Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-524163EnglishN-0001November30HealthcareMORPHOMETRIC ANALYSIS OF CHORDA TENDINAE OF MITRAL VALVE IN HUMAN HEARTS
English0106S. KavithaEnglish A. AnandEnglish K.Y. ManjunathEnglishObjective: Chorda tendinae are the connecting link between heart valves and the ventricular chambers. In valve replacement surgeries, the effective post surgical performance of the valve depends on the length of the chordae which are anchored. This study aims at giving a definitive measurement of the chordae from the papillary muscle of the ventricle to the leaflet of the valve so that an operating surgeon can have a fair idea of how much length of the chordae can be sacrificed. Materials and Methods: 50 human hearts were studied and the length, breadth and the numerical count and the position of the chordae of the mitral valve were studied. Results: The mean length of anterior chorda was 12.77mm ±4.03 S.D and posterior 12.33mm ±3.89 S.D. The mean breadth of anterior chorda was .28mm± .20 S.D and posterior .25mm ± 0.14 S.D .The anterior papillary muscle contained around an average of 10.42 ± 2.48 S.D chordae and the posterior papillary muscle contained around an average of 9.72 ± 2.39 S.D chordae. Based on the position of attachment, lateral chordae were approximately 73.22% and apical chordae 19.06% and marginal chordae was 7.72%. Conclusion: The length and the breadth of the chordae of the anterior papillary muscle were more than the length and breadth of the posterior papillary muscle. Approximate measurements of chordae will give a definitive value in mitral valvular replacement surgeries.
Englishchorda, chordae, tendinae, mitral valve, ventricle, leaflet, papillary muscleINTRODUCTION Cardiovascular diseases are an important cause of morbidity and mortality in our modern world. This is due to affluent practices of the population with associated factors like obesity and a sedentary lifestyle combined with a diet rich in fats. Whatever be the underlying cause which results in a cardiac affliction, it has affected the quality of life and a significant reduction in the life expectancy of individuals with cardiac ailments. Advancements in cardiothoracic surgery has resulted in replacement of heart valves using artificial heart valves which has a definitive life span and issues of compatibility. Of late, cadaveric heart valve replacements have gained precedence. In cadaveric heart valve replacement surgeries, thorough knowledge of valvular anatomy and dimensions of the valves is a must before planning a surgical procedure. Preservation of chordae either in artificial heart valve and cadaveric heart valve replacement surgeries has been advocated for efficient post surgical valvular function of the heart. This study pertains to supplementing the preservation of chordae of the valvular apparatus in replacement surgeries. Detailed approximate measurements of the dimensions of the mitral valve apparatus will serve as a guideline for determining the architecture of chordae during valve replacements. Preservation of chordae is advocated as it contains extensions of ventricular conducting tissues1 . A detailed classification of the types of chordae is not possible because of variations in individuals and in places of free edge of the valve where the chordae are not attached become prone to prolapse1 . This assumes significance because anatomical or near anatomical alignment of the mitral valve has to be maintained post surgically for efficient function of the heart. The opening of the mitral valve is smaller than the tricuspid opening and the annulus itself is made of fibrocollagenous elements of different consistencies which help the annulus change in the shape and dimensions during the phases of cardiac cycle which ensures an optimal efficiency in valvular action2 . Near normal alignment of mitral valve in cadaveric heart valve and artificial heart valve replacements can be achieved by paying attention to the presurgical dimensions of the fibrocollagenous structure of the mitral valve. This study aims to give the operating surgeon a guideline for approximate measurements of chordae tendinae of the mitral valve. Many studies have elaborated on the importance of preservation of the chordae which reduces the risk of catastrophic ventricular rupture3 . Approximate measurements of the chordae have to be known because there is always a possibility of over shortening of the chordae during valve replacement procedures which can result in rupture of papillary muscle4 . This study assumes significance since in conventional operative procedures for replacement of the mitral valve as there is a loss of continuity between the annulus of the mitral valve and the papillary muscles. This loss of continuity results in a decrease in left ventricular ejection fraction and an increase in after load by eliminating regurgitation in patients with mitral regurgitation5 . Left ventricular function in post mitral valvular surgical procedures depend on preservation of chordae tendineae6 because preserved chordae is bound to promote effective valvular function. Moderator bands are structures which connect the septum of the ventricles to the ventricular free wall. These moderator bands histologically are made of cardiac muscle fibres, blood vessels and purkinje fibres and are directly involved in the conduction of electrical impulses in the heart. Presence of conducting structures in the chordae tendinae as an extension of ventricular conducting system will have to be taken into consideration in valve replacement surgeries 7, 8. Hence histological study of the chordae was also performed to demonstrate the presence of conducting tissues of the chordae in the present study.
MATERIALS AND METHODS
The specimens for the study were obtained from the departments of Forensic medicine and Anatomy of VMKV Medical College & Hospitals, Salem after obtaining necessary clearances from the Institutional Review Board and Ethical Committee for conducting the study. The specimens were fresh cadaveric human hearts numbering 50. The equipments utilized for the study were digital vernier calipers, hand lens, measuring scale and digital photographic equipment. Tissue sample was taken from a few chordae and paraffin wax blocks were prepared after processing, sections were taken and stained and examined under microscope for evidence of any conducting structures present in the chordae. The study pertained only to the chordae and papillary muscles attached to the mitral valve. The requisite parameters of the study were length (fig – 3) and breadth of the chordae, the number of chordae and the position of attachment of the chordae (fig – 4) between the papillary muscle and the annulus was documented. The length of the chorda was measured from the point of origin from the papillary muscle to the point of attachment to the leaflet. The breadth of the chorda was taken at the midpoint of the chorda after measuring the length. The number of chordae was enumerated for both anterior (fig – 1) and posterior papillary (fig – 2) muscles. The measurements obtained from the set parameters were documented.
Statistical Analysis
The measurements were subjected to statistical analysis using SPSS software version 16. The range, standard deviation and mean were calculated. Chi square test was done to compare the variables.
RESULTS
The length of the chordae in anterior papillary muscle ranged from a minimum of 5.80mm to a maximum of 25.17mm with a mean of 12.77mm. The length of chordae in posterior papillary muscle ranged from a minimum of 5.57mm to a maximum of 22.05mm with a mean of 12.33mm. The length of the chordae of anterior papillary muscle was marginally higher than that of the posterior papillary muscle. The breadth of the chordae in anterior papillary muscle ranged from a minimum of .02mm to a maximum of .89mm with a mean of .28mm.The breadth of chordae in posterior papillary muscle ranged from a minimum of .07mm to a maximum of .68mm with a mean of .25mm. The breadth of the anterior papillary muscle was marginally higher than that of posterior papillary muscle. The total number of chordae in anterior papillary muscle ranged from a minimum of 07 to a maximum of 16 with a mean of 10.42 chordae. The total number of chordae in the posterior papillary muscle ranged from a minimum of 06 to a maximum of 17 with a mean of 9.72chordae. In the present study the anterior papillary muscle contained more chordae than the posterior papillary muscle. The position of attachment of chorda tendinae in both anterior and posterior papillary muscles, the laterally attached chordae were 73.22%, apical chordae 19.06% and marginal chordae were 7.72%. The percentage of lateral chordae was more in anterior and posterior papillary muscles The chordae tendinae were stained with haematoxylin and eosin (fig – 5) which showed evidence of collagen fibres and cardiac muscle which collates that the chordae does contain conducting tissue.
DISCUSSION
Although numerous studies have elaborated on the position and attachment of chordae, enumerates the length and breadth of chordae from the tip of papillary muscle to the septal leaflet. These measurements will give a definitive value for preserving the chordae in mitral valvuloplasty performed for mitral valve insufficiency. Post surgical efficiency of the left ventricular function improved after preservation of chordae in patients who underwent mitral valvuloplasty9 . In mitral incompetence the length, location and number of chordae are required to estimate the reattachment of the chordae to the ventricular wall10. The chordal length and breadth measurements play an important role as a long length or short length would lead to mitral valve prolapse syndrome11. The length and breadth of the chordae of anterior and posterior papillary muscles will provide an approximate guideline value for determining the position and attachment of a prosthetic valve. Various authors have elaborate classifications for attached position of the chordae to the papillary muscle. The length and breadth of the chordae is determined based on the position of attachment to the papillary muscle. Based on the position of chordae, the attachments can be apical, marginal and lateral. This inequality in the arrangement of chordae determines the efficacy and robust function of the mitral valve. Hypothetically if all chordae were of equal length and with the same positional attachment, mechanical function of the mitral valve would be inefficient. Too many chordae also will interfere with left ventricular function12 . The length and breadth of the chordae determines the positional attachment of a prosthetic mitral valve in cases where the preservation of chorda papillary complex is advocated. Any laxity in reattachment of the chorda leads to discrepancy of effective valvular function and leads to mitral valve prolapse11 . The measurement of parameters obtained from the present study correlates with a study performed in the United Kingdom13. The sections taken from the chordae when analyzed under microscope after staining with haematoxylin and eosin showed evidence of wavy collagen and cardiac muscle tissue similar to the study performed earlier13. The presence of collagen and some amount of elastic fibres in the chordae is suggestive of the enormous amount of force required for repetitive movements of the valve and optimal performance of the left ventricle. Preservation of the chordae in mitral valve replacement procedures caused significant reduction in the operative mortality14. The preservation of chordae of both anterior and posterior papillary muscles of left ventricle during mitral valve replacement provides a tethering effect of chordal support and totally prevents mitral regurgitation5 . In conventional mitral valve replacements, the mitral regurgitation is more.
CONCLUSION
To summarize as evident from the analysis of the set parameters, it is observed that the length and breadth of the chordae of anterior papillary muscle are higher than the length of the chordae of the posterior papillary muscle. Numerically the chordae of the anterior papillary muscle were significantly more than the chordae of the posterior papillary muscle. In both anterior and papillary muscles of the left ventricle, laterally attached chordae were more in number than the apical and marginal chordae. In the present study the number of apical chordae was more in anterior papillary muscle and the percentage of marginal chordae was more in posterior papillary muscle of the left ventricle. The results obtained from the present study can be used as guideline for measurements in prosthetic valve replacements for mitral valve and will help the surgeons in planning a procedure and have a reduction in morbidity and mortality in critical situations.
ACKNOWLEDGEMENTS
The authors sincerely wish to thank the management, administrators and the Professor and Head of the department of Anatomy and Forensic Medicine of Vinayaka Missions Kirupananda Variyar Medical College, Salem for their whole hearted support and permissions to utilize their resources and conduct this study. The authors acknowledge the great help received from the scholars whose articles cited and included in references of this manuscript. The authors are also grateful to authors/editors/publishers of all those articles, journals and books from where the literature for this article has been reviewed and discussed. Authors are grateful to IJCRR editorial board members and IJCRR team of reviewers who have helped to bring quality to this manuscript.
Englishhttp://ijcrr.com/abstract.php?article_id=957http://ijcrr.com/article_html.php?did=957REFERENCES
1. Susan Standring. Gray’s Anatomy, 39th Edition. Anatomical basis of clinical practice, Churchill Livingstone, London. 2005; 39:1008.
2. Susan Standring. Gray’s Anatomy, 40th Edition. Anatomical basis of clinical practice, Churchill Livingstone, London. 2008; 40:970.
3. Miller DW Jr, Johnson DD, Ivey TD. Does preservation of the posterior chordae tendinae enhance survival during mitral valve replacement? Ann Thoracic Surg 1979; 28:22- 7.
4. Trites PN, Kiser JC, Johnson C, et al. Occlusion of Medtronic Hall mitral valve prosthesis by ruptured papillary muscle and chordae tendinae. J Thorac Cardiovasc Surg 1984; 88:301-2.
5. Shigehito Miki, Kenji Kusuhara, Yuichi Ueda et al. Mitral valve Replacement with preservation of chordae tendinae and papillary muscles in Ann Thorac Surg Jan 1988; 45: 28- 34.
6. Spence PA, Peniston CM, David TE, Mihic N, et al. Toward a better understanding of etiology of left ventricular dysfunction after mitral valve replacement: An experimental study with possible clinical implications. Ann Thorac Surg 1986; 41:363-371.
7. Gulyaeva AS, Roshchevskaya IM. Morphology of moderator bands (Septomarginal Trabecula) in porcine heart ventricles. J Vet.Med – Anatomia, Histologia, Embryologia, 2012; 41:1-9.
8. Roberts WC, Cohen LS .Anomalous left ventricular band. An unemphasized cause of a precordial musical murmur. Am.J.Cardiol, 1969; 23:735-8.
9. Jose Luiz Dancini, Pablo Maria Alberto Pomerantzeff,Guilherme Sobreira Spina, et al. Valve Replacement with Chordal preservation and Valvuloplasty for Chronic Mitral Insufficiency. Arq Bras Cardiol 2004; volume 82: 243-50.
10. Gerda L Van Rijk et al. Evolving significance of Human chordae Tendineae. Am. J. of Card Surg 1994; 9:255 – 261.
11. Harken D. E, Ellis L.B, Ware P.F, Norman L.R. The surgical treatment of mitral stenosis, valvuloplasty in New England J Med 1948; 238: pp: 801 – 806.
12. Silverman M E, Hurst J.W, et al. The mitral complex: interaction of anatomy, physiology and pathology of the mitral annulus, mitral valve leaflets, Chordae tendineae and papillary muscle. Am heart J 1968; 76: 399.
13. Millington-Sanders C, Meir A, Lawrence L, Stolinski C. Structure of chorda tendineae in the left ventricle of the human heart. Journal of Anatomy. 1988; 192:573 – 581.
14. Lillehei CW, Levy MJ, Bonnabeau RC. Mitral valve replacement with preservation of papillary muscles and chordae tendineae. J Thorac Cardiovasc Surg 1964; 47:532.
Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-524163EnglishN-0001November30HealthcareA COMPARATIVE STUDY OF EARLY ONSET VERSUS LATE ONSET NEONATAL SEPSIS WITH SPECIAL REFERENCE TO BACTERIOLOGICAL, DEMOGRAPHIC AND CLINICAL PROFILE
English0715Kuhu PalEnglish Arnab Kumar SamantaEnglish Ritesh SinghEnglishIntroduction: Neonatal sepsis is classically divided into early onset sepsis (EOS) and late onset sepsis (LOS) depending upon the time of onset of sepsis. Aims: To determine the differences in bacteriological, demographic and clinical profile between EOS and LOS, if any. Setting and Design: A prospective study was carried out in a tertiary care centre of Eastern India from March 2011 to February 2012. Methods: Neonates with less than 28 days age, associated with a clinical diagnosis of sepsis were included in the study. Blood was collected aseptically for culture and isolates were identified in conventional way and antibiotic sensitivity test was done by Kirby Bauer disc diffusion method. Epidemiological parameters, clinical features were compared in the light of culture report. Statistical analysis used: Chi square test, ANOVA and multivariate regression analysis. Results: Out of 230 clinically suspected septic neonates, 72.6% of neonates presented with EOS and 27.4% with LOS. None of the bacteria except Klebsiella sp. was significantly associated with EOS (P=0.043). Almost identical result was observed between EOS and LOS, when demographic parameters including gender, birth weight, gestational age, mode of delivery, outcome and clinical presentation were compared. Caesarean section was significantly associated with increased morbidity in cases of EOS (multivariate P value 0.018). Conclusion: There was no statistically significant difference in epidemiological, clinical and bacteriological profile between EOS and LOS except a few. So it is better to judge the two groups of neonatal sepsis as one and universal.
EnglishDemography, Early onset sepsis, Late onset sepsis, Microbiological spectrumNTRODUCTION
Neonatal Sepsis is defined as a clinical syndrome characterized by signs and symptoms of systemic infections accompanied with bacteraemia in the first month of life.[1] Symptoms occurring within the age of 3 days are considered as early onset sepsis (EOS). Late onset sepsis (LOS) usually occurs after 3 days of life. This distinction has thought to have a clinical relevance, due to difference in peripartum pathogenesis.[2] In EOS neonates acquire infection by the organisms colonized in mothers’ genitourinary tract. But late onset sepsis is either nosocomial or community acquired.[3] But, this conventional difference may not be relevant today, as alteration in genital flora by intrapartum medication or prolonged hospital stay and failure to maintain proper asepsis during delivery may lead to intermixing of causative agents of EOS and LOS.[4,5] The aim of the study was to evaluate the microbiological, demographic, and clinical characteristics of EOS and LOS and to determine the differences in these two groups of neonates if any.
MATERIALS AND METHODS
Study design: This was a hospital based singlecentred prospective study. Study area: Study was carried out in the Neonatal Care Unit, Department of Paediatrics of a tertiary care centre of eastern part of Bengal. Study population: Neonates with less than 28 days age, with clinical signs of sepsis, including refusal of feed, lethargy, respiratory distress, abdominal distension, vomiting, hypothermia etc born inside this hospital as well as referred from different centres were enrolled in this study. Exclusion criteria: Babies who had received antibiotics before collection of blood samples, having surgical problems, chromosomal or congenital anomalies were excluded from the study. Neonates who were referred to higher centres or who left the hospital against medical advice before proper diagnosis were also excluded from the study. Study period: March 2011 to February 2012. Study was carried out after getting permission from Institutional Ethics Committee. Written informed consent was obtained from parents/guardians of neonates who participated in this study. Procedure: Each neonate was assessed by a paediatric resident. Medical records of all neonates with suspected sepsis were reviewed for demographic characteristics (birth weight, gestational age, gender, type of delivery, associated risk factors) and babies were followed till recovery or death. 2 ml of blood was collected maintaining proper aseptic technique and 1ml was inoculated in 5 ml of brain heart infusion broth with 0.025 % sodium polyanethol sulfonate. Another 1ml was used for testing other haematological parameters. The blood culture broth was incubated aerobically at 370 C. Blind subculture was done on 24hrs, 48 hrs, 72 hrs, then on 7th day. Any sign of growth in between was followed by subculture. Media used for subculture were 5% sheep blood agar, chocolate agar and Mac Conkeys agar (Himedia Laboratories). In suspected cases of meningitis CSF was collected and cultured in chocolate agar and blood agar media. Isolates were identified by colonial morphology, Gram staining as well as standard biochemical tests. Aerobic spore bearers and diphtheroids, wherever grown were regarded as contaminants. The remaining isolates were included in the analysis. Antimicrobial sensitivity test was done by Kirby Bauer disc diffusion method following guidelines provided by Clinical and Laboratory Standards Institute (CLSI).[6] The clinical manifestations and demographic parameters were compared with blood culture reports. Presence of any of the clinical features was considered as indicator of morbidity. Statistical analysis: Percentages and Mean ± SD (Standard Deviation) were used to express the categorical variables and quantitative variables respectively. Differences in distribution were analysed by Chi square test and ANOVA. By multivariate regression analysis, Odds Ratio (OR) and 95% confidence interval for OR was also calculated. The statistical analysis was done using Statistical Package for Social Sciences (SPSS) version 16.0.
RESULTS
Two hundred and thirty neonates with suspected sepsis were followed. Incidence of EOS (167/230=72.6%) was more than LOS (63/230=27.4%) in this institution. Gram negative bacilli (GNB) were the major offender 45/65=69.2%) of EOS, whereas Gram positive cocci (GPC) were responsible for 50% (13/26) of LOS. Staphylococcus aureus was in the leading role (9/13=69.2%) in LOS. Klebsiella sp was the most common organism responsible for EOS followed by Staphylococcus aureus , E.coli , Enterobacter sp . The contribution of Klebsiella sp was more in EOS than LOS [29(44.62%) versus 5(19.23%), P value= 0.043]. The spectrum of organisms causing neonatal sepsis has been shown in Table 1. No particular organism was exclusively isolated from either EOS or LOS. Five CSF samples sent for bacteriological culture were found to be sterile. Demographic characteristics of EOS and LOS have been shown in Table2. There was no significant difference in different parameters of EOS and LOS except place of delivery. The neonates who had been referred from other centres were more prone to develop LOS. Male female ratio was 1.6:1 in both EOS and LOS. Mean Birth weight of babies with EOS was 2.3 ± 0.49 kg. Mean gestational age was 36 ± 2 weeks and these were almost similar in LOS group. Majority of newborns were delivered by vaginal delivery (76.9%) compared to caesarean section(CS) (23.1%) in EOS but reverse situation was observed in LOS, where 42.1% babies were delivered by CS. Mortality rate due to EOS and LOS remained almost equal. Majority of newborns with neonatal sepsis presented with refusal of feed (52.7%) and respiratory distress (24.1%). These features were true for both EOS and LOS (Table 3). Multivariate logistic regression analysis showed that morbidity was significantly higher in babies delivered by caesarean section in EOS group (P=0.018) only. No significant differences were seen in morbidity indicators based on the male gender, low birth weight, pre term status of the neonates (Table 4). Antimicrobial susceptibility testing from blood culture isolates revealed that Gram negative bacilli were highly resistant to ampicillin (83.64%), cefixime (78.18%), gentamicin (70.91%) and cefotaxime (70.91%). Pseudomonas sp and Acinetobacter sp showed resistance to almost all the common drugs used. Four of the Klebsiella sp and one E.coli were identified phenotypically as extended spectrum beta lactamase (ESBL) producers (9.09%). Almost 50% of the GNB were resistant to piperacillin – tazobactam, and amoxycillin- clavulanic acid. Imepenem was found to be the most sensitive drug followed by amikacin and chloramphenicol (Table 5). Gram positive cocci were also quite resistant to ampicillin (87.10%), azithromycin (64.52%) and gentamicin (67.74%). Thirteen (61.9%) Staphylococcus aureus were screened as Methicillin resistant Staphylococcus aureus (MRSA). But no Vancomycin resistant Staphylococcus aureus (VRSA) was reported (Table 6).
DISCUSSION
Among 230 suspected cases of neonatal sepsis blood culture was positive in 91 (39.56%) neonates. Of which incidence of EOS was 71.4%. This was in accordance with the studies done by V Sundaram,[5] M.N Shah,[7] A Hafsa,[8] but reverse result was seen in studies in Nepal,[9] South India,[10] Taiwan[11] and Johannesburg.[12] The causes of this discordance might be due to the fact that those centres were running as referral centres without facility of delivery and consideration of timeframe of EOS as 48hrs. Another possible explanation for a lower incidence of LOS in this study might be the early discharge policy of the neonatal unit. The most common organism causing EOS was found to be Klebsiella sp followed by Staphylococcus aureus, E. coli, whereas predominant organism in LOS was Staphylococcus aureus. This result was similar to a study done in northern India.[5] Pivotal role of enteric Gram negative bacilli especially Klebsiella sp in EOS was also ascertained in a couple of studies in Eastern India,[13,14] Bangladesh[8] and Nepal.[9] Along with Staphylococcus aureus, E.coli was the found to be responsible for LOS in a study in Nepal.[9] Studies from different parts of the world,[11,12,15,16] showed that Gram positive organisms were responsible for more than 60% of LOS and coagulase negative staphylococcus was the major culprit. But a study in Iran[17] showed that Enterobacter sp was the predominant organism of both EOS and LOS. Disagreement of bacteriological profile of EOS and LOS in different regions was the essence of a continuous surveillance of the aetiology of neonatal sepsis. Types of organisms isolated in EOS and LOS were more or less similar except Klebsiella sp which was closely associated with EOS (P value=0.043). In the present study, neonatal sepsis was significantly higher in low birth weight and preterm babies (P value 0.027 and 0.00 respectively). But no significant diversity of demographic parameters including birth weight, gestational age, gender, mode of delivery, outcomes, were observed between EOS and LOS except place of delivery. Referred neonates were more prone to develop LOS. This was in concordance with a study in South India,[10] Iran[17] and Nepal.[18] Birth weight served as a differentiating factor for LOS and EOS in a study in South India [10] but that was not prominent in this study. Mortality in EOS was higher than LOS group in Bangladesh[8] and Iran[15] but in present study it was almost same in EOS and LOS. Overall case fatality rate due to sepsis is 16.4% which was found to be quite similar to a studies done in Johannesburg Hospital (20.8%)[12] and Iran (19%).[15] Clinical presentation of EOS and LOS were similar in this study simulating the result of an Iranian study.[17] Refusal to feed was the most common presenting feature followed by respiratory distress, lethargy and hypothermia in our study and studies from Nepal[18] and West Bengal[13] whereas respiratory distress was the chief complaint of the study in Iran.[19] Multivariate regression analysis showed caesarean section was associated with increased morbidity in cases of EOS but no significant differences were seen in morbidity rates based on gender, birth weight, and preterm status. This was comparable to a Study in Tehran.[17] Though Signore C[20] et al showed that caesarean delivery was associated with increased morbidity in neonates, but not associated directly with sepsis. So this might be due to co-morbidity that had occurred after Caesarean delivery. None of the demographic factors was found to be associated with increased risk of LOS. As seen across the rural India [13] and Bangladesh[8],result of this study also revealed that most of the cases of sepsis were caused by multidrug resistant bacteria. Among them, Gram negative bacteria showed high resistance to ampicillin, gentamicin and cephalosporin group of drugs. Emergence of ESBL (9.09%) was a threat to the clinician but it was less than the incidence of ESBL found in a NICU of Iran (44%).[15] In contrast to a study in rural Bengal[6],where more than 80% cases were found to be resistant to amikacin and ciprofloxacin, 50% of GNB were resistant to these drugs in this hospital. Imepenem remains as the only effective drug against these notorious pathogens. Incidence of MRSA was 61.9 %. Similar finding has been reported elsewhere.[15] Vancomycin, teicoplanin and linezolid were recovered as the most effective drugs for gram positive cocci. Similar findings have been reported in previous studies done in eastern India.[14] The high prevalence of multidrug resistant organisms in this study indicates an association of neonatal sepsis with treatment failure, higher morbidity and mortality and increased cost. Limitation of this study was that, we did not consider maternal genital flora or any maternal risk factors which might have a major contribution to the outcome of babies.
CONCLUSION
This study reflected the fact that neonates were suffering from EOS and LOS in this hospital with almost same epidemiological and clinical presentation with very little differences in microbiological profile. Gram negative organisms were predominant isolates of sepsis so, their source is to be searched for and necessity of infection control measures is to be emphasized. Collection of up-to-date data would strengthen local information system, ultimately enabling policy-makers and programme planners to use existing resources more effectively to achieve a better and effective goal to reduce mortality as well as morbidity caused by neonatal sepsis
ACKNOWLEDGEMENTS
We would like to thank the colleagues and staffs of Department of Paediatrics and Microbiology for their help to pursue this work. We want to acknowledge the immense help received from the scholars whose articles are cited and included in references of this manuscript and also grateful to authors/editors/publishers of all those articles, journals and books from where the literature for this article has been reviewed and discussed
Englishhttp://ijcrr.com/abstract.php?article_id=958http://ijcrr.com/article_html.php?did=958REFERENCES
1. Klein Jo. Bacterial Sepsis and Meningitis. In: Remington JS, Klein Jo.eds. Infectious diseases of fetus and newborn and infants. 5 theds. W B Saunders Co. Philadelphia; 2001:943-984.
2. Guha D.K, Saili A. Guha R, Aggarwal A. Common Infections in the Newborn. In: Neonatology – Principles and Practice. Guha D.K; 3rd eds. Jaypee Brothers Medical Publishers (P) Ltd. New Delhi, India; 2005: 654 – 672.
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4. A.K. Zaidi, W.C. Huskins, D. Thaver et al., Hospital acquired neonatal infections in developing countries, Lancet 2005; 365 : 1175–1188.
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7. Shah M. N. and. Desai P. B. Clinical and bacteriological profiles of blood culture positive sepsis in newborns.IntJ. of Pharm. and Life Sci. 2011; 2(9):1041-1045
8. Hafsa A, Fakruddin M, Hakim MA, Sharma JD. Neonatal bacteremia in a neonatal intensive care unit: analysis of causative organisms and antimicrobial susceptibility. Bangladesh Journal of Medical Science 2011; 10 (3) ;187-194
9. Shrestha NJ, Subedi KU, Rai GK. Bacteriological Profi le of Neonatal Sepsis: A Hospital Based Study. J Nepal Paedtr Soc 2011;31(1):1-5.
10. Kuruvilla A K, Pillai S, Jesudason M and Jana A K. Bacterial profile of sepsis in a Neonatal Unit in South India. Indian Pediatrics 1998; 35:851-858
1. Jia-Horng Jiang, Nan-Chang Chiu, Fu-Yang Huang, Hsin-An Kao, Chyong-Hsin Hsu, Han-Yang Hung et al . Neonatal sepsis in the neonatal intensive care unit: characteristics of early versus late onset. J Microbiol Immunol Infect 2004;37:301-306
12. Motara F, Ballot DE, Perovic O. Epidemiology of neonatal sepsis at Johannesburg hospital. The Southern African journal of Epidemiology and Infection 2005;3: 90-93
13. Viswanathan R, Singh Arun K, Ghosh C, Dasgupta S, MukherjeeS,and Basu S. Profile of Neonatal Septicaemia at a District-level Sick Newborn Care Unit. J Health Popul Nutr 2012;30(1):41-48
14. Pal K, Samanta A K. Bacteriological profile and antimicrobial resistance pattern in early onset neonatal sepsis: challenge to the physician. Int J Cur Res Rev 2013; 5 (6):51- 58
15. Yilmaz N O, Agus N, Helvaci M, Kose S, Ozer E, and Sahbudak Z. Change in Pathogens Causing Late-onset Sepsis in Neonatal Intensive Care Unit in Izmir, Turkey. Iranian Journal of Pediatrics 2010; 20 (4): 451-458
16. Joseph CJ, Wee Bin Lian W B, Yeo C L. Nosocomial Infections (Late Onset Sepsis) in the Neonatal Intensive Care Unit (NICU). Proceedings of Singapore Healthcare 2012; 21(4): 238-244
17. Afsharpaiman S,Torkaman M, Saburi A,Farzaampur A, Amirsalari S, Kavehmanesh Z. Trends in incidence of neonatal sepsis and antibiotic susceptibility of causative agents of two neonatal intensive care units in Tehran.I.R. Iran. Journal of Clinical Neonatology. 2012;1(3):124-130.
8. Khinchi Y. R, Kumar A, Yadav S. Profile of Neonatal sepsis. Journal of college of Medical Sciences-Nepal, 2010; 6 (2): 1-6
19. Mosayebi Z, Movahedian AH, Soori T. Clinical and bacteriological chartacteristics of neonatal sepsis in an intensive care unit of Kashan, Iran: A 2 years descriptive study. Arch Pediatr Infect Dis. 2013;1(2):61-64
20. Signore C.Klebanoff M. Neonatal morbidity and mortality after elective caesarean delivery. Clin Perinatol 2008; 35: 361-371
Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-524163EnglishN-0001November30HealthcareHIGH LEVEL AMINOGLYCOSIDE RESISTANT ENTEROCOCCUS SPECIES : A STUDY
English1621Niharika LallEnglish Silpi BasakEnglishBackground and Objectives: The emergence of Enterococcus species as a causative agent of Health care Associated Infections, poses therapeutic challenge to clinicians. Enterococci are well equipped with intrinsic resistance to multiple antibiotics. Acquired resistance to commonly used antibiotics such as Penicillin, Aminoglycoside and Vancomycin have made the situation even worse. Detection of High Level Aminoglycoside Resistance (HLAR) in Enterococcus species can predict the loss of synergy between cell wall active antimicrobial agents and Aminoglycosides. Hence, the present study was undertaken to detect the incidence of High level Aminoglycoside Resistant (HLAR) Enterococcus species isolated in a rural hospital and to study their antibiotic susceptibility profile. . Method: HLAR in Enterococcus species was detected by disk diffusion test using High level Streptomycin (HLS - 300 μg) disk and High level Gentamicin (HLG - 120 μg) disk and Agar dilution method as per CLSI guidelines. High Level Gentamicin Resistance (HLGR) was also confirmed by HiMedia Ezy MIC Strip. Antibiotic sensitivity test was done by Kirby Bauer Disc diffusion method as per CLSI guidelines. Result: Out of total 190 Enterococcus species isolated from different clinical samples, High Level Aminoglycoside Resistance was detected in 115 (60.5%) Enterococcus strains. Amongst 115 HLAR Enterococcus strains, 59 (51.4%) were E. faecalis and 56 (48.6%) were E. faecium. Conclusion: We hereby conclude that Enterococcus strains, isolated from different clinical specimens must be screened routinely for HLAR (High Level Aminoglycoside Resistance) by all Clinical Microbiology Laboratories to improve the therapeutic outcome.
EnglishEnterococci, High Level Aminoglycoside Resistant (HLAR), Multi-drug resistant.INTRODUCTION
Enterococci are Gram positive bacteria that normally inhabit the gastrointestinal tract of many animals including humans. However, when they colonize the sites where they are not normally found, they can become pathogens1 . Enterococci are now the second most common cause of nosocomial urinary tract and wound infections and third most common cause of nosocomial bacteremias2 . Serious enterococcal infections (e.g., bacteremia and endocarditis) require treatment with a bactericidal combination of antibiotics that should include penicillin group of drugs (e.g., Ampicillin or Penicillin G) to which the Enterococcus isolate is susceptible and an Aminoglycoside (e.g., Gentamicin or Streptomycin) to which the Enterococcus isolate does not exhibit high-level resistance3 . High-level Aminoglycoside resistance (HLAR) among Enterococci is increasingly being reported worldwide. The presence of HLAR is predictive of the loss of synergy between a cell-wall-active agent (e.g. Penicillin, Ampicillin or Vancomycin) and an Aminoglycoside, which makes the treatment of serious enterococcal infections difficult4 . Hence, the present study was undertaken to detect the incidence of High-level aminoglycoside resistant (HLAR) Enterococcus species isolated from different clinical samples and their antibiotic susceptibility profile.
MATERIAL AND METHODS
A total number of 190 Enterococci were isolated from various specimens like urine, blood, pus and wound swab, body fluids, etc from July 2011 to August 2013. The specimens were collected from patients attending Indoor and Outdoor Patients Department of our Hospital and sent to Department of Microbiology. Our hospital is a tertiary care hospital in a rural set up. All Enterococcus species were identified by conventional methods2 . The speciation of Enterococcus species was done by scheme proposed by Facklam and Collins5 . Antibiotic susceptibility test was done for all 190 strains by Kirby- Bauer disk diffusion method, according to CLSI guidelines6 . Antibiotics like Ampicillin(10µg), Linezolid (15µg),Vancomycin (30µg), Erythromycin (15µg), Tetracycline (30µg), Quinupristin-dalfopristin (15µg) were used. Additionally, for urine samples only, Nitrofurantoin disc (300µg) was used. HLAR (High Level Aminoglycoside Resistance) was detected by disk diffusion test using High level Streptomycin (HLS - 300 µg) disk and High level Gentamicin (HLG - 120 µg) disk and Agar dilution method as per CLSI guidelines6 . As per CLSI guidelines, for detection of High Level Gentamicin Resistance by Agar dilution method, Gentamicin concentration was taken as 500 µg/ml. 10 µL of 0.5 Mc Farland suspension of test strain was spotted onto BHI (Brain Heart Infusion) agar surface containing Genatamicin 500 µg/ml6 . High Level Gentamicin Resistance (HLGR) was also confirmed by putting the Himedia Ezy MIC strip. A lawn culture of the test strain (turbidity adjusted to 0.5 Mc Farland) was done on Mueller Hinton (MH) agar plate and Himedia Ezy MIC strip was put and MIC (Minimum Inhibitory Concentration) was detected after incubation at 370C for 18 hours. MIC range of the strip was from 0.064-1024 µg/ml. E.faecalis ATCC 29212 was used as quality control for all the tests.
RESULTS
Out of 190 Enterococcus strains isolated, High level aminoglycoside resistance (HLAR) was detected in 115(60.5%) strains. HLAR strains were detected by disc diffusion test, agar dilution method and HLGR by Gentamicin Ezy MIC strip (photo1,2 and 3). Amongst 115 HLAR strains, 59 (51.4%) strains were E.faecalis and 56 (48.6%) were E.faecium (fig1). Only High level Gentamicin resistance (HLGR), only High level Streptomycin resistance (HLSR) and both HLGR and HLSR producing E.faecalis strains were detected as 23(38.9%), 7 (11.9%) and 29 (49.2%) respectively out of total 59 HLAR positive E.faecalis strains (fig 2). Similarly, out of total 56 HLAR producing E.faecium strains, 34(60.7%) produced only High level Gentamicin resistance (HLGR), 5(8.9%) produced only High level Streptomycin resistance (HLSR) and 17 (30.4%) produced both High level Gentamicin resistance (HLGR) and High level Streptomycin resistance (HLSR) (fig 3). Out of 93 HLAR Enterococcus strains isolated from urine samples, 49 (52.7%) were E.faecalis and 44 (47.3%) were E.faecium. 11(68.8%) HLAR strains were isolated from total 16 pus and wound swab samples. Out of 11 HLAR strains, E. faecalis and E. faecium were 7 and 4 strains respectively. Out of total 25 blood samples, 9(36%) strains produced HLAR and E. faecium strains were shown to exhibit more HLAR than E. faecalis. Amongst 9 HLAR Enterococcus strains, only 3(33.3%) strains were E. faecalis whereas 6(66.7%) were E. faecium. Only 2 HLAR strains were detected from other specimens, which were identified to be E. faecium (fig 4). Maximum 36 (31.3%) HLAR strains were isolated from Surgery ward followed by Pediatrics (22), Medicine (16) and Gynaecology ward (10) (fig 5). Out of 36 HLAR strains isolated from Surgery ward, 23 (63.9%) were E. faecalis and rest 13 were E. faecium. Only 7 (6.1%) HLAR strains were isolated from different ICUs (MICU-3, NICU-3 and PICU-1). No HLAR strain was isolated from OT ICU . Out of 115 HLAR strains, 90 Enterococcus strains were MDR (Multi-drug Resistant). MDR Enterococcus strains were detected on the basis of resistance (acquired) to Erythromycin, Tetracyline and High level aminoglycosides. All (100%) HLAR Enteococcus strains were sensitive to Vancomycin and Linezolid. Out of 115 HLAR strains, only 7 (6.1%) and 23 (20%) were sensitive to Quinupristin-dalfopristin and Ampicillin respectively. Nitrofurantoin sensitivity was tested for 93 HLAR Enterococcus strains isolated from urine sample only and 85(91.4%) HLAR strains were sensitive to Nitrofurantoin.
DISCUSSION
A common regime for treatment of serious enterococcal infections is the combination of cellwall inhibitors, such as penicillin, ampicillin or vancomycin; with aminoglycosides, such as streptomycin or gentamicin. The addition of cellwall inhibitor agent helps in the penetration of the aminoglycoside into the bacterial cytoplasm, making the intrinsically resistant organism aminoglycoside sensitive. The presence of HLAR in Enterococci makes the synergism of cell-wall inhibitor and aminoglycoside ineffective7 . In our study, 60.5% HLAR producing Enterococcus strains were isolated. Our study correlated well with Deshpande et al who had reported 58.8% HLAR producing Enterococcus strains8 . Mendiratta et al in 2008 reported 46% HLAR producing Enterococcus strains9 , whereas Vinod kumar et al in 2008 reported 65.6 % HLAR producing Enterococcus strains10. The study conducted by Mendiratta et al also reported that 69.6 % of their HLAR strains were E.faecalis and 30.4 % were E. faecium9 , whereas in the present study 51.4 % strains were E. faecalis and 48.6 % were E. faecium. As per χ 2 test, when only HLGR producing E. faecalis (23) and only HLGR producing E. faecium (34) are considered, we conclude, that isolation of only HLGR producing E.faecium is significantly higher than only HLGR producing E. faecalis11. In our study, out of 115 HLAR strains, 90 (78.3%) were MDR which is quite higher than reported by Deshpande et al (57%) in 20088 . MDR is defined as acquired nonsusceptibility to at least one agent in three or more antimicrobial categories12. Nitrofurantoin sensitivity was tested for 93 HLAR Enterococcus strains and it showed good activity (total 91.4% sensitivity) against both E.faecalis and E.faecium. Probably, the resistance could not be acquired by Enterococcal strains as Nitrofurantoin is not used commonly nowadays. HLAR (High level aminoglycoside resistance) i.e., Streptomycin MICs >2000 µg/ml or Gentamicin MICs > 500 µg/ml is an acquired resistance2 . HLAR occurs due to the presence of AME ( Aminoglycoside modifying enzymes). The most frequently encountered enzyme include dual function 2?phosphotransferase and 6?acetyl transferase conferring HLR to all available Aminoglycoside (Kanamycin, Gentamicin, Amikacin, Netilmicin, Tobramycin) except streptomycin13. Hence, gentamicin resistance is a good predictor of resistance to other aminoglycosides except streptomycin14 . 6?adenyl transferase is another AME which is responsible for HLR to streptomycin but does not inactivate other useful aminoglycosides13 .
CONCLUSION
We hereby conclude, that as High level Aminoglycoside resistant (HLAR) Enterococcus species can predict the loss of synergy between cell-wall active antimicrobial agents and Aminoglycosides, all Enterococcus strains must be screened routinely for HLAR by all Clinical Microbiology Laboratories to get an effective therapeutic outcome. Administration of Penicillin –Aminoglycoside combination without knowing HLAR status of the causative Enterococcus species can be fatal in life threatening Enterococcal infections.
ACKNOWLEDGEMENT
Authors are thankful to DMIMS (DU) for the help rendered, while conducting the study. Authors also acknowledge the immense help received from the scholars whose articles are cited and included in references of this manuscript. The authors are also grateful to authors/editors/publishers of all those articles, journals and books from where the literature for this article has been reviewed and discussed.
Englishhttp://ijcrr.com/abstract.php?article_id=959http://ijcrr.com/article_html.php?did=959REFERENCES
1. Tendulkar PM, Baghdayan AS, Shankar N. Pathogenic Enterococci: New developments in the 21st Century. Cell Mol Life Sci. 2003;60: 2622-2636.
2. Washington Winn, Jr., Stephen Allen, William Janda et al.Koneman’s Color Atlas and Textbook of Diagnostic Microbiology. 6th ed. Philadelphia: Lippincott Williams And Wilkins; 2006. Chapter 13, Gram –Positive Cocci Part II: Streptococci, Enterococci, and the “Streptococcus-Like” Bacteria; p.672-764.
3. Herman, D. J., and Gerding DN. Screening and treatment of infections caused by resistant enterococci. Antimicrob. Agents Chemother. 1991; 35: 215–219.
4. Papaparaskevas J, Vatapoulos A, Tassios PT, Avlami A, Legakis NJ and Kalapothaki V. Diversity among high-level aminoglycosideresistant enterococci. Journal of Antimicrobial Chemotherapy 2000;45: 277-283.
5. Facklam RR, Collins MD. Identification of Enterococcus species isolated from human infections by conventional test scheme. J Clin Microbiol. 1989;24: 731-734.
6. Clinical and Laboratory Standards Institute (2012) Performance standards for antimicrobial susceptibility testing: 22th informational supplement. CLSI document M100-S22. Clinical Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA.
7. Adhikari L. High-level Aminoglycoside Resistance and Reduced Susceptibility to Vancomycin in Nosocomial Enterococci. J Glob Infect Dis. 2010; 2(3): 231–235.
8. Deshpande VR, Karmarkar MG, Mehta PR. Prevalence of multidrug-resistant enterococci in a tertiary care hospital in Mumbai, India. J Infect Dev Ctries. 2013;7 (2): 155-158.
9. Mendiratta DK, Kaur H, Deotale V, Thamke DC, Narang R, Narang P. Status of High level aminoglycoside resistant Enterococcus faecium and Enterococcus faecalis in a rural hospital of Central India. Indian Journal of Medical Microbiology. 2008; 26 (4): 369-71.
10. Vinodkumar C. S, Srinivasa H., Basavarajappa K. G., Geethalakshmi S., Bandekar N. Isolation of bacteriophages to multi-drug resistant Enterococci obtained from diabetic foot: A novel antimicrobial agent waiting in the shelf? Indian Journal of Pathology and Microbiology. 2011; 54 ( 1 ):90-95.
11. Park K. Park’s Textbook of Preventive and Social Medicine. 22nd ed. Jabalpur: Banarsidas Bhanot; 2013. Chapter 19, Health information and basic medical statistics: p.782-796.
12. Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, Harbarth S, Hindler JF, Kahlmeter G, Olsson-Liljequist B, Paterson DL, Rice LB, Stelling J, Struelens MJ, Vatopoulos A, Weber JT, Monnet DL. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect. 2012; 18(3):268-81.
13. Marothi YA, Agnihotri H, Dubey D. Enterococcal resistance:an overview. Indian Journal of Medical Microbiology. 2005; 23(4):214-219.
Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-524163EnglishN-0001November30HealthcareVALIDATION OF NEWLY FORMULATED LAPORTEA ARISHTA BY USING DIFFERENT ANALYTICAL METHODS
English2234Deepa P.English SeenaEnglishTraditional medicines have nurtured the knowledge of natural remedies against diseases since ages. Growing awareness about harmful side effects of modern medicine has led to interest in ayurveda. Laportea interrupta (L) Chew of family Urticaceae having common name Hawaii woodnettle is an herb having many traditional uses. As a preliminary step physicochemical analysis of crude drug, Laportea interrupta-leaves were performed. Physicochemical analysis includes Rodent Contamination, Foreign Organic Matter, Insect Infestation Total Ash Value, Acid Insoluble Ash, Water Insoluble Ash, Sulphated Ash and Moisture Content. Laportea interrupta leaves extracted in soxhlet apparatus using ethyl acetate, petroleum ether, chloroform, methanol and water for identification of constituents by qualitative phytochemical analysis (tests for protein, carbohydrates, phenols and tannins, flavanoids, saponins, glycosides, steroids, terpenoids and alkaloids) and quantitative phytochemical analysis (total phenol content and total flavanoid content). The collected leaves of Laportea interrupta was used to prepare arishta. After preparing the arishta the organoleptic characteristics (colour, odour, taste and appearance), the physicochemical analysis (pH, acid value, alcohol content, total solid content, viscosity and refractive index), quantitative analysis (alcohol content by spectrophotometry by dichromate method, total reducing sugar, total phenol content and total flavanoid content), anti-oxidant activity determination ( Ferric Thiocyanate method, Thiobarbituric acid method and Total Antioxidant Activity by FRAP method) were performed. LCMS was performed for determining various constituents (Qualitative analysis) and standardisation of formulation was done by using UV and HPLC. The above formulated Laportea Arishta was validated by using different analytical equipment’sas per ICH guidelines.
EnglishArishta, Physicochemical analysis, Organoleptic characteristicsINTRODUCTION
Herbal medicine (or "Herbalism") is the study and use of medicinal properties of plants. They have the ability to synthesize a wide variety of chemical compounds that are used to Herbal medicine (or "Herbalism") is the perform important biological functions, and to defend against attack from predators such as insects, fungi and herbivorous mammals. Many of these phytochemicals have beneficial effects on long-term health when consumed by humans, and can be used to effectively treat human diseases. Herbal medicines do not differ greatly from conventional drugs in terms of how they work. This enables herbal medicines to be as effective as conventional medicines, but also gives them the same potential to cause harmful side effects. Ayurveda - The Natural Way of Treating Disease Ayurveda is a form of alternative medicine that uses different parts of herbs, plants and parts of animals to cure diseases and ailments. The word Ayurveda comes from two Sanskrit words, 'ayus' meaning life and 'Veda' meaning knowledge. Ayurveda is a complete and highly integrated science of life. It is a holistic and complex spiritual and philosophical system which has been developed over thousands of years and is a subtle body of knowledge. This medical system is based on the system of prevention which means that people are often treated before the symptoms of the illness become evident. Arishta and Asava have been used as medicines for over 3000 years to treat various disorders and are also taken as appetizers and stimulants. Due to their medicinal value, sweet taste, and easy availability people are prone to consume higher doses of these drugs for longer periods. Arishta are medicinal preparations made by soaking the drugs in coarse powder form or in the form of decoction (Kashaya), in a solution of sugar or jaggery, as the case may be, for a specified period of time, during which it undergoes a process of fermentation generating alcohol, thus facilitating the extraction of the active principles contained in the drugs. Laportea interrupta (L) Chew of family Urticaceae having common name Hawaii woodnettle is an herb having many traditional uses like Allergies, Alopecia, Amenorrhea, Arthritis, Asthma, Bedwetting/incontinence, Female Hormones, Fibromyalgia, Libido, Longevity/tonics, Menorrhagia, Nutrition, Osteoporosis, PMS, Prostate, Rheumatoid arthritis. Flavanoids are natural products widely distributed in plant kingdom and currently consumed in large amounts in the daily diet. Flavanoids have been reported to exert wide range of biological activities like antiinflammatory, antibacterial, antiviral, antiallergic, cytotoxic antitumor, treatment of neurodegenerative diseases, vasodilatory action. In addition they are known to inhibit lipid-peroxidation, platelet aggregation, capillary permeability and fragility, cyclooxygenase and lipoxygenase enzyme activities. They exert these effects as antioxidants, free radical scavengers, chelators of divalent cation. These are also reported to inhibit variety of enzymes like hydrolases, hyalouronidase, alkaline phosphatise, arylsulphatase, cAMP phosphodiesterase, lipase, α-glucosidase, kinase. Standardization is the development and implementation of concepts, doctrines, procedures and designs to achieve and maintain the required levels of compatibility, interchangeability or commonali ty in the operational, procedural, material, technical and administrative fields to attain interoperability. These standardization processes create compatibility, similarity, measurement and symbol standards. Validation is documenting that a process meets its pre-determined specifications and quality attributes the objective of validation of an analytical procedure is to demonstrate that it is suitable for its intended purpose. A tabular summation of the characteristics applicable to identification, control of impurities and assay procedures is included. Other analytical procedures may be considered in future additions to this document. Typical validation characteristics which should be considered are accuracy, precision, repeatability, intermediate precision, specificity, detection limit, quantisation limit, linearity, range.
MATERIALS AND METHOD
Collection of plant material The leaves of Laportea interrupta were collected and washed well to remove any adhering foreign particles and soil materials. The washed plant leaves were dried under shade and coarsely powdered. Physicochemical analysis of crude drug Total ash value: About 1 gm dried powder ignited to constant weighed. Acid insoluble ash: Boiled the total ash with 25ml of hydrochloric acid ignited, cooled and weighed. Water insoluble ash: Boiled the total ash for 5 min with 25 ml water, ignited, cooled and weighed.
Extraction of plant material
The plant material was dried in the shade, then coarsely powdered and extracted in Soxhlet Apparatus using various solvents according to their polarity. (Harborne, J.B., 2007) Preparation of the Extracts: The coarse leaf powder of Laportea interrupta was taken about 20gm and extracted with 250ml of ethyl acetate (70-80°c), 250ml of Petroleum Ether
Qualitative phytochemical analysis
The extracts were subjected to qualitative tests for identification of phytochemical constituents present in it. (RNS Yadev et al. 2011)
Test for carbohydrates
Fehling’s Test: Equal volume of Fehling's A and B reagent mixed together and 2ml of it added to crude extract and heated gently. Benedict’s test: Crude extract mixed with 2ml Benedict reagent and boiled. Molisch’s Test: Crude extract was mixed with 2ml molisch reagent and added 2ml concentrated sulphuric acid along the sides of test tube. Iodine Test: Crude extract was mixed with 2ml iodine solution. additions to this document. Typical validation characteristics which should be considered are accuracy, precision, repeatability, intermediate precision, specificity, detection limit, quantisation limit, linearity, range. The washed plant leaves were dried under shade and coarsely powdered. Sulphated ash: 2gm of powdered drug was taken in 3 ml of sulphuric acid incinerated until free from carbon. Moisture content: 10 gm. sample was dried in oven at 100° C, cooled and weighed . (60-80°c), 250ml of Chloroform (50.5-61.5°c ), 250ml of methanol (60-70°c) and 250ml of Distilled water (0.25%) by continuous hot percolation using soxhlet apparatus.
Test for phenols and tannins
Ferric Chloride Test: Crude extract was mixed with 2ml of 2% solution of ferric chloride. Lead Acetate Test: Crude extract was mixed with 2ml of 10% lead acetate solution.
Test for flavanoids
Shinoda Test: Crude extract was mixed with few fragments of magnesium ribbon and concentrated hydrochloric acid was added drop wise. Alkaline Reagent Test: Crude extract was mixed with 2ml of 2% solution of sodium hydroxide
Test for saponins
: Crude extract was mixed with 5ml distilled water in a test tube and shaken vigorously. Test for glycosides Liebermann’s Test: Crude extract was mixed with each of 2ml of chloroform and 2ml of acetic acid. The mixture was cooled in ice and carefully added concentrated sulphuric acid.
Salkowski’s Test: Crude extract was mixed with 2ml of chloroform and then 2ml of concentrated sulphuric acid was added carefully and gently shaken. Keller-Kilani Test: Crude extract was mixed with 2ml of glacial acetic acid and containing 1-2 drops of 2% solution of ferric chloride, the mixture was poured into another test tube containing 2ml of concentrated sulphuric acid. Test for steroids: Crude extract was mixed with 2ml of chloroform and concentrated sulphuric acid was added sidewise. Mixing crude extract with 2ml chloroform, then 2ml of concentrated sulphuric acid was added and heated for 2 minutes.
Quantitative phytochemical analysis Total Phenol Content:
The amount of phenol in the ethyl acetate extract was determined by Folin-ciocalteu reagent method with some modification.5ml of 10% Folin-ciocalteau reagent and 4ml of 1M sodium carbonate added to 0.1ml extract and to 1ml of standard serial dilution of standard Gallic acid, then incubated at dark for 30 minutes and then absorbance were measured at 765nm. Total Flavanoid Content: The amount of phenol in the ethyl acetate extract was determined by Aluminium chloride colorimetric method with some modification. 0.1ml of extract was mixed with 3ml of methanol, 0.2ml of 10% aluminium chloride, 0.2ml of potassium acetate and 5.6ml of distilled water, then incubated at room temperature for 30 minutes and then absorbance were measured at 420nm
PREPARATION OF FORMULATION
The earthen pot sufficiently large and strong is chosen. The proportion of the different ingredients are 32 seers of distilled water, boiled and added 1.25 seers dried plant leaf powder to make decoction,to this decoction added jaggery 12.5 seers, honey 6.25 seers and sugar 6.25 seers, mixed well to form a uniform solution and then poured to the prepared earthen pot mixture upto its three by forth, then lid is closed and sealing is done by cleaned ribbon. Pot is buried in a pit made in the soil for 30 days, filtered through a muslin Test for terpenoids: Crude extract was dissolved in 2ml of chloroform and evaporated to dryness. To this, 2ml of concentrated sulphuric acid was added and heated for about 2 minutes
Test for alkaloids
Mayer’s Test: Crude extract was mixed with 2ml of 1% hydrochloric acid and heated gently. Mayer’s reagent was added to the mixture. Wagner’s Test: Crude extract was mixed with 2ml of 1% hydrochloric acid and heated gently. Wagner’s reagent was added to the mixture loth and stored in tightly stoppered glass bottles.
Physicocemical analysis of formulation Determination of PH of formulation:
The digital PH meter was used and calibrated using buffer tablets of PH 4.0 and PH 7.0. Determination of acid value: 10g of formulation was dissolved in 50ml of equal volume of ethanol and ether previously neutralized with 0.1M KOH to phenolphthalein solution. To it 1ml of phenolphthalein added and titrated with 0.1M KOH.
Determination of alcohol content:
25ml of formulation transferred to distillation flask and added equal volume of water into it. Afterwards it distilled and distillate less than 2ml was collected and make up with distilled water. Specific gravity of this distillate determined and alcohol content analysed using relative density. Total solid content: 10 g of formulation was allowed to evaporate so that only solid content remained. Then it weighed to find out the solid content Determination of viscosity of formulation: Viscosity determined with help of Ostwald viscometer. Determination of refractive index: Refractive index of formulation was found out using Abbes' Refractometer.
QUANTITATIVE ANALYSIS OF FORMULATION
Alcohol content by spectrophotometry (Dichromate Method): Alcohol was estimated by caputi et al (1968). For calibration a standard curve prepared using absolute alcohol without distillation.1ml sample with 25 ml dichromate reagent taken in a distillation flask and distilled to obtain about 25ml distillate, Incubated the flask at 60° C for 29 min and Measured the absorbance at 620nm. Total reducing sugar: 50ml distilled water and 3ml HCL was added to 5ml of sample then cooled and neutralized with sodium carbonate and Volume was adjusted to 500ml with distilled water and titrated against 5ml Fehling’s A and 5ml Fehling’s B followed by mixing them and making final volume 500ml using methylene blue a indicator. Total phenol content: The amount of phenol in the formulation was determined by Folinciocalteu reagent method with some modification. 5ml of 10% Folin-ciocalteau reagent and 4ml of 1M sodium carbonate added to 1ml extract and to 1ml of standard serial dilution of standard Gallic acid, then incubated at dark for 30 minutes and then absorbance were measured at 765nm and the results were determined from the standard curve and expressed in gallic acid equivalents (mg/g of extracted compound).. Total flavanoid content: The amount of phenol in the formulation was determined by Aluminium chloride colorimetric method with some modification. 1ml of extract was mixed with 3ml of methanol, 0.2ml of 10% aluminium chloride, 0.2ml of potassium acetate and 5.6ml of distilled water, then incubated at room temperature for 30 minutes and then absorbance were measured at 420nm and the results were determined from the standard curve and expressed in Quercetin equivalents (mg/g of extracted compound).
ANTI-OXIDANT ACTIVITY DETERMINATION OF FORMULATION Ferric Thiocyanate (FTC)
method: 4ml of formulation and 4mg of Vitamin C were mixed with 4ml of absolute ethanol, 4.1ml of 2.52% linoleic acid in absolute ethanol, 8ml of 0.02M phosphate buffer (pH 7.0) and 3.9ml of distilled water, the mixture was placed at 40°c (0.1ml) and was then mixed with 9.7ml of 75% (v/v) ethanol and 0.1ml of ammonium thiocyanate, after 3 minutes 0.1ml of 0.02M ferrous chloride were added and the absorbance was measured at 500nm. Thiobarbituric Acid (TBA) method: 2ml formulation and 2ml standard on the final day of the FTC assay was added to 1ml of 20% aqueous Thiobarbituric acid. After boiling for 10 minutes, the sample was cooled and absorbance was measured at 532nm. Total Antioxidant activity by FRAP method: Ferric reducing antioxidant potential (FRAP) assay was carried out using modified method of benzie and strain (1996).the result was expressed in Fe (II)/g dry mass. FRAP reagent was prepared freshly by mixing 25ml acetate buffer pH 3.6,2.5ml of TPTZ, 2.5ml of 20mM ferric chloride hexahydrate. 2850µl of FRAP reagent were heated to attain 37°c and then allowed to react 150 µl formulation for 30 minutes in dark. Then the absorbance was measured in 593nm
Qualitative analysis of formulation by lcms
Specifications of LCMS are LC Column: REVERSE PHASE C-18, Pump: SPD 10 AVP, Mobile Phase: 2% OPA IN WATER: METHANOL (50:50), Ionisation Mode: ELECTRONIC SPRAY IONIZATION, Mode: BOTH POSITIVE, Injection Volume: 10 µL, Flow Rate: 2 ML/MIN, Column Temperature: 250°C, Column: PHENOMENEX RP 18, Column Dimension: 25CM 2.5 MM,LC Detection: 254NM,m/z range: 50-1000,Soft Ware: CLASS V P INTEGRATED AND Library: METWIN 2.0. Standardisation of formulation using UV Specifications of UV are Model: ELICO SL 164, Instrument: UV-Visible Double Beam Spectrophotometer, Wavelength Range: 190- 999.9 nm, Accuracy: +/- 0.5nm, Repeatability: +/- 0.2nm, Resolution: 0.1nm, Bandwidth: 0.5- 6.0 nm (Variable at an Interval of 0.1nm), Photometric range: -2.5 TO +2.5 Abs, Accuracy: +/- 0.005 Abs at 0.1Abs from 200- 850nm, Repeatability: +/- 0.002 Abs at 1.0 Abs from 200-850nm, Stray light: less than 0.05% at 220-340nm,Base line correction: Automatic Base Line Correction, Scan speed: Slow, Medium and Fast, Data interval: Depend Upon Wavelength, Sample holder: 5 Position, Specification of HPLC is Instrument: Schimadzu, LC Column: Reverse Phase C-18 Phenomenex C18, Pump: SPD 10 AVP, Mobilephase: Acetonitrile: Methanol (40:60), Injection volume: 10 µl, Flow Rate: 2.0 ml/min, Column Temperature: 25°C, Column: Automatic positioning far 10mm and Sample Cuvette and Fixed position for Reference Cuvette, Source: Tungsten- Halogen lamp (310-999.9nm) and Deuterium lamb with Quartz window (190-340nm) and Detectors: Photomultiplier Tube (PMT).Validation experiments were performed to demonstrate linearity, precision, accuracy, robustness, ruggedness, LOD and LOQ as per ICH guidelines.
Standardisation of formulation using HPLC
Phenomenex RP 18, Column Dimension: 5cm 1.5 cm,LC detection: 254nm and Soft Ware: Class V P Integrated.Validation experiments were performed to demonstrate system suitability, linearity, precision, accuracy study and robustness as per ICH guidelines
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Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-524163EnglishN-0001November30HealthcarePROTEIN ISOLATES : PRODUCTION, FUNCTIONAL PROPERTIES AND APPLICATION
English3545Umar GarbaEnglish Sawinder KaurEnglishProtein isolates are refined form of protein containing the greater amount of protein with greater digestibility. Nowadays it’s the major source of cheap proteins especially for athletes, bodybuilders, vegetarians, and has gained wide application in various beverages and dairy industries, and infant foods due to its different functional properties. It is widely produced from deoiled cake of legumes such as peanuts, cowpeas, soybeans, etc and animal sources like fish and milk by a combination of isoelectric and alkaline precipitation using H2SO4 and NaOH, followed by centrifugation to obtain the pure isolates which can further be dried to powdered form of about 90% protein. The problem of antinutritional factors in legumes can be successfully reduced by these processing methods.
EnglishProtein Isolates, isoelectric precipitation, Ultrafiltration, antinutritional factorINTRODUCTION
Proteins are nitrogen-containing compounds made of up amino acids unit. They are the major structural component of muscles and other tissues in the body. They are also component of hormones, enzymes and haemoglobin (Jay et al. 2004). Proteins composed of twenty different amino acids linked together by a peptide bond and the resulting chain is called polypeptide. They have similar basic structure but differ in their side chains. This difference in side chains gives the proteins their specificity and functionality. These amino acids are classified as essential (those that cannot be synthesis by body but rather taken as supplement from diet), and nonessential (that can be synthesis by the body) (Clark 2003). Demand for the relatively cheap sources of protein that can be incorporated to value –added food products is increasing worldwide, and numerous researches are still going on various sources of plant proteins that may help in improving the nutritional value of food products at low cost (Gurpreet et al. 2006). Proteins are available in different varieties of dietary sources including animals, plants origin, and from highly marketed spot supplement industry. Typically, all dietary animal proteins (e.g. eggs, milk, meat, fish and poultry) are considered complete protein because they contain all essential amino acids. Proteins from vegetable sources (such as legumes, nuts and soy) are incomplete proteins since they are lacking one or two essential amino acids (Hoffman and falvo, 2004). Assessing the quality of protein is important when considering the nutritional benefit that it can provide. Protein quality of food is the ability of the food to meet the nutritional requirement of an individual species. It is indicated by how well the protein is digested, absorbed, and utilised for the growth and sustenance of the body (Wardlaw et al. 1996). The quality of protein can be determined by assessing its essential amino acid profile. Numerous methods to determine the quality of proteins include : ( 1) Protein efficiency ratio (PER) which is achieved by measuring the animal growth after feeding with the test protein. The weight gained in grams per gram of protein consumed is calculated and then compared with the standard value of 2.7 (standard value of casein protein). If the value is greater than 2.7, the food is regarded as excellent source of protein. However, the result mostly provides a measure in rat but does not provide a strong correlation to human growth needs. (2)Biological value is calculated by dividing the nitrogen used for tissue formation and the nitrogen absorbed from the food and then multiplied by 100 and express as percentage of nitrogen utilised. This gives an idea of how efficient the body utilises the protein consumed in the diet. Higher value indicates that food supplies high essential amino acids. (3) Net protein utilisation (NPU) is almost identical to BV except that direct measure of retention of absorbed nitrogen is involved. However, the BV is calculated from nitrogen absorbed while NPU is calculated from nitrogen ingested (Jay et al. 2004). (4) Protein digestibility Corrected Amino acids Score, this, according to joint (FAO/WHO, 1990) could be determined by expressing the content of the first limiting essential amino acid of the test protein as a percentage of the content of the same amino acid content in a reference pattern of essential amino acid. The reference values used were based on essential amino acid requirement of preschool-age children, which was corrected for true faecal digestibility of the test protein. The value obtain was known as protein digestibility corrected amino acid score (PDCAAS).
Protein isolates
Isolate are the most refined form of protein products containing the greatest concentration of protein but unlike flour and concentrates contains no dietary fibre. Isolates originated from United State around 1950s (Jay and Michael 2004). They are very digestible and easily incorporated into different food products. Protein isolates are nowadays believed to have played a major role in the development of new class of formulated foods. It is high concentration of protein with the advantage of colour, flavour and functional properties make it an ideal raw ingredient for used in beverages, infant foods and children milk food, textured protein products and certain types of specialty foods (Olaofe 1998). Protein isolates have been developed from a variety of legumes among which are soy bean, peanut, canola, cashew nut, almonds, sesame, pinto and navy beans (Seyam et al. 1983). Extraction Methods Isoelectric precipitation: - - The isoelectric point (pI) of a protein is the pH where the net charge on the protein is zero. At this point, there exist no electrostatic repulsion keeping them apart, thus protein aggregate and precipitate. Proteins have different isoelectric point because of their different amino acid sequence, and therefore, they can be separated by adjusting the pH of a solution. When the pH is adjusted to the isoelectric point of a particular protein it precipitates leaving the other protein in the solution (McClements 2013). A more recent study on isoelectric precipitation of soya protein confirmed that both extraction condition and the type of acid used for isoelectric precipitation had little effect. However, it was found that an isoelectric point of around pH 4.2 leads to high proportion of protein precipitated out (Smith et al. 1939). Alkaline extraction: It was found that alkaline reagents were more effective in extraction of protein from food legumes. However, several changes such as destruction of lysine, formation of lysine-alanine, racemisation may occur during alkali extraction and reduce the protein quality (Tobin et al. 1978). Hence, a better technology to produce protein isolate and concentrate with maximum yield and minimum or no antinutritional factors need to be developed. Ultrafiltration method: - This is a potential membrane processing for extraction of protein, a solution of protein is placed in a cell containing a semi-permeable membrane, and pressure is applied. Small molecules pass through the membrane, whereas the larger ones remain in the solution. The semi-permeable membranes with cut-off points ranging between 500 and 300,000 are mostly available (McClements 2013). The extraction of soy protein isolate by this method was first reported over twenty years ago. The Ultrafilteration system provides a commercially feasible alternative to the existing isoelectric precipitation method of soya beans
Protein isolate from different plant and animal sources
animal sources Proteins that are utilised in food processing are of various origins, and can roughly be classified into animal proteins (gelatins), vegetable proteins (e.g. peanut protein, soy protein, wheat proteins, Almond protein, canola meal protein etc.), and animal derived protein (e.g. milk proteins). However, many vegetable proteins require processing to provide food material having acceptable functional properties such as emulsification, oil and water absorption, texture modifications, colour control and whipping properties, which are primarily attributed to the protein characteristics. Many plants have attracted a deal of interest as a source of lowcost protein to supplement human diet, this include among others soybeans and peanut (Ogunwolu et al. 2009).
Whey protein isolates (WPI)
Whey is the liquid by-product of cheese which can further be processed into a spray dried products like instance whey protein concentrates (WPC), whey protein isolate (WPI) or whey protein hydrolysate (WPH) (Brucic et al. 2009).By definition from the code of Federal Regulation, is the liquid substances obtained by separating the coagulum from milk or cream in the cheese making. During cheese making, the whey protein remains in the serum phase which represents about 20 percent of the milk protein (Kimberlee 2012). In the production of whey protein isolates, significant amount of fat and lactose get removed, as a result of which individual with lactose-intolerant can safety consume these products (Akintayo et al 1999).Whey proteins are widely used as ingredients in different foods (dairy, meat and bakery products) due to their unique functional and nutritional properties (Brucic et al. 2009). The heat sensitivity of whey protein may present a major challenge in some formulations. Consequently, the Dairy Research Institute, established under the leadership of America’s dairy farmers through the dairy check off program, has supported a variety of researches that aims to improve whey protein’s performance in higher heat processing (Kimberlee 2012). Beta-lactoglubin and α- lactalbumin are the major whey protein responsible for heat stability characteristics of ingredients such as WPC and WPI (Vardhanabhuti and Foegeding 2008).The relationship between sugar addition and heat stability of whey protein isolate have been studied by many researchers. Sucrose addition was found to increase gelation temperature and gel strength of WPI and bovine serum albumin (Rich and Foegeding 2000). Adding glycerol improved heat stability of WPI and decreases turbidity and protein gelation (Kulmyrzaev et al. 2000).Thermal denaturation temperature of WPI was also found to increased by addition of sorbitol more effectively than glycerol (ParedesLopez 2006).Many food application contains sugars or sugar alcohols, and their presence can help to improve the heat stability of whey protein ingredients by preventing the formation of large aggregates and providing better clarity in application such as beverages (Kimberlee 2012). Fish Protein isolates (FPI) To solve the problem of utilisation of unconventional raw material (dark muscle fish, fatty fish) and also fish by-products (fish trims, fish frames etc.) a process was developed to economically develop a functional protein isolates from these kind of raw materials. This technology uses the pH dependant’s solubility properties of fish muscle proteins for their separation and recovery from other components of muscle that are not desirable in a final product. Fish protein isolate is a protein concentrate which is prepared from fish muscle without retaining the original shape of the muscle. It is not generally consumed directly, but used as raw material for production of other value added products. Fish protein isolate does not retain the original shape of muscle, and is normally utilized as ingredient for the production of value added products. It is still a good source of protein for the production of ready to eat fish products. The overall processes involved are simple. The proteins of the muscle tissue are first solubilised. The solubilisation can be accomplished by addition water with alkali added to approximately pH 10.5 or higher, or with acid added to about pH 3.5 or lower. It is usually necessary to choose the pH at which the consistency of the solution decreases to a value that allows the removal of undesirable material. The mixture is then centrifuged, and due to density differences the oil rises to the top and can then be removed. Other insoluble impurities such as bone or skin are also sedimented at this stage. The muscle protein are then precipitated and collected by a process such as centrifugation (Shaviklo 2006)
Peanut protein isolates (PPIs)
Peanut contains 26-29% protein with good nutritional quality. Peanut proteins are used for their functional properties (emulsification, forming) or for their nutritional properties in different food products. They are also used for human nutrition in developing countries to supplement cereals, beverages and skim milk. Peanut protein isolate can be prepared from the defatted peanut cake or powdered by macerating with high salt phosphate buffer (20 mM Na2HPO4, 2 mM KH2PO4, 5.4 mM KCl, 1M NaCl, pH 7.4), followed by centrifugation and supplementing the supernatant with (NH4)2SO4 to 90% saturation. After centrifugation, the pellet can be dialyse against distilled water overnight at 4oC and then freeze-dried (Mouecoucou 2004). Soy protein isolates (SPIs) Soy protein isolate is a common isolate. It has high protein content of about 90%. It is made out of defatted soy meal by removing most of the fat and carbohydrates (Seyam 1983). Soybean is crushed into oil and defatted meal. The meal is usually used as animal feed, while smaller amount is further processed into food ingredients including soy flour, protein concentrate, protein isolates and textured protein (Kinsella 1976). Soy protein isolate is usually combined with other food ingredients such as vitamins, minerals and flavour in preparation of soy protein shake powder (Seyam 1983). Advance in food technology resulted in the development of a variety of soy product such as concentrates, isolate and extruded-expanded products, this consequently leads to increased utilisation by technically developed regions of the world (Young 1979). The production of soy protein isolate involve solubilising the protein and carbohydrate at neutral or alkaline pH and the recovery of the solubilised protein, by separation and optionally washing and neutralization before drying (Moure et al. 2006). Three steps involved in the processing of soy protein isolates (SPI) are (1)The soy flakes are slurried with water under alkaline conditions (pH 6.8-10 at 27-66 oC using sodium hydroxide and other alkaline substances approved for food used) so that the protein and the oligosaccharides can dissolve into the solution. The protein solution is then separated from the insoluble residue by centrifugation, (2) the supernatant containing the protein and sugars is then acidified to isoelectric pH 4.5 ( where the solubility of proteins is minimal), using hydrochloric acid (HCl). This leads to the precipitation of protein as curd, (3) the solubility of the precipitated protein is restored by neutralizing to alkaline pH of 6.5-7.0 after re
diluting with fresh water or spray dried in its acidic form and packed in multilayer paper bags (Lusas et al. 1995, and Anon 2008). Canola protein isolates (CPIs) According to USDA 2010, Canola meal has been the second largest feed meal after soybean meal. It has a good amino acid profile with a well balance amino acid composition although it has found only marginal used in the food industry, due to the presence of anti-nutritional factors. The vast majority of canola protein isolates are prepared by alkaline extraction method fallowed by isoelectric precipitation. Although, this extraction method generates high yield of nitrogen, but the isolate produced by this method have been found to have poor solubility and digestivity, this is most probably due to the nature of proteins constituent of canola meal which consist of alkaline-soluble fraction that can be easily denatured during the extraction process. CPI thus possesses generally an unacceptable food-functional properties including poor water holding, gelling and oilbinding, foaming and emulsification properties. Many studies have been carried out to modify the properties of this isolate e.g. by succinylation, acylation and enzymatic hydrolyses. However, enzymatic hydrolyses has been prepared because it’s less effect than chemical method (Alashi 2011).
Chick pea protein Isolate
Chick pea protein is the world’s third largest pulse crop in term of area, grown mostly in West Asia and Mediterranean region. It is one of the major vegetable proteins. Many functional properties of this protein isolate has been studied whereas, information on gelation properties of chick pea protein isolate was scares. The chick pea protein isolate dispersed with sodium and calcium salts showed different rheological behaviour at different ionic strength and pH. Increasing the ionic strength of dispersion could strengthen the gelation properties of CPI under acidic conditions, however reduced the elastic parameters of CPI at pH of 7.0 (Zhang 2007).Cashew nut protein isolate Cashew has considerable economic importance because its components have numerous economic uses. The kernel has high food value with about 40-57% oil and 21% protein content. A cashew kernel meal contains about 42% crude protein, a low crude fibre and 0.5% and 0.2% calcium and phosphorous, respectively, which is comparable to that of peanut composition, which has been used for peanut protein isolate and concentrate. Protein isolates and concentrates can be obtained from defatted cashew nut powder by both alkaline extraction-isoelectric precipitation (IP) and alkaline extractionmethanol precipitation (MP). Cashew nut protein isolates has water and oil absorption capacities of 2.20ml/g and 4.42ml/g respectably, emulsifying stability index (447%), foam capacity and stability (45% and 55%, respectively ), and low gelation capacity of (13.5%) (Ogunwolu 2009).
Functional Properties of Protein Isolate Bulk density:-
- Bulk density is an important parameter that determines the packaging requirement of a product. It signifies the behaviour of a product in dry mixes and can varied with fineness of particles (Butt and Batool 2010).The bulk density of protein isolate of sources can be determined by taking 10 grams of protein isolate into 100 ml graduated cylinder and taping several time on a laboratory bench until the isolate settled. The values recorded can be express as g/cm (Olaofe 1998, and Okaka and Potter 1977).Protein solubility :- Protein solubility is a useful indicator for the performance of protein isolates incorporated in the food system and also the extent of protein denaturation because of heat and chemical treatment at different pH (Horax 2004).To determine the protein solubility, the protein isolates (250 mg) should be homogenized in 20 ml of 0.1 M NaCl at pH 0f 7.0 for 1h fallowed by centrifugation at 10,000×g for 30 min. Nitrogen content should then be determined in the soluble fraction and the solubility can be expressed as the percentage total nitrogen of the original sample to that of soluble fraction (Morr 1985). Foaming Capacity and stability: - The foaming properties are used as an index of the whipping characteristics of the protein isolate (Mwasaru 1999). The capacity and stability of foams can be determined by dispersing 50ml of 3% (w/v) of protein isolate sample in distilled water and transfer immediately into a graduated cylinder, the volume should be recorded before and after whipping. The foaming capacity can be expressed as the percentage volume induced by whipping. The change in volume of foam after 60 min of standing at room temperature is recorded as foam stability (Lin 1974). Water/oil absorption: Proteins has both hydrophilic and hydrophobic properties therefore, can interact with water and oil in foods (Butt and Batool 2010). The lower water absorption capacity can be due to less availability of polar amino acids (Kuntz 1971), and the low fat absorption may be due to the presence of large proportion of hydrophilic and polar amino acids on the surface of the protein molecules (Sathe et al.1982). Water absorption is determine by mixing approximately 3g of protein isolate with 25ml distilled water in a pre-weighed centrifuge tubes, the tubes are stirred and centrifuge for 25 min at 3000×g for 30 min interval. The supernatant is carefully decanted and the centrifuge tubes containing the protein isolate are re-weighed. Water absorption is express as the number of grams of water absorbed per gram of sample (Sosulski et al. 1976). High water absorption of protein isolates help to reduce moisture loss in package bakery products (Lin 1974). Oil absorption can also be estimated by mixing 0.5g of protein isolates with 6ml corn oil in preweighed centrifuge tubes. The tubes are stirred for like one minutes to get complete dispersion of the sample in oil. After 30 min holding time, samples are centrifuge at 3000×g for 25 min, the supernatant be decanted and the tubes inverted for 25 min to drain the oil before re-weighing. The oil absorption is express as gram of oil absorbed per gram of protein isolate (Sosulski 1976). The high oil absorption is essential in the formulation of food systems like sausages, cakesbatters, and mayonnaise and salad dressing (Butt and Batool 2010).
Emulsifying activity and stability:
Proteins are surface active agents that can form and stabilize the emulsion by creating electrostatic repulsion on oil droplet surface (Makri et al. 2005). To determine the emulsifying activity, Protein isolate (3.5g) is homogenised for 30 seconds in 50ml water using homogenizer at approximately 10,000rpm. Corn oil (25ml) should is then be added to the mixture and homogenised for 30 sec. The emulsion is divided into two equal volume aliquots and centrifuged at 1100×g for 5 min, the aliquots is heated for 15 min at 85oC. The ratio of the height of emulsion to the high of liquid layer is noted to calculate emulsion activity. The emulsion stability remains after heating (Naczk et al. 1985). Least gelation concentration: - This is a qualitative parameter that expresses the minimum protein concentration at which the gel does not slide along the test tube walls in inverted position (Young and Scrimshaw 2006).The lower the gelation concentration, the better is the gelling ability of proteins, because protein gels are aggregates of denatured molecules (Akintayo 1999).
Antinutritional factor in protein isolate
Several studies have reported the preparation and functional properties of protein isolates and concentrate (Sathe and Salunkhe 1981). Legumes contain some anti-nutritional factors such as lectins, saponin, haemagglutinin, protease inhibitor, oxalate, goitrogen, phytates, trypsin inhibitor and tannin (Amarowicz and Pegg 2008). These compounds reduce protein digestibility and availability. Some antinutritional in legumes have been reported to have health benefits. Tannin, a polyphenolic compound is reported to possess antioxidative property (Apata and Ologhobo 1997).Very extensive work has been carried out on optimising the conditions for trypsin inhibitor destruction during processing of soya protein isolate (Smith and Circle 1977). Studies had also shown that trypsin and chymotrypsin inhibitory activities were reduced to 75.3% and 84.8% in protein concentrates, 98.0% and 85.4% in protein isolates of Great Northern beans respectively. However, these protein fractions did not contain oligosaccharides of the raffinose family (such as raffinose, stachyose, and verbascose) and hemagglutinating activities. Therefore, most of the antinutritional factors can possibly be eliminated by preparation of protein isolates and concentrates (Sathe and Salunkhe 1981).Specific studies were carried out on isoelectric process for protein isolate, where trypsin inhibitors levels were found to be dependent on the degree of heat treatment to which the raw materials had been subjected and on the pH used for isoelectric precipitation. Trypsin inhibitor levels in isolate produced by ultrafiltration process have also been studied and found to be fairly high; this might possibly be due to complexing of the relatively low weight trypsin inhibiting protein fractions with higher molecular weight proteins (Honig and Wolf 1987). Application of protein isolate in food systems. Protein isolates are the acceptable ingredients for dairy application due to their fine particle size and dispersibility, Emulsification, emulsion stability, colour and flavour are critical in dairy application. Isolates (especially soy proteins) are being used to fortify all type of pasta products such as macaroni, spaghetti, to improve the nutritional value etc (Sipos 2013). Protein isolates are important sources of protein with high lysine content. Isolates from different legumes varied slightly in physiochemical and thermal properties. They are used as proteinaceous ingredients in many food products such as salad dressing, meat products and dessert. Whey proteins are mainly used in beverage applications, due to their health benefits (Kudre 2013). The problem of poor flavour, mouth feel, texture, dryness and flavour associated with the use of soy flour and soy concentrate above 10% has been resolved by using soy isolate in meat loaves, sausage-type products for their emulsion-stabilizing effect, gelation, and moisture retention and improved effects on texture (Kinsella 1976). Soy protein is regarded as textured protein products use both in meat and vegetarian meat analogue industry and thus, has good water holding capability (Riaz 2006). It is often used as meat extenders in comminuted meat products such as patties, fillings, meat sauces, meat balls, etc (Berk 1992). Many soy protein isolates have been developed for providing different functional or physical properties to meet the requirement of various food systems. Soy protein isolates form firm, hard, resilient gels, unlike soy flour and concentrates that form soft and fragile gels (Riaz 2006). The firm-forming ability of soy protein isolates is important in meat products. When heat and pressure are applied the protein films fused together to form a firm, continuous, textured mass that can be sliced and used as meat substitutes. They also vary in their ability to form gels. Some are designed to form gel while others will not form gel at 14% solid content. Protein isolates are utilised in meat and baking application due to their water and fat absorption properties, the value for these properties for protein isolates ranges from 150 to 400% (Sipos 2013). Neutralised protein isolates are often highly dispersible and form gel under appropriate aqueous conditions. They have both emulsifying and emulsion stabilising properties, and are excellent fat and water binders, For this reason, they are widely used in processed meat products, as well as both coarse and fine emulsions (e.g., patties, loaves and sausages) (Sipos 2013). Different protein products such as whey protein, soy protein isolates, wheat gluten, rice bran protein, peanut protein, and cottonseed proteins where investigated for film development (Rhim 1998).
CONCLUSION
Protein isolates shows a lot of potential to combat the problem of malnutrition. The underutilised plant and animal sources can be exploited in order to extract the proteins and make them available for used as food supplements. Research is needed compared the benefit of different protein isolates and their benefit as food supplements.
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Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-524163EnglishN-0001November30HealthcareRISK FACTORS FOR STROKE : A CASE CONTROL STUDY
English4652Vijaya SorganviEnglish M. S. KulkarniEnglish Deepak KadeliEnglish Suhasini AthargaEnglishBackground: Stroke is global health problem and is a leading cause of disability. It is one of the leading causes of mortality and morbidity worldwide. In rapid increase in burden of stroke in coming years and limited availability of stroke care in India, it would be better to study stroke prevention strategies. The current hospital based case-control study was undertaken with aim, to identify the risk factors for stroke. Material and Method: Study was carried out in BLDEU?s Shri B M Patil Medical College, Hospital & Research Centre. A prospective case-control, age and sex matched study was designed to find the risk factors. Cases: The study consisted of 100 hospitalized computed demography (CT) scan proved cases of stroke. Controls: The controls were selected from patients who attended the study hospital for conditions other than stroke (confirmed with CT scan). For each case of stroke, one control was selected. The controls were matched to cases in respect to age (± 5 years), sex. One hundred controls were selected in the same manner. Results: Bivariate analysis included odds ratio and its 95% confidence intervals were indentified, hypertension (0R=3.807), diabetes (0R=3.473), hypercholesteremia (OR=3.768), obesity (0R=2.471), smoking (0R=2.42), family history of stroke (OR=2.359), and transient ischemic attack (OR=2.006) as a risk factors for stroke. Conclusion: This is a study that quantifies the contribution of different factors to the overall risk of stroke. Finding that, hypertension, hypercholesterolemia, diabetes are the biggest risk factors for all type of strokes is important because like many other factors, these are modifiable risks, that can be treated by appropriate medication and life style changes.
EnglishStroke, Risk factors, case-control study, odds ratio, 95% confidence interval.INTRODUCTION
It has been suggested that no single medical measure could makes as much as contribution to the quality of life in old age as prevention of cerebro-vascular disease. An understanding of the etiological factors that contribute to the onset of stroke is required in order to assess the potential for stroke prevention (1). Stroke is global health problem and is a leading cause of disability. It is one of leading causes of mortality and morbidity worldwide. Approximately 20 million people each year will suffer from stroke (2). And of these 5 million will not survive. Developing countries accounts for 85% of global deaths from stroke (3). Stroke is also a leading cause of impairment with 20% of survivors requiring institutional care after 3 months and 15% - 30% being permanently disabled (4). It is a life changing event that affects not only the person who may be disabled, but their family and caregivers. India with more than 1 billion inhabitants is undergoing remarkable economic and demographic changes in recent years, resulting in a transition from poverty - related infections and nutritional deficiency diseases towards lifestyle related cardiovascular and cerebro-vascular diseases(5,6). Despite rapid economic boom, a large segment of the Indian population still lives in poverty. Given the anticipated increase in burden of stroke incoming years. The prevalence of stroke in India varies in different regions of country and the estimated prevalence rates increases from 0.3/1000 in Englishhttp://ijcrr.com/abstract.php?article_id=962http://ijcrr.com/article_html.php?did=962REFERENCES
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Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-524163EnglishN-0001November30HealthcareVEIN OF GALEN MALFORMATION IN A 47 YEAR OLD MALE-A RARE ENTITY
English5356Subrata Chakrabarti English Koushik PanEnglishVein of Galen Malformation (VGM) is a rare vascular malformation representing less than 1% of vascular intracranial abnormalities. It is a congenital anomaly and frequently detected during the last trimester of gestational age, during neonatal period or early childhood. Clinical manifestations can appear at any age but mainly in the neonatal or infancy stage in the form of heart failure, delayed neuropsychomotor development, hydrocephalus and seizures. First presentation in adult age is very rare. We report a case where a patient remained symptom free until presentation following generalized tonic clonic seizures at the age of 47 years.MRI brain and MR Angiography confirmed the presence of VGM.
EnglishVein of Galen malformation, generalized tonic clonic seizuresINTRODUCTION
The vein of Galen malformation is a rare cerebrovascular disorder which is characterized by an abnormal direct communication between one or several cerebral arteries and the vein of Galen. Posterior choroidal artery, anterior cerebral artery and transmesencephalic artery are respectively the most common arteries that join the malformation1 . During the neonatal or infancy period, the disease presents with congestive heart failure, intracranial hemorrhage, seizures or focal neurologic symptoms2 . Thereafter, progressive hydrocephalus or headache are the most frequent presentations of the VGM. Rare manifestation of VGM as occurs in adult may include seizures or hemiparesis3 .We report such a case of VGM in which the patient presented with seizures first at the age of 47 years.
CASE REPORT
A 47 year old non diabetic, non hypertensive, occasional smoker and alcoholic male was brought to the emergency room because of generalized tonic-clonic seizures which started 1 hour ago. The seizures were associated with urinary incontinence and tongue biting. No antecedent history of head trauma or ingestion of any toxin or recent alcoholic binge or any febrile episode was noted. No history of seizures was present in the past and no similar family history was found.He was born as a normal vaginal delivery and achieved normal developmental milestones at appropriate age and had no history of scholastic performance. During childhood, there was no neuropsychomotor developmental delay. Past medical history was insignificant. Physical examination on admission revealed a blood pressure 130/76, heart rate 112/minute, regular and respiratory rate 26/minute. He was frothing at the mouth and had urinary incontinence. No evidence of any head trauma was noted. No sign of limb abnormality or any skin tag or any facial deformity was noted. No sign of meningeal irritation was found. Fundoscopy revealed features compatible with mild papilledema bilaterally. On admission, he was treate Lorazepam and injection Phenytoin on the lines of standard management as a case of status epilepticus. Routine blood parameters were within normal limits except evidence of lactic acidosis. CSF study was within normal limits. MRI brain was ordered after patient was stable which revealed features compatible with partly thrombosed Vein of Galen Malformation. Subsequently MR Angiography was done which confirmed the diagnosis of VGM. As the aneurysm was already partly thrombosed and also as there were no signs or symptoms of intracranial hypertension or hydrocephalus, the patient was not referred for surgery or endovascular treatment. Improvement in the level of consciousness and absence of seizures episodes were noted during the follow-up period while the patient was kept on therapy for seizures.
DISCUSSION
VGM is a vascular abnormality1 typically found in children2 . The two most used classifications for vein of Galen malformations are those proposed by Lasjaunias et al.4 . and Yasargil5 . According to Lasjaunias et al.4 , these malformations can be divided into mural and coroidal types depending on the fistula localization. The coroidal type is characterized by multiple fistulas at the anterior and terminal segment of the median prosencephalic vein. This type usually presents at the neonatal period causing serious heart failure leading to multiple organ failure and death. The mural type has the fistula at the vascular wall usually at the lateral-inferior wall of the median prosencephalic vein. They are of slow flow and asymptomatic. Yasargil's lesions types 1, 2 and 3 are direct fistulas between the malformations and the vein of Galen. Lesion type 4 are parenchymal arteriovenous malformations, which drain directly into the vein of Galen. It is typically diagnosed during the neonatal period or in childhood with features of heart failure , macrocephaly. Adults may present with slow-flow fistulas, headaches, seizures, hydrocephalus6 . Angiography is the gold standard for the diagnosis of VGM which shows the relationship of the cerebral venous system and vascular relationships to the fistula7,8. Because of its rarity in adults, there is still insufficient information about the course of this disease during adult age. In adults, isolated case reports have been mentioned by some authors9-12. Use of oral contraceptives13, postpartum status14, sickle cell anemia15, and aseptic meningitis16 are considered risk factors related to thrombosis of the vein of Galen. Treatment of VGM is case-based. No standard treatment guideline is available. Even considering recent micro-neurosurgery advances, the lesion resection usually is not possible. However, recently it has been shown, interventional neuroradiology can lead to good results. Nowadays, endovascular techniques are the procedures of choice17. However, conservative treatment may be considered, especially for the elderly with co-morbidities, and neurological stable patients without severe neurological signs or symptoms. The presence of partial thrombosis in the malformation, absence of signs or symptoms of intracranial hypertension, and pharmacological control of seizures led us to conservative treatment of the patient presented here.
CONCLUSION
Possibility of VGM as the underlying etiology in a case of a 47 year old patient presenting for the first time with generalized tonic-clonic seizures is relatively remote but appropriate investigations namely MRI brain and MRAngiography can unravel this rare but fascinating entity.
ACKNOWLEDGEMENT
We wish to thank the faculty of Radiology Department, SSKM for prompt performance of the MRI and MRA of the patient which helped us
in reaching this interesting diagnosis and also providing us with valuable inputs regarding the disease which helped us in preparing the manuscript. CONFLICT OF INTEREST: NIL
Englishhttp://ijcrr.com/abstract.php?article_id=963http://ijcrr.com/article_html.php?did=963REFERRENCES
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4. Lasjaunias P, Ter Brugge K, Ibor LL, et al. The role of dural anomalies in vein of Galen aneurysms: report of six cases and review of the literature. AJNR 1987;8:185-192
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12. Ribeiro VT, Botelho LF, Lopes AC, et al. Choroidal type aneurysmal malformation of the vein of Galen associated with DandyWalker malformation in an adult. Acta Med Port 2003;16:217-220.
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17. Shin M, Kurita H, Tago M, Kirino T. Stereotactic radiosurgery for tentorial dural arteriovenous fistulae draining into the vein of Galen: report of two cases. Neurosurgery 2000;46:730-733