant promoting a more active and alert body reflexes. CHAPTER 1 INTRODUCTION Background of the Study Long before the introduction of modern medicines and Western curative methods herbal medicines had been widely used in the Philippines. This attributed to the fact that the Philippines an archipelago rich in natural resources, including herbal medicines. The curative effects of the herbs have been tested by traditional healers on their patient on trial-and-error basis.

The knowledge and skills on the curative application of any given herbal medicine has been handed down from generation to generation. Apart from prescribing herbal medicines, the traditional healers were known to give psychological comfort and moral support to their patients. Through generations of selective process, the herbs that were known to be effective were kept alive and the ineffective ones were soon forgotten. In the days when drugs from the West just started to be used in the Philippines, they were not only too expensive but also too scarce.

The rural folks and the poor then continued to rely on herbal medicines available in abundance, locally. Moreover, the Western-style medical care has reached a very limited number of people, mostly in urban areas. As modern drugs were increasingly available at much cheaper prices, the popularity of herbal medicines waned considerably and faced extinction. Despite the continuous evolution of modern science, Filipinos are still able to utilize the manifestations of using herbal remedies in curing some of the common diseases.

Sometimes the long processes of mixing certain potions and recipes containing common plants found around the garden which they believe have medicinal contribution in the treatment of the signs and symptoms of different known sicknesses. Weeping Willow, scientifically known as Salix sepulcralis, has been widely introduced throughout Europe, Asia and some North America. It is practically a purely ornamental tree. It grows from 30 to 80 feet in height with many round, widely spreading branches, silky when young, and thick, brown bark, full of cracks, small branches smooth and greenish.

One benefit to willow use is that the natural salicylic acid present in Weeping Willows produces fewer side effects than the synthetically produced acetylsalicylic acid of aspirin. With the increasing integration of traditional and modern medicines to improve health care delivery system, the use of medicinal plants has motivated the researchers to conduct a study on the utilization of Weeping Willow as a stimulant agent. Stimulants are used for the treatment of certain psychiatric conditions and also used (and abused) for recreational purposes, enhanced levels of energy, and weight loss.

These may be prescription or over-the-counter medications, illegal street drugs, or ingredients in commonly ingested substances, such as the caffeine in coffee or the nicotine in cigarettes. Whatever their forms, stimulants increase respiration, heart rate, and blood pressure, and their abuse can cause adverse physical effects and endanger a person’s health and even his or her life. A particular overdose of stimulants can result in chest pains, convulsions, paralysis, coma, and death. Statement of the Problem This study aimed to determine the stimulant activity of Weeping Willow (Salix sepulcralis).

More specifically, it sought answers to the following questions: 1. What extraction process is used in obtaining salicylates from Weeping Willow bark? 2. What test is used to determine the presence of salicylates in Weeping Willow bark? 3. How are salicylates isolated? 4. What dose of the salicylates obtained from Weeping Willow bark exhibits the stimulant effect? 5. What are the parameters in determining the effectiveness of salicylates as stimulating agent? 6. Is there a significant difference between the stimulant effect of salicylates obtained from Weeping Willow bark and Kapeng Barako (Caffeine)?

Significance of the Study Plants are widely and commonly used as medicinal products which can treat and prevent disease, thus, these may serve for life extension. Anent to this belief, the researchers aimed to determine and prove that the salicylates obtained from the bark of Weeping Willow have the ability to increase physical activity, thus producing a stimulant effect. If this ornamental tree particularly the bark has the ability to produce a stimulant effect, this would be used as a raw material in coming up with a stimulant drug. Scope and Limitations

The research was limited on the use of salicylates obtained from the Weeping Willow bark (Salix sepulcralis, Salicaceae). It was conducted to seek the efficacy of the plant material as a stimulant agent. Moreover, the researchers focused on the extraction, identification of the salicylates found in the crude extract and the isolation of salicylates from the Weeping Willow bark. The extraction, identification of the salicylates and the isolation of salicylates and caffeine were conducted at the Pharmacy Department Laboratory Room. The study was restricted to the investigation of he stimulant activity of the salicylates obtained from the Weeping Willow bark. The stimulant effect was tested on male Swiss mice and was compared to commercially prepared stimulant caffeine (Kapeng Barako) on December 22, 2009 at the Mariano Marcos State University Pharmacy Department Laboratory Room. The parameters performed in determining the efficacy of stimulant activity were increased motor coordination, increase in respiratory rate, and swimming endurance. Operational Definition of Terms To enhance proficiency in understanding this paper, the following terms are defined operationally and conceptually: Crude Drug.

It refers to a vegetable or animal drug that consists of natural substances that undergo the processes of collection and drying only. Crude Extract. It is a solution of the active ingredients obtained from the bark of Weeping Willow (Salix sepulcralis, Salicaceae). Decoction. It is a method of boiling the dissolved herbal or plant material, which may include stems, roots, bark and rhizomes. Duration of Action. It is the length of time that a drug continues to produce its effect. Onset of Action. It is the time of appearance of the first observable effect. Salicylates. This pertains to salt or ester of salicylic acid.

Stimulant. It is an agent that promotes the activity of a body system or function by increasing the motor activity, respiratory rate, body temperature and swimming endurance. CHAPTER II Review of Literature This chapter presents the description and information about the Weeping Willow specifically its bark as principal material for this study. The foreign and local literatures and studies of the said plant are clearly stated to enhance the systematic acquaintance to the plant. Figure 1 Weeping Willow (Salix sepulcralis) tree Literature The Weeping Willow is an introduced, deciduous tree which grows up to 12 meters tall.

It is instantly recognized by its slender ‘weeping’, golden-yellow twigs. The bark is grayish-brown, deeply and coarsely fissured. It is widely planted for ornament on river banks, pond margins and in parks and gardens. It is known for its drooping leaves and branches. The leaves of the willow appear long and narrow and are whitened underneath. The leaves are narrow, alternate, 7-12 cm long and up to 18 mm wide. They end in a very slender, tapering point, often turned in one direction and have a fine, regularly toothed margin. They are bright green above, bluish-green below, and only hairy, on both surfaces, when young.

The leaf-stalk is very short which is less than 8 mm in length. Corollary to this, Weeping Willow is of rapid growth, native of Europe, and introduced into this country. Its flowers appear from March to June. The bark, which is the medicinal part, is readily removed from the stem during the months of July, August, and September. The dried bark is met with more or less tough, with a faint odor, and a bitter taste, combined with some astringency. Water takes up its medicinal properties, the decoction having a dark-reddish color, and which is precipitated abundantly by gelatin, carbonates of potassium, and ammonium.

Lime-water gives at first a blue, and then a buff-colored precipitate. Ferric chloride throws down a dark-green tannate of iron. If the decoction contains much salicin, concentrated sulphuric acid reddens it (http://www. henriettesherbal. com/electic/kings/sali x. html) . Locally, Weeping Willow is found in the provinces of Bulacan, Baguio, Rizal, Bohol, and in Mindanao. It is a tree flowering after leafing. The bark is rough, with deep, vertical fissures. The young shoots and young leaves are silky. The leaves are lanceolate, 8 to 15 centimeters long, with minutely and regularly toothed margin.

Kirtikar and Basu quoted Dalziel and Gibson, who statedthat the bark is used as a febrifuge. They further cited Long, who said that the bark yields out tonic substances. (Quisumbing 1978) The use of Willow bark dated back thousands of years, to the time of Hippocrates (400 BC) when patients were advised to chew on the bark to reduce fever and inflammation. The Willow bark has been used throughout the centuries in China and Europe, and is continued today for the treatment of pain (particularly low back pain and osteoarthritis), headache, and inflammatory conditions such as bursitis and tendinitis. Historically, the

Willow bark has been used as an antipyretic, anti-inflammatory, and analgesic. Despite a lack of clinical trials, these uses have persisted for several hundred years. This, along with willow bark’s similarities to aspirin, suggests these indications are substantiated. In modern herbal therapy, willow bark is predominantly used as an anti-inflammatory for relief of gouty arthritis and as an analgesic for mild neurologic pain, toothaches, and headaches. It is not widely used for its fever-fighting activity, having been replaced by diaphoretics (agents that cause sweating) and other antipyretics affective at lowering fevers.

Of primary importance when addressing the general level of efficacy of willow bark preparations is evaluating whether to use anti-inflammatory at all. Inflammation is a homeostatic response to pathogens and tissue injury. Inhibiting such processes may do more harm good and may be associated with some degree of cellular and organ system toxicity. The use of anti-inflammatories should therefore be carefully considered, restricted to a limited time, and followed up with more appropriate therapies to address the underlying cause of inflammation (http://www. newhope. om/nutrionsciencenews/nsn_box/su n_01/willowbark. cfn? path=ex). Moreover, Willow bark has been used for its pain relieving qualities since ancient times. The willow contains salicin, which is converted to salicylic acid in the body. Salicylic acid is closely related to aspirin the synthetic drug that has displaced Willow bark from popular use. Willow bark reduces fever and relieves rheumatism, a common ailment in cold and damp regions like the British Isles. On the other hand, salicin preparations from crude willow bark do not present a hemorrhagic risk and may clinically advantageous for some.

More clinical studies are needed to establish the therapeutic effectiveness of willow bark preparations. Upon determination of efficacy, additional questions need to be investigated. These include the possible pharmacokinetic advantage of the glycosidic form present in the bark in sustaining plasma salicylate levels and the possibility if activity of constituents other the salicylates. In the meantime, due to the wide variation of salicin content in willow bark, care should be taken to obtain products that deliver an effective concentration of salicin. All willows produce salicin, which is closely related chemically to aspirin.

Native Americans used various preparations from willows to treat tooth ache, diarrhea and dysentery, and dandruff. Native Americans also used flexible willow stems for making baskets, bowa, arrows, scoops, fish traps, and other items. However, studies have identified several other components of willow bark that have antioxidant, fever-reducing, antiseptic, and immune-boosting properties. Recent studies have shown that the Willow is as effective as aspirin for reducing pain and inflammation, and at a much lower dose. Researchers think that maybe due to the other compounds in the herb. More research is needed.

The bark of willow contains salicin, which is a chemical similar to aspirin (acetylsalicylic acid). In addition, it is thought to be responsible for the pain-relieving and anti-inflammatory effects of the herb. In fact, in the 1800s, salicin was used to develop aspirin. However, the Willow appears to be slower than aspirin to bring pain relief, but its effects may last longer (http://www. umn. edu/altmed/articles/willow-bark-000281. html). The active ingredient in the willow bark is a bitter glycoside called salicin, first isolated in a pure form in 1829 by Leroux, who also demonstrated its anti pyretic effect.

On Hydrolysis, salicin yields glucose and salicylic alcohol. The latter can be converted into salicylic acid, either in vivo or by chemical manipulation. Meanwhile, Sodium salicylate was first used for the treatment of rheumatic fever and as an antipyretic in 1875, and the discovery of its uricosuric effects and of its usefulness in the treatment of gout soon followed. During the 1970s in Europe, willow bark preparations were used intermittently with NSAIDs, with the NSAIDs used only when acute arthritis pain did not respond to salicin-containing preparations such as willow bark.

Such a therapeutic strategy may lessen the side effects associated with NSAIDs. Improvement from willow bark treatment is usually observable within one to four weeks, sometimes preceded by a transient worsening of symptoms, then followed by a significant decrease in discomfort, swelling, and inflammation. In some cases, improvement is seen within the first few days of therapy. A decoction can be used for gum and tonsil inflammations and as footbath for sweaty feet. The bark of young tree branches is harvested during the early spring.

The grayish bark is separated from the tree, then either dried or used fresh. Willow is commercially available in tincture, tablet, capsule, powder, or tea forms. According to Pelletier and Caventou(1998), the chief constituents of willows, is the glucosid salicin. Tannin is also prominent. The Major Constituents of Weeping Willow are Salicin, tannins, phenolic, flavonoid, glycosides, salicottin, and triandrin (Thomas S. C. 2000). Salicin (C13H18O7) is an alcoholic ? -glycoside which contains D-glucose. Salicin is an anti-inflammatory agent that is produced from the Willow barks.

Salicin is closely related in chemical make-up to aspirin and has a very similar action in the human body. When consumed, it is metabolized to salicylic acid. The systematic (IUPAC) name of the molecule is 2-(Hydroxymethyl)phenyl ? -D-glucopyranosides. (http://en. wikipedia. org/wiki /Salicin) Salicylic acid (from the Latin word for the willow tree, Salix, it is a beta hydroxy acid. This colorless crystalline organic acid is widely used in organic synthesis and functions as a plant hormone. It is derived from the metabolism of salicin.

In addition, it is a compound that is chemically, similar but not identical to the active component of aspirin (acetylsalicylic acid). It is probably best known for its use in anti-acne treatments. The salts and esters of salicylic acid are known as salicylates (http://en. wikipedia. org/wiki/Salicylic _acid). According to Goodman and Gillman (1996) salicylates have miscellaneous neurological effects. In high doses, salicylates have toxic effects on the CNS consisting of stimulation followed by depression, confusion, dizziness, tinnitus, high-tone deafness, delirium, psychosis, stupor, and coma may occur.

Salicylates, including aspirin are drugs that bring down the fever, reduce minor pain, reduce inflammation and prevent blood clots. The antipyretic and analgesic actions are mediated by an action in the CNS. Prevents heart attacks, probability due to its anti clotting action via inhibition of the production of thromboxane A (Stringer, J. L. 1996). Aspirin on the otherhand, inhibits prostaglandin synthesis and release and, probably through this mechanism, reduce the duration of stages 3 and 4 NREM sleep. Shneerson 2001) Coffee Coffee contains caffeine, which acts as a stimulant. For this reason, it is often consumed in the morning and when feeling tired. Students preparing for examinations with late-night cram sessions or code jams frequently use coffee to stay awake. Many office workers take a coffee break when they have low energy. Furthermore, Coffee also stimulates the bowels. Recent research has uncovered additional stimulating effects of coffee which are not related to its caffeine content.

Coffee contains an as yet unknown chemical agent which stimulates the production of cortisone and adrenaline, two stimulating hormones. For occasions when one wants to enjoy the flavor of coffee with almost no stimulation, decaffeinated coffee (also called decaf) is available. This is coffee from which most of the caffeine has been removed, by the Swiss water process (which involves the soaking of raw beans to absorb the caffeine) or the use of a chemical solvent such as trichloroethylene (“tri”), or the more popular methylene chloride, in a similar process.

Another solvent used is ethyl acetate; the resultant decaffeinated coffee is marketed as “natural decaf” because ethyl acetate is naturally present in fruit. Extraction with supercritical carbon dioxide has also been employed. The effects of caffeine as a stimulant have been one of the most studies aspects of coffee, particularly in studies concerning the relationship between coffee consumption and human health. While caffeine is found naturally in tea, kola nuts, Yerba mate, guarana, and cacao beans, it is predominantly associated with coffee beans.

The main pharmacological properties of caffeine include its stimulating action on the central nervous system, in which it also has psychotropic effects. Additionally, caffeine has a stimulating effect on respiration and the human heart rate. Furthermore,  Caffeine also contains a diuretic effect. It is believed that the way that caffeine affects the brain is by blocking adenosine receptors, which slows down nerve cell activity. When the caffeine molecule binds to the receptors f nerve cells, it causes the release of hormone epinephrine, which results in increased heart rate, increased blood flow to the muscles, increased blood pressure, decreased blood flow to the skin, decreased blood flow to the inner organs, a release of glucose by the liver, and increase in the levels of the neurotransmitter dopamine in the brain. While many are worried about the addictive properties of caffeine, one has to follow the maxim that “too much of a good thing becomes bad”, thus, one is able to avoid the negative effects of caffeine that results from an over consumption of coffee.

Moreover, since coffee is a stimulant, it is the principle reasons why it is such a popular drink throughout the world. While there are many social traditions that result from drinking coffee, the use of coffee as a stimulant is almost as old as coffee. For instance, the Monks in the African desert where coffee plants were first discovered had historically used coffee beans as a way to stay fully awake so they can prolong their own meditations. So coffee and its stimulating principles have lived on for centuries and will continue to live on as one of the benefits of drinking coffee. http://www. ringsurf. com/online/2115stimulant. html) Studies In the research conducted by Ester and Gabrys (2004), they tested the swimming capacity of mice after prolonged treatment with psycho stimulants. The study found out that the effect of long-term treatment with fecamfamine on the swimming endurance and availability of metabolic substrates in mice. They used Fencamfamine (14 micrograms/gram per day orally for 6 weeks) which reduced maximum swimming capacity by more than 40%.

They concluded that this effect could not be attributed to motor in coordination or diminution of pre-swimming level of metabolic substrates such as liver and muscle glycogen stores were depleted more rapidly in fencamfamine-treated animals. Thus, it appeared that fencamfamine leads more rapidly to a shortage of combustile substrates in the swimming animals (http://www3. interscience. wiley. com/journa l/121674827/abstract). Recently in 2008, a study was conducted by Costa et. al to determine the effects of caffeine in mice.

In their study, the effects of caffeine on mice performance in the object recognition task were tested in different intertrial intervals. In addition, they analyzed the effects of caffeine on brain derived neurotrophic factor (BDNF) and its receptor, TrkB, immunocontent to try to establish a connection between the behavioral finding and BDNF, one of the neurotrophins strictly involved in memory and learning process. CF1 mice were treated during 4 consecutive days with saline (0. 9g%, i. p. ) or caffeine (10mg/kg, i. . , equivalent dose corresponding to 2-3 cups of coffee). Caffeine treatment was interrupted 24 hour before the object recognition task analysis. In the test session performed 15 minutes after training session, caffeine-treated mice recognized more efficiently both the familiar and the novel object. In the test session performed 90min and 24h after training session, caffeine did not change the time spent in the familiar object but increased the object recognition index, when compared to control group.

Western blotting analysis of hippocampus from caffeine-treated mice revealed an increase in BDNF and TrkB immunocontent, compared to their saline-matched controls. Phospho-CREB immunocontent did not change with caffeine treatment. Their results suggest that acute treatment with caffeine improves recognition memory, and this effect may be related to an increase of the BDNF and TrkB immunocontent in the hippocampus (http://www. ncbi. nlm. nih. gov/pubme d/18620014? ordinalpos=13;itool=EntrezSystem2. PEntrez. Pubmed.

Pubmed_ ResultsPanel. Pubmed_ DefaultReportPanel. Pubmed_RVDo cSum. ) Meanwhile, in an earlier study conducted by Yacoubi (2000), et. al on the stimulant effects of caffeine on locomotor behavior in mice they found out that the locomotor stimulatory effects induced by caffeine (1,3,7-trimethylxanthine) in rodents have been attributed to antagonism of adenosine A1 and A2A receptors. The roles of adenosine A1 and A2A receptors in these activities were investigated using a Digiscan actimeter in experiments carried out in mice.

Besides caffeine, the A2A antagonist SCH 58261 (5-amino-7-(? -phenylethyl)-2-(8-furyl)pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine), the A1 antagonist DPCPX (8-cyclopentyl-1,3-dipropylxanthine), the A1 agonist CPA (N6-cyclopentyladenosine) and A2A receptor knockout mice were used. They found out that Caffeine had a biphasic effect on locomotion of wild-type mice not habituated to the open field, stimulating locomotion at 6. 25–25 mg kg? 1 i. p. doses, while depressing it at 100 mg kg? 1.

In sharp contrast, caffeine dose-dependently decreased locomotion in A2A receptor knockout mice over the whole range of tested doses. The depressant effects induced by high doses of caffeine were lost in control CD1 mice habituated to the open field. The A1 agonist CPA depressed locomotion at 0. 3–1 mg kg? 1 i. p. doses. The A1 antagonist DPCPX decreased locomotion of A2A receptor knockouts and CD1 mice at 5 mg kg? 1 i. p. and 25 mg kg? 1 i. p. respectively. DPCPX (0. 2–1 mg kg? 1 i. p. ) left unaltered or even reduced the stimulant effect of SCH 58261 (1–3 mg kg? 1 i. p. ) on CD1 mice.

These results suggest therefore that the stimulant effect of low doses of caffeine is mediated by A2A receptor blockade while the depressant effect seen at higher doses under some conditions is explained by A1 receptor blockade (http://www3. intersci ence. wiley. com/journal/121674827/abstract). The research study differs from other research in such a way that instead of utilizing synthetic preparations it utilized isolated salicylates from Weeping Willow bark as an alternative stimulant agent. Conceptual Framework The research materials in the study are the salicylates obtained from Weeping Willow bark.

The stimulant activity of the salicylates obtained from Weeping Willow bark was compared to Coffee, a commercially prepared stimulant agent to determine its level of efficacy. The mechanism of salicylates in the inhibition of prostaglandin has the ability to reduce the duration of stage 3 and 4 NREM of sleep, which increases stage zero (stage of wakefulness). The focus of this study specifically outlined in the research paradigm shown in Figure 2. Isolation process for Salicylates Bark Crude Extract of Weeping Willow (Salix sepulcralis) Stimulant Activity

Independent VariableIntervening Variable Dependent Variable Figure 2 Research Paradigm Research Hypotheses This study was guided by the following hypotheses: First, there was a white crystalline precipitate which determines the presence of salicylates in Weeping Willow bark Extract; Second, the salicylates obtained from Weeping Willow Bark Extract is effective in increasing motor activity, respiratory rate and swimming endurance; and Third, there is no significant difference on the salicylates obtained from Weeping Willow bark and caffeine from “Kapeng Barako”. CHAPTER III

METHODOLOGY This chapter presents the methods and procedures that were used in the study. Furthermore, the techniques, instruments used, different parameters, and data analysis employed in the study are discussed. Research Design The study employed experimental procedures of research which investigated the effect of the salicylates obtained from Weeping Willow (Salix sepulcralis) bark as a stimulant In the representation of the process, the extracted constituents and isolated salicylates were subjected to a chemical test in establishing the confirmation of salicylates.

Twenty-eight (28) healthy Swiss mice with an age of 3-4months and approximate weight of 25 grams were used in the study. The test animals were divided into three groups, with the following groupings; (1) Negative Control (distilled water); (2) Positive Control, caffeine containing beverage (Kapeng Barako); (3) Sample Control (salicylates obtained from Weeping Willow bark). The parameters that were observed after sample administration included the increase in motor coordination, increase in respiratory rate, and swimming endurance. Data Gathering Procedures Collection and Preparation of Materials

The dried barks of Weeping Willow (Salix sepulcralis) were gathered in Agricultural Training Institute, Brgy. Tabug, Batac City, Ilocos Norte. The barks were collected early in the morning and air dried for 14 days. The barks were cut into small pieces using scissors, after which, there were subjected for extraction through decoction method. Extraction by Decoction For the extraction procedure, 500g of dried pieces of Weeping Willow (Salix sepulcralis) bark was weighed and placed in an Erlenmeyer flask 3000 mL of Distilled water was added and the mixture was subjected to decoction process.

Determination of Salicylates The crude extract was treated with Ferric Chloride (25g/L) TS. An intense reddish violet color appeared, which remained upon the addition of small amount of acetic acid (~300g/L) TS; but disappeared upon the addition of Hydrochloric acid (~70g/L) TS, with the separation of white crystalline precipitates. The presence of white crystalline precipitate indicates the presence of salicylates. Isolation of Salicylates Ten (10) mL of crude extract was mixed to 200 mL of 25% solution of sodium hydroxide in a round bottom flask.

After which it was transferred in a reflux condenser. The mixture was subjected to reflux for 1 hour. It was cooled, and acidified with concentrated hydrochloric acid. The mixture was then filtered and then washed with distilled water. Finally it was placed in a watch glass and dried in an oven. White crystalline compound were obtained. Isolation of Caffeine from Coffee beans The 10 grams of ground coffee beans were weighed and placed in a 150 mL beaker and was mixed with 30 mL of distilled water and 6. 67 grams of anhydrous sodium carbonate.

The mixture was placed in electric burner with a watch glass on top of the beaker. It was boiled for 10 minutes and stirred using a stirring rod. After which, the hot liquid was decanted into a 50 mL Erlenmeyer flask and 66. 67 mL of distilled water was added and boiled. Again, the liquid was decanted in the beaker into the 50 mL Erlenmeyer flask. The extract obtained was then cooled at room temperature. The extract was transferred from the 50 mL Erlenmeyer flask to a 125 mL separatory funnel that was supported by an iron ring on a ring stand. 16. 67 mL of dichloromethane was added to the separatory funnel.

While holding the stopper tightly into the neck of the funnel, the funnel was inverted so the liquid is no longer in contact with the stopcock. To release any pressure that may have built up inside the funnel, the stopcock was opened. The stopcock was closed and agitated without shaking the mixture vigorously. The contents of the separatory funnel were allowed to settle. Two distinct clear layers were observed. The lower layer was drained into 25 mL Erlenmeyer flask. The aqueous (upper) layer was not included. The steps were repeated from the addition of 16. 7 mL of dicholoromethane to the separatory funnel to the draining of the lower layer. Approximately 1. 67 g of anhydrous sodium sulfate was added to the combined dichloromethane extracts in the 25 mL Erlenmeyer flask. The contents of the flask then swirled. As it was swirled, the anhydrous sodium sulfate absorbed the small amount of water that was dissolved in the dichloromethane. The liquid from the flask into the 25 mL beaker was also decanted. The beaker was placed on a hot plate and petroleum ether was added by means of a dropper until the volume of the solution was between 3 -5 mL.

The solution in the beaker began to be cloudy the beaker was removed from the heat and was cooled at room temperature. As it cooled, crystals of caffeine was formed in the solution. Pharmacological Assay a. Test Animals Twenty-eight (28) male healthy Swiss mice with an age of 3-4 months and an approximate weight of 25 grams were used in the study. The test animals were divided into three groups. The first group the Negative control group consisted of four (4) male Swiss mice, the second group the Positive control group consisted of twelve (12) mice and the last group, and the Sample group consisted of twelve (12) mice.

All the test animals were allowed to adjust themselves to the new environment for one week before the test. The test animals were given pellets and water throughout the duration of the experiment. b. Doses Administration Twenty-eight (28) Swiss mice were weighed at the time of administration of the salicylates. The Sample Control group which consisted of twelve (12) mice, was administered orally with four different doses of the tst drug. An initial dose of 1mg/kg of the salicylates using a tuberculine syringe was given and the dose was increased by computing the increasing doses with the use of 0. logarithmic scales methods. The Positive group which consisted of twelve (12) mice, was induced orally with four different doses of caffeine. The negative group which consisted of four male Swiss mice and distilled water were administered orally. The following parameters were observed: Increase in Motor Coordination- For the estimation of motor coordination the mice were placed on a rota-rod the time until the animals fell of the rod were recorded. Both the normal coordination and the coordination after the administration of treatments were recorded.

Respiratory Rate- The mice were palpated and the time for fifty (50) breaths were measured with a stop watch. The number of seconds and minutes were recorded. Both the respiratory rates before and after the administration of treatments were recorded. Swimming Endurance- The mice were forced to swim in a warm water (25oC) where a detergent had been added to ensure thoroughly wetting the hairs of the mice. The time after total exhaustion were recorded. Statistical Treatment of Data To determine the constituent present in the crude extract, descriptive and qualitative analyses were employed.

The Two-way Analysis of Variance (ANOVA) was used to compare the different stimulant activity tested in the male Swiss Mice. The mean and F-value were computed. The test was applied to determine if there are significant differences in the different doses of the salicylates from the bark of the Weeping Willow (Salix sepulcralis) and if there is no significant difference between the positive control and the salicylates obtained from the Weeping Willow bark (Salix sepulcralIs). The most common measure of central tendency is the mean.

X symbolizes the score on a variable is ? x/N, which is the sum of raw scores divided by N. M= _? x_ N Where: N is the number of doses X is the number of treatments Analysis of Variance(ANOVA) was used to compare the stimulant activity. The data below summarizes the formula for the Analysis of Variance. Table 1. Analysis of Variance Table Sources of Variation| Degrees of Freedom| Sum of Squares| MeanSquares| F-value| Treatment| T-1| SSV| MSB=SSB T-1| F=MSV MSW| Expt’l Error | T(n-1)| SSW| MSW=SSW| | Total| T+E| SST| T(n-1)| |

Legend: T-Number of treatments; E=experimental Errors: N-number of rows Where: SST=? x2 –(? x)2 N SSB=? xc2 – (? x)2 nN SSW=SST – SSB The mean squares for treatments, denoted by MSB, measures the disparity among the sample means. The value of MSB can be obtained from dividing the sum of squares for treatment (SSB) by the degrees of freedom for treatment, which is T – 1. The means square for error, denoted by MSW, measures the variation within the samples. MSW is equal sum of squares for experimental error divided by the degrees of freedom for experimental error, which is T(n – 1).

Collection and Drying Extraction Method by Decoction Crude Extract Isolation of Salicylates Chemical Test Determination of Saicylates * Ferric Chloride Test * Reaction with Acetic Acid * Reaction with hydrochloric Acid Pharmacological Test Stimulant Effect: * Increase in Motor Coordination * Increase in Respiratory Rate * Increase in Swimming Endurance Figure 3 Flowchart of Methods CHAPTER IV RESULTS AND DISCUSSIONS This Chapter presents the results as well as the analysis and interpretation of data gathered in the study. Identification test

Table 2: Results of the Identification Test for Salicylates Test Conducted| Positive Result| Plant Extract Trial| Ferric Chloride TS| intense reddish violet color| Positive| Hydrochloric acid| white crystalline precipitate| Positive| As shown in Table 1 the Weeping Willow Bark extract yielded positive results for the presence of salicylates. The appearance of intense reddish-violet color with Ferric Chloride TS and the formation of white crystalline precipitates with Hydrochloric acid confirmed the presence of salicylates. Pharmacological Screening

Table 3: Concentration and Treatment Means of Motor coordination of the Three Groups of Swiss Mice Doses| T1(seconds)| T2(seconds)| T3(seconds)| Concentration Means| 1mg/kg| 20. 00| 39. 33| 37. 33| 32. 22| 3. 98mg/kg| 21. 00| 43. 33| 43. 00| 35. 78| 15. 85mg/kg| 20. 00| 47. 33| 50. 00| 39. 11| 63. 10mg/kg| 23. 00| 53. 33| 56. 67| 44. 33| Treatment Means| 21. 00| 45. 83| 46. 75| | Legend: T1= Negative Control (); T2=Positive Control (Caffeine);T3=Sample Control (Salicylates obtained from the weeping willow bark) Table 3 shows that the Concentration Means of the three groups of Swiss mice treated with 1mg/kg, 3. 8mg/kg, 15. 85mg/kg, and 63. 10mg/kg are 32. 22, 35. 78, 39. 11 and 44. 33 respectively. The Treatment Means of the three groups of Swiss mice treated are T1 (distilled water), T2 (Caffeine), T3 (Salicylates from Weeping Willow) are 21. 00, 45. 83, and 46. 7. Meanwhile, comparing the Row and Treatment Means of Motor coordination of the three groups of Swiss mice it can be deduced from the results of the Two-way ANOVA displayed in table 4. Table 4: Results of the Two-way ANOVA on the Motor Coordination Source of Variation| Sum of Squares| Degree of Freedom| Mean Score| F-ratio| Prob. Concentration| 238. 849| 3| 79. 616| 5. 560*| 0. 036| Group| 1707. 250| 2| 853. 625| 59. 611**| 0. 000| Error| 85. 919| 6| 14. 320| | | Total| 2032. 018| 11| | | | Legend:*-Significantly different; **-Highly significantly different Table 4 shows the comparison of the stimulant activity between the different concentrations and treatments of the test animals. The analysis of variance reveals that there are significant differences among the means of the concentrations as indicated by the F-ratio of 5. 560 with an associated probability of 0. 36 which is lower than 0. 05 level of probability. Furthermore, implies that there are highly significant differences observed among the means of the treatments with an F-ratio of 59. 611 and a probability of 0. 000 which is lesser than the 0. 01 level of significance. Table 5. Results of the Least Significant Difference (LSD) test for Motor Coordination | Concentration| Mean| | 1mg/kg3. 98mg/kg15. 85mg/kg63. 10mg/kg| 32. 22a35. 78a39. 11ab43. 33b| | Treatments| Mean| | Negative ControlPositive ControlSample Control| 21. 00a45. 83b46. 5b| Note: Means with the same letter superscript are not significantly different Table 5 shows that there is no significant difference between the concentrations raging from 1mg/kg to 15. 85mg/kg. As for 63. 10mg/kg compared with 1mg/kg and 3. 98mg/kg shows a significant difference but shows that there is no significant difference with that of 15. 85mg/kg. The findings implies that the higher the concentration is an increasing motor coordination is observed. It also reveals that the Positive and the Sample groups of Swiss mice have no significant difference in increasing the motor coordination.

Table 6: Concentration and Treatment Means of Respiratory Rate of the Three Groups of Swiss mice Doses| T1(seconds)| T2(seconds)| T3(seconds)| Concentration Means| 1mg/kg| 27. 20| 20. 20| 20. 73| 22. 71| 3. 98mg/kg| 26. 50| 18. 53| 18. 63| 21. 22| 15. 85mg/kg| 26. 30| 18. 33| 17. 67| 20. 77| 63. 10mg/kg| 25. 90| 16. 20| 15. 50| 19. 20| Treatment Means| 26. 48| 18. 32| 18. 13| | Legend: T1= Negative Control (); T2=Positive Control (Caffeine);T3=Sample Control (Salicylates obtained from the weeping willow bark) Table 6 shows that the Concentration Means of the three groups of Swiss mice treated with 1mg/kg, 3. 8mg/kg, 15. 85mg/kg, and 63. 10mg/kg are 22. 71, 21. 22, 20. 77 and 19. 20 respectively. The Treatment Means of the three groups of Swiss mice treated are T1 (distilled water), T2 (Caffeine), T3 (Salicylates from Weeping Willow) are 26. 48, 18. 32, and 18. 13. Comparing the Row and Treatment Means of Respiratory Rate of the three groups of Swiss mice it can be deduced from the results of the Two-way ANOVA displayed in table 7. Table 7: Results of the two-way ANOVA on the Respiratory Rate Source of Variation| Sum of Squares| Degree of Freedom| Mean Score| F-ratio| Prob. Concentration| 18. 793| 3| 6. 264| 8. 724*| 0. 013| Group| 181. 622| 2| 90. 811| 126. 478**| 0. 000| Error| 4. 306| 6| 0. 718| | | Total| 204. 720| 11| | | | Legend:*-Significantly different; **-Highly significantly different Table 7 shows the comparison of the stimulant activity between the different concentrations and treatments of the test animals. The analysis of variance reveals that there are significant differences among the means of the concentrations as indicated by the F-ratio of 8. 724 with an associated probability of 0. 013 which is lower than 0. 5 level of probability. It also implies that there are highly significant differences observed among the means of the treatments with an F-ratio of 126. 478 and a probability of 0. 000 which is lesser than the 0. 01 level of significance. Table 8: Results of the Least Significant Difference (LSD) Test for the Respiratory Rate of the Swiss Mice After the oral Administration of the different Treatments. | Concentration| Mean| | 1mg/kg3. 98mg/kg15. 85mg/kg63. 10mg/kg| 22. 71a21. 22ab20. 77bc19. 20c| | Treatments| Mean| | Negative ControlPositive ControlSample Control| 26. 8a18. 32b18. 13b| Note: Means with the same letter superscript are not significantly different Table 8 shows that there is no significant difference between the concentrations 1mg/kg and 3. 98mg/kg. The concentrations 3. 98mg/kg and 15. 85mg/kg show no significant difference. As for 63. 10mg/kg compared with 1mg/kg and 3. 98mg/kg shows a significant difference but shows that there is no significant difference with that of 15. 85mg/kg. it is interesting to note that the higher the concentration the faster the respiratory rate.

Moreover, it reveals that the Positive and Sample control groups of Swiss mice have no significant difference in the respiratory rate. Table 9: Concentration and Treatment Means of Swimming Endurance of the Three Groups of Swiss Mice Doses| T1(minutes)| T2(minutes)| T3(minutes)| Concentration Means| 1mg/kg| 13. 00| 19. 20| 15. 06| 15. 75| 3. 98mg/kg| 15. 00| 23. 41| 22. 29| 22. 29| 15. 85mg/kg| 14. 06| 26. 15| 26. 16| 26. 16| 63. 10mg/kg| 15. 12| 31. 57| 32. 03| 26. 24| Treatment Means| 14. 30| 25. 08| 23. 89| | Table 9 shows that the Concentration Means of the three groups of Swiss mice treated with 1mg/kg, 3. 8mg/kg, 15. 85mg/kg, and 63. 10mg/kg are 15. 75, 22. 29, 26. 16 and 26. 24 respectively. The Treatment Means of the three groups of Swiss mice treated are T1 (distilled water), T2 (Caffeine), T3 (Salicylates from Weeping Willow) are 14. 30, 25. 08, and 23. 89. Comparing the Row and Treatment Means of Respiratory Rate of the three groups of Swiss mice it can be deduced from the results of the Two-way ANOVA displayed in table 10. Table 10: Results of the Two-way ANOVA on the Time After Exhaustion of the Test Animals Source of Variation| Sum of Squares| Degree of Freedom| Mean Score| F-ratio| Prob. Concentration| 170. 412| 3| 56. 804| 5. 238*| 0. 041| Group| 279. 696| 2| 139. 848| 12. 895**| 0. 007| Error| 65. 067| 6| 10. 845| | | Total| 515. 175| 11| | | | Legend:*-Significantly different ; **-Highly Significantly different| Table 10 shows the comparison of the stimulant activity between the different concentrations and treatments of the test animals. The analysis of variance reveals that there are significant differences among the means of the concentrations as indicated by the F-ratio of 5. 238 with an associated probability of 0. 041 which is lower than 0. 05 level of probability.

In addition, it implies that there are highly significant differences observed among the means of the treatments with an F-ratio of 12. 895 and a probability of 0. 007 which is lesser than the 0. 01 level of significance. Table 11. Results of the Least Significant Difference (LSD) Test for the time after total exhaustion of the Swiss Mice After the oral Administration of the different treatments. | Concentration| Mean| | 1mg/kg3. 98mg/kg15. 85mg/kg63. 10mg/kg| 15. 75a20. 23a22. 12ab26. 24b| | Treatments| Mean| | Negative ControlPositive ControlSample Control| 14. 30a25. 08b23. 9b| Note: Means with the same letter superscript are not significantly different Table 11 shows that there is no significant difference between the concentrations raging from 1mg/kg to 15. 85mg/kg. As for 63. 10mg/kg compared with 1mg/kg and 3. 98mg/kg shows a significant difference but shows that there is no significant difference with that of 15. 85mg/kg. It implies that the higher the concentration the longer the time of exhaustion observed. It also reveals that the Positive and Sample control groups of Swiss mice have no significant difference in increasing the motor coordination.

CHAPTER V SUMMARY, CONCLUSIONS AND RECOMMENDATIONS This chapter summarizes the experimental procedures used in the study. Furthermore, it presents the findings, conclusions, and recommendations derived from the study. This study aimed to determine if the Salicylates obtained from the Weeping Willow Bark (Salix sepulcralis) is effective as a stimulant. Three groups were used to test its stimulant effect: (1) Negative Control Group (distilled water); (2) Positive Control Group (Caffeine); and (3) Sample Control Group (Salicylates obtained from Weeping Willow bark).

An experimental design was used in this study. The procedure has the following phases explicitly: 1) Collection of the bark; 2) Confirmatory test for the presence of salicylates in the Weeping Willow bark extract; 3) Isolation of salicylates and caffeine; and 4) Pharmacological Screening to determine which dose exerts the greatest stimulant effect and whether the salicylates obtained from the Weeping Willow bark exert the same stimulant effect compared to caffeine found in “Kapeng Barako”.

Twenty-eight (28) male Swiss mice were used as specimens. The reagents used to test the presence of salicylates on the crude extract of the Weeping Willow Bark include Ferric Chloride TS, Acetic Acid and Hydrochloric acid. In the pharmacologic screening, twenty-eight (28) healthy male Swiss mice with an age of 3-4 months and an approximate weight of 25 grams were used.

The study has three treatments these were: T1=Distilled water consisting of four (4) mice, T2=Caffeine which consists of twelve (12) mice and T3= salicylates obtained from the Weeping Willow bark which also contained another twelve (12) mice. The data were gathered through observation and the results were statistically analyzed through mean and two-way analysis of variance (ANOVA). Summary of Findings Based from the findings, the crude extracts of the Weeping Willow (Salix sepulcralis) bark showed a positive result on the confirmatory test for the presence of salicylates.

Numerically, the salicylates obtained from the Weeping Willow (Salix sepulcralis) bark showed an increase in motor coordination, increase in respiratory rate and increase in the swimming endurance but there is no significant difference in terms of the stimulant effects compared to the Caffeine. Conclusions Based from the findings, the following conclusions were derived:First, the salicylates obtained from the Weeping Willow bark possess stimulant activity in varying doses- the greater the dose the greater the stimulant activity.

Second, since it Is comparable to the positive control group and there is no significant difference between the two groups. Third, though it is not comparable to the negative control, the salicylates obtained from the Weeping Willow bark (salix sepilcralis) still serves as a good stimulant promoting environmental alertness. Recommendations Based on the findings and considering the limitations of the study, the researchers recommend the following: First, determination of its stimulant ffect should be done in the other parts of the Weeping Willow (Salix sepulcralis) tree; Second, the use of other test animals is also recommended so as to determine the efficacy of the salicylates obtained from the Weeping Willow bark; Third, more parameters should be added in order to further test the stimulant activity of the Weeping Willow (Salix sepulcralis) bark; Fourth, in order to determine the safety level of the salicylates obtained from the Weeping Willow bark for human consumption bio-assay is recommended; Fifth, other methods of isolation and extraction maybe used in order to fully obtain the salicylates responsible for its stimulant effect; Sixth, further studies should be conducted to determine if the mechanism of the salicylates in the inhibition of prostagalindin has the ability to reduce the duration of stage 3 and 4 NREM of sleep thus producing wakefulness; and Finally, further studies should be conducted in determining other medicinal uses of the Weeping Willow (Salix sepulcralis) tree. BIBLIOGRAPHY Books Hardman, J. G. (1996). Goodman and Gillman’s The Pharmacological Basis of Therapeutics. 9th Edition. USA:The McGraw-Hill Companies, Inc. Miller, L. G. et al. (1998). Herbal Medicinal: A Clinician’s Guide. New York:Haworth Press, Inc. Quisumbing, E. (1978). Medicinal Plants of the Philippines. Quezon Avenue, Quezon City, Philippines: JMC Press, Inc. Shneerson, J. M. ( 2001). Sleep Medicine Guide to Sleep and its Disorders. 2nd Ed. USA:Blackwell publishing.

Stringer, J. L. et al. (1996). Basic Concepts in Pharamacology: A Student’s Survival Guide. USA: McGraw-Hill Companies, Inc. Thomas, S. C. (2000). Medicinal Plants: Culture, Utilization, and Phytopharmacology. USA: Technomic Publishing Company, Inc. Unpublished Thesis Mendoza, J. P. et. al. (2007). The Sedative and Hypnotic Properties of the Semi-Purified Alkaloidal Extract From the Leaves of Water Hyacinth(Eichhornia crassipes). Undergraduate Thesis. Mariano Marcos State University. College of Health Sciences. Department of Pharmacy. Batac, Ilocos Norte. Online Information Costa, M. S. (2008). Caffeine improves adult mice performance in the object ecognition task and increases BDNF and TrkB independent on phospho-CREB immunocontent in the hippocampus. http://www. ncbi. nlm. nih. gov/ pubmed/18620014? ordinalpos=13;itool=EntrezSystem2. PEntrez. Pubmed. Pubmed_ResultsPanel. Pubmed_DefaultReportPanel. Pubmed_RVDocSum Ester, C. J and Gabrys, M. K. (2004). Swimming Capacity of Mice after Prolonged Treatment with Psycostimulants. http://www. springerlink. com/content /q64p3851027wg250/ Thomson G. (1998). Stimulant Drugs. http://www. healthline. com/galecontent/ stimulant-drugs Yacoubi, M. E. et. al. (2000). The stimulant effects of caffeine on locomotor behaviour in mice are mediated through its blockade of adenosine A2A receptors. http://www3. interscience. wiley. om/journal/121674827 /abstract Coffee. http://www. ringsurf. com/online/2115stimulant. htmilliliters) Salicin. http://en. wikipedia. org/wiki /Salicin Salicylic acid. http://en. wikipedia. org/wiki/Salicylic_acid The Weeping Willow. http://www. umn. edu/altmed/articles/willow-bark-00028 1. htm Weeping willow. http://www. henriettesherbal. com/electic/kings/salix. htmilliliters) Weeping Willow Bark. http://www. newhope. com/nutrionsciencenews/nsn_ box/su n_01/willowbark. cfn? path=ex APPENDICES APPENDIX A Preparation of Reagents Used in the Confirmatory Test of Salicylates 1. Acetic acid- approximately 300g of Acetic Acid are added to 1000mL of distilled water 2.

Ferric Chloride TS- 25 g Ferric Chloride are added to 1000mL of distilled water 3. Hydrochloric Acid –Approximately 70g of Hydrochloric acid in 1000mL. 4. Weeping Willow Bark Extract About 500g of dried pieces of weeping willow bark were equally divided into three (3) 1000mL beaker. Approximately 1000mL of distilled water was poured in each beaker and then these subjected to decoction for one (1) hour. APPENDIX B Weight of the Test Animals and Dose Computation Table 12. Weight and Dose of the Sample Control Group for the oral administration of Salicylates Obtained from the Weeping Willow (Salix sepulcralis) Bark. Swiss Mice| Initial dose| Weight (grams)| Dose(mL)| 1| 1mg/kg| 23. 9| 0. 0239=0. 02| 2| 1mg/kg| 24. | 0. 0242=0. 02| 3| 1mg/kg| 24. 3| 0. 0243=0. 02| 4| 3. 98mg/kg| 24. 9| 0. 099102=0. 10| 5| 3. 98mg/kg| 23. 9| 0. 095122=0. 10| 6| 3. 98mg/kg| 23. 7| 0. 094326=0. 09| 7| 15. 85mg/kg| 24. 2| 0. 38357=0. 38| 8| 15. 85mg/kg| 24. 8| 0. 39308=0. 39| 9| 15. 85mg/kg| 23. 6| 0. 37406=0. 37| 10| 63. 10mg/kg| 22. 9| 1. 44499=1. 44| 11| 63. 10mg/kg| 23. 9| 1. 50809=1. 51| 12| 63. 10mg/kg| 23. 0| 1. 4513=1. 45| Computation: a. Initial doses using logarithmic scales methods (0. 6 log intervals). Log 1mg/kg + 0. 6= 0. 6 mg/kg antilog = 3. 98mg/kg Log 3. 98mg/kg + 0. 6= 1. 20mg/kg antilog = 15. 85mg/kg Log 15. 85mg/kg + 0. 6= 1. 80mg/kg antilog=63. 10mg/kg b.

Weight(kg) was converted to Weight(g) 1kg=1000g c. Computation of Dose per mice Initial dose:1000g= X: weight of the mice 1mg:1000g=X:23. 9g X=0. 0239mg=0. 02mg *1mg of the salicylates was diluted to 1 mL of distilled water. Table 13. Weight and Dose of the Negative Control Group for the oral administration of Distilled Water. Swiss Mice| Initial dose| Weight (grams)| Dose(mg)| 1| 1mg/kg| 23. 2| 0. 0232=0. 02| 2| 3. 98mg/kg | 24. 1| 0. 095918=0. 10| 3| 15. 85mg/kg| 23. 9| 0. 378815=0. 38| 4| 63. 10mg/kg| 23. 4| 1. 47654=1. 48| Computation: a. Initial doses using logarithmic scales methods (0. 6 log intervals). Log 1mg/kg + 0. 6= 0. 6 mg/kg antilog = 3. 98mg/kg Log 3. 8mg/kg + 0. 6= 1. 20mg/kg antilog = 15. 85mg/kg Log 15. 85mg/kg + 0. 6= 1. 80mg/kg antilog=63. 10mg/kg b. Weight(kg) was converted to Weight(g) 1kg=1000g c. Computation of Dose per mice Initial dose:1000g= X: weight of the mice 1mg:1000g=X:23. 2g X=0. 0232mg=0. 02mg *1mg is equal to 1 mL of distilled water. Table 14. Weight and Dose of the Positive Control Group used for the oral administration of Caffeine. Swiss Mice| Initial dose| Weight (grams)| Dose(mL)| 1| 1mg/kg| 23. 7| 0. 0237=0. 02| 2| 1mg/kg| 23. 6| 0. 0236=0. 02| 3| 1mg/kg| 25. 0| 0. 025=0. 03| 4| 3. 98mg/kg| 24. 8| 0. 098704=0. 10| 5| 3. 98mg/kg| 23. 2| 0. 092336=0. 09| 6| 3. 98mg/kg| 24. | 0. 098306=0. 10| 7| 15. 85mg/kg| 24. 6| 0. 38991=0. 39| 8| 15. 85mg/kg| 23. 7| 0. 375645=0. 38| 9| 15. 85mg/kg| 24. 2| 0. 38357=0. 38| 10| 63. 10mg/kg| 24. 3| 1. 53333=1. 53| 11| 63. 10mg/kg| 23. 9| 1. 50809=1. 51| 12| 63. 10mg/kg| 23. 7| 1. 49547=1. 50| Computation: a. Initial doses using logarithmic scales methods (0. 6 log intervals). Log 1mg/kg + 0. 6= 0. 6 mg/kg antilog = 3. 98mg/kg Log 3. 98mg/kg + 0. 6= 1. 20mg/kg antilog = 15. 85mg/kg Log 15. 85mg/kg + 0. 6= 1. 80mg/kg antilog=63. 10mg/kg b. Weight(kg) was converted to Weight(g) 1kg=1000g c. Computation of Dose per mice Initial dose:1000g= X: weight of the mice 1mg:1000g=X: 23. 7 X=0. 237mg=0. 02mg *1mg of the salicylates was diluted to 1 mL of APPENDIX C Data on the Motor Coordination of the Swiss Mice Table 15. Time until the mice stop running on the rota-rod before the oral administration of the different treatments. Swiss Mice| T1| T2| T3| 1| 19 seconds| 18 seconds| 19 seconds| 2| 23 seconds| 21 seconds| 23 seconds| 3| 21 seconds| 21seconds| 18 seconds| 4| 23 seconds| 23 seconds| 22 seconds| 5| | 19 seconds| 20 seconds| 6| | 20 seconds| 20 seconds| 7| | 18seconds| 23 seconds| 8| | 19 seconds| 18 seconds| 9| | 22 seconds| 19 seconds| 10| | 20 seconds| 23 seconds| 11| | 18 seconds| 21 seconds| 12| | 23 seconds| 20 seconds|

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