Challenges in integrating component level technology and system level information from Ayurveda: Insights from NMR phytometabolomics and anti-HIV potential of select Ayurvedic medicinal plants

Background: Information from Ayurveda meeting the analytical challenges of modern technology is anarea of immense relevance. Apart from the cerebral task of bringing together two different viewpoints,the question at the pragmatic level remains ‘who benefits whom’.Objective: The aim is to highlight the challenges in integration of information (Ayurvedic) and technology using test examples of Nuclear Magnetic Resonance (NMR) metabolomics and anti-HIV-1 potential of select Ayurvedic medicinal plants. The other value added objective is implications andrelevance of such work for Ayurveda.Materials and methods: Six medicinal plants (Azadirachta indica, Tinospora cordifolia, Swertia chirata,Terminalia bellerica, Zingiber officinale and Symplocos racemosa) were studied using high resolutionproton NMR spectroscopy based metabolomics and also evaluated for anti-HIV-1 activity on threepseudoviruses (ZM53 M.PB12, ZM109F.PB4, RHPA 4259.7).Results: Of the six plants, T. bellerica and Z. officinale showed minimum cell cytotoxicity and maximumanti-HIV-1 potential. T. bellerica was effective against all the three HIV-1 pseudoviruses. Untargeted NMRprofiling and multivariate analyses demonstrated that the six plants, all of which had different Ayurvedicpharmacological properties, showed maximum differences in the aromatic region of the spectra.Conclusion: The work adds onto the list of potential plants for anti-HIV-1 drug molecules. At the sametime, it has drawn attention to the different perspectives of Ayurveda and Western medicine underscoring the inherent limitations of conceptual bilinguism between the two systems, especially in thecontext of medicinal plants. The study has also highlighted the potential of NMR metabolomics in studyof plant extracts as used in Ayurveda.© 2017 Transdisciplinary University, Bangalore and World Ayurveda Foundation. Publishing Services byElsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Effect of Progressive Resistance Exercise Training on Hepatic Fat in Asian Indians with Non-Alcoholic Fatty Liver Disease.

Introduction: Nonalcoholic fatty liver disease (NAFLD) is closely associated with obesity and insulin resistance and lifestyle measures form the cornerstone of therapy. Objective: To study the effect of progressive resistance training (PRT) on hepatic fat content, body composition and insulin sensitivity in patients with NAFLD. Methods: This study included 24 adult patients with NAFLD diagnosed on ultrasonography. Subjects with alcohol intake >140 gm/week and any secondary cause of fatty liver were excluded. Patients underwent thrice weekly sessions (40 minutes each) of resistance exercises including flexion at biceps, triceps, and hip flexion, knee extension and heel rise for 12 weeks. Pre- and post-intervention evaluation included anthropometry, BIA analysis, short insulin tolerance test (SITT), lipid profile and hepatic fat quantification by MRI. Results: Twenty four patients (17 males, 7 females, mean age 39.8±10.5 yrs) completed the study protocol with 78.7% compliance to PRT protocol. There was significant decrease in waist, hip and mid-thigh circumferences and skinfold thicknesses at biceps, triceps, subscapular and suprailiac regions (p<0.05), with no significant change in BMI and WHR. Insulin sensitivity improved significantly at 12 weeks as indicated by increase in k-value (rate of change of glucose) on SITT (0.84 vs 1.3, p=0.002). A decrease in total cholesterol and LDL-c with increase in HDL-c was noted after 12 weeks (p<0.05). Hepatic fat content also decreased at 12 weeks (22.3±3.9 vs 21.4±4.0 %, p=0.01). Conclusion: Moderate intensity PRT is associated with significant improvement in hepatic fat, truncal subcutaneous fat and insulin sensitivity in patients with NAFLD.

Differential response to sustained stimulation by hCG & LH on goat ovarian granulosa cells.

Background & objectives: Chapekar established a model of ovarian tumourigenesis in mice by splenic transplantation of ovaries, resulting in sustained luteinizing hormone (LH) levels because of absence of feedback inhibition. There is increasing evidence of the differential response to LH or hCG under various experimental conditions. The effect of sustained hormonal stimulation in long term cultures is sparsely investigated. The study is aimed to determine the role of hCG and LH stress on caprine ovarian granulosa cells and their downstream signaling in short and long term cultures. Methods: To study the response of hCG and LH stress and downstream signaling, short term cultures were set up by exposing goat ovarian granulosa cells in primary cultures to hCG and LH stress (levels beyond their physiological doses) for 5 days (P0). Cells were sub-cultured at sixth day and subjected to prolonged LH/ hCG stress for two weeks in passage 1(P1) (long term cultures). Downstream cell signaling molecules were assessed. Intracellular cAMP was estimated by ELISA. For PKA and PKC, activity assays were performed. pERK protein expressions in short term cultures were assessed by Western blot and flowcytometry; in long term cultures, pERK expression was analyzed by flowcytometry. Results: Differential effects on cell proliferation were observed in long term cultures, where the untreated and hCG exposed cells showed markedly reduced cell proliferation after second week of exposure while LH treated cells continued to proliferate. Different levels of cAMP, PKA, PKC and phosphorylated ERK1/2 were observed on short term and long term LH stimulation. On sustained hormonal stimulation, cAMP levels were significantly (P<0.05) higher in hCG treated cultures as compared to controls and LH treated cultures. LH led to maximal elevation of ERK in long term cultures. Interpretation & Conclusions: As pERK1/2 promotes cellular proliferation, activation of ERK1/2 in LH treated cultures may be responsible for sustained growth. Prolonged LH treatment promoted growth and proliferation in caprine ovarian granulosa cells whereas prolonged exposure to hCG led to elevated levels of cAMP and decreased the rate of proliferation. Defining the signals and second messengers that act as survival or apoptotic mediators may help in elucidation of the mechanisms controlling proliferation or programmed cell death in granulosa cells.

Apo E genotyping from blood stored on filter paper.

Background & objectives: Dried blood spotted on to filter paper has been found suitable for a large number of studies. In tropical countries with varying temperature conditions the use of dried blood needs to be validated. We carried out this study to assess the use of blood spotted filter paper as a transport system to study genotyping of Apo E gene. Methods: Fifty five patients visiting Cardiothoracic Neuroscience Centre (CNC) OPD at the All India Institute of Medical Sciences (AIIMS), New Delhi, and referred for lipid investigations to Cardiac Biochemistry Laboratory were selected at random. Blood was spotted on to Whatman 3 MM filter paper, dried and stored at room temperature. Genomic DNA was extracted and genotyping was carried out at the end of 0, 3 and 12 months. The study was further validated using samples collected on to filter paper from four centres and stored for eight years at room temperature. The temperature and humidity conditions of the centre varied widely. Results: Fifty five samples collected on to filter paper showed exact match of the genotyping when compared to fresh blood. In dried blood samples collected and stored for 1 yr at room temperature DNA extraction and apo E genotyping was done successfully. Interpretation & conclusions: The present results showed the feasibility of using dried blood samples on filter paper for apo E genotyping in tropical temperature. The findings need to be validated on a large sample before being recommended for use.

Apolipoprotein E polymorphism in cerebrovascular & coronary heart diseases.

The role of apolipoprotein E (apo E) in lipid metabolism and cholesterol transport is well established. About 14 per cent of the variation in plasma cholesterol levels is attributed to polymorphisms in apo E gene (APOapo E). E consists of three common alleles, designated as ε2, ε3 and ε4 which code for E2, E3 and E4 proteins respectively resulting in three homozygous (E2/E2, E3/E3, E4/E4) and three heterozygous (E3/E2, E4/E2 and E4/E3) phenotypes. Different populations studied worldwide inherit variable frequencies of the E alleles and genotypes, with the most frequent allele being ε3.The ε4 allele has been consistently shown to be associated with Alzheimer’s disease, coronary heart disease and cerebrovascular disorders. In this review, we have discussed the role of apo E polymorphisms in cerebrovascular and coronary heart diseases. The status of apo E polymorphisms and their disease associations in Asian Indians besides, other populations has also been discussed. Further, studies elucidating the pathophysiology of apo E deficiency conducted in knock-out mice have been reviewed.

Study of dihydropteroate synthase (DHPS) gene mutations among isolates of Pneumocystis jiroveci.

BACKGROUND & OBJECTIVES: Pneumocystis jiroveci (also known as P. carinii) causes fatal pneumonia in patients with AIDS and other immunocompromised patients. Co-trimoxazole (trimethoprim + sulphamethoxazole, TMP-SMZ) is the drug of choice for treatment and prophylaxis. Widespread use of sulpha medication has raised the possible selection of resistant P. jiroveci strains worldwide. Non-synonymous polymorphisms associated with sulpha resistance have been observed in P. jiroveci dihydropteroate synthase (DHPS) gene at codons 55 and 57. In view of this, we investigated mutation at DHPS locus amongst P. jiroveci isolates obtained at a tertiary care hospital in north India. METHODS: Microscopic examination of P. jiroveci in 69 clinical samples obtained from patients suspected to have P. carinii pneumonia (PCP), was performed by Grocott's Gomori methenamine silver and direct fluorescent antibody staining. Molecular studies were carried out by polymerase chain reaction (PCR) using major surface glycoprotein (MSG) as the target gene. Investigations for DHPS mutations were carried at specific 55th and 57th codon using PCR-RFLP (restriction fragment length polymorphism) assay. RESULTS: Microscopic examination detected P. jiroveci in four cases and MSG gene was amplified in five cases. Further, amplification of DHPS gene was successful in four of the five cases positive by MSG gene PCR. No point mutation was observed and all four isolates presented wild-type sequences at DHPS gene by RFLP analysis. INTERPRETATION & CONCLUSION: Although our findings suggest that in Indian subpopulation, point mutations in DHPS gene of P. jiroveci are not as common as in other parts of the developed world, further studies are needed.

Distribution and cutoff points of fasting insulin in Asian Indian adolescents and their association with metabolic syndrome.

AIM: To evaluate the levels and appropriate cutoff points of fasting insulin, and their association with the metabolic syndrome (MS) in Asian Indian adolescents. METHODS: This cross-sectional, population based study included 948 (527 males & 421 females) adolescent subjects aged 14-19 years selected randomly from New Delhi, India. Cutoff points of fasting insulin were defined using Receiver Operating Characteristics curve analysis against overweight, abdominal obesity and high subscapular skinfold thickness. The MS was defined according to NCEP, ATP III and IDF criteria using age-, gender- and ethnicity-specific cutoff points. RESULTS: Fasting insulin levels peaked at 16 y and reduced subsequently in both genders. The derived cutoff points for fasting insulin (pmol/L) were: 14-15 y- 128.5 and 164.8; 16-17 y- 126.1 and 152.8; 18-19 y- 121.2 and 162.4 in males and females, respectively. Prevalence of fasting hyperinsulinemia (39.1%) and MS (NCEP 2.2%, IDF 1.5%) was highest in age group 16-17 years. CONCLUSION: The data from this first study describing the distribution and cutoff points of fasting insulin in Asian Indian adolescents may be helpful for detection of and application of primary prevention strategies for fasting hyperinsulinemia and the metabolic syndrome in this population.

Use of different primer directed sequence amplification by polymerase chain reaction for identification of Pneumocystis jirovecii in clinical samples.

BACKGROUND: Pneumocystis carinii pneumonia (PCP), caused by opportunistic agent Pneumocystis jirovecii (formerly, Pneumocystis carinii is one of the most serious respiratory infection in immunocompromised patients. AIM: The present study was conducted to compare polymerase chain reaction (PCR) assays targetting three different genes of Pneumocystis to study their application in its diagnosis. METHODS: One hundred and eighty (n = 180) clinical samples from 145 immunocompromised patients with clinical suspicion of PCP and 35 samples from control group of 30 immunocompetent individuals with respiratory infections other than PCP were prospectively examined for the presence of Pneumocystis jirovecii (P. jirovecii). All the samples were subjected to microscopic examination, one single [major surface glycoprotein, (MSG)] and two nested [mitochondrial large subunit ribosomal ribonucelic acid, (mtLSU rRNA) and internal transcribed spacer (ITS) region], polymerase chain reaction assays. RESULTS: Microscopic examination was positive in only six (n = 6) patients, whereas single round MSG PCR detected P. jirovecii deoxyribonucleic acid (DNA) in 16 cases. When the clinical samples were tested by mtLSU rRNA and ITS nested PCR assays, it was possible to detect seven additional cases of PCP, making it to a total of 23 cases. None of the clinical specimens in control group (n = 30) were positive by any of the above-mentioned techniques. Amongst the 81 bronchoalveolar lavage (BAL) samples tested, 16 were positive by MSG PCR, while 20 were positive by both nested, i.e., mtLSU rRNA and ITS PCR assays. Similarly, out of 50 sputum samples, only three were positive by MSG, seven by mtLSU rRNA and six by ITS nested PCR assays. CONCLUSION: It has been observed that MSG is relatively more sensitive when single round PCR assay is used for detection of human Pneumocystosis compared to the first (single) rounds of either ITS or mtLSU rRNA nested PCRs. However, the two nested PCRs using ITS and mtLSU rRNA have been found to be more sensitive. On comparison of two nested PCR assays, the results have been more or less comparable.

Association between subclinical inflammation & fasting insulin in urban young adult north Indian males.

BACKGROUND & OBJECTIVES: Elevated levels of c-reactive protein (CRP) are known to be associated with insulin resistance and metabolic syndrome in adults. A substantial prevalence of hyperinsulinaemia and elevated CRP levels have been shown in Indian young adults. We therefore studied the association of serum high-sensitivity C-reactive protein (hs-CRP) with fasting insulin and insulin resistance in urban adolescent and young adult males in north India. METHODS: In this cross-sectional study 324 healthy males, 14-25 yr of age were selected randomly and their clinical and anthropometric profile [body mass index (BMI), waist and hip circumferences, waist-to-hip circumference ratio (W-HR), and skinfold thickness at four sites], percentage of body fat (%BF) and biochemical (fasting blood glucose, lipoprotein profile, fasting insulin and hs-CRP) parameters were recorded. Insulin resistance was assessed by the homeostasis model of assessment (HOMA-IR). RESULTS: Fasting insulin and hs-CRP levels correlated significantly with BMI, waist circumference, and triceps and subscapular skinfold thickness. Fasting insulin also correlated with %BF, and hs- CRP correlated with W-HR. No correlation was observed between hs-CRP and fasting insulin levels or insulin resistance. In multiple logistic regression analysis different independent risk factors for hyperinsulinaemia and elevated hs-CRP levels were observed; hypercholesterolaemia, overweight and high subscapular skinfold thickness for the former, and high triceps skinfold thickness for the latter. INTERPRETATION & CONCLUSION: Lack of correlation between hs-CRP and surrogate markers of insulin resistance and different risk factors for each, in young Indian males are unique observations of our study. Further studies on a larger sample of both genders need to be done to confirm these findings.

Dyslipidemia in Asian Indians: determinants and significance.

Data suggest that lipid fractions other than total cholesterol, i.e. serum triglycerides (TG) and high-density lipoprotein (HDL) cholesterol are important for the pathogenesis of atherosclerosis. A combination of hypertriglyceridemia, low levels of HDL-cholesterol and high levels of small dense low-density lipoprotein, termed as "atherogenic dyslipidemia', is particularly seen in Asian Indians. Although precise reason for such dyslipidemia is unknown, genetic predisposition and characteristic body composition (excess truncal subcutaneous fat and intraabdominal fat) may be important contributors. A common interface between such body composition and dyslipidemia in Asian Indians is high tendency to develop insulin resistance, more than the other ethnic groups. The general guidelines for the management of dyslipidemia in Asian Indians should be according to National Cholesterol Education Program, Adult Treatment Panel III. However, optimal management requires consideration of ethnic-specific dietary, lifestyle and management factors to formulate individual treatment guidelines.