Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-52411111EnglishN2019June10HealthcareCorrelation between Pelvic Alignment and Trunk Control in Children with Spastic Diplegia - A Cross-Sectional Study English0104Feba Sara OommenEnglish Suchetha PSEnglish Dhanesh Kumar KUEnglishAim: To evaluate the correlation between pelvic alignment and trunk control in children with spastic diplegia of the age group 8-15 years. Methods/Design: The study used a cross-sectional research design. 24 children with spastic diplegic cerebral palsy were included in the study. Pelvic asymmetry of each child was measured using palpation meter device (PALM) and trunk control using a trunk control measurement scale (TCMS) on the same day with 5 minutes break in between. Results: The results show that there is a negative correlation between pelvic asymmetry and trunk control. The p-value is EnglishPelvic alignment, Spastic diplegia, Trunk controlINTRODUCTION Cerebral palsy (CP) defines a group of permanent disorders of the development of movement and posture causing activity limitations that are attributed to non- progressive disturbances that occurred in the developing fetal or infant brain[1]. Cerebral palsy is one of the commonest physical dysfunction that children face during their childhood. The prevalence of CP is 1.5 to 4 per 1000 live births of the age group 10 to 14 years. The overall birth prevalence of CP is 2 per 1000 live births[2]. Spastic diplegia is the predominant type of CP in preterm children, especially in the most immature preterm children[3]. Children with spastic diplegia demonstrate a normal tone or hypotonia in the first four months. Spasticity gradually develops and is then progressive in the leg during the initial stages of life[4]. Impaired trunk control, pelvic asymmetry, balance, and gait asymmetry are some of the common impairments in spastic diplegia[5, 6, 7]. Trunk control is defined as control of proximal muscles of the spine that stabilize the trunk when the body is responding to gravity, either in quiet balance patterns or during a movement or position change in space[8]. From retrieved works of literature, it is proved that pelvic asymmetry is a common impairment in CP [9]. Children with CP often show weakened trunk control, which can, in turn, affect their daily functional tasks including reaching, sitting and walking[10]. The pelvis acts as a base in the sitting position and as a connecting link between the trunk and lower limbs while standing. It is assumed that pelvis and trunk are in close relation concerning impairment and association. But there is a scarcity of literature which shows the impact of the trunk on pelvis and pelvis on the trunk. If the correlation is proved between pelvic alignment and trunk control, the inclusion of pelvic training strategies in the early stages of rehabilitation of children with spastic diplegia will yield promising results. As there is a dearth of literature that establishes correlation linking trunk control with pelvic malignment, this study is been taken up to establish a correlation between the two, using the outcome measures PALM and TCMS in children diagnosed as spastic diplegic CP of the age group 8-15 years. METHODOLOGY A total of 24 children with spastic diplegia were recruited from the out-patient physiotherapy department of Justice K S Hegde Charitable Hospital Mangalore and Kapikad special school, Thokkottu, Mangalore. Approval from the scientific committee and the Institutional Ethics Committee was obtained before the commencement of the study. All subjects were screened before participating in this study, using the following inclusion criteria: Either gender between 8-15 years of age, children diagnosed with spastic diplegia, children having pelvic asymmetry (measured by palpation meter device), children who are able to follow commands (paediatric mini-mental state examination), children who can sit independently at least for 15minutes, children with gross motor function classification system level between ?-???. Children undertaking botox injection since 6months, who underwent surgery involving the spine and lower limbs in the past 6months, those having uncontrolled seizures since past 6months, those children with fixed deformities in the spine and with any fractures and dislocations of the spine and lower extremities were excluded from the study. Before commencing the study, consent and assent forms were obtained. Pelvic asymmetry of each child was measured using palpation meter device and trunk control using a trunk control measurement scale on the same day. A break of 5 minutes was given between the two. Instructions were given to the children regarding the scale and device. RESULTS For this study, 24 children with spastic diplegia had been recruited. Of these, 70.8% were males and 29.2% were females. Since the data is not following a normal distribution, Spearman‘s correlation coefficient was used to calculate the correlation between pelvic alignment and trunk control. The p-value less than 0.001 considered significant for the study. The trunk control measurement scale has a mean value of 38.04 + 11.58 and PALM has a mean value of 0.69 + 0.54. The correlation coefficient is -0.918 and p-value is < 0.001, which shows that there is a negative correlation between pelvic asymmetry and trunk control in children with spastic diplegia as shown in the scatter diagram below. DISCUSSION The purpose of the current work was to inquire the correlation between pelvic asymmetry utilizing palpation meter device and trunk control using a trunk control measurement scale (TCMS) in children with spastic diplegia. It was a cross-sectional study and no intervention was given. 24 children diagnosed with spastic diplegia were included in the study. Socio-demographic details revealed that 70.8% of the total sample included were males and 29.2% were females. Researches show that the occurrence of cerebral palsy is greater in males compared to females with a ratio of 1.33:1[11]. The results of the present study indicated that there is a correlation between pelvic asymmetry and trunk control in children with spastic diplegia. Non-parametric correlation test, Spearman‘s correlation was used, as the data was not following a normal distribution. The correlation coefficient is -0.918 and the p-value is Englishhttp://ijcrr.com/abstract.php?article_id=2605http://ijcrr.com/article_html.php?did=2605 Rosenbaum P, Paneth N, Leviton  A, Goldstein  M, Bax  M, Damiano  D,  et al. A report: the definition and classification of cerebral palsy April 2006. Dev Med Child Neurol Suppl. 2007;49:8-14. Stavsky M, Mor O, Mastrolia SA, Greenbaum S, Than NG, Erez O. Cerebral Palsy—Trends in Epidemiology and Recent Development in Prenatal Mechanisms of Disease, Treatment, and Prevention. Front Pediatr. 2017;5:21. Leppert M. Developmental-behavioral pediatrics. 4th ed. Philadelphia, Pa: Saunders/Elsevier; 2009. Fenichel G. Clinical pediatric neurology. 6th ed. Philadelphia, Pa: Saunders/Elsevier; 2009.  Shepherd R. Physiotherapy in pediatrics. 3rd ed. London, Pa: Elsevier/Butterworth- Heinemann; 1995.  El Basatiny HM, Abdel aziem A. Effect of Trunk Exercises on Trunk control, Balance and Mobility Function in Children with Hemiparetic Cerebral Palsy. Int J Ther Rehabil. 2015; 4(5):236-43. Niklasch M, Döderlein L, Klotz M, Braatz F, Wolf S, Dreher T. Asymmetric pelvic and hip rotation in children with bilateral cerebral palsy: Uni- or bilateral femoral derotation osteotomy?. Gait Posture. 2015; 41(2):670-5. Umphred D. Neurological rehabilitation. 5th ed. Edinburgh, Pa: Elsevier/Mosby; 2006. Niklasch M, Döderlein L, Klotz M, Braatz F, Wolf S, Dreher T. Asymmetric pelvic and hip rotation in children with bilateral cerebral palsy: Uni- or bilateral femoral derotation osteotomy?. Gait Posture. 2015; 41(2):670-5. El Basatiny HM, Abdel aziem A. Effect of Trunk Exercises on Trunk control, Balance and Mobility Function in Children with Hemiparetic Cerebral Palsy. Int J Ther  Rehabil. 2015; 4(5):236-43. Agarwal A, Verma I. Cerebral palsy in children: An overview. J Clin Orthop Trauma. 2012; 3:77-81. Pathak S, Vijaya Kumar K, Nayak A, Kedambadi RC. The relationship between pelvic alignment and trunk control in stroke subjects: a cross-sectional study. Int J Res Med Sci. 2014; 2(4):1483-7. Kluszczynska A, Kluszczynski A, Raczkowski J, Siwik P. Evaluation of pelvic asymmetry and lower limb functional shortening in a cohort of children re-examined after a ten-year observation. Scoliosis. 2014; 9(1):01. Panibatla S, Kumar V, Narayan A. Relationship between trunk control and balance in children with spastic cerebral palsy: a cross-sectional study. J Clin Diagn Res. 2017; 11(9):5-8.  Yildiz A, Yildiz R, Elbasan B. Trunk control in children with cerebral palsy and its association with upper extremity functions. J Dev Phys Disabil. 2018; 30(5):669-76. Acknowledgments My sincere and heartfelt gratitude to Mr. Kovela Rakesh Krishna, Associate Professor, Vikas College of Physiotherapy, Mangalore for his constant support and guidance throughout the study. I am very grateful for the support provided by Dr. Purusotham Chippala, Clinical in charge, Department of Physiotherapy, Justice KS Hegde Charitable Hospital, Deralakatte, Mangalore. My heartfelt gratitude to Dr. Sanal TS, Statistician, for his guidance in the statistical analysis of the study.       Ethical clearance obtained from Nitte (deemed to be University)        Ref: NIPT/IEC/Min//015/2017-18/dated 21-01-2018     Conflicts of interest: None     Funding: Nil

Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-52411111EnglishN2019June10HealthcareCombined Effectiveness of Mirror Therapy and Motor Imagery on Gait in Stroke Patients English0510Neha HatwarEnglish Suchetha PSEnglish Dhanesh Kumar KUEnglishAim: The purpose of the study was to evaluate the combined effectiveness of lower limb mirror therapy and motor imagery on gait in stroke patients. Methods: A total number of 38 patients were recruited for the study. They were divided into two groups: Group A (n=19) and Group B (n=19). Group A was given motor imagery and conventional rehabilitation while Group B was given lower limb mirror therapy, motor imagery, and conventional rehabilitation. The interventions were given for 2weeks (5days/week). The values of Dynamic Gait Index (DGI) and Lower limb component of Fugl Meyer Assessment (FMA-LE) were taken both at baseline and after 2 weeks of intervention. Result: Both Group A and Group B showed significant improvements in DGI and FMA-LE after 2 weeks of intervention (p valueEnglishGait disturbances, Mirror Therapy, Motor Imagery, StrokeINTRODUCTION According to WHO, stroke is defined as ‘rapidly developing clinical signs of focal (or global) disturbance of cerebral function, lasting more than 24 hours or leading to death, with no apparent cause other than the vascular origin [1]. It is one of the leading causes of disability in India, the prevalence being 84-262/100000 in rural and 334-424/100000 in urban areas[2]. It is estimated that nearly 80% of stroke patients have an upper limb or lower limb functional deficit[3]. The inability to walk properly due to lower limb impairment is a major problem confronted by many stroke patients [4]. The common abnormal gait patterns seen in stroke patients are circumduction, genu recurvatum, and spastic paretic stiff-legged gait. The deviations include decreased gait speed, decreased cadence, decreased stride length, increased step width and increased time spent in double limb support compared to healthy adults [5]. Gait restoration is one of the major goalsin post-stroke rehabilitation.For the planning of appropriate rehabilitation, the mechanism behind gait control should be understood. Spinal cord (central pattern generators) initiates rhythmic walking and the voluntary modifications are made by the cerebral cortex. In stroke patients, the cerebral cortex function is disrupted while that of the spinal cord is protected. Thus, the ability of the spinal cord of producing rhythmic walking can be used to reorganize the cerebral cortex to improve walking ability in post-stroke patients[6]. Mirror therapy and motor imagery are such methods which help in reorganizing the motor cortex. Mirror therapy is a type of intervention which creates a reflection of the non-affected limb by using a mirror to trick the brain to think that movement has occurred in the affected extremity[3]. Mirror therapy is based on the principle of visual illusion. Mirror therapy works by activating the ipsilateral motor cortex and the premotor and sensorimotor areas which control contralateral movements. This enhances communication between the hemispheres and results in appropriate motor recovery. Mirror therapy has proved to be effective for the recovery of the upper limb after stroke. Studies regarding the effectiveness of mirror therapy on lower limb recovery after stroke are few[7]. Motor imagery is a type of therapy in which the patient rehearses the movement without actually performing it[8]. More recovery is seen when motor imagery is followed by the performance of physical activities. Systematic reviews done earlier have demonstrated that motor imagery enhances upper extremity functions in stroke patients. Recently motor imagery associated with lower limb functions has also been investigated and is found to be effective[9]. The retrieved works of literature have proved that mirror therapy and motor imagery are effective in improving the motor functioning of the upper limb and lower limb after stroke[3,8]. Mirror therapy and motor imagery are proved to be effective in improving gait function when given isolation[10,11]. Though, there are several studies done on the use of mirror therapy on upper limb function in a stroke patient, very few studies done on the effectiveness of lower limb mirror therapy. Also, there is paucity in the literature on the combined effect of mirror therapy and motor imagery on gait in post-stroke patients. Therefore, this study has been taken up to evaluate whether mirror therapy has additional effectiveness combined with motor imagery in the improvement of post-stroke gait. METHODOLOGY Approval from the scientific committee and institutional ethics committee was obtained before the commencement of the study. Participants with stroke were recruited from Justice K S Hegde charitable hospital, Mangalore. These participants were screened based on inclusion criteria and consent was taken from selected participants. The participants were included based on the following criteria: a) Patients who are less than 6 months post-stroke b) Age- 45-65 years c) Ability to walk with minimal assistance (functional ambulation category less than or equal to 3.) d) No remarkable cognitive deficit (an outcome more than 25 on mini-mental status examination). The exclusion criteria were: a) Any musculoskeletal disorder impeding lower limb functionb)Any neglect of space on the affected side, or any other neurological disease or auditory or visual c) Any psychiatric disorders. The participants were divided into two groups. Group A was given 15 minutes of mirror therapy,15 minutes of motor imagery and 30 minutes of conventional treatment whileGroup B was given 15 minutes of motor imagery and 45 minutes of conventional treatment. The interventions were given for 2 weeks (5 days a week).  Fugl Meyer assessment of lower extremity (FMA-LE) and Dynamic Gait Index (DGI) values were taken at baseline and after 2 weeks of intervention (Figure 1). In mirror therapy, the patient was made to sit and a mirror was mounted in between the legs of the patient in such a way that the paretic leg was on the non-reflective side of the mirror while the non-paretic limb was facing the reflective side of the mirror. The patient was made to perform the following exercises using the non-paretic leg (Figure 2 and 3): Hip flexion knee flexion ankle flexion Knee extension with ankle dorsiflexion Full knee flexion Components of gait which were missing were focussed more. In motor imagery therapy, the patient was made to sit on a chair with eyes closed in a quiet room. He was then made to listen to motor imagery scripts through headphones. The patients were made to imagine all gait specific lower limb movements (Figure 4). The scripts were recorded in local languages. Conventional treatment was given according to patient’s requirements which included stretching, active and passive range of motion exercises, weight bearing, balance and coordination exercises. RESULTS SPSS software version 16.0. was used to assess the data obtained. To compare the pre and post measurements for Dynamic Gait Index(DGI) andFugl Meyer Assessment -lower extremity (FMA-LE), paired ‘t&#39; test was used for group A and Group B. To compare the effectiveness of intervention between two groups, independent sample ‘t&#39; test was used. The p-value less than 0.05, was regarded as significant for the study. The comparison of age distribution between the two groups was done using the independent t-test. The mean age for group A is 57.42±8.03 and for group B is 52.95±9.16.The p-value is 0.118 which is not statistically significant. Hence, age was homogenously distributed (Table1) The comparison of gender distribution between group A and group B was done utilizing chi-square test. The p-value is 0.118 which is not statistically significant. Hence, gender was homogenously distributed between the two groups (Table 2). Within the group analysis for the outcome measures DGI and FMA-LE was done using a paired t-test (Table3)  For Group-A, Pre DGI, mean value is 9.31±4.40 and Post DGI mean value is 18.84±3.26. On comparison of the mean values of Pre DGI and Post DGI, the mean values of Post DGI are higher with a difference of 9.125.  The p-value is less than 0.001 which is statistically significant. Pre FMA-LE mean value is 74.44±3.18 and Post FMA-LE mean value is 81±4.25.On comparison of the mean values of Pre FMA-LE and Post FMA-LE, the mean values of Post FMA-LE are higher with a difference of 6.563 and the p-value is less than 0.001 which is statistically significant. For Group-B, Pre DGI mean value is 18±10.11 and Post DGI mean value is 18±21.28. On comparison of the mean values of Pre DGI and Post DGI, the mean values of Post DGI are higher with a difference of 11.167 and the p-value is less than 0.001 which is statistically significant.Pre FMA-LE mean value is 75.67±4.39 and Post FMA-LE mean value is 83.06±3.94. On comparison of the mean values of Pre FMA-LE and Post FMA-LE, the mean values of Post FMA-LE are higher with a difference of 7.389 and the p-value is less than 0.001 which is statistically significant. Between the group comparison for DGI was done using an independent t-test (Table 4). Comparison of the Pre DGI between the two groups shows that Pre DGI is higher in group B (mean=10.42±5.54) with a t value of -0.319. The p-value is 0.752 which is not statistically significant.A comparison of the Post DGI between the two groups shows that Post DGI is higher in group B (mean=21.28±3.73) with a t value of -2.345. The p-value is 0.025 which is statistically significant. A comparison of the DGI difference between the two groups shows that DGI difference is higher in group B (mean=11.17±4.55) with a t value of -1.725. The p-value is 0.097 which is not statistically significant (Graph 1) Between group comparison for FMA-LE was done using independent t-test (table 5). Comparison of the Pre FMA-LE between the two groups shows that Pre FMA-LE is higher in group B (mean=75.54±4.28) with a t value of -1.044. The p-value is 0.303 which is not statistically significant. Comparison of the Post FMA-LE between the two groups shows that Post FMA-LE is higher in group B (mean=83.06±3.94) with a t value of -1.46. The p-value is0.154 which is not statistically significant. A comparison of the FMA-LE difference between the two groups shows that the FMA-LE difference is higher in group B (mean=7.39±3.18) with a t value of -0.831. The p-value is 0.412 which is not statistically significant (Graph 2). DISCUSSION Cerebrovascular accidents or stroke may occur either due to blood clots which impair blood flow to the brain or any trauma which ruptures the blood vessels causing leakage of blood around the brain. It can adversely disrupt the functioning of the limbs which in turn causes restriction of activity and participation in day to day life. Disability caused by stroke may persist lifelong if proper rehabilitative measures are not taken. In the present study, Group A received only motor imagery for lower limbs while group B received both motor imagery and mirror therapy for lower limbs for 2 weeks. Previously, studies have been done in which mirror therapy was combined with other forms of therapies such as action observation therapy, neuromuscular electrical stimulation. Lee et al suggested that action observation therapy and mirror training with conventional rehabilitation significantly improved patient’s gait[12]. Xu et al demonstrated that mirror therapy combined with neuromuscular electrical stimulation caused improvement in spasticity and walking ability of stroke patients[13]. No study is being done till now combining mirror therapy with motor imagery. So, this study was taken up. In group A (MI), there was significantly improved both gait and motor function of lower limbs after 2 weeks of intervention. Both DGI and FMA-LE showed statistically significant differences (p-value 0.005). Hence, it can be concluded that the combination of mirror therapy with motor imagery has not caused any additional improvement on gait and lower limb function in stroke patients. Thus, it can be inferred that the combination of mirror therapy with motor imagery is equally effective as motor imagery alone in improving gait and function in stroke patients. CONCLUSION The present study demonstrates that motor imagery combined with mirror therapy improved gait and lower limb function in stroke patients. Also, motor imagery alone improved gait and lower limb function in post-stroke patients. When the results of both the groups were compared with each other, there was no statistically significant difference. But on comparing the mean difference of both the groups, motor imagery combined with mirror therapy was superior to motor imagery alone.  Hence, it can be inferred that the combination of mirror therapy with motor imagery is equally effective as motor imagery alone in improving gait and function in stroke patients. LIMITATIONS The study had a small sample size The intervention period was only for two weeks long term follow up was not taken SCOPE FOR FUTURE WORK A similar kind of study can be carried out with large sample size. Also, the interventions can be given for a longer period. A follow-up study can be conducted to determine the long-term effects of the interventions. Further studies can be conducted comparing mirror therapy with motor imagery SOURCES OF FUNDING Nil CONFLICTS OF INTEREST None Englishhttp://ijcrr.com/abstract.php?article_id=2606http://ijcrr.com/article_html.php?did=2606 Sacco R, kasner S, Broderick J, Caplan L, Culebras A, Elkind M et al. An Updated Definition of Stroke for 21st Century. A Statement for Health Care Professionals from the American Heart Association/American Stroke Association.Stroke. 2013;44:2064-89. Pandian J, Sudhan P. Stroke Epidemiology and Stroke Care Services in India. J Stroke. 2013;15(3):128-34. Thieme H, Mehrholz J, Pohl M, Behrens J, Dohle C. Mirror Therapy for Improving Motor Function After Stroke. Stroke. 2012;44(1):e1-e2. Beyaert C, Vasa R, Frykberg G. Gait post-stroke: Pathophysiology and rehabilitation strategies. Clin Neurophysio. 2015;45(4-5):335-55. Oh Y, Kim H, Woo Y. Effects of Rhythmic Auditory Stimulation Using Music on Gait With Stroke Patients. Phys Ther Korea. 2015;22(3):81-90. Verma R, Arya K, Sharma P, Garg R. Understanding gait control in post-stroke: Implications for management. J Body Mov Ther. 2012;16(1):14-21. Arya K, Pandian S, Kumar V. Effect of activity-based mirror therapy on lower limb motor-recovery and gait in stroke: A randomized controlled trial. NeuropsycholRehabil. 2017;1-18. García D, Aboiti J. Effectiveness of motor imagery or mental practice in functional recovery after stroke: a systematic review. Neurología. 2016;31(1):43-52. Li R, Li Z, Tan J, Chen T, Lin W. Effects of motor imagery on walking function and balance in patients after stroke: A quantitative synthesis of randomized control trials. Complement Ther Clin Pract. (2017); 28:75-48. Ji S, Kim M. The effects of mirror therapy on the gait of subacute stroke patients: a randomized controlled trial. Clin Rehabil. 2014;29(4):348-54. Oostra K, Oomen A, Vanderstraeten G, Vingerhoets G. Influence of motor imagery training on gait rehabilitation in sub-acute stroke: A randomized controlled trial. J Rehabil Med. 2015;47(3):204-09. Lee H, Kim Y, Lee D. The effects of action observation training and mirror therapy on gait and balance in stroke patients. J Phys Ther Sci. 2017;29(3):523-26. García D, Aboiti J. Effectiveness of motor imagery or mental practice in functional recovery after stroke: a systematic review. Neurología. 2016;31(1):43-52. Lacourse M, Orr E, Cramer S, Cohen M. Brain activation during execution and motor imagery of novel and skilled sequential hand movements. NeuroImage. 2005;27(3):505-19. Guerra Z, Lucchetti A, Lucchetti G. Motor Imagery Training After Stroke. J Neurol Phys Ther. 2017;41(4):205-14. Li Y, Wei, Q, Gou W, He C. Effects of mirror therapy on walking ability, balance and lower limb motor recovery after stroke: a systematic review and meta-analysis of randomized controlled trials. Clin Rehabil. 2018;32(8):1007-21 Sacco R, kasner S, Broderick J, Caplan L, Culebras A, Elkind M et al. An Updated Definition of Stroke for 21st Century. A Statement for Health Care Professionals from the American Heart Association/American Stroke Association.Stroke. 2013;44:2064-89. Pandian J, Sudhan P. Stroke Epidemiology and Stroke Care Services in India. J Stroke. 2013;15(3):128-34. Thieme H, Mehrholz J, Pohl M, Behrens J, Dohle C. Mirror Therapy for Improving Motor Function After Stroke. Stroke. 2012;44(1):e1-e2. Beyaert C, Vasa R, Frykberg G. Gait post-stroke: Pathophysiology and rehabilitation strategies. Clin Neurophysio. 2015;45(4-5):335-55. Oh Y, Kim H, Woo Y. Effects of Rhythmic Auditory Stimulation Using Music on Gait With Stroke Patients. Phys Ther Korea. 2015;22(3):81-90. Verma R, Arya K, Sharma P, Garg R. Understanding gait control in post-stroke: Implications for management. J Body Mov Ther. 2012;16(1):14-21. Arya K, Pandian S, Kumar V. Effect of activity-based mirror therapy on lower limb motor-recovery and gait in stroke: A randomized controlled trial. NeuropsycholRehabil. 2017;1-18. García D, Aboiti J. Effectiveness of motor imagery or mental practice in functional recovery after stroke: a systematic review. Neurología. 2016;31(1):43-52. Li R, Li Z, Tan J, Chen T, Lin W. Effects of motor imagery on walking function and balance in patients after stroke: A quantitative synthesis of randomized control trials. Complement Ther Clin Pract. (2017); 28:75-48. Ji S, Kim M. The effects of mirror therapy on the gait of subacute stroke patients: a randomized controlled trial. Clin Rehabil. 2014;29(4):348-54. Oostra K, Oomen A, Vanderstraeten G, Vingerhoets G. Influence of motor imagery training on gait rehabilitation in sub-acute stroke: A randomized controlled trial. J Rehabil Med. 2015;47(3):204-09. Lee H, Kim Y, Lee D. The effects of action observation training and mirror therapy on gait and balance in stroke patients. J Phys Ther Sci. 2017;29(3):523-26. García D, Aboiti J. Effectiveness of motor imagery or mental practice in functional recovery after stroke: a systematic review. Neurología. 2016;31(1):43-52. Lacourse M, Orr E, Cramer S, Cohen M. Brain activation during execution and motor imagery of novel and skilled sequential hand movements. NeuroImage. 2005;27(3):505-19. Guerra Z, Lucchetti A, Lucchetti G. Motor Imagery Training After Stroke. J Neurol Phys Ther. 2017;41(4):205-14. Li Y, Wei, Q, Gou W, He C. Effects of mirror therapy on walking ability, balance and lower limb motor recovery after stroke: a systematic review and meta-analysis of randomized controlled trials. Clin Rehabil. 2018;32(8):1007-21

Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-52411111EnglishN2019June10HealthcareRole of Cartridge Based Nucleic Acid Amplification Test of Cerebrospinal Fluid in the Diagnosis of Tubercular Meningitis English1117Lavanya S.R.English Mahesh DaveEnglish Archana GokhrooEnglishIntroduction: Tubercular meningitis is the commonest form of neurotuberculosis. Diagnosis is challenging because of paucibacillary nature, lack of specific sign and symptoms. CBNAAT is a real time Polymerase Chain Reaction (PCR) test for the diagnosis of tubercular meningitis. So we used this rapid test to assess its role in diagnosis of Tubercular Meningitis(TBM). Material and Methods: This study was done over a period of one year in a tertiary centre. Patients with symptoms suggestive of tubercular meningitis were our study population. Their detailed clinical history, followed by a thorough general physical & systemic examination were done and documented in a predesigned proforma. Chest x-ray as well as neuroimaging were done in patients whose condition permitted. 3ml CSF fluid was drawn by lumbar puncture, 2 ml was sent for routine and bacteriological examination test and 1 ml for CBNAAT. Results: 100 patients were included, with a male to female ratio of 1.7:1. Mean age of the affected population was 37.53 years. 47% had radiological finding suggestive of tuberculosis.18 out of 100 were HIV reactive. MRI brain in 64 patients showed meningeal enhancement as the most common finding (60.93%). In Cerebrospinal Fluid (CSF) analysis mean CSF protein was 136.5 mg/dl, mean CSF glucose was 56.4 mg/dl and CSF cell count was 66.7 cells/microliter. Mean Adenosine Deaminase (ADA) was 11.22 U/L.CSF CBNAAT was positive in 9 patients out of which 8 were sensitive to rifampicin and one resistant to it. Conclusion: Even though CSF cytology gives good estimate of suspected TBM patient the test is not confirmative for bacilli demonstration. CBNAAT being a rapid accurate test would play a major role in the diagnosis, treatment, as well as for estimating rifampicin resistance of one of the common medical emergency in India by clear guidance from WHO. EnglishCBNAAT, Tubercular Meningitis, Cerebrospinal Fluid, Rifampicin resistance, WHOINTRODUCTION Tuberculosis remains one of the deadliest communicable diseases. Worldwide, TB is one of the top 10 causes of death and the leading cause from a single infectious agent (above HIV/AIDS)1. India is the highest TB burden country in the world, accounting for about 23.3% of the global prevalence and estimated incidence being 2.84 million cases2. About 2.2% of new cases and 15% of previously treated cases have MDR-TB in India. Tuberculosis was first recognized as a clinical entity in the early 19thcentury by schonlein, who used the term tuberculosis in 1830, which was derived from the English term “tubercle” or “lesion of consumption”3. According to World Health Organization (WHO), TB is a worldwide pandemic. While pulmonary tuberculosis is the most common presentation, extra-pulmonary tuberculosis (EPTB) is also an important clinical problem4,5.     CNS Tuberculosis includes three clinical categories, tubercular meningitis, tuberculoma and spinal arachnoiditis6. Tubercular meningitis is the commonest form of neurotuberculosis in Indian subcontinent accounting for 70%-80% of cases7. The burden of CNS TB is directly proportional to the prevalence of TB infection. Tubercular meningitis is the most devastating form of extra-pulmonary TB with 30% mortality and disabling neurological sequelae in >25% survivors7. Important risk factors includes HIV, overcrowding of urban population, poor nutritional status, appearance of drug resistant strains of tuberculosis7 The diagnosis of EPTB is challenging because of its paucibacillary nature, lack of specific signs symptoms and often negative acid fast bacilli smear of biological specimens8. Patients who demonstrate a subacute clinical course with headache, vomiting, pyrexia and anorexia should be suspected of having TBM. Diagnosis is based on the clinical symptoms and cerebrospinal fluid changes (increased protein, low glucose and mononuclear pleocytosis)9. Definitive diagnosis of tuberculosis involves demonstration of MTB by microbiological, cytological or histo-pathological methods Gene Xpert or CBNAAT (Cartridge Based Nucleic Acid Amplification Test) is a real time PCR test approved by WHO Policy in 2010, initially used in diagnosing MDR-TB and HIV associated TB. RNTCP policy update in 2013 expanded its uses, including for the diagnosis of TB in children, on selected specimens for the diagnosis of EPTB and for all individuals suspected of having pulmonary TB.10 Based on systematic review, WHO recommends Xpert over conventional tests for diagnosis of EPTB which permits rapid TB diagnosis through detection of the DNA of mycobacterium TB and simultaneous identification of a majority of the mutations that confirm Rifampicin resistance which is highly predictive of MDR TB. In the present study we prospectively determined the utility of this test in detection of MTB in CSF samples obtained from the patients suspected to have tubercular meningitis. Aim and Objective. To determine utility of Gene Xpert (CBNAAT) test in detection of MTB in CSF obtained from the patients who are clinically diagnosed case of tubercular meningitis. Materials and Methods. The present study was conducted for a period of one year at R.N.T. Medical College and attached group of hospitals, Udaipur. After obtaining approval from the institutional ethical committee and written informed consent from patient, 100 patients with symptoms and signs suggestive of TBM were included. Their detailed clinical history, previous history of tuberculosis, history of contact with pulmonary tuberculosis, past history of medical illness were taken. After general physical and central nervous system examination, Chest x-ray as well as neuroimaging (CT / MRI brain) were done if patients condition permitted. Sputum samples from study population, who had cough for any duration, was sent for AFB examination. 3ml CSF fluid was drawn by lumbar puncture, 2 ml was sent for routine biochemical and bacteriological examination and 1 ml for CBNAAT. Bacterial, viral and fungal meningitis were ruled out by clinico-radiological basis & biochemical and bacteriological examination of CSF. All the information was recorded in predesigned proforma formed in Microsoft excel for final analysis.            Inclusion Criteria 1.All patients with suspicion of TBM admitted in MBG (Maharana Bhupal Government) hospital in medicine and neurology ward. 2.Age group >18yrs. Exclusion Criteria 1.Age less than 18yrs 2.Patients with other causes of altered sensorium such as dyselectrolytemia, cerebrovascular accident, bacterial /viral meningitis/meningoencephalitis etc 3.Not willing to give consent. RESULTS In this study of 100 patients 63 patients (63%) were male and 37 patients (37%) were females. Male to female ratio was 1.7:1. Majority of them were in adolescent/early adult age group. Mean value for age was 41.3 years. Most common co-morbidity associated with study population was past history of pulmonary tuberculosis and diabetes mellitus.18 patients were HIV positive. Fever was the most common symptom followed by altered sensorium headache and vomiting. Chest x-ray suggestive of pulmonary tuberculosis was 39 (39%) out of which only 4 patients were positive for sputum AFB.8(8%) patients had x-ray chest suggestive of old healed lesion and 53(53%) patients had a normal chest x-ray. CSF analysis showed mean CSF protein value of 136.5 mg/dl, CSF cell count value of 58.8 cells/microliter (90% lymphocytes) CSF glucose value of 56.4mg/dl and CSF ADA level had a mean value of 11.3 U/L. MTB was detected in 9 CSF samples out of 100 sent for CBNAAT. Out of 9 samples 8 were Rifampicin sensitive and 1 showed resistance. Among 8 positive and rifampicin sensitive result one patient was HIV positive. Neuroimaging (MRI) was done on 64 patients out of which 55(85.97%) was abnormal and normal in 9 patients. Most common finding was meningeal enhancement (60.93%) followed by hydrocephalus(11.76%). 39 patients recovered completely, 20 patients recovered with residual morbidity and 16 patients had mortality. We lost the track of 25 patients as they did not show up in further follow ups. In this study of 100 patients final outcome was variable, 39% cases were completely recovered from the disease and 20% patients had some residual morbidity, while 16% mortality was observed. 25% cases outcome could not be documented as we lost the follow up. DISCUSSION: In this study 63 patients (63%) were male and 37 patients (37%) were females. Male to female ratio was 1.7:1 and mean age of population was 37.53 years, Most of the patients were in the age group of 8 to 30 yrs (49%). In a study by Rakesh Bhatia et al11. 20 were males and 14 were females and male to female ratio was 1.4:1. Patients of productive age groups were frequently involved by this clinical entity. Co-morbidities in our study group included 18 patients of diabetes mellitus, 14 of hypertension, 24 with past history of tuberculosis/contact and 2 of bronchial asthma. In Inês Sanches et al study, HIV, DM and cancer were frequent co-morbidities associated with extra-pulmonary tuberculosis and seen in 15.8% (20), 6.3% (8) and 4.8% (6) patients respectively12. DM is a well known risk factor for Tuberculosis. depressed cellular immunity, dysfunction of alveolar macrophages, low levels of interferon gamma, pulmonary microangiopathy, and micronutrient deficiency have been implicated in the occurrence of tuberculosis in Diabetic patients. 18 (18%) patients out of 100 were HIV positive in our study population. In a Study by Nathan C Bahr et al out of 257 patients with meningitis 105 (40%) patients were HIV positive13. HIV is a major risk factor for tuberculosis. Patients with HIV and active tuberculosis have an increased risk of extrapulmonary tuberculosis, and this risk will also increase with declining CD4+ count. In symptomatology of our study population, all patients (100%) had fever followed by altered sensorium in 58 (58%) patients. Headache and vomiting had a prevalence of 41 (41%) and 46 (46%) respectively among patients, followed by seizure (30%), cough (17%), focal neurological deficit (9%,one with facial nerve involvement) and loss of consciousness(5%). In study by Modi M et al14 out of 209 patients 195 patients had fever followed by headache (199), vomiting(169), loss of appetite(139), altered sensorium (101), loss of weight (94), focal deficit (67) and seizure (49). In our study chest x-ray suggestive of pulmonary tuberculosis was 39, out of which only 4 patients were positive for sputum AFB. 8 patients had x-ray chest suggestive of old healed lesion and 53(53%) patients had a normal chest x-ray. Chest x-ray lesions included apical infiltrates (unilateral >bilateral), miliary infiltrate and old healed calcified lesion and pleural thickening. In a study conducted by Solomons R S et al out of 84 children 37 (44%) TBM patients had CXR findings suggestive of TB, 9 (11%) with disseminated (miliary) TB.  In a study conducted by Sidra Aurangzeb et al15 out of 100 TBM patients radiographic findings of pulmonary TB was found only in 30(30%) patients .The predominant patterns on CXR were apical infiltration (26.6%), miliary mottling (20%) and hilar enlargement (16.6%).The relationship between pulmonary and cranial miliary lesions is controversial and there is a paucity of work done on adults in this regard. CSF analysis conducted in our study population had mean CSF protein value of 136.5 mg/dl and CSF cell count value of 58.8 cells/microliter ( 90% lymphocytes) and CSF glucose value of 56.4mg/d. In the study conducted by Modi M et al14 out of 203 patients 179 (88%) patients had lymphocytic predominant (>90%) pleocytosis,101 (44%) patients had glucose 50%). High protein (>45mg/dL) was seen in 173 patients. Characteristic CSF findings of TBM include the following: (i)lymphocytic-predominant pleocytosis. Total white cell counts are usually between 100 and 500 cells/μL. Very early in the disease, lower counts and neutrophil predominance may be present, (ii)elevated protein levels, typically between 100 and 500 mg/dL, (iii)low glucose, usually less than 45 mg/dL or CSF: plasma ratio 6 U/l had a sensitivity of 60% and was 94% specific. ADA is released by T cells during cell mediated immune response (CMI) to the tubercle bacilli. Raised levels of ADA in CSF are not specific to meningeal inflammatory disease but it can be a test for confirming its etiology with good predictive value. CSF ADA level 10 μ/L is sensitive and can suggest the diagnosis of TBM, especially if the clinical suspicion is high20,21. In this study MTB was detected in 9 CSF samples out of 100 sent for Gene Xpert technique and 1 with error. Out of 9 samples 8 were Rifampicin sensitive and 1 showed resistance. Among 8 positive and rifampicin sensitive  result, one patient was HIV positive. In study by Nguyen Thi Quynh Nhu et al22 X-pert MTB/ RIF was positive in 108 (59.3%) patients with sensitivity of 59.3% and specificity 99.5%. 4 cases of RIF resistance(4/108) was identified by Xpert. Patel and colleagues23 report the diagnostic performance of the Gene Xpert system&#39;s Xpert MTB/RIF assay for the diagnosis of TBM assay&#39;s overall sensitivity was 62%, and specificity was 95%. In the study conducted by Sharma Kusum et al24 multiplex PCR was positive in 84.78% cases. The overall sensitivity and specificity was 86.63% and 100 % respectively. In CSF, the pooled sensitivity from metaanalysis of Xpert MTB/RIF compared against culture as a reference standard was 79.5% (95% CI, 62.0-90.2%) (16 studies, 709 specimens)25.  Various studies conducted worldwide has varied sensitivity and specificity depending on various factors such as volume, centrifugation. Despite improved diagnostic accuracy using centrifuged CSF for Xpert compared with un-centrifuged CSF, the ideal CSF volume to collect is unknown. Xpert has an analytical sensitivity detection threshold of approximately ~100 CFU/mL M. tuberculosis organisms. In our study neuroimaging (MRI) was done on 64 patients out of which 55(85.97%) showed abnormalities, 39 (60.93%) had meningeal enhancement, 8 patients (11.76%) had hydrocephalus, 5 patients (7.81%) had vasculitic infarct, 2 patients(3.12%) had meningoencephalitis and 1 patient (1.56%) had dural sinus thrombosis.MRI brain was normal in 9 patients. In study conducted by Modi M et al14. Exudates were present in 82.3% patients Hydrocephalus was present in 52.1% and infarcts were seen in 23.9% of  patients. Tuberculomas were present in 45.9% of patients. R. Abdelmalek et al16 in their retrospective study reviewed 29 patients out of which 26 patients showed abnormalities in their MRI and concluded that Cerebrospinal MRI performed when TBM is suspected aids in its diagnosis and is also a useful means of monitoring the course of the disease under treatment. Common findings on imaging are abnormal meningeal enhancement in the basal cisterns, hydrocephalus, and vascular complications. Out of 100 patients in our study population 39 patients (39%) recovered completely, 20 patients (29%) recovered with residual morbidity and 16 patients(16%) had mortality. We lost the track of 25 patients as they did not show up in further follow ups. Prognosis of TBM largely depends on neurologic status at the time of presentation, and time-to-treatment initiation. While the course of TBM is generally not as rapid or fulminant as meningitis due to pyogenic bacteria, empiric treatment should be initiated as soon as the diagnosis is suspected as any delay in treatment can worsen outcome18 Mortality risk is highest in those with comorbidities, severe neurologic involvement on admission, rapid progression of disease, and advanced or very young age.            LIMITATIONS The main limitation of our study was a small study population and a significant loss of case follow up. The other being overburden of number of samples (both pulmonary and extrapulmonary) as the test was facilitated by a single machine for a tertiary centre. Due to which there was delay in processing of the sample and hence a high false negative result. The third limitation being our inability to repeat CSF samples for comparing different factors such as volume and centrifugation which could have further decreased false negative result of CSF samples study population. CONCLUSION: In order to reach a quick diagnosis using CSF specimens, CBNAAT should be preferentially used as rapid diagnosis and treatment is a strong prognostic indicator for reduced death and neurologic deficit. Eventhough CSF cytology gives good estimate of suspected TBM patient the test is not confirmative for bacilli demonstration. Hence CBNAAT has to be endorsed in every centres as the test gives rapid result and also detects rifampicin resistance which is the major concern for every clinician. To increase the value of this test which has gained popularities in detection of MTB and its resistance in sputum samples, a good amount as well as centrifuged CSF sample has to be considered. Clear guidance should be given by WHO regarding CBNAAT testing of CSF samples in suspected TBM patients so that this rapid test would play a major role in diagnosis and treatment of one of the most common medical emergency in India. Abbreviations TBM-Tubercular Meningitis CSF-Cerebrospinal Fluid CBNAAT-Cartridge Based Nucleic Acid Test WHO-World Health Organisation HIV-Human Immunodeficiency Virus MRI-Magnetic Resonance Imaging AIDS-Acquired Immune Deficiency Syndrome MDR-TB-Multi Drug Resistant Tuberculosis ADA-Adenosine Deaminase CT-Computerised Tomography PLHA-People living With HIV/AIDS EPTB-Extra Pulmonary Tuberculosis MTB/RIF-Mycobacterium Tuberculosis/Rifampicin resistance RNTCP-Revised National Tuberculosis Control Programme CFU-Colony Forming Unit DM-Diabetes Mellitus CXR-Chest Xray ACKNOWLEDGEMENT Authors 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=2607http://ijcrr.com/article_html.php?did=26071.Tuberculosis key facts-World Health Organisation, cited at https://www.who.int 2.Gobal Tuberculosis report 2018.Geneva :World Health Organisation 2018 3.Kligman, Stanton, Stgeme, Schor, Paediatric Infectious Disease. Nelson textbook of pediatrics. Volume 2, Ch 215, pp 1445-1453. 4.Fanning A. Tuberculosis: Extrapulmonary disease. Canadian Med Asso J. 1999; 160: 1597-603. 5.Iscman MD. Tuberculosis in relation to human immunodeficiency virus and acquired immunodeficiency syndrome. In: Iseman MD, editor. A clinician’s guide to tuberculosis. Philadelphia: Lippincott Williams and Wilkins; 2000 p. 199-252 6. John M Leonard et al, Central Nervous System Tuberculosis. Aminoff’s neurology and General medicine, cited at www.sciencedirect.com, 5th edition, 2014. 7. Virendra Kr. Goyal,  Approach to Neurotuberculosis,  cited at www.apiindia.org, Ch 24 Medicine updates 2016. 8.Sunil Kumar Komanapalli, Uma prasad, Bhagyalakshmi Atla, Vasundhara nammi et al. Role Of CBNAAT in diagnosing extrapulmonary tuberculosis in correlation with FNA in a tertiary care centre, International Journal of Research in Medical sciences, Vol 6 9. Suzuki Y, Nihon Rhinso et al .Tuberculosis infection in the nervous system. www.ncbi.nlm.nih.gov/m/pubmed/21838041/, 2011 Aug ;69(8):1422-6. 10.Xpert MTB/RIF implementation manual Technical and operational ‘how-to’: practical considerations, 2014,  cited at www.who.int 11. Rakesh Bhatia, Dayal R, Jindal S, Agrawal D, Goyal A ; Gene Xpert for Diagnosis of Tubercular Meningitis. Indian J Pediatr.2016 Nov;83(11):1353-1355 [pubmed] 12. InêsSanches a, Aurora Carvalho, Raquel Duarte  Who are the patients with extra-pulmonary tuberculosis? Rev Port Pneumol. 2015; 21(2):90--93. https://journalpulmonology.org 13. Nathan C Bahr, Lillian Tugume, David R Boulware; Improved Diagnostic Sensitivity for TB Meningitis with Xpert MTB/RIF of centrifuged CSF : A Prospective study. Int J Tuberc Lung Dis.2015 Oct;19(10):1209-1215. 14. M Modi, Sharma, Prabhakar, Goyal MK, Takkar A et al. Clinical and radiological predictors of outcome in tubercular meningitis: A prospective study of 209 patients. Clinical Neurology and Neurosurgery 161 (2017) 29–34.[pubmed] 15. Sidra Aurangzeb et al. Chest Radiographic Findings in Neurotuberculosis without Pulmonary Signs and Symptoms. Cited at www.jcpsp.com Journal of College of Physicians and Surgeons Pakistan, 2008; vol.18(1);27-30. 16. Ali Moghtaderi, Niazi A, Alavi-Naini R, Yaghoobi S, Naroule B et al. Comparative analysis of CSF ADA in Tuberculous and Nontuberculous meningitis. Clinical Neurology and Neurosurgery.112(2010) 459-462.[pubmed] 17. Kumar K et al. Diagnosis and treatment of tuberculosis :latest developments and future priorties. ARH. Cited at www.arh.amegroups.com/article/view/3820/4592, 22 Aug 2017. 18. Grace E Marx et al. Tuberculous Meningitis: Diagnosis and Treatment Overview. Tuberculosis Research and Treatment Volume 2011, cited at https://www.hinawi.com/journals/trt/2011/798764, Article ID 798764, 9 pages. 19. Lely Solari et al. The Validity of CSF parameters for the diagnosis of tuberculous meningitis. Int J of Infect Dis. Vol 17:issue 12,Dec 2013; e1111-e1115. https://doi.org/10.1016/j.ijid.2013.06.003 20. Bharat Kumar Gupta et al. Adenosine Deaminase Levels in CSF of Tuberculous Meningitis Patients. J Clin Med Res. 2010 Oct; 2(5): 220–224. doi:10.4021/jocmr429w 21. Gopal Chandra Gosh et al.CSF ADA Determination in Early Diagnosis of Tuberculous Meningitis in HIV-Infected Patients. Scientifica Volume 2016. doi:10.1155/2016/5820823. 22. Nguyen ThiQuynhNhu, Dorothee Heemskerk, Do Dang Anh Thu. Evaluation of GeneXpert MTB/RIF for Diagnosis of Tuberculous Meningitis. Journal of Clinical Microbiology p. 226–233 January 2014 .doi:10.1128/JCM.01834-13. 23. Vinod B Patel, Grant Theron, Laura Lenders, Brian Matinyena, Cathy Connolly, Ravesh Singh. Diagnostic accuracy of quantitative PCR (Xpert MTB/RIF) for Tuberculous Meningitis in a high burden setting: A Prospective Study. PLoS Med.2013 Oct. doi:10.1371/journal.pmed.1001536 24. Sharma Kusum et al. Multiplex PCR for rapid diagnosis of tuberculous meningitis. J Neurol (2011) 258:1781–1787. J Glob Infect Dis. DOI:10.4103/0974-777X.112272. 25. Xpert MTB/RIF implementation manual Technical and operational ‘how-to’: practical considerations. www.who.int, SBN: 978 92 4 150670 0 © World Health Organization 2014.

Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-52411111EnglishN2019June10Life SciencesAnti-cancer effect of Gymnema sylvestre Leaf Extract against MG63, Human Osteosarcoma cell line - An in vitro analysis English1824B. PackialakshmiEnglish S. Raga SowndriyaEnglishBackground: Gymnema sylvestre also known as ‘GURMAR’ is one of the important herbs in the Ayurvedic system of medicine and Indian proprietary medicines. Objective: In the present investigation, phytochemical screening, free radical scavenging, anti-inflammatory and anti-cancer activities were evaluated in vitro in the aqueous extract from the leaves of Gymnema sylvestre (AGS). Methods: Leaf samples were subjected with four different solvents (ethanol, acetone, ethyl acetate and distilled water) and the four extracts were screened for phytochemicals using different biochemical tests. AGS was subjected to fourier-transform infrared (FT-IR) spectroscopy to analyze its functional groups. Antioxidant activity of AGS was determined by measuring nitric oxide (NO) radical scavenging activity. The anti-inflammatory activity was studied using the inhibition of protein denaturation assay and anti-cancer activity was evaluated using MTT assay against MG63 cell lines. Results: Phytochemical screening confirmed the presence of phytoconstituents like alkaloids, carbohydrates, triterpenoids, proteins, phenols and flavonoids. The FT-IR spectrum with different peak values revealed the presence of various functional compounds in AGS and it showed significant NO radical scavenging activity with the EC50 value of 401.66 μg/mL. AGS displayed strong anti-inflammatory activity (IC50 = 17.5 μg/mL). Moreover, AGS also exhibited significant dose-dependent anti-cancer activity against MG63 cell line and the IC50 value was 19.5 μg/mL. Conclusion: The results indicate that Gymnema sylvestre can act as a promising free radical scavenger, anti-inflammatory and anticancer agent against MG63 human osteosarcoma cell lines. Hence, G. sylvestre can be used as natural antioxidants for preventing the oxidative stress related degenerative diseases.. EnglishAnti-cancer, Anti-inflammatory, Free radicals, Gymnema sylvestre, MG63, OsteosarcomaINTRODUCTION Many important valuable medicinal compounds are present in the herbal plants which were proven in the ancient time by some traditional methods1. There are about 45,000 plant species are found in India, in that majority of plant species possess pharmacological significance2. Many plant species consist of bioactive constituents which are used directly as drugs or pharmacological agents. Phytochemicals act as antioxidants by neutralizing free radicals which damage DNA, proteins and lipids. 80% of human population relies on traditional system of medicine for primary health needs and these systems are mainly based on medicinal plants3, 4. Due to the rise in population, there is demand in supply of drugs and also cost for treatments are increased. Sometimes, several synthetic drugs cause side effects and development of resistance to presently using drugs by microbes also increased, recently5. To overcome these problems, now a day, plant materials are used as a source of medicine for a wide range of human ailments. Today plant compounds provide the models for 50% of western drugs6. Gymnema sylvestre (G. sylvestre) is a large tropical plant which is native to central and western India and also found in Africa and in Australia. G. sylvestre is one of the ayurvedic and medicinal herb, have been used for more than 2,000 years in India to treat diabetes. Now a day, there is a demand for G. sylvestre leaves are in pharmaceutical trade because it is an important anti-diabetic medicinal plant. This plant contains pentriacontane, hentriacontane, phytin, chlorophyll a, chlorophyll b, tartaric acid, formic acid, butyric acid, anthraquinone derivatives, inositol, d-quercitol, gymnemic acids7. Osteosarcoma, the primary bone malignancy, is one of the most common cancers worldwide. Generally, chemotherapy with agents such as cisplatin, methotrexate, and cyclophosphamide is widely used for treating osteosarcoma8. Chemotherapy may result in drug resistance, as well as several side effects including drug-cytotoxicity which causes damage to normal tissues. Therefore, alternative treatments for osteosarcoma need to be considered. At present, cancer-fighting foods are being discussed as potential therapeutic products against osteosarcoma. Various nutritional and functional phytochemicals have been extracted from plants. These plant-derived substances also act as natural anticancer agents9. Pharmacological studies reveal that G. sylvestre has some medicinal properties such as anti-diabetes, anti-microbial effect, anti-inflammatory, anti-cancer, anti-atherosclerotic effect, larvicidal effect and anti-hepatoprotective activity10,11. However, the anticancer effects of G. sylvestre on human osteosarcoma have not been studied so far. Therefore, in the present study, we investigated No radical scavenging activity and anti- inflammatory activity of aqueous extract of G. sylvestre (AGS). This study was also focused on investigating the anticancer effects of AGS on human osteosarcoma MG63 cells. MATERIALS AND METHODS Plant material The healthy and fresh leaves of G. sylvestre were collected from the farms of Coimbatore in the month of December 2018, Tamil Nadu. Preparation of extracts The collected leaves of G. sylvestre were shade dried and powered, and extracted in a Soxlet extractor using four different solvents such as distilled water (AGS), ethanol (EGS), acetone (AcGS) and ethyl acetate (EaGS). The solvent was evaporated under reduced pressure using a rotary evaporator at 45°C and the dried extracts obtained were stored at −20°C until further use. Cell lines and Chemicals MG63 cell lines (Human Osteosarcoma cell) were procured from National centre for cell sciences (NCCS), Pune, India. Cell lines were cultured in minimal essential medium supplemented with fetal bovine serum (FBS). Antibiotics, EDTA and glucose were purchased from Hi-Media laboratories Ltd., Mumbai. 3-(4,5–dimethyl thiazol–2–yl)–5–diphenyl tetrazolium bromide (MTT), MEM medium, trypsin, Fetal bovine serum, Phosphate buffered saline and other chemicals were purchased from Sigma-Aldrich (Bangalore, India).. Qualitative analysis of phytochemicals For preliminary phytochemical analysis, standard procedures were performed in different extracts of G. sylvestre. Phytoconstituents such as alkaloids, flavonoids, triterpenoids, phenols, carbohydrates, proteins, glycosides, saponins and steroids were examined by performing different phytochemical tests12 [Edeoga et al., 2005). Fourier Transform Infrared spectral analysis: Fourier Transform Infrared Spectrophotometer (FT-IR) is the most powerful tool to identify the types of chemical bonds or functional groups present in the phytochemicals. The wavelength of light absorbed is the salient feature of the chemical bonds seen in the annotated spectrum. Dried powder of AGS was used for FT-IR analysis (IR-affinity 1, Sigma, Japan) using KBr pellet method13 with a Scan range from 4000 to 400cm−1. Estimation of total phenolic content: The total phenol in AGS was measured according to the method of Singleton and Rossi14. 1.0 mL of the extract was mixed with 1.0 mL of Folin-Ciocalteu’s phenol reagent and 1.0 mL of saturated sodium carbonate (35%) was added to the mixture after 3 min. The mixture was made up to 10 mL by adding deionised water and kept for 90 min at room temperature in the dark. The absorbance was measured immediately against the prepared blank at 725 nm. Gallic acid was used as the reference standard. The total phenol content is expressed as milligrams of gallic acid equivalents (GAE) per gram of extract. Estimation of total flavonoid content:         Total flavonoid content in AGS was evaluated as described by Jia et al15. 0.25 mL of extract was diluted with 1.25 mL of distilled water and 75 µL of a 5% sodium nitrite were added. After 6 min, 150 µL of 10% aluminium chloride was added and mixed. After 5 min, 0.5 mL of 1 M sodium hydroxide was added. The absorbance was measured at 510 nm against the blank. Rutin was used as the reference standard. The total flavonoid content is expressed as milligrams of rutin equivalents (RE) per gram of extract. Total antioxidant activity assay (Phosphomolybdenum assay): The antioxidant activity of the sample was determined by phosphomolybdenum method16. An aliquot of 0.1 mL of sample solution was mixed with 1 mL of the reagent solution (0.6 M sulphuric acid, 4 mM ammonium molybdate and 28 mM sodium phosphate). The tubes were capped with silver foil and kept at 95°C for 90 min and cooled to room temperature. The absorbance was measured at 695 nm against a blank. Ascorbic acid was used as a standard and total antioxidant capacity was expressed as ascorbic acid equivalents (GAE) per gram of extract. Nitric oxide scavenging activity assay Nitric oxide (NO) released from sodium nitroprusside was evaluated using Griess reaction17. Different concentrations of AGS (dissolved in dimethyl sulfoxide) was added with sodium nitroprusside (5 mM) in phosphate?buffered saline and incubated at room temperature for 2.5h. After incubation, 500 μL of sample was diluted with 500 μL of griess reagent. The absorbance was measured at 695 nm against a blank. Gallic acid was used as the positive control. The percentage nitrite radical scavenging activity of AGS and gallic acid were calculated using the following formula: [(Acontrol-Atest)/ Acontrol]×100, where Acontrol was the absorbance of the control, and Atest was the absorbance of the test. In vitro anti?inflammatory activity – Protein denaturation method Aqueous extract of G. sylvestre (0.05 mL) at various concentrations (20, 40, 60, 80, 100 µg/mL) and standard drug, diclofenac sodium (0.05 mL) at different concentrations (20, 40, 60, 80, 100 µg/mL) were taken separately and 0.5% BSA (0.45 mL) was added to both test and standard drug solution. Test control consisted of 0.05 mL of distilled water and 0.45mL of BSA. The samples were incubated at 37°c for 20 min and the temperature was increased progressively up to 57°C for 3 min. After cooling, add 2.5 mL of phosphate buffer to the above solutions after 20 min. The absorbance was measured at 416 nm using UV-Visible spectrophotometer18. The results were compared with the standard drug, diclofenac sodium. The percentage inhibition of protein denaturation can be calculated as. Percentage Inhibition = 100 – [{(Absorbance of test – Absorbance of standard drug) / Absorbance of test control}/ × 100]. In vitro anti-cancer activity – MTT assay MTT assay is a cytotoxic assay which is frequently used to identify the toxicity level of the test sample. The antiproliferative activity of AGS was measured using the 3?(4,5?dimethylthiazol?2?yl)?2,5?diphenyltetrazolium bromide (MTT) assay. MG63 cell lines were used to evaluate the anti-cancer activity. The different concentrations of AGS (10, 20, 30, 40, and 50 μg/mL) were supplemented to cell lines in 96-well plates. After the removal of exhausted media, the MTT reagent (5 μg/mL) added in all wells, and plates were incubated at 37 °C for 3 h. Subsequently, the MTT solution was replaced with dimethyl sulfoxide. The absorbance was measured at 540 nm using a microplate reader19. The graph for percentage of cell viability was plotted to calculate IC50 value. Statistical analysis All analyses were carried out in triplicates and the results are presented as mean±SD. Data were analysed using Analysis of variance (ANOVA) and Duncan’s multiple range test (DMRT) with least significance difference (LSD), pEnglishhttp://ijcrr.com/abstract.php?article_id=2608http://ijcrr.com/article_html.php?did=2608REFERENCES Nair SS, Kavrekar V, Mishra A. Evaluation of in vitro anti diabetic activity of selected plant extracts. Int J Pharm Sci Invention 2013;2(4):12-19. Rikke Stamp Thorsen. 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