Cover created by: Gerrick James M. De Leon University oftheImmaculateConception The OfficialJournalofPharmacy/ChemistryResearch Volume 3March201 gatherers root Online ISSN Online ISSN Print Print ISSN ISSN 2 2345-8569 1656-8362 2244-6540 2244-1263 root gatherers The Official Journal of Pharmacy / Chemistry Research

University of the Immaculate Conception Volume 3 • March 2012 Root Gatherers UIC PHARMACY/CHEMISTRY RESEARCH JOURNAL The Official Journal of Pharmacy / Chemistry Research University of the Immaculate Conception

Volume 3 • March 2012 EDITORIAL BOARD

About the Title Editor in Chief Judee N. Nogodula Root Gatherers is an annual research publication of the Pharmacy/Chemistry Program of the University of the Immaculate Conception. The “Root Gatherers” depicts the primary interest of the Pharmacy/Chemistry in drug discovery and development undertakings utilizing plants. Associate Editor Kathleen G. Bersabal This journal expresses the scholarly endeavors of the Pharmacy & Chemistry faculty and students. Managing Editors Renan P. Limjuco

About the Cover Emma V. Sagarino

The cover shows the roots of the plants in the hands of the pharmacists and chemists as these are utilized as possible sources of drugs for man’s health and wellness.

The panoramic view of hand below depicts the people who are sick and in quest for plant’s cure for their ailment. Editorial Assistants Staff Editor in Chief Neil John M. Galagar Francis Kenneth D. Canono

Students TITLE Copyright © 2012 by the individual authors and the University of the Immaculate Conception. All rights reserved. Gerrick C. De Leon Jose Marlo B. Mañosa Printed in the Philippines. Reproduction without permission from the publisher is strictly prohibited

Published by: University of the Immaculate Conception Editorial Consultants Bonifacio Street, Davao City 8000 Philippines Dr. Ma. Eva C. San Juan Telefax: (+6382) 227-3794 Website: http://www.uic.edu.ph Dr. Adorico M. Aya-ay

Print ISSN 1656-8362 Online ISSN 2345-8569 inflammatory effect of the formulated tablet in albino rats (Canete, PREFACE et al.). On the other hand, trompang elepante (Heliotropium indicum Linn) was formulated into syrup in order to assess its antitussive effect in citric acid cough-induced guinea pigs. This scholarly work was done t is with great pleasure and honor to present the Volume 3 of by Obra et al. One study dealt with pharmacognosy, and microbiology, Root Gatherers, the Official Journal of Pharmacy/Chemistry the kalamansi (Citrofortunella microcarpa) fruit extract was evaluated IResearch. This issue contains the research studies of the Bachelor on the phytochemicals, its antibacterial and mutagenic properties of Science in Pharmacy and Bachelor of Science in Chemistry faculty (Langreo, et al.) and students. This installment encompasses the different fields of pharmacy such as pharmacognosy, pharmacology, pharmacokinetics, Other studies focused on the quality control such as the physical pharmacoeconomics, quality control, toxicology, and microbiology. stability of commercially available lagundi (Vitex negundo) syrup at Likewise, this journal showcases a lot of environmental chemistry issues varied storage temperatures (Aquino, et al.). Likewise, a comparative around us. study on the percentage content of synthetic and herbal supplements of Vitamin C available in Davao City (Alinsasaguin et al.) was also Several studies dealt with the phytochemical screening of different discussed. The physical properties of formulated semisolid dosage plants. One of them was the screening of flavonoid from ripe jackfruit form (ointment, gel and cream) were evaluated by the use of Hauli (Artocarpus heterophyllus) fruit extract and evaluation of the syrup (Ficus septica) leaf extract (Egar, et al.) The cost-effective analysis of formulation. This study was done by Ceniza, et al. On the other hand, the extracted mucilaginous substance of okra (Hibiscus esculentus) and cabbage (Brassica oleracea) and kangkong (Ipomoea aquatica) were corn starch as tablet binders was also explained by Pascua, et al. evaluated on the antioxidant level by Dequito, et al. Few studies represented the environmental chemistry and toxicology such as the With this issue, we would like to express our gratitude to the work of Roces, et al. on the determination of mercury level in three Research and Publication Center headed by Dr. Renan P. Limjuco who selected canned tuna in the Philippines. Likewise, the nitrate contents patiently supervised and mentored and inspired the Root Gatherers have been evaluated from the three tuna products of selected branded Editorial Board. It is our hope that this collection of articles may serve and homemade producers in General Santos City (Gualvez, et al.) and as valuable reference materials for the readers and especially, future pesticide residue of the isolated and formulated ascorbic acid (Vitamin researchers of the University of the Immaculate Conception. C) from overripe mango (Mangifera indica) by Flores, et al.

Rafael, et al., studied the Green Caviar (Caulerpa lentillifera J. Agardh). The formulated tablet was evaluated on the cholesterol Judee N. Nogodula lowering activity using the hypercholesterolemia-induced rabbits. Editor in Chief Likewise, dalanghita (Citrus nobilis) was evaluated on the anti- Root Gatherers Determination of Mercury Level in Three CONTENTS Selected Canned Tuna in the Philippines 70 Adorico M. Aya-ay, Ritzmer Jay C. Roces, Patrizzia Marie R. Cebrero, Karrah M. Go Cost-Effective Analysis of the Extracted Mucilagenous Substance of Okra Cholesterol Lowering Activity of Formulated (Hibiscus esculentus) and Corn Green Caviar (Caulerpa Lentillifera J. Agardh., Starch as Tablet Binders 1 Caulerpaceae) Seaweed Extract Tablet Cherie D. Gasendo, Julmarie Claire C. Pascua, in Hypercholesterolemia-Induced Rabbits 82 Clair J. Supangan, Rhea A. Cañal Ma. Corazon S. Loquellano, Dune Vienis Karen N. Rafael, Melanie Bat-ao, Lovely Edchelle Bermudez, Danmarie Inting Physical Stability of Commercially Available Lagundi (Vitex negundo) Determination of the Nitrite Contents Syrup at Varied Storage Temperatures 27 as a Food Preservative in Three Tuna Florie C. Casalan, Beberlie D. Aquino, Products from the Selected Branded and Jocelyn R. Cotiangco, Windel Jane T. Demavivas Homemade Producers in General Santos City 100 Venchie C. Badong, Lloyd Jason H. Gualvez, Phytochemical, Antibacterial and Judy S. Manliguis Mutagenic Analyses of Commercially Available Kalamansi (Citrofortunella Pesticide Residue Analysis of the Isolated microcarpa) Fruit Extract 35 and Formulated Ascorbic Acid (Vitamin C) Judee N. Nogodula, Nathalie Grace C. Langreo, from Overripe Mango (Mangifera Cleo P. Palaca, Karen Mae B. Padernal indica, Linn. Anacardiceae) 110 Annabelle A. Ribo, Mariz Kay M. Flores, Anti-inflammatory Effect of the Formulated Maribelle C. Hao, Camille F. Selgas Tablet of Dalangita (Citrus nobilis) Peel Extract in Albino rats 46 Flavonoid Screening and Evaluation of Ma. Corazon S. Loquellano, Rochell V. Canete, Formulated Syrup of Ripe Jackfruit Hazel M. Gargoles, Janson Jesson B. Palarisan, (Artocarpus heterophyllus) Fruit Extract 122 Joyce A. Pelaez May Florence D. Bacayo, Sydney Paole Anne G. Ceniza, Sheana Marie M. Montaño, Rose Anne V. Ubalde, Phytochemical Screening and Determination of the Antioxidant Levels of Cabbage A Comparative Study on the Percentage (Brassica Oleracea) and Kangkong Content of Synthetic And Herbal Supplements (Ipomoea Aquatica) Leaf Extracts 60 of Vitamin C Available in Davao City 137 Annabelle C. Ribo, Angelica Mae L. Dequito, Donnah D. Nahial, Chresyl C. Alinsasaguin, Lewey Joy G. Lunio, Kay Maieze B. Mato Jenny R. Edulan, Arie Mae E. Radaza, Liza A. Tomimbang PHARMACY/CHEMISTRY

Physical Properties of Formulated Semisolid Dosage Forms (Ointment, Gel And Cream) of Cost-Effective Analysis of the Extracted Hauli (Ficus septica, Moraceae) Leaf Crude Extract 151 Mucilagenous Substance of Okra Florie C. Casalan, Romma Ninnah O. Egar, (Hibiscus esculentus) and Corn Joseph Pet F. Lusay, Ma. Lady Dianne A. Paccial, Starch as Tablet Binders Michelle A. Peñafiel Cherie D. Gasendo, Julmarie Claire C. Pascua, Clair J. Supangan, Rhea A. Cañal The Antitussive Effect of Formulated Syrup from Trompang Elepante (Heliotropium Indicum Linn.) Leaf Extract in Citric Acid Abstract Cough-induced Guinea Pigs 177 Donnah D. Nahial, Rocel Joy C. Obra, Cost-effective pharmaceutical excipients are always desirable and Jennifer Gwen L. Santiago, Emily R. Vasquez economical because they are coming from the natural resources. Thus, this study aimed to isolate mucilage from okra (Hibiscus esculentus) fruits and to explore its utility as a pharmaceutical excipient such as binding agent. Acetaminophen tablets containing cornstarch as a binding agent was used as standard for comparison with the Acetaminophen tablets containing okra mucilage. On the other hand, okra mucilage and cornstarch were compared as individual binders in Paracetamol tablet formulations. Formulated tablets were prepared following the Paracetamol tablet of USP-NF (1995) protocol. Results revealed no significant difference between the okra mucilage and cornstarch in post-compression analysis of the formulated tablet in terms of the weight variation test, hardness, thickness and friability test. Meanwhile, disintegration time was less than 10 minutes. These test results conformed to the specification and standard of USP/NF. However, moisture content test of both formulated tablets failed to conform to the 10% specification of USP/NF since the result exhibited 2.80% of okra mucilage and 3.00% of cornstarch. Test for solubility revealed that both sources were insoluble in cold water; however, okra mucilage was slightly soluble while cornstarch was readily soluble in hot water. Study found that the price per tablet of the formulation had a P0.000012 difference. Formulated Paracetamol tablet with okra mucilage was cheaper than the cornstarch. Okra mucilage had the same efficacy yet was less expensive as tablet binder than cornstarch.

Keywords: Pharmacoeconomics, excipient, okra mucilage, friability test, disintegration time, weight variation test, table thickness.

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Introduction tablet using the extracted mucilage from okra and cornstarch as tablet binder. Specifically, this aimed to determine the significant difference Excipients, the materials that go into drugs other than the active between okra mucilage and cornstarch in post-compression analysis in ingredient, comprise a market value at nearly $800 million in the U.S., terms of weight variation test, tablet hardness and thickness; to assess if Western Europe, and China. According to estimates from the global the formulated tablets conform to the standard in the USP/NF in terms specialty excipient market for oral solid-dosage form pharmaceuticals, of friability, moisture content and disintegration time; and to identify 2005-2015, the mature markets of the U.S. and Western Europe are the cost factor of okra mucilage and corn starch in terms of market growing at 2.3% and 2.6%, respectively, while China’s emerging price. market is growing at 8%. Wet granulation accounts for more than 90% of tablet production in China and drives the specialty excipient product mix towards cost-competitive, locally produced starches and other Materials and Method products. Chinese drug makers have been slow to adopt the direct- compression method because of the abundance of inexpensive labor This investigation was basically a cost effectiveness study. Cost- (Arnum, 2007). effectiveness analysis is a tool used to aid decisions about which medical care should be offered. It is a method of comparing the cost and Natural excipients show lack of the physical, chemical and toxicity, effectiveness of two or more alternatives. Such comparisons are most easy availability and economic considerations in biologic characteristics useful when one of the alternatives being considered is standard care, of the drug substances and pharmaceutical industry as compared to as this allows the decision maker to consider whether an innovation is their synthetic pharmaceutical ingredients to be used in fabricating better than the status quo. Convenience sampling was used in gathering the counterparts (Nnoli, 2010). However, with the increasing number the plant materials from Bankerohan Public Market, Davao City. of new drug moieties with varying physicochemical and stability properties, there is a growing pressure on formulators to search for new excipients to achieve the desired set of functionalities. Other factors Research Procedure driving the search for new excipients are the growing popularity of the direct-compression process and a demand for an ideal filler-binder that A. Collection and Authentication of Experimental Okra can substitute two or more excipients, tableting machinery’s increasing (Hibiscus esculentus) Immature Pods speed capabilities and low weight variation, high moisture sensitivity (Bansai & Nachaegari, 2004). The immature pods of the Okra plant were collected at Bankerohan Public Market, Davao City. Collection was done With the increase in demand for natural mucilages, it has become in the morning with proper handling. The plants were placed necessary to explore the newer sources of mucilages to meet the in black bag and were preserved for authentication by the industrial demands. Okra, originally an Indian plant, is now grown botanist stationed in Davao City. in many other areas of the world. The stems, leaves and pods of this plant have mucilage. Okra mucilage is water-soluble which produces B. Extraction of Mucilage from Okra fruits slippery, aqueous colloidal suspensions (Deveswaran, et al., 2011). Hence, this study determined the more cost-effective in formulating a Immature pods were selected because they contain more

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mucilage compared to mature fruits. Thirty grams of okra D. determination of the Percentage Yield of Extracted fruits were washed, sliced, soaked and boiled in 400 mL of Mucilage water for fifteen minutes, cooling and passing sufficient cold water through the drug to produce the required volume. Sticky To determine the percentage yield of extracted mucilages swollen mass arising from the fruit of Okra was separated from okra immature pods, the formula below was used: by filtration through muslin cloth. The resulting thick slimy solution was made ready for confirmatory test. % Yield = wt. of extract x 100% wt. of sample C. Confirmatory Test E. Procedure in Making the Paracetamol Tablet (USP- 1. Solubility Test NF, 1995)

One mL of extracted mucilage was allowed to dissolve The entire ingredient was mixed except for the binder in a in 5 mL cold water and another 1 mL in 5 mL hot water. suitable mixer. It was granulated into a damp mass through the addition of a binding solution. Mass was screened by forcing 2. pH of 1% solution through a 10’ mesh screen and was dried in an oven at 120oF. After drying, the mixture was screened through a 14’ mesh The pH of the mucilage solution was measured using screen. It was then lubricated in a suitable blender followed by a digital pH meter by dispersing the black gram mucilage compressing into a tablet machine to create the final tablets. in 25 mL of distilled water. The procedure was also applied to cornstarch.

3. Iodine Solution Test F. Evaluation of Tablet

Three drops of iodine solution were added to 5 mL 1. Post-Compression Analysis extracted mucilage in a test tube. a. Weight Variation (USP/NF, 1995) 4. alcohol Test Each of the ten (10) tablets from the formulation of Five mL of ethyl alcohol was added to 3 mL extracted Paracetamol was weighed; their average then determined. mucilage in a test tube. The weight variation per tablet was computed using the formula: 5. Fehlings Test % wt. variation = actual wt. – average wt. x 100% Three drops of Fehlings solution were added to 5 mL average wt. extracted mucilage in a test tube.

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b. Hardness (USP/NF, 1995) g. Statistical Treatment

The procedure being followed was the United States This study employed t-test in determining the Pharmacopeia/National Formulary (USP/NF) protocol. significant difference between the Paracetamol tablets with The machine used in measuring the hardness of the tablet cornstarch and with okra mucilage as tablet binders. was the Vanderkamp 200. h. Cost-Effective Analysis c. Tablet Thickness A list of cost of the cornstarch, Paracetamol powder, The procedure was done following the hardness methyl paraben sodium, propyl paraben sodium, lactose, step. USP, distilled water, talcum, and magnesium stearate was provided. Price of the plant sample for the preparation of d. Friability Test (Deveswaran, et al., 2011) the extracted mucilages was also determined (Davey & Walley, 2004). Friability test measures the tablet strength. Reading of the sample was done using Friabilator (Erweka Tablet tester Type TA Model NR. 67671). The following is the Results and Discussion formula used for calculations. Table 1 Friability (%) = ((Initial wt. of tablets – Final wt. Test for the Significant Difference between Okra Mucilage of tablets) / Initial wt. of tablets) X 100% and Cornstarch when Analyzed According to Weight Variation

e. Moisture Content Computed Probability Decision Tablets Mean t-value value on Ho The moisture content of the tablet was measured by Okra Mucilage 583.41 AD-4714A. This followed the protocol of The United State 0.342 0.734 Accepted Pharmacopeia National Formulary, 2005). Cornstarch 584.77

f. disintegration time If probability value is less than or equal to 0.05, reject Ho.

One tablet was placed in each of the six tubes of the basket and apparatus was operated using water There was no significant difference in weight variation between maintained at 37 + 2 degrees as the immersion fluid. The formulated Paracetamol tablets of okra mucilage and of cornstarch. protocol being followed was from the USP/NF (1995). This result indicates that okra mucilage is a good binder and it can be used as a substitute for cornstarch in the formulation of tablet. A good binder has the ability to bind sufficient quantity of pharmaceutical

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excipients during tablet compression that prevents chipping, capping Table 3 and other errors in tablet formulation, which results in uniform weight Test for the Significant Difference between Okra Mucilage of the prepared tablets. and Cornstarch when Analyzed According to Tablet Thickness

Computed Probability Decision Tablets Mean Table 2 t-value value on Ho Test for the Significant Difference between Okra Mucilage Okra Mucilage 4.02 and Cornstarch when Analyzed According to Tablet Hardness 1.537 0.133 Accepted Cornstarch 4.07 Computed Probability Decision Tablets Mean t-value value on Ho If probability value is less than or equal to 0.05, reject Ho.

Okra Mucilage 5.95 0.327 0.745 Accepted Cornstarch 5.89 There was no significant difference in tablet thickness between the formulated Paracetamol tablets of okra mucilage and of cornstarch.

If probability value is less than or equal to 0.05, reject Ho. This shows that the okra mucilage is a good binder and can substitute cornstarch in the compression of the tablet since it produces uniform compression force and uniform die to fill which leads to uniform There was no significant difference in tablet hardness between the thickness of the tablets (Gothoscar, et al., 2011). formulated Paracetamol tablets of okra mucilage and of cornstarch. This finding indicates that okra mucilage is again a good binder. A good tablet binder has the ability to bind pharmaceutical ingredients Table 4 hard enough to pass USP/NF specifications and to resist chipping and Formulated Tablets in Terms of Friability abrasion during storage, transportation and handling before usage. It must be noted that according to Bari, et al. (2004), tablet hardness increases with increase concentration of binding agent and vice versa. Tablets Friability Standards

Okra Mucilage 0.50% not more than 0.50% Cornstarch 0.47%

The results of friability test for the formulated Paracetamol tablets of cornstarch and okra mucilage conform to the specification of USP/ NF, which is not more than 0.50%. This indicates that okra mucilage is a good binder and can replace cornstarch in the formulation since it contains less friability values. According to Ghasemi, et al. (2004),

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friability value decreases when there is an increase in concentration of Disintegration time increases with increase in concentration binder due to tablet hardness. In addition, low moisture content and of binder since binding agent can provide sufficient strength of the insufficient binder may also contribute for a tablet to become friable. tablets during its compression (Bari, et al., 2010). Therefore, poor disintegration of tablet may be due in too much binding agent or when it is compressed too hard (Pujeda, et al., 1994). Table 5 Formulated Tablets in Terms of Moisture Content Table 7 Cost Factor of the Cornstarch and Okra Mucilage Tablets Moisture Content Standards

Difference between the Okra Mucilage 2.80% Tablet Binder Price Per Tablet Price of the Two Tablet 10% Cornstarch 3.00% Cornstarch 0.12879376 peso 0.000012 peso The results of the moisture content test for both formulated tablets Okra Mucilage 0.12878176 peso failed to conform to the 10% specification set by USP/ NF since the result was 2.80% for okra mucilage and 3.00% for cornstarch. According to Gothoscar (2011), moisture content increases with binder concentration When it comes to the price per tablet, okra mucilage shows lesser due to the ability of the binder to retain moisture on drying. amount of cost compared to the cornstarch with the difference of 0.000012 peso. In the real manufacturing setting, this indicates that okra mucilage is beneficial as a tablet binder since it can produce tablets Table 6 which pass the aforementioned tests at a lower cost. This can help the Formulated Tablets in Terms of Disintegration Time local manufacturer to lessen their cost during production.

Furthermore, the result of the cost-effective analysis of both Tablets Disintegration Time Standards Paracetamol tablet formulations does not coincide with the four (4) quadrant parameters of the cost-effective analysis by Davey & Walley Okra Mucilage 2.17-3.01 minutes Less than 10 minutes (2004). This is due to the reason that the researchers used the prepared Cornstarch 2.37-2.59 minutes cornstarch from the market.

The results of disintegration time test for both formulated tablets conformed to the specification set by USP/NF, which is less than 10 minutes. This indicates that okra mucilage is a good binder to produce a tablet, which is hard enough, yet can still conform to the disintegration time specification set by USP/NF.

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Table 8 Table 9 Summary of Test and Total Cost per Tablet Solubility Test between Okra Mucilage and Cornstarch

USP/NF Solubility Results Test Cornstarch Okra Mucilage Results Sample Standard Cold water Hot water

1. Weight Variation Okra Mucilage Insoluble Slightly soluble Test 598.1 mg 589.72 mg 600mg Passed Cornstarch Insoluble Readily soluble 2. Tablet Thickness 4.02mm 4.07mm 4mm Passed (USP Standard)

3. Tablet Hardness 57N 56.8N 5N/kg Passed Based on Table 9 data, okra mucilage and cornstarch showed insolubility in cold water. On the other hand, okra mucilage showed 4. Friability Test 0.50% 0.48% nmt 0.50% Passed slight solubility while cornstarch was readily soluble using hot water.

5. Moisture Content 2.8% 3.0% <1% Failed This result indicates that when okra mucilage is to be used as binder, it must be dissolved in hot water first to solubilize and mix 6. Disintegration 2.37-2.59 2.17-3.01 Less than 10 Passed freely with other pharmaceutical ingredients (Ghasemi, et al., 2004). Test mins mins mins.

Based on the different tests performed, both cornstarch and okra Table 10 mucilage passed the 5 tests of USP/NF standard except for moisture Confirmatory Test for Okra Mucilage and Cornstarch content test. Test Sample Using cornstarch as a binder, result showed that three (3) out pH of 1% Iodine test Alcohol test Fehling’s test of five (5) tests conducted exhibited precise similarity to USP/NF solution solution standard Paracetamol tablet. On the other hand, using okra mucilage, Okra Mucilage pH 7 Brown Formation of No change result showed that two out of five test conducted also registered precise solution precipitate in color similarity to aforementioned standard Paracetamol tablet. Cornstarch pH 4-7 Deep blue Formation of Brick red (USP Standard) solution precipitate precipitate

Table 10 shows that the pH of 1% solution of okra mucilage was 7 while the pH of cornstarch (USP standard) was 4-7. Both results conform to the 6-9 pH requirement of USP/NF.

In the iodine solution test for okra mucilage, solution yielded brown color when 3 drops of iodine solution was added. It confirmed

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the absence of starch (Deveswaran, et al., 2011). On the other Percentage Yield Determination hand, cornstarch (USP Standard) yielded deep blue colored solution confirming the presence of starch. In this study the researchers determined percentage of mucilage present in okra fruit (Hibiscus esculentus). Furthermore, the result for okra mucilage indicates that it can be used as a tablet binder for drugs against diabetes mellitus since it From 30 grams of fresh okra fruit sample, 15 grams of mucilage does not contain starch, which is metabolized into glucose in the body was extracted. The formula below was used in the percentage yield leading to increase blood sugar (Olasotan, 2011). determination of mucilage extract.

In the result of the alcohol test, okra mucilage and cornstarch %Yield = weight of extract x 100% formed precipitate due to the presence of mucilage in okra and starch weight of sample in cornstarch. According to Olasotan (2011), okra fruit is abundant in pectins, mucilage and some fats, which precipitate in the presence = 15 grams x100% of alcohol. With such phytochemical contents, okra mucilage can be 30 grams considered as a good remedy for throat irritation caused by coughing because of its soothing and demulcent effect. = 50%

In Fehling’s solution test, no formation of brick red precipitate was Result shows that 30 grams of okra fruit with 400 ml of water observed in the okra mucilage, which indicates absence of reducing contained 50% mucilage. According to Woolfe (2006), okra seed must sugar. On the other hand, cornstarch produces brick red precipitate only contain approximately 0.2-0.3% of mucilage. The result obtained confirming the presence of reducing sugar. was relatively higher due to the presence of water in the extracted mucilage.

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References Olasotan, F.O. (2001). Optimum Population Density for Okra (Abelmoschus esculentus (L) Moench) in a Mixture with Cassava Bari, Abdul, et al. (2010). Formulation of Okra-Natural Mucilage (Manihot esculentus) and its Relevance to Rainy Season-Based as Drug Reducing Agent in Different Sizes of Galvanized Iron Cropping System in South-Western Nigeria. Jr. of Agric. Sc. pipes in Turbulent Water Flowing System. J Applied Science. 136:207-214. 3105-3110. Pujeda L., et al. (1994). Extraction of Mucilagenous Substance Arnum, Van P. (2007). Expanding Opportunities for Specialty From the Fruit of Okra (Hibiscus esculentus) as Plasma Expander. Excipients. Retrieved April 2, 2011, from http://www.phexcom. The United States Pharmacopea – National Formulary. 1995. cn/en/admin/UploadFiles/Technology/Expanding%20Oppor tunities%20for%20Specialty%20Excipients%20.pdf. Woolfe, M.L., et al. (2006). Studies on the mucilages extracted from Okra fruits (Hibiscus esculentus) and baobab leaves (Adansonia Bansal, A.K. & Nachaegari, S.K. (2004). Coprocessed Excipients digitata L.). Retrieved May 10, 2011 from http:/onlinelibrary. for Solid Dosage Forms. Pharmaceutical Technology. 42, 52- wiley.com/dol/10.1002/jsfa.2740280609.references 64.

Davey P. & Walley T. (1995). Pharmacoeconomics: a challenge for Clinical Pharmacologists. Br J Clin, Pharmacol. 40, 199- 202.

Deveswaran, R., et al. (2011). Studies on Hibiscus esculentus Mucilage as a Pharmaceutical Excipient. Vol. 2 (1), 8-17.

Gardner, H.J., et al. (1995). Confidence-Intervals for Cost- Effectiveness Ratios. Medical Decision Making. 15(3), 254-63.

Ghasemi N., et al. (2004). Evaluation of Okra Gum as a Binder in Tablet Dosage Forms. Iranian J Pharm Res. 2, 47.

Gothoscar, A., et al. (2011). Study of Effect of Custard Apple Pulp Powder Asan Excipient on the Properties of Acetaminophen Tablet. World Applied Sciences Journal. 12 (3), 364-371.

Nnoli, I.O. (2010). Film coating potential of Okra Gum Using Paracetamol Tablets as a Model Drug. Asian J Pharm. 4, 130- 134.

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Physical Stability of Commercially Available strength, quality and purity. It can be interpreted as the length of Lagundi (Vitex negundo) Syrup at Varied time under specific conditions and storage that a product will remain Storage Temperatures within the pre-defined limits for all its important characteristics. Each Florie C. Casalan, Beberlie D. Aquino, Jocelyn R. Cotiangco, ingredient, whether therapeutically active or inactive, in a dosage form Windel Jane T. Demavivas can affect stability. Environmental factors such as temperature, light, air (specifically oxygen, carbon dioxide and water vapors) and humidity can affect stability. Similarly, factors such as particle size, pH, the properties Abstract of water and other solvents employed the nature of container and the presence of other chemicals resulting from contamination or from the Quality of crude drug material, plant preparations and finished intentional mixing of different products can influence stability. Stability products depend upon the content variation and stability during storage. testing on typical natural extracts such as flavonoid containing herbal The physical and chemical stability of a pharmaceutical product in the drugs (Drug, in 1998) draft guidance for stability testing. container in which it is to be marketed should be tested under defined storage conditions and the shelf-life should be established. This study Stability considerations, for example selection of excipients, was intended to determine the physical stability of three commercially determination of their level and process development, should therefore available Lagundi (Vitex negundo) syrup products by exposing the be given high priority in the developmental stage of the product. The different brands into various temperatures, level of pH. In order to possible interaction of the drug product with the packaging material in determine the changes, which occurred in the product, positive control which it will be delivered, transported and stored throughout its shelf- syrup was used. The viscosity, density, its color and microbial load of life must also be investigated. The storage conditions recommended the product were also tested. Results show there that there was an by manufacturers on the basis of stability studies should guarantee existing significant difference (p<0.05) on the level of pH at 5oC, 15oC the maintenance of quality, safety and efficacy throughout the shelf- and 25oC. This means that three brands have different pH level and life of a product. The effect on products of the extremely adverse dependent on temperature. However, there was no significant difference climatic conditions in certain countries to which they may be exported on the density and color of the drug at specified temperatures but an calls for special consideration (Pharmaceutical stability, 2000). These existing significant difference on the viscosity of the syrup. Lastly, considerations may help the study to determine the stability of the the microbial growth does not affect with the temperature during the product, especially that today there are many new pharmaceutical storage condition. This study further suggests investigate at the 35oC products that consist of many additives, and this additives has its capacity storage temperature and sensitivity testing of the new commercially to make the product unstable particularly in storage condition. available lagundi product. The primary goal of this study was to find out the physical stability Keywords: Pharmacology, Vitex negundo, physical stability, storage of commercially available lagundi (Vitex negundo) syrup when exposed temperatures to three different temperatures 5oC, 15oC and 25oC. Specifically, this aimed to determine the effect of temperature in terms of pH, density, Determination of stability of herbal drug in formulations is color, viscosity and microbial content. important. The stability is aimed at assuring that the drug/drug product remains within the specifications established to ensure its identity,

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Materials and Method D. Color Determination of the Lagundi syrup

An experimental method, specifically a variation test of group The applicable method used was through comparison of design was used. The commercially available lagundi was used as the the true color of lagundi syrup and the color presents after experimental drug. The temperature was used as a variation. There exposing to the different temperatures. were three commercially available products of lagundi syrup in Davao City. E. determination of Viscosity

The prepared set up of viscometer was placed in a stable Physical Stability Procedure worktable. After taking a fixed volume of lagundi syrup, using an aspirator, aspirate the liquid until it reached the other line A. Measurement of the Desired Storage condition by allowing it to flow under controlled conditions. The time of flow of liquid was recorded. All the drug products were stored at suitable temperatures. Sixty (60) mL of commercially-available lagundi was prepared and exposed to the following temperature for 4 weeks, in a Microbial Assay very cold temperature at 5oC, in cool temperature at 15oC and in control room temperature at 25oC. Each test analysis was Aerobic Plate Count (FDA, 1998) observed and recorded. The decimal dilutions of 10-2, 10-3, 10-4 were prepared by using B. Measurement of the pH separate sterile pipets then 10mL of previous dilution was transferred to 90ml of diluent. Appearance of foam was avoided. All dilutions were To measure the pH of the lagundi sample, a calibrated shaken 25 times in 30cm (1ft) arc within 7s. One (1) mL of each dilution pH meter was used using buffer solution. The calibrated pH was transferred into separate, marked Petri dishes. The dilution was meter was dipped into the lagundi syrup and the results were reshaken for 25 times in 30cm arc within 7s if it stands more than 3 recorded. minutes, before it was pipetted into Petri dish. Twelve (12)- fifteen (15) mL plate count agar (cooled to 45+-1oC) was added to each plate C. determination of Density of the Lagundi Syrup within 15 min. of original dilution. The agar was allowed to solidify. Solidified plates were inverted and incubated promptly for 48+-2h at The mass of the empty graduated cylinder was measured. 35oC. It was noted not to stack plates when pouring the agar or when Twenty (20) mL of lagundi syrup was weighed. The mass of agar is solidified. the syrup was determined by subtracting the mass of empty graduated cylinder.

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Results and Discussion Table 2 Testing the Significant Difference on the Three Brands of Lagundi Syrup by Density at Three Specified Temperatures Table 1 Testing the Significant Difference of the Three Brands of Lagundi Test Variables Density Computed F Tabular F p Syrup by Level of pH on Three Specified Temperatures Brand A 1.21

Test Variables pH Computed F Tabular F p Brand B 1.22 0.4286 4.459 0.6655 Brand C 1.23 Brand A 2.54 Brand B 2.82 3.9431 3.6823 0.0421 Note: *Significant at the p<0.05 level of significance Brand C 2.88

Note: *Significant at the p<0.05 level of significance Results show that there is no existing significant difference (p>0.05) on the density among the three brands of lagundi syrup on three specified temperatures. This indicates that in terms of density, Results show that there is an existing significant difference lagundi syrup, is not affected by temperature. Thus, the three brands (p<0.05) on the level of pH among the three brands of lagundi syrup are comparable in density and are independent of temperature. on three specified temperatures: 5oC, 15oC, and 25oC. This indicates that in terms of level of pH among the three brands of lagundi syrup, is To determine whether temperature in a storage condition affect the affected by temperature. Thus, the three brands have different levels of stability of the commercially available lagundi syrup, two-way Analysis pH and are dependent on temperature. of Variance (ANOVA) was utilized. This was employed to test on two independent variables, viscosity and brand. To determine whether temperature in a storage condition affect the stability of the commercially available lagundi syrup, the two-way Analysis of Variance (ANOVA) was utilized. This was employed to test for the stability of the lagundi syrup on three specified temperatures, on two independent variables, density and brand.

According to Henderson Hasselbalch Equation, the pH applies to all buffer systems formed from single conjugated acid-base pair, regardless of the nature of the salts. It should be pointed out that the buffer of higher concentration of each component will have a much greater capacity for neutralizing added acid or base. Therefore a simple solution of a weak acid like lagundi syrup will exhibit a temperature dependent.

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Table 3 Table 4 Testing the Significant Difference on the Three Brands of Lagundi Testing the Significant Difference on the Three Brands of Lagundi Syrup by Viscosity at Three Specified Temperatures Syrup by Color at Three Specified Temperatures

Test Viscosity Test Variables χ2 df p Variables (milliseconds) Computed F Tabular F p Brand A Brand A 153,731 Brand B 3.50 4 0.479 Brand B 196,737 13.738 5.143 0.0058 Brand C Brand C 230,705 Note: *Significant at the p<0.05 level of significance Note: *Significant at the p<0.05 level of significance

Results show that there is no existing significant difference Results show that there is an existing significant difference (p>0.05) on the color among the three brands of lagundi syrup. This (p<0.05) on the viscosity of the syrup. This indicates that it is affected means that it is not affected by temperature. Thus, the three brands do by temperature. Thus, the three brands differ in viscosity and are not differ in color and are independent of temperature. dependent on temperature.

To determine whether temperature in a storage condition affect Table 5 the stability of the commercially available lagundi syrup, the chi-square Testing the Significant Difference on the Three Brands of Lagundi was utilized. This was employed to test for color and brand. Syrup by Microbial Growth at Three Specified Temperatures

According to Poiseuilles Law, the temperature of liquid increases Test Microbial as its viscosity decreases. In the liquid, the flow between the molecular Variables Growth Computed F Tabular F P predominant, the molecular momentum transfer between molecules, when the liquid is heated the viscosity will reduce. Brand A 266.66 Brand B 203.33 0.8453 3.4633 0.4749 Brand C 366.66

Note: *Significant at the p<0.05 level of significance

Results show that there is no existing significant difference (p>0.05) on the microbial growth among the three brands of lagundi syrup on three specified temperatures: 5oC, 15oC, and 25oC. This indicates that

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in terms of microbial growth temperature does affect the condition. Phytochemical, Antibacterial and Mutagenic Thus, the three brands are comparable in microbial growth and are Analyses of Commercially Available Kalamansi independent of temperature. (Citrofortunella microcarpa) Fruit Extract Judee N. Nogodula, Nathalie Grace C. Langreo, Overall, the temperatures in a storage condition affect the stability Cleo P. Palaca, Karen Mae B. Padernal of the three commercially available lagundi syrup in terms of pH level and viscosity. However, density and microbial growth are not affected by temperature. Abstract

With the emerging diseases and the economic turmoil in the society, people tend to take herbal plants instead of synthetic drugs. Majority of References the Filipinos regularly drink kalamansi juice, in different preparations, for boosting the immune system. This scenario had encouraged the Drug. (1998). (FDA) draft guidance for stability testing. researchers to conduct phytochemical and antimicrobial screening of Kalamansi juice since commercial products have evolved in the market. FDA. (1998). Bacteriological Analytical Manual. CFU. 8th edition. A commercially prepared Kalamansi fruit extract had evaluated the Pharmaceutical stability of drugs in the Philippines. presence of Alkaloid, Tannins and Saponin while Kirby-Bauer disk diffusion method and Minimum inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) and Potency tests were performed for the antimicrobial evaluation. Likewise, Ames test was utilized for the mutagenic analysis. Results reveal that none of the secondary metobolite is detected. A zone of inhibition of 10.63mm ± 0.6844 is measured against Escherichia coli, while 11.94mm ± 1.1480 of Staphylococcus aureus and 10.25 mm ± 0.4945 for the Pseudomonas aeruginosa. Antimicrobial analysis shows that the fruit extract has a lesser strength to have an antibacterial property. Furthermore, MIC value is identified at 31.25 mg/ml (P. aeruginosa), 15.63 mg/ml for E. coli, and 500 mg/ml towards S. aureus. MBC test reveals that at 31.23 mg.mL concentration can effectively inhibit the growth of P. aeruginosa only. Kalamansi extract has a comparable strength with Azithromycin using E. coli and P. aeruginosa. However, it is not equipotent with Oxacillin in S. aureus. Ames test shows that the extract and/or other ingredients of the product have mutagenic property using Salmonella typhimurium TA98.

Keywords: Pharmacognosy, Microbiology, Staphylococcus aureus; Pseudomonas aeruginosa; Escherichia coli; Citrofortunella microcarpa

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Introduction one local industrial company recommended its kalamansi product as the sample for studying its phytochemical contents and antibacterial Some infections or diseases somehow affect the body’s immune property. Hence, the researchers aimed to conduct specifically, the response. In return, the agent may physiologically or genetically change presence of secondary metobolites, the level of antibacterial activity its chemical component thus developing resistance. It may even develop with use of Escherichia coli, Pseudomonas aeruginosa and Staphylococcus resistance from different drug treatments. Globally, people are suffering aureus, to identify the Minimum Inhibitory Concentration (MIC) and from different ailments depending on the degree of severity both in Minimum Bactericidal Concentration (MBC); compare the potency of Industrialized and developing countries. The disease may sometimes the fruit juice with the commercial drugs such as Oxacillin, Tetracycline differ due to environmental condition such as tropical and temperate and Azithromycin and to examine the mutagenic property. zones.

In 2011, Northern Germany had experienced a serious outbreak of Materials and Method foodborne illness caused by Escherichia coli 0104:H4. This characterized by bloody diarrhea, with serious complications such as hemolytic- The study employed experimental design. The concentrated uremic syndrome. According to epidemiological fieldwork, it suggested kalamansi (Citrofortunella microcarpa) fruit extract was recommended that the source was taken from fresh vegetables. (http://en.wikipedia. by a local manufacturer to undergo phytochemical and antimicrobial org/wiki/2011_E._coli_O104:H4_outbreak). In the Philippines, screenings. several outbreak of diseases were experienced by Filipinos. One reported outbreak was typhoid fever happened in Iloilo City however, The test organisms (Escherichia coli, Pseudomonas aeruginosa and a high incidence occurred in Cebu City. Moreover, leptospirosis was Staphylococcus aureus) were procured from Department of Science and increasingly devastating after a flood. Furthermore, with an improper Technology (DOST) XI. Salmonella typhimurium TA98 was procured hygiene, several food or waterborne diseases are developed such as from Philippine National Collection of Microorganism, UP Biotech, Los cholera in Zamboanga City, hepatitis A in Iloilo City, diarrhea from Baños, Laguna. Northern Luzon and Cotabato and food poisoning due to improper food preparation from few areas in Metro Manila (http://en.wikipilipinas. The researchers performed at the Organic Chemistry & org/index.php?title=Top_10_Disease_Outbreaks_in_the_Philippines_ Pharmaceutical Microbiology Laboratory of University of the Immaculate in_2010). Recently, Department of Science and Technology XI (Sunstar Conception, Fr. Selga St., Davao City. This started in June 2011 until Davao, 2012) conducted sampling from street foods in Davao City and January 2012. found out that vended foods were not safe from microbial load in which can lead to stomach pains and even food poisoning.

Generally, a typical Filipino opted to take herbal plants available in the market or even in the backyard and one of these plants is kalamansi. In the market, several commercially available kalamansi juices are sold yet the efficacy and safety have not regulated by the government authority, thus products are labeled “no approved therapeutic claim”. Fortunately,

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Phytochemical Screening thirty seconds. It was allowed to stand and observed for ten minutes. When the froth was greater than three centimeter from the surface of Test for Alkaloid the liquid and persisted after thirty minutes, the sample was considered positive for saponin and was compared with the standard. For plant

An aliquot portion of the extracted plant material that is equivalent with pour frothing effect, a little of 5% Na2CO3 solution was added to 20 grams was taken from the stock plant extract and evaporated to the extract. The formation of a stable and dense froth was used to a syrupy consistency over a steam bath. Five (5) milliliters of 2M to indicate the presence of the free fatty acid. Capillary method was hydrochloric acid was added heated with stirring for about five minutes performed following the protocol of Guevara, et al. (2005). and allowed to cool. About 0.5 g sodium chloride was then added, stirred and filtered, and the residue was washed with enough 2M HCL to bring Bacteriological Assay the filtrate to a volume of five milliliters. One milliliter of the filtrate was taken and tested with two or three drops of Dragendorff’s reagent. The susceptibility test was performed with the use of Escherichia Another one milliliter of the filtrate was taken and tested with two to coli, Pseudomonas aeruginosa and Staphylococcus aureus. These were three drops of Mayer’s reagent. A positive result was indicated by an inoculated to Mueller Hinton Broth, respectively and incubated at 35oC. orange precipitated with Dragendorff’s reagent and a white precipitate After incubation period, each culture tube was adjusted its turbidity with the Mayer’s test. The confirmatory test was performed following to 0.5 McFarland standard. The extract was dispensed onto the sterile the protocol of Guevara, et al. (2005). paper disk and subjected for air-drying. Mueller-Hinton Agar plate was swabbed with the standardized inoculum. The dried sterile paper Test for Tannin disks were placed into the seeded agar plate. Plates were incubated for 24 hours at 35oC. The zone of inhibition was measured in millimeter Plant material of the plant extract was taken and evaporated to (mm). incipient dryness over a water bath. The residue was extracted with 20 milliliters of hot distilled water. Five (5) drops of 10% NaCl was added, Minimum inhibitory concentration employed the two-fold broth filtered and divided the filtrate into three test tubes. One portion was dilution method. Likewise the Minimum Bactericidal Concentration taken as control and an aqueous solution of tannic acid was used as was performed based upon the result of the MIC. The protocol was reference standard. The gelatin and ferric chloride tests were employed followed from Guevara, et al. (2005). in the study following the protocol of Guevara, et al. (2005) Stock solution of antibiotics such as Oxacillin and Azithromycin Test for Saponin were prepared for potency test. The test organisms were inoculated to tubes with Mueller-Hinton Broth, respectively and incubated at Frost test was first performed with use of one gram of bark of 35oC. Then, these were adjusted following the 0.5 McFarland Standard Entada phoseobides or locally known as “gogo”. It was soaked in 10 (Guevarra, et al., 2005). ml of 80% ethyl alcohol. It was allowed to stand for 30 minutes. The plant material was taken in a separate test tube. One (1) milliliter of Ames test was performed by the use of Salmonella typhimurium the “gogo” extract was used as the standard. Ten milliliter of water TA98. This employed the direct mutagen assay only. Top agar was was added to each test tube. It was covered and shaken vigorously for prepared by mixing NaCl and agar to distilled water. The solution was

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mixed thoroughly and divided in 100 aliquots. Afterwards, sterilization Table 1 was done for twenty (20) minutes at 121oC (Engelhardt, et al., 2004). Phytochemical Screening of Kalamansi Fruit Extract The L-histidine and biotin were added to the melted agar before use. Thirty milliliter (30 ml) of base agar was poured unto the plate Trial Alkaloid Tannin Saponin o while maintained in a temperature of 45 C. Top agar, with Salmonella Plant P Test C Test typhimurium TA98 and plant extract, were poured into the base agar. FeCl3 Cap Frth D M D M The plates were incubated at 37oC for 24-48 hours (Engelhart, et al., 2004). 1 ------

Kalamansi Fruit Extract 2 ------Results and Discussion 3 ------The result of the phytochemical screening for Alkaloid was not detected in kalamansi fruit extract in the preliminary screening and this Legend : was further confirmed using the confirmatory test for alkaloid having P Test = preliminary test cap = capillary method - = negative C Test = confirmatory Frth = froth testFeCl3 = Ferric chloride test a no definite orange and yellow turbidity in Dragendorff’s and Mayer’s D = Dragendorff’s reagent + = positive tests. The presence of the saponin was not detected in kalamansi fruit M = Mayer’s reagent extract. The persistent foam was not formed upon the addition of FeCl3 = Ferric chloride test aqueous solution with agitation. Furthermore, this plant extract was also negative in the capillary method for the saponins in which the level of extract was more than that of water. This is due to the ability Table 1 shows that the fruit extract of kalamansi does not contain of the saponin to lower the surface tension of water. Thus, presence of the alkaloid, tannin and saponin. This means the commercial product saponins in kalamansi Extract was not observed. has no secondary metabolites. This probably eliminated during the process or the added ingredient may destroy the components of the There was no formation of precipitate in gelatin test which gave said secondary metabolites. a negative result for tannic acid and polyphenolic compound and was further confirmed in the ferric chloride test of kalamansi Extract indicating no Blue-black color and Brownish green for hydrolysable and Descriptive Analysis of Susceptibility test condensed tannins respectively. In the susceptibility test of kalamansi towards Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa, the researchers performed in-vitro analyses using the agar method. The tests were performed under standardized condition so that the results are reproducible. In this manner, the researchers can be guided on the potency of the kalamansi extract to inhibit the growth of test microorganism. Overall results are shown preceding tables of this chapter.

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Table 2 Table 3 Overall Data on the Antimicrobial Property of Kalamansi Overall Data on the Antimicrobial Property of Kalamansi Fruit Extract Towards Escherichia coli Extract Towards Staphylococcus aureus

Trial Zones of inhibition, mm Treatment Trial Number Descriptive Treatment Trial Number Descriptive Grand equivalent Grand equivalent S.D. S.D. 1 2 3 Mean 1 2 3 Mean

Positive 9.14 21.52 21.2 17.28 7.0552 Active Positive 17.3 15.66 13.38 15.45 1.9671 Active

Negative 7.62 7.62 7.62 7.62 0.0000 Inactive Negative 7.62 7.62 7.62 7.62 0.0000 Inactive

Extract 11.1 10.91 9.85 10.63 0.6844 Partially Active Extract 11.33 13.27 11.23 11.94 1.1480 Partially Active

In-vitro susceptibility analysis showed that the Citrofortunella Results of the in-vitro analysis showed that the kalamansi extract microcarpa extract partially inhibits the growth of the test organism was potent to partially inhibit the growth of the test organism by 11.94 by 10.63 ± 0.6844. This revealed that it possesses an antibacterial ± 1.1480 mm. However, the positive control showed the highest property towards E. coli. On the other hand, the positive control drug inhibition, which obtained a clear zone of about 15.45 ± 1.9671. obtained a clear zone of inhibition of 17.28 ± 7.0552, which is higher compared to the other treatments (kalamansi extract and Negative This clearly indicates that the positive control drug is more effective control). This demonstrates the efficacy of the positive control drug to as an antibacterial agent than the test drug (kalamansi extract). On the inhibit the growth of the test microorganism. other hand, the negative control showed no clear zone of inhibition on S. aureus.

Even if the phytochemical screening was limited to alkaloid, saponin and tannin testing, there might be other reason to partially inhibit the growth like the presence of Vitamin C and citric acid. The latter may render its antimicrobial property.

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Table 4 Table 5 Overall Data on the Antimicrobial Property of Kalamansi Independent t-test Analysis Between the Mean Zones Fruit Extract Towards Pseudomonas aeruginosa of Inhibition of Positive Control and Kalamansi Extract Against TestMicroorganisms Zones of inhibition, mm Treatment Trial Number Descriptive Test t- P- Grand equivalent Treatment Grand Mean Decision S.D. Microorganism value value 1 2 3 Mean Positive 17.28 ± 7.0552 Positive 14.71 13.69 14.32 14.24 0.5162 Active control Not E. coli 1.63 0.179 significant Negative 7.62 7.62 7.62 7.62 0.0000 Inactive Plant 10.63 ± 0.6844 extract

Extract 10.26 9.75 10.74 10.25 0.4945 Partially Active Positive 15.45 ± 1.9671 control Not S. aureus 2.661 0.056 significant Table 4 reveals that Pseudomonas aeruginosa was found to be susceptible Plant on both the positive control (tetracycline) and test drug (kalamansi fruit extract 11.94 ± 1.1480 extract). Results show that these moderately obtain a clear zone of inhibition of about 14.24 ± 0.5162 and 10.25 ± 0.4945, respectively. This proved Positive 14.24 ± 0.5162 the efficacy of the kalamansi fruit extract as an antibacterial agent against control P. aeruginosa 9.677 .0006 Significant Pseudomonas aeruginosa. This may mean that the extract has an antibacterial Plant 10.25 ± 0.4945 property towards Gram-negative P. aeruginosa. extract

Inferential Analysis of Susceptibility test Statistical analyses showed that there is no significant difference (p>0.05) on the mean zones of inhibition between the positive control Descriptive analysis of the susceptibility test proved that both and kalamansi fruit extract against the Escherichia coli and Staphylococcus positive control and kalamansi extract inhibit the growth of Escherichia aureus. This signifies that both test drugs have similar efficacy in coli, Staphylococcus aureus and Pseudomonas aeruginosa. To determine inhibiting the growth of the test organismsin, which confirmed the which of these treatments showed the highest inhibition against the test potential antibacterial characteristic of the kalamansi extract. microorganisms, independent t-test analysis was conducted and results were interpreted based on the p-value obtained in the calculation. Note On the other hand, results showed that there is a significant however, that the negative control was not included in the statistical difference (p<0.05) between the mean zones of inhibition of positive process since it did not show any clear zones of inhibition in the in vitro and negative controls against the P. aeruginosa. This means that positive susceptibility test. Overall result of the statistical computation is shown control is more effective antibacterial agent than the kalamansi fruit in Table 5. extract.

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Minimum Inhibitory Concentration Test Results showed that the MIC of kalamansi extract against Pseudomonas aeruginosa was 31.25 mg/mL, 15.63 mg/mL against E. The minimal inhibitory concentration aimed to describe the coli and 500 mg/ml towards S. aureus. The plant extract is more effective minimum concentration of plant material that showed inhibition on to inhibit the growth of E. coli than the rest of the test organisms because the test organism. This test will likewise assess the resistance of the test of the least concentration that exhibited. Meanwhile, the extract is microorganism on the assigned treatment. Results of the test are shown effective towards S. aureus at higher concentration. It shows that S. below: aureus has a stronger capability to resist the extract. This means killing or inhibiting the bacterial growth needs higher concentration compared to the other bacteria. Table 6 Minimum Inhibitory Concentration of Kalamansi Extract Using the Selected Bacterial Strains Table 7 Minimum Bactericidal Concentration of Kalamansi Fruit Plant Pseudomonas Escherichia Staphyloccocus Extract against Selected Bacterial Strains Concentration aureus mg/mL aeruginosa coli 500 - - - Plant Staphyloccocus Concentration Pseudomonas Escherichia aeruginosa coli aureus 250 - - + mg/mL 125 + + + 500 - - - 62.5 - - + 250 + + + 31.25 - - + 125 + + + 15.63 + - + 62.5 + + + 7.81 + + + 31.25 + + + 3.91 + + + 15.63 + + + 1.95 + + + 7.81 + + + 0 + + + 3.91 + + + 1.95 + + + Negative + + + Control Antibiotic + + + standard Positive + + + Contol Legend: (-): There is no indication bacterial growth Legend: (+): There is indication of bacterial growth Negative Control: Water + presence of growth; - absence of growth Positive Control: Tetracycline for E. coli & P. aeruginosa Oxacillin for S. aureus

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Table 7 depicts the minimum bactericidal concentration exhibited In the potency test towards Escherichia coli, the researchers by Kalamansi fruit extract. This is done based from the MIC value if it obtained an equation of the line equivalent to: y = 11.463x + 6.6366. can accurately inhibit the growth at a specific concentration. It found In this case, the y-axis represents the mean zones of inhibition of out that at 31.23 mg/mL, the extract has clearly inhibited P. aeruginosa. the 9 replicate tests while the x-axis represents the concentration in However, S. aureus is inhibited but on higher concentration of 500 mg/ milligrams (mg) of the antibiotic. Using this formula, the researchers m. This result did not conform to the MIC value. Meanwhile, the extract obtained an equivalent concentration of the plant extract against the with a concentration of 15.63 mg/mL is not effective against E. coli. This positive control of 0.52 mg. See figure below: may mean that the fruit extract has a specific concentration to inhibit or kill microorganisms. This may relate to the different characteristics of 12.00 the organisms such as its virulence factors and genetic composition. 11.00

10.00 9.00 Potency Test 8.00 y = 11.463x + 6.6366 2 7.00 R = 0.7742 In order to determine the equivalent concentration of kalamansi

Zones of inhibition, mm 6.00 extract against the positive control towards growth inhibition of the 0.15 0.2 0.25 0.3 0.35 0.4 0.45 three microorganisms tested in this study, the researchers used the Concentration, mg equation of the line in testing the results. Results of the potency test are Legend: Observable Effect Expected Effect presented in the succeeding tables below. Figure 1. Graphical Presentation of the Potency Test of Kalamansi Extract against the Positive Control towards Growth Inhibition of Escherichia coli Table 8 Potency Test of Kalamansi Juice against the Positive Control Towards Growth Inhibition of Escherichia coli

Concentrations of Grand mean zones Standard Deviation Antibiotic, mg/mL of inhibition (mm) 0.2 8.90 0.7437 0.24 10.05 0.5188 0.28 9.35 0.8544 0.32 9.85 0.6889 0.36 10.66 0.5336 0.4 11.64 0.9243 Plant Extract 13.21 0.5802 Equivalent concentration, 0.57 mg

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Table 9 Table 10 Potency Test of Kalamansi Extract against the Positive Control Potency Test of Kalamansi Extract against the Positive Control Towards Inhibition of Pseudomonas aeruginosa Towards Growth Inhibition of Staphylococcus aureus

Concentrations of Grand mean zones Standard Deviation Concentrations of Antibiotic, mg Grand mean zones Standard Deviation of inhibition (mm) Antibiotic, mg of inhibition (mm) 0.20 8.01 0.3701 0.20 7.62 0.0000 0.24 8.21 0.1790 0.24 7.62 0.0000 0.28 8.76 0.3154 0.28 7.62 0.0000 0.32 9.14 0.4292 0.32 7.62 0.0000 0.36 9.88 0.2386 0.36 7.62 0.0000 0.40 10.11 0.3010 0.40 7.62 0.0000 Plant Extract 13.42 0.5621 Plant Extract 7.62 0.4945 Equivalent concentration, 0.69 10.25 mg Equivalent concentration, Cannot be computed: None of the antibiotic concentrate mg has shown inhibition against the Staphylococcus aureus

Figure 2. Graphical Presentation of the Potency Test of Kalamansi Extract against the Positive Control towards The results in Table 9 showing the activity of the extract against Growth Inhibition of Pseudomonas aeruginosa S. aureus cannot be computed since the antibiotic concentration does not show any inhibition against Staphylococcus aureus. There 10.50 10.00 was no growth of the bacterium using the plant extract even at the 9.50 highest concentration. This may mean that S. aureus is resistant or the 9.00 kalamansi juice has no phytochemical component that considered as an 8.50 8.00 antimicrobial against the said bacterium. That may relate to the result 7.50 y = 11.328 + 5.62 2 of phytochemical screening that has no presence of either alkaloid, 7.00 R = 0.9793 6.50 saponin or tannin. Zones of inhibition, mm 6.00 0.15 0.2 0.25 0.3 0.35 0.4 0.45 Concentration, mg

Legend: Observable Effect Expected Effect Results showed that the equation of the line obtained was y = 11.328x + 5.62. Calculation showed that equivalent concentration of the plant extract against the positive control of 0.69 mg. The table shows the point where the observable effect meets the expected effect. Based on the result, 20 mcg of the extract is equipotent with 0.69 mg of the positive control (Tetracycline).

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Table 11 References Mutagenicity (Ames)Test of Kalamansi Extract Using Salmonella typhimurium TA98 Guevara, B., et al. (2005). A Guide to Plant Screening: Phytochemical and Biological (p68-98). Manila: Research Number of Revertant Colonies Center for the Natural Sciences – University of Santo Tomas Test Trial 1 Trial 2 Trial 3 Mean Descriptive Variable r1 R2 R3 r1 R2 R3 r1 R2 R3 Engelhart, G., Jacob, E. & Jäckh, R. (2004). Assessment of the Performance of the Ames II TM assay: a Collaborative Study with POSITIVE CONTROL 559 648 743 478 412 715 653 532 764 1101.2 Mutagenic 19 Coded Compounds. Aniara Mutation Research. (Benzene) NEGATIVE E. Coli. Retrieved: 12-20-10. http://wikipedia.org/wiki/ CONTROL Not Escherichia_coli (Distilled 0 0 0 0 0 0 0 0 0 0 mutagenic water) Ross, Z.M., O’Gara, E.A., Hill, D.J., Sleightholme, H.V. & PLAIN 0 0 0 0 0 0 0 0 0 0 Not Maslin, D.J. (2001). Antimicrobial Activity Herbal extracts on AGAR mutagenic Staphylococcus aureus and Propionibacterium acnes. TEST PLATE (Plant 358 302 283 234 259 289 350 140 256 358 Mutagenic Street Food. 2012. Sun Star News. Issued on January 25, 2012. extract) http://www.sunstar.com.ph/davao/local-news/2012/01/25/ davao-street-food-contaminated-study-202360 Table 11 shows the result of mutagenicity test on S. typhimurium TA98. It reveals that both the positive control (benzene) and plant extract are the only ones trigger the growth of the bacterium. This means that S. typhimurium TA98 has the ability to revert its protein content due to the presence of the chemical tested. However, since the kalamansi is not a pure extract, it may have been the other components that triggered the mutagenic activity of the bacterium.

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Anti-inflammatory Effect of the Formulated Introduction Tablet of Dalangita (Citrus nobilis) Peel Extract in Albino rats In the progress of synthetic chemistry and biotechnology, hundreds Ma. Corazon S. Loquellano, Rochell V. Canete, Hazel M. Gargoles, of plant species are recognized as having therapeutic value. Many of Janson Jesson B. Palarisan, Joyce A. Pelaez those are commonly used to treat and prevent specific ailments and disease. The production and processing of medicinal plant offers the possibility of fundamentally upgrading the lives and well-being of Abstract people (Mills, 2007). Plants with its medicinal property alleviate the suffering of many people, in as much as they are use to treat many Plants with its medicinal property alleviate the suffering of many diseases like, rheumatoid arthritis. people, in as much as they are used to treat many diseases like, rheumatoid arthritis. With this study, it aimed to determine the inhibitory effect For most people, arthritis pain and inflammation is a normal process of the formulated tablet from dalangita peel extract (Citrus nobilis) in as the body ages. In fact, most people over the age of 50 show some the paw edema of carrageenan-induced albino rat. Experimentation signs of this disease. According to WHO (2011) that forty percent (40%) utilized three Swiss mice administered with the dose level of 2000 mg/ of people over seventy (70) years suffer from osteoarthritis, a type of kg body weight following the Acute Single Toxicity Test. Likewise, six rheumatoid arthritis of the knee. It was found out that eighty percent healthy male albino rats were with these doses starting at 10mg/kg (80%) suffering from this diseased have some degree of limitation of to 630.96 mg/kg body weight for Approximate Effective Dose (AED). movement. Rheumatoid arthritis has reached worldwide epidemic Formulated tablet for bioassay contained 40 mg/kg dose of dalangita proportions. In 1990, an estimated of 1.7 billion people have fractures peel extract. Bioassay results show that the percentage inhibition in because of these diseases. It is one of the most common autoimmune formulated ointment is 32%, Etoricoxib (positive control) is 45.32% diseases affecting one percent of global population. The greatest incidence while negative control (placebo) is 0.73%. This reveals that at 0.05% of theses diseases were found in countries such as Brazil, Chile, China, level of significance, the test group animal treated with formulated tablet Pakistan, India, Indonesia, Malaysia, Mexico, Thailand, and Philippines. show decrease in paw edema which only developed for 4 hours after In the developed countries, the United States, European countries, Japan, treatment and similar result was observed with Etoricoxib. However, New Zealand and Australia all have high rates (WHO, 2011). test animals that received placebo drug manifested a minimal decreased in paw edema. Analysis of variance and Tukey’s multiple comparison In the Philippines, The Philippine Rheumatology Association test proved that both positive control and dalangita peel extract did not warned that the number of arthritis sufferers and those with soft tissue differ significantly with each other. This implies that the peel extract rheumatism, which now stands at 2.6 million adult Filipinos, will rise as tablet has an equal effect with Etoricoxib in lowering the formation of the average life span of Filipinos increases. The incidence of arthritis in paw edema on the albino rats. the country is high primarily because of our diet here in the Philippines. Filipinos are fond of eating “laman-loob” (internal organs of animals) Keywords: Pharmacology, dalangita, anti-inflammatory, Approximate used as ingredients in native recipes like adobo and paksiw. Among Effective Dose, acute single toxicity, Eterocoxib, Philippines the internal organs, a separate study conducted reveal that liver has the highest amount of uric acid which if deposited can cause arthritis (Sahelian, 2007).

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With these findings, the major challenge for researchers is to B. Preparation of Plant Extract provide alternative cheap treatment which is effective by conducting research studies involving the efficacy of Dalangita (Citrus nobilis) as The freshly collected Dalangita peels were washed an anti-inflammatory agent which contains a flavonoid called nobiletin thoroughly with tap water to remove dirt and to be freed from that is effective in treating inflammation and alleviating pain (Morton, contamination. The peels were removed from the pericarp 1987). and finely cut into pieces. About 200 grams of finely cut peel was placed in Erlenmeyer flask; soaked in 95% ethyl alcohol This study aimed to determine the percent inhibition of the for seventy-two (72) hours. After macerating, the peels were formulated tablet from Dalangita (Citrus nobilis) peel extract on paw concentrated using the rotary evaporator. The concentration edema formation of the carrageenan induce paw edema of albino of the extract was computed using a formula. rats. Specifically, it sought to determine the acute single dose toxicity of Dalangita peel extract in Swiss mice; to identify the Approximate Concentration of extract = weight of extract in, grams Effective Dose (AED) as an anti-inflammatory agent in Albino Rats; to Volume of extract in, mL statistically determine the significant difference in the percent inhibition of edema formation among the test animals treated with the formulated C. Extract Determination of OECD 423 Acute Single tablet from the Citrus nobilis peel extract, Etoricoxib tablet (positive Dose Toxicity control) and placebo tablet (negative control). In determination of toxicity test, twelve (12) male Swiss mice were used. Each animal at the commencement of its Methodology dosing was between 8-12 weeks old. The Swiss mice underwent fasting with food for 3 to 4 hours but not with water. The Swiss In this study the researchers employed the pretest-posttest through mice were marked to permit individual identification and were experimental research design. In this design, the paw edemas of the kept in their cages for 5 days prior to the administration of the three groups of test animals were observed prior to and after treatment Dalangita peel extract. Each computed dose was administered administration. through oral gavage. The initial doses used were 5mg/kg, 50mg/ kg, 300mg/kg, 2000mg/kg and 5000mg/kg body weight.

Procedure The test animals were observed individually after dosing at least once during the first 30 minutes periodically during A. Collection of Plant Material the first 24 hours with special attention given during the first 4 hours and daily thereafter for a total of 14 days. The volume of Dalangita (Citrus nobilis) peels were collected from the test substance administered to the animals was calculated Basak, Sultan Kudarat. The collected peel of the plant material using the dose level, respective weight and concentration of were brought to the University of the Immaculate Conception the test substance (http//iccvam.niehs.nih.com/ suppdocs/ laboratory for its preparation into crude extract. feddocs/OECD/OECD_GLA423.pdf).

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D. Determination of Approximate Effective Dose agent, Magnesium Stearate and Talcum powder were added to the dried granules and compressed into tablets (Ansel, 2005). Four (4) male albino rats were used since they do not undergo estrogenic cycle and weighed individually and placed G. Anti-Inflammatory Determination to designated cages. The albino rats were given different doses 1st dose 0.001 ml, 2nd dose 0.002 ml, 3rd dose 0.008 ml, Eighteen (18) randomly selected healthy male albino rats and 4th dose 0.026 ml to determine which among the dose weighing 150-400 grams were assigned into three groups, level exhibits an anti-inflammatory activity by inhibiting the each group was composed of six test animals. Group one was carageenan induced edema of the hind paw. treated with formulated tablet of Citrus nobilis extract, the experimental drug, while the other two groups underwent E. Standard Preparation of Phlogistic Agent for Edema treatment with Etoricoxib as positive control, and placebo as induction negative control. Prior to administration, the baseline paw thickness was measured using Vernier caliper. An hour after A 0.5 grams of Carrageenan was dissolved in 50 milliliter induction of edema into albino rats, the formulated tablet of of distilled water, to make the one percent (1%) Carrageenan Dalangita, etoricoxib tablet, and placebo drug respectively, solution. After injecting 0.5 mL of 1% carrageenan to the plantar were immediately administered (Sevilla, 1990). surface of the hind paw of the albino rats, the consequent inflammation was measured by using Vernier caliper. H. Biological Assay

F. Tablet Formulation Paw thickness Inhibition

Wet Granulation Six healthy albino rats were weighed individually and placed in designated cages. The albino rats were divided The Citrus nobilis peel extract was formulated into a tablet into three pair. The initial paw thickness of each albino rat using wet granulation method. The ingredients added were was measured using Vernier Caliper for every pair. About the peel extract, starch, Methyl Paraben Sodium, and Propyl 0.5 milliliters of one percent carrageenan was injected Paraben Sodium. subcutaneously on the hind paw of each albino rat to induce inflammation. The powder mixture was converted into a wet mass using gelatin as the binding solution. Then, the wet mass underwent The paw thickness of each albino rat was measured after granulation process. the granules were screened using sieve induction of inflammation. Then the dose of the Dalangita #80. These granules were dried in an oven that is thermostically peel tablet was administered orally. The paw thickness of each controlled at 75°F. Albino rat was again determined, recorded and computed its percentage inhibition (Sevilla, 1990). This procedure was also After drying, the granules were screened again for performed using Etoricoxib and placebo treatments. uniformity of the powder using sieve #20. Then the lubricating

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Results and Discussions Determination of Approximate Effective Dose (AED)

Determination of Single Dose Acute Toxicity In this experiment, the test for the determination of approximate effective dose of the plant material starts at the administration of 10 mg/ In this experiment, the researchers used three test animals kg body weight of the plant material. The succeeding four doses were: administered with the dose level of 2000 mg/kg body weight. The 39.81 mg/kg, 158.49 mg/kg and 630.96 mg/kg which were calculated selection of a high dose was based on the fact that the plant material is at the 0.6 logarithmic intervals. Results of the test are presented in known to be non-toxic and will likely not cause mortality to male swiss Table 2. mice. Results of this test are presented in Table 1.

Table 2 Table 1 Determination of Dalangita Peel Crude Extract as Acute Single Dose Toxicity Test of the Dalangita Anti-inflammatory Agent of the Carrageenan (Citrus nobilis) Peel Extract to Swiss Mice Induced Paw among Albino Rats

Administered amounts Dose Weight of Paw thickness in millimeter (mm) Dose Level Mice Number Results Albino Milligram (mg) Volume (ml) Level, Test Results Rats # mg/kg animals, kg Baseline Induced Treated Diff.* M1 44 0.037 Survived 1 10 0.070 2.73 5.21 3.80 1.41 + 2000 mg/kg M2 44 0.037 Survived 2 39.81 0.081 2.47 6.59 3.67 2.92 + M3 34 0.029 Survived 3 158.49 0.060 3.03 5.80 3.64 2.16 + 4 630.96 0.049 2.60 4.58 2.98 1.60 + As the researchers observed the individual test animal for the first 30 minutes to 24 hours, results show that no swiss mice have Legend: *Diff. = Induced – treated; (+): decrease on the paw edema of test animals experienced severe pain or enduring signs of severe distress. During the observation period, no changes in the skin, eyes and discoloration were observed. Tremors, convulsions, salivation and diarrhea were likewise Results of the AED determination proved the potentiality of the not detected. This was then interpreted that the plant material is not plant material as an anti-inflammatory agent after the tested animals toxic at the dose levels of 2000 mg/kg body weight. showed decreased of paw edema four hours after administration of dalangita peel extract. It further suggested that the plant materials can effectively carry out anti-inflammatory action towards carrageenan paw edema on albino rats at dose level ranging from 10-630.96 mg/kg.

From these results, the researchers have chosen the 39.81 mg/kg dose for tablet formulation.

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Determination of Bioassay Table 3 Bioassay Determination of the Anti-inflammatory Agents A. Anti-inflammatory Activity toward Carageenan Induced Paw Edema Albino Rats

Paw Thickness in millimeter (mm) The comparative bioassay technique wherein the anti-inflammatory Group Albino Rat effect of formulated tablet of dalangita peel extract that contained the Number Baseline Induced Treated Difference dose level of 39.81 mg/kg was compared to the positive control group 1 3.22 7.92 5.20 2.72 which received the etoricoxib tablet and negative control group which 2 4.01 7.96 5.64 2.32 3 3.91 7.93 5.29 2.64 receive the placebo drug. Each group was composed of six carrageenan Experimental induced paw edema albino rats. Results of the test are presented in 4 3.89 7.90 5.16 2.74 table 3. 5 3.25 7.30 4.97 2.33 6 3.23 7.28 4.90 2.38 As depicted in the results, it has been showed that both groups Mean 2.52 of test animals that received the treatment and positive control S.D. 0.1992 group which received the etoricoxib tablet had significant decreased 1 3.90 7.15 4.01 3.14 inflammation by 2.52 mm ± 0.1992 and 3.42 ± 0.2810, respectively. 2 3.79 7.65 3.86 3.79 From these two test drugs, it was observed that etoricoxib caused the Positive 3 4.05 7.89 4.16 3.73 greatest decreased in the inflammation of the carrageenan induced paw Control 4 3.95 7.18 4.03 3.15 edema among albino rats than those that received the experimental 5 3.98 7.54 4.22 3.32 drug (formulated tablet). On the other hand, the group of test animals 6 4.02 7.68 4.28 3.40 that received the placebo drug recorded that least decreased in paw Mean 3.42 edema which was only at 0.05 mm ± 0.013. S.D. 0.2810 1 4.08 7.92 7.86 0.06 Therefore, the positive control (Etoricoxib) has the highest anti- 2 3.94 7.31 7.28 0.03 inflammatory activity, second was the experimental drug (formulated Negative 3 3.85 7.22 7.16 0.06 tablet) and the negative control (placebo tablet) showed the least anti- Control 4 3.67 7.01 6.97 0.04 inflammatory activity. 5 3.98 7.42 7.36 0.06 6 3.75 7.13 7.07 0.06 Mean 0.05 S.D. 0.0133

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B. Comparison of the Percent Inhibition Table 5 One-Way Analysis of Variance on the Percentage Inhibition of the To clearly depict which of the three test drugs is a good anti Three Test Drugs on the Carragenan Induced Edema in Albino Rats inflammatory agent, the researchers expressed the results from table 4 in terms of percentage of inhibition (IE%) as shown in Table 4. Source of Df Sum of Mean Calculated Tabular F Decision Variation Squares Square F Calculation of percentage inhibition was based from the difference of induce and treated paw edema test animals divided by the edema after Column 2 6296.37 3148.19 758.46 3.68 Reject Ho* induction with carrageenan multiplied by 100. Error 15 62.26 4.15 Total 17

*Calculation was performed at the 0.05 level of significance Table 4 Percentage Inhibition of the Edema Formation of the Test Drugs Toward Carageenan Induced Paw Edema Albino Rats As shown in Table 5, results of the calculation show that the calculated F is 758.46, which is higher than the tabular F value of Percentage Inhibition Group Mean S.D. 3.68, therefore the null hypothesis stating that: There is no significant 1 2 3 4 5 6 difference on the Percentage inhibition of the three test drugs on Experimental 34.34 29.15 29.24 34.68 31.91 32.69 32.00 2.4034 the carrageenan induced paw edema on albino rats is rejected. This Positive 43.92 49.54 47.28 44.63 42.48 44.27 45.32 2.5787 signifies that the three different test drugs have different capability in Negative 0.75 0.41 0.83 0.71 0.81 0.84 0.73 0.1622 lowering the paw edema of test animals. To further assess, which of the three drugs is best in inhibiting the paw edema of test animals, Tukey’s Results in Table 4 show that positive control can greatly inhibit multiple comparison test was used and presented on the next page. the paw edema of test animals by 45.32% ± 2.5787 as compared with the experimental drug which can inhibit the paw edema of test animals by about 32.00% ± 2.4034 while the placebo drug can only inhibit at Table 6 0.73% ± 0.1622. Tukey’s Multiple Comparison Test of Mean Percentage Inhibition of Three Test Drugs on the Paw Edema of Albino Rats

Statistical Evaluation Mean Comparison Mean Critical Decision* Difference value

To determine if there is a significant difference existing among Etoricoxib Formulated tablet 13.32 2.52 Reject Ho the three test drugs in terms of their percentage inhibition capacity Etoricoxib Placebo tablet 44.59 2.52 Reject Ho on the paw edema of carrageenan induced albino rats, One-Way Formulated tablet Placebo tablet 31.27 2.52 Reject Ho Analysis of Variance and Tukey’s Multiple Comparison test were used *Calculation was performed at the 0.05 level of significance for the statistical evaluation. Results are presented in Table 5 and 6, respectively. Results of the test presented in Table 6 proved that Etoricoxib can significantly inhibit the paw edema of test animals which is higher

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by 13.32% as compared to the formulated tablet. On the other hand, REFERENCES it further shows that test animals that received Etoricoxib has higher percentage inhibition by 44.59% compared with those receiving the Ansel, et al. (2005). Wet granulation, Ansel’s pharmaceutical placebo drug. In this case, it can be established that the Etoricoxib dosage forms and drug delivery systems (8th ed., pp240-242) (positive control drug) is the most effective among the three test drugs used in this study. Mills, S. Y. (2007).The Dictionary of Modern Herbalism (3rd ed., pp189-198) On the other hand, results of the test also suggested that though the capacity of formulated tablet to inhibit the paw edema of test animals is Morton. (1987). Complement Therapeutic Medicine (Volume 2, not significantly similar to Etoricoxib tablet. It can however significantly pp76-84) inhibit the paw edema of test animals by 31.27% compared with the group that received the placebo drug. This established the capacity of the OECD 423. (2001). Retrieved December 2001, from http// formulated tablet to significantly lower the paw edema of carrageenan iccvam.niehs.nih.com/ suppdocs/feddocs/OECD/OECD_ induced edema of albino rats thus proving its potentiality to be used as GLA423.pdf). an anti-inflammatory test drug. Ohigashi, et al. (2007). Retrieved. July 19 2009 from http:// www.ncbi.nlm.nih.gov/nobiletin/medscape/12212096

Quisumbing, E. (1978). Medicinal plants of the Philippines: citrus nobilis (pp445-456). Katha publishing Co., Inc. Philippines.

Rheumatoid arthritis. (n. d.) Retrieved July 02 2009 from http:// en.wikipedia.org/wiki/Rheumatoid_arthritis

Sahelian. (2007). Retrieved July 19 2009 from http://www.ncbi. nlm.nih.gov/nobiletin/medscape/12211342

Sevilla, E. (1990). Anti-Inflammatory Determination, Acta Manilana Volume 38, UST press for the Research Center for the Natural Sciences. Philippines: Manila

World Health Organization. 2011. http//iccvam.niehs.nih. com/ suppdocs/feddocs/OECD/OECD_GLA423.pdf

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Phytochemical Screening and Determination Introduction of the Antioxidant Levels of Cabbage (Brassica Oleracea) and Kangkong Free radicals and other reactive oxygen species are derived either (Ipomoea Aquatica) Leaf Extracts from normal essential metabolic processes in the human body or from Annabelle A. Ribo, Angelica Mae L. Dequito, Lewey Joy G. Lunio, external sources such as exposure to X-rays, ozone, cigarette smoking, Kay Maieze B. Mato air pollutants and industrial chemicals (Montez, 2005). On the other hand, the highly significant correlation between consumption of fats and oils and death rates from leukemia and malignant neoplasia of the Abstract breast, ovaries and rectum among persons 55 years may be a reflection of greater lipid peroxidation. Studies on the atherosclerosis reveal the The highly significant correlation between consumption of fats probability that the disease may be due to free radical reactions involving and oils and death rates from leukemia and malignant neoplasia of diet- derived lipids in the arterial wall and serum to yield peroxidases the breast, ovaries and rectum among persons of 55 years may be a and other substances. These compounds induce endothelial cell injury reflection of greater lipid peroxidation. To reduce the possible effect of and produce changes in the arterial walls (Bagchi, et al., 1998). lipid peroxidation, this study focused on the phytochemical screening and determination of the antioxidant level of cabbage (Brassica Members of the Cruciferous family seem to be rich in antioxidants oleracea) and kangkong (Ipomoea aquatica) leaf extract. The two plants and are in the first line of defense against cancer. Cabbage (Brassica were selected based on their antioxidant property. The phytochemical oleracea) is one of the vegetables that are highly promoted for prevention tests include the Wagner’s test for Alkaloid; Wilstater Cyanidin Bate- of cancer. Kangkong (Ipomoea aquatica) is also rich in antioxidants that Smith and Shinoda Tests for Flavonoid; and Froth test for Saponin. On ward off free radicals from the body, which causes diseases such as cancer. the other hand, the reducing power method for antioxidant level was Cabbage and kangkong leaves yield flavanols-Quercetin, Kampferol, performed using UV-VIS spectrophotometer and Vitamin C was used as Myricetin and Isorhamnetin and flavones - Apigenin and Luteolin. The the standard. Results reveal that cabbage and kangkong leaf extracts flavonoids have aroused considerable interest recently because of their are both positive for flavonoid and saponin however no detection for potential beneficial effects on human health-they have been reported to alkaloid. The level of Vitamin C in kangkong is 0.34 ± 0.0525 while have antiviral, anti-allergic, antiplatelet, anti-inflammatory, antitumor cabbage is at 0.30 ± 0.0453. Independent t-test analysis shows that and antioxidant activities (Buhler, et al., 2011). there is no significant difference (p>0.5) between the level of vitamin C and the two plant extracts. This indicates that both plant extracts People are very particular and compulsive when it comes to showed potential amount of Vitamin C and may exhibit antioxidant symptoms but which limit them because of financial incompatibilities property. and bad effect of synthetic drugs. Thus, this study aimed to conduct a phytochemical screening and to measure the antioxidant levels of Keywords: Pharmacognosy, phytochemical screening, Brassica cabbage and kangkong leaf extracts. Specifically, this is to assess the oleracea, Ipomoea aquatica, reducing power method, antioxidant level, antioxidant level of the two leaf extracts using reducing power method; Philippines to determine if there is a significant difference between the plant leaf extract of cabbage and kangkong and the coated ascorbic acid as antioxidant.

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Methodology Determination of Antioxidant level

The researchers used the true experimental design for this study. The kangkong and cabbage extracts were subjected for antioxidant The plants were collected at Ulas and Baguio districts in Calinan, level determination. One (1) mL of plant extract was added with 2.5 Davao City, respectively. These were submitted to the Botanist for mL phosphate buffer and 2.5 mL potassium ferricyanide. This was identification and authentication. The entire experimentation was incubated at 50°C for 20 minutes afterwards 2.5 mL Trichloroacetic conducted at the Dispensing Laboratory of the University of the acid (100g/L) was added and centrifuged at 800 rpm/ 10 mm. After Immaculate Conception. centrifugation, 2.5 ml of supernatant liquid was taken. Distilled water (2.5 mL) and ferric chloride (0.5 mL) were added. The samples were Extraction and Phytochemical analyzed using the UV-VIS spectrophotometer having the absorbance Screening of the Plant Materials of 700 nm.

Two hundred (200) g of fresh kangkong and cabbage leaves were Determination of Vitamin C Level placed in an Erlenmeyer flask. Ninety five (95%) ethyl alcohol was added separately to the kangkong and cabbage leaves. The plants were The determination of the Vitamin C content of the two plant submerged and covered with funnel so as to minimize the evaporation materials made use of the conversion of the acquired absorbance of of the solvent. The flask was placed over a hot water bath and it was both plant extract multiplied to their standard concentration over the refluxed for an hour. Pouring of mixture was done while still hot absorbance standard. Results in the level of Vitamin C were based from through the Bucher funnel lined with filter paper. The extracts were the overall data for the determination of antioxidant property of the concentrated and volume was measured. These were kept in a tightly two plant extracts. It was computed through the formula: stoppered container for the preceeding tests. Concentration of Unknown = Concentration of Standard An equivalent of 10 g plant material was evaporated into incipient (Absorbance of unknown) dryness over water bath. It was cooled to room temperature. The Absorbance Standard residue was defatted by treating with hexane or petroleum ether until it was almost colorless. The residue with 10ml of 80% of ethyl alcohol was taken up. On the other hand, the filtrate was filtered into four test Statistical Treatment tubes and one portion was taken as control. The t-test was used to determine if there is an existing significant Phytochemical screening of plant materials include the Wagner’s difference between the concentrations of Vitamin C on the two plant test for alkaloids, Froth test for saponins, and Bate-Smith and Metcalf extracts. test, Wilstater “Cyanidin” test, and Shinoda test for flavonoids. These tests were followed by the protocol set by Guevara, et al. (2005).

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Results and Discussions Table 2 Results of the Phytochemical Screening of Phytochemical analysis of the cabbage plant extract reveals the Kangkong (Ipomea aquatica) Plant Extract presence of the following secondary metabolites: flavonoids and saponins. The presence of flavonoids and saponins are manifestations Component Methods Results that this plant species can be a very good source of medicine. However, Alkaloid Wagner’s Reagent Test – cabbage plant extract did not show positive screening on alkaloids. An Wilstater Cyanidin Test + article from Tara green of Nutrition data supported that per cup of Flavonoid Bate-Smith Method + cruciferous plants like cabbage contains high levels of Vitamin C (54%) Shinoda Test + and it also provides a rich source of phytonutrient antioxidants like Saponins Froth Test ++ quercitin and flavonols. A saponin content known as sarsasapogenin is mostly found in crucefirous plants like cabbage (WHFoods, 2012). The results of the phytochemical screening of the ethanol extract on the leaves of kangkong show the presence of flavonoids and saponins. This indicates that the leaves are the most suitable for a good Table 1 supply of flavonoids and saponins. The presence of this metabolite may Results of the Phytochemical Screening of also indicate that the plant is a potential antioxidant and may exhibit Cabbage (Brassica oleracea) Plant Extract different pharmacological properties. On the other hand, alkaloids are not detected in kangkong extract. A report released from the Journal Component Methods Results of Applied Science in 2007 indicated that green leafy vegetable like Alkaloid Wagner’s Reagent Test – kangkong has high concentrations of flavonoid- flavonol and quercetin. Wilstater Cyanidin Test + Saponin content is due to its belongingness to the Cruciferous family. Flavonoid Bate-Smith Method + Shinoda Test + Accordingly, the key benefits of saponins include: Improvement of Saponins Froth Test ++ vitality, enhances the immune system and strengthens the function of the heart. While flavonoids can improve blood circulation and stamina, LEGEND : (++) intense change in color (+) slight change of color may provide protection against oxidative and free-radical damage and (-) no appearance of color change lowers fat build-up in blood stream (Montez, 2005).

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Table 3 Table 4 Concentration of Vitamin C on the Two Plant Extracts Testing the Significant Difference between the Using Reducing Power Method Level of Vitamin C of the two Plant Extracts

Level of Vitamin C, µg/mL (ppm) Trial Plant Mean S.D. Degrees of t value P value Decision* Cabbage Kangkong Extract freedom 1 0.35 0.39 Cabbage 0.30 0.0453 2 0.29 0.34 4 1.0215 0.3648 Not Significant 3 0.26 0.29 Kangkong 0.34 0.0525 Mean 0.30 0.34 *Statistical computation was done at the 0.05 level of significance S.D. 0.0453 0.0525

As such, this study also aimed to determine the level of Vitamin Results of the statistical computation showed that there is no C on the collected plant extract to determine its potential capacity as significant difference (p>0.05) between the level of Vitamin C of the antioxidant agent. Table 3 shows the summary of the level of Vitamin two studied plant extract. This clearly indicates that both cabbage and C on these extracts. Results reveal that kangkong extract has higher kangkong plant extracts showed potential amount of Vitamin C and Vitamin C concentration than cabbage. It was shown that the level of may exhibit antioxidant property. Likewise, demonstrated the fact that Vitamin C in kangkong was 0.34 ± 0.0525 while cabbage was 0.30 both plant materials are good sources of these vitamins. An article from ± 0.0453. To determine if there is an existing significant difference Tara green of Nutrition data supported that per cup of cruciferous plants between the concentrations of Vitamin C on the two plant extracts, like cabbage contains high levels of Vitamin C (54%) while kangkong independent t-Test was utilized and summary of report is shown in contains 325 mg of flavonoid per cup. Table 4.

Vitamin C is a potent water-soluble antioxidant in humans. The antioxidant effects have been demonstrated in many experiments in vitro (Padayatty, et al., 2011).

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REFERENCES Sahelian, R. M.D. (2011). Antioxidant Validity for Diseases.

Acworth, I.N., et al. (1997). Reactive Oxygen Species: The Stuart, R. Herbal plants. (2011). Constituents, Properties and Handbook of Oxidative Cultivation.

Metabolism. Massachusetts: ESA Inc., p1-1 to 4-4. Yamamoto, K. (1988). Free Radical-Mediated Damage of Blood and its Inhibition by Antioxidants, 34: 507-512. Bagchi, K., et al. (1998). Free Radicals and antioxidants in health and disease, vol. 4, issue 2, 1998, p 350-360. http://whfoods.org/genpage.php?dbid=19&tname=foodspice

Buhler, D.R., et al. (1980). Antioxidant Activities of Flavonoids: http://www.herbs2000.com/h_menu/alkaloids.htm Department of Environmental and Molecular Toxicology, Oregon State University, 2011 http://www.herbs2000.com/h_menu/saponins.htm

Guevara, B., et al. (2005). A Guidebook to Plant Screening http://www.nal.usda.gov/fnic/foodcomp Phytochemical and Biological. Manila: UST Publishing House. http://www.phytochemicals.info/abstracts/saponins-antioxidant. Harley, E., et al. (2011). Anticancer Attributes of Desert Plants: php A Review, American University of Health Sciences. http://runetwork.org/html/en/articles/5670/preview_to_print. Karlsson, J. (1997). Introduction to Nutraology and Radical html Formation. In: Antioxidants And Exercise. Illinois: Human Kinetics Press. p 1-143. http://healthrecipes.com/author/cindy-pa-ac

Montez, H. (2005). Metabolic Processes in the Human Body: Review on Considerations in Metabolism. p 345-387.

Padayatty, S.J., et al. (2011). Vitamin C: A Concentration- Function Approach Yields Pharmacology and Therapeutic Discoveries, vol. 2:78-88, 201.

Roger, C.R. (1998). The Nutritional Incidence of Flavanoids: some Physiologic and Metabolic Considerations, p 725-804, Experentia.

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DETERMINATION OF MERCURY LEVEL IN THREE methylmercury (EPA, 2010). SELECTED CANNED TUNA IN THE PHILIPPINES Adorico M. Aya-ay, Ritzmer Jay C. Roces, One of the most widely eaten fishes worldwide is tuna. It is rich Patrizzia Marie R. Cebrero, Karrah M. Go in protein, low in fat and calories and is an excellent source of the essential omega-3 fatty acids, which help to lower blood pressure and cholesterol. While tuna provides great source of protein and inexpensive, Abstract the mercury content of canned tuna can pose great health risk for those who consume them often. One of the most widely eaten fishes worldwide is tuna. While it is rich in protein, low in fat and calories and is an excellent source of At the University of Nevada Las Vegas where researchers analyzed the essential omega-3 fatty acids which help to lower blood pressure more than 300 samples of canned tuna for mercury content, more than and cholesterol, the mercury content of canned tuna can pose great half (55%) of the samples contained levels of mercury higher than the health risk for those who consume them often. In this study, mercury EPA standard of 0.5 parts per million (ppm) for safe fish consumption. was determined after digestion by the standard methods of Association Five percent of the samples contained more than 1.0 ppm of mercury, of Official Analytical Chemists. Mercury contents of canned tuna were double the EPA standard. All three of the largest tuna brands had determined by cold vapor atomic absorption spectrophotometer. The samples with mercury levels in excess of the recommendations (Mercury metal content, expressed in µg/g wet weight for mercury varied with In Canned Tuna, 2010). an average of <0.0003, 0.0421 and <0.0003 in canned tuna A, B and C, respectively. The values were comparable and in the range of the In the United States, tuna has affected one-in-six children born literature values. The results of this study indicate that canned tuna fish every year due to exposure to high mercury levels. These newborn manufactured and sold in Philippines have concentrations below the babies were at risk for learning disabilities, motor skill impairment and standards of WHO for this toxic metal. short-term memory loss. One government analysis shows that 630,000 children each year are exposed to potentially unsafe mercury levels in Keywords: Canned Tuna, Mercury the womb (The Mercury Story, 2005).

In the Philippines, where per capita fish consumption for the Introduction Filipino adult was reported as 75 g d-1 in 1998 (UNEP 2003) and 69 g d-1 in 2003 (FNRI 2003a), there are two clear risk concerns. First, Fish and fish products are lean, low-calorie source of protein. They adults and children who eat greater-than-average amounts of fish may contain healthy fats that will reduce cholesterol and improve health get excessive methylmercury exposure even if the average mercury (Stibich,2009). However, nearly all fish and shellfish contain traces level in their fish is relatively modest and secondly people who prefer of mercury. Some fish may contain methylmercury or other harmful to eat predatory, mercury-accumulating species can easily be exposed chemicals at sufficiently high levels to be a concern. They contain high to excessive methylmercury doses if they eat those fish often. amounts of mercury - enough to damage a fetus or newborn. The FDA recommends that women who are pregnant, or may become pregnant Since Philippines is situated in a region of greatly abundant tuna and nursing mothers avoid fish that may contain unsafe levels of resources and many tuna industries are flourishing in different part of

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the country, it is very important to examine this fish species in terms of was based on the three largest fish canning corporation its mercury content. operating in the Philippines. The researchers considered the expiration date of the product, batch or kit lot number, and This study aimed to determine the mercury (Hg) level of three coded the samples with brand A, brand B, and brand C, to selected canned tuna in Philippines. Specifically, this study sought to avoid mismatching and also to protect the name of the product identify the highest level of mercury from the three canned tuna; to being tested. assess if the level of mercury is within allowable limit as based on CODEX and EU Standards and evaluate if there is a significant difference in the B. Validation of Selected Canned Tuna Products mercury (Hg) level among three selected canned tuna. The selected canned tuna products were submitted to the Bureau of Fisheries and Aquatic Resources XI. They validated Materials and Methods and approved the said sample for digestion following mercury determination. Research Design C. digestion of Validated Canned Tuna Products This study is descriptive in nature. The researchers were studying the level of mercury in the selected canned tuna products in the Philippine The researchers performed the wet digestion with reflux market today, which was based on the allowable limit of mercury in distillation set-up, wherein approximately 2.5 grams of wet canned products as provided by the World Health Organization. sample was weighed into the digestion flask. Then, 3-5 pieces

of boiling chips, 10-20 mg of vanadium pentoxide (V205) and

The researchers selected three canned tuna and coded the samples 20 ml concentrated sulfuric acid (H2SO4) – Nitric acid (HNO3) as brand A, brand B, and brand C. (1+1) were added to the digestion flask. The flask was then connected to the condenser immediately, swirled and mixed Research Instruments and heated gently for six minutes. Heating was continued with a strong boil for about 20 minutes. When the solution was clear, For the determination of mercury in the selected canned tuna, the flask was washed with 15 ml of de-ionized water through

the researchers made use of the standard AOAC Official Method of the condenser and a few drops of H2O2, and then another 15 digestion and determination of mercury using Atomic Absorption ml of de-ionized water was washed through the condenser. Spectrophotometer. The digested sample was transferred in 100 ml volumetric flask and diluted to volume with de-ionized water. Research Procedure The researchers performed a blank first and three replicates A. Collection and Selection of Canned Tuna Products in each brand of selected canned tuna samples.

The researchers selected three canned tuna products at the grocery store of one of the malls in Davao City. The selection

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D. determination of the Level of Mercury Using Atomic means that though canned tuna contain trace amounts of mercury, Absorption Spectrophotometer Hg content was established to be at tolerable level. Several agencies around the world have established reference dose for mercury. The U.S. The level of mercury in selected canned tuna products EPA’s 2001 Reference Dose (RfD) for methylmercury was calculated was analyzed and determined at the Bureau of Fisheries and to protect the developing nervous system. It was estimated that the Aquatic Resources (BFAR). They made use of the Atomic daily dose of mercury that can be consumed safely over a lifetime Absorption Spectrophotometer. was at 0.1 micrograms per kilogram of body weight per day (FDA, 2010). While in 2003 the World Health Organization (WHO) revised After the sample has been digested, it was analyzed using its recommendation for safe intake levels for mercury in food to 0.4 the AAS with corresponding standard specific for mercury micrograms per kilogram of body weight per week. This means that analysis. daily intake of methylmercury at a level of 0.1 and 0.40 micrograms per kilogram body weight per day for extended periods up to a lifetime presents no risk of adverse health outcomes in even the most sensitive Results and Discussions human populations (pregnant women, developing fetuses, and young children). Atomic absorption spectrophotometry with vapor generation accessory was the standard method used in the analysis of mercury In the study conducted by EPA (2001), recent human biological in three selected canned tuna. To ensure accuracy of testing, standard monitoring shows that most people have blood mercury levels below solutions were used as calibrators of the instrument. Results of the a level (5.8 µg/L or parts per billion of whole blood) associated with analyses were compared with the standard specification for compliance possible health effects. Consumption of fish (e.g. tuna) with higher with CODEX and European Union standards set at ≥ 1.00 ppm Hg. To methylmercury levels can lead to elevated levels of mercury in the obtain high precision of analyses, six trials analyses were conducted bloodstream of unborn babies and young children and may harm their for each type of tuna. Overall results of the test are shown in the table developing nervous system. These disabilities have been documented below. in ability to use language, to process information, and in visual/motor integration. Table 1 Overall Data of Mercury Level in Three Selected Canned Tuna However, concentration of Hg in studied canned products is far below these concentrations. Results showed that the range concentration Type of Mercury level in parts per million, (µg/g) of Hg in the studied product was <0.0003 - 0.0421 ppm. The highest tuna 1 2 3 4 5 6 concentration was shown in Canned B but its level is lower than the Can A <0.0003 <0.0003 <0.0003 <0.0003 <0.0003 <0.0003 tolerable limit set by health authorities. As such, the products tested in Can B 0.0356 <0.0003 0.0904 0.0356 <0.0003 0.0904 tested research are safe for human consumption. See Table 2 for the Can C <0.0003 <0.0003 <0.0003 <0.0003 <0.0003 <0.0003 summary of results.

Results showed that none of the three selected tuna exceeded to the safe level set by CODEX and EU standard (≥ 1.00 ppm Hg). This

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Table 2 Table 3 Summary of Mercury Level in Three Selected Canned Tuna One-Way Analysis of Variance on the level of Mercury on the Three Selected Canned Tuna Type of tuna Mean S.D. Tolerable limit Description

Can A <0.0003 0.0000 ≥1.000 Conform Source of SS Df MS F p Decision Variation Can B 0.0421 0.0406 ≥1.000 Conform Between 0.003 2 0.002 3.180 3.180 Accept Can C <0.0003 0.0000 ≥1.000 Conform Within 0.003 6 0.001 Ho To clearly depict the level of mercury in these products, graphical Total 0.007 8 presentation is shown below. Note that the red grid line indicates the maximum allowable limit of mercury in fish products. Results of the statistical test showed that there is no significant difference (p>0.05) on the level of Hg in three selected tuna. This means that the level of Hg among the three products were statistically similar which conforms to the standard set by various government organization. This result further suggests that the products tested in this research will likely not yield adverse health outcomes when consumed based on the FDA reference standard (FDA, 2011).

Figure 1. Graphical Presentation of Hg Analyses in Selected Canned Tuna Inferential Statistics

To statistically evaluate the level of Hg in three selected tuna, One-Way Analysis of Variance (ANOVA) was conducted to test the null hypothesis stating that: There is no significant difference in the Hg level of three selected canned tuna. The calculation was done at the 0.05 level of significance and results of statistical test are presented in Table 3.

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REFERENCES Long, H., Nelson, L.S. Metals and metalloids. In: Tintinalli JE, Kelen GD, Stapczynski JS, Ma OJ, Cline DM, eds. Emergency Ammushijo, J. (2011). Tuna. Retrieved August 28, 2011, from Medicine: A Comprehensive Study Guide. 6th ed. New York, NY: http://ammushijo.hubpages.com McGraw-Hill; 2004:chap 184.

Bryant, P. (2010). Mercury in Fish. Retrieved August 28, 2011, Lyons, S., et al. (2006). Mercury Calculator, updated FDA and from http://www.betterhealth.vic.gov.au/bhcv2/bhcarticles. EPA standard. Retrieved December 22, 2011, from http:// nsf/pages/mercury_in_fish?open www.gotmercury.org/article.php?list=type&type=75

Chua, P. (2008). Toxic Mercury in Fish. Inquirer Entertainment. Magos, L., Clarkson, T.W. (2006). Overview of the clinical Retrieved September 5, 2011, from Inquirer Global Nation. toxicity of mercury. Ann Clin Biochem. Jul;43(Pt 4):257-268. Review Monterey Bay Aquarium Seafood Watch (2011). Tuna. Coldiron, D. (2009). Tuna: Underwater World. United States: New York: McGraw. Minnesota. Mercury Contamination. (n.d.). Retrieved December 2, 2011 Ebrahim, R. (2010). Analysis and determination of mercury, cadmium and lead in canned tuna fish marketed in Iran. Mercury in Canned Tuna. (2010). Retreived December 13, African Journal of Biotechnology 9(31), 4938-4941 2011

Emamikahansari, et al. (2004). Heavy Metals Content of Mercury in Fish. (2011). Retrieved November 12, 2011 from Canned Tuna Fish. Retrieved December 23, 2011 from http:// http://www.betterhealth.vic.gov.au/bhcv2/bhcarticles.nsf/ medicine.tums.ac.ir/FA/Users/mahmood_ghazi/Heavy%20m pages/mercury_in_fish?open etals%20content%20of%20canned%20tuna%20%EF%AC%8 1sh.pdf Mercury in the Environment. (2009). Retrieved January 28, 2012 from http://www.usgs.gov/themes/factsheet/146- Gonzales M. (2010). Retrieved January 8, 2012 from http:// 00/#environment www.nrdc.org/health/effects/mercury/sources.asp Mercury Policy Report. (2009). Mercury in Fish: A Global Laliberte, R. (2010). Special Report: Mercury in Fish. United Health Hazard. New York: Pelham. States: Department of Agriculture. Moore, C.J. (2001). A Review of Mercury in the Environment. Langmhyr, S., et al. (1998). Cold Vapor Atomic Absorption South Carolina: Department of Natural Resources. Spectrophotometer Narvaez, D. (2002). Human Exposure in Mercury in Fish in Mining in the Philippines

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Ocean Mercury on the Increase. (2009). Retrieved September Terio, V., et al. (2010). Identification of Tuna 10, 2011 from http://www.nature.com/news/2009/090331/ full/news.2009.218.html Tyson, K., et al. (1998). Determination of Mercury in Urine

Perkin Elmer Manual. (2011). Atomic Absorption. Retrieved U.S. Environmental Protection Agency. (2010). Retrieved December 21, 2011 fromhttp://www.perkinelmer.com/PH/ August 15, 2011, from http://www.epa.gov/hg/effects.htm Search/Search.aspx?Ntt=Atomic%20Absorption&N=0&buck et=PopularSearch U.S. Food and Drug Administration. (2009). While You’re Pregnant – Methylmercury. Retrieved August 15, Rogers, Keith. Las Vegas review Journal 2010 2011, from http://www.fda.gov/Food/ResourcesForYou/ HealthEducators/ucm083324. Science and Health. (2005). The Mercury Story. Retrieved September 2, 2011 http//www.pbs.org/now/science/ U.S. Geological Survey. (2000). Mercury in the Environment. mercuryinfish.html United States: California.

Shum, et al. (1998). Fish Toluene Extraction Uthe, et al. (1998). Extraction of Mercury from Fish Tisssue

Smith, S.E. (2011). What is a Tuna? Retrieved August 28,2011, Vera, C.A. & Hipolito, Z. (2006). The Philippines Tuna Industry: from http://www.wisegeek.com/what-is-a-tuna.htm A Profile. Retrieved September 5,2011, from http://www.icsf. net/icsf2006/uploads/publications/monograph/pdf/english/ South Atlantic Fishery Management Council .(2010). San issue_38/ALL.pdf Fransisco: W.H. Freeman. Viñas J., et al. (2009). A Validated Methodology for Genetic “SPSS”. (2009). Retrieved September 12, 2011 from http:// Identification of Tuna Species (Genus Thunnus). PLoS ONE www.spss.com.hk/ 4(10): e7606. doi:10.1371/journal.pone.0007606

Status of the World Fisheries for Tuna. (2009). Retrieved Voegborlo, R.B., El-Methnani, A.M., Abedin, M.Z. (1997). November 6, 2011 Mercury, Cadmium and Lead Content of Canned Tuna Fish. Food Chemistry 67 (1999) 341-345. Stibich, M. (2009). The Best Types of Fish for Health. Retrieved September 2, 2011, from http://longevity.about.com/od/ WHFoods: TUNA. (2011). Retrieved December 19, 2011. From lifelongnutrition/a/fish_mercury.htm http://www.whfoods.com/genpage.php?tname=foodspice&d bid=112 Sunderland, E.M. (2009). Ocean Mercury on Increase. Retrieved August 15, 2011 from http://www.nature.com/ news/2009/090331/full/news.2009.218.html

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Cholesterol Lowering Activity of Formulated Introduction Green Caviar (Caulerpa Lentillifera J. Agardh., Caulerpaceae) Seaweed Extract Tablet in Elevated cholesterol levels are estimated to cause eighteen percent Hypercholesterolemia-Induced Rabbits (18%) of strokes and fifty six percent (56%) of ischemic heart disease, Ma. Corazon S. Loquellano, Dune Vienis Karen N. Rafael, accounting for 4.4 million or seven point nine percent (7.9%) of total Melanie Bat-ao, Lovely Edchelle Bermudez, Danmarie Inting deaths worldwide (WHO, 2002). Prevalence of hypercholesterolemia among Filipino adults based on total cholesterol of equal or more than 240 milligrams per deciliter (mg/dL) is 8.5 percent (8 out of 100) in Abstract 2003. These figures are from the National Nutrition and Health Survey (NNHeS) 2003-2004. In 2009, heart diseases ranked as the top one (1) This study was designed to investigate the effects of the formulated of the causes of death in which the most common is the coronary artery Green Caviar (Caulerpa lentillifera J. Agardh) seaweed extract in lowering disease (DOH, 2005). total cholesterol, triglyceride and low density lipoprotein levels in 15 rabbits fed on high-cholesterol/high-fat diets. Ethanolic extract was determined As core of health care, medicines are used to relieve pain, cure on its Acute Oral Toxicity using female albino rabbits and Approximate diseases, preventive or substitute for endogenous compounds. Despite Effective Dose (AED) using male rabbits. Meanwhile, Effective Dose enormous progress in medicinal chemistry, the development of a novel (ED90) was determined within AED range using Algebraic Probit analysis drug has become more difficult. Nature is an important source for using hypercholesterolemia-induced rabbits. Bioassay for formulated developing novel leads for medicines (Verpoorte, Kim, & Choi, 2006). tablet was obtained through dividing 3 groups of rabbits. Group 1 was Since Davao City is rich with natural resources, Green Caviar (Caulerpa administered with the formulated Green caviar tablet, group 2 with lentillifera J. Agardh) seaweed, otherwise known as Lato in the Cebuano placebo and 5 mg/kg dose of Simvastatin (positive control) for group 3. dialect, is one of the favored species of edible Caulerpa due to its soft Rabbits on the high cholesterol fat diet had significantly increased plasma and succulent texture. Green Caviar is abundant of a phytosterol called total cholesterol (TC), plasma low-density lipoprotein cholesterol (LDL-C), clionasterol. β-Sitosterol which is an epimer of clionasterol, is known to plasma triglycerides (TG) in 30 days. Animals treated with Simvastatin reduce blood levels of cholesterol as it inhibits cholesterol absorption in showed decrease in total cholesterol, triglycerides and LDL level at 1.1475 the intestine(Matsuoka, et al., 2008). Likewise, Moreau (2002) proves ± 0.7030, 1.0775 ± 01.2224 and 0.76 ± 0.7109 respectively.Those that that this species is most effective at reducing plasma total cholesterol. received the formulated test drug have shown positive response towards the treatment as indicated in the decreased of values in these parameters. With these bases, the researchers decided to engage and focus their Total cholesterol, triglycerides and LDL lowered by the experimental group study on the effects of Green Caviar seaweed extract and formulating was found to be at 1.36 ± 0.1627, 1.1725 ± 0.7444 and 0.13 ± 0.4567. tablet in lowering total cholesterol, triglyceride and low density Statistical analysis showed that there is no significant difference in the lipoprotein levels in hypercholesterolemia-induced rabbits. This aimed total cholesterol, triglyceride and low density lipoprotein lowered by the to determine the Acute Oral Toxicity (AOT) Category in female albino formulated Green Caviar tablet and Simvastatin. rats; Approximate Effective Dose (AED) and Effective Dose (ED90) of Green Caviar seaweed extract in hypercholestolemia-induced rabbits; Keywords: Pharmacology, Green Caviar, Lipid Profile, Rabbit, physical tablet specifications of the formulation of Green Caviar tablet. Hypercholesterolemia, Simvastatin

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Materials and Methods cholesterol levels 1, 2.5, 4 hours after administration of the tablets.

The protocol for animal care was followed during the The data from the assay were analyzed using t-Test to determine experimentation. Green Caviar seaweed was procured fresh at Toril the significant difference of the cholesterol lowering activity of Green Public Market, Davao City. It was extracted by macerating into 95% Caviar seaweed extract tablet and Simvastatin. ethanol for twenty four hours and filtered through Buchner funnel. The final concentration of the extract was done by means of rotary In the determination of blood cholesterol level, 1 mL of blood was evaporation followed by the determination of Acute Oral Toxicity taken from the rabbit’s jugular vein by intravenous route. Blood collection Testing based on OECD Guidelines 423 using three female albino rats. was done by a licensed doctor of veterinary medicine. Cholesterol testing for the blood samples was done at Penlab Diagnostic Center located at Approximate Effective Dose was conducted using seven male Omnor Building, Diversion Road, Buhangin, Davao City. rabbits procured from PFPF Rabbitry farm in Panabo City.These were subjected to lipid profiling before and after feeding. The starting dose of 10 mg/kg was logarithmically increased to 0.6 log intervals. The extract Results and Discussions was administered orally on each rabbit. After 48 hours, the rabbits were subjected for another lipid profiling. After the data has been tabulated and analyzed, the researchers have chosen the Approximate Effective Table 1 Dose range. It is done by identifying the dose levels that was obtained Determination of Acute Oral Toxicity Using the Rabbits within the AED range. Fifteen male hypercholesterolemia-induced rabbits used for 30 days and subjected to lipid profiling before and Dose Weight Volume of Animal Administered after the induction of hypercholesterolemia and fasted with food. In Level of Mice Administered *Remarks (mg/kg Number (kg) Dose (mg/kg) Extract (mL) contrast to the determination of AED, the starting dose was based on 2000 1 0.034 68 0.067 S the lowest calculated dose level derived from the AED range and was 2000 2 0.036 72 0.071 S logarithmically increased to 0.2 log intervals. The ED90 was determined 2000 3 0.035 70 0.069 S by using Probit Algebraic method of analysis. *S – Survived; D – Died Green Caviar tablets were formulated through wet granulation method with a strength based on the calculated ED90. This method was followed from Allen (2005). In the pharmacological assay, twelve rabbits The results show that Green Caviar belongs to Category 5. The fed with butter for 30 days in order to induce hypercholesterolemia. test animals in this experimentation were given the highest dose to These were categorized into three groups with four replicates using the be administered in determining the acute oral toxicity. All of the parallel group design. Baselines of cholesterol levels were determined test animals were able to survive. No changes in skin and fur, eyes in a population of experimental group treated with Green Caviar and mucous membranes, tremors, convulsions, salivation, diarrhoea, seaweed extract tablet (group 1), negative or placebo control (group lethargy, sleep and coma were observed during the observation period 2) and positive control (group 3) where Simvastatin 5 mg/kg dose was of 14 days. Green Caviar possesses low acute oral toxicity and is free for administered. Blood samples were obtained and analyzed for blood further experimentation.

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On the other hand, there was an obvious increase of body weight The data above shows that there is a decrease of total cholesterol in all albino rats used in the experimentation. During the 14-day levels on 10 mg/kg and 158.5 mg/kg to 39,810.70 mg/kg dose of C. observation after administration, the rats were able to eat their regular lentilliferaexcept for 39.81 mg/kg dose where there is an increase of rations. 0.26 mmoL/L in the total cholesterol level in a hypercholesterolemia - induced male rabbit. Musculoskeletal necropsy findings have shown no evidence of fracture or muscle trauma. They have clean and clear nares. Trachea It was reported in a study by Matanjun (2010) that the extract was found no exudates and lungs are pinkish. Gastrointestinal necropsy was most effective at reducing plasma total cholesterol. This supports findings shown the esophagus, stomach, and intestinal segments are the decrease of total cholesterol levels in six out of seven male rabbits pinkish; no gross lesions in the mucosa such as ulcerations and bleeding used in this experimentation. Based on the results, seaweed extract has were noted. The heart was in normal size with healthy arteries and a hypocholesterolemic effect. veins. Bladder and ureters were found normal. The overall impression was that the animals are healthy at the time they were examined. The cause of death was due to asphyxiation from chloroform. Overall result Table 3 of this necropsy suggests that Green Caviar is not harmful to all vital Determination of Approximate Effective Dose (AED) of Triglyceride organs of the albino rats. Levels in Hypercholesterolemic - Induced Male Rabbits

Weight Volume Triglyceride Level of of Admi- (mmoL/L) Table 2 Dose Level nistered Rabbit (mg/kg) Extract Pre- Post- Post-

Determination of Approximate Effective Dose (AED) of Total (kg) *Remarks Difference Animal No. Animal (mL) test Induction test Cholesterol Levels in Hypercholesterolemia - Induced Male Rabbits 667 0.9 10 0.0095 2.11 5.15 2.76 -2.39 + 664 0.9 39.81 0.038 2.24 2.38 2.16 -0.22 + Weight Volume Total Cholesterol Level of of Admi- (mmoL/L) 665 1 158.5 0.151 3.06 5.81 2.09 -3.72 + Dose Level nistered Rabbit (mg/kg) Extract Pre- Post- Post- 659 1 630.95 0.6 2.38 4.54 2.03 -2.51 +

(kg) *Remarks Difference Animal No. Animal (mL) test Induction test 660 1.1 2,512.06 2.624 2.62 2.48 1.45 -1.03 + 667 0.9 10 0.0095 3.65 5.23 4.89 -0.34 + 661 1.1 10,000 10.45 2.80 5.69 2.44 -3.25 + 664 0.9 39.81 0.038 3.41 5.27 5.53 0.26 – 669 1.1 39,810.70 41.6 2.45 3.07 2.52 -0.55 + 665 1 158.5 0.151 3.42 6.83 5.94 -0.89 + Legends: (-) Negative Result; (+) Positive Result 659 1 630.95 0.6 3.47 6.11 4.85 -1.26 + 660 1.1 2,512.06 2.624 3.64 4.19 3.79 -0.4 +

661 1.1 10,000 10.45 4.13 7.11 6.7 -0.41 + Based on the Table 3, there is a decrease of triglyceride levels from 669 1.1 39,810.70 41.6 2.85 5.11 4.68 -0.43 + 10 mg/kg to 39,810.70 mg/kg dose of crude seaweed extract. A dose Legends: (-) Negative Result ; (+) Positive Result of 39.81 mg/kg has the lowest decrease in triglyceride levels with a difference of 0.22 mmoL/L. On the other hand, a dose of 158.5 mmoL/ L has the highest decrease in triglyceride level. Consumption of plant

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sterols reduces plasma and hepatic triglycerides (Rideout, 2010). Based Table 5 on the results, seaweed extract has hypotriglyceridemic effect. Determination of Approximate Effective Dose (AED) of High Density Lipoprotein (HDL) Levels in Hypercholesterolemic - Induced Male Rabbits Table 4 Determination of Approximate Effective Dose (AED) of Low Density Weight Volume High Density Lipoprotein of of Admi- (HDL) Levels Level (mmoL/L) Lipoprotein (LDL) Levels in Hypercholesterolemic - Dose Level nistered Rabbit (mg/kg) Extract Pre- Post- Post-

Induced Male Rabbits (kg) *Remarks Difference Animal No. Animal (mL) test Induction test 667 0.9 10 0.0095 1.44 1.09 1.04 -0.05 – Weight Volume Low Density Lipoprotein of of Admi- (LDL) Level (mmoL/L) 664 0.9 39.81 0.038 1.06 1.82 1.6 -0.22 – Dose Level nistered Rabbit (mg/kg) Extract Pre- Post- Post- 665 1 158.5 0.151 0.82 1.57 1.19 -0.38 –

(kg) *Remarks Difference Animal No. Animal (mL) test Induction test 659 1 630.95 0.6 1.01 1.43 1.41 -0.02 – 667 0.9 10 0.0095 1.25 1.8 2.6 0.8 – 660 1.1 2,512.06 2.624 1.16 1.18 1.07 -0.11 – 664 0.9 39.81 0.038 1.33 2.37 2.95 0.58 – 661 1.1 10,000 10.45 0.91 3.94 1.05 -2.89 – 665 1 158.5 0.151 1.21 2.62 3.8 1.18 – 669 1.1 39,810.70 41.6 0.67 1.32 1.29 -0.03 – 659 1 630.95 0.6 1.38 2.62 2.52 -0.1 + Legends: (-) Negative Result; (+) Positive Result 660 1.1 2,512.06 2.624 1.29 1.88 2.06 0.18 – 661 1.1 10,000 10.45 1.95 0.58 4.54 3.96 – 669 1.1 39,810.70 41.6 1.07 2.39 2.24 -0.15 + Based on the data, there is a decrease of high density lipoprotein Legends: (-) Negative Result; (+) Positive Result (HDL) from 10 mg/kg dose to 39,810.70 mg/kg dose of crude seaweed extract administered in all test animals in this experimentation. The 10 mg/kg dose has the lowest decrease in HDL level of -0.05 mmoL/L The data on the Table 4 present that there is an increase of low while a dose of 10,000 mg/kg has the greatest decrease in HDL level of density lipoprotein levels from 10 mg/kg to 158.5 mg/kg dose of Green -2.89 mmoL/L. Caviar crude seaweed extract. This is followed by the increase of low density lipoprotein levels from 2,512.06 mg/kg to 10,000 mg/kg dose The test animals were not able to have enough physical activities of the said extract. There is a decrease of low density lipoprotein (LDL) during the course of the experimentation. Phytosterols may inhibit the levels in 630.95 mg/kg and 39,810.70 mg/kg dose of crude plant uptake of dietary and biliary cholesterol, thus decreasing cholesterol extract. The data suggest that Green Caviar is not an excellent LDL levels (Moreau, et al., 2002) but the data shows the contrary. reducing agent. The concentration to be used in the determination of ED90 is based from the ability of the Green Caviar crude seaweed extract to lower total cholesterol and triglyceride levels. The dose of 39.81 mg/kg was not able to lower total cholesterol but was able to lower triglyceride level. However, doses from 158.5, and 630.95 mg/kg were able to lower total

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cholesterol and triglyceride levels. In the Probit algebraic method of computing the ED90 of the plant extract, this transformed both the variables dose and observed effect It was arbitrarily chose doses within 39.81 to 630.95mg/kg and response to logarithmic dose and Probit Y value, respectively. Using increased logarithmically by 0.2 intervals. These doses of Green Caviar the equation of the line obtained, it calculated the ED90 of the plant crude seaweed extract were used in the determination of ED90. material which is equivalent to 304.06 mg/kg. To further depict the ED90 determination in a more vivid and easy presentation, graphical method was used to locate the ED90 as presented in Figure 1 and 2. Determination of Effective Dose 90 (ED 90)

This determines the effect of seaweed extract in total cholesterol, triglyceride, low density lipoprotein (LDL) and high density lipoprotein (HDL) in hypercholesterolemia - induced male rabbits. The total cholesterol lowered by the experimental group was found to have positive results except for triglycerides, high density lipoprotein and low density lipoprotein. In this case, results on triglycerides, high density lipoprotein and low density lipoprotein were not incorporated on the statistical analysis. The analysis showed that the effective dose 90% of the population is 304.06 mg/kg.

Table 6 Figure 1. Graphical Presentation of ED 90. Probit Algebraic Method of Analysis in Determining Effective Dose 90 (ED 90)

Dose Number of % Response Level Log No. test Animals w/ Probit (mg/kg dose animals effects/tested Observed Expected Y 63.09 1.80 3 2/3 67 53 5.08 100.00 2.00 3 1/3 33 67 5.43 158.49 2.20 3 2/3 67 78 5.78 251.18 2.40 3 3/3 100 87 6.13 398.10 2.60 3 2/3 67 93 6.49

* Calculated ED 90 = 304.06 mg/kg

Figure 2. Graphical Presentation of ED 90 with Emphasis on the Unknown X Value

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Manual Computation of ED 90 the average force applied of four point seventy two kilograms (4.72kg). The minimum requirement for a satisfactory tablet Using the equation of the line y = 1.76x + 1.91 wherein y is considered of about four to seven (4-7) grams (Allen, 2005). the probit y value and x is the log dose, the ED90 can be obtained by The results tested and gathered for tablets were within the locating the 90% response equivalent in the probit conversion table. limit. The 90% response is equivalent to 6.28 therefore: B. Friability Test 6.28 = 1.76 (x) + 1.91 Log dose, x = (6.28 – 1.91)/1.76 x = 2.4829545; thus the ED90 concentration is: Table 8 x = antilog (2.4829545) Tablet Friability Results on Four (4) Samples of Formulated x = 304.06 mg/kg Green Caviar Seaweed Extract Tablet

Initial Weight After Sample Weight testing Weight Loss Percent Tablet Specification Tests (mg) (mg) (mg) Loss 1 470 467.88 2.12 0.45% These tests were used to determine if the tablet is within the 2 450 447.78 2.22 0.49% minimum satisfactory requirement as emphasized by the guideline for 3 410 407.57 2.43 0.59% tablets. 4 430 427.68 2.32 0.54% Average 452.50 437.73 2.27 0.52% A. Tablet Hardness

Table 7 Friability test shows that the average percent loss of four Tablet Hardness Test Results on Five (5) Samples of the samples was 0.52%. This indicates that for four tablet samples Formulated Green Caviar Seaweed Extract Tablet having an average weight of 452.50mg, the average weight loss of the tablet sample was 2.27mg. The average weight of Sample Hardness (kg) the tablet samples after testing had the average of 437.73mg. 1 4.6 This may conform the study of Allen (2005) stating that the 2 3.8 maximum weight loss of the tablet must not exceed to one 3 5.0 percent (1%) is generally considered acceptable. The results 4 5.3 obtained for Green Caviar tablets tested was within the 5 4.9 acceptance level. Average 4.72

Presented above are results for tablet hardness for the formulated Green Caviar tablets. The five tablet samples gave

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C. Tablet Uniformity Table 10 Summary of Results of the Bioassay The average results for Green Caviar tablet for the weight Test Variables uniformity test (Table 8) was 455mg. According to Allen Sample (2005), the average weight of 10 tablets shall within ± 5% Cholesterol Triglycerides HDL LDL of the desired weight which was 450mg. The upper limit was Experimental 1.36 ± 1.1725 ± 0.4825 ± 0.13 ± 495 mg, and the lower limit was 405 mg. The results for ten Drug 0.1627 0.7444 0.5258 0.4567 formulated Green Caviar tablets were within the specification. Positive 1.1475 ± 1.0775 ± 0.2325 ± 0.76 ± Control 0.7030 1.2224 0.1931 0.7109 Negative -1.03 ± -1.29 ± 0.11 ± -0.42 ± Table 9 Control 0.3640* 0.9156* 0.2281 0.3207* Tablet Uniformity Results on Ten Samples of Formulated Green Caviar Seaweed Extract Tablet *(-) values indicate increase in cholesterol, triglycerides and LDL level

Sample Weight (mg) Sample Weight (mg) 1 450 6 460 As indicated in the Table 9, a noticeable decreased on the blood 2 480 7 450 cholesterol, triglycerides and LDL level have been recorded both in 3 440 8 450 the experimental and positive control group after treatment. Test 4 450 9 460 animals treated with Simvastatin showed decrease in total cholesterol, 5 460 10 430 triglycerides and LDL level at 1.1475 ± 0.7030, 1.0775 ± 01.2224 Average: 454 and 0.76 ± 0.7109, respectively. Those that received the formulated test drug have shown positive response towards the treatment as indicated in the decreased of values in these parameters. The total Biostatistical Analysis cholesterol, triglycerides and LDL lowered in the experimental group was found at 1.36 ± 0.1627, 1.1725 ± 0.7444 and 0.13 ± 0.4567. To scientifically investigate the effect of the formulated test drugs Except for the LDL level, the total cholesterol and triglyceride levels from Green Caviar extract on the hyperlipidemic, hypercholesterolimic, lowered in the experimental group were found higher than the positive and hypertriglyceridemic test animals, the researchers conducted the control group. On the other hand, though there were decreased in the experimental in vivo bioassay. Summary of results of the bio assay are cholesterol, triglycerides and LDL level of the hypercholesterolemic, presented in Table 9. hypertriglyceridemic and hyperlipidemic test animals after treatment, the HDL level did not elevate in all of the three groups. In this case, statistical analysis was not incorporated in this parameter. Furthermore, since the negative control showed incresed in cholesterol, triglycerides and LDL level, the researchers did not include this variable in the statistical test since its effect was found to be different from activity of both the formulated test drug and Simvastatin.

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Table 11 Table 12 Testing the Significant Difference on the Level of Total Cholesterol Testing the Significant Difference on the Level of Triglycerides Lowered by Experimental and Positive Control Group Lowered by Experimental and Positive Control Group

Mean Mean Mean Mean Df Calculated t Tabular t Decision* Comparison values Df Calculated t Tabular t Decision* Comparison values Experimental 1.36 Not Experimental 1.17 Not 6 0.59 2.45 6 0.13 2.45 Positive control 1.15 significant Positive control 1.08 significant *Calculation was performed at the 0.05 level of significance *Calculation was performed at the 0.05 level of significance

The above data indicates that the blood cholesterol level lowered between the two treatments did not significantly differ from each Table 13 other. This further proved that the formulated test drug showed Testing the Significant Difference on the LDL Level Lowered antihypercholesterolemic activity similar to the effect of the commercially by Experimental and Positive Control Group available Simvastatin. Mean Mean Comparison values Df Calculated t Tabular t Decision* On the other hand, statistical analysis of the triglycerides level Experimental 0.13 lowered by the two treatments on the groups of test animals showed Not 6 1.49 2.45 significant that calculated t value is lower than the tabular t, therefore the null Positive control 0.76 hypothesis indicating that there is no significant difference in the *Calculation was performed at the 0.05 level of significance Triglycerides level lowered by formulated seaweed extract tablet (test drug) and Simvastatin (Vidastat) as the positive control is accepted. This proved that the formulated test drug demonstrate can significantly Further statistical analysis on the LDL level lowered by both lower the triglycerides level of hypercholesterolemia-induced rabbits formulated test drug and Simvastatin showed that that the calculated since its effect was found to be not significantly different to the group t is lower that tabular t which implies that the null hypothesis of study that received Simvastatin as treatment. is accepted. These results signify that after treatment, both groups test animals showed similar decreased in the LDL level. This further demonstrated the capacity of the formulation to effectively lower the LDL level of the hypercholesterolemia-induced rabbits.

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REFERENCES Verpoorte, R., Kim, H., & Choi, Y. (2006). Plants as Source of Medicines. In R. Bogers, L. Craker, & D. Lange, Medicinal and Allen, J.L. (2005). Ansel’s Pharmaceutical Dosage Forms and Drug Aromatic Plants (p 261-273). Netherlands: Springer. Delivery Systems. Philadelphia: Lippincott Williams & Wilkins. WHO. (2002). World Health Organization: World Health Report American Heart Association. (2010). What Your 2002: Reducing Risks, Promoting Healthy Life. Geneva: World Cholesterol Levels Mean. Retrieved January 06, 2011 from Health Organization. americanheart.org: http://www.americanheart.org/presenter. jhtml?identifier=183

DOH. (2005). Mortality: Ten (10) Leading Causes Number And Rate/100,000 Population Philippines 5-year Average (2000- 2004) & 2005. Retrieved August 10, 2010 from Department of Health: http://www.doh.gov.ph/kp/statistics/leading_ mortality

Matsuoka, et al. (2008). Study of Thermodynamic Parameters for Solubilization of Plant Sterol and Stanol in Bile Salt Micelles. Chemistry and Physics of Lipids 154, 87-93.

Moreau, et al. (2002). Phytosterols, Phytostanols, and Their Conjugates in Foods: Structural Diversity, Quantitative Analysis, and Health-Promoting Uses. Progress in Lipid Research 41, 457-500.

Rideout, T.C. (2010). Consumption of plant sterols reduces plasma and hepatic triglycerides and modulates the expression of lipid regulatory genes and de novo lipogenesis in C67BL/6J mice. Molecular nutrition and food research, S7-13.

Singh, V.N. (2009, August 04). Low HDL Cholesterol (Hypoalphalipoproteinemia). Retrieved January 06, 2011 from eMedicine from WebMD: http://emedicine.medscape.com/ article/127943-overview

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DETERMINATION OF THE NITRITE CONTENTS AS A FOOD Introduction PRESERVATIVE IN THREE TUNA PRODUCTS FROM THE SELECTED BRANDED AND HOMEMADE The formation of carcinogenic nitrosamines in meats containing PRODUCERS IN GENERAL SANTOS CITY sodium nitrite when meat is charred or overcooked is the primary Venchie C. Badong, Lloyd Jason H. Gualvez, concern about the additives sodium nitrite in foods. Such carcinogenic Judy S. Manliguis nitrosamines can be formed from the reaction of nitrite with secondary amines under acidic conditions (such as occurs in the human stomach) as well as during the curing process used to preserve meats (Paika, 2000). Abstract In the early 1970’s, outbreaks of liver disorders (including cancer) were recorded in various farm animals in Norway. Intensive investigations This study was conducted to determine the level of nitrites in revealed that all of the affected animals had consumed rations selected commercial and homemade tuna products such as chorizo, containing herring meal, which had been preserved by the addition of embutido and nuggets from General Santos City; to assess the levels of relatively large amounts of sodium nitrite. Their investigation showed nitrites based on the tolerable limit of 10 ppm under FDA standard; and that dimethylnitrosamine was present as a result of a chemical reaction to evaluate levels of nitrite content in terms of types of tuna products. between dimethylamine (commonly occurring amine in fish meal) Determination of nitrites in the predetermined samples was done using and nitrosating agent that formed from the sodium nitrite (Scanlan, Spectroquant Pharo 300 Photometer. Analyses conducted to three types 2010). of tuna products of two different brands.Findings show that branded tuna products (brand A) have lower concentration of nitrites than In the Philippines, sodium nitrite has been used extensively in Homemade (brand B). Of the three types of tuna tested in Brand A, curing meat and meat products, particularly pork products such as ham, chorizo showed the highest concentration of nitrite. Whereas, Embutido bacon and frankfurters. The process includes dry curing, immersion in brand B showed almost 10 times higher nitrite concentration. curing, or direct addition or injection of the curing ingredients, which Moreover, Brand A conformed to the allowable limit while brand B includes potassium salts of nitrite and nitrate and seasonings (Ingested have exceeded the allowable concentration. There is no significant Nitrate and Nitrites, 2011). Cured meats can contain nitrosamines difference in the levels of nitrite concentration on the three types of because meats contain amines, and sodium nitrite is a good source of

tuna products in Brand A. This indicates that nitrite concentrations do nitrosating agents. The tested level of NaNO2 in the finished products not differ significantly with each other. However, an existing significant will be assessed on the 10 ppm allowable limit (for finished products) difference on the nitrite concentrationof Brand B products. This showed by the Food and Drugs authority. Any concentration of the test samples higher concentrations of nitrites. Higher than 10 ppm may pose risk on higher than 10 ppm maybe concluded to impose risk on the health of the health of the consumer since this likely produces carcinogenic N- the consumer (Food and Drugs Administration, 2010). nitrosamines by the reaction of sodium nitrite with amino acids in heat and acidic environments. Therefore, standard specification of nitrites With the changing lifestyle of the people this scenario must be must be monitored by Government authorities. addressed. Hence, this aimed to determine the level of sodium nitrite in the selected tuna products in General Santos City. Specifically it Keywords: Environmental chemistry, toxicology, tune products, determined by photometric method the level of nitrite content as a nitrites concentration, FDA standard food preservative from branded and homemade products such as tuna

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chorizo, tuna embutido and tuna nuggets; to assess the levels of nitrites Data Gathering Procedures if significantly exceed the tolerable limit of 10 ppm under FDA standard; and to evaluate the significant difference in the levels of nitrite content A. Preparation of Materials in terms of types of tuna products. Sealable clean plastic containers were used in collecting the samples. The date, time of the sampling and the sampling Materials and Methods site were indicated in the sampling material to ensure that there were no cross contamination and misclassification of Research Design samples.

The researcher utilized the quantitative research design, specifically B. Laboratory Analysis descriptive-comparative. The quantitative measurement of the nitrites used the standard laboratory procedures intended for fish and fish After the samples were bought on site, the test samples products. Descriptive interpretation of results was done by comparison (not more than 24 hours from time of it was bought) were with the existing standard criteria provided by the Food and Drug brought to the laboratory for analyses using the Spectroquant administration in the Philippines. Pharo 300 Photometer were conducted at the Science Resource Center, UIC. Sampling Design Analysis was done by quantitatively weighing 10 mg of The researcher utilized non-probability purposive sampling design. the sample and mix with about 80 ml deionized water. Then, Both the homemade and branded three types of processed tuna products homogenize the sample with the Ultra-Turrax homogenize the in General Santos City were collected for evaluation of the nitrite level. mixture for 60 seconds. Then, with 50 ml hot deionized water, To obtain highly precise results, the collection was done in three trials homogenizer was rinsed into the flask, adding it to the mixture. for each type of tuna products. The processed tuna products collected Then pH-value of the solution was adjustedto 7-7.2 with 1 mol/ were Tuna Embutido, Chorizo and Nuggets. L sodium hydroxide solution, using the pH-meter, and heat for 15 minutes in a bath of boiling water, while occasionally Research Locale shaking (the pH was controlled to ensure optimum analysis of nitrites). It was allowed to cool in room temperature. The research was conducted in General Santos and Davao City. The samples of processed tuna products both homemade and Branded Prepared sample was transferred into a 200 mL standard were in General Santos City. Sampling was done in three consecutive volumetric flask and successively mixed with 2 ml Carrez- weeks in three trials. The obtained samples were then transported to solution 1 and 2 at a time. The protocol was followed as the Science Resource Center, UIC, Davao City for immediate testing of described from Merck incorporated (2011). nitrites level using standard procedure of analysis.

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Statistical Analysis Results revealed that the level of nitrite on the three types of tuna products in brand A - Branded have conformed to the acceptable limit The average concentration of nitrite tested was computed and for nitrite. It was demonstrated that the chorizo showed the highest compared to the criteria provided by Food and Drug administration of concentration of nitrite than embutido and nuggets. However, the 10ppm. One-Way Analysis of Variance (ANOVA) was also utilized to concentration obtained was found lower than acceptable limit of <10 determine if there is an existing significant difference on the level of ppm nitrite. This suggests that the products tested are unlikely to nitrites from the three types of samples collected namely: Embutido, produced high concentration of nitrosamines, which are found to be Chorizo and Nuggets. Further statistical computation using Post detrimental to health once present in excess amount. Hoc Multiple comparison test was done to determine which of these products showed the highest and the lowest concentration of nitrites. Furthermore, the independent t test computation was done to determine Table 2 if there is an existing significant difference on the nitrite concentration Level of Nitrite in Three Types of Tuna of samples regardless of its brand. Products in Brand B - Homemade

Types of Tuna Mean S.D. Allowable Limit Interpretation Results and Discussions Chorizo 24.06 0.4612 <10 ppm Outside acceptable limit To determine if the mean concentration of nitrites in the three types Embutido 125.86 3.9864 <10 ppm Outside acceptable limit of tuna product samples conformed to the acceptability concentration Nuggets 17.25 1.6543 <10 ppm Outside acceptable limit for nitrite, the mean and standard deviation concentrations of the three trials test per type of tuna products on each selected brand was The three types of tuna products tested for nitrite in Brand B - computed. The determination of nitrite was very important since higher Homemade did not conform to the acceptable limit of <10 ppm. It concentration of nitrites (usually above the allowable limit) could results can be shown the Embutido showed the highest level of nitrites with a to the formation of nitrosamine, which are known to be carcinogenic. mean value of 125.86 ± 3.9864, which is almost 13 times higher than Results are shown in table 1 for Brand A -Branded and table 2 for brand the acceptable limit.On the other hand, chorizo and nuggets in brand B B - Homemade. - Homemade products were found to have mean nitrites values of 24.06 ± 0.4612 and 17.25 ± 1.6543, respectively. These values were found to Table 1 be almost 2 times higher than the allowable limit for nitrite in finished Level of Nitrite in Three Types of Tuna food products. Consequently, the products tested from homemade Products in Brand A - Branded processed tuna (Brand B) are likely to produce high concentrations of nitrosamine, which is harmful to human health. Types of Tuna Mean S.D. Allowable Limit Interpretation Chorizo 2.20 1.0148 <10 ppm Within acceptable limit Embutido 1.17 0.4996 <10 ppm Within acceptable limit Nuggets 1.09 0.2489 <10 ppm Within acceptable limit

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Inferential Statistics Table 4 Testing the Significant Difference among the To determine if there is an existing significant difference on the Three Tuna Products in Brand B - Homemade level of nitrites among the three products tested, One-Way Analysis of variance was computed and results are shown in Table 3 to 4. Source of Sum of Mean variation square df square F P value Decision Between 22205.75 2 11102.874 Within 37682.00 6 6280 1767.899 0.000 Significant Table 3 Total 22243.43 8 Testing the Significant Difference among the Three Tuna Products in Brand A - Branded *Calculation was performed at the 0.05 level of significance

Source of Sum of Degrees of Mean variation square Freedom square F P value Decision The computed p value which equal to 0.000 is lower than the p value Between 2.299 2 1.150 Not of 0.05 therefore the null hypothesis was rejected. This means that the Within 2.683 6 0.447 2.571 0.156 significant nitrite concentration among the three products in brand B - Homemade Total 4.982 8 significantly differs between each other. To determine which of this *Calculation was performed @ 0.05 level of significance product showed the highest concentration of nitrites, post hoc multiple comparison tests was done and results are shown in table.

Results of the statistical analysis showed that since computed p valuewhich is equal to 0.156 was higher than 0.05 therefore the null Table 5 hypothesis indicating that there is no significant difference on the Multiple Mean Comparisons of Nitrite Levels between concentration of nitrites in the three types of tuna products in Brand Types of Tuna Products in Brand B - Homemade A (Branded products) was accepted. This means that none of three products is significantly higher in nitrite concentration than the other. Mean Multiple Comparison Difference T value P value Decision* Chorizo Embutido 101.80 43.9022 0.0010 Significant On the other hand, similar statistical computation was done for the Chorizo Nuggets 6.81 6.8665 0.0024 Significant three types of tuna products obtained from brand B - Homemade, the Embutido Nuggets 108.61 43.55 0.0001 Significant results are shown in Table 4. *Calculation was performed @ 0.05 level of significance

Post hoc multiple comparison tests proved that there is an existing significant difference (since computed p value is <0.05) between the tested tuna products in brand B. Embutido showed the highest nitrite concentration than Chorizo and Nuggets by 101.80 and 108.61 ppm,

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respectively. This means that the nitrite concentration in Embutido was References found to be significantly 10 times higher than the other two products tested. On the other hand, between Chorizo and Nuggets tuna, the Food and Drugs Administration. (2010). Retrieved September chorizo tuna products showed higher concentration by 6.81 ppm than 2, 2011 from http://www.fda.gov/FDAC/features/1998/398_ nuggets. These clearly indicate that high nitrosamine concentration can pain.html likely occur. Of which, these compounds have been linked with health issues such as gastric cancer, colon cancer, pancreatic cancer, and Chronic Ingested Nitrate and Nitrites. (2011). Retrieved January Obstructive Pulmonary Disease (COPD) (Mark Daily Apple, 2011). 2, 2012 from http://monographs.iarc.fr/ENG/Monographs/ vol94/mono94-6A.pdf To determine if there is an existing significant difference on the level of nitrites between the brands of tuna tested regardless of its tuna products, Mark Daily Apple. (2011). Retrieved September 5, 2011 from independent t-test was computed and results are shown in table 6. http://www.marksdailyapple.com/sodium-nitrite-meat/

N.A. Food Preservatives Synthetic Vaccines. Retrieved Table 6 January 5, 2012 from http://www.appliedozone.com/ Testing the Significant Difference of Nitrite Levels between Brand A riskstwo.html -Branded and Brand B - Homemade Regardless of the Tuna Products N.A. 2012. Nitrosamines. Retrieved January 2, 2012 from Test http://en.wikipedia.org/wiki/Nitrosamine Variables Mean S.D. T value P value Decision*

Brand A 1.49 0.7890 3.0851 0.0071 Significant Paika, D., et al. (2000). The epidemiological enigma of gastric – Branded cancer rates in the US: was grandmother’s sausage the cause? Brand B - 55.71 52.7194 Oxford Journals. International Journal of Epidemiology. Homemade Volume 30, Issue 1, p 181-182 *Calculation was performed at the 0.05 level of significance Scanlan, R. (2010). Nitrosamines and Cance. Retrieve September 1, 2011 from http://lpi.oregonstate.edu/f-w00/nitrosamine. The computed p value which is equal to 0.0071 is lower than the html p value of 0.05 therefore the null hypothesis indicating that there is no existing significant difference on nitrite level between the two brands of tuna tested in this research was rejected. This clearly suggests that the amount of sodium nitrites added in both Brand A (Branded) and B (Homemade) samples significantly vary with each other. As such, the mean nitrites level in Brand A, which is equal to 1.49 ppm is significantly lower than the nitrites concentration in Brand B (homemade) which is equal to 55.71 ppm.

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PESTICIDE RESIDUE ANALYSIS OF THE ISOLATED Introduction AND FORMULATED ASCORBIC ACID (Vitamin C) FROM OVERRIPE MANGO (Mangifera indica,Linn. Anacardiceae) According to Casis (2008), the definition of climate change by the Annabelle A. Ribo, Mariz Kay M. Flores, Maribelle C. Hao, United Nations Framework Convention on Climate Change (UNFCCC) Camille F. Selgas is “a change of climate which is attributed directly or indirectly to human activity that alters the composition of the global atmosphere and which is in addition to the natural climate variability observed over Abstract comparable time periods.” A major evidence of climate change today is global warming. The global warming phenomenon is the accumulation This study focused on pesticide residue analysis of isolated ascorbic of greenhouse gases (GHG) - carbon dioxide, methane and nitrous oxides acid from overripe mango (Mangifera indica), to evaluate the amount of in the atmosphere; these gases trap the sun’s heat energy thus resulting Vitamin C and assess the significant difference in the Vitamin C content in the increase of the average global temperature and escalating the of formulated syrup and commercial product. Overripe mangoes were risk of different diseases brought about by the changing climate. collected from Bankerohan public market, Davao City. These were subjected to extraction and isolation. Resulting isolate was utilized As a contribution to the global warming awareness campaign, the for formulation of Vitamin C syrup and granular powder. Lastly, assay researchers conducted this study about the isolation of the vitamin C via 0.1 N iodine solution was used to evaluate percent (%) Vitamin C content of the overripe mango (M. indica) fruit which is usually neglected present in the isolate, formulated syrup and granular powder. Pesticide in the market, waiting to be disposed of or left decomposing in the residue analysis was performed at National Pesticide Analytical waste bins. These overripe mangoes were chosen in the formulation of Laboratory of the Bureau of Plant Industry, Davao City. The isolate has Vitamin C syrup and granular powder since they provide a good source an amount of 23 to 24 milligrams Vitamin C per milliliter. Analysis of calories and especially Vitamin C (Dorette, 2010). revealed no pesticide residues present per sample of Vitamin C via GLC method. Assay of formulated syrup were 99.47% and granular powder This study sought to determine amount of vitamin C present in the were 100.21% content Vitamin C, passing USP limit of 99.5 to 100.5%. isolate from overripe mango; to identify the pesticide residue present in Comparison of Vitamin C content between commercial and formulated the isolate Vitamin C isolate from M. indica; to evaluate the amount of vitamin C syrup showed a significant difference of 2.439 as subjected Vitamin C in the formulated syrup and granular powder; and assess the to t-test analysis. This affirms that the utilization of overripe mangoes significant difference in the Vitamin C content of the formulated syrup as safe Vitamin C product preparations. Furthermore, quantitative from overripe mango and commercial Vitamin C syrup. analysis shows compliance with USP standard and assures possibility of preparing quality product from trash material. Materials and Methods Keywords: Ascorbic acid, Gas liquid Chromatography, Pesticide Residue Analysis, Mangifera indica Gathering of fruit material was limited to Manga Cebu variety collected from Bankerohan public market in Davao City.Ripeness stage of mango fruit was characterized and classified according to color, softness, wrinkled and darkened skin. Approximately one kilo of

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mango fruit was analyzed by its overripe characteristics. Peel and pulp For pesticide residue analysis, the researchers went to Bureau color were also evaluated.It was then cleaned, washed and prepared of Plant and Industry, National Pesticide Analytical Laboratory, for experimentation.The fruit sample submitted for identification and Bago Oshiro, Tugbok District, Mintal, Davao City for Gas Liquid certification was approved and validated as Mango (Mangifera indica) Chromatography isolate analysis. The 500 ml of isolated Vitamin C Linn. Anacardiaceae. syrup from overripe mango stored in an amber bottle container was required for the analysis. For isolation, fruits were sliced and frozen into deep freezer and were stored at -20ºC for Vitamin C determination. Frozen pulverized For formulation of Vitamin C syrup, 85 g of sugar was added into fruit samples were weighed for desired quantity and were mixed with distilled water, a sufficient quantity to make 100 mL of simple syrup. 2.5 mL of the extractant solution (3% MPA and 8% acetic acid for MPA- The sugar was dissolved with the aid of heat. Liquid was strained acetic acid extraction and 0.1% oxalic acid for oxalic acid extraction). and enough distilled water passed through the strainer to make the The mixture was homogenized in a high-speed blender for 1 minute product, when cold, 100 mL. Then the product was thoroughly mixed. and then centrifuged for 20 min. The procedure was repeated twice After preparing the simple syrup, 50 mL of the isolate was introduced and the two resulting supernatants were mixed together. All extractions in every 60 mL formulation to contain 100mg Vitamin C in every 5 mL were carried out in quintuplicate. Several precautions were observed of the isolate. Then the rest of the ingredients were added. in order to perform all the operations under reduced light and at 4ºC temperature. To stabilize Vitamin C in the extractant solutions addition In the formulation of granular powder, 227.5 g of both tartaric acid of 1 mL EDTA solution is recommended. and citric acid was added to 50 ml of Vitamin C isolate from overripe mango and was mixed in a mortar and pestle. After mixing, it was To determine ascorbic acid (Vitamin C) from isolated overripe transferred to evaporating dish. Then it was heated upon a water bath, mango requires first a preparation of Vitamin C standard solution. at 60-71 degrees Celsius under constant stirring with wooden spatula, About 0.250 grams of ascorbic acid was dissolved in 100 mL distilled until dry & uniformly granular. water. Then, the solution is diluted to 250 mL with distilled water in a volumetric flask and it was labeled as the Vitamin C standard solution. For the formulation of Ascorbic Acid (Vitamin C) syrup and granular powder, 25 mL of formulated vitamin C syrup solution was added to Twenty five mL of Vitamin C isolate solution was added to 100mL 100mL distilled water in a 125 mL Erlenmeyer flask. Then 10 drops distilled water in a 250 mL Erlenmeyer flask to determine Vitamin C of 1% starch solution was added. The burette was rinsed with a small content in the isolate. Then 10 drops of 1% starch solution was added. volume of the 0.1 N iodine solution and it was filled. The initial volume The burette was rinsed with a small volume of the iodine solution and was recorded. The solution was titrated until the endpoint is reached. it was filled. The initial volume was recorded. The solution was titrated The final volume of iodine solution was recorded. The titration was until the endpoint is reached. This was done when the first sign of blue repeated in five trials. color is seen that remains after 20 seconds of swirling the solution. The final volume of iodine solution was recorded. The volume that is For the Comparison of commercial Ascorbic acid (Vitamin C) required is the starting volume minus the final volume. The titration syrup and formulated Ascorbic acid (Vitamin C) syrup, about 25 mL was repeated at least twice more. of commercialize and formulated vitamin C syrup solution was added separately to 100 mL distilled water in a 125 mL Erlenmeyer flask.

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Then 10 drops of 1% starch solution was added to each solution. The Table 1 shows the results of the determination of Vitamin C from burettes were rinsed with a small volume of the 0.1 N iodine solutions. overripe mango isolate, presented in milligrams per millilitre done in The initial volumes were recorded. The solution was titrated until the five trials with three replicates. endpoint is reached. Same protocol was followed on the abovementioned procedure. The response to iodine titrations of the isolate confirms that Vitamin C is still present in overripe mangoes. From the numerous determinations made, the isolate contains an average range of 23 to Results and Discussions 24 mg/mL of Vitamin C via direct titration using 0.1 iodine solution. According to the National Nutrient database, fresh mango fruit (M. Table 1 indica) has a national value of 100 g is 27.7 mg vitamin C per 100 g. Data and Results of the Assay of Vitamin C in mg/ml In the analysis, it was revealed that 100 mL obtained from overripe Fruit Isolate from Overripe Mango (M. indica) mangoes contains 2300 mg of vitamin C isolate. This confirms that the amount Vitamin C from overripe mango (M. indica) is a good source Iodine used (ml) Average Vitamin C (mg) of vitamin C, establishing it as an appropriate and valuable material Iodine Average No. of mL Vitamin in formulating different dosage forms and preparations found in the Trials used used Replicate mL (mL) Replicate mg (mg) market such as powders, tablets and syrups (Dorette, 2010). It was also stated that the utilization of mangoes can be expanded the needs of the Rep 1 9.2 Rep 1 23 population. Trial 1 10 Rep 2 9.3 9.4 Rep 2 23 23. 3 Rep 3 9.7 Rep 3 24 Rep 1 9.5 Rep 1 23 Table 2 Trial 2 10 Rep 2 9.5 9.5 Rep 2 23 23 General Analysis for Pesticide Residue of the 500mL Rep 3 9.5 Rep 3 23 Isolated Vitamin C from Overripe Mango (M. indica) Rep 1 9.6 Rep 1 24 using Gas Liquid Chromatography Trial 3 10 Rep 2 9.0 9.2 Rep 2 23 23. 3

Rep 3 9.0 Rep 3 23 Limit of Rep 1 9.1 Rep 1 23 mL sample Analyze Pesticide Determination Remarks Residue Trial 4 10 Rep 2 9.5 9. 3 Rep 2 23 23 (LOD in ppm/ppb) Rep 3 9.2 Rep 3 23 Organophosphates 0.01ppm (parts per 500 (OPs) million) ND Rep 1 9.2 Rep 1 23 Trial 5 10 Rep 2 9.0 9.2 Rep 2 23 23.2 Organochlorines 0.5ppb (parts per 500 (OCs) billion) ND Rep 3 9.4 Rep 3 23.5 0.002ppm(parts Pyrethoids 500 per million) ND

Legend: ND= none detected

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The results presented upon analysis of the overripe mango isolate via From the results, the percent (%) Vitamin C ranges from 98.28% to gas liquid chromatography method on the presence of pesticide residues 100.53% with an average of 99.47%. Results revealed that from the six namely organophosphates (OPs), organochlorines (OCs) and pyrethoids assays made, trials two and three did not pass the USP specification of showed the absence of these pesticide residues in the sample. This indicates 99.5 to 100.5% Vitamin C with 98.28% and 98.72% respectively both that the pesticides utilized as plant growth regulator, defoliant desiccant lower than the USP limit. This indicates that errors may have occurred or agent for thinning fruit or preventing the premature fall of fruit and during titration. Furthermore, the Vitamin C molecule from overripe substances applied to crops to protect commodity from deterioration mango may have possibly undergone oxidation since it is the most during storage and transport were removed during isolation. One possible rapidly oxidized vitamin. Solutions of Vitamin C are strongly reducing reason is that once the harvested mango is cleaned off of any exuding latex and thus, the Vitamin may be rapidly oxidized (Remington, 2005). and treated with hot water and treated with fungicide to extend storage, thoroughly cleaned and washing,all pesticide residue are sufficiently The other four trials namely trial one, four, five and six have passed removed (Dorette, 2010). Likewise, peeling (and trimming) the fruit USP specification. Results obtained from the assays of the formulated material reduces the residues. Pesticide compounds that penetrate the syrup revealed that Vitamin C isolate from overripe mango could be epidermis may not be completely removed by washing, but by peeling, formulated into syrup formulation containing a standardized Vitamin C pesticide residues are comprehensively removed. This is confirmed by a content. Lastly, the tabular results presents that the average content of recent NCA study revealing peeling to be very effective in the removal of 99.7% Vitamin C obtained from six trials of assay passed the USP limit carbaryl, malathion and DDT from tomatoes, and of DDT from potatoes. content percent (%) Vitamin C indicating that the formulated Vitamin Similar observations have been made with other vegetables and fruits C syrup from overripe mango complies with USP standard assuring (Street, 1989). product content purity (Jenkins, 1995).

Table 3 Table 4 Assay of Formulated Ascorbic Acid Syrup (Vitamin C) Assay of Formulated Vitamin C (Ascorbic acid) Granular from Overripe Mango Powder from Overripe Mango (M. indica)

Formulated syrup % Vitamin C Formulated % Vitamin C No. of trials Iodine used No. of trials Granular powder Iodine used (99.5-100.5 USP Remarks 60mL (Sample (99.5-100.5 USP Remarks of titration (mL) of titration used mL) (mL) specification) (mg) specification) Trial 1 25 22.1 99.62 + Trial 1 50 44.1 99.84 + Trial 2 25 22.4 98.28 - Trial 2 50 43.7 100.76 - Trial 3 25 22. 3 98.72 - Trial 3 50 43.9 100.3 + Trial 4 25 21.9 100.53 + Trial 4 50 44.5 98.94 - Trial 5 25 22.1 99.62 + Trial 5 50 43.5 101.21 - Trial 6 25 22.0 100.07 + Average % Vitamin C 100.21 + Average % Vitamin C 99.47 + Legend: (+) passed; (-) failed Legend: (+) passed; (-) failed

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Results from the assay of formulated Vitamin C (ascorbic acid) Table 5 granular powder range from 98.94% to 101.21% of Vitamin C via Comparison of Commercial Ascorbic Acid (Vitamin C) Syrup and determination by direct titration of iodine.From the five trials conducted, Formulated Ascorbic Acid (Vitamin C) Syrup from Overripe mango three determinations did not pass the USP standard. Trials 2 and 5 at 100.76% and 101.21% respectively, were higher than USP limits Commercial Vitamin C Syrup Formulated Vitamin C Syrup while result from trial 4 at 98.94 was lower than USP specification. The Number Brand X of Trial noncompliant results indicate that the content of isolated Vitamin C may mL % mL % vary significantly when prepared as a granular powder. The processes sample Iodine Vitamin C sample Iodine Vitamin C involve in preparing the granular powder may affect the results of the Trial 1 25 21.9 100.53 25 22.1 99.62 assay. Further studies are required to obtain precise results. The lower Trial 2 25 22.1 99.62 25 22. 3 98.72 result of one assay may be due to oxidation of Vitamin C, where it is Trial 3 25 21.9 100.53 25 22. 0 100.07 rapidly oxidized by the presence of air, accelerated by heat (Remington, Trial 4 25 21.9 100.53 25 21.9 100.53 2005). Trial 5 25 21.9 100.53 25 22.0 100.07 Trial 6 25 22.1 99.62 25 22.1 99.62 However, trials 1 and 3 passed the specified limit of the USP for Trial 7 25 21.9 100.53 25 22.3 98.72 percent Vitamin C wherein it was recorded as 99.84% and 100.3%. Trial 8 25 21.9 100.53 25 22.1 99.62 This indicates that the formulated Vitamin C granular powder contains Trial 9 25 22.1 99.62 25 22.0 100.07 an allowable percentage of Vitamin C complying with the USP. Lastly, Trial 10 25 22.1 99.62 25 22.1 99.62 the average percent Vitamin C from the five trials, which passed USP Trial 11 25 21.9 100.53 25 21.9 100.53 standard indicate that the prepared granular powder can be a sufficient Trial 12 25 22.1 99.62 25 21.9 100.53 product form that complies with USP limits. Trial 13 25 22.1 99.62 25 22.3 98.72 Trial 14 25 21.9 100.53 25 22.3 98.72 Trial 15 25 21.9 100.53 25 22.1 99.62 Average % vitamin C 100.16 Average % vitamin C 99.56

The commercial Vitamin C syrup was used as the positive control to establish comparison from the researchers formulated Vitamin C syrup from overripe mango (M. indica). The results of the assay of the two Vitamin C syrup showed that formulated Vitamin C syrup from overripe mango (M. indica) had an average of 99.56 % Vitamin C while assay of the commercial vitamin C syrup labeled as Brand X= yielded an average of 100.16% Vitamin C. This implies that both vitamin C preparations passed the standard for Vitamin C syrup as presented by the allowable limit of the USP specification (USP-NF, 1995).

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Table 6 References Independent Sample Test of the Commercial and Formulated Vitamin C Syrup Levene’s Test for Equality of Casis, R. J. (2008). “The Climate Change Crisis: Global Legal Variances t-test for Equality of Means Framework, Policy Initiatives and the Philippine Response, Philippine Climate Change Policy: Mitigation and Adaptation Levene’s Test Measures”. for Equality t-test for Equality of Means of Variances 95% Dorette. (2010). Postharvest Handling Systems Perishable Food Confidence Crops F Sig. T Df Interval of the

Mean Difference Std. Error Difference Difference Jenkins. (1995). Jenkins Quantitative Pharmaceutical Chemistry

Sig. (2-tailed) Lower Upper

Remington, Gennaro. (2005). The Science and Practice of Pharmacy. 21st ed. Street. “Post-harvest Pathology”. .621 .437 2.439 28 .021 .51000 .20908 .08172 .93828 assumed

Percentage USP. (1995). The United States Pharmacopeia/National Formulary Equal variances

Legend: Ho: x1 = x2 Ha: x1= x 2

The researchers used t-test for independent samples to determine the difference in the means of percent Vitamin C between formulated and commercial Vitamin C syrup. Independent sample test showed that computed value of t = 2.439 is greater than the tabular value of t= 2.408, thus rejecting the null hypothesis (Ho) and accepting the alternative hypothesis (Ha). Results indicate that there is a significant difference between the two samples in terms of percent Vitamin C. Natural and synthetic ascorbic acid are chemically identical. As assessed by at least two studies, there appears to be no clinically significant difference in the bioavailability and bioactivity of natural ascorbic acid and synthetic ascorbic acid (Dorette, 2010).

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FLAVONOID SCREENING AND EVALUATION OF showed the highest viscosity value compared with syrup 1, 3 and plain FORMULATED SYRUP OF RIPE JACKFRUIT syrup. This suggests that syrup 2 may have higher soluble matters of (Artocarpus heterophyllus) FRUIT EXTRACT the extract present than syrups 1 and 3. May Florence D. Bacayo, Sydney Paole Anne G. Ceniza, Sheana Marie M. Montaño, Rose Anne V. Ubalde, Keywords: Pharmacology, phytochemical screening, flavonoid, formulated syrup, Artocarpus heterophyllus

Abstract

Flavonoids or bioflavonoids are basically polyphenols that have INTRODUCTION antioxidant, anti-inflammatory, anti-viral, anti-cancer, anti-tumor and anti-diarrheal properties. The study focused on the formulation and The plant kingdom has been the best source of remedies for curing characterization of syrup dosage form prepared with the extracted a variety of disease and pain. This is why medicinal plants have played flavonoids from ripe jackfruit (Artocarpus heterophyllus) fruit. The fruit a key role in the worldwide maintenance of health. Traditional herbal extract was obtained by maceration using 95% ethanol as solvent and medicine is intimately related to the Philippines popular culture; its separated from solvent by rotary evaporation. The gathered extract was use has origins based on ancestral knowledge. Natural products of tested for the presence of flavonoids. All three trials showed yellow higher plants are an important source of therapeutic agents; therefore, solution when the extract was reacted with diluted NaOH and turned many research groups are currently screening the different biological to colorless solution when reacted with diluted HCl, which implies activities of plants. Plants (fruits, vegetables, medicinal herbs, etc.) the presence of flavonoids in the extract. The extract was formulated may contain a wide variety of free radical scavenging molecules, such into a syrup and characterized by determining its color, odor, taste, as phenolic compounds (e.g. phenolic acids, flavonoids, quinones, pH and viscosity. The formulated syrup has a dark yellowish- brown coumarins, lignans, stilbenes, tannins), nitrogen compounds (alkaloids, color, sweet taste and odor, which resemble the physical properties of amines, betalains), vitamins, terpenoids (including carotenoids), and a medicated syrup. The pH of three syrups conforms to the specified some other endogenous metabolites, which are rich in antioxidant range which is greater than 5.4 but not less than 6.9. The researchers activity (Zheng and Wang, 2001 and Cai, et al., 2003). Epidemiological determined the viscosities of syrups in three trials. The viscosity of studies have shown that many of these antioxidant compounds possess plain syrup was also determined for comparison. The formulated syrup anti-inflammatory, antiatherosclerotic, antitumor, antimutagenic, 1, 2 and 3 took an average of 60.51 centipoise, 67.48 centipoise and anticarcinogenic, antibacterial, or antiviral activities to a greater or 65.06 centipoise to flow respectively, while the plain syrup took only lesser extent (Owen, et al., 2000; Sala, et al., 2002). Flavonoids or an average of 37.98 centipoise to flow. It shows that formulated syrup bioflavonoids are basically polyphenols that have antioxidant, anti- containing ripe jackfruit (Artocarpus heterophyllus) fruit crude extract is inflammatory, anti-viral, anti-cancer, anti-tumor and anti-diarrheal more viscous than the plain syrup. The statistical test proved that there properties. They are believed to improve natural immune response is an existing significant difference on the viscosity values of prepared of the body to fight against disease causing agents, allergens and also syrup, which means that the four types of syrup significantly differ in carcinogens. Therefore, they have been a subject of large number of viscosity. Post Hoc using Tukey’s HSD Multiple Comparison Test was researches, aimed towards unveiling the truths about flavonoids health conducted and results revealed that of the four types of syrup, syrup 2 benefits (Bora, 2011).

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New drugs may be discovered from a variety of natural sources or expansion of knowledge has been accompanied by an ever-increasing synthesized in the laboratory. They may be discovered by accident or as acceptance by orthodox medicine of the potential of plant products, the result of many years of tireless pursuit. Throughout history, plant as well as a growing demand for herbal medicines by the public at materials have served as a reservoir of potential new drugs. Yet only a large. Undoubtedly, the plant kingdom still holds may species of small portion of the known plants thus far have been investigated for plants containing substances of medicinal value which have yet to be medicinal activity. Certain major contributions to modern drug therapy discovered; large numbers of plants are constantly being screened for may be attributed to the successful conversion of botanic folklore their possible pharmacological value (Trease and Evans, 1983). remedies into modern wonder drugs (Ansel, 2005). Flavonoids are becoming very popular because they have many Herbal medicines are also known as natural medicines. health promoting effects. Some of the activities attributed to flavonoids Phytomedicines are according to the World Health Organization include: anti-allergic, anti-cancer, antioxidant, anti-inflammatory and (WHO), “Finished labelled medicinal products that contain as active anti-viral. The flavonoids quercetin is known for its ability to relieve ingredients, aerial or underground parts of plants, or other plant hay fever, eczema, sinusitis and asthma (Top Cultures, 2011). material, or combinations thereof, whether in the crude state or as plant preparations”. Plant material includes juices, gums, fatty oils, essential The researchers are interested in the formulation and evaluation oils, and any other substance of this nature. A World Health Organization of syrup with flavonoids containing crude fruit extract of ripe jackfruit (WHO) survey indicates that about 70-80% of the world population (Artocarpus heterophyllus). Specifically, it aimed to determine the particularly in the developing countries rely on non-conventional presence of flavonoids from the ripe fruit extract; determine the medicines mainly of herbal sources in their primary healthcare. WHO characteristics of the formulated syrup based on color, odor, taste, pH has described traditional medicine as one of the surest means to achieve and viscosity; identify if the syrup can conform to the specified range total health care coverage of the world’s population. In pursuance of its of pH. goal of providing accessible and culturally acceptable health care for the global population, WHO has encouraged the rational use of traditional plant based medicines by member states and has developed technical Materials and Methods guidelines for the assessment of herbal medicine. Research Design The phytochemical investigation of plant may thus involve the following: authentication and extraction of the plant material; This study employed an experimental type. The presence of separation and isolation of the constituents of interest; characterization flavonoids from ripe jackfruit (Artocarpus heterophyllus) fruit was the of the isolated compounds; investigation of the biosynthetic pathways basis for formulating into syrup. The pH and viscosity formulated syrup to particular compounds; and quantitative evaluations. Parallel to this were also determined. may be the pharmacological assessment of the separated components (Trease and Evans, 1983). Sampling Design

Rapid developments in phytochemistry and pharmacology have The selected medicinal plant, Jackfruit (Artocarpus heterophyllus) enourmously expanded the boundaries of pharmacognosy. This was grown and cultivated in Calinan, Davao City. It was then purchased

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by the researchers in Bankerohan, Davao City. The plant fruits were diluted HCl. The yellow solution with NaOH, which will turn randomly selected from one source. colorless with diluted HCl confirms flavonoids.

Locale of the Study Formulation of Syrup

The preparation of the ripe jackfruit (Artocarpus heterophyllus) fruit Ingredients Amount for extraction, formulation of dosage form and evaluation was performed Crude fruit extract 26.5 ml in the laboratory of University of the Immaculate Conception. Citric acid 0.72 g Sucrose 52.2 g Glycerin 3 ml Data Gathering Procedure Methyl Paraben Sodium 0.06 g Propyl Paraben Sodium 0.024 g Collection and Authentication of Saccharins 0.24 g Experimental Selected Medicinal Plants Distilled Water, qs ______Volume of dilution as needed 60 ml The selected medicinal plant, ripe jackfruit was purchased in Bankerohan, Davao City. The plant was collected with proper The ingredients for the formulation of Artocarpus heterophyllus handling and was placed in a black bag. The plant samples fruit extract formulated syrup were citric acid, saccharin, propyl were preserved for authentication by a botanist in Davao City. paraben sodium, methyl paraben sodium, glycerin, sugar and distilled water weighed and prepared. Distilled water was boiled Preparation of Selected Medicinal Plants in a clean beaker and allowed to stand until marked 60-70°C. Citric acid and sugar was then added and stirred to completely The freshly collected Artocarpus heterophyllus fruit was melt in water. When completely dissolved, the mixture was washed with running water. Two hundred grams (200 g) filtered while hot. This served as first solution. of plant material was cut into small pieces and placed in an Erlenmeyer flask and macerated in 95% ethanol for forty- In a separate beaker, glycerin was boiled and allowed eight hours (48 hours). The plant material was allowed to to stand until it reached 60°C. Then, methyl paraben sodium submerge completely in the solvent. After 48 hours, it was and propyl paraben sodium were added and dissolved into the filtered through a Buchner funnel and was subjected to Rotary boiling glycerin mixing this solution to the first solution served Evaporation machine at 40-60°C. as the main syrup.

Test for Flavonoids Saccharin sodium was dissolved into the distilled water. When dissolved completely, it was then added to the main The water extract of the selected medicinal plants was syrup to obtain 60 ml of fruit extract. The solution was stirred evaporated to dryness over a boiling water bath. The residue well and was allowed to cool at room temperature. Another was treated with diluted NaOH, followed by the addition of syrup was formulated but eliminating the extract.

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Evaluation of Formulated Syrup C. Viscosity Determination

A. Physical Properties

Color

The formulated syrup was placed in a clear bottle container and its color was evaluated against a white background. The clarity of the liquid dosage form was also observed.

Odor

A small amount of sample of formulated syrup was placed in a small beaker and the odor was evaluated and described by the researchers thru smelling. Figure 1. Ostwald Viscometer

Taste The Ostwald viscometer was clamped to an iron stand. A droplet of the formulated syrup was placed on the The plain syrup was pipetted to arm C until to the line above tongue of the researchers then the taste was evaluated and bulb A. Suction was applied to arm D and allowed the sample described. to flow up to bulb B. When the sample reached the bottom line of bulb B, the timer was started. The time was stopped until bulb B was already filled with the sample until the line above B. pH Determination it. The time was then recorded. After the formulated syrup was then tested for its viscosity. pH paper was used to determine the pH of the formulated Artocarpus heterophyllus crude fruit extract syrup. The pH range of the syrup should not be less than 5.4 and not more Statistical Treatment than 6.9. After all the needed data was gathered, statistical treatment was done. The statistical treatment used Post Hoc using Tukey’s HSD Multiple Comparison Test for the viscosity values.

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Results and Discussions In testing the physical quality of the formulated syrup, the researchers did the laboratory analysis for pH and viscosity. Table 1 Test for Presence of Flavonoids in Ripe Jackfruit (Artocarpus heterophyllus) Table 3 pH Values of Three Types of Formulated Syrup Reagent Positive Trial No. Used Result 1 2 3 pH values Trial No. Syrup 1 Syrup 2 Syrup 3 USP Diluted Yellow Positive Positive Positive Specification NaOH Solution 1 6.00 6.00 5.50 Diluted Colorless Greater than HCl Solution Positive Positive Positive 2 6.50 6.00 6.00 5.4 and less 3 6.00 6.50 6.00 than 6.9

The researchers conducted three trials for the determination of The researchers formulated three different batches of syrup, presence of flavonoids in ripe jackfruit (Artocarpus heterophyllus) fruit namely syrup 1, syrup 2 and syrup 3 using the same ingredients and extract. Based on the results gathered, all three trials showed positive procedures. In determining the pH of the three syrups formulated, three results. In all three trials had the same observation in which solution trials were performed. The pH results of all syrups were almost equal. turned into yellow upon the addition of diluted sodium hydroxide and The pHs of all the syrups were slightly acidic. The specified pH range turned into a colorless solution when diluted hydrochloric acid was was greater than 5.4 but less than 6.9. All of the formulated syrups added. It also shows the presence of flavonoids. conform to the specified range.

Evaluation of Formulated Syrup Table 4 Overall Data for the Viscosity Determination Table 2 Property and Observations of Formulated Syrup Viscosity (Centipoise) Trial No. Plain Syrup Syrup 1 Syrup 2 Syrup 3 Property Observation/s 1 38.51 60.61 66.90 65.31 Color Dark yellowish- brown 2 38.61 60.40 68.28 64.01 Odor Sweet 3 36.81 60.53 67.25 65.85 Taste Sweet Mean 37.98 60.51 67.48 65.06 S.D. 1.0116 0.1060 0.7174 0.9458 The results showed that the formulated syrup’s color was dark yellowish-brown. It has almost the same color as the extract of ripe The researchers formulated three different batches of syrup, namely jackfruit. It has a sweet odor and a sweet taste. syrup 1, syrup 2 and syrup 3 using the same ingredients and procedures.

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A plain syrup was also formulated for comparison. In determining their Table 6 viscosities, three trials were performed. The viscosities of the three Post Hoc Multiple Comparison Test of Viscosity formulated syrups were higher than the plain syrup, which means that of the Four Types of Syrup they are more viscous. Mean Tukey’s Critical HSD Mean Comparison Difference HSD (q) (q) value Decision The table above showed that syrup 2 has higher viscosity value than Plain syrup 1 and 3. This means that syrup 3 may have more soluble solids Syrup 1 22.53 49.92 4.53 Significant Syrup than the other prepared syrup. On the other hand, plain syrup showed Plain the east viscosity value of 37.98 when compared to the experimental Syrup 2 29.5 65.37 4.53 Significant Syrup syrup 1 to 3. Plain Syrup Syrup 3 27.08 60.01 4.53 Significant Table 5 Syrup 1 Syrup 2 6.97 15.44 4.53 Significant Testing the Significant Difference of the Viscosity Syrup 1 Syrup 3 4.55 10.08 4.53 Significant Values of Three Kinds of Formulated Syrup Syrup 2 Syrup 3 2.42 5.36 4.53 Significant

Source of Sum of Degrees of Mean Results revealed that of the four types of syrup, syrup 2 showed the variation Squares Freedom Square F P Decision highest viscosity value compared with syrup 1, 3 and plain syrup. This Between 1639.738 3 546.579 suggests that syrup 2 may have higher soluble matters present than Within 0.500 6 0.083 894.649 0.000 Significant syrup 1 and 3. On the other hand, plain syrup showed the least viscosity Total 0.731 8 values among the prepared syrup in this experiment. It also shows that the Ripe Jackfruit (Artocarpus heterophyllus) fruit extract could greatly Results of the statistical proved that there is an existing significant affect the viscosity of plain syrup. difference on the viscosity of prepared syrup. This means that the four types of syrup significantly differ in viscosity. To determine which of this syrup showed the highest or lowest viscosity value, Post Hoc using Tukey’s HSD Multiple Comparison Test was conducted.

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REFERENCES Halperin, M., Liemee, J., Matrick, N. (1995). USP XXII/ NF XVII. 22nd Ed. Phillips. p 1,982 Ansel, et al. (2005), Ansel’s Pharmaceutical Dosage Forms and Drug Delivery Systems. Lippincott Williams and Wilkin’s, Kokate, C.K., Purohit, A.P., Gokhale, S.B. (2002). Textbook Chicago of pharmacognosy, Nirali prakasan: Pune. 18:1-4 p.

Baliga, M. (2011). Phytochemistry, nutritional and Kotowaroo, M., et al. (2006). Screening of traditional pharmacological properties of Artocarpus heterophyllus antidiabetic medicinal plants of mauritius for possible α- Lam (jackfruit): A review. Retrieved last November 2011 amylase inhibitory effects in vitro. Retrieved November 2011 from http://www.sciencedirect.com/science/article/pii/ from http://onlinelibrary.wiley.com/doi/10.1002/ptr.1839/ S0963996911001372 abstract

Bora, C. (2011). Flavonoids Health Benefits. Retrieved from Lange, N.A., Ph.D. (1967). Lange’s Handbook of Chemistry 10th December 2011 from http://www.buzzle.com/articles/ Edition, p 1199 & 1680 flavonoids-health-benefits.html Morton, J. (1987). Jackfruit. p 58-64. In: Fruits of warm climates. Cai, Y., et al. (2004). Antioxidant activity and phenolic compounds Julia F. Morton, Miami, FL. of 112 traditional Chinese medicinal plants associated with anticancer. Life Sci., 74: 2157-2184 Oino Food Ltd. (2001). Glucose Syrup. Retrieved September 2011 from http://www.oinofood.com/Glucose%20Syrup.htm Department of Agriculture. (2010). Retrieved September 2011 from http://www.da.gov.ph/tips/jackfruit.pdf Owen, R. (2000). The antioxidant/anticancer potential of phenolic compounds isolated from olive oil. Eur. J. Cancer, 36: Elert, G. (2011). The Physics Hypertextbook. Retrieved September 1235-1247 2011 from http://physics.info/viscosity/ Peter, K. (2008). Underutilized and Underexploited Horticultural Feng, N. (1997). Scavenger and Antioxidant Properties of Crops. Retrieved November 2011 from http://www. Prenylflavones Isolated From Artocarpus Heterophyllus. vedamsbooks.com/no94814/cart.php Retrieved November 2011 from http://www.sciencedirect. com/science/article/pii/S0891584998000318 Prakash, O., et al. (2009). Artocarpus heterophyllus (Jackfruit): An Overview. Phcog Rev. 2009 Guevara, B., et al. (2005). A Guide to Plant Screening: Phytochemical & Biological (p 67-98). Manila: Research Center Quisumbing, E. (1951). Medicinal Plants of the Philippines, for the Natural ciences- University of Santo Thomas Department of Agriculture and Natural Resources Technical Bulletin 16, Manila.

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Sala, A., et al. (2002). Anti-inflammatory and antioxidant A COMPARATIVE STUDY ON THE PERCENTAGE CONTENT properties of Helichrysum italicum. J. Pharm. Pharmacol., 54: OF SYNTHETIC AND HERBAL SUPPLEMENTS OF 365-371 VITAMIN C AVAILABLE IN DAVAO CITY Donnah D. Nahial, Chresyl C. Alinsasaguin, Jenny R. Edulan, Sinko, P. (2006). Martin’s Physical Pharmacy and Pharmaceutical Arie Mae E. Radaza, Liza A. Tomimbang, Sciences. Lippincott Williams & Wilkins, 5th Edition

Stuart, G. (2011). Langka. Retrieved September 2011 from Abstract http://stuartxchange.com/Langka.html Vitamin plays in the normal functioning of the body. One of the Trease and Evans. (2002). Pharmacognosy Phytochemistry vitamins that the human body significantly need is ascorbic acid, Medicinal Plants, Lavoisier Pubs, Paris. commonly known by its biologically active form, as Vitamin C. This study sought to obtain the percentage content analysis of Vitamin C Top Cultures. (2011). Phytochemicals. Retrieved August 2011 supplements available in the market today. This included the range of from http://www.phytochemicals.info/phytochemicals/ synthetic and herbal Vitamin C supplements. In so doing, the researchers flavonoids.php proposed to broaden the knowledge on informed use of Vitamin C supplements. The researchers employed the alkaline cupric tartrate test University of Hawaii. (1996). Viscosity. Retrieved September to collect the Vitamin C and the iodimetric titration to further determine 2010 from http://www.spacegrant.hawaii.edu/class_acts/ the percentage content of ascorbic acid (Vitamin C) based on the United ViscosityTe.html States of Pharmacopeia/National Formulary method. Essentially, the percentage content analyses obtained from the sampled synthetic and Weng, R., et al. (2005). Antiinflammatory Flavonoids from herbal Vitamin C supplements do not conform with the United States Artocarpus heterophyllus and Artocarpus communis. Retrieved Pharmacopoeia standards. Most of the obtained results for percentage December 2011 from http://pubs.acs.org/doi/abs/10.1021/ content of Vitamin C supplement both for synthetic (Class X) and jf047873n herbal (Class Y) fall below the USP reference standard for Vitamin C (99-100.5). Necessary recommendations were then formulated after Zheng and Wang. (2001). Journal of Ethnopharmacology. the findings of this study. Elsevier Ireland Ltd., Volume 137, p 16-26 Keywords: Pharmacology, percentage content, iodimetric titration, 101 Healthy Recipes. (2010). About Jackfruit. Retrieved from synthetic ascorbic acid, herbal ascorbic acid http://www.101healthyrecipes.com/health-benefits-of-fruit/ jackfruit-fruit-facts-health-benefits-101.php

Introduction

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Vitamins are organic substances essential for the health, growth, in dietary supplements. Misleading information on supplement labels development and maintenance of the body. Additionally, vitamins happen to be the most common reason for confusion over these must be included in the diet since they cannot be fully synthesized ingredients. Consuming these potentially dangerous chemicals is much in the body. Each vitamin performs specific functions, if any of these more serious than once thought. We should be concerned about taking vitamins is lacking in the diet, biochemical alterations result in changes synthetic vitamins and other unnatural nutrients (Maffetone, 2008). in tissue/organ structure/function that subsequently results in clinical manifestations known as deficiency diseases (Remington, 2005). According to U.S. Dietary Supplement Health and Education Act (DSHEA) of 1994, herbal supplements are classified as dietary Long since has human beings been able to survive with the supplements and it can be sold without safety and efficacy testing vitamins acquired from natural foods they consume. In the advent (Gabardi,et. al, 2007). This paved the way to the growth of herbal of synthetic vitamins, however, this has expanded the wide array of medicines in the United States, which grew by 380% and the use of sources of vitamins that maybe acquired to maintain well-being. Today, vitamin therapy by 130% between 1991 to 1997. In Australia, 57% of a significant number of these vitamins are currently available in the the population now use some form of alternative medicine, in Germany, different pharmacies, which are made synthetically through chemical 46% and in France 49%. Today, 40% of American adults take vitamin processes rather than derived directly from plants or other materials supplements regularly and in the Western States, 66% of the adults are (Obikoya, 2010). vitamin users (Larsen, 2011).

Natural food complex vitamins are not all chemically identical to In the Philippines, the Public Health Ministry has taken up a policy isolated United States Pharmacopeia (USP) vitamins. Some synthetic to revitalize the use of herbal medicines, as the number of hospitals USP vitamins are analogous of the natural agents found to have vitamin in the country are not sufficient enough to provide services to people action, while, others have been shown to have no vitamin action. and solve health problems in the rural areas (Sandhyarani, 2011). Moreover, a few acts as vitamin antagonists and a number, which are Evidently, there is widespread appeal to the general population on use analogues of certain vitamins, produce deficiency symptoms specific to of traditional medicine. their vitamin analogues (Thiel, 2000). Traditional medicine is the sum total of knowledge, skills and Most supplement manufacturers claim that vitamins derived from practices based on the theories, beliefs and experiences indigenous natural sources have incomparable properties than synthetic vitamins, if to different cultures. Additionally, traditional medicines are used not are more superior. All supplements whether derived from natural or to maintain health, as well as to prevent, diagnose, improve or treat synthetic sources are created chemically in a laboratory. The difference physical and mental illnesses. Traditional medicines include herbs, in quality can be accounted to the percentage content, dosage amount herbal materials, herbal preparations, and finished herbal products that and the range of ingredients used in production. Synthetic vitamins are contain parts of plants or other plant materials as active ingredients. usually sold in the market at a lower cost due to the lack of necessary It is estimated that 80% of the earth’s population uses some form of molecular structure of the natural version (Berry, 2008). traditional medicine in their health care (WHO, 2008).

Health-conscious consumers choose to avoid artificial, unnatural The Food and Drug Administration (FDA) which is under the and synthetic chemicals, but often and unknowingly consume these Department of Health (DOH) warned the public to always check the

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product they are buying. Ensuring that before use, these products have Sampling Design been duly approved by the FDA and marked with the disclaimer “NO APPROVED THERAPEUTIC CLAIMS” (DOH, 2010). This research randomly collected samples from neighboring drugstores of various vitamin C supplements, which were synthetic and Remington (2005) emphasized the critical function that each herbal. In so doing, also obtained label claim of the formulations of the vitamin plays in the normal functioning of the body. One of the vitamins certain vitamins for further analysis and interpretation. that the human body significantly need is Ascorbic Acid, commonly known by its biologically active form, as Vitamin C. It is a water-soluble Locale of the Study vitamin with over 300 metabolic functions and is non-toxic in mega doses. In addition, it is a powerful antioxidant and has a positive effect The researchers collected synthetic and herbal Vitamin C for eradicating radiation from the body (USP/NF, 2007). supplements in selected drugstores in Davao City. The synthetic and herbal vitamin C supplements were brought to the Analytical Chemical With the above-mentioned issues, the researchers of this study Laboratory at the University of Immaculate Conception for the were inspired to determine the percentage content analysis of Vitamin preparation purposes and percentage content analyses. C supplements available in the market today. This included the range of synthetic and herbal Vitamin C supplements. In so doing, the researchers Data Gathering Instruments proposed to broaden the knowledge on informed use of synthetic and herbal supplements of vitamin C. Specifically, this aimed to determine the Synthetic and herbal Vitamin C supplements were gathered from percentage content analyses of the Vitamin C supplement from synthetic different drugstores in Davao City. Using iodimetric titration, the and herbal samples; to identify the distribution of data obtained for the percentage content of the samples was obtained. The instrument to be percentage content analyses of the Vitamin C supplement samples and used for the percentage content analyses of the sample supplements to evaluate the percentage content analyses obtained from the sampled were burette, iron stand, iron clamp, pipette and Erlenmeyer flask in synthetic and herbal supplements of Vitamin C compare with the United performing the iodimetric titration. States Pharmacopoeia (USP) reference standard for Vitamin C.

Vitamin C: Iodimetric Titration Materials and Methods A. Identification/Iodimetry Titration Research Design Ascorbic acid solution (1 g in 50 ml water) was prepared. This study utilized the experimental design to determine the Reduced the solution by alkaline cupric tartrate test solution percentage content of synthetic and herbal supplements of Vitamin at room temperature but more readily upon heating (USP, C and its comparability to reference standards by the United States 2007). Pharmacopeia (USP).

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B. Assay Results and Discussions

Dissolved about 400 mg Ascorbic Acid accurately weighed in a mixture of 100 ml of water and 25 mL 2 N sulfuric acid. Table 1 Add 3 ml of starch test solution and titrate at once with 0.1 N Percentage Content of Synthetic Vitamin C iodine VS. Each ml of 0.1 N iodine is equivalent to 8.806 of Supplements (Class X) C6H8O6 (USP, 2007). Synthetic Vitamin C Trial 1 Trial 2 Trial 3 Products (% content) (% content) (% content) C. Standardization of 0.1N Iodine Class X – 01 97.47 97.34 98.96 A 0.2 g of arsenic trioxide was weighted and dissolved Class X – 02 29.13 25.74 26.14 in 20 ml of 1N NaOH. This was dilute to 40 ml of distilled Class X – 03 53.02 52.91 53.74 water. Two drops of methyl orange was added and followed Class X – 04 90.35 90.33 81.20 by a gradual addition of 6 N HCl until yellow was changed to Class X – 05 96.59 95.66 97.25 pink. Then, 2 g of sodium bicarbonate was added and dilute Class X – 06 96.46 94.68 96.71 with 50 ml of distilled water. Three ml of starch TS was added. Class X – 07 105.34 104.07 104.32 Titration was done with iodine to a blue end point (USP, Class X – 08 102.04 101.79 101.79 2007). Class X – 09 101.02 100.26 103.31 Class X – 10 102.30 100.77 103.06 Class X – 11 97.39 98.98 94.88 Tools for Analysis Class X – 12 93.51 89.18 91.46 Class X – 13 77.55 75.72 78.00 The frequency distribution scale was utilized to show a summarized Class X – 14 76.91 76.18 81.19 grouping of data divided into mutually exclusive classes and the number Class X – 15 41.63 40.62 37.80 of occurrences in a class. Each entry in the table contains the count of the Class X – 16 92.76 96.80 96.00 occurrences of values within a particular group or interval, in this way, Class X – 17 98.62 94.58 94.99 data obtained in the study were clustered according to the following Class X – 18 86.70 87.71 89.53 intervals: above reference standard range, within reference standard Class X – 19 75.18 74.78 76.39 range and below reference standard range. This was facilitated using Class X – 20 50.12 51.13 49.92 the calculation of the range of the obtained percentage content data Class X – 21 91.53 94.82 94.82 from the experimentation. Percentage content analysis simply was used to represent raw streams of data as percentages on the content of active ingredient present in the sample for analysis. As shown in Table 1, the percentage content analyses of twenty- one (21) synthetic vitamin C supplements on three examined trials were relatively variable, from one production sample to another of the assayed synthetic vitamin supplement samples (n=21).

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Table 2 Table 3 Percentage Content of Herbal Vitamin C Frequency Distribution of the Percentage content Supplements (Class Y) of Class X Vitamin C Supplements

Naturally-derived Trial 1 Trial 2 Trial 3 Percentage Content Trial 1 Trial 2 Trial 3 Vitamin C Products (% content) (% content) (% content) (% content) Intervals ƒ % ƒ % ƒ % Class Y – 01 30.96 30.19 30.96 29 – 54 4 19 4 19 4 19 Class Y – 02 52.64 54.96 54.57 55 – 80 3 14 4 19 3 14 Class Y – 03 12.19 11.42 11.61 81 – 106 14 67 13 62 14 67 Class Y – 04 54.18 52.25 53.02 Total 21 100 21 100 21 100 Class Y – 05 103.82 105.85 105.85 *Range = 77.21, 78.33, 78.18; Width=26 Class Y – 06 105.09 104.07 105.59 Class Y – 07 106.10 106.61 104.83 Class Y – 08 95.95 94.17 93.66 Table 3 depicts the frequency distribution of the percentage content Class Y – 09 105.85 103.82 103.82 of Class X Vitamin C supplements (n=21). Clearly, the data revealed Class Y – 10 43.97 43.33 43.79 that majority of Class X Vitamin C supplements, fall on the upper interval Class Y – 11 43.56 43.11 46.76 close to the reference standard of USP. The values obtained for range of Class Y – 12 86.67 86.90 84.39 the three trials were 77.21, 78.33, and 78.18, which were approximated Class Y – 13 0.61 0.40 0.40 to 78 in order to group the three trials into three intervals. Class Y – 14 55.17 56.18 55.98 Class Y – 15 88.24 89.21 88.44 Table 4 Frequency Distribution of the Percentage content As shown in Table 2, the percentage content analyses of 15 herbal of Class Y Vitamin C Supplements vitamin C supplements on three examined trials were likewise relatively Trial 1 Trial 2 Trial 3 variable, from one production sample to another of the assayed herbal Percentage Content (% content) Intervals ƒ % ƒ % ƒ % vitamin supplement samples (n=15). 0 – 35 3 20 3 20 3 20 Second, the researchers of this study organized the data obtained 36 – 71 5 33 5 33 5 33 from the results of the assays of synthetic and herbal Vitamin C 72– 107 7 47 7 47 7 47 supplements. The grouped frequency distribution is shown on Tables 3 Total 15 100% 15 100 15 100 and 4 to depict the distribution of percentage content of Vitamin C of *Range = 105.49, 106.21, 105.38; Width=36 the two sample groups.

Table 4 shows the frequency distribution of the percentage content of Class Y Vitamin C supplements (n=15). Clearly, the data revealed

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that majority of Class Y Vitamin C supplements, fall on the upper interval As shown in Table 5, clearly on three trials conducted to assay the close to the reference standard of USP. However, a considerable number percentage content Vitamin C in the Class X Vitamin C Supplements belong to middle interval dose. The values obtained for range of the (Synthetic), did not meet the reference standard as indicated by United three trials were 105.49, 106.21 and 105.38, which were approximated States Pharmacopeia Specifications. Only on the first trial of Class X-17 to 106 in order to group the three trials into three intervals. depicted a value within reference standard, but inconsistently posted percentage content values on succeeding trials. The percentage content analyses for both Class X and Class Y Vitamin C supplements obtained range values of 78 and 106, respectively. Range of Class Y Vitamin C supplements is higher in comparison with Table 6 range of Class X Vitamin C supplements. Therefore, the percentage Frequency Distribution of the Percentage content of content of synthetic Vitamin C supplements are less dispersed and Class Y Vitamin C Supplements by United States revealed consistent percentage content analyses than herbal Vitamin C Pharmacopoeia Specifications supplements. Percentage Content Trial 1 Trial 2 Trial 3 Range (% content) Intervals ƒ % ƒ % ƒ % Third, the researchers sought to compare the percentage content analyses obtained from the sampled synthetic and herbal Vitamin C Below Reference Standard < 98 11 73 11 73 11 73 supplements with the United States Pharmacopoeia reference standard Within Reference Standard 99-100.5 ------Vitamin C content value. Tables 4.5 and 4.6 clustered the data obtained Above Reference Standard >100.5 4 27 4 27 4 27 in the study into the following intervals: above reference standard Total 15 100 15 100 15 100 range, within reference standard range and below reference standard *USP Reference Standards (99-100.5) range by United States Pharmacopoeia specifications.

As shown in Table 4.6, clearly on three trials conducted to assay Table 5 the percentage content Vitamin C in the Class Y Vitamin C Supplements Frequency Distribution of the Percentage content of (herbal), did not meet the reference standard as indicated by United Class X Vitamin C Supplements by United States StatesPharmacopeia Specifications, consistently on succeeding trials. Pharmacopoeia Specifications Evidently, the Vitamin C supplements available in the market do not qualify within the reference standard of USP specifications. Percentage Content Range (% content) Intervals ƒ % ƒ % ƒ % Below Reference Standard < 98 16 76 16 76 16 76 Analysis and Interpretation Within Reference Standard 99-100.5 1 5 - - 1 5 Above Reference Standard >100.5 4 19 5 24 4 19 Essentially, the percentage content analyses obtained from the Total 21 100 21 100 21 100 sampled synthetic and herbal Vitamin C supplements do not conform *USP Reference Standards (99-100.5) with the United States Pharmacopoeia standards. By U.S. Pharmacopeia (USP) standards which certifies that supplements meet certain quality,

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strength and purity standards as embodied in the Good Manufacturing REFERENCES Practices (GMP), the assayed Vitamin C supplements in this study have been found to be non conforming to the reference standards. Most of Berry, Lynn. (2008). Knowing the Difference Between Natural the obtained results for percentage content of Vitamin C supplements and Synthetic both for synthetic (Class X) and herbal (Class Y) fall below the USP reference standard for Vitamin C (99-100.5). Vitamins Is Vital to Good Health, from http://www. naturalnews.com/0241716 Natural Vitamins Synthetic. The Nutritional Supplement industry must therefore be closely html#ixzzlZdRPy6sf. regulated. This is apparent from the results obtained in this study which may be due to rapid appearance of Vitamin C supplements in the market Cvetković, Biljana R., et al. (2009). Effect of Preservation which fail to comply with USP reference standards and poor regulatory Method and Storage Condition on Ascorbic Acid Loss laws and surveillance. in Beverages, from http://www.doiserbia.nb.rs/img/ doi/14508/2009/1450_71880940001C.pdf However, one reason may play in part of why the results obtained were as such, considering that although USP sets standards for dietary Herbal medicine’s great strength. (2009). In The Herbarium. supplements, regulations for dietary supplements are very different than Retrieved. August 8, 2010 from http://theherbarium. those for drugs; manufacturers may voluntarily choose to meet USP wordpress.com/2009/02/01/transition-herbal medicine/ standards but are not required to do so. Nonetheless, under the law, products that claim to conform with USP standards on their labels must Herbal Supplements Are. (2009). In NCAM National Center for do so or they may be deemed to be misbranded per FDA regulations. Complementary and Alternative Medicine. Retrieved August 10, 2010 from http://nccam.nih.gov/health/supplements/ United States Pharmacopeia (USP) plays an important role in wiseuse.htm. peoples’ general health and well-being. It sets standards so that there is uniform, consistent quality that consumers can confidently expect when Larsen, H.R. (2011). Alternative Medicine: Why so popular? they buy and use these products. In the light of the findings obtained in from http://www.yourhealthbase.com/alternative medicine. this study, about sub-standard dietary supplement products, this added htm assurance of quality is thus important. More importantly, to consumers who may rely on supplements to generally enhance their health or to Lipasek, Rebecca A., et al. (2011). “Effects of Anticaking meet a specific dietary need based on a vitamin deficiency. Agents and Relative Humidity on the Physical and Chemical Stability of Powdered Vitamin C”, from http://www. healthandwellness360.com/summaries/anti-caking-agents- added-to- powders-hasten-degradation-of-vitamin-c.html.

Marshall, Charles W. (2002). Vitamin C: Do High Doses Prevent Colds? From http://www.quackwatch.com/ 01QuackeryRelatedTopics/DSH/colds.html

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Obikoya, G. (2010). Natural Vitamins vs. Synthetic from PHYSICAL PROPERTIES OF FORMULATED SEMISOLID http://www.vitamins-nutrition.org/vitamins/natural/natural- DOSAGE FORMS (OINTMENT, GEL AND CREAM) vitamins- synthetic.html OF HAULI (Ficus septica, Moraceae) LEAF CRUDE EXTRACT Pauling, Linus. (2002). “Vitamin C,” - Vitamin Intake and Florie C. Casalan, Romma Ninnah O. Egar, Joseph Pet F. Lusay, Health: A Scientific Review, from http://www.healingdaily. Ma. Lady Dianne A. Paccial, Michelle A. Peñafiel com/detoxification-diet/vitamin-c.htm.

Limuaco, O.M. & Cruz, M.J.C. (2009). Pharmaceutical Abstract Jurisprudence and Ethics. The study investigated if Hauli (Ficus septica, Moraceae) leaf crude Qiao J., et al. (2011). Proteomic identification of the related extract can be suitably formulated into gel, cream, and ointment semi- immune- Enhancing proteins in shrimp Litopenaeus vannamei solid dosage forms. Researchers formulated semisolid dosage forms and stimulated with vitamin C and Chinese herbs, from http:// determined the following: conformation of the product formulations to www.ncbi.nlm.nih.gov/pubmed/21767650. the USP guideline in terms of the minimum fill test, physical properties of the three formulated semisolid dosage forms and skin sensitivity Sia Su, G.L. and Kayali, S. (2008). Blood vitamin C levels effect to human subjects. Minimum fill test was performed to compare of motorized tricycle drivers in Parañaque, Philippines, from the weight or volume of the product filled into each container with their http://www.ncbi.nlm.nih.gov/pubmed/18716387. labeled weight or volume. This helps in assessing the content uniformity of the products. Results showed that 73% of the formulations passed Thiel, R.J. (2000). Natural Vitamins May be Superior to Synthetic the USP guidelines in relation to the minimum fill test. Identification of Ones from http://www.ncbi.nlm.nih.gov/pubmed/11090291 the Physical Properties such as texture, melting point, and consistency of the three formulations was also conducted. Results showed that the United States Pharmacopeia 30 – National Formulary 25 three formulations conformed to the ideal physical properties of semi- Page 1441. 2007. solid dosage forms, in terms of texture, melting point, and consistency. Determination of seven-day cumulative irritancy patch test of the three Williams, Lippincott, et al. (2005). Remington “The Science formulations was also conducted. Results demonstrated that all of the and Practice of Pharmacy” 21st Edition. respondents showed no visible reaction towards the three formulated dosage forms within seven days. Therefore, the three formulated semisolid dosage forms when used on human skin for seven days, was proven to be gentle and non-irritating.

Keywords: Ficus septica, semisolid preparations, minimum fill test

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INTRODUCTION cycle to ensure that the sampled filled products will meet quality control specifications based on the USP ^755 & Minimum Fill or ^698& In Massachussetts, there are problems encountered in Deliverable Volume tests. pharmaceutical creams and ointments: When added to water, thickening, stabilizing and emulsifying ingredients can form agglomerates which The common acceptance criterion of the two USP tests is that the agitators cannot break down. Similarly oil phase ingredients can average content of all samples tested must not be less than 100% of form lumps which require shear to disperse, Ingredients must be fully the labeled amount. Such a requirement will lead to a filling volume hydrated to obtain the required viscosity and develop yield, Partially target greater than 100% of the labeled amount. This article proposes a hydrated materials can build up on the vessel wall, in-tank baffles criterion for establishing an appropriate target fill such that a sample will and parts of the agitator, Agitators cannot sufficiently reduce droplet have a 95% probability of passing these USP tests at 95% confidence, size to form a stable emulsion. Active ingredients can be temperature i.e., that the established target fills will guarantee with 95% confidence sensitive, cooling of the product before adding the active ingredient that 95 out of 100 samples will pass the USP tests. further increases processing time. Poor dispersion of the active impairs product effectiveness, Long mixing times and additional equipment This study aimed to determine whether the Hauli (Ficus septica, may be required to obtain a homogeneous and stable finished product Moraceae) extract could be suitably formulated to semisolid dosage (Silverson, 2008) forms such as cream, gel and ointment, against fungal infection. Specifically, this aimed to determine the physical properties of the Semisolid pharmaceutical systems comprise a body of products, formulated cream, gel, and ointment in terms of appearance, odor, which when applied to the skin or accessible mucous membranes tend consistency, texture, melting point, solubility, and dermal toxicity; to to alleviate or treat a pathological condition or offer protection against assess if dosage forms conform to USP compendial requirement and to a harmful environment. They have the property to cling to the skin evaluate the irritating effect on the skin of human subjects. or mucous membrane for a protracted period of time to exert their therapeutic effect through protection and occlusion. The adhesion is due to their plastic rheologic behavior, which allows semisolid to Materials and Methods retain their shape and cling as film until acted upon by an outside force. Semisolid dosage forms can be applied topically to the skin, cornea, Research Design rectal tissue, nasal mucosa, vagina, buccal tissue, urethral membrane, and external ear lining. This research utilized the experimental design in the determination of the physical properties of the three formulated semisolid dosage The USP (755) Minimum Fill test applies to liquids, semisolids, and forms solids such as creams, gels, jellies, lotions, ointments, pastes, powders, and aerosols, including pressurized and nonpressurized topical sprays Data Gathering that are packaged in containers in which the labeled amount is not more than 150 g or 150 mL. Meeting the USP requirements for minimum In this study the data investigated through various measures fill and deliverable volume is a serious concern in pharmaceutical depending on the test. For minimum fill test was measured through the production. Filling operations must be controlled throughout the filling weight content of the product and container.

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A. Collection of Plant materials E. Cream Formulation of Hauli leaf extract

A non-probability purposive sampling design was used in The effective concentration that was incorporated in the selection of plant sample. The plant material was procured the formulation of Hauli cream was based on the minimum from Don Isidro, Bankerohan, Davao City. The leaves were inhibitory concentration of the past study having the same washed with distilled water to remove the dirt and dust and plant material. A mixture of 2 grams of beeswax, 2.4 ml of cut into pieces. mineral oil, and 0.04 grams of methyl paraben with distilled water were heated. With rigorous stirring, water was added B. Preparation and Extraction of Plant part to the obtained solution. 1.6 ml of Propylene glycol and 0.05 grams of Hauli leaf extract was heated until it was dissolved, Six hundred (600) grams of Hauli leaves were collected mixed with aqueous solution, and continued to stir during with 1000 ml of 80% ethanol in 1000 ml Erlenmeyer flask, cooling at room temperature. stoppered with aluminum foil for 24-48 hours, filtered in Buchner funnel using a vacuum pump and the solvent was F. gel Formulation removed under vacuum using a rotary evaporator (Vital, et al., 2009). The formulation of Hauli gel will be based on the preparation of Carbomer gel. The effective concentration that C. Preparation of Yellow Ointment Base was incorporated in the formulation of Hauli gel was based on the minimum inhibitory concentration of the past study Five (5) grams of yellow wax was melted in a suitable dish having the same plant material. The parabens were dissolved and fifteen (15) grams of white petrolatum are added. The in 19 mL of water with the aid of heat and allowed to cool. mixture was heated until it liquefies. This was continuously The gelatin (in replacement of carbomer) was added in small stirred until it congealed. amounts to the solution until a smooth dispersion is obtained. The preparation was allowed to stand, permitting entrapped D. Ointment formulation of Hauli leaf extract air to separate. Then the neutralizing agent, triethanolamine, was added very slowly to avoid entrapping air. The effective concentration that was incorporated in the formulation of Hauli ointment was based on the minimum G. Minimum fill test inhibitory concentration of the past study having the same plant. A 0.05 grams of Hauli crude leaf extract was added with This test was performed to compare the weight or volume sufficient quantity of white ointment as base to make 20 grams of product filled into each container with their labeled weight and stirred until the mixture was congealed. The ointment or volume. This helped in assessing the content uniformity of was preserved in a well-closed container for physical tests product. There were 10 containers used having the same weight (Guevara, et al., 2005). of 20 grams. Initially, labels from the product containers were removed. After washing and drying the surface, the weights were recorded. Second step, the entire product from each

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container was removed. After cleaning and drying, the weights Results and Discussions were recorded. The difference between the total weight (W1) and empty container weight (W2) gives the weight of the product. Table 1 The Physical Properties of Three Formulated Dosage Forms H. Melting Point Physical Cream Gel Ointment Three capillary tubes were used separately having the properties three formulated semisolid dosage forms with a height of 0.3 Appearance White Transparent Translucent cm. The capillary tubes were tied together with the 360 degrees Odor Odorless Odorless Waxy odor thermometer and submerged in a heated vegetable oil. Consistency homogeneous homogeneous homogeneous

Texture Smooth, Non Smooth, Non Smooth, greasy I. Test for solubility greasy greasy Melting point 59°C 10°C 59°C Two (2) mL of water was placed in three test tubes Solubility Slightly soluble Soluble Insoluble separately with a pinch of the different formulated semisolid dosage forms (cream, gel and ointment) with vigorous shaking for at least three minutes. The table above presents the physical properties of the three formulated semisolid dosage forms (cream, gel and ointment) of Hauli J. 7-day cumulative irritancy patch test (Ficus septica, Moraceae).

In this test, the formulated semisolid dosage forms (cream, Results show that the three semisolid dosage forms (cream, gel gel and ointment) are applied on the back or volar forearm. and ointment) meet the ideal properties of semisolid dosage forms for The formulations were applied topically. The applications were it follows the requirements based on USP/NF. repeated daily for 7 days. Twenty-four subjects were used in this test. In the formulation of a vehicle for topical drug application, many factors must be considered. Drug stability, intended product use, site of application, and product type must be in combined in a dosage form or delivery system that will release the drug readily when place in contact with the skin. Further, the release characteristic of the vehicle depend on the physical - chemical properties of the specific drug substance to be delivered to the skin: the drug release from a vehicle is a function of the drug concentration and solubility in the vehicle, and the drugs partition coefficient between the vehicle and the skin (Remington, 2006).

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Table 2 Table 3 Minimum Fill Test Data for Ointment Minimum Fill Test Data for Cream

Ointment Cream No. of No. of Weight of Empty Average Net Weight of Empty Total weight Average Net Total weight Container Container Content (90%) Container Container Content (90%) 1 8.37 29.96 19.431% 1 8.55 28.32 17.793% 2 8.63 29.93 19.170% 2 8.26 29.40 19.026% 3 8.46 28.62 18.144% 3 8.38 28.60 18.198% 4 8.43 29.65 19.098% 4 8.31 29.70 19.251% 5 8.79 29.40 18.549% 5 8.77 29.20 18.870% 6 8.72 28.87 18.135% 6 8.58 29.60 18.918% 7 8.37 28.65 18.252% 7 8.25 28.15 17.910% 8 8.97 29.09 18.108% 8 8.78 28.18 19.400% 9 8.80 28.70 17.910% 9 8.97 28.26 17.667% 10 8.96 29.67 18.639% 10 8.29 29.20 18.819%

The table above represents the different weights of containers with The table above represents the different weights of containers with and without the product that are used in the formulation of ointment of and without the product that are used in the formulation of cream of Hauli leaf crude extract. Hauli leaf crude extract.

Results show that the net content of nine out of ten containers Results show that the net content of seven out of ten containers having the formulated semisolid product (ointment) are less than 90% having the formulated semisolid product (cream) are less than 90% of of the labeled amount where the labeled amount is 20g. the labeled amount where the labeled amount is 20 g.

There are factors to be considered that affects the original volume of the products (cream, gel and ointment), factors like, in weighing variations - while doing the weighing of ingredients, it was oversaw; human errors - unintentional mistakes; and small amount in production.

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Table 4 Table 5 Minimum Fill Test Data for Gel Day 1 Cumulative Irritancy Patch Test

Results Gel No. of Remarks No. of Subjects Weight of Empty Total weight Average Net Cream Gel Ointment Container Container Content (90%) 1 8.47 28,26 18.711% 1 0 0 0 no visible reaction 2 8.68 29.30 18.588% 2 0 0 0 no visible reaction 3 8.75 28.40 17.685% 3 0 0 0 no visible reaction 4 8.35 28.38 18.027% 4 0 0 0 no visible reaction 5 8.23 29.70 19.323% 5 0 0 0 no visible reaction 6 8.43 29.10 18.603% 6 0 0 0 no visible reaction 7 8.97 29.12 18.135% 7 0 0 0 no visible reaction 8 8.50 28.10 17.640% 8 0 0 0 no visible reaction 9 8.42 28.12 17.730% 9 0 0 0 no visible reaction 10 8.46 28.05 17.631% 10 0 0 0 no visible reaction 11 0 0 0 no visible reaction 12 0 0 0 no visible reaction The table above represents the different weights of containers with 13 0 0 0 no visible reaction and without the product that are used in the formulation of gel of Hauli 14 0 0 0 no visible reaction leaf crude extract. 15 0 0 0 no visible reaction 16 0 0 0 no visible reaction Results show that the net content of six out of ten containers having 17 0 0 0 no visible reaction the formulated semisolid product (gel) are less than 90% of the labeled 18 0 0 0 no visible reaction amount where the labeled amount is 20 g. 19 0 0 0 no visible reaction 20 0 0 0 no visible reaction 21 0 0 0 no visible reaction 22 0 0 0 no visible reaction 23 0 0 0 no visible reaction 24 0 0 0 no visible reaction

Legends: 0 - No visible reaction 1 - Minimal erythema, barely perceptible 2 - Definite erythema, readily visible; minimal edema or minimal papular response 3 - Erythema and papules 4 - Intense erythema with edema vesicular erosion.

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Lanman, et al. (1998), reported that several days of repeated Table 6 exposures to mildly irritating cosmetic products produced a method Day 2 Cumulative Irritancy Patch Test to discriminate among low-level irritants. With modifications, 7-day cumulative irritancy patch test remains the gold standard test for Results No. of determining a product’s potential for inducing cutaneous irritation. Subjects Remarks Cream Gel Ointment The table above represents the results obtained from 24 subjects 1 0 0 0 no visible reaction who were then applied with the formulated semisolid dosage forms 2 0 0 0 no visible reaction (cream, gel and ointment) of Ficus septica crude extract. 3 0 0 0 no visible reaction 4 0 0 0 no visible reaction Results show that all of the respondents showed no visible reaction 5 0 0 0 no visible reaction towards the three formulated dosage forms (cream, gel and ointment) 6 0 0 0 no visible reaction on day 1. Having a rating of zero (0) indicates no visible reaction. 7 0 0 0 no visible reaction 8 0 0 0 no visible reaction Therefore, the three formulated semisolid dosage forms (ointment, 9 0 0 0 no visible reaction gel, and cream) of the leaf crude extract when used on human skin for 10 0 0 0 no visible reaction a day, is proven to be gentle and non-irritating. 11 0 0 0 no visible reaction 12 0 0 0 no visible reaction 13 0 0 0 no visible reaction 14 0 0 0 no visible reaction 15 0 0 0 no visible reaction 16 0 0 0 no visible reaction 17 0 0 0 no visible reaction 18 0 0 0 no visible reaction 19 0 0 0 no visible reaction 20 0 0 0 no visible reaction 21 0 0 0 no visible reaction 22 0 0 0 no visible reaction 23 0 0 0 no visible reaction 24 0 0 0 no visible reaction

Legends: 0 - No visible reaction 1 - Minimal erythema, barely perceptible 2 - Definite erythema, readily visible; minimal edema or minimal papular response 3 - Erythema and papules 4 - Intense erythema with edema vesicular erosion.

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The table above represents the results obtained from 24 subjects Table 7 who were then applied with the formulated semisolid dosage forms Day 3 Cumulative Irritancy Patch Test (cream, gel and ointment) of F. septica crude extract. Results No. of Results show that all of the respondents showed no visible reaction Subjects Remarks towards the three formulated dosage forms (cream, gel and ointment) Cream Gel Ointment on day 2. Having a rating of zero (0) indicates no visible reaction. 1 0 0 0 no visible reaction 2 0 0 0 no visible reaction Therefore, the three formulated semisolid dosage forms (ointment, 3 0 0 0 no visible reaction gel, and cream) of the leaf crude extract when used on human skin for 4 0 0 0 no visible reaction 2 days, is proven to be gentle and non-irritating. 5 0 0 0 no visible reaction 6 0 0 0 no visible reaction 7 0 0 0 no visible reaction 8 0 0 0 no visible reaction 9 0 0 0 no visible reaction 10 0 0 0 no visible reaction 11 0 0 0 no visible reaction 12 0 0 0 no visible reaction 13 0 0 0 no visible reaction 14 0 0 0 no visible reaction 15 0 0 0 no visible reaction 16 0 0 0 no visible reaction 17 0 0 0 no visible reaction 18 0 0 0 no visible reaction 19 0 0 0 no visible reaction 20 0 0 0 no visible reaction 21 0 0 0 no visible reaction 22 0 0 0 no visible reaction 23 0 0 0 no visible reaction 24 0 0 0 no visible reaction

Legends: 0 - No visible reaction 1 - Minimal erythema, barely perceptible 2 - Definite erythema, readily visible; minimal edema or minimal papular response 3 - Erythema and papules 4 - Intense erythema with edema vesicular erosion.

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The table above represents the results obtained from 24 subjects Table 8 who were then applied with the formulated semisolid dosage forms. Day 4 Cumulative Irritancy Patch Test Results show that all of the respondents showed no visible reaction towards the three formulated dosage forms (cream, gel and ointment) Results No. of on day 3. Having a rating of zero (0) indicates no visible reaction. Subjects Remarks Cream Gel Ointment

Therefore, the three formulated semisolid dosage forms when used 1 0 0 0 no visible reaction on human skin for 3 days, is proven to be gentle and non-irritating. 2 0 0 0 no visible reaction 3 0 0 0 no visible reaction 4 0 0 0 no visible reaction 5 0 0 0 no visible reaction 6 0 0 0 no visible reaction 7 0 0 0 no visible reaction 8 0 0 0 no visible reaction 9 0 0 0 no visible reaction 10 0 0 0 no visible reaction 11 0 0 0 no visible reaction 12 0 0 0 no visible reaction 13 0 0 0 no visible reaction 14 0 0 0 no visible reaction 15 0 0 0 no visible reaction 16 0 0 0 no visible reaction 17 0 0 0 no visible reaction 18 0 0 0 no visible reaction 19 0 0 0 no visible reaction 20 0 0 0 no visible reaction 21 0 0 0 no visible reaction 22 0 0 0 no visible reaction 23 0 0 0 no visible reaction 24 0 0 0 no visible reaction

Legends: 0 - No visible reaction 1 - Minimal erythema, barely perceptible 2 - Definite erythema, readily visible; minimal edema or minimal papular response 3 - Erythema and papules 4 - Intense erythema with edema vesicular erosion.

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Results show that all of the respondents showed no visible reaction Table 9 towards the three formulated dosage forms (cream, gel and ointment) Day 5 Cumulative Irritancy Patch Test on day 4. Having a rating of zero (0) indicates no visible reaction. Results Therefore, the three formulated semisolid dosage forms when used on No. of human skin for 4 days, is proven to be gentle and non-irritating. Subjects Remarks Cream Gel Ointment 1 0 0 0 no visible reaction 2 0 0 0 no visible reaction 3 0 0 0 no visible reaction 4 0 0 0 no visible reaction 5 0 0 0 no visible reaction 6 0 0 0 no visible reaction 7 0 0 0 no visible reaction 8 0 0 0 no visible reaction 9 0 0 0 no visible reaction 10 0 0 0 no visible reaction 11 0 0 0 no visible reaction 12 0 0 0 no visible reaction 13 0 0 0 no visible reaction 14 0 0 0 no visible reaction 15 0 0 0 no visible reaction 16 0 0 0 no visible reaction 17 0 0 0 no visible reaction 18 0 0 0 no visible reaction 19 0 0 0 no visible reaction 20 0 0 0 no visible reaction 21 0 0 0 no visible reaction 22 0 0 0 no visible reaction 23 0 0 0 no visible reaction 24 0 0 0 no visible reaction

Legends: 0 - No visible reaction 1 - Minimal erythema, barely perceptible 2 - Definite erythema, readily visible; minimal edema or minimal papular response 3 - Erythema and papules 4 - Intense erythema with edema vesicular erosion.

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Results show that all of the respondents showed no visible reaction Table 10 towards the three formulated dosage forms on day 5. Having a rating of Day 6 Cumulative Irritancy Patch Test zero (0) indicates no visible reaction. Therefore, the three formulated Results semisolid dosage forms of leaf crude extract when used on human skin No. of for 5 days, is proven to be gentle and non-irritating. Subjects Remarks Cream Gel Ointment 1 0 0 0 no visible reaction 2 0 0 0 no visible reaction 3 0 0 0 no visible reaction 4 0 0 0 no visible reaction 5 0 0 0 no visible reaction 6 0 0 0 no visible reaction 7 0 0 0 no visible reaction 8 0 0 0 no visible reaction 9 0 0 0 no visible reaction 10 0 0 0 no visible reaction 11 0 0 0 no visible reaction 12 0 0 0 no visible reaction 13 0 0 0 no visible reaction 14 0 0 0 no visible reaction 15 0 0 0 no visible reaction 16 0 0 0 no visible reaction 17 0 0 0 no visible reaction 18 0 0 0 no visible reaction 19 0 0 0 no visible reaction 20 0 0 0 no visible reaction 21 0 0 0 no visible reaction 22 0 0 0 no visible reaction 23 0 0 0 no visible reaction 24 0 0 0 no visible reaction

Legends: 0 - No visible reaction 1 - Minimal erythema, barely perceptible 2 - Definite erythema, readily visible; minimal edema or minimal papular response 3 - Erythema and papules 4 - Intense erythema with edema vesicular erosion.

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Results show that all of the respondents showed no visible Table 11 reaction towards on day 6. Having a rating of zero (0) indicates no Day 7 Cumulative Irritancy Patch Test visible reaction. Hence, the three formulated semisolid dosage forms Results when used on human skin for 6 days, is proven to be gentle and non- No. of irritating. Subjects Remarks Cream Gel Ointment 1 0 0 0 no visible reaction 2 0 0 0 no visible reaction 3 0 0 0 no visible reaction 4 0 0 0 no visible reaction 5 0 0 0 no visible reaction 6 0 0 0 no visible reaction 7 0 0 0 no visible reaction 8 0 0 0 no visible reaction 9 0 0 0 no visible reaction 10 0 0 0 no visible reaction 11 0 0 0 no visible reaction 12 0 0 0 no visible reaction 13 0 0 0 no visible reaction 14 0 0 0 no visible reaction 15 0 0 0 no visible reaction 16 0 0 0 no visible reaction 17 0 0 0 no visible reaction 18 0 0 0 no visible reaction 19 0 0 0 no visible reaction 20 0 0 0 no visible reaction 21 0 0 0 no visible reaction 22 0 0 0 no visible reaction 23 0 0 0 no visible reaction 24 0 0 0 no visible reaction

Legends: 0 - No visible reaction 1 - Minimal erythema, barely perceptible 2 - Definite erythema, readily visible; minimal edema or minimal papular response 3 - Erythema and papules 4 - Intense erythema with edema vesicular erosion.

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Results show that all of the respondents showed no visible reaction REFERENCES towards the three formulated dosage forms on day 7. Having a rating of zero (0) indicates no visible reaction. Therefore, the three formulated Amander, B.S.R., et al. (2008). Mutagenicity Study of Tawa- semisolid dosage forms when used on human skin for 7 days, is proven tawa (Euphorbia hirta Euphotbiae) Plant Decoction. Unpublished to be gentle and non-irritating. Thesis: University of the Immaculate Conception.

Dwyer, J. and Rattray, D. Magic. (1990). Medicine of Plants. 5th edition. November. The Reader’s Digest Association in the United States of America.

Espina, A.R., et al. (2009). Formulations of antimicrobial ointment from Kamatigi (Impatients balsamina) crude leaf extract against Trichophyton mentagrophytes and Streptococcus pyogenes.

Faergemann J. Retrieved from http://www.ncbi.nlm.nih.gov/ pubmed/8255067

Gouda. 2010. Retrieved from http://www.sciencedirect.com/ science/article/pii/S1319016411000089

Goun, E., Cunningham, G., Chu, D., Nguyen, C. & Miles, D. (2003). Antibacterial and antifungal activity of Indonesian ethnomedical plants. Department of Chemistry, University of Central Florida, Orlando, FL 32816, USA.

Gran, P.F., et al. (2011). Antifungal and Mutagenic properties of dried Lagnub (Ficus septica, Moraceae) leaf crude extract.

Herbs. (2000). Retrieved from http://www.herbs2000.com/ herbs/herbs_maidenhair_fern.htm

Jain, Sourabh. (2010). In Process Quality Control in Pharmaceutical http://www.authorstream.com/Presentation/ srbmch-1219158-sourabh-jain/

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Micromedex™ (updated Aug. 11th, 2011), Cerner Multum™ THE ANTITUSSIVE EFFECT OF FORMULATED SYRUP FROM (updated Aug. 10th, 2011), Wolters Kluwer™ (updated Aug. TROMPANG ELEPANTE (Heliotropium indicum Linn.) 2nd, 2011) and others. Retrieved from http://www.drugs.com/ LEAF EXTRACT IN CITRIC ACID COUGH-INDUCED sfx/ketoconazole-sideeffects.html GUINEA PIGS Donnah D. Nahial, Rocel Joy C. Obra, Jennifer Gwen L. Santiago, Mosby’s Dictionary (Medical, Nursing and Allied Health). 3rd Emily R. Vasquez Edition. The C.V. Mosby Company. 18830 Wesline Drive, St. Louis, Missouri, 63146, 1990. ABSTRACT Quisumbing, Eduardo. (1978.) Medicinal Plants of the Philippines. Quezon city, Philippines: The Katha Publishing The study endeavored to validate the antitussive activity of Co, Inc., Remington: The Science and Practice of Pharmacy. Trompang Elepante (Heliotropium indicum Linn.) leaf extract. It contains Easton, Pennsylvania 18042: Mack Publishing Company, alkaloidal principle, specifically heliotrine, a pyrrolizidine alkaloid 1399:1400:1585-1588:1589. that inhibits the cough reflex by the inhibition of acetylcholine to bind at M3 receptors, thus smooth muscle contraction is suppressed. The Steiner, et al. (2002). Retrieved from http://www.scielo.br/ study shows that Trompang elepante leaf extract does not contain toxic scielo.php?pid=S0365-05962002000500012&script=sci_ substances and free for further exploration of therapeutic effect based arttext&tlng=en from the result of the Acute Oral Toxicity Testing. In the determination of Approximate Effective Dose, ten healthy Guinea pigs (5 males and 5 http://edge.silverson.com/assets/PDFs/AppReports/Pharma/ females) previously weighed and acclimatized inside the cough chamber PPharmaCreamsOintments.pdfUSP XXII, NF XVII. R601 was used in the experimentation. After acclimatizing, the guinea pigs Twinbook Parkway, Rockville, MD 20852: The United States were exposed to citric acid by enclosing the animals in cough chamber, Pharmacopeial Convention, Inc., 1989, 1692. nebulized to saturation with citric acid for them to inhale for 5 minutes. The number of coughs within 10 minutes was recorded. After recording Zamora, P. M. and Co, L. (1986). Guide to Philippine Flora the number of cough of each male and female guinea pigs, test animals and Fauna, Vol. II. Natural Resources Management Center. received orally the determined dose in mg/kg body weight of Trompang JMC Press, Inc. 388 Quezon Avenue, Quezon City. elepante (Heliotropium indicum) leaf extract and then increased the dose logarithmically by 0.6 intervals (Bernas, 2004). After administration it International Journal of Trichology. (2008). Retrieved from was enclosed again in cough chamber and the number of coughs for 10 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3129121/ minutes was recorded. The results showed that the cough induction of Philippine Herbal Medicinal Plant. Retrieved from http:// citric acid was inhibited after the administration of Trompang Elepante www.philippineherbalmedicine.org/;07-21-2009 leaf extract. From the experimental results, the Approximate Effective Dose is found to be between 158.49 to 2511.90 mg/kg dose level. http://www.fda.gov/ohrms/dockets/98fr/990236Gd.pdf Keywords: Antitussive Activity, Approximate Effective Dose, Acute http://www.fda.gov/ohrms/dockets/98fr/990236Gd.pdf Oral Toxicity test, Trompang Elepante

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INTRODUCTION trompang elepante contains an antitussive property.

Cough is a common condition experienced by many people and it Cough medications are very common in the U.S. since coughs are a is the most common reason why people consult a doctor. It is readily very common symptom of many illness, it is understandable that people transmitted through direct inhalation of droplets from a patient. No would want to prevent the risks and as well as to ease the discomfort of wonder why cough has high prevalence rate each year carrying potential coughing brings. (Laleh Gharahbaghian, Cough and Cold Preparation underlying risks. This can be caused by many factors and it is ideal to Toxicity) treat the underlying cause (Madhura Pandit, 2010). There are approximately 40,000,000 cases of whooping cough Chronic cough lasting for more than 8 weeks is common in reported annually across the world. In 2009, approximately 17,000 the community. The causes include cigarette smoking, exposure to cases were reported in the United States (Cassievk, 2010). According cigarette smoke, and exposure to environmental pollution, especially to National Statistical Coordination Board in Makati, last 2008 there particulates. Diseases causing chronic cough include asthma, eosinophilic are 615 notifiable cases of whooping cough (National Statistical bronchitis, gastro-oesophageal reflux disease, postnasal drip syndrome Coordination Board, 1997-2012). or rhinosinusitis, chronic obstructive pulmonary disease, pulmonary fibrosis, and bronchiectasis. Doctors should always work towards a clear With this, the researchers aimed to find courses in diminishing the diagnosis, considering common and rare illnesses. In some patients, underlying risks of cough, the researchers come up with this experimental no cause is identified, leading to the diagnosis of idiopathic cough. based approach in order to evaluate the promising benefits of trompang Moreover, structural and inflammatory airway mucosal changes in non- elepante to provide an alternative medicine that can cure coughing. This asthmatic chronic cough could represent the cause or the traumatic aimed to formulate syrup from the leaf extract of trompang elepante response to repetitive coughing. Effective control of cough requires not (Heliotropium indicum Linn.) as an anti cough. Specifically, this aimed only controlling the disease causing the cough but also desensitisation to identify the chemical constituents present in trompang elepante of cough pathways (The Lancet, 2008). (Heliotropium indicum Linn.); to determine the acute oral toxicity dose based on OECD Guidelines of formulated cough syrup of trompang Some cough medicines help reduces inflammation and loosen elepante to Swiss mice; to determine the Approximate Effective Dose mucus so it can be expelled easily. Most synthetically prepared (AED) of formulated cough syrup of the plant extract using the Swiss medicines in the market today are getting costly as they gain popularity mice; and to evaluate the pH of the formulated syrup if conforms to the to the mob. According to Ladion (1985), a return to natural healing, standards and specification stated in the USP/NF. the use of organic medicines is in need of our time. The prices of manufactured drugs are high; their availability is not always assured. We must rediscover the healing elements of nature. Materials and Methods

Several accounts of folkloric uses of trompang elepante Research Design (Heliotropium indicum Lin.) are noted throughout different points around the world. There are recent studies concerning the plant’s This study was experimental in nature. The researchers used phytochemical results lead the researchers to a conclusion that a true experimental design. The researchers conducted an actual

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experimentation on the different methods in collecting the data and of Data Gathering Procedures the research instrument in order to investigate if the formulated syrup of the trompang elepante leaf extract can be an alternative antitussive I. Collection of Experimental Trompang Elepante drug from the commercialized drugs. To meet the objectives of the Plant study, the researchers used the Swiss mice for the determination of acute oral toxicity dose. In the determination of Approximate Effective Fresh leaves of trompang elepante plant (Heliotropium Dose the guinea pigs were used as the test animals. indicum Linn) were collected from Banay-banay, Davao Oriental. These were collected with proper handling and placed Collection of Plant Material and Test Animal in a clean container. The collection of the plant material was done early in the morning. The researchers gathered the trompang elepante plant (Heliotropium indicum Linn.) at Banay-banay, Davao Oriental. The experimental II. Preparation of Trompang Elepante Leaf Extract animals (mice and guinea pigs) were bought in a certified and accredited pet shop or seller. Healthy, randomized and properly identified adult The researchers washed and chopped the gathered leaf mice weighing 17 to 33 grams were used. Healthy guinea pigs (male into small pieces. The 200 grams of the leaf was soaked with and female) weighing 300 to 500 grams were sampled and properly 95% ethyl alcohol in an Erlenmeyer flask for 48 hours. After identified (Vogel, 1997). 2 days, the ethanolic extract was strained through a muslin cloth. Lastly, the resulting filtrate was submitted for Rotary Research Locale evaporation for pure extract collection. The concentration of extract was computed using the formula: The researchers prepared trompang elepante leaf Extract inclusive of Rotary Evaporation at the Science Resource Center, syrup formulation Concentration of the extract= weight of the extract at the Dispensing Laboratory and the different research procedures Volume of the extract were done at the Animal house of the University of the Immaculate Conception. III. Phytochemical Screening of Leaf Extract

Sampling Design

In this study, purposive design was used. The experimental animals Research Procedures for Alkaloid Analysis that were used are male and female Swiss mice for OECD acute toxicity dose, male and female guinea pigs for the Approximate Effective Dose. Preliminary Test The condition of the animals in the experiment was controlled by acclimatization in order to accustom animals to new conditions. Aliquot portions of trompang elepante leaf extract equivalent to a twenty (20) gram of the plant material was evaporated over heated water bath to a syrupy consistency. Five (5) ml of 2M Hydrochloric acid was then added to the latter. About five (5) minutes, the evaporated

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extract was heated, stirred, and cooled thereafter. After which, 0.5 gram filtrate was divided into three (3) test tubes and one portion from which of Sodium Chloride was added, stirred and filtered. A sufficient amount was taken as control. An aqueous solution of tannic acid was used as of 2M Hydrochloric acid was used to wash the residue and brought the reference standard. filtrate to a volume of five (5) ml. Gelatin Test One (1) ml of the filtrate was tested with two (2) to three (3) drops of Dragendorff’s reagent and another one (1) ml was tested with The researchers used two separate tubes. One portion of the filtrate two (2) to three (3) drops of Mayer’s reagent. A positive result was obtained from previous procedure was treated with three (3) drops of indicated by an orange precipitate with Dragendorff’s reagent and a gelatin salt solution. In a separate tube, a portion of tannic acid was white precipitate with Mayer’s test. The preliminary test was performed treated with three (3) drops of gelatin solution. A positive result was into three trials (Guevarra, et al., 2005). indicated by formation of precipitate. The test was conducted in three (3) trials. (Guevara, et al., 2005).

Research Procedures for Saponin Analysis Ferric Chloride Test

The Capillary Method The researchers used two separate tubes. One portion of the filtrate was treated with three (3) drops of Ferric Chloride solution and another Four capillary tubes were prepared for the experimentation. A three (3) drops to tannic acid in a separate tube. A positive result was height of ten (10) mm of Trompang Elepante leaf extract was loaded indicated if a blue-black colour appeared, which indicates the presence in three (3) capillary tubes. The fourth tube was loaded with a height of hydrolyzable tannins while brownish-green colour was indicated for of ten (10) mm of distilled water. All tubes were kept in a vertical the presence of condensed tannins. The test was conducted in three (3) position and the liquid was allowed to flow out freely. All tubes were trials (Guevara, et al., 2005). compared according to the level of the liquid present. A positive result was indicated if the level of the plant extract within the tube is half IV. Determination of Acute Oral Toxicity (OECD or less than that of water. The test was performed into three trials Guidelines 423) (Guevarra, et al., 2005). A. Selection of Animal Species

Research Procedures for Tannin Analysis The OECD guidelines state that for oral acute toxicity test, the preferred species are rodents, normally, females were Preparation of Plant extract used. Although there is a little difference in sensitivity between sexes, in these cases where differences are observed, females An equivalent of ten (10) gram plant material was taken up and are generally slightly more sensitive than males. Females evaporated to incipient dryness over a heated water bath. The residue should not be pregnant. Each animal, at the initiation of its was extracted with twenty (20) ml of hot distilled water; five (5) drops dosing should be between 8-12 weeks old (OECD, 2001). In of 10% Sodium Chloride (NaCl) was added and filtered thereafter. The this study therefore, female Swiss mice were used.

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B. Preparation of Animals After acclimatizing, the guinea pigs were exposed to citric acid by enclosing the animals in cough chamber. They were Following the OECD guidelines, the animals were marked nebulized and allowed citric acid to saturate cough chamber to permit individual identification and kept in their cages for at for 5 minutes. The number of coughs within 10 minutes will least 5 days prior to dosing to allow for acclimatization to the be recorded. After recording the number of cough of each male environment conditions (OECD, 2001). and female guinea pigs, test animals had received orally the determined dose in mg/kg body weight of Trompang elepante C. Administration of Dose (Heliotropium indicum) leaf extract and then increased the dose logarithmically by 0.6 intervals (Bernas, 2004). After Animals were fasted prior to dosing. Swiss mice were administration, test animals were enclosed again in cough fasted with food but not water for 3-5 hours. Following the chamber and recorded the number of coughs for 10 minutes. period of fasting, the mice were weighed and the test substance was administered. After the substance had been administered, VI. Cough Syrup Formulation food will be given after 1-2 hours (OECD, 2001). A. Preparation of Simple Syrup D. Number of Animals and Dose Levels In the preparation of simple syrup, the sucrose used is Three animals were used for each step. The dose level of crystalline white variety. Eighty-five grams of sucrose was were used on the starting dose and was selected from one of dissolved in 100 mL distilled water. Moderate heat is applied the four fixed levels, 5, 50, 300 and 2000 mg/kg body weight. to sucrose solution (Abdon, 1995). The starting dose level was that which is most likely to produce mortality in some of the dosed animals (OECD, 2001). B. Cough Syrup Formulation

The formulation for the Trompang Elepante cough syrup V. determination of Approximate Effective Dose (AED) is based from the dose level mg/kg body weight. The following were the formula used in the formulation of Trompang Elepante A. Preparation of 7.5% Citric Acid (Inducing Agent) syrup. Leaf extract – 0.0094 g In preparing 7.5% citric acid, 7.5 grams of citric acid was Na benzoate - .06g Saccharin Na - .114g weighed and dissolve in about 15 ml of distilled water and Citric acid - .06g sufficient quantity of water was added to make 100-ml solution. Sorbitol solution – 19.44ml Syrup – 7.29g NaCl - .45g B. Administration of doses Liquid glucose – 2.64ml Glycerin – 3ml Color – q.s Ten healthy Guinea pigs (5 males and 5 females) Flavor – q.s Distilled water to make – 60 ml previously weighed and acclimatize inside the cough chamber.

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In 7.5 ml of distilled water, leaf extract, saccharin Na, and NaCl Table 2 were dissolveed. Then add the sorbitol solution, liquid glucose, and Results for Test for the Presence of Saponins glycerin to the solution. The Na benzoate, syrup, colorant, and flavor will be added also and agitate to dissolve. Sufficient quantity of distilled Tests Positive Test Trial 1 Trial 2 Trial 3 water will be added to make 60 ml preparation. The formulated syrup Capillary Level of the plants half or + + + will be placed in a sterilized amber colored bottle and labeled properly less than that of water (Ansel, 1995). Legend: (-) Negative; (+) Level of the plant extract in the capillary tube is half or less than that of water

Results and Discussion The presence of saponins was detected in trompang elepante leaf extract using capillary method for saponins in which levels of the plant Table 1 extract were half and less than that of the water. This is due to the Results for Test for the Presence of Alkaloids ability of saponins to lower surface tension of the water.

Tests Reagent Positive Test Trial 1 Trial 2 Trial 3 Orange ppt. + + + Preliminary Test Dragendorff Table 3 Mayer’s White ppt. + + + Results for Test for the Presence of Tannins Legend: (-) negative; (+) slight turbidity; (++) definite turbidity; (+++) heavy turbidity Tests Positive Test Trial 1 Trial 2 Trial 3 Gelatin Formation of ppt. + +

Table 1 shows the results of the phytochemical screening. Alkaloid was detected trompang elepante leaf extract in the preliminary screening having a definite orange and white precipitate in Dragendorfff’s and The table also presents that there were formation of precipitates in Mayer’s tests respectively. gelatin test of trompang elepante leaf extracts indicated as positive for the presence of tannins.

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Determination of Acute Oral Toxicity Hence, leaf extract used in this study does not contain toxic substances and is free from further exploration on the possible The Acute Oral Toxicity study of trompang elepante leaf extract is pharmacological use exhibit by this plant. based on OECD guidelines 423. The guideline is a step wise procedure with the use of 3 animals of a single sex per definite dose level. The results allow trompang elepante to be marked and classify as a substance Determination of Approximate Effective Dose which can cause acute toxicity. In the determination of the Approximate Effective Dose of leaf extract, the researchers used 5 pairs of test animals. In this experiment, Table 4 the guinea pigs were exposed to 7.5% citric acid by enclosing the Determination of the Acute Oral Toxicity Dose of animals in cough chamber, nebulized to saturation with citric acid for TrompangElepante Leaf Extract in Female Swiss Mice them to inhale for 5 minutes. The number of coughs within 10 minutes was recorded. After recording the number of cough of each male and Weight of Administered Volume of female guinea pigs, test animals received orally the determined dose Dose level Animal the mice Dose Administered Remarks mg/kg # (kg) (mg/kg) extract (mL) in mg/kg body weight of Heliotropium indicum leaf extract and then 1 0.021 0.105 0.000105 S increased the dose logarithmically by 0.6 intervals (Bernas, 2004). After 5 mg/kg 2 0.023 0.115 0.000115 S administration it was enclosed again in cough chamber and record the 3 0.025 0.125 0.000125 S number of coughs for 20 minutes. 1 0.022 1.100 0.0011 S 50 mg/kg 2 0.021 1.050 0.00105 S 3 0.022 1.100 0.0011 S 1 0.024 7.200 0.0072 S 300 mg/kg 2 0.026 7.800 0.0078 S 3 0.022 6.600 0.0066 S 1 0.024 48.00 0.048 S 2000 mg/kg 2 0.020 40.00 0.04 S 3 0.025 50.00 0.08 S Legend: S- survived; D- died

The results indicated that all test animals had survived the testing after administration of leaf extract. It was observed during critical period stated in OECD guidelines 423 that there were no signs of toxic reactions such as tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma.

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Table 5 Graph 1 Determination of Approximate Effective Dose of Trompang Cough Inhibition of Male Guinea Pigs Elepante Leaf Extract in Citric Acid Cough Induced

Frequency Frequency Volume of of Cough Guinea Dose Body Desired of Cough Administered after Pig level Weight dose Exhibited Extract Induction Number (mg/kg) (kg) (mg) after (ml) of Citric Acid Administration M1 10 0.24 2.4 0.0023 9 (+) 3 (+) F1 10 0.34 3.4 0.0034 10 (+) 2 (+) M2 39.8 0.40 15.92 0.0157 11 (+) 2 (+) F2 39.8 0.30 11.94 0.0118 9 (+) 1 (+) M3 158.49 0.30 47.55 0.0470 9 (+) 0 (-) F3 158.49 0.33 52.30 0.0517 7 (+) 0 (-) M4 630.96 0.31 195.60 0.1934 6 (+) 0 (-) F4 630.96 0.25 157.74 0.1560 7 (+) 0 (-) Legend: Pink - Frequency of cough exhibited after administration of Trompang M5 2511.90 0.32 803.81 0.7947 8 (+) 0 (-) Elepante Leaf Extract F5 2511.90 0.27 678.21 0.6705 8 (+) 0 (-) Blue - Frequency of cough after induction of Citric Acid

The graph shows that after the induction of citric acid to 5 The results above shows that the cough induction of citric acid was male guinea pigs there were coughing that were observed. After the inhibited after the administration of trompang elepante leaf extract. administration of the trompang elepante leaf extract the frequency of It is evident that inhibition of the leaf extract on citric acid induced coughing in each guinea pigs was reduced. guinea pigs started at the dose level of 158.49 mg/kg.

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Graph2 Table 6 Cough Inhibition of Male Guinea Pigs Results for the Determination of pH of the Cough Syrup

pH of the Cough Syrup According to pH of the Trompang Elepante Cough USP/NF Syrup 5.0 – 5.5 5

The pH of the Trompang Elepante Cough Syrup was within the range of the pH of the cough syrup as sated by USP/NF. Hence, the formulated syrup conforms to the standard.

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AHFS® Consumer Medication Information. (2011). Legend: American Society of Health-System Pharmacists. Last Reviewed Pink - Frequency of cough exhibited after administration of Trompang Elepante Leaf Extract - 10/01/2010. Retrieved August 14, 2011 from http://www. Blue - Frequency of cough after induction of Citric Acid nlm.nih.gov/medlineplus/druginfo/meds/a682159.html

Ansel, H. C., et al. (2005). Ansel’s Pharmaceutical Dosage Forms The graph shows that after the induction of citric acid to 5 and Drug Delivery Systems (8th Ed.). Philadelpia: Lippincott female guinea pigs there were coughing that were observed. After the Williams & Wilkins. administration of the Trompang Elepante leaf extract the frequency of coughing in each guinea pigs was reduced. Daniel More. (2010). Causes of cough. from http://allergies. about.com/od/lungallergies/a/cough.htm

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Duda, K. (2010). The problem with prescription cough suppressants. Kurian, J.C. (2010). Amazing healing plants. Manila; Philippine Retrived: May 20, 2011 from http://coldflu.about.com/od/ Publishing House. medications/a/rxcoughmeds.htm. Madhura Pandit. (2010). Cough and sore throat. Retrieved: Evans, W.C. (2005). Pharmacognosy (15th Ed.). Singapore: August 20, 2011, from http://www.buzzle.com/articles/ Elsevier PTE LTD. cough-and-sore-throat.html\

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