In vitro and In vivo Validation of Folk Lore Claims of Thymus serpyllum

Traditional medicinal plants have a long history of therapeutic use. The beneficial health effects of medicinal plants is often attributed to their potent antioxidant activities due to the presence of secondary metabolites like the polyphenols, since diets rich in antioxidants are epidemiologically associated with a decreased incidence of age-related diseases in humans. Thymus serpyllum commonly known as Jawand in Kashmir is used as a culinary herb, as well as for aromatizing and traditional medicinal purposes. In the present study, the hexane, ethyl-acetate, ethanol, methanol and the aqueous extracts of Thymus serpyllum were studied for the antioxidant, antimicrobial and the anti inflammatory activities. The antioxidant activity was checked by four different methods- DPPH assay, lipid peroxidation assay of liver microsomes, lipid peroxidation assay and hydroxyl radical scavenging assay. The ethyl-acetate, butanol, ethanol, methnol and the aqueous extracts of Thymus serpyllum showed good antioxidant activities, but the methanolic extract being the most active one causing 89.84%, 57.32%, 66.8% and 77.14% inhibition of the radical activity in DPPH assay, lipid peroxidation assay of liver microsomes, lipid peroxidation assay and the hydroxyl radical scavenging assay respectively. The antimicrobial activity was checked by broth micro dilution method. The ethyl-acetate and the methanolic extract were found to be active against bacteria (both gram positive and gram negative) and fungi with MIC values ranging from 2000 to 4000μg/ml. The anti inflammatory activity was assessed by using the model of carregeenen induced edema in rats. The butanolic extract was found to beneficial against inflammation, causing a reduction of 25.23% and 56.07% of edema at doses 250mg/Kg body weight and 500mg/Kg body weight respectively

In Vitro

Arnebia benthamii is a major ingredient of the commercial drug available under the name Gaozaban, which has antibacterial, antifungal, anti-inflammatory, and wound-healing properties. In the present study, in vitro antioxidant and anticancer activity of different extracts of Arnebia benthamii were investigated. Antioxidant potential of plant extracts was evaluated by means of total phenolics, DPPH, reducing power, microsomal lipid peroxidation, and hydroxyl radical scavenging activity. The highest phenolic content (TPC) of 780 mg GAE/g was observed in ethyl acetate, while the lowest TPC of 462 mg GAE/g was achieved in aqueous extract. At concentration of 700 µg/mL, DPPH radical scavenging activity was found to be highest in ethyl acetate extract (87.99%) and lowest in aqueous extract (73%). The reducing power of extracts increased in a concentration dependent manner. We also observed its inhibition on Fe2+/ascorbic acid-induced lipid peroxidation (LPO) on rat liver microsomes in vitro. In addition, Arnebia benthamii extracts exhibited antioxidant effects on Calf thymus DNA damage induced by Fenton reaction. Cytotoxicity of the extracts (10–100 µg/mL) was tested on five human cancer cell lines (lung, prostate, leukemia, colon, and pancreatic cell lines) using the Sulphorhodamine B assay

Elucidation of etiology of colorectal cancer: A study on silencing of p16 gene by promoter hypermethylation

Colorectal cancer (CRC), commonly known as bowel cancer is the third most common cause of cancer-related deaths in the western world. Colorectal cancer is one of the leading malignancies worldwide. CRC has been reported to show geographical variation in its incidence, even within areas of ethnic homogeneity. The usual treatment is surgery and subsequent chemotherapy and radiotherapy. Cancer development and progression is dictated by series of alterations in genes such as tumor suppressor genes, DNA repair genes, oncogenes and others. In colorectal carcinogenesis disturbances different from mutations called an epigenetic regulation are also taken into consideration. The aim of this study was to study the promoter hypermethylation of CpG islands of p16 gene in colorectal cancer patients among the Kashmiri population. The study included 70 surgically obtained colorectal samples among which 50 were obtained from colorectal cancer patients and 20 were histopathologically normal colorectal samples. All the samples were histopathologically confirmed before further processing. DNA was extracted from all the samples and was modified using bisulphite modification kit. Methylation-specific polymerase (MSP) chain reaction was used for analysis of the promoter hypermethylation status of p16 gene. The genetic analysis of the cases and controls by MSP- PCR method, for checking the promoter hypermethylation of CpG islands of p16 gene revealed that unlike other high risk regions, Kashmiri population has a different promoter hypermethylation profile of p16 gene. 66% of the cases showed p16 promoter hypermethylation while as 34% of the cases were nonhypermethylated. The study also revealed that 20% of the normal cases also had promoter hypermethylation of p16 gene and 80% did not showed promoter hypermethylation of p16 gene. The association of promoter hypermethylation with colorectal cancer was evaluated by χ2 (Chi square) test with Odds ratio and was found to be significant. Among 29 male cases and 21 female cases, the association of promoter hypermethylation with colorectal cancer was evaluated using Fisher’s exact test and was found to be significant in both males and females. Occurrence of p16 promoter hypermethylation was found to be unequally distributed in males and females with more frequency in males than in females but the difference was not statistically significant. When the frequency of p16 promoter hypermethylation was compared with clinical staging of the disease, p16 promoter hypermethylation was found to be certainly higher in Stage III/IV (83.33%) compared to Stage I/ II (56.25%) but the difference was not statistically significant. Also, the degree of p16 promoter hypermethylation increased with the increasing severity of the lesion but the difference was not again statistically significant. These results clearly suggest that p16 aberrant promoter hypermethylation in Kashmiri population contributes to the process of carcinogenesis in colorectal cancer and is reportedly one of the commonest epigenetic changes in the development of human CRC. It also demonstrates that hypermethylation of p16 gene can be designated as epigenetic biomarker for the screening, diagnosis and prognosis of colorectal cancer. The data gives a clue that p16 gene expression can be readily and fully restored and growth rate of cancer cells decreased by treatment of cancer cells with demethylating agents and DNA methylation inhibitors

Insulin Receptor Substrate 1 Gly972Arg (rs1801278) Polymorphism Is Associated with Obesity and Insulin Resistance in Kashmiri Women with Polycystic Ovary Syndrome

Background: Polycystic ovary syndrome (PCOS) is commonly associated with metabolic abnormalities such as hyperinsulinemia, insulin resistance and obesity. The genetic variants of genes regulating insulin action, expression and regulation are suggested as possible factors involved in development and severity of clinical manifestations in PCOS. Aim: We investigated whether IRS-1Gly972Arg (rs1801278) polymorphism is associated with increased risk of PCOS in Kashmiri women. The correlation of various clinical, metabolic and hormonal markers with rs1801278 single nucleotide polymorphism was analyzed. The genotypic–phenotypic association of clinical manifestations of PCOS with the tested genetic variant was also assessed. Results: There were no significant differences in allele frequency (OR = 0.87, CI = 0.59–1.29, χ2 = 0.456, p = 0.499) or genotypic distribution (χ2 = 3.73, p = 0.15) between PCOS women and controls. No significant association was also found in the dominant (OR = 1.63, χ2 = 0.377, p = 0.53), recessive (OR = 0.79, χ2 = 1.01, p = 0.31) or heterozygote vs. homozygote (OR = 1.34, χ2 = 1.53, p = 0.22) genotype model analysis. The genotype–phenotype correlation analysis showed that the Arg allele was significantly associated with increased central adiposity markers hip circumference (p = 0.012), and body adiposity index BAI (p = 0.002) in the recessive model in PCOS women. The two-hour glucose (p = 0.04) and insulin resistance marker HOMA (p = 0.44) were significantly higher in Arg allele carriers. The androgen excess markers dehydroepiandrosterone sulfate DHEAS (p = 0.02), Ferriman–Gallwey score (p = 0.012), prevalence of acne, alopecia and hirsutism (all p < 0.01) were significantly elevated in the wild-type GG genotype. Conclusions:IRS-1Gly972Arg genetic variant does not increase the risk of PCOS in Kashmiri women. However, this polymorphism is associated with clinical manifestations of insulin resistance, obesity and hyperandrogenism, suggesting its possible role in variable phenotypic manifestations of PCOS

XRCC1 Arg194Trp polymorphism is no risk factor for skin cancer development in Kashmiri population

Background: Recently, three coding polymorphisms in X-ray cross complementing gene 1 (XRCC1) have been identified; with probable effect on DNA repair capacity and thus modulation of cancer susceptibility. Moreover, association of these polymorphisms with the cancer risk are reported to be population dependent. Therefore, in this case control study we aimed to investigate the polymorphism at codon 194 (Arg to Trp) in XRCC1 gene and the possible association of its polymorphic genotypes with skin cancer in the ethnically different population of Kashmir. Aim: To study if there is any possible association of Arg194Trp XRCC1 polymorphism with risk of developing skin cancer in ethnically different Kashmir population. Subjects and methods: For this study 68 skin cancer patients and 60 healthy controls, matched for age and gender were recruited. PCR-RFLP followed by statistical analysis was employed to check for the C194T polymorphism and its possible association with the skin cancer risk in the population. Result: An insignificant association among skin cancer patients with respect to the wild (Arg/Arg) versus variant (Trp/Trp) genotypes (OR = 0.34, 95% CI = 0.10–1.05, p = 0.06) was observed. However, individually homozygous and heterozygous variant alleles were observed to be associated with risk of developing skin cancer. As far as, individual allelic ratio among cases and controls is concerned Trp allele of codon194 showed a remarkably high frequency in cases (67.7% vs. 32.3%) in comparison with controls (OR = 1.94, 95% CI = 1.22–3.0, p = 0.004). Discussion: These findings suggest that the combined homozygous and heterozygous variants of each codon and the 194Trp allele are associated with the disease, however when genotypes were compared individually, the association turned out to be insignificant. Keywords: Skin cancer, X-ray cross complementing 1 gene (XRCC1), Single nucleotide polymorphism (SNP), Kashmi

Effect of six-month use of oral contraceptive pills on plasminogen activator inhibitor-1 & factor VIII among women with polycystic ovary syndrome: An observational pilot study

Background & objectives: Polycystic ovary syndrome (PCOS) is an endocrinopathy warranting lifelong individualized management by lifestyle and pharmacological agents mainly oral contraceptive pills (OCPs). This study was aimed to report the impact of six-month OCP use on plasminogen activator inhibitor-1 (PAI-1) and factor VIII (FVIII) in women with PCOS. Methods: PCOS women diagnosed on the basis of Rotterdam 2003 criteria, either treated with OCPs (ethinyl estradiol-0.03 mg, levonorgestrel-0.15 mg) for a period of six months (n=40) or drug-naïve (n=42), were enrolled in this study. Blood was drawn to estimate glucose, insulin levels and lipid profile. Chemiluminescence immunoassays were used to measure hormones (LH, FSH, PRL, T4). Plasma levels of PAI-I and FVIII were measured by commercially available kits. Results: Menstrual regularity, Ferriman-Gallwey score and serum total testosterone significantly improved in the OCP group compared to drug-naïve group (P<0.01). No significant difference was observed in PAI-1 levels of the two groups; however, significant decrease in FVIII levels was observed in OCP group as compared to drug-naïve group. PAI-1 levels of OCP group correlated positively with blood glucose two hours, triglycerides and insulin two hours, while FVIII levels of OCP group correlated negatively with fasting insulin and homoeostatic model assessment-insulin resistance. Interpretation & conclusions: OCPs use has differential effect on pro-coagulant markers among women with PCOS. Well-designed, long-term, prospective, large-scale studies are prerequisite to elucidate the efficacy and safety of OCP in the treatment of PCOS