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Archive for May, 2010

Sounds like weird, right? What is the relationships between AURA, ANTISEPTIC and RED-OX titration???…..he3x….

Actually this story occurred last year when me and my co-workers (Pn. Suryati, lecturer from Padang, Indonesia) did orientation for the new topic of ANALYTICAL-PHARMACEUTICAL CHEMISTRY PRACTICAL (The Most Favorite Subject, isn’t it??? He3x) i.e. RED-OX titration and VISIBLE SPECTROSCOPY. We chosen Determination of Vitamin C using Dichlorophenol Indophenol (DCPIP) method as an experiment for red-ox titration and Determination of Acriflavine using Visible Spectroscopy one as an experiment for spectroscopy.

As we have known that vitamin C (ascorbic acid) is a reducing agent. It is because DIENOL groups will be very easy suffering OXIDATION process, i.e. losing electron (LEO =Losing Electron-à Oxidation) while DCPIP were gaining electron, it undergoes REDUCTION process (GER = Gaining Electron –à Reduction). The dienol groups of vitamin C will be oxidized become diketone by Chlorine (Cl) group of DCPIP via electronic withdrawl effect of Cl. Therefore, the C=N bridge of DCPIP will be hydrogenised (reducing double bond) by vitamin C.

    redox reaction of vit.C with DCPIP

In this kind of titration we don’t need an indicator to detect the END POINT of titration since the blue color of DCPIP will play as AUTOINDICATOR. The discoloration of blue DCPIP after wisely dropping of vitamin C will be assigned as the end point of titration. In this titration, the acid pH must be maintained by adding a few drops of strong acid like HCl to avoid the pre-oxidation process by O2 from the air.

blue DCPIP

dropping vit. c altering the discoloration of blue DCPIP

end point of titration

ACRIFLAVINE is one of drug which is sold as ANTISEPTIC at pharmacy retail. This compound is a reddish brown  powder but when it is dissolved in water, it will give yellowish green solution. Later, when I illuminate the acriflavine solution under Na lamp, it  produced a green FLUORESCENCE that looks like AURA  of sweet angel. Why acriflavine is able producing fluorescent? Please check its molecule structure. Any compounds that is having chromophore (benzene like structure), auxochrome (functional group which is bond to chromophore), and rigid atomic bonding. How can we recognize whether atomic bonding is rigid or not? Rigid character will be occurred when the compound has HETEROCYCLIC RING or non-heterocyclic but having INTRAMOECULAR HYDROGEN BONDING.

dosage form, powder and structure of acriflavine

When the rigid compound being exposed to UV/Visible light, it will absorb energy from there. The Energy absorbed will transfer the electron of compound from the GROUND STATE to the EXCITED state. As we knew that the EXCITED state is unstable condition so the electron tends to turn back to the ground state while emitting the energy absorbed previously. Before grounding back the electrons will get a vibrational relaxation in the lower different step, after that the energy will be emitted as FLUORESCENCE.

energy transition from excited state to the lower level altering the vibration mode & produce fluoresence

After the orientation finished, I still kept in lab while playing around the rest of my reagents. I was just dropping the DCPIP into acriflavine solution, and surprisingly I saw the FLUORESCENCE being disappeared spontaneusly. What an amazing phenomenon I never imagined before. Later, I thought if the fluorescence of acriflavine solution can be LIGHTED OFF by DCPIP, it should be used as internal indicator in Determination of Vitamin C using IODIMETRIC method. The C=N of acriflavine who is responsible to its fluorescence character will be oxidized by I2 after all molecule of vitamin C completely consumed by these oxidizing agent.

vit. C & iodine solution before titration

vit.c added by acriflavine (look at the yellow fluorescence) & iodine solution before titration

acriflavine solution, used as indicator in determination of vit. C using iodimetric method

vit. c after end poit, look at the dissappearing of yellow fluorescence

Iodimetric titration is a redox titration in which analyte will be titrated using Iodine solution (I2) as an oxidizing agent (titrant). So far, starch 10% has been commonly used as indicator in this kind of titration by producing blue color at the end point of titration. Next, I made my own trial and error experiment using acriflavine solution as indicator in determination of vitamin C using Iodimetric method. And DONE!!! The yellow fluorescence of analyte solution suddenly disappeared at the volume of standard solution as exact as THE TRUE VALUE. I made 3 replications (TRIPLO) and gave the sharp Standard Deviation statistically.

I compared the titration using starch 10% as indicator

blue complex of iod-amylum after end point of titration, right side: acriflavine solution

So, what can be learned from that??? Even though Titration is the oldest method of drug determination but it still important to study as the basic concept of analytical chemistry. Nowadays, HPLC has been favorable to be applied in drug determination but without studying TITRATION, it is difficult to understand the sophisticated one. So, this experiment will enrich us about the availability of redox indicators.

I remembered that in my country INDONESIA, one of ACRIFLAVINE derivate, i.e. ETHACRIDINE LACTATE (Popular as RIVANOL solution) also free sold in Pharmacy Retail (APOTEK) as external ANTISEPTIC. So, hey INDONESIAN!! Why don’t you try to explore more about THE AURA of that such ANTISEPTIC (Rivanol) in RED-OX TITRATION????

Rivanol, antiseptic freely sold in Indonesia

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One of my obsessions??? sounds like too much, isn’t it? That’s me, the one who always make a controversy against environment. When most of my classmates prefer to study pharmacology, I was the only one who highly  interested in organic chemistry. I didn’t care what people thinking about me, weird, abnormal and mad,  may be!!! He..he..he

I don’t know why I never feel comfortable  what people normally did. I love to walk away to anywhere while people enjoying their cool air conditioner in their cars and I prefer to buy a book when people busy to upgrade their ‘hardware’.  Laboratory is my favorite room to spend the whole of my week end while people having fun with their couples and families.

When I was teaching in School of Pharmaceutical Science, STIFAR”Yayasan Pharmacy” Semarang, Indonesia, I saw the monotone activity done every day. Students just had a class, practical, lab report and exam…that’s all. No activity relevant with their studies conducted to improve their knowledges, skills, networks and experiences,…it was not effective and sooo flat I thought. Actually, I saw some students had a talent to develop themselves but just nobody encourages them.

Later, I had initiative to do something, yaa…..a changing!!! And my goodness, at same time I got information letter from Indonesian High Education Board that they invited all high education institution to contribute by sending their students to apply the 2008 STUDENT CREATIVITY PROGRAM GRANT, one of area was research. No need to think longer, I went straight to announce this on information board. At the commencement of lecture, I always promoted and campaigned this to the class.

First time, no body aware, what a fuck of Mr. Maywan!!! I told to my co-staff about this, some of them supported but also doubted what I would to do, “Are you sure Maywan, our students??? Come on, wake up guys, we are private university, don’t to be TOO HIGH EXPECTATION!!”. I was just smiling such a fool, but I went through my way”Nothing impossible if we have a good will to do”.

No term”tired” for me to promote and campaign during one week, two weeks…. Finally, 3 students came to my office and asked regarding that program. What a happy I was!!! They told me that actually they were interested but they didn’t have experiences what they had to do while nobody could be asked. I told them”if you are serious I will be outstanding for you”. Later, I tried to motivate them slowly, step by step, to make sure that they could do it. And….. confirmed, they would!!!

Next, I gave an intensive briefing after office hours. What a so tiring, I have to start with INTRODUCTION TO RESEARCH METHODOLOGY & PHARMACY RESEARCH AREA. Even though chemistry was my major field but I won’t force them to ‘stereotype ‘ me. I said”Just be yourselves! Even you interest in Pharmacology, Pharmaceutics or Biology Pharmacy, it doesn’t matter, I will back up you by all out. “ Finally, they chosen Pharmaceutical Technology as their interested one, but no idea there was.

Later, I searched internet to inspire what they could do, and done!!! FORMULATION OF LOZENGES FROM ETHANOLIC EXTRACT OF MENIRAN HERBS (Phyllanthus niruri) AS IMUNOMODULATOR DOSAGE FORM. Then, I gave them an intensive tutorial about Pharmaceutical Technology, Pharmacognosy and Drug Delievery System. We searched a lot of literature and they start to compose their proposals. The next day, I was so surprised that 12 students more, came to my office with the high spirits to join that program. OK…OK…OK!!! Suddenly, I was burning with my own spirits to create 4 research groups (3-4 members each group) representative of Pharmacology, Pharmaceutical Technology, Organic Chemistry and Phytochemistry.

Some students of STIFAR who involved in Student Creative Program & my research project (REUNIAN, September 2009)

Next, it was born 4 research titles that ready to submit to Board, 1 title existed already and 3 more are:

  1. Effect of Soya (Soja max) Juice and Its Suspension toward Spermatogenesis of White Rats Orally as a New Chance of Male Oral Contraception (PHARMACOLOGY)
  2. Structure Elucidation of Potential Anticancer Chemical Constituent of Tembelekan Herbs (Centella asiatica) (PHYTOCHEMISTRY)
  3. Synthesis of Salicylic Acid Derivatives as Artificial Fragrants. (ORGANIC CHEMISTRY).

the stigmasterol constituent of soya bean has been regonized having male infertility effect

terpenoid constituent of lantana herbs can be proposed as a new anticancer drug

flavonoide glycoside presented in Phyllnatus herbs has been established as imunomodulator

some salycylic acid ester can be used as artificial fragrants

It was so tiring, I had to handle 4 groups with 4 different interestings, finally 3 of co-lecturers confirmed to help me assisting them, and I focused on Chemistry Groups. Last but not least, 4 proposals ready to be submitted, and MY GOODNESS all of them are qualified to get THE GRANT. And surprisingly, one (PHARMACEUTICAL TECHNOLOGY GROUP) of them was selected to be a candidate of NATIONAL HIGHEST EDUCATION STUDENT COMPETITION 2008 and STUDENT TECHNOPRENEURSHIP, whaoowww!!!

 

letter of acceptance my 4 groups in Student Creativity Program Grant, High Education Board, Indonesia

Nowadays, I have been 1.5 years teaching in UniKL-RCMP and but nothing I can do to improve and develop them. I was just teaching, teaching and teaching. So far, I have seen a positive activities run by students like INDIAN CULTURE NIGHT, RUMAH ANAK YATIM, SPORT COMPETITION, STUDENT ORIENTATION. That were good and I love it so much!!!

Pharmacy UniKL-RCMP student orientation

 But regarding with the UniKL Motto “WHERE KNOWLEDGE IS APPLIED”, it will be better if students also balance their activities with their relevant studies. I am sure that a few of them are very talented to do it, but the same situation occurred with what I got in Indonesia. But here, I am just A FOREIGNER, I have a limited movement to do it. So, What can I do to produce my EXCELLENT STUDENT that will be recognized locally, moreover internationally in the future ??? Shall my obsession come true??? ZZZZZHHH,,,,I  am still dreaming…………….

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So many beautiful things in the world, one of them is DAHLIA, a flower which is classified as Compositae family. This herb usually well growth up in the plateau with the cool climate and people just benefit it to make their gardens beautifully and also for business because dahlia is one of preserved flower which can be made as a wedding bouquets.

In spite of that usage, actually Dahlia saves benefits more than that. Dahlia is a tuberous and so many advantages are able to be obtained from this part of plant. It has been well known that Dahlia TUBER contains one of chemical constituent which is called as INULIN. In health, inulin has been used as a prebiotic in health foods, a chemical which stimulates the micro flora growth in our intestine in order to increase our normal food digestion. So, prebiotic will prevent us from gastrointestinal disorder such as constipation, diarrhea, colon cancer, and so on.

structure of inulin

Inulin is a polymer which is composed by β (2→1) and β (2→6) fructosyl fructose chain with less than 200 DEGREE of POLYMERYZATION (DP). Its DP is categorized as a short polymer, means it is easier to decompose this polymer become the small units of monomer. This characteristic can be beneficial for us as we have known that the longer polymer chain such as undegradable plastics having a big problem in our current environmental issues. Beside Dahlia, some plants such as chicory, yacon, garlic, onion, and banana also known containing this constituent.

chicory roots

  

yacon (Taraxacum officinale)

Regarding the advantages mentioned above, I was interested to study more about Inulin. In my hometown (SOLO, CENTRAL JAVA, INDONESIA), I have 2 nearest plateau which Dahlia was easily found, i.e. TAWANGMANGU and BANDUNGAN. I collected this herb from Bandungan and I selected two varieties only, i.e. WHITE & PURPLE DAHLIA.

Later, I took the tuber part and extract it using water-ethanol as a solvent. The extraction method was using centrifugation followed by purification using precipitation method and charcoal adsorption. The white powder was obtained as a product and I had to analyze it to confirm its molecule structure. I had a PURE CHICORY INULIN  got from SIGMA-ALDRICH and used it as a STANDARD REFERENCE. I compared the physical appearance that looked like similar, but the product from white dahlia seemed a bit browning. The solubility of both compounds was also same, and majority were soluble in polar solvent due to the a lot of hydroxyl group presence in the structure

Precipitation of inulin & the obtained products (left (white dahlia); middle (purple dahlia); inulin standard)

The melting point and TLC profile of both my products and standard inulin was exactly similar. Next, the structure was confirmed by using IR  spectroscopy. IR spectrum showed the exact same spectra   whether at fundamental or fingerprint region between my products & standard inulin. Therefore, I was sure the extraction of inulin from dahlia tuber was done already.

IR spectrum of inulin standard (left) and my product (right),look at the smooth broad band at 4000 cm-1 shows OH group

It was easy to recognize the presence of HYDROXYL GROUP by checking a very smooth broad spectrum at 3000-4000 cm-1  region. Due to this OH group, I interest to modify the structure at this part of molecule in order to decrease the polarity. By acetylating of OH group I predicted that the product would be having Hydrophyl-Lipophyl Balance (HLB). So, it is possible to develop this polymer become a new surface active agent that has been well known to stabilize the disperse system such as emulsion and suspension.

My further research would be acetylating inulin using acetyl chloride as a resource of acetyl group. I was synthesize in two different solvents, i.e. Dimethylformamide (DMF) & PYRIDINE. I avoid the use of polar protic solvent such as water or ethanol due to the instability of acetyl chloride toward hydrolysis or ethanolysis. Thereafter, I reacted my inulin with acetyl chloride under Foam Hoed and  the product was isolated using liquid-liquid extraction with ethyl acetate as a solvent and followed by distillation to remove the ethyl acetate from the product.

SNA reaction of inulin with acetyl chloride produces acetylated inulin

The semi-synthetic product was brownish liquid, a bit sticky, insoluble in water, soluble in alcohol and chloroform. Compared to the starting material (Inulin), we can see that it was already different in physical properties, means the product was done. I also run TLC test, IR and H1-NMR spectroscopy to confirm its structure, and done!!! My product showed the sharp band at 1800 cm-1 that was recognized as a STRETCHING VIBRATIONS of -C=O whereas this band was absence at inulin IR spectrum, means the acetylation was successfully done. The 1H-NMR also supported the presence of methyl proton which is adjacent to carbonyl groups.

acetylated inulin (semi-synthetic product)

 

Comparison 1H-NMR of inulin (starting material) and acetylated inulin (product)

IR NMR of acetylated inulin (product), look at the SHARP BAND at 1800 cm-1 shows the carbonyl group

It is interesting to develop my products become the new suspending agent to enrich the availability of this pharmaceutical inactive ingredients. And surprisingly, when I heated my product at 200C it changed to a kind of plastic matter, and I think being logic since my product was less polar polymer that looks like a plastic. So, may we develop further with these findings? Yah, my product can be developed as BIODEGRADABLE plastics coz only less than 200 of DP it has, therefore it is more friendly to environment. So, ANYBODY interested????

check my published fulltext journal at http://mnh20.wordpress.com/2010/04/12/the-9th-national-symposium-of-polymeric-materials-upm-malaysia/

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It has been 3 a.m now. Last night I slept early coz I felt so tired after all day activities. I wake up from my dream wherein I met with my parents, yah, my mom and my daddy. Not too clear what the story  was , it so fast to wake up. But, I feel so happy being able to see them even though just in a dream. I just realize that this early morning (2 May) is the same date when my daddy passed away 19 years ago. He died at the night after my birthday,  and I think he want to give me a birthday wishing in this early morning, he3x….

My daddy was a person who had a straight and discipline character. He would undoubtedly hit me or my brothers when we made a mistake.  Although I felt  unguilty and my friend initiate me fighting first, he still punished me. I was not too close with him since we have in contrast character.  I tend to be melancholies and I prefer to share with my mom rather than him. I thought he didn’t love me, because he never showed it.  He never praised me when I got 1st rank in my school, he won’t give me a money to buy a book. I was really disappointed with him and jealous when I saw my friends having fun with their fathers.

But what we saw doesn’t always describe what the truth.  I really felt what a huge he loved me when I got DENGUE fever and should be in a hospital patient. I saw what a panic he was more than my mom. I felt so peace when he hold me alike don’t want let me went away. Thereafter, I changed my perception and proven what a noble he was. He just wanted us to be an independent person, so we will be survived even in the critical situation.

My daddy was a hard smoker and had a complicated cardiovascular disease then it be worsted by his chronic ASTHMA.  His physic was dropping triggered by asthma attack and it became worst when it modulated other cardiovascular system such as hypertension. Only in 6 hours after he dropped, he died at  a night after my birthday. Ya…that was a destiny and nobody could avoid this. My big brother told me one last message he gave us that we had to be survive even not together anymore. I was crying so much and feel so sorry that I was thinking he never loved me.

Life must go on, and my goodness we have been survived even my mom being a single parent until now. She growths up us such as she never thinking herself. She dedicates the whole of her life to take care us. And step by step we can recover and being a compact family.

My carrier was getting better when I took a course in Master Degree. I chosen drug discovery and development as my field and specialized in SYNTHETIC MEDICINAL CHEMISTRY. Then I was inspired to study the drug discovery in ASTHMATIC disease regarding what my daddy was suffered.

Asthma is a common chronic inflammatory disease of the airways characterized by variable and recurring symptoms, airflow obstruction, and bronchospasm. Symptoms include wheezing, cough, chest tightness, and shortness of breath.

Certain key steps in the pathophysiology of asthma are depicted, emphasizing the role of the cysLTs C4, D4, and E4. The contributions of many cells (such as dendritic cells, mast cells, macrophages and epithelial cells) and mediators (such as proteases, eotaxin and endothelin) have been omitted. As shown, antigen challenge results in the accumulation and activation of TH2 lymphocytes, which secrete eosinophil-activating cytokines. Eosinophils in turn secrete cysLTs and other pro-inflammatory mediators. A variety of cell types also contribute to the generation of LXA4. CysLTs and LXA4 bind to target cells, including leukocytes, smooth-muscle cells, epithelial cells and endothelial cells. LXA4 can interact with its cognate receptor, ALX, to generate suppressive signals that are anti-asthmatic. In addition, LXA4 can compete with the binding of cysLTs at their cognate receptor, cysLT1, thereby blocking the pro-asthmatic actions of cysLTs. Inset: Metabolism of arachidonic acid (AA) to prostaglandins, cysLTs and LXA4. Other abbreviations: PLA2, phospholipase A2; COX, cyclooxygenase; LO, lipoxygenase.

One of classic asthmatic drug has been known is theophylline. Its mechanism action in asthmatic disease treatment was known by inhibiting Phosphodiesterase inhibitor (PDE4), one of enzymes in our bodies who catalyses the hydrolysis CAMP (Cyclic-Adenosine Mono Phosphate) become AMP (Adenosine Mono Phosphate). Bu inhibiting the hydrolysis pathway, the increasing of CAMPO will cause the smooth muscle in trachea or bronchus relax back.

Other study showed that theophylline can treat asthmatic disease by adenosine antagonism. The blocking of Adenosine Receptor with its natural ligand will prevent the smooth muscle constriction become worst.

As we knew that one of asthmatic trigger is allergen and it alters the releasing histamine from mast cell. Theobromine is a chemical constituent which is the same class with theophylline, xanthine, but didn’t show antiasthmatic action. Different with theophylline, theobromine has no alkyl group in N1  position, so it could be a high chance to alkylate this part by a bulky alkyl group.

 

         Theophylline                                     Theobromine

Based on the Structure-Activity Relationship (SAR), the elongation of alkyl group in N1 position of theobromine can cause it having dilatation effect.

Structure-Activity Relationship of Alkylxanthine Derivatives

Then, I created the N1-alkyltheobromine by substituting isopropyl and sec-butyl group to the N1 position of theobromine. The synthesis was undergoing reaction between theobromine and alkyl bromide derivatives and catalyzed by NaOH in the ethanol as a solvent. The reaction took 36 hours heating and I have to stay in laboratory all night along.

reflux (reaction taking 36 hours)

The proton withdrawal from NaOH to imide group of Theobromine

SN2 Reaction Mechanism of Theobromine & sec-butyl bromide

After isolation using Preparative Thin Layer Chromatography (TLC) then I got the crystal as my product. Refer to the preliminary test (melting point, solubility and TLC), I confident that my product was done. I confirmed its molecule structure by running with UV, IR, NMR and GC-MS Spectroscopy method, and my goodness!!! It was absolutely perfect as what I expected.

Thin Layer Chromatography, the highest spots are my products (less polar)

UV Spectrum, any absorption in 200-40o nm showed the chromophore of my structures

IR Spectrum, showed the functional groups suitable with my structures

NMR Spectrum, showed the C-H (Hydrocarbon) environment of my structures

Mass Spectrum, confirmed my structure as N1-sec-butyl-theobromine with molecule weight = 236

After the product being confirmed, I tested its tracheospasmolytic activity against histamine in a New Guine Pig (Cavia porcella). First, I killed the animal by hitting its back head to the porcelain sink. I was shaking and nervous to do that (my first time to kill the cute animals, OMG, forgive me), especially the bleeding one. Next, I surged the neck and evacuated the trachea.

I was a ‘murder’, he3x….

The most difficult step was a trachea preparation. I had to sew the small organ without damage the smooth muscle inside.

the trachea was so small, how could I sew it??

Thereafter, the trachea was ready to set up in the instrument. The instrument name is LEVER TRANSDUCER which contains a bath which is filled by KREBS SOLUTION, that is installed to the detector and recorder. The trachea was set into the bath and added by histamine. Since histamine is an allergen, it might cause the trachea constriction which was shown by the KIMOGRAPH (the curve shows the increasing of trachea respon when the concentration of histamine being increased). Therefore, the adding of the product will make the trachea relax back and the curve will decrease gradually, proportional with the increasing of product concentration.

LEVER TRANSDUCER

Kimograph, look the adding of my product decrease the curve line gradualy

The comparison of tracheospasmolytic activity between my products (N1-isopropyltheobromine & N1-sec-butyltheobromine) and theophylline. N1-sec-butyltheobromine> theophylline > N1-isopropyltheobromine

The result of this test showed that both of my product able to inhibit histamine and cause the trachea relaxing back better than theophylline. It’s the challenge to develop my product become a new antihistamine drug.

 DADDY, I DEDICATE MY PRODUCTS JUST FOR YOU, MISS U ALWAYS AND WISH U  REST IN PEACE…….

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