Journal of Clinical and Diagnostic Research, ISSN - 0973 - 709X

Users Online : 273

AbstractMaterial and MethodsResultsConclusionReferences
Article in PDF How to Cite Citation Manager Readers' Comments (0) Audio Visual Article Statistics Link to PUBMED Print this Article Send to a Friend
Advertisers Access Statistics Resources

Dr Bhanu K Bhakhri

"The Journal of Clinical and Diagnostic Research (JCDR) has been in operation since almost a decade. It has contributed a huge number of peer reviewed articles, across a spectrum of medical disciplines, to the medical literature.
Its wide based indexing and open access publications attracts many authors as well as readers
For authors, the manuscripts can be uploaded online through an easily navigable portal, on other hand, reviewers appreciate the systematic handling of all manuscripts. The way JCDR has emerged as an effective medium for publishing wide array of observations in Indian context, I wish the editorial team success in their endeavour"



Dr Bhanu K Bhakhri
Faculty, Pediatric Medicine
Super Speciality Paediatric Hospital and Post Graduate Teaching Institute, Noida
On Sep 2018




Dr Mohan Z Mani

"Thank you very much for having published my article in record time.I would like to compliment you and your entire staff for your promptness, courtesy, and willingness to be customer friendly, which is quite unusual.I was given your reference by a colleague in pathology,and was able to directly phone your editorial office for clarifications.I would particularly like to thank the publication managers and the Assistant Editor who were following up my article. I would also like to thank you for adjusting the money I paid initially into payment for my modified article,and refunding the balance.
I wish all success to your journal and look forward to sending you any suitable similar article in future"



Dr Mohan Z Mani,
Professor & Head,
Department of Dematolgy,
Believers Church Medical College,
Thiruvalla, Kerala
On Sep 2018




Prof. Somashekhar Nimbalkar

"Over the last few years, we have published our research regularly in Journal of Clinical and Diagnostic Research. Having published in more than 20 high impact journals over the last five years including several high impact ones and reviewing articles for even more journals across my fields of interest, we value our published work in JCDR for their high standards in publishing scientific articles. The ease of submission, the rapid reviews in under a month, the high quality of their reviewers and keen attention to the final process of proofs and publication, ensure that there are no mistakes in the final article. We have been asked clarifications on several occasions and have been happy to provide them and it exemplifies the commitment to quality of the team at JCDR."



Prof. Somashekhar Nimbalkar
Head, Department of Pediatrics, Pramukhswami Medical College, Karamsad
Chairman, Research Group, Charutar Arogya Mandal, Karamsad
National Joint Coordinator - Advanced IAP NNF NRP Program
Ex-Member, Governing Body, National Neonatology Forum, New Delhi
Ex-President - National Neonatology Forum Gujarat State Chapter
Department of Pediatrics, Pramukhswami Medical College, Karamsad, Anand, Gujarat.
On Sep 2018




Dr. Kalyani R

"Journal of Clinical and Diagnostic Research is at present a well-known Indian originated scientific journal which started with a humble beginning. I have been associated with this journal since many years. I appreciate the Editor, Dr. Hemant Jain, for his constant effort in bringing up this journal to the present status right from the scratch. The journal is multidisciplinary. It encourages in publishing the scientific articles from postgraduates and also the beginners who start their career. At the same time the journal also caters for the high quality articles from specialty and super-specialty researchers. Hence it provides a platform for the scientist and researchers to publish. The other aspect of it is, the readers get the information regarding the most recent developments in science which can be used for teaching, research, treating patients and to some extent take preventive measures against certain diseases. The journal is contributing immensely to the society at national and international level."



Dr Kalyani R
Professor and Head
Department of Pathology
Sri Devaraj Urs Medical College
Sri Devaraj Urs Academy of Higher Education and Research , Kolar, Karnataka
On Sep 2018




Dr. Saumya Navit

"As a peer-reviewed journal, the Journal of Clinical and Diagnostic Research provides an opportunity to researchers, scientists and budding professionals to explore the developments in the field of medicine and dentistry and their varied specialities, thus extending our view on biological diversities of living species in relation to medicine.
‘Knowledge is treasure of a wise man.’ The free access of this journal provides an immense scope of learning for the both the old and the young in field of medicine and dentistry as well. The multidisciplinary nature of the journal makes it a better platform to absorb all that is being researched and developed. The publication process is systematic and professional. Online submission, publication and peer reviewing makes it a user-friendly journal.
As an experienced dentist and an academician, I proudly recommend this journal to the dental fraternity as a good quality open access platform for rapid communication of their cutting-edge research progress and discovery.
I wish JCDR a great success and I hope that journal will soar higher with the passing time."



Dr Saumya Navit
Professor and Head
Department of Pediatric Dentistry
Saraswati Dental College
Lucknow
On Sep 2018




Dr. Arunava Biswas

"My sincere attachment with JCDR as an author as well as reviewer is a learning experience . Their systematic approach in publication of article in various categories is really praiseworthy.
Their prompt and timely response to review's query and the manner in which they have set the reviewing process helps in extracting the best possible scientific writings for publication.
It's a honour and pride to be a part of the JCDR team. My very best wishes to JCDR and hope it will sparkle up above the sky as a high indexed journal in near future."



Dr. Arunava Biswas
MD, DM (Clinical Pharmacology)
Assistant Professor
Department of Pharmacology
Calcutta National Medical College & Hospital , Kolkata




Dr. C.S. Ramesh Babu
" Journal of Clinical and Diagnostic Research (JCDR) is a multi-specialty medical and dental journal publishing high quality research articles in almost all branches of medicine. The quality of printing of figures and tables is excellent and comparable to any International journal. An added advantage is nominal publication charges and monthly issue of the journal and more chances of an article being accepted for publication. Moreover being a multi-specialty journal an article concerning a particular specialty has a wider reach of readers of other related specialties also. As an author and reviewer for several years I find this Journal most suitable and highly recommend this Journal."
Best regards,
C.S. Ramesh Babu,
Associate Professor of Anatomy,
Muzaffarnagar Medical College,
Muzaffarnagar.
On Aug 2018




Dr. Arundhathi. S
"Journal of Clinical and Diagnostic Research (JCDR) is a reputed peer reviewed journal and is constantly involved in publishing high quality research articles related to medicine. Its been a great pleasure to be associated with this esteemed journal as a reviewer and as an author for a couple of years. The editorial board consists of many dedicated and reputed experts as its members and they are doing an appreciable work in guiding budding researchers. JCDR is doing a commendable job in scientific research by promoting excellent quality research & review articles and case reports & series. The reviewers provide appropriate suggestions that improve the quality of articles. I strongly recommend my fraternity to encourage JCDR by contributing their valuable research work in this widely accepted, user friendly journal. I hope my collaboration with JCDR will continue for a long time".



Dr. Arundhathi. S
MBBS, MD (Pathology),
Sanjay Gandhi institute of trauma and orthopedics,
Bengaluru.
On Aug 2018




Dr. Mamta Gupta,
"It gives me great pleasure to be associated with JCDR, since last 2-3 years. Since then I have authored, co-authored and reviewed about 25 articles in JCDR. I thank JCDR for giving me an opportunity to improve my own skills as an author and a reviewer.
It 's a multispecialty journal, publishing high quality articles. It gives a platform to the authors to publish their research work which can be available for everyone across the globe to read. The best thing about JCDR is that the full articles of all medical specialties are available as pdf/html for reading free of cost or without institutional subscription, which is not there for other journals. For those who have problem in writing manuscript or do statistical work, JCDR comes for their rescue.
The journal has a monthly publication and the articles are published quite fast. In time compared to other journals. The on-line first publication is also a great advantage and facility to review one's own articles before going to print. The response to any query and permission if required, is quite fast; this is quite commendable. I have a very good experience about seeking quick permission for quoting a photograph (Fig.) from a JCDR article for my chapter authored in an E book. I never thought it would be so easy. No hassles.
Reviewing articles is no less a pain staking process and requires in depth perception, knowledge about the topic for review. It requires time and concentration, yet I enjoy doing it. The JCDR website especially for the reviewers is quite user friendly. My suggestions for improving the journal is, more strict review process, so that only high quality articles are published. I find a a good number of articles in Obst. Gynae, hence, a new journal for this specialty titled JCDR-OG can be started. May be a bimonthly or quarterly publication to begin with. Only selected articles should find a place in it.
An yearly reward for the best article authored can also incentivize the authors. Though the process of finding the best article will be not be very easy. I do not know how reviewing process can be improved. If an article is being reviewed by two reviewers, then opinion of one can be communicated to the other or the final opinion of the editor can be communicated to the reviewer if requested for. This will help one’s reviewing skills.
My best wishes to Dr. Hemant Jain and all the editorial staff of JCDR for their untiring efforts to bring out this journal. I strongly recommend medical fraternity to publish their valuable research work in this esteemed journal, JCDR".



Dr. Mamta Gupta
Consultant
(Ex HOD Obs &Gynae, Hindu Rao Hospital and associated NDMC Medical College, Delhi)
Aug 2018




Dr. Rajendra Kumar Ghritlaharey

"I wish to thank Dr. Hemant Jain, Editor-in-Chief Journal of Clinical and Diagnostic Research (JCDR), for asking me to write up few words.
Writing is the representation of language in a textual medium i e; into the words and sentences on paper. Quality medical manuscript writing in particular, demands not only a high-quality research, but also requires accurate and concise communication of findings and conclusions, with adherence to particular journal guidelines. In medical field whether working in teaching, private, or in corporate institution, everyone wants to excel in his / her own field and get recognised by making manuscripts publication.


Authors are the souls of any journal, and deserve much respect. To publish a journal manuscripts are needed from authors. Authors have a great responsibility for producing facts of their work in terms of number and results truthfully and an individual honesty is expected from authors in this regards. Both ways its true "No authors-No manuscripts-No journals" and "No journals–No manuscripts–No authors". Reviewing a manuscript is also a very responsible and important task of any peer-reviewed journal and to be taken seriously. It needs knowledge on the subject, sincerity, honesty and determination. Although the process of reviewing a manuscript is a time consuming task butit is expected to give one's best remarks within the time frame of the journal.
Salient features of the JCDR: It is a biomedical, multidisciplinary (including all medical and dental specialities), e-journal, with wide scope and extensive author support. At the same time, a free text of manuscript is available in HTML and PDF format. There is fast growing authorship and readership with JCDR as this can be judged by the number of articles published in it i e; in Feb 2007 of its first issue, it contained 5 articles only, and now in its recent volume published in April 2011, it contained 67 manuscripts. This e-journal is fulfilling the commitments and objectives sincerely, (as stated by Editor-in-chief in his preface to first edition) i e; to encourage physicians through the internet, especially from the developing countries who witness a spectrum of disease and acquire a wealth of knowledge to publish their experiences to benefit the medical community in patients care. I also feel that many of us have work of substance, newer ideas, adequate clinical materials but poor in medical writing and hesitation to submit the work and need help. JCDR provides authors help in this regards.
Timely publication of journal: Publication of manuscripts and bringing out the issue in time is one of the positive aspects of JCDR and is possible with strong support team in terms of peer reviewers, proof reading, language check, computer operators, etc. This is one of the great reasons for authors to submit their work with JCDR. Another best part of JCDR is "Online first Publications" facilities available for the authors. This facility not only provides the prompt publications of the manuscripts but at the same time also early availability of the manuscripts for the readers.
Indexation and online availability: Indexation transforms the journal in some sense from its local ownership to the worldwide professional community and to the public.JCDR is indexed with Embase & EMbiology, Google Scholar, Index Copernicus, Chemical Abstracts Service, Journal seek Database, Indian Science Abstracts, to name few of them. Manuscriptspublished in JCDR are available on major search engines ie; google, yahoo, msn.
In the era of fast growing newer technologies, and in computer and internet friendly environment the manuscripts preparation, submission, review, revision, etc and all can be done and checked with a click from all corer of the world, at any time. Of course there is always a scope for improvement in every field and none is perfect. To progress, one needs to identify the areas of one's weakness and to strengthen them.
It is well said that "happy beginning is half done" and it fits perfectly with JCDR. It has grown considerably and I feel it has already grown up from its infancy to adolescence, achieving the status of standard online e-journal form Indian continent since its inception in Feb 2007. This had been made possible due to the efforts and the hard work put in it. The way the JCDR is improving with every new volume, with good quality original manuscripts, makes it a quality journal for readers. I must thank and congratulate Dr Hemant Jain, Editor-in-Chief JCDR and his team for their sincere efforts, dedication, and determination for making JCDR a fast growing journal.
Every one of us: authors, reviewers, editors, and publisher are responsible for enhancing the stature of the journal. I wish for a great success for JCDR."



Thanking you
With sincere regards
Dr. Rajendra Kumar Ghritlaharey, M.S., M. Ch., FAIS
Associate Professor,
Department of Paediatric Surgery, Gandhi Medical College & Associated
Kamla Nehru & Hamidia Hospitals Bhopal, Madhya Pradesh 462 001 (India)
E-mail: drrajendrak1@rediffmail.com
On May 11,2011




Dr. Shankar P.R.

"On looking back through my Gmail archives after being requested by the journal to write a short editorial about my experiences of publishing with the Journal of Clinical and Diagnostic Research (JCDR), I came across an e-mail from Dr. Hemant Jain, Editor, in March 2007, which introduced the new electronic journal. The main features of the journal which were outlined in the e-mail were extensive author support, cash rewards, the peer review process, and other salient features of the journal.
Over a span of over four years, we (I and my colleagues) have published around 25 articles in the journal. In this editorial, I plan to briefly discuss my experiences of publishing with JCDR and the strengths of the journal and to finally address the areas for improvement.
My experiences of publishing with JCDR: Overall, my experiences of publishing withJCDR have been positive. The best point about the journal is that it responds to queries from the author. This may seem to be simple and not too much to ask for, but unfortunately, many journals in the subcontinent and from many developing countries do not respond or they respond with a long delay to the queries from the authors 1. The reasons could be many, including lack of optimal secretarial and other support. Another problem with many journals is the slowness of the review process. Editorial processing and peer review can take anywhere between a year to two years with some journals. Also, some journals do not keep the contributors informed about the progress of the review process. Due to the long review process, the articles can lose their relevance and topicality. A major benefit with JCDR is the timeliness and promptness of its response. In Dr Jain's e-mail which was sent to me in 2007, before the introduction of the Pre-publishing system, he had stated that he had received my submission and that he would get back to me within seven days and he did!
Most of the manuscripts are published within 3 to 4 months of their submission if they are found to be suitable after the review process. JCDR is published bimonthly and the accepted articles were usually published in the next issue. Recently, due to the increased volume of the submissions, the review process has become slower and it ?? Section can take from 4 to 6 months for the articles to be reviewed. The journal has an extensive author support system and it has recently introduced a paid expedited review process. The journal also mentions the average time for processing the manuscript under different submission systems - regular submission and expedited review.
Strengths of the journal: The journal has an online first facility in which the accepted manuscripts may be published on the website before being included in a regular issue of the journal. This cuts down the time between their acceptance and the publication. The journal is indexed in many databases, though not in PubMed. The editorial board should now take steps to index the journal in PubMed. The journal has a system of notifying readers through e-mail when a new issue is released. Also, the articles are available in both the HTML and the PDF formats. I especially like the new and colorful page format of the journal. Also, the access statistics of the articles are available. The prepublication and the manuscript tracking system are also helpful for the authors.
Areas for improvement: In certain cases, I felt that the peer review process of the manuscripts was not up to international standards and that it should be strengthened. Also, the number of manuscripts in an issue is high and it may be difficult for readers to go through all of them. The journal can consider tightening of the peer review process and increasing the quality standards for the acceptance of the manuscripts. I faced occasional problems with the online manuscript submission (Pre-publishing) system, which have to be addressed.
Overall, the publishing process with JCDR has been smooth, quick and relatively hassle free and I can recommend other authors to consider the journal as an outlet for their work."



Dr. P. Ravi Shankar
KIST Medical College, P.O. Box 14142, Kathmandu, Nepal.
E-mail: ravi.dr.shankar@gmail.com
On April 2011

Important Notice

Experimental Research
Year : 2010 | Month : October | Volume : 4 | Issue : 5 | Page : 3289 - 3297

Auto Fluorescence And Fourier Transform – Infra Red (FTIR) Spectral Investigation On Di Ethyl Nitrosamine (DEN) Induced Hepatocellular Carcinoma, Treated With Pericarp Extract Of Garcinia Mangostana Linn In Rats

VISHNU PRIYA V*, SANKARI G**, MALLIKA JAINU***, SURAPANENI K M****, AISHWARYA T S*****, SARASWATHI P******, CHANDRA SADA GOPAN V S******* Correspondence Address :
Surapaneni Krishna Mohan,
Assistant Professor,
Department of Biochemistry,
Saveetha Medical College & Hospital,
Saveetha University, Saveetha Nagar,
Thandalam, Chennai – 602 105, Tamilnadu, INDIA.
E-mail: krishnamohan_surapaneni@yahoo.com

Abstract

The aim of the present study was to analyze the tumour activity of diethyl nitrosamine (DEN); a chemical carcinogen which induced hepatocellular carcinoma in the liver tissue samples of male albino rats. The analysis has been carried out in the following conditions; normal, tumour induced and tumour treated liver tissue samples. In all these cases, the action of the diethyl nitrosamine drug in inducing tumours in the liver has been investigated. Garcinia mangostana pericarp extract was the Ayurvedic drug which was chosen for the treatment of hepatocellular carcinoma. The control liver tissues, those in which tumour formation was induced by using diethyl nitrosamine and those in which the tumour was treated with the Garcinia mangostana pericarp extract after inducing tumor formation were analyzed by using auto fluorescence spectroscopy and fourier transform infra red (FTIR) spectroscopy. The qualitative spectral analysis was done by using auto fluorescence spectroscopy and the quantitative study was carried out by using FTIR spectroscopy. The tissue samples were experimented with auto fluorescence spectroscopy at the excitation wavelengths of 280nm, 325nm and 405nm, which exhibits emission due to tryptophan, collagen and porphyrin, respectively. It was observed that in all the tissue samples, the effect of diethylnitrosamine in inducing tumours was predominant. However, on treating the tumours with the pericarp extract of Garcinia mangostana, they responded positively. FTIR spectra have been recorded in the mid frequency region of 4000 – 450 cm-1 for all the liver tissue samples. The samples were analyzed quantitatively by using intensity ratio calculation among the selected absorption peaks to study the biochemical changes in the tissue samples. From this study, it was found that the total protein content was decreased in the liver tissues after inducing them with diethyl nitrosamine, thus causing the hepato cellular carcinoma. During the recovery phase, the decreased levels of the bio-chemical constituents were restored to near normal levels.

Keywords

Auto fluorescence spectroscopy, Fourier Transform Infra Red (FTIR) spectroscopy, diethyl nitrosamine, hepatocellular carcinoma, pericarp extract, Garcinia mangostana Linn.

INTRODUCTION
Optical techniques such as FTIR and fluorescence spectroscopy, Raman and light scattering can be used to distinguish between cancerous and non-cancerous tissues (1)(2)(3)(4). The underlying physical basis is that they are strongly influenced by the cellular structure and the chemical composition of the tissues. Due to its sensitivity to minute variations, fluorescence spectroscopy can provide quantitative biochemical information about the state of the tissues, which may not be obtained by using standard pathology. A number of fluorophores ranging from structural proteins to various enzymes and co-enzymes, are present in the human tissue and can be excited by ultra- violet and visible light. The fluorescence emission can differ significantly in normal and cancerous tissues due to the differences in the concentrations of the absorbers and the scatterers and also due to the size of the scatterers (5) – (7). Infrared spectroscopy is a powerful method for the study of the molecular structure and the intermolecular interaction in biological tissues and cells. Feride et al (8) studied the effect of sreptozotocin (STZ) induced diabetes on rat liver and heart tissues by using FTIR spectroscopy. Chiriboga et al (9) studied the infrared spectra of normal and cancer liver tissues such as glycogen, DNA and RNA. Patrick et al (10) studied human colon tissues at the molecular level from the normal epithelium to the malignant tumour by pressure tuning FTIR spectroscopy.

Medicinal plants which are commonly included in the ayurvedic recipes for liver ailments have drawn much attention, as no reliable hepato - protective drug is available in modern medicine. Research investigations conducted on several natural plant products which are used for liver protection are well documented. Garcinia mangostana Linn. which is commonly known as "mangosteen", is a tropical evergreen tree and is an emerging category of novel which are sometimes called as "super fruits" and are presumed to have a combination of appealing subjective characteristics such as taste, fragrance and visual qualities, nutrient richness antioxidant strength (11) and a potential impact for lowering the risk of human diseases (12). The pericarps of G. mangostana have been widely used as a traditional medicine for the treatment of diarrhoea, skin infections and chronic wounds in South East Asia for many years (13). These are nature’s most abundant sources of xanthones, which are natural chemical substances possessing numerous bio-active properties that help to maintain intestinal health, which neutralize free - radicals, which help and support joints and cartilage functions and promote immunomodulation systems (14). These are extracted from the rind of mangosteen and contain 95% xanthones and also isoflavones, tannin and flavonoids (15). In the present communication, the safe use of the G. mangostana pericarp extract has been shown by conducting an oral toxicity study in rats.

With this point of view, the present experimental work has been designed to study the effect of diethylnitrosoamine on the bio-chemical profile of the liver tissue of rat and also the protective effect of the pericarp extract of Garcinia mangostana against DEN induced hepatocellular carcinoma by using Fourier Transform Infra Red (FTIR) and auto fluorescence spectroscopy (16) – (19).

Material and Methods

The study was conducted in the Department of Biochemistry, Saveetha Dental College, Saveetha Medical College and Hospital, Saveetha University and Department of Biomedical Engineering, SSN Engineering College, Chennai, Tamil Nadu, India.
1. Animals
Adult Male Wistar Rats weighing 180 – 220 grams, who were maintained under asceptic conditions in the Department of Biomedical Engineering, SSN Engineering College, were used throughout the experiments and were fed with standard laboratory chow and water ad libitum. All animal procedures were performed according to approved protocols and in accordance with the recommendations for the proper care and use of laboratory animals. Due clearance was obtained from the Institutional Animal Ethics Committee (IAEC) before the start of the study.

2. Drugs and chemicals
Diethyl nitrosamine (DEN) was purchased from Sigma Chemicals, St. Louis, MO and the G. mangostana pericarp extract powderobtained from Avasthagen Company, California, USA, as a compliment, was used in the present study. All other chemicals which were used were of analytical grade.

3. Animal treatment
The adult male Wistar rats were randomly divided into four groups: the Normal control group (Group A), the Diethylnitrosoamine (DEN) induced organ injury control group (Group B), the DEN induction with pericarp extract of the Garcinia Mangostana Linn treatment group (Group C) and six rats who were treated with the pericarp extract of the Garcinia Mangostana Linn alone to find out the effect of the drug on the liver if any (Group D). Each group consisted of 6 male Wistar rats (n = 6). The rats of groups B and C were administered orally with 0.01% diethyl nitrosamine which was dissolved in drinking water upto 16 weeks, which causes hepatocellular carcinoma. Simultaneously, the animals of group C were co - treated orally with G. Mangostana pericarp extract (400 mg / kg body weight) which was dissolved in drinking water as an intervention. The rats of control (group A) were on the standard laboratory diet alone and tap water ad libitum. The group D rats were administrated the Garcinia mangostana pericarp extract alone and tap water ad libitum. This was done to ensure whether the Garcinia mangostana pericarp extract by itself produced any side - effects in the liver. All the studies were conducted according to the guidelines which were described in the NIH Guide for the care and use of laboratory animals. The total weight of the diet was kept constant throughout the experimental period. After the scheduled treatment, the animals were sacrificed after obtaining complete ethical clearance from IAEC. The whole liver tissue was isolated immediately and was used for auto fluorescence and FTIR spectral studies. The development of hepatocellular carcinoma (HCC) in group B and the reversal of hepatocellular carcinoma to normal upon treatment with the pericarp extract of the G. mangostana in group C animals were confirmed by performing the histological studies of the liver tissues.

4. Sample Preparations for the Spectroscopic Study:

The whole liver tissue samples of each group of rats were isolated. The isolated whole liver tissue samples were then used for spectral analysis by the auto fluorescence and the FTIR spectroscopic methods. The tissue samples were put in separate plastic containers which were filled with clinical saline completely. Both the spectroscopic studies were conducted at the Sophisticated Analytical Instrument Facility (SAIF), Indian Institute of Technology, Madras (IITM). The samples were stored in a deep freezer at a temperature of -17°C till the spectral measurements were carried out.

5. Auto Fluorescence Spectral Measurement:

The fluorescence emission spectrum (FES) was recorded by using an ISA-Spex FluoroMax-2 spectro fluorometer. The main source that supplies UV radiation to this instrument is a xenon arc source. The main advantage of the FluoroMax-2 instrument is that it measures high sensitivity regardless of sample volume (20), (21). The unique wavelength drive scans the grating at speeds as high as 200nm/s. The grating grooves are blazed to provide maximum light in the UV and visible regions. The fluorescence emission spectra (FES) were recorded in the region of 400-700nm. The tissue samples to be recorded were cut into thin slices and were placed directly in the sample holder of the instrument.

6. FTIR spectral Measurement:

The whole liver tissue samples of each group of mice were isolated. The isolated whole liver tissue samples were lyophilized and were made into fine powder. The tissue powder samples and KBr (all in the solid dry state) were again lyophilized in order to remove most bound water, which could interfere with the prominent group frequencies. Five milligram of liver tissue sample was mixed with 100 mg of dried KBr and this was subjected to a pressure of 5x106 Pa and was made into a clear pellet of 13 mm diameter and 1mm thickness. Mid Infrared spectra in the region of 400 – 4000 cm-1 were recorded on a PERKIN – ELMER Spectrum One FTIR spectrophotometer which was equipped with a KBr beam splitter and an air - cooled DTGS (Deuterated Triglycine Sulfate) detector at SAIF, IIT Madras. The sampling window was scanned as the background and 32 scans were co -added with a spectral resolution of 1cm-1. The spectrometer was continuously purged with dry nitrogen. The absorption intensity of the peak was calculated by using the base line method.

Results

1. Auto Fluorescence Spectral Analysis:

The liver tissue samples of each of the groups were first analyzed by auto fluorescence spectroscopic study. The fluorescence emission spectrum of the liver tissue samples were recorded at excitation wavelengths of 280nm, 325nm and 405nm, respectively. According to Beer - Lambert’s law, the optical absorption is directly proportional to the concentration of the sample. From each spectra which was obtained, it was distinctly seen that there was a marked difference between the normal, the tumour induced and the tumour treated sample groups. The liver tissue samples were recorded individually for the four groups. Since the spectra obtained for the samples under a single group were alike, an average was calculated for their intensities and thus, an average emission spectrum was drawn for all the three groups separately.

(Table/Fig 1) shows the superimposition of the averaged fluorescence emission spectra (FES) of the liver tissue samples of groups A – D at 280nm excitation wavelength, which shows a prominent fluorescence emission peak at a wavelength of 327nm.


(Table/Fig 1): Average Fluorescence spectra of four different liver sample groups - at Excitation wave length 280nm.

From the spectra, it was observed that the spectral characteristics of the control group (group A) tissue samples differed significantly from those of the groups B,C and D. Upon excitation at 280nm, the observed emission peak at 327nm was due to the fluorescence of the amino acid, tryptophan, as shown in (Table/Fig 1). It is clearly inferred that the concentration and absorption of the amino acid goes very high, than that observed in normal healthy tissue, when diethyl nitrosamine drug is induced. This is indicated by the proliferation in the intensity corresponding to the tryptophan emission peak at 327nm. This confirms the denaturation of tryptophan due to the influence of the carcinogenic nature of diethyl nitrosamine, thus causing tumours in the tissue. When the Garcinia mangostana pericarp extract was administered after inducing diethyl nitrosamine to the tissue (group C), it was seen that there was a controlled revival of tryptophan, which was indicated by the intensity value which was obtained, corresponding to 327nm, nearing the intensity which was obtained for the control group. Similarly, the spectrum corresponding to the Garcinia mangostana pericarp extract alone treated liver tissue samples (group D) indicated that there were no side - effects due to the extract. This was indicated from the intensity at 327nm for group D samples, which were almost closer to the intensity at 327nm for the control group samples.

In order to find more about the behaviour of other proteins, the fluorescence emission spectra of the samples were studied at 325nm excitation, which occurs due to the fluorescence of the fibrous protein, collagen. (Table/Fig 2) shows the superimposition of the averaged fluorescence emission spectra (FES) of all the four groups at 325nm excitation wavelength, which shows a prominent fluorescence peak at 370nm wavelength.

The well defined emission peak at 370nm indicates the concentration and absorption of the fibrous protein, collagen in the tissue. It was observed that there was deterioration of the collagen content, as diethyl nitrosamine was induced to the tissue, which was marked by a decrease in the fluorescence intensity when it was compared with the intensity which was obtained for the control group tissue samples. This confirms the toxic activity of diethyl nitrosamine more effectively.

(Table/Fig 2): Average Fluorescence spectra of four different liver sample groups – at Excitation wave length 325nm.

However, it was seen that when the Garcinia mangostana pericarp extract was administered to the diethyl nitrosamine induced tissue (group C), there was no regeneration of the collagen protein in the tissue. This was observed from the intensity values corresponding to the emission peak at 370 nm for group B and group C liver tissue samples, as obtained from the spectrum. However, it was also observed that the spectrum corresponding to the Garcinia mangostana pericarp extract alone treated liver tissue samples (group D) indicated that there were no side-effects due to the extract. This was indicated from the intensity at 370 nm for group D samples, which was almost closer to the intensity at 370 nm for the control group samples. Thus, the Garcinia mangostana pericarp extract was observed to be not much significant in the regeneration of the fibrous protein in the liver tissue.

In order to find about the behaviour of the heme content in the tissue, fluorescence emission spectra of the liver tissue samples were studied at 405nm excitation, which occurs due to the fluorescence of the porphyrin content of the samples. [Table/Fig.3] shows the superimposition of the averaged fluorescence emission spectra (FES) of the tissues of groups A to D at the 405nm excitation wavelength, which shows two prominent fluorescence peaks at 588nm and 636nm wavelength respectively.

(Table/Fig 3): Average Fluorescence spectra of four different liver sample groups-405nm.

From [Table/Fig.3], the well defined emission peaks at 588nm and 636nm indicate the presence of the heme group of the porphyrin in the tissue samples. It was observed that when diethyl nitrosamine carcinogen is induced to the liver tissue samples (group B), there was a sudden increase in the concentration of porphyrin, which was seen from increase in the intensity corresponding to the band peaks at 588nm and 636nm respectively, when compared with that of the control group (group A). It was observed that when the Garcinia mangostana pericarp extract was administered as a treatment drug to the diethyl nitrosamine induced tissue samples (group C), the abnormal rise in the intensity which occurred at both the emission peaks disappeared and it approached the actual value of the concentration of the amino acid to a great precision, which is shown in (Table/Fig 3). The spectrum peak corresponding to the Garcinia mangostana pericarp extract alone treated liver tissue samples (group D) indicates that there were no side - effects due to the extract. The sudden rise in the intensity peak may be attributed to the carcinogenic activity of diethyl nitrosamine, which leads to the excessive cell proliferation of the tumour cells. When the Garcinia mangostana pericarp extract was administered, the abnormal cell proliferation was curtailed and there was a controlled reformation of the porphyrin compound.

2. FTIR Spectroscopic Analysis:

The FTIR spectra of the normal liver tissues (group A), the diethyl nitrosamine induced liver tissues (group B), the diethyl nitrosamine followed by Garcinia mangostana pericarp extract treated liver tissues (group C) and the Garcinia mangostana pericarp extract alone treated tissue samples (group D) are shown in Fig.4. The relative intensities (log Io / I) and tentative assignments of the fundamental Infrared absorption frequencies are shown in (Table/Fig 4).

(Table/Fig 4): Infrared absorption frequencies (cm-1), relative intensities (log Io / I) and tentative assignments of fundamental frequencies of liver tissue samples.

Note: Io corresponding to ~1653 cm-1 (amide I) and I corresponding to the bands mentioned in the first column.

The FTIR spectrum of a sample exhibits characteristic absorption frequencies due to the specific functional groups which are present in the sample. FTIR spectroscopy offers the capability of identifying biochemical substances because of the highly distinctive features of the characteristic molecular vibrations which are rendered in the spectra. A vast amount of substances exist within the cells and most of those substances contribute specifically to the vibrational spectra. Changes in the composition of the cells’ biochemistry should therefore be detectable by FTIR spectroscopic investigation. Although it is most likely that the variety of all those changes appear ambiguously in the spectra, multivariate data evaluation tools should provide access to the diagnostic features which are hidden among the abundance of spectroscopic information (22) – (27).

It is known that tissue proteins, carbohydrates and lipids play a major role as energy providers for animals who are exposed to stress conditions. Shaw et al (23) indicated that a majority of toxic substances initiate biochemical alterations acting at the molecular level by anyone of the following mechanisms:
i inhibition of the enzyme system,
ii altering the level of the enzymes and specificity or by
iii altering the permeability properties of body membranes.

The infrared spectra of the proteins were characterized by a set of absorption regions which are known as the amide and the C-H regions. The most widely used modes in protein structure studies in the amide region are amide I, amide II and amide III. The amide I band arises principally from the C=O stretching vibration of the peptide group. The amide II band is primarily N-H bending with a contribution from C-N stretching vibrations. The amide III absorption is normally weak and arises primarily from the N-H bending and C-N stretching vibrations.

The amide absorptions are considered to be sensitive to protein conformation; hence, an increase or a decrease in the ratio of the intensities of the bands at ~1548 cm-1 (amide II) and ~1653 cm-1 (amide I) could be attributed to a change in the composition of the whole protein pattern. The bands observed at ~1461 cm-1 and ~1396 cm-1 are mainly due to asymmetric and symmetric CH3 bending modes respectively of the methyl groups of the proteins. The medium intensity band observed at ~1235 cm-1 is due to the PO2- asymmetric stretching modes of the phospho di ester indication of phospholipids and the amide III / CH2 wagging vibration from the glycine backbone and protein side chain. The band at 1065cm-1 has been assigned to the PO2- symmetric stretching symmetry phosphates; the stretching of glycogen also makes a contribution to the intensity of this band.


(Table/Fig 5): FTIR Spectra of liver tissues of rats, (a) control, (b) diethyl nitrosamine induced, (c) diethyl nitrosamine followed by Garcinia mangostana pericarp extract, (d) Garcinia mangostana pericarp extract alone

The relative intensities (log Io / I) and tentative assignments of the fundamental Infrared absorption frequencies are shown in (Table/Fig 4). The liver synthesizes a great amount of protein and glycogen, which is needed ostensibly for the repair of damaged cell organelles and tissue regeneration. Stressful situations mainly disturb the rate of carbohydrate metabolism through the levels of glycogen and protein profiles in toxicant exposed animals. Glycogen, a reserve energy source is decreased during the induction of diethyl nitrosamine, which is seen in (Table/Fig 4). A fall in the glycogen profile in the liver tissue indicates the possibility of glycogenolysis. The depletion of the protein profile has also been observed in the liver tissue of rats, when treated with diethyl nitrosamine.

It was observed in this study, that the liver tissues of the rats showed a remarkable recovery from the tumour effect of diethyl nitrosamine. When the rats were exposed to diethyl nitrosamine and Garcinia mangostana pericarp extract treatment, they showed a restoration in the levels of biochemical constituent profiles in the liver tissues. The recovery could be attributed to the restoration of the regulatory function of the proteins and glycogen by elimination of the tumour causing toxicants.

Conclusion

In the present study consisting of the spectral analyses, the following facts have been deduced; when diethyl nitrosamine was induced into the liver tissue, the carcinogenic activity of diethyl nitrosamine led to the denaturation of tryptophan, collagen and fluorescence completely, which signifies cellular, metabolic and pathological disorders. When Garcinia mangostana pericarp extract was administered to the tumour tissues, there was a regeneration of the normal cells in the tissues, thus enabling proper fluorescence, as shown in the fluorescence emission spectral graphs.

The decreased band areas of symmetric and asymmetric CH2 stretching modes which are observed in the diethyl nitrosamine induced tissues, suggests the decreased composition of the lipid chains in the liver tissues of the rats. The decrease in the intensities of the amide bands in the tissues indicate a decrease in the protein quantity of the system and the destructive effect of the diethyl nitrosamine in the liver tissues. The decrease in the intensities of the symmetric and asymmetric stretching modes of the phosphodiester groups suggest a decrease in the relative content of the nucleic acids in the liver tissues, thus indicating the carcinogenic nature of diethyl nitrosamine. It was noted that when the Garcinia mangostana pericarp extract was administered to the tumour tissues, there was a revival in the amide and glycogen contents prominently, thus reversing the tumour activity in the liver tissues.

References

1.
Alfano R, TataD, Cordero J, Tomashefsky P, Longo F, and Alfano M. Laser induced fluorescence spectroscopy from native cancerous and normal tissues. IEEE J. Quantum Electron. 1984; 20 (12): 1507 – 1511.
2.
Alfano R, Tang G, Pradhan A, Lam W, Choy D, and Opher E. Fluorescence spectra from cancerous and normal human breast and lung tissues. IEEE J. Quantum Electron. 1987; 23 (10): 1806 – 1811.
3.
Wagnieres G A, Star WM, and Wilson BC. In vivo fluorescence spectroscopy and imaging for oncological applications. Photochem. Photobiol. 1998; 68 (5): 603 – 632.
4.
Richards-Kortum R and Servick-Muraca E. Quantitative optical spectroscopy for tissue diagnosis. Annu. Rev. Phys. Chem. 1996; 47: 555 – 606.
5.
Ramanujam N. Fluorescence spectroscopy of neoplastic and nonneoplastic tissues. Neoplasia. 2000; 2 (1): 89-117.
6.
Mahadevan–Jansen A and Richards-Kortum R. Raman spectroscopy for the detection of cancers and precancers. J. Biomed. Opt. 1996; 1 (1): 31 – 70.
7.
Tata D B, Foresti M, Cordero J, Tomashefsky P, Alfano MA, and Alfano RR. Fluorescence polarization spectroscopy and time resolved fluorescence kinetics of native cancerous and normal rat kidney tissues. Biophys. J. 1986; 50 (3): 463 – 469.
8.
Severcan F, Toyran N, Kaptan N and Turan B. Fourier transform infrared study of the effect of diabetes on rat liver and heart tissues in the C-H region. Talanta. 2000; 53 (1): 55 – 59.
9.
Chiriboga L, Yee H and Diem M. Infrared spectroscopy of human cells and tissue: Part VI: A comparative study of histopathology and infrared microscopy of normal, cirrhotic and cancerous liver tissue. Applied Spectroscopy. 2000; 54 (1): 1 – 8.
10.
Wong PTT, Lacelle S and Yazdi HM. Malignant human colonic tissues investigated by pressure-tuning FTIR spectroscopy. Applied Spectroscopy, 1993; 47 (11): 1830–1836.
11.
Moongkarndi P, Kosem N, Kaslungka S, Luanratana O, Pongpan N and Neungton N. Antiproliferation, antioxidation and induction of apoptosis by Garcinia mangostana (mangosteen) on SKBR3 human breast cancer cell line. J. Ethnopharmacol. 2004; 90 (1):161 - 166.
12.
Pedraza-Chaverri J, Cárdenas-Rodríguez N, Orozco-Ibarra M and Pérez- Rojas JM. Medicinal properties of mangosteen (Garcinia mangostana). Journal of food and toxicology, 2008; 3: 24 - 27.
13.
Mahabusarakam W, Wiriyachitra P and Taylor WC. Chemical constituents of Garcinia mangostana. J Nat Products 1987; 50 (3): 474 - 478.
14.
Suksamrarn. S, Komutiban O, Ratananukul P, Chimnoi N, Lartpornmatulee N and Suksamrarn A. Cytotoxic prenylated xanthones Journal of Clinical and Diagnostic Research 6 from the young pericarp of Garcinia mangostana. Chem. Pharm. Bull. (Tokyo). 2006; 54 (3): 301 - 305.
15.
Yu L, Zhao M, Yang B, Zhao Q and Jiang Y. Phenolics from hull of Garcinia mangostana pericarp and their antioxidant activities. Food Chemistry, 2007; 104 (1): 176 – 181.
16.
Perromat A, Melin A-M and Deleris G. Pharmacologic application of Fourier Transform Infrared Spectroscopy: The in vivo toxic effect of Carrageenan. Applied Spectroscopy. 2001; 55 (9): 1166-1172.
17.
Manoj K and Ragothaman G. Effect of mercury, copper and cadmium on the Red blood cells of Boleophthamus duosumieri. Pollution Research. 1999; 18 (2): 149–152.
18.
Margarat A and Jagadeesan G. Effect of Tribulus terrestris extract on mercuric chloride poisoning in mice, Mus musculus – a biochemical study. Indian J Environ Toxicol. 2000; 10: 14 – 15.
19.
Rigas B and Wong PTT. Human colon adenocarcinoma cell lines display infrared spectroscopic features of malignant colon tissues. Cancer Research 1992; 52 (1): 84 – 88.
20.
Chien GL, Anselone CG, Davis RF and Van-Winkle DM. Fluorescent vs. radioactive microsphere measurement of regional myocardial blood flow. Cardiovasc Res. 1995; 30 (3): 405 – 412.
21.
Guilbault GG. Practical Fluorescence. Modern Monographs in Analytical Chemistry, 1990; 3: 2nd Edition, Marcel Dekker, INC, New York, pp: 41 – 74.
22.
Shaw RA and Mantsch HH. Vibrational biospectroscopy: from plants to animals to humans. A historical perspective. J Mol Struct. 1999; 480–481: 1-13.
23.
Shaw RA, Kotowich S, Leroux M and Mantsch HH. Multianalyte serum analysis using mid-infrared spectroscopy. Annals Clin. Biochem. 1998; 35: 624 - 632.
24.
Khanmohammadi M, Ansari MA, Garmarudi AB and Garoosi G. Cancer diagnosis by discrimination between normal and malignant human blood samples using attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy. Cancer Investigation 2007; 25 (6): 397 – 404.
25.
Gunasekaran TS, Devi R, Sreenivasakumar M and Santhosam K. Analysis of renal failure blood sera – a spectroscopic approach. Asian J Microbiol. Biotechnol. Environ. Science 2007; 9: 281 – 286.
26.
Akkas SB, Severcan M, Yilmaz O and Severcan F. Effects of lipoic acid supplementation on rat brain tissue: An FTIR spectroscopic and neural network study. Food Chemistry 2007; 105 (3): 1281 – 1288

JCDR is now Monthly and more widely Indexed .
  • Emerging Sources Citation Index (Web of Science, thomsonreuters)
  • Index Copernicus ICV 2017: 134.54
  • Academic Search Complete Database
  • Directory of Open Access Journals (DOAJ)
  • EBSCOhost
  • Google Scholar
  • HINARI Access to Research in Health Programme
  • Indian Science Abstracts (ISA)
  • Journal seek Database
  • Google
  • Popline (reproductive health literature)
  • www.omnimedicalsearch.com