sign in sign up
<<
Home , Adrenosterone, Etiocholanolone, Testosterone glucuronide

Accepted Manuscript

Moderate consumption in chronic form enhances the synthesis of cho- lesterol and C-21 , while treatment with Tinospora cordifolia modulate these events in men

Suman Kumari, Ashwani Mittal, Rajesh Dabur

PII: S0039-128X(16)00083-0 DOI: http://dx.doi.org/10.1016/j.steroids.2016.03.016 Reference: STE 7953

To appear in:

Received Date: 10 December 2015 Revised Date: 14 March 2016 Accepted Date: 22 March 2016

Please cite this article as: Kumari, S., Mittal, A., Dabur, R., Moderate alcohol consumption in chronic form enhances the synthesis of and C-21 steroid hormones, while treatment with Tinospora cordifolia modulate these events in men, Steroids (2016), doi: http://dx.doi.org/10.1016/j.steroids.2016.03.016

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Moderate alcohol consumption in chronic form enhances the synthesis of cholesterol and C-21 steroid hormones, while treatment with Tinospora cordifolia modulate these events in men

Suman Kumari1, Ashwani Mittal3, Rajesh Dabur1,2 *

1Department of Biochemistry, Maharshi Dayanand University, Rohtak, Haryana - 124001. India

2National Research Institute of Basic Ayurvedic Sciences, Nehru Garden, Kothrud, Pune,

Maharastra-411038. India

3Department of Biochemistry, University College, Kurukshetra University, Kurukshetra -136119,

Haryana, India.

*Corresponding Author, Present Address: Dr. Rajesh Dabur Department of Biochemistry, Maharshi Dayanand University, Rohtak, Haryana-124001, India. E-Mail: [email protected] Phone: +911262393070.

The declaration: There is no conflict of interests among authors

1

Abstract

Chronic and heavy alcohol consumption disrupts lipid and hormonal balance including levels. However, studies doubt the relationship between moderate alcohol intake and sex levels. Therefore, the aim of the present investigation was to establish the direct impact of chronic and moderate alcohol intake on cholesterol homeostasis and synthesis. Asymptomatic chronic and moderate alcoholics (n=12) without chronic liver disease and healthy volunteers (n=14) were selected for the study. Furthermore, effects of standardized water extract of Tinospora cordifolia (Willd) Mier. (Menispermaceae) (TCJ), a well reported anti-alcoholic herbal drug, on urinary steroids was studied. This study included four groups, i.e. a) healthy; b) healthy+TCJ; c) alcoholic; d) alcoholic+TCJ. The blood and urine samples from each group were collected on day 0 and 14 of the post-treatment with TCJ and analyzed. Alcoholic blood samples showed the significantly higher values of traditional biomarkers γ-GT and MCV along with cholesterol, LDL, TGL and urinary methylglucuronide compared to healthy. Qualitative analysis of steroids showed that moderate alcohol intake in a chronic manner increased the cholesterol synthesis and directed its flow toward C-21 steroids; shown by increased levels of (2.456 fold) and (3.7 fold). Moreover, alcohol intake also increased the synthesis of and clearance rate of other steroids through the formation of glucuronides. Therefore, it decreased the synthesis and increased the clearance rate of testosterone (T) and (A). Quantitative analysis confirmed decreased T/A ratio from 2.31 to 1.59 in plasma and 2.47 to 1.51 in urine samples of alcoholics. TCJ intervention normalized the levels of steroids and significantly improved the T: A ratio to

2.0 and 2.12 in plasma and urine. The study revealed that TCJ modulated lipid metabolism by inhibiting cholesterol and glucuronides synthesis.

Keywords: Alcoholism, Tinospora cordifolia, Testosterone, Androstenedione, Metabolomics, Biosteroids

2

Introduction

Chronic and heavy alcohol intake led to impotence and infertility. These effects of alcohol were due to inhibition of testosterone synthesis in testis (Fernandes et al, 2014). Decreased levels of testosterone have been observed in short as well as long term heavy drinkers (Valimaki et al., 1984). Chronic (moderate and heavy) alcohol intake has been reported to be associated with a degree of elevation in plasma levels in male alcoholic by extending the conversion of androgenic hormone into estrogen (Kenna, 2012). However, reports available on the effects of low or moderate dose alcohol intake contradict these findings (Sarkola and Eriksson, 2003; Sierksma et al, 2004). This shows that alcohol consumption may have different effects on both genders and according to age (Orentreich et al, 1984). Moderate alcoholism has been reported to prevent atherogenesis through change in lipid metabolism (Agarwal, 2002), inflammation (Imhof et al, 2001) and hemostasis (Dimmitt et al, 1998). An inverse relationship between aortic atherosclerosis and levels of testosterone has been reported in a 55 year old man (Hak et al, 2002). Subsequently, aortic stiffness have been related inversely to testosterone (Frances et al, 2002). Therefore, to understand the effects of alcohol on steroid homeostasis, it is important to study the effects of moderate alcohol intake on the steroid levels, in a controlled environment.

Presently few drugs are available in the market with various side effects to prevent alcoholism or disorders of alcoholism (Litten et al, 2012). Herbal medicines may be the alternate option of these drugs or to develop new therapies. In Ayurveda, Tinospora cordifolia (Willd) Mier. (Menispermaceae) has been reported to possess rejuvenating properties with no in vivo toxic effect (Srivastava, 2011; Gahlaut et al., 2012). Several bioactive compounds, including berberine, cordifoliosides, tinosporoids etc have been reported from the plant (Singh et al, 2003). Berberine and other metabolites have been reported as anti-hyperlipidemic and anti- diabetic agents (Hao et al, 2010; Sangeethaa et al, 2013). Hence, it is the drug of choice for hepatic aliments (Bishayi et al, 2002), the most affected part in alcoholics.

The objectives of this study were to analyze the effect of moderate alcohol consumption on urinary steroids in men and co-relation between urinary with plasma testosterone levels. Furthermore, efficacy and the mechanism of T. cordifolia water extract (TCJ) in prevention of alcoholic effects on the steroid metabolism in moderate alcohol consumers male was also studied.

3

Material and Methods

Chemicals: Standards of steroidal compounds, their conjugates and solvents i.e. water, acetonitrile, formic acid, were purchased from Sigma (St. Louis, MO). Internal and external calibrates and standards were purchased from Agilent Technologies. All other reagents used in the study were LC-MS grade.

Study subjects: Twelve alcoholic and fourteen non-alcoholic male volunteers of age 40.5±3.8 (Mean+SD) years, having a BMI 30.9±3.5 (Mean+SD) were selected for this study from the local areas of Pune, Maharastra, India. All were apparently healthy and hadn’t recorded any disease or any kind of regular medication. All of them reported a typical alcohol consumption of 60 ml of 80 proof liquor (40% alcohol) (24-30 gm) per day from last 4-5 years, and were classified as moderate chronic drinkers. An informed written consent was taken from all the individuals who entered into the study. Exclusion criteria included body mass index >30 kg/m2, blood pressure >160/90 mm Hg, total cholesterol >7.5 mmol/L, present or prior history of cardiovascular disease, diabetes mellitus, respiratory, gastrointestinal, hepatic, renal, endocrine, or reproductive disorders; or use of lipid-lowering agents, antihypertensive agents. All the volunteers underwent the questionnaire of alcohol use disorders identification test (AUDIT) for assessment of alcohol abuse. The Human Ethics Committee of the DYP Ayurveda College, Pune, India has approved the study protocols wide letter no. NRIBAS/2011/HEC/2023 dated 18-11-2011.

Drug preparation: Spot identified fresh stems of T. cordifolia plants were collected in September 2011 from garden of National Research Institute of Basic Ayurvedic Sciences, CCRAS (Department of AYUSH), Nehru Garden, Kothrud, Pune. The voucher specimens (No. 207) were kept at the medicinal plant museum of the Institute. The stems were washed 3 times with reverse osmosis (RO) water and homogenized with RO water in 1:5 ratio and kept overnight at room temperature under septic conditions. Stem juice (TCJ) was prepared under the guidance of an Ayurvedic physician. The LC-MS profiles of the at least three extracts prepared at different time were analyzed for the standardization purpose and no significant difference was observed in the chemical constituents of stems (Mittal and Dabur, 2015; Shirolkar et al, 2012).

Study design: All volunteers were asked to maintain their usual vegetarian diet throughout the study and not to take any medicine without consulting. Figure 1 shows the workflow of treatment and analysis period. After one week pre-watch analysis period, volunteers further divided into four groups (Healthy; Healthy+TCJ; Alcoholic; Alcoholic+TCJ). Volunteers were given 100 ml TCJ (3.0 gm solid extract) early in the morning with an empty stomach for 14

4 days in the presence of a physician. Most of the standard drugs for treatment of alcohol induced disorders take 2 weeks to synthesize sufficient unbound enzyme to metabolize alcohol adequately. TCJ has also been reported to lower the cholesterol levels after two week treatment (Prince et al, 2008). Therefore, the 14 day trial period was chosen to analyze the effect of TCJ on moderate alcohol intake. The alcoholic subjects continued 60 ml of 80 proof liquor (40% alcohol) consumption per day (0.5 gm/kg/body weight corresponding to two to three standard drinks) during the TCJ treatment period of 14 days. All volunteers admitted to the day care center were given standard vegetarian diet. Fasting blood samples for analysis were collected at day 0 (after 7 day observation) and 14 after administration of TCJ. In the same way, first pass urine samples were collected on day 0 and 14 after 12 hrs of alcohol intake and treated with sodium azide to a final concentration of 2.5 mM. The entire urine sample was centrifuged filtered through 0.2 m filters and stored at -80 °C until analysis.

Hematology and blood biochemistry: Blood samples were collected into cold tubes containing EDTA and reduced glutathione and centrifuged immediately at 1000 g for 10 min at 4°C. The plasma samples were added with butylated hydroxytoluene (40 µg/ml plasma) to protect them from oxidation and stored at −80 °C until analysis. γ-GT, serum glutamic oxaloacetic transaminase (SGOT), serum glutamic-pyruvic transaminase (SGPT) and the red blood cell volume (mean corpuscular volume: MCV) were measured to confirm alcohol intake and the effect of treatment. Hematology and blood biochemistry was performed at the start (seven days after alcohol consumption), end of the baseline (0 day) and end of the treatment period (14th day) using auto analyzer D-280 (Sinnowa, China Republics). The blood pressure of all volunteers was measured thrice a day using an automatic blood pressure monitor.

Liquid chromatography coupled with mass spectrometry: Unprocessed urine samples were analyzed by RRLC coupled with QTOF mass spectrometry. The HPLC system consisted of Poroshell 120-SB C18 column (3 X 100 mm, 2.7 µm particle size), a model 1100 binary pump, with auto sampler (Agilent Technologies, Germany). The solvent system had (A) 0.1 % formic in water and (B) 0.1 % formic in acetonitrile: water (80:20). The gradient mode {%/min} for solvent B 20% /0; 50% / 18; 90% /21; 90% /25; 20% /29; 20%/35}, with a flow rate of 0.2 ml/min was used. Q-TOF mass spectrometer was operated in positive and negative polarity modes and total ion chromatogram (TIC) in the range of 100-1200 m/z with acquisition rate 3 spectra s-1 was acquired with fragmentor voltage 195 V. Samples infused with standards of lidocaine (235.3 m/z) and 5, 7-isoflavone (285.3 m/z) were used with continuous internal calibration of the machine. Mass data were analyzed as described previously with Qualitative MassHunter and MassProfiler Professional (MPP) software (Agilent Technologies, Germany).

5

Quality assurance: A mixture of standard compounds was injected to check the operational conditions of the RRLC–MS/MS device before sample analysis. Steroidal hormones were identified on the basis of their retention time and the product . Quality control of urine samples was also performed by mixing the urine samples of chronic alcoholics in the group a group of three so that actual levels of steroidal hormones can be compared with individual of that group. Another quality control check was kept by mixing the urine samples with standard steroids (2.5 ppm) to check the peak volumes and intensity. After identification, comparative concentration of the analytes was calculated by its area and intensity ration. Area ratios were determined by integration of the area of an analyte in the chromatograms with reference to the integrated area of the standard.

Quantization of metabolites: Mass spectrometric detection was performed in selected ion monitoring (SIM) mode after electron impact ionization (EI) at 70 eV electron energy to quantize testosterone (T), androstenedione (A) and ethylglucucronide in plasma and urine samples. Major ions for all the three metabolites were monitored i.e. 289.3, 109.1 for testosterone, 287.2, 109.1 for androstenedione and 223.1, 205.1 and 115.06 for ethylglucuronide.

Statistics: Values for biochemistry were calculated as mean ± standard error. Values for γ-GT, SGOT, SGPT and MCV were expressed as the mean ± SD. Data was analyzed and compared for Non-alcoholics (healthy), healthy plus TCJ treatment, alcohol consumers and alcohol consumers plus TCJ treatment groups by Oneway ANOVA. Mass spectroscopic data sets were analyzed using Mass Profiler Professional (Agilent, version B.02.02). Initial alignment of data was carried out by keeping retention time window and mass difference 0.1 min and 0.05 mDa with internal standards. Data was further screened by keeping the molecular features which were present in 100% of samples in at least one type of data set, p<0.05, fold change >2.0 and CV <15. To distinguish different groups and explore unique features present across the groups, partial least square discrimination analysis (PLS-DA) was performed. PLS-DA data were subjected to Wilcoxon paired t-test, One way ANOVA with Bonferroni multiple testing corrections, keeping up value cutoff p<0.05 and fold change >2.0. Differential molecular features in the groups have been identified using Metalin accurate mass library and standards.

6

Results

Drug preparation and standardization: Figure 2 shows the mass profiles and total ion chromatogram of TCJ prepared, which is collected at different time interval. General observation as well as analysis with Mass Profiler Professional (Agilent Technologies) didn’t show much variation in the TCJ samples. Identified metabolites like magnoflorine, columbin, citramalic acid, diferuloylquinic acid, tinocordifoliside, sulfocysteine, reservatol and abscisic acid were found to be present in the entire sample in the same abundance (Figure 2). Some major unidentified metabolite of m/z 104.108, 149.02, 160.139, 174.154, 297.122, 342.181,

382.363 and 775.564 were also found to be present universally and in the same abundance. Fresh extracts were given to the subjects by keeping in view degradation of compounds in TCJ, already been reported from our lab (Shirolkar et al, 2012).

Hematology and blood biochemistry: As shown in Table 1, there was a significant decrease (p< 0.01) in the mean of γ-GT, SGOT, SGPT and MCV (fl) i.e. 23.3, 20.9, 32.1 and 3.01 respectively in the TCJ treated alcoholic as compared to the alcoholics (Table 1). Changes in γ- GT levels were found to be consistent with reported in alcohol intake by univariate regression analysis (R2=0. 912). GSH levels of plasma in alcoholics were depleted by nearly 50% as compared to normal individuals and TCJ treatment increased the levels to 87% in alcoholics (Table 1). An inverse correlation in increased GGT activity and depleted levels of GSH was observed. Levels of plasma MDA in the alcoholics were found to be significantly increased to 9.7±1.4 mmol/L, as compared to healthy controls (3.3±0.7 mmol/L). Uric acid levels were also observed to be significantly elevated in alcoholics (4.1±0.7 to 5.7± 0.3 mg/dL). TCJ treatment decreased the MDA and uric acid levels to 4.2±0.5 mmol/L and 4.7± 0.1 mg/dL on day 14. Most of other hematological parameters in TCJ treated alcoholics were also recorded significant low values (p<0.05) in comparison to the alcoholic consumers (Table 1). In response to prolonged and moderate alcohol consumption, an increase in TC, TGL, and HDL levels was observed that depleted after treatment with TCJ, with exception for HDL (Table 1). HDL levels were increased in alcoholics as well as treated alcoholics and healthy individuals given TCJ. In addition, the mean blood alcohol concentration of the alcoholics determined after the drink was found to be 43.7 mg/L. There was no significant difference in the body weight after the treatment period. However, a minor increase in body weight of treated groups (alcoholics and healthy) was observed. At the same time, no toxic effects were observed probably due to the presence of antioxidant compounds in TCJ.

7

Partial least square–discrimination analysis: MassHunter Qualitative Analysis B.04.00 (Agilent Technologies) was used to extract molecular features from mass spectroscopic data sets of healthy, alcoholics and alcoholics treated with TCJ using Personal Compounds Database Library of 98 steroidal compounds with mass fragments and retention times (Figure S1). Extracted molecular features were imported into MPP for further analysis. Mass and retention time alignment was performed as described in Material and Method section. Molecular features with less than 5000 cycles per second were filtered out from all the samples. Retention time and m/z were aligned using standards, present throughout the sample, i.e. compound of m/z 287.06 and 317.2. Data were further normalized using Z transform. Box Whisker plot in Figure 3A of normalized data demonstrating altered expression of metabolites in alcoholic in comparison to healthy. Most of the variation was observed in upper quartile and Whisker of alcoholics. Alcoholics treated with TCJ have shown less variation and found near to the healthy group. Healthy group treated with TCJ didn’t show major variations. The plot shows opposite effects of alcohol and TCJ on the steroid profiles. In the further processing of data, molecular features present 100% at least in one dataset were kept. Out of these insignificant molecular features (p<0.05) were filtered out using asymptotic one way ANOVA with Benjamini- Hochberg correction and keeping coefficient variation <15. Further data were subjected to PLS- DA and support vector machine (SVM) analysis (Table 2). The results of the sample classification presented in terms of discrimination and recognition abilities were found to be 91.6% accurate, with r2= 0.97 representing the percentage of the samples correctly classified during model training and cross-validation. Androstendione was found in the top ten metabolites found to highly discriminate in PLS-DA model. PLSDA PCA plot among healthy, healthy treated with TCJ, alcoholics and alcoholics treated with TCJ groups showed 27.94, 11.3, and 9.61% separation along with X, Y and Z axis in positive ion modes (Figure 3B). PLSD PCA showed the closeness in TCJ treated and healthy groups showing the recovery of alcoholic group. PLS-DA and SVM data were subjected to Wilcoxon matched paired test (p<0.05) in pairs conditions with fold change <1.5. Analysis has shown 35 significantly differentially expressed metabolites across the samples (Table 2).

Effect of moderate alcohol intake on steroidal compounds: Table 2 shows the changed levels of steroidal compounds from baseline across study groups obtained after Wilcoxon matched paired test. Thirty seven steroidal compounds were identified by comparing mass fragmentation and retention time from personal compounds database library constructed from standard compounds. Thirty steroids and seven steroidal glucuronides were found to have altered fold change <2.0 and significant p< 0.05. Qualitative analysis of urine showed decreased

8 ratio of urinary T: A along with increased levels of hydroxytestosterone (1.5 folds). By fold change analysis T: A ration was found to be decreased by 1.574. Decreased levels of 21- deoxycortisol (4.1 folds), testosterone (3.4 folds), (2.6 folds), corticosterone (2.4 folds), (2.3 folds), (1.3 folds), and cortexolone (2.1 folds) were found in the alcoholics (Table 2). Moderate alcoholism was found to increase the urinary levels of (4.2 folds), 20-hydroxycholesterol (3.5 folds), cholesterol (3.5 folds), dehydrocholesterol (2.1 folds), , β-estradiol (1.4 folds) and (1.5 folds). Most of the glucuronides were found to be increased more than 2.0 folds in urine samples of alcoholics except glucuronide and androstenedione- glucuronide (Table 2).

Effects of TCJ treatment on steroids in chronic alcoholics: TCJ treatment significantly depleted the urinary levels of cholesterol, 20-hydroxycholesterol, dehydrocholesterol, dehydrocholesterol glucosiduronate, hydroxytestosterone, pregnanediol, β-estradiol and testosterone to the normal levels. Table 2 shows the decreased levels of glucuronides more than normal levels, including and corticosterone levels, which were amplified over normal level demonstrating the reversion effect of TCJ in moderate alcohol consumers in fourteen days. T: A ration was also increased by 2.01 whereas cholesterol and its intermediate levels were decreased by the significant level indicated its function as alcohol metabolism blocker. Plasma cholesterol level was decreased significantly in the biochemistry test (Table 1). When compared to TCJ treated healthy and non-alcoholics, it was found to be a significant revitalization (Table 2).

The effect of TCJ treatment on steroidal compounds in healthy men: TCJ treatment decreased the urinary levels of 2-methoxyestrone 3-sulfate, -16-glucuronide and β- estradiol glucuronide significantly (p<0.05) by more than 2 fold. It increased hydroxycorticosterone, etiocholanolone, pregnenalone, hydroxypregenalone, estriol-16- glucuronide, and dehydrocorticosterone levels in urine more than 2 fold. It also increased the level of corticosterone, estrone and testosterone by 1.5 folds in healthy individuals. Levels other steroids were not affected by any means after TCJ treatment in healthy men (Table 2).

T: A ratio and ethylglucuronide: Qualitative analysis didn’t find out the exact concentrations of testosterone and androstendione therefore, quantitative analysis of both the steroids along with standard ethylglucuronide (a marker of alcoholism) was carried out using SIM mode. The chromatograms of selective ion monitoring transitions of androstenedione, testosterone and ethylglucuronide extracted from urine samples from all the three groups. The criteria for the

9 limit of quantification (LOQ) was total precision of <15% %CV. Limit of detection (LOD) was a signal-to-noise ratio of greater than 3:1. The LOD for androstenedione and testosterone were observed to be 1.5 ng/mL with linearity (r2≤0.98). In case of ethylglucuronide LOD was found to be 3.0 pg/mL with linearity (r2=0.99) (Table 3). The assay was linear to 40, 25 and 30 µg/L for androstenedione, testosterone and ethylglucuronide respectively. The blood alcohol concentration was highly variable between the individuals and after one hour of drinking as it was found to be 3.2 to 12.8 mg/L. The individual results for ethylglucuronide levels in the 12 hr urine after drinking during the treatment on 14th day were variable between the individuals ranging from 8.4–66.2 mg/L. After 12 hrs alcohol consumption and on 14th day, a significant decrease in plasma testosterone (from 3.2±0.42 to 2.44±0.84 ng/L, p < 0.05) and an increase in androstenedione (from 1.48± 0.22 to 1.53±0.15 ng/L, p < 0.05) were observed (Table 3). Overall a decline in the T: A ratio from 2.31±0.19 to 1.59±0.44, p < 0.01was observed. After treatment with TCJ, T: A ratio was observed to be 2.25±0.35, which is a significant increase, as compared to alcoholics. A small non-significant increase was also observed in TCJ treated healthy individuals (Table 3). Urine testosterone levels were decreased in alcoholics (1.23±0.15 ng/L) as compared to healthy (1.78±0.12 ng/L) with p < 0.05. An increased level of androstenedione (0.81± 0.03 ng/mL) was observed in alcoholics as compared to healthy (0.72±±0. 04 ng/L) (Table 3). Therefore, a decrease in T:A ratio from 2.47±0.3 to 1.51±0.5, p < 0.01, was observed. Result showed almost equivalent rational decrease in both in urine and plasma T:A ratio in alcoholics. Treatment of healthy individuals with TCJ shifted the T: A ratio of 2.90±0.28 which shows its efficiency and direct effect on steroid metabolism to normalize the T: A ratio significantly.

Discussion

In the present study, it was found that moderate alcohol consumption in chronic form affected the steroid hormone synthesis at multiple points starting from cholesterol. Increased levels of urinary cholesterol in the alcoholics were consistent with enzymatic measurement of plasma cholesterol (Table 1). The heavy alcohol exposure is known to cause hypercholesterolemia by enhancing cholesterol biosynthesis, LDL and TGL (Aviv et al, 1997, Crouse and Grundy, 1984). Alcohol exposure increased levels of plasma cholesterol, required for the synthesis of steroid hormones (Etelalahti et al, 2011). Elevated levels of hydroxylated and keto steroids, i.e. hydroxycholesterol, hydroxycortosterone, hydroxypregenanlone, hydroxytestosterone, pregnanediol, and androstenediol, in the alcoholics indicates that alcoholism direct the synthesis of hydroxylated and keto steroids. Keto and hydroxy steroids persist in the body fluids for a

10 long time, even after the alcohol withdrawal (Stig et al, 1986). Observations indicated that alcohol consumption increased the rate of reductive reactions and slow down the oxidative reactions. It may be due to presence of ready stock of NADPH produced at the cost of NAD+ during the conversion of alcohol to acetaldehyde. Eventually, this led to the synthesis of C-21 steroids rather than C-18 and C-19 steroids, as clearly indicated from the data (Figure 4). The increased levels of corticosterone have been reported to be associated with the amount of alcohol consumed by male rats ( Fahlke et al., 1995; Silveri and Spear, 2004). Excessive intake of alcohol is reported to impair memory and cognition due to elevated levels of corticosterone (Piazza et al, 1993). 11-Deoxycortisol and cortexolone are reported to synthesize cortisol (Mohamed et al., 2011). Decreased levels of 11-deoxycortisol and cortexolone and increased levels of cortisol indicated the increased conversion reaction rate in the alcoholics. A positive association between cortisol and units of alcohol intake has been reported by Badrick et al, (2007). Increased cortisol levels are related to hypertension, impairment of immune function, and alteration of metabolism (Plat et al, 1997; Whitworth et al, 2005). Our results support the observations as alcoholics were found to have high blood pressure. Increased cortisol concentration has detrimental effects on insulin and glucose function and predicted to help in the development of type 2 diabetes (Perry et al, 1995). We also found hypoglycemic condition in alcohol consumers in the morning, which may be due gluconeogenesis inhibition by alcohol as reported earlier (Sporer et al, 2007).

Testosterone and corticosterone were found to be affected in the inverse manner in moderate alcohol consumers. Healthy males have increased testosterone levels following intake of a low dose of alcohol (Sarkola and Eriksson, 2003). On the contrary, acute alcohol intoxication has been reported to decrease testosterone levels (Frias et al., 2002). Moreover, a number of studies reported to reduce the levels of peripheral testosterone in chronic alcoholics (Maneesh et al., 2006). Testosterone levels are reported to be normalized after the alcohol withdrawal (Heinz et al., 1995). However, in contradiction, Marc et al. (2007) did not find significant differences in free testosterone levels between alcohol-dependent patients and healthy controls. In this diet controlled study, we found decreased levels of testosterone and androstenedione and T: A ratio in the moderate chronic alcoholic consumers (Table 3; Figure 4), similar to most of the earlier reports. Testosterone is an and required for the growth of muscle, bone and body hair. Alcohol consumption slows down the conversion of DHEA and androstenedione to testosterone and directed it toward the formation of cortisol and corticosterone; C-21 steroids. The conversion of DHEA to androstenedione and then estrone and estradiol (C-18 steroids) to some extent also contributed in the decreased levels of testosterone. However, synthesis of other

11

C-18 steroids, i.e. , etiocholanole and was not affected in alcohol consumers. Etiocholanolone was reported to have thermogenic effects, normalize blood sugar and maintain islet integrity. Therefore, in the study, moderate alcohol consumers didn’t gain or loss their weight, which might be due to unaffected etiocholanalone levels. However, etiocholanolone levels were increased in TCJ treated healthy groups indicates TCJ increased the etiocholanolone levels, which is effective to control hyperglycemia and excessive accumulation of fat.

Moreover, testosterone itself is reported to convert into in alcoholics. This shows that alcohol lower T: A ratio by directing androstenedione, the precursor of testosterone, to be converted into estrone and testosterone into estrodiol through aromatization. Increased aromatization of testosterone and androstenedione to estrogens due to activation of aromatase has been reported by Gordon et al (1979). Increased cortisol levels and stress are reported to increase estradiol levels by increasing the expression of aromatase via corticotropin-releasing hormone and human chorionic gonadotropin (Dickens et al, 2011; Wang et al, 2014). Furthermore, levels were found to be increased with other glucuronides indicated the increased clearance rate of steroids that contributed to deplete the testosterone levels. Increased levels of estradiol in blood of alcoholics have been previously reported in several studies (Van Thiel et al. 1979; Gordon et al. 1978). Estrone and estradiol plays important role in the development of bones and sexual functioning in men and women. Increased excretion or production of estardiol glucuronide also observed in this study may be one of the factors to keep the levels of estradiol in limit due to high clearance rate. The level of urinary dehydrocholesterol glucosiduronate, a provitamin D3 derivative, was observed to be increased, which supports the fact of inverse effect on Ca2+ metabolism in alcoholics.

Treatment with TCJ normalized the levels of most of steroids affected by chronic moderate alcohol consumption (Table 2). Decreased cholesterol levels in both plasma and urine in TCJ treated alcohol consumers has indicated the regulation of the statin pathway by TCJ (Figure 4). TCJ contains several metabolites having hypolipidemic properties (Vetrivadivelan et al, 2012). Berberine is the best example, which possesses anti-hyerlipidemic activity (Weijia et al, 2004). A current study indicated that palmatine, a compound present in TCJ acts as PPARα agonist and regulates cholesterol homeostasis (Sangeetha et al, 2013). It also depletes cholesterol concentration by reducing the abundance of nuclear SREBP-2. TCJ also increased the conversion of 20-hydroxycholesterol into controlled by CYP 11A and regulated the flow of pregnenolone to , corticosterone, and cortisol. However, reports are not

12 available to support the finding and need to be explored. However, inflow of pregnenalone toward testosterone has been confirmed by elevated levels of testosterone in both TCJ treated alcoholic and healthy groups.

Treatment with TCJ also reduced oxidative burden caused by alcohol intake, clearly indicated by reduced levels of MDA and increased levels of GSH. Decreased activities of liver function enzymes, i.e. GGT, SGOT, SGPT also supported the anti-oxidant activity and reduced liver stress in TCJ treated alcohol consumers. Decreased levels of estradiol and steroidal glucuronides levels along with ethylglucuronide, a standard marker of alcoholism in the urine of TCJ treated alcohol consumers confirmed the increased synthesis of testosterone by reducing its conversion to estradiol and clearance rate. Decreased oxidative stress reduced the activity of aromatase and therefore the conversion of testosterone to estradiol and testosterone glucuronide. Further, it regulated the rate of reduction reactions by decreasing free radicals and therefore, oxidative stress caused by alcohol intake and restored the normal course of steroidal hormones. A number of in vivo and in vitro anti-oxidant compounds have been reported from TCJ, including magnoflorine, apigein magnoflorine, and syringin (Jayaprakash et al, 2015). TCJ treatment increased the T: A ratio in alcoholics as well in healthy individuals, which shows its independent property with disrespect to alcohol effects and need to be studied (Table 2). Depleted levels of glucuronides and restoration of steroid levels in the plasma and urine of alcoholics indicate that TCJ suppressed metabolism of ethanol and regulated cholesterol synthesis in the liver.

Moreover, it is suggested that the corticosterone, dehydrocholesterol, cotisol, 2-methoxyestrone 3-sulfate, dehydrocholesterol glucosiduronate, androstenediol, 20-hydroxycholesterol, pregnanediol and increased levels of glucuronides in the urine of alcoholics might be of value as biochemical markers in alcoholism and recent alcohol intake.

Conclusion:

Alcoholism is a preventable cause of mortality across the globe. Currently, the numbers of drugs to treat alcoholism induced disorders are limited and available drugs have serious side effects. Moderate alcohol consumption is supposed to be harmless and sometime beneficial. However, the study shows that moderate alcohol consumption in chronic form increase the reaction rate of reductive enzymes and therefore, directs the synthesis of C-21 steroids, including cortisol, corticosterone, and pregnanediol. It slows down the reaction rate of oxidative reactions and therefore, decreases the synthesis of C-19 steroids, i.e. testosterone. No effect was observed on the synthesis of C-18 steroids except estradiol, which was not very significant.

13

Moderate alcohol consumption also increases the glucuronidation and therefore, the clearance rate of steroids. Cumulative impacts greatly affect the testosterone levels and decrease it significantly. Therefore, a direct relationship between T: A ratio of urine and plasma was observed in this study (Figure 5). Decreased levels of cholesterol in alcohol consumers after TCJ treatment indicate the inhibition of statin pathway that regulated the cholesterol synthesis. Decreased cholesterol levels impacted the C-21 steroid synthesis. TCJ also acted as anti- oxidant, as indicated by decreased levels of MDA and increased levels of GSH and therefore, regulated the redox potential of cells. Normalization of redox potential decreased the activity of aromatase and balanced the NADPH/NADP+ ratio and testosterone synthesis. This data further depict the possibility that that TCJ inhibit alcohol metabolism along with activation of PPARα, which increase the fatty acid and cholesterol metabolism and ameliorate hyperlipidemic conditions. Overall, present study strongly illustrates that the T. cordifolia water extract may be used to alleviate the toxic effects of alcohol and to modulate testosterone and cholesterol biosynthesis. Further investigations on this strategy are mandatory.

Acknowledgement: The author acknowledges DST-FIST (Grant No. SR/LSI-534/2012(C)) and HSCST (HDCST/R&D/2015/2380) programs for their financial assistance for some of the research instruments.

References:

1. Agarwal, D. P. (2002) Cardioprotective effects of light–moderate consumption of alcohol: a review of putative mechanisms. Alcohol and alcoholism 37, 409-415. 2. Allam, R. F., Shafei, M. S., Refai, A. E., Ali, M. I., & Mohamed, S. S. (2011). 11β- Hydroxylation of cortexolone (Reichstein's compound S) to hydrocortisone using immobilized Cunninghamella elegans spores Malaysian Journal of Microbiology, 7(1), 7 13. 3. Aviv, S., Pape, M., Rea, T., Rai A., Srivastava, K., Mickey A. Latour, Hopkins, D., and Schonfeld, G. (1997) Alcohol Increases Plasma Levels of Cholesterol Diet–Induced Atherogenic Lipoproteins and Aortic Atherosclerosis in Rabbits. Arteriosclerosis, thrombosis, and vascular biology17, 1091-1097 4. Bishayi, B., Roychowdhury, S., Ghosh, S. and Sengupta, M. (2002) Hepatoprotective and immunomodulatory properties of Tinospora cordifolia in CCl4 intoxicated mature albino rats. The Journal of toxicological sciences 27, 139-146.

14

5. Boris, T., and Yanai, J. (1979) Cortexolone antagonizes development of alcohol tolerance in mice. Psychopharmacology 64, 123-124. 6. Crouse, John R., and Grundy, S. M. (1984) Effects of alcohol on plasma lipoproteins and cholesterol and triglyceride metabolism in man. Journal of lipid research 25, 486-496. 7. Dimmitt, S. B., Rakic, V., Puddey, I. B., Baker, R., Oostryck, R., Adams, M. J., Chesterman, C. N., Burke, V. and Beilin, L. J. (1998) The effects of alcohol on coagulation and fibrinolytic factors: a controlled trial. Blood coagulation & fibrinolysis 9, 39-46. 8. Etelalahti, Tiina J., Sirkku T. Saarikoski, and Eriksson, C. J. (2011) Associations of corticosterone and testosterone with alcohol drinking in F2 populations derived from AA and ANA rat lines. Alcohol 45, 421-426. 9. Fahlke, C., Hård, E., Hansen, S., Eriksson, C. J. P. and Engel, J. A. (1995) Consequence of long-term exposure to corticosterone or dexamethasone on ethanol consumption in the adrenalectomized rat, and the effect of type I and type II receptor antagonists. Psychopharmacology 117, 216-224. 10. Fernandes, A., and De Sousa, A. (2014) Alcohol Dependence and Sexual Dysfunction: a clinical review. Medical Science 3. 11. Frances, D., Christopher J. Bulpitt, Agarwal, S., Donaldson, M. and Rajkumar, C. (2003) Testosterone suppression in men with prostate cancer leads to an increase in arterial stiffness and hyperinsulinaemia. Clinical Science 104, 2: 195. 12. Frias, J., Torres, J. M., Miranda, M. T., Ruiz, E. and Ortega, E. (2002) Effects of acute alcohol intoxication on pituitary–gonadal axis hormones, pituitary–adrenal axis hormones, β-endorphin and prolactin in human adults of both sexes."Alcohol and Alcoholism 37, 169- 173. 13. Gahlaut, A., Gothwal, A., and Dabur, R. (2012) TLC Based Analysis of Allelopathic Effects on Tinosporoside Contents in Tinospora cordifolia." Journal of Chemical & Pharmaceutical Research 4. 14. Gordon, Gary G., Louis Southren, A. and Charles S. Lieber. (1978) The effects of alcoholic liver disease and alcohol ingestion on levels. Alcoholism: Clinical and Experimental Research 2, 259-263. 15. Hak, A., Jacqueline, E., Witteman, C. M., Frank H. de Jong, Mirjam I. Geerlings, Hofman, A. and Huibert AP Pols. (2002) Low levels of endogenous increase the risk of atherosclerosis in elderly men: the Rotterdam study. The Journal of Clinical Endocrinology & Metabolism 87, 3632-3639.

15

16. Hao, Z., Wei, J. Xue, R., Wu, J. D., Zhao, W., Wang, Z. Z., Wang S. K. et al. (2010) Berberine lowers blood glucose in type 2 diabetes mellitus patients through increasing insulin receptor expression. Metabolism 59, 285-292. 17. Heinz, A., Rommelspacher, H., Klaus-Jurgen Graf, Kürten, I., Otto, M. and Baumgartner, A. (1995) Hypothalamic-pituitary-gonadal axis, prolactin, and cortisol in alcoholics during withdrawal and after three weeks of abstinence: comparison with healthy control subjects. Psychiatry research 56, 81-95. 18. Imhof, A., Froehlich, M. Brenner, H., Boeing, H., Mark B. Pepys, and Koenig, W. (2001) Effect of alcohol consumption on systemic markers of inflammation. The Lancet 357, 763- 767. 19. Jayaprakash R, Ramesh V, Sridhar MP, Sasikala C. Antioxidant activity of ethanolic extract of Tinospora cordifolia on N-nitrosodiethylamine (diethylnitrosamine) induced liver cancer in male Wister albino rats. J Pharm Bioallied Sci. 2015 Apr;7(Suppl 1):S40-5. doi: 10.4103/0975-7406.155791. 20. Kenna, G.A., Swift, R. M., Hillemacher, T., Leggio, L. (2012) The relationship of appetitive, reproductive and posterior pituitary hormones to alcoholism and craving in humans. Neuropsychology review 22, 211-228. 21. Kono, H., Rusyn, I., Yin, M., Gäbele, E., Yamashina, S., Dikalova, A., Kadiiska, M. B. et al. (2000) NADPH oxidase–derived free radicals are key oxidants in alcohol-induced liver disease. Journal of Clinical Investigation 106, 867-872. 22. Litten, Raye Z., Egli, M., Heilig, M., Cui, C., Joanne B. Fertig, Megan L. Ryan, Daniel E. Falk, Moss, H., Huebner, R., and Noronha, A. (2012) Medications development to treat alcohol dependence: a vision for the next decade. Addiction biology 17, 513-527. 23. Maneesh, M., Dutta, S., Chakrabarti, A. and Vasudevan, D. M. (2006) Alcohol abuse- duration dependent decrease in plasma testosterone and antioxidants in males. Indian journal of physiology and pharmacology 50, 291-295. 24. Marc, W., Gerhard, U., Gerlach, M., Heinz-Gerd Weijers, Boening, J. and Gerhard A. Wiesbeck. (2007) Controlled study on the combined effect of alcohol and tobacco smoking on testosterone in alcohol-dependent men. Alcohol and Alcoholism 42, 19-23. 25. Mittal A, Dabur R. Detection of new human metabolic urinary markers in chronic alcoholism and their reversal by aqueous extract of Tinospora cordifolia Stem. Alc Alc 2015; 50: 271-281

16

26. Perry IJ, Wannamethee SG, Walker MK, Thomson AG, Whincup PH, Shaper AG 1995 Prospective study of risk factors for development of non-insulin dependent diabetes in middle aged British men. BMJ 310:560–564 27. Piazza, P.V., Deroche, V., Deminière, J. M., Maccari, S., Le Moal, M., Simon, H. (1993) Corticosterone in the range of stress-induced levels possesses reinforcing properties: implications for sensation-seeking behaviors. Proceedings of National Academy of Science of U. S. A., 90, 11738–11742 28. Plat L, Leproult R, L’Hermite-Baleriaux M, Fery F, Mockel J, Polonsky KS, Van Cauter E1999 Metabolic effects of short-term elevations of plasma cortisol are more pronounced in the evening than in the morning. J Clin Endocrinol Metab 84:3082–3092. 29. Sangeetha, M. K., Priya, C. D. and Hannah R. Vasanthi. (2013) Anti-diabetic property of Tinospora cordifolia and its active compound is mediated through the expression of Glut-4 in L6 myotubes. Phytomedicine 20, 246-248. 30. Sarkola, T., and Eriksson, C. J. (2003) Testosterone increases in men after a low dose of alcohol. Alcoholism: Clinical and Experimental Research 27, 682-685. 31. Sharma B, Dabur R. Protective effects of Tinospora cordifolia on hepatic and gastrointestinal toxicity induced by chronic and moderate alcoholism. Alc Alc 2015; Accepted. doi: 10.1093/alcalc/agv130. 32. Shirolkar, A., Gahlaut, A., Hooda, V. and Dabur, R. (2013) Phytochemical composition changes in untreated stem juice of Tinospora cordifolia (W) Mier during refrigerated storage. Journal of Pharmacy Research 7, 1-6. 33. Sierksma, A., Sarkola, T., Eriksson, C. J., Martijn S. Gaag, Diederick E. Grobbee, and Henk FJ Hendriks. (2004) Effect of Moderate Alcohol Consumption on Plasma Dehydroepiandrosterone Sulfate, Testosterone, and Estradiol Levels in Middle‐Aged Men and Postmenopausal Women: A Diet‐Controlled Intervention Study." Alcoholism: Clinical and Experimental Research 28, 780-785. 34. Silveri, Marisa M., and Spear P. L. (2004) Characterizing the ontogeny of ethanol- associated increases in corticosterone. Alcohol 32, 145-155. 35. Singh, S. S., S. C. Pandey, S. Srivastava, V. S. Gupta, B. Patro, and A. C. Ghosh. (2003) Chemistry and medicinal properties of Tinospora cordifolia (Guduchi). Indian journal of pharmacology 35, 83-91. 36. Sporer KA, Ernst AA, Conte R, Nick TG. The incidence of ethanol-induced hypoglycemia. Am J Emerg Med. 1992 Sep;10(5):403-5.

17

37. Srivastava, P. (2011) Tinospora cordifolia (Amrita)-A miracle herb and lifeline to many diseases. International Journal of Medicinal and Aromatic Plants 1, 57-61. 38. Stig, A., Cronholm, T. and Sjovall, J. (1986) Redox effects of ethanol on steroid metabolism. Alcoholism: Clinical and Experimental Research 10, 55S-63S. 39. Valimaki, M., Tuominen, J. A., Huhtaniemi, I., Ylikahri, R. (1990) The pulsatile secretion of gonadotropins and growth hormone, and the biological activity of luteinizing hormone in men acutely intoxicated with ethanol. Alcohol and Clinical Experimental Research 14, 928– 931. 40. Van Thiel, David H., and Lester, R. (1979a) The effect of chronic alcohol abuse on sexual function. Clinics in endocrinology and metabolism 8, 499-510. 41. Van Thiel, David, H., Loriaux, D. L. (1979) Evidence for an adrenal origin of plasma estrogens in alcoholic men. Metabolism 28(5), 536-541. 42. Vetrivadivelan, K., Venkateswaran, K. V., Selvasubramanian, S., and Sesh, P. S. L. (2012). Antihyperlipidemic effect of Tinospora cordifolia extract in streptozotocin induced diabetic Rats. International Journal of Pharmaceutical Sciences & Research 3, 5: 1423-1429. 43. Wang WS, Liu C, Li WJ, Zhu P, Li JN, Sun K. Involvement of CRH and hCG in the induction of aromatase by cortisol in human placental syncytiotrophoblasts. Placenta. 2014 Jan;35(1):30-6. doi: 10.1016/j.placenta.2013.10.018. 44. Weijia, K., Wei, J., Abidi, P., Lin, M., Inaba, S., Li, C., Wang Y. et al. (2004) Berberine is a novel cholesterol-lowering drug working through a unique mechanism distinct from statins. Nature medicine 10, 1344-1351. 45. Whitworth JA, Williamson PM, Mangos G, Kelly JJ 2005 Cardiovascular consequences of cortisol excess. Vasc Health Risk Manag 1:291–299.

18

Table 1: Blood biochemistry of different groups after a week of usual alcohol intake and treatment with TCJ. All the values are expressed in Mean+SD.

Parameters Healthy Alcoholic Alcoholic + TCJ Healthy+ TCJ [Healthy Vs Treated Treated Alcoholic] p value Participants, n 14 12 12 14 - Age (Years) Mean±SD 40.3±4.5 40.6±3.2 40.6±3.2 40.3±4.5 - Smoker, n (%) - - - - - BMI (kgm-2) 30.4±2.8 31.4±4.2 --- 30.4±2.8 - SBP (mm Hg) 120±2.9 140±4.1 122±2.4 122±1.2 <0.05 DBP (mm Hg) 80 ±3.5 77±6.0 79±3.01 82.33±1.04 >0.05 TGL (mg/dl) 119.3± 2.05 195.8±1.10 121.3±1.01 101.25±0.8 <0.05 CHL(mg/dl) 173.9± 1.12 241.5±0.86 183.1±3.0 168.64±3.10 <0.05 HDL (mg/dl) 58.2± 5.01 83.4±4.2 63.2± 1.0 74.8±2.11 <0.01 LDL (mg/dl) 172.4± 0.8 238.3± 3.05 144.1± 2.0 167.24±2.01 <0.01 γ-GT (U/L) 20.6±2.10 38.9± 2.45 22.1 ± 0.7 21.8±1.91 <0.01 SGOT(U/L) 28.6±1.6 48.3± 0.55 32.8± 1.10 27.0±0.38 <0.01 SGPT(U/L) 18.1 ±2.66 48.2± 3.00 29.3± 0.95 23.4±1.0 <0.01 MCV (fl) 83.2 ±1.2 105.7± 2.4 94.6± 1.9 86.3±4.9 <0.01 Glucose 92.3± 0.9 70.1± 2.3 98.4± 3.0 88.2±3.5 <0.05 Uric acid (mg/dL) 4.1±0.7 5.7± 0.3 4.7± 0.1 4.2± 0.1 >0.05 Plasma GSH (mmol/L) 1.5± 0.2 0.85±0.3 1.2±0.2 1.6±0.5 <0.05 Plasma MDA (mmol/L) 3.3±0.7 9.7±1.4 4.2±0.5 3.1±0.6 <0.05

19

Table 2: Table is showing significantly altered metabolites in different groups after discrimination analysis data subjected to Wilcoxon paired t-test, One way ANOVA with Bonferroni multiple testing corrections. The negative and positive sign is showing down and up regulation of metabolites. All the fold changes are as compared to the healthy group.

S. Tentatively Identified Compounds KEGG p Fold Change Fold Change Fold Change Healty Mass Retention No. ID Alcoholic Alcoholic Treated Treated with TCJ Time with TCJ 1. Dehydroepiandrosterone 3-glucuronide - 0.012141 5.212 1.233 -1.134 464.167 28.117 2. Pregnanediol C05484 1.36E-04 4.234 1 -1 320.241 33.650 3. Cortisol C00735 0.001 3.7 -1.5 1.15 363.09 25.65 4. 11-β-Hydroxyandrosterone-3-glucuronide - 5.40E-05 3.536 1 1 482.251 21.532 5. Cholesterol C00187 5.52E-06 3.525 1 -1.217 386.188 38.857 6. 20-Hydroxycholesterol C05500 3.95E-09 3.524 -1 1 402.221 31.608 7. Androstenediol C04295 4.06E-06 3.523 1.234 1 290.194 33.606 8. Provitamin D3 glucosiduronate - 7.84E-04 3.452 -1 -1 560.332 31.687 (Dehydrocholesterol glucosiduronate) 9. Etiocholanolone 3-glucuronide C11136 2.76E-06 3.235 1.232 -1 466.190 14.388 10. 11-Hydroxyprogesterone 11- - 1.98E-04 3.235 1 1 506.251 25.140 glucuronide 11. 2-Methoxyestrone 3-sulfate C08358 1.08E-04 3.071 -1.121 -2.787 380.090 17.234 12. Androsterone 3-glucuronide C11135 0.010479 2.522 -2.041 -1.235 466.130 32.302 13. Corticosterone C02140 5.52E-06 2.456 1.5145 1.547 346.220 26.895 14. Hydroxyprogestrone C01176 0.002145 2.359 1.385 1 330.228 26.807 15. Dehydrocholesterol C01164 2.31E-10 2.125 -1 -1 384.294 31.579 16. Testosterone glucoronide C11134 1.35E-06 2.089 -1.037 -1.348 464.266 30.236 17. 11β-Hydroxyisoandrosterone glucuronide - 1.52E-26 2.054 1 1 482.145 23.663 18. Estrone C00468 6.01E-04 1.574 1.281 1.054 270.105 28.352 19. β-Estradiol glucuronide C05503 0.0073 1.520 -2.013 -2.052 448.160 23.913 20. Hydroxytestosterone C05291 3.08E-11 1.445 1 1 304.090 25.650

21. β-Estradiol C00951 5.02E-06 1.370 1.122 -1 272.204 32.825 22. Androstenedione- glucuronide - 5.92E-04 1.131 -2.337 1 462.261 31.649 23. Pregenolone C01953 2.27E-06 -1 -1 3.161 316.207 32.075 24. Hydroxypregenolone C05138 6.48E-08 -1 -1 3.212 332.237 26.359

25. Dehydrocorticosterone C05490 0.009065 -1 -1 3.652 344.125 26.426 26. Androstanediol C07632 9.10E-05 -1.370 -1.190 -1 292.163 29.954 27. Cortexolone C05488 8.55E-06 -2.145 1.521 -1 346.216 26.967 28. Androstenedione C00280 2.63E-06 -2.163 -1.860 -1 286.076 29.164 29. Tetrahydrocortisone C05470 9.38E-04 -2.314 1.425 1.121 364.224 26.982 30. Dehydroepiandrosterone C01227 5.52E-06 -2.563 -1.865 -1 288.189 22.848 31. Testosterone C00535 0.051225 -3.424 1.222 1.535 288.185 31.568 32. 21-Deoxycortisol C05497 0.002701 -4.110 2.213 1.212 346.207 27.722 33. Etiocholanolone C04373 1.98E-08 - 1.524 2.121 290.226 32.926 34. Estriol-16-Glucuronide C05504 1.98E-08 - -1 -3.213 464.204 28.124

20

Table 3: LOD, LOQ, regression coefficient, quantifier ions, collision energy (CE) and quantified amounts of testosterone, androstenedione and ethylglucuronide in urine (U) and plasma (P) samples.

Hormone LOQ LOD r2 Q1 Q2 CE Healthy Healthy Alcoholics Alcoholics (ng/mL) (ng/mL) (ng/mL) treated (ng/mL) Treated with TCJ with TCJ (ng/mL) (ng/mL) Testosterone (P) 1.5 20 0.98 289.3 109.1 30 3.42±0.42 3.5±0.34 2.44±0.84 2.52±0.42 Testosterone (U) 1.5 20 0.99 289.3 109.1 30 1.78±0.12 1.89±0.14 1.23±0.15 1.68±0.24 Androstenedione (P) 1.5 30 0.98 287.2 109.1 28 1.48 ±0.22 1.41±0.13 1.53±0.15 1.12±0.12 Androstenedione (U) 1.5 30 0.98 287.2 109.1 28 0.72±0.04 0.65±0.05 0.81±0.03 0.79±0.05 Ethylglucuronide (U) 0.3 50 0.99 223.2 205.2, 25 1.12±1.51 2.32±1.44 37.77±1.65 4.92±1.84 115.1 (µg/mL (µg/mL) (µg/mL) (µg/mL)

21

Figure

Figure 1: Work flow diagram showing pre-watch, TCJ treatment, sample collection time timelines, mass profiles, discrimination analysis and quantization in the samples against control or healthy group.

Alcoholics Healthy n=12 n=14

One Week Washout Period Day -7

Alcoholics Healthy

Sample Collection Day 0 TCJ Treatment Started

Alcoholics Healthy Sample Collection Day 14

Urine Samples Urine Samples

RRLC-Q-TOF-MS

Mass Profiles

Alcoholics Alcoholics + TCJ Healthy Healthy +TCJ

Discrimination Analysis

Fold Change in Steroids Figure 2: Total ion chromatograms (A, B, C) and their mass profiles (D, E, F) of TCJ preparation from three different batches.

A A

B

C

Citramailc acid Abscisic acid Magnoflorine Columbin Diferuloylquinic acids D

E Sulfocysteine Tinocordifolioside

Resveratrol F Figure 3: (A) BoxWhiker plot of different groups after mass data normalization with 75% percentile score and Z transformations. No significant difference was observed in healthy (HLT) and healthy treated with TCJ (HLT_T), whereas alcoholics (ALC) treated with TCJ (ALC_T) showed less variations as compared to healthy (HLT). (B) PCA diagram, after parial least square discrimination analysis, showing 27.94, 11.3, and 9.61% variations along with x, y and z axis in positive mode in different groups, i.e. healthy (Brown), healthy treated with TCJ (Blue), alcoholics (Red) and alcoholics treated with TCJ (Gray).

A

HLT HLT_T ALC ALC_T

B Figure 4: Steroid metabolism affected by moderate alcohol intake. Green, red and light red boxes are showing the depleted, elevated, and insignificantly elevated and observed steroids in the urine samples of moderate alcohol consumers. Red and light red arrows indicated the increased inflow of cholesterol. Pathways blocked and activated by TCJ are also depicted in the picture.

Acetyl-CoA

HMG-CoA

TCJ Mevalonic acid

Cholesterol Deoxycorticosterone Corticosterone

20-Hydroxycholesterol Hydroxypregnenolone Hydroxyprogesterone

TCJ Pregnenolone Progesterone Pregenadiol Cortolone

TCJ

17-Hydroxypregenolone 17-Hydroxyprogesterone 11-Deoxycortisol Cortisol Cortol

Andro 4-ene 3,17-dione Adrenosterone DHEA Etiocholanolone

3,17-Dihydroxyandrost-5-ene Testosterone Androsterone Estrone Hydroxyestrone

TCJ Estradiol Estriol TCJ

Testosterone-Glu 5α- 5β-Dihydrotestosterone TCJ

Androstan-3, 17-diol Estradiol-Glu