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Indian Journal of Gastroenterology https://doi.org/10.1007/s12664-017-0815-8

ORIGINAL ARTICLE

Clinical outcomes, histopathological patterns, and chemical analysis of Ayurveda and herbal medicine associated with severe liver injury—A single-center experience from southern India

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Cyriac Abby Philips 1 & Rajaguru Paramaguru 2 & Adarsh K. Joy 3 & K. L. Antony 4 & Philip Augustine 5 Received: 21 June 2017 / Accepted: 25 December 2017 # Indian Society of Gastroenterology 2018

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Abstract Introduction Ayurvedic and herbal medicines (AHM) are known to cause varying degrees of drug-induced liver injury (DILI). Clinical, biochemical, histological spectrum and outcomes of AHM linked to severe DILI are not well studied. Methods Out of 1440 liver disease patients, 94 were found to have a severe liver injury and associated AHM intake. Thirty-three patients were suspected to have AHM-DILI on Roussel Uclaf Causality Assessment Scoring Method. Forty-seven and 30 of retrieved AHM samples were analyzed for heavy metals and hepatotoxic volatile organic compounds (hVOCs), respectively. Eleven patients ingested AHM from unregistered traditional healers (UTH). Clinicopathological outcomes were analyzed in 27 patients (who underwent liver biopsy) and outcomes with respect to chemical analyses were studied in 33 patients. Results Males predominated (70.4%) with mean age 46.9±15.8 years. Mean follow up was 119.2±81.4 days. The median duration of drug intake was 28 days (10 – 84). Five patients died (18.5%). Hepatic encephalopathy, hypoalbuminemia, and hepatic necrosis were significantly associated with mortality (p < 0.005). Arsenic and mercury ingestion was significantly associated with death (p < 0.005). hVOCs were detected in more than 70% of samples. AHM intake from UTH was associated with higher mortality. Conclusion Adequate regulation and scrutiny regarding AHM use among the general population is an unmet need. Early liver biopsy after clinical identification of at-risk patients can expedite definitive treatment with a liver transplant.

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Keywords Ayurveda . Drug-induced liver injury . Fibrosis . Heavy metals . Hepatotoxicity . Herbal medicines . Histopathology . Liver biopsy . Liver injury . Liver necrosis . Volatile organic compounds

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Parts of this study results were presented as Parallel Oral and Presidential Plenary at the American Association for the Study of Liver Diseases annual conference, The Liver Meeting, held at Washington D.C. on 22nd and 24th October 2017, respectively. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12664-017-0815-8) contains supplementary material, which is available to authorized users. * Cyriac Abby Philips [email protected] 1

Department of Hepatology and Liver Transplant Medicine, PVS Memorial Hospital, Kochi 682 025, India

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Department of Pathology, PVS Memorial Hospital, Kochi 682 025, India

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Sophisticated Test and Instrumentation Centre, Cochin University of Science and Technology, Kochi 682 022, India

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Envirodesigns Eco Labs, Kochi 682 025, India

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Department of Gastroenterology, PVS Memorial Hospital, Kochi 682 025, India

Indian J Gastroenterol

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Drug-induced liver injury (DILI) is challenging to the clinician and the pathologist as there is no gold standard for diagnosis. Liver histology in DILI patients is not part of current causalityassessment algorithms. In clinical practice, a liver biopsy could help in identification of other causes of liver injury by careful assessment of clinical, biochemical, and histological patterns and may help define the severity of the injury and prognosis. The classic histopathological patterns of DILI associated with prescription drugs are well described [1]. Ayurvedic and herbal medications (AHM) are known to cause varying degrees of liver injury that range from asymptomatic liver function test abnormalities to acute liver failure requiring liver transplantation. Due to the variety of herbal components and complexity in preparations of AHM, it is difficult to pinpoint a compound related to hepatotoxicity [2, 3]. Large DILI series, including one from India, shed light on incidence, clinical, and investigational aspects and final outcomes associated with AHM but clinical outcomes in relation to histopathological patterns of liver injury due to AHM are not well studied [4, 5]. Hepatotoxicity and heavy metal analysis of AHM have been shown in studies ranging from case reports to large patient series [6]. In the current study, we describe clinical and investigational parameters and discuss histopathological findings and chemical analysis in relation to outcomes in AHMrelated severe liver injury.

Methodology, chemical standards, reagents, and vials were acquired as per standards set by the United States Environmental Protection Agency (USEPA), method 5021A, 8015, 8021, and 8260. Briefly, 1 g of the given sample was digested with nitric acid and perchloric acid, made up to 50 mL, and filtered. The filtrate was aspirated into the plasma chamber and the corresponding emission line of the characteristic wavelength by each element was recorded. The intensity of the emission line corresponded to the quantity of the elements introduced. Values were expressed in milligrams per kilogram. Hepatotoxic volatile organic compounds’ (hVOC) qualitative and quantitative analyses were performed in 30 samples (22 solid, 8 liquid formulations) using gas chromatography coupled to tandem mass spectrometry method (GC/ MS-MS; Varian Saturn 2200; Agilent Technologies, USA). Head space analysis and purge and trap methods were utilized for solid and liquid samples, respectively. Briefly, approximately 1 g of solid sample was taken in a vial containing 5 mL of matrix modifying solution that was mechanically shaken for at least 2 min before placing the vial in head space analyzer. The sample was then heated to 1000 °C for 25 min and 1 μL of the prepared solution mixture was injected into the GC and identification and quantification done by dual MS. For liquid samples, methodology, chemical standards, reagents, and vials were acquired as per standards set by the USEPA method 5030B coupled to GC/MS for separation and analysis. Briefly, an inert gas (nitrogen) was bubbled through 5 mL of the aqueous sample at ambient temperature and the volatile compounds were efficiently transferred from the aqueous phase under mount temperature 90 °C, purge ready temperature 45 °C, and flow rate 10 mL/min for 0.25 min. The sample was allowed to enter into the vapor phase after a purge time of 11 min at flow rate 40 mL/min. Post-purge and after 1 min, the sorbent column was heated and back flushed with inert gas to allow desorption of the vaporized sample at 250 °C at 200 mL/min and the vaporized sample injected at 180 °C within 1 min on to the GC for separation and subsequent identification and quantification were done by dual MS. Detailed methodology for heavy metal and hVOC analysis is shown in Supplementary Table 1. We also analyzed qualitative heavy metals and hVOC detection in ten samples of prescription drugs used commonly in hepatology practice (ursodeoxycholic acid, N-acetyl cysteine, S-adenosyl methionine, vitamin E and vitamin C tablet formulations, multivitamin B complex syrups, and injectable glutathione) as a control group. Outcomes with respect to chemical analyses were studied in the 33 patients diagnosed with AHM-associated DILI by Roussel Uclaf Causality Assessment Method (RUCAM). Twenty-seven patients underwent percutaneous or trans-jugular liver biopsy depending on presence or absence of ascites/coagulation failure after informed consent. Clinical, biochemical, and histopathological analysis related to outcomes was performed in 27 patients

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Introduction

Methods

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Out of 1440 liver disease patients (September 2016 to March 2017) seen in the outpatient and emergency departments, 94 patients (6.5%) with severe liver injury (defined as aspartate aminotransferase [AST] and/or alanine aminotransferase [ALT] > 5 times upper limit of normal and total bilirubin > 5 mg/dL and/or presence of hepatic encephalopathy) had ingested AHM. We excluded patients who were taking other medications such as analgesics and antimicrobials and those who were on antiepileptic drugs in the preceding 3 months. Type of liver injury was determined using R ratio [7]. Sixty-one patients (23—significant alcohol consumption; 18—acute viral hepatitis A/E; 9—chronic hepatitis B infection; 6—classical autoimmune hepatitis; 5—underlying cirrhosis) were excluded and 33 patients (2.3%) were found to have possible or probable (including tests for hepatitis C and B virus RNA and DNA, respectively) AHM-induced liver injury on Roussel Uclaf Causality Assessment Scoring Method (RUCAM) [8]. Forty-seven AHM samples were retrieved from these 33 patients. Heavy metal concentration was determined in all 47 samples by inductively coupled plasma atomic emission spectrometer (ICP-AES; Thermo Electron, IRIS Intrepid II XSP Duo, Munich, Germany).

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Biochemical characteristics and outcomes

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The baseline investigational parameters of the patients in the study are shown in Table 1. Baseline total leukocyte count, total serum bilirubin, albumin, and international normalized ratio were found significantly associated with mortality (p < 0.05). On multivariate analysis, only serum albumin was significantly associated with mortality (p = 0.004; cutoff ≤ 2.9 mg/dL; sensitivity 83.3%, specificity 90.5%, area under the curve 0.833 [95% CI 0.641– 0.948]). Autoantibodies were found in 29.6% (n = 8) of patients in the study. Anti-nuclear antibodies (ANA) were seen in 25.9% (n = 7), anti-liver-kidney-microsomal-1 (LKM-1) antibody in 7.4% (n = 2), anti-mitochondrial antibody (AMA) in 3.7% (n = 1), and anti-smooth muscle antibody (ASMA) in none. The presence of high concentrations of serum immunoglobulin G and autoantibodies did not affect mortality.

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Results

joint pains in 22.2% (n = 6), abdominal pain in 7.4% (n = 2), and hypersensitivity rash in none. Hepatomegaly was noted in 88.9% (n = 24) and splenomegaly 40.7% (n = 11). Eight patients (29.6%) had hepatic encephalopathy (HE) at admission while ascites was seen in 37% (n = 10). Only the presence of HE at admission was associated with higher mortality (p = 0.04; relative risk, RR 2.53 [1.02–6.21]).

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with AHM-induced liver injury. We have adhered to the minimal elements for reporting DILI as described by Agarwal et al. [9]. Chi-square and Fisher’s exact tests were used to compare nominal variables. Mann-Whitney’s U test was used to evaluate continuous variables. In view of small sample size, exact logistic regression was applied to identify independent parameters associated with mortality using the Cox-Snell R2 method. The probability of patients surviving up to the study end point was calculated using the Kaplan-Meier method and graphically represented by the survival time curve. Plotting the cumulative proportion surviving against the survival times gave the stepped survival curve. Patients’ survival times were plotted on the x-axis, and the probability of survival calculated according to the Kaplan-Meier method was plotted on the yaxis. Comparison of survival curves was done using the logrank test and p-value < 0.05 was considered significant. If less than 50% of the observations were uncensored and the largest observation was censored, the survival time (in days) was described in mean within text, limited to a given time (t = 180 days, end of study observation).

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Clinical, biochemical, and histopathology-related outcomes (n = 27)

Histopathology and outcomes (Supplementary Table 2)

Patient characteristics

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Males predominated (70.4%, n = 19) with mean age 46.9 ± 15.8 years. The median duration of drug intake was 28 days (range 10–84) and time to onset of liver injury from the start of AHM was 51 days (14–112). Fifty-two percent (n = 14) had single, and 11.1% (n = 3) had multiple comorbidities (diabetes mellitus, hypertension, hypothyroidism, obesity, dyslipidemia, and chronic obstructive lung disease). The most frequent indication for using AHM was dyspepsia and nonspecific abdominal complaints (29.6%, n = 8) followed by appetite enhancement (22%, n = 6). Hepatocellular, cholestatic, and mixed patterns of liver injury were seen in 55.6%, 14.8%, and 29.6%, respectively. As per RUCAM scoring, possible, probable, and definite AHM-related liver injury was seen in 59.2% (n = 16), 40.7% (n = 11), and none, respectively. Patients were followed up for mean period of 119.2 ± 81.4 days. Five patients died (18.5%) including one who underwent liver transplantation (at 168 days). Age, sex, duration of drug intake, time to onset of liver injury, associated comorbidities, and type of liver injury were not significantly associated with mortality. Clinical characteristics and outcomes Fatigue, anorexia, and jaundice were seen in 95.8% (n = 26) patients, pruritus in 44.4% (n = 12), fever in 29.6% (n = 8),

Chronic hepatitis was the commonest type of inflammation seen in 81.5% (n = 22). Lobular inflammation was observed in 85.2% (n = 23) patients and portal inflammation was seen in 88.9% (n = 24). Interface hepatitis was seen in 70.4% (n = 19). Mixed cellular inflammation was the predominant activity 59.2% (n = 16) in AHM-related liver injury. Necrosis was observed in 55.6% (n = 15) and chiefly of bridging type (40.1%, n = 11). Four patients (14.8%) had pan acinar submassive and massive type of necrosis. Fibrosis was seen in 77.8% (n = 21) patients, predominantly of the bridging type (40.7%, n = 11) (Fig. 1). Cholestasis was seen in 66.7% (n = 18) mostly mixed type (51.8%, n = 14). The most common associated finding was ballooning of hepatocytes (18.5%, n = 5) and Mallory hyaline in 14.8%. Iron deposition (siderosis) was seen in 18.5% of patients with AHM-related liver injury while steatosis was seen in 44.4% (n = 12, mixed type 25.9%). Bile duct injury was seen in 37% of patients with AHMinduced liver injury. Presence of necrosis (p = 0.02, RR 1.5 [1.04–2.14]; Fig. 2a) and steatosis (p = 0.02, RR 0.67 [0.46– 0.95]) on liver biopsy correlated significantly with outcomes with patients in the latter group showing better survival. Patients with pan acinar sub-massive and massive necrosis had significantly higher mortality (mean survival 77 vs.

Indian J Gastroenterol

Parameters (n = 27)

Values

p-value^

95% CI^

Age (years)

50 (5–70)

0.07



Hemoglobin (g/L)* Total leukocyte count (/L) Platelet count (× 106/L)*

12.6 ± 2.7 8400 (2200–14,400) 2.1 ± 1.1

0.302 0.006 0.499

– 5.12–8.71

Total bilirubin (mg/dL)

8.2 (2.8–53.5)

0.025

4.21–10.92

Aspartate aminotransferase (IU/L)

367.5 (40–1894)

0.413



Alanine aminotransferase (IU/L)

297 (23–1447)

0.393



Alkaline phosphatase (IU/L) Gamma-glutamyl transpeptidase (IU/L)

128 (34–431) 116 (20–688)

0.644 0.675

– –

Albumin (g/dL)*

3.4 ± 0.6

0.002

3.20–4.02

International normalized ratio Blood urea (mg/dL)

1.4 (1.05–5.91) 17.5 (10–44)

0.006 0.455

1.13–1.53 –

Creatinine (mg/dL)

0.7 (0.4–2.6)

0.432



Sodium (meq/L)*

131 ± 7.7

0.213



Immunoglobulin G (mg/dL)

1018.5 (628–3428)

0.722



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Table 1 Baseline features of the patients with Ayurveda- and herbal medicine-related severe injury

*Values are represented in mean ± SD; others are in median (minimum–maximum) ^

Italicized significance on univariate analysis

histopathology had better outcomes in comparison to other types of liver injury (p = 0.03; mean survival, 180 vs. 133.6 days, respectively, Fig. 2c).

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171.8 days; p = 0.001, hazard ratio HR 10.9 [95% CI 0.74– 146.36]; Fig. 2b) than those without, with focal, or multifocal necrosis. Patients with a predominantly steatotic type of

Fig. 1 Common patterns of liver injury seen in patients with Ayurveda and herbal medication consumption. a Massive hepatic necrosis (in a patient who took unknown herbal medication from a traditional healer; hematoxylin & eosin stain × 10). b Sub-massive hepatic necrosis (in a patient who had ingested multiple Ayurveda products for joint pains; hematoxylin & eosin stain × 20). c Bridging necrosis (in a patient

exposed to herbal appetite enhancers; hematoxylin & eosin stain × 20). d Severe mixed steatosis in a patient who took Ayurvedic Blehyam^ for weight gain (hematoxylin & eosin stain × 10). e Mixed cellular inflammation with a few eosinophils (hematoxylin & eosin stain × 40). f Bridging fibrosis (Masson’s trichrome stain, × 20)

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Fig. 2 a Kaplan-Meir graph showing that patients without necrosis had a better survival than those with necrosis. b Patients with sub-massive or massive necrosis had higher mortality than those with no or another type of necrosis. c Patients with steatosis as the predominant type of injury had a better prognosis in comparison to other types of injury

Indian J Gastroenterol

Drug

Company

Yog Sallaki

Legend Pharma

Bonsil

Ballan Pharma Ayurchem

Swarna Guggulu Bitana Forte

Dabur HV Pharma

Bacfo Joint Care Ikshwadi Sankara Bati

Bacfo Pharma Balakishna Pharma Vyas Pharma

Unknown traditional healer Everest Co Oushadi

Pankaja Kasturi Charak Ayamodakam Raja Health Faiz Unani Kottakal Reckeweg Reckeweg Nagarjuna Pharma Kerala Ayurveda Athreya

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Orthoherb Livomyn Ayamodaka Sathaal Kaisoragulu Vatika Najati Powder Vilwadi Gulika Testes Siccati Oleum Jeconis Arethaki Lehyam* Drakshati Kath* G H Evandam*

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Hriden Plus Vasaguluchyadi Prameoushadi

Gasna Syrup* Noni* Rasna Sallaki* Ginseng* Unknown^

Most patients were on multiple medications *Liquid formulations ^

Discussion

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Ostina Forte

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Table 2 Ayurveda and herbal medications retrieved from patients who developed severe liver injury

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Among 33 solid samples retrieved from 33 patients (Table 2), lead (Pb) was detected in 72.7% (n = 24, range 7317.2 mg/kg [minimum to maximum 0.25 to 7318.1]); mercury (Hg) in 63.6% (n = 21, 751.3 [0.2–751.5]), arsenic (As) in 57.6% (n = 19, 110.6 [0.4–111]); cadmium (Cd) was seen in 36.4% (n = 12, 1.29 [0.30–1.32]); and antimony (Sb) in 9% (n = 3, 6.9 [0.46– 37.4]). Detection of heavy metals in 14 liquid formulations (Table 2) were less frequent and in lower concentrations—as in 7.1% (n = 1, range 0.1 mg/L, minimum–maximum [0.01– 0.11]); Cd in 28.5% (n = 4, 3 [0.1–3.1]); Hg in 35.7% (n = 5, 3 [0.1–3.1]); and Pb in 35.7% (n = 5, 0.54 [0.02–0.56]). Among solids, the highest arsenic content was found in (product name concentration [mg/kg] - company name) Swarna Guggullu - 111 - Dabur Pharma, followed by Bitana Forte - 77.6 - HV Pharma

and Hriden Plus - 54 - unregistered traditional healers (UTH). The highest mercury content was seen in Hriden Plus - 751.5 UTH, Swarna Guggullu - 128.4 - Dabur Pharma, Sankara Bati 83.7 - Vyas Pharma; the highest Pb in Sanakara Bati - 7318.1 Vyas Pharma, Hriden Plus - 229.8 – UTH, and KaisoraguluVatika - 95.9 - Raja Health. Arsenic and mercury levels were significantly associated with mortality (n = 33, p = 0.001; risk ratio, RR 1.56 [0.50–4.86] and p = 0.017; RR 2.188 [0.72– 6.62], respectively). Complete heavy metal analysis of all retrieved AHM samples and highly represented levels of important quantification is shown in Supplementary Table 3A and 3B. Regarding hVOCs, pentanes were detected in 46.6% of AHM, cyclopentane in 30%, cyclobutane in 23.3%, dimethylamine in 16.6%, and heptane, ethylamine, cyclobutylamine, phenol, and propanol in 3.3%. On quantification, the highest levels detected were that of cyclobutane (115.9 ppb) and methylamine (24.78 ppb), both from traditional healer preparations. The detailed qualitative and quantitative hVOC analyses and associated samples are shown in Supplementary Table 3C. No heavy metals or hVOCs were detected from any of the prescription drugs that were used as controls. Consumption of AHM from UTH was significantly associated with mortality (n = 33, p = 0.009; hazard ratio 5.1 [1.2–22.1]) (Fig. 3).

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Chemical analysis of Ayurveda and herbal medications and outcomes (n = 33)

Both solid and liquid formulations

Das Classic Apollo DC Pharma Reckeweg Traditional healers

We describe the clinical, biochemical, and histopathological patterns of AHM-induced severe liver injury and shed light on chemical analyses and parameters related to outcomes. Based on available data of DILI cohorts from the West and Southeast Asia, Ayurveda and herbal products are implicated as a cause of hepatotoxicity in 2% to 11% of patients with only one study from India, showing a prevalence of 1.3% which was comparable to our cohort. Females predominated in most of the DILI Western studies, in contrast to males seen in our AHM group [5, 8]. Like other studies, the most common type of liver injury in our cohort was hepatocellular; prodrome was seen > 90% of patients, but none showed features of eosinophilia or hypersensitivity. Studies on DILI including a recent Swedish study have shown that female sex, hepatocellular type of injury at onset, and high serum bilirubin predicted mortality [4, 10]. In a study by Ou and colleagues, higher albumin was accompanied by a decrease in mortality in DILI patients [11]. In our AHM cohort, only HE and hypoalbuminemia at admission were independently associated with mortality. Björnsson et al., in their series, reported 9.2% patients with d r u g - i n d u ce d a u t oi m m u ne h ep a t i t i s ( A I H ) [ 12 ] . Autoantibodies were seen in 29.6% patients in our study. Autoantibodies are seen more frequently with drug-induced AIH in contrast to classical AIH [13]. Our findings suggest that autoimmune markers may be associated with

Indian J Gastroenterol

presence of necrosis was associated with a poor prognosis and the panacinar sub-massive, and massive sub-types were associated with 100% mortality in comparison to other types. In previous studies, patients with histologic evidence of a hypersensitivity type reaction with eosinophils and granulomas had a milder clinical course and better recovery [18]. We did not find eosinophilic infiltrates nor granulomatous changes on liver histology in our cohort of patients. Saper et al. demonstrated toxic levels of lead, mercury, and arsenic in the USA- and Indian-manufactured AHM sold via the Internet [19] and severe liver injury with herbals and Ayurveda are well described [20]. None have analyzed heavy metals and hVOC components in the suspected offending drug. In our study, we demonstrate toxic levels of heavy metals and presence of hVOCs in AHM that are approved for sale by regulatory bodies in India and in those prescribed by UTH. Most heavy metals were 10 to 1000 times higher than acceptable regulatory levels [21]. It was recently demonstrated that liver injury during experimental fatty liver disease was enhanced by concomitant arsenic exposure in mice suggesting that the relative risk of hepatic damage caused by arsenic exposure could be dependent on underlying liver condition. Case studies and reports involving fulminant hepatic failure or hepatitis following mercury exposure have been reported. Methylated mercury compounds have been shown to cause liver injury. A recent mouse study of inhaled lead acetate documented centrilobular hepatic steatosis, hepatocyte proliferation, apoptosis, inflammatory infiltrate, and fibrosis [22]. Acute hepatitis, chronic hepatitis, steatosis, and portal fibrosis have been associated with volatile organic and inorganic compounds including halogenated and aromatic hydrocarbons like those detected in our study. Volatile organic compounds have been associated with necrosis and severe steatohepatitis [22, 23]. A study from Brazil showed that 20

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complementary medications like those described with prescription drugs. Sebode et al. stated that unrecognized and continuous herbal intake could lead to chronic hepatitis and liver damage and that such chronic DILI could mimic AIH which is probably more frequently seen with herbal use than classical DILI which could have been the case with our study cohort [14]. In the Drug-Induced Liver Injury Network (DILIN) study, five patterns—acute hepatitis, chronic hepatitis, acute or chronic cholestasis, and cholestatic hepatitis, were the predominantly described histological entities [4]. In our patients, we found that chronic hepatitis was the predominant type of inflammation. Inflammation site and type were not associated with mortality. Fibrosis noted in our series could be part of DILI or an underlying chronic liver disease. In our study, common chronic liver diseases were ruled out explicitly. However, the presence of metabolic syndrome in more than half of our patients could have led to high frequency of fibrosis on histology. Fibrosis associated with chronic hepatitis like features due to DILI has been described by Kleiner and colleagues [15] in their DILIN study. Non-alcoholic fatty liver disease (NAFLD) in patients with DILI was associated with higher mortality in the study by Chalasani and colleagues. We did not find a significant association between mortality and metabolic syndrome or fibrosis on liver biopsy in patients with AHM-DILI [16]. Several histological features are known to be associated with outcomes in DILI in general. The degree of necrosis, fibrosis, microvesicular steatosis, cholangiolar cholestasis, and the presence of ductular reaction portends increased in risk of liver failure and death [15, 17]. In the current study, a dominant steatotic type of injury (mixed macro- and microvesicular) was associated with better survival. Associated findings of ballooning and Mallory hyaline in our patients are not a sine qua non for NAFLD and can also be seen with DILI [17]. The

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Fig. 3 Kaplan-Meir graph showing that patients who ingested Ayurveda and herbal medications prescribed by unapproved/unregistered traditional healers had significantly higher mortality than those who ingested drug from approved alternative medicine practitioners

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Compliance with ethical standards Conflict of interest CAP, RP, AKJ, KLA, and PA declare that they have no conflict of interests.

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Ethics statement The authors declare that the study was performed in a manner to conform to the Helsinki Declaration of 1975, as revised in 2000 and 2008, concerning human and animal rights. The protocol was approved by the Institutional Ethics Committee and informed consent was obtained from the study subjects. The authors are responsible for the findings and the content of the paper.

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non-obese, non-drinking petrochemical workers with abnormal liver enzymes had hepatic steatosis on biopsy following exposure to 18 industrial volatile organic compounds (VOCs) including benzene, toluene, xylene, styrene, and vinyl chloride, and in a study conducted at a petrochemical plant in Argentina, 27 of 92 workers exposed to VOCs had elevated transaminase levels [24, 25]. A follow up study on an Austrian cohort of chemical workers involved in herbicide production suggested that high polychlorinated dibenzo compound exposures at a younger age led to chronic liver disease [26]. Halogenated biphenyls, organochlorines, and alkanes are known to cause jaundice, hepatomegaly, portal inflammation, fibrosis, glycogenated nuclei, and lipofuscin accumulation in prior studies. Nitro aliphatic compounds, such as nitrosopiperazine derivatives detected in our study, can cause severe hepatic injury in humans including fatty change, centrilobular necrosis, bile duct proliferation, cholestasis, fulminant hepatic failure, and death [22]. Even though data is lacking on VOC aspect in DILI, we believe that heavy metals, along with hVOCs, and complex drug-drug interactions at the organ level could have potentiated severe liver injury along with unknown metabolites from herbal components [27]. Our study has many strengths as well as limitations. Previous studies on AHM-related liver injury—mostly on clinical and biochemical patterns—were a conglomeration of case reports and small series analyzed from specific regions and presented as a review, in contrast to the current study. Ours was a single-center study and beckons future large multicenter studies to confirm findings and associations. The sample size is small, but arguably significant when extrapolated to the intervention performed and rarity of disease in the current discussion. We did not analyze specific components of AHM that could have led to the liver injury. We also did not perform blood levels of heavy metals in view of multiple AHM intake, differences in time to referral/ admission to our facility, and ongoing treatment in many patients. Pharmaco-pathological or pharmaco-investigational outcomes were difficult to ascertain because of heterogeneity in AHM content and use in patients. With multiple AHM use, each with multiple herbal and non-herbal components, categorization of all organic and inorganic compounds and identifying specific injury patterns based on components within each sample were not feasible at a practical level. Patients with severe liver injury due to Ayurvedic herbal medications have high mortality in the presence of HE, hypoalbuminemia, and sub-massive/massive necrosis at baseline. Early clinical recognition and histopathology stratification of at-risk patients with AHM-related liver injury can help expedite definitive treatment with a liver transplant or salvage therapies. Appropriate regulations in AHM use within India and overseas are an unmet need. Unregistered traditional AHM practitioners need supervision by government and medical authorities.

8.

9.

10. 11.

12.

13. 14.

15.

16.

17.

Indian J Gastroenterol Björnsson E, Kalaitzakis E, Olsson R. The impact of eosinophilia and hepatic necrosis on prognosis in patients with drug-induced liver injury. Aliment Pharmacol Ther. 2007;25:1411–21. 19. Saper RB, Phillips RS, Sehgal A, et al. Lead, mercury, and arsenic in US- and Indian-manufactured ayurvedic medicines sold via the internet. JAMA. 2008;300:915–23. 20. Navarro VJ, Barnhart H, Bonkovsky HL, et al. Liver injury from herbals and dietary supplements in the US drug induced liver injury network. Hepatology. 2014;60:1399–408. 21. Sharma B, Singh S, Siddiqi NJ. Biomedical implications of heavy metals induced imbalances in redox systems. Biomed Res Int. 2014;2014:640754. 22. Wahlang B, Beier JI, Clair HB, et al. Toxicant-associated steatohepatitis. Toxicol Pathol. 2013;41:343–60.

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Tolman KG, Sirrine RW. Occupational hepatotoxicity. Clin Liver Dis. 1998;2:563–89. Cotrim HP, Andrade ZA, Parana R, Portugal M, Lyra LG, Freitas LA. Nonalcoholic steatohepatitis: a toxic liver disease in industrial workers. Liver. 1999;19:299–304. Perez CA, Bosia JD, Cantore MS, et al. Liver damage in workers exposed to hydrocarbons. Gastroenterol Hepatol. 2006;29:334– 7. Neuberger M, Rappe C, Bergek S, et al. Persistent health effects of dioxin contamination in herbicide production. Environ Res. 1999;81:206–14. Malaguarnera G, Cataudella E, Giordano M, Nunnari G, Chisari G, Malaguarnera M. Toxic hepatitis in occupational exposure to solvents. World J Gastroenterol. 2012;18:2756–66.

FO

R

A

P P

R

O V

A L

18.

Supplementary Table 1: Histopathological findings on liver biopsy in 24 patients with liver injury due to Ayurveda and herbal medications Patterns of liver injury * Inflammation

Lobular

Portal

Mixed

Interface

Neutrophilic

Lymphocytic

Plasmacytic

Lympho-

(site) Inflammation (type)

Mixed

plasmacytic

Type of

Acute hepatitis

Chronic hepatitis

inflammation Necrosis

Focal

Fibrosis

Bridging

Periportal and portal

Associated

Steatosis

Binucleation/

findings

(Micro/Macro/Mixed)

multinucleation

Ballooning and

Rossettes

Mallory hyaline

Multifocal

Panacinar

Panacinar

submassive

massive

Perisinusoidal

Bridging

Ballooning

Iron pigments

Eosinophils

Cholestasis (intrahepatic/ intraductal)

*All the identified findings of AHM related liver injury on histology are shown in the table. The most common patterns of injury in the study are shown as ‘shaded’ boxes; acute hepatitis - lobular predominant inflammation and apoptosis with lobular disarray ± necrosis; chronic hepatitis - portal predominant inflammation; chronic active hepatitis – portal and lobular inflammation.

Supplementary Table 3A: Complete heavy metal quantification of solid samples of Ayurveda and herbal medications causing severe liver injury. SS

Company

As

Cd

Hg

Pb

Sb

Drug

1

Legend Pharma

26.1

BDL

2.2

2.57

BDL

Yog Sallaki

2

Ballan Pharma

BDL

0.97

BDL

17.74

BDL

Bonsil

3

Ayurchem

0.4

BDL

1.7

2.25

BDL

Ostina Forte

4

Dabur

111

BDL

128.4

7.55

BDL

Swarna Guggulu

5

HV Pharma

77.6

BDL

50.8

7.65

BDL

Bitana Forte

6

Bacfo Pharma

BDL

0.03

4.3

BDL

BDL

Bacfo Joint Care

7

Balakishna Pharma

BDL

0.04

3.9

BDL

BDL

Ikshwadi

8

Vyas Pharma

9

0.08

83.7

7318.06

37.39

Sankara Bati

9

Unknown

3.49

1.32

3.9

35.31

BDL

Unknown

10

Unknown

0.63

BDL

0.35

3.39

BDL

Unknown

11

Unknown

54

0.16

751.5

229.79

BDL

Hriden Plus

12

Unknown

1.4

0.22

2.6

18.59

0.46

Unknown

13

Home remedy

1.4

BDL

0.9

BDL

0.46

Home Remedy

14

Unknown

1.9

BDL

6

22.66

BDL

Unknown

15

Unknown

BDL

BDL

0.8

4.1

BDL

Unknown

16

Everest Co

1

BDL

BDL

0.27

BDL

Vasaguluchyadi

17

Everest Co

2.2

0.06

1.5

2

BDL

Unknown

18

Oushadi

1.2

BDL

BDL

BDL

BDL

Prameoushadi

19

Pankaja Kasturi

2

0.22

0.4

6.56

BDL

Orthoherb

20

Unknown

BDL

0.22

1.8

6.53

BDL

Unknown

21

Charak

BDL

BDL

1.3

2.17

BDL

Livomyn

22

Home remedy

BDL

BDL

BDL

0.82

BDL

Home remedy

23

Unknown

BDL

BDL

BDL

10.25

BDL

Unknown

24

Unknown

3.1

BDL

4.4

0.67

BDL

Unknown

25

Unknown

BDL

BDL

BDL

6.83

BDL

Unknown

26

Ayamodakam

BDL

BDL

0.2

BDL

BDL

Ayamodaka Sathaal

27

Raja Health

1.2

BDL

BDL

95.95

BDL

Kaisoragulu Vatika

28

Faiz Unani

BDL

BDL

BDL

0.25

BDL

Najati Powder

29

Kottakal

0.68

0.11

2.55

BDL

BDL

Vilwadi Gulika

30

Home remedy

BDL

0.2

BDL

BDL

BDL

Home remedy

31

Reckeweg

0.85

BDL

BDL

1.44

BDL

Unknown

32

Reckeweg

BDL

BDL

BDL

BDL

BDL

Testes Siccati

33

Reckeweg

BDL

BDL

BDL

BDL

BDL

Oleum Jeconis

*BDL – below detection range; As – Arsenic; Pb – Lead; Hg – Mercury. Cd – Cadmium; Sb – Antimony; Unknown – traditional healers

Supplementary Table 3B: Concise representation of heavy metals in samples of Ayurveda and herbal medications causing severe liver injury. Heavy metals

Quantification (mg/kg)

Solid samples (n=33)

n

% of samples

Range

Mean

Standard error of mean

Min

Max

Limit of detection

Arsenic

19

57.6

110.6

15.74

4.27

0.4

111

0.09

Mercury

21

63.6

751.3

47.87

22.9

0.2

751.5

0.009

Lead

24

72.7

7317.2

325.14

221.4

0.25

7318.1

0.02

Cadmium

12

36.4

1.29

0.31

0.05

0.3

1.32

0.009

Antimony

3

9

6.9

12.77

1.13

0.46

37.4

0.04

Liquid samples (n=14)

Quantification (mg/kg)

Arsenic

1

7.1

0.28

0.37

0.02

0.09

0.37

0.09

Mercury

6

42.8

3

1.12

0.24

0.1

3.1

0.009

Lead

7

50

0.47

0.23

0.04

0.09

0.56

0.02

Cadmium

4

28.5

0.1

0.05

0.008

0.1

0.11

0.009

Supplementary Table 2C: Qualitative and quantitative analysis of hepatotoxic volatile organic compounds detected from Ayurveda and herbal medications causing severe liver injury.

Compounds Detected

Detection in percentage of samples

Highest quantification (ppb)

Drug implicated

Manufacturer

Methyl amine

16.6

24.78

Unknown

Traditional healer

Ethyl amine

3.3

0.64

Unknown

Traditional healer

Pentane

46.6

9.33

Unknown

Traditional healer

Cyclopentane

30

0.42

Drakshati Kath

Kerala Ayurveda

Heptane

3.3

0.24

Ayamodhaka Sathaal

Ayamodhakam

Cyclobutane

23.3

115.9

Unknown

Traditional healer

Propanol

3.3

3.42

Unknown

Traditional healer

Phenol

3.3

0.002

Ayamodhaka Sathaal

Ayamodhakam

Cyclobutylamine

3.3

0.941

Drakshati Kath

Kerala Ayurveda

Other chemical and organic compounds with significant peak activity detected on mass spectroscopy analysis, but not quantified

Carbamic acid-ammonium salts, chloro-acetic acid derivatives, nitroso derivatives of piperazine, furfural, acetic acid anhydrates, hydrazide, menthol and borneol

*Only those drugs with positive findings of hepatotoxic volatile organic compounds are shown here; ppb – parts per billion;

Indian Journal of Gastroenterology https://doi.org/10.1007/s12664-018-0820-6

EDITORIAL

Ayurvedic and herbal medicine-induced liver injury: It is time to wake up and take notice Harshad Devarbhavi 1 Received: 29 December 2017 / Accepted: 4 January 2018 # Indian Society of Gastroenterology 2018

Sir William Osler, the renowned physician and one of the founding professors of Johns Hopkins Hospital once remarked, Bthe desire to take medicine is perhaps the greatest feature which distinguishes man from animals.^ A number of prescription drugs used by mankind, including but not limited to antibiotics and antiviral agents, anesthetics, antihypertensives, and analgesics, have undoubtedly enhanced the quality of life and helped cure many chronic diseases. Anecdotal reports of the side effects and ineffectiveness of modern or allopathic drugs have led to the false and fallacious belief in the safety and puissance of complementary and alternative medicine (CAM) worldwide. The notion that these compounds are Bnatural^ and Bsafe^ and hence bereft of side effects has contributed to their popularity and widespread use. In the United States of America (USA), the annual expenditure on CAM in 2007 was about a third of the total spent on prescription drugs, which was estimated to be 33.9 billion US dollars [1]. Increased use of CAM has also led to increased concerns about their safety and potential for hepatotoxicity. Although prescription drugs are an important cause of liver injury, the contribution of herbal and botanical medicationinduced liver injury has increased over the years and has varied considerably across the world ranging anywhere from 17% in Japan [2], 20% in the USA [3], and 31% in China [4] to 70% in Korea [5]. Furthermore, the incidence of CAMinduced severe liver injury in the USA has almost doubled from 12.4% in the last decade to 21% now [6]. This has resulted in a higher rate of liver transplantation, and a lower transplant-free survival, highlighting the increased morbidity and mortality from these agents [6]. Similar studies are unavailable in India, although final results of the study by the Indian Network of Drug-Induced Liver Injury are awaited. * Harshad Devarbhavi [email protected] 1

Department of Gastroenterology and Hepatology, St. John’s Medical College Hospital, Sarjapur Road, Bengaluru 560 034, India

While CAM in the West is often used for weight loss, body building, or performance enhancement, traditional and alternative forms of medicines in India, China, Korea, and other Asia-Pacific regions are primarily used for treatment of myriad symptoms and syndromes, and at times solely for the purpose of Bimproving general health or well-being.^ Comparisons between western medicines and traditional medicines are scarce. One retrospective study from China identified 1985 drug-induced liver injury (DILI) cases (2.05%) among 96,857 patients hospitalized for liver dysfunction from 2009 to 2014 [7]. DILI cases were stratified and compared between traditional Chinese medicines (TCM) vs. Western medicines (WM). The investigators observed a significantly greater number of females ingesting TCM vs. WM (71% vs. 51%). Hepatocellular injury (88% vs. 62%) and severe disease leading to mortality (4.8% vs. 2.8%) were also higher in the TCM group [7]. The lower probability score on causality assessment due to multiple ingredients and/or polypharmacy attests to the challenge of ascribing causality to a specific agent [7]. The use of Ayurvedic herbal medicines (AHM) dates back to ancient times, as far back as 3000 years ago. They are widely advertised and promoted as natural products and hence considered safe. Aside from anecdotal case reports, large series or studies of hepatotoxicity from AHM are lacking in India. Reports of hepatotoxicity from the use of AHM procured from local stores or on the internet have been published or presented. Given that a vast majority of the population in India is exposed to AHM for centuries, the Breported^ lack of adverse reports is both misleading and intriguing. Nevertheless, medical providers, particularly gastroenterologists and hepatologists, will attest to its occurrence as patients seek medical care only when they develop severe liver disease. A major reason for the unsubstantiated lack of hepatotoxicity is the uncertainty in ascribing causality, especially when using multi-ingredient and often unlabeled products and when all other causes of liver disease have not been meticulously ruled out (Fig. 1). The lack of investigational and

Indian J Gastroenterol

Fig. 1 Multi-ingredient Ayurvedic formulations from a patient who developed severe liver injury (twice) with re-challenge. The culprit ingredient is difficult to impute among them

financial resources, the inability to obtain comprehensive medical history due to patient ignorance or reluctance to divulge pertinent information, and the relative unfamiliarity of CAM products among mainstream health care providers often lead to a missed diagnosis. In this context, the article by Philips et al. published in this issue of the Journal is of major significance [8]. It adds to the growing body of knowledge in the area of AHM-induced liver injury by reporting the clinical, laboratory, liver biopsy findings, toxicological profile, and outcome in 34 patients exposed to AHM. In their study which spans over less than a year, 94 patients with severe liver injury who presented to outpatient clinics or emergency rooms were observed to be exposed to AHM. After excluding patients with other contributing causes of liver dysfunction, 2.3% of the cases were ascribed to AHM. The investigators reviewed the clinicopathologic features of 27 patients and analyzed the levels of heavy metal and highly volatile organic compounds (hVOC) of 47 and 17 AHM formulations respectively from 33 patients. The primary reason for AHM use was for relief of Bdigestive issues.^ About two thirds of the patients had other co-morbidities. Other unique features in this patient population included the presence of liver fibrosis in 21 out of 27 patients, and the presence of autoantibodies in the remaining six patients (implying the possibility of a drug-induced autoimmune-like hepatitis which is a novel finding). Mortality was 22% (6/27 patients), and some patients with liver fibrosis and steatosis also had evidence of hepatic decompensation such as hepatic encephalopathy and ascites. This observation supports a diagnosis of acute-on-chronic liver failure (ACLF) in this subset of

patients. This observation is very relevant in that it lends further support to the finding of the ACLF APASL research consortium (AARC) where CAM or AHM was the second most common cause of drug-induced ACLF [9]. Philips et al. additionally reported higher than permissible levels of arsenic and mercury in the AHM formulations which were significantly associated with mortality [8]. They also identified the presence of hVOC in 70% of samples and reported scientific links to liver injury. Higher levels of arsenic, mercury, and lead in Ayurvedic drugs were reported earlier by Saper et al. in a seminal paper [10]. Philips and colleagues should be commended for systematically compiling the data in their study as there is little information available regarding AHM producing DILI, although case reports of DILI from AHM were anecdotally reported or presented before. While high levels of arsenic and mercury have been found in AHM, they have been hitherto associated with extrahepatic toxicity and hence the active ingredients that caused hepatotoxicity are still at large unknown. Unacceptably high levels of metals and hVOC suggest contamination or adulteration during the production or delivery process and needs to be investigated by regulatory bodies. More recently, Navarro and colleagues from the DrugInduced Liver Injury Network (DILIN) reported that mislabeling of ingredients was rampant, occurring in more than 50% of the products tested particularly in weight loss and body building supplements [11]. A similar finding is reproduced in this study reported by Philips et al. in the Journal. Philips et al. study is a timely reminder of the hepatotoxic potential of at least some AHM. What can we learn from the already published studies to help minimize the adverse effects of AHM? (I) It is time to get rid of the wrongly held notion that alternative systems of medicines have no side effects. Acknowledgement of this fact is imperative and should not be held off any further for lack of publication of more studies. The Chinese were early to acknowledge and recognize the association of hepatotoxicity with traditional Chinese medicine (TCM) [7] and measures were implemented to identify the culprit ingredient and publish reports comparing hepatotoxicity between different systems of medicine. A similar line of action should be implemented in India. (II) More research and controlled studies are needed to better clarify the efficacy and safety of CAM for the countless diseases they are claimed to benefit. (III) Regulatory oversight should be instituted for all such products akin to what is employed for the modern/ western/allopathic medicines. (IV) Health care providers should be mandated to report to regulatory agencies of instances of adverse effects from CAM so that remedial measures including restriction/termination of products can be undertaken. (V) There should be a greater collaboration between health care providers of different systems of medicine for a

Indian J Gastroenterol

better understanding of the medications used for various diseases, including their propensity to produce adverse effects. And finally, to paraphrase William Osler, Bone of the first duties of the physician is to educate the masses not to take medicines^ particularly for trivial ailments.

5.

Compliance with ethical standards

7.

6.

Conflict of interest HD declares that he has no conflict of interest. 8.

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4.

Nahin RL, Barnes PM, Stussman BJ, Bloom B. Costs of complementary and alternative medicine (CAM) and frequency of visits to CAM practitioners: United States, 2007. Natl Health Stat Report. 2009;30:1–14. Takikawa H, Murata Y, Horiike N, Fukui H, Onji M. Drug-induced liver injury in Japan: an analysis of 1676 cases between 1997 and 2006. Hepatol Res. 2009;39:427–31. Navarro VJ, Barnhart H, Bonkovsky HL, et al. Liver injury from herbals and dietary supplements in the U.S. Drug-Induced Liver Injury Network. Hepatology. 2014;60:1399–408. Zhao P, Wang C, Liu W, et al. Causes and outcomes of acute liver failure in China. PLoS One. 2013;8:e80991.

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Suk KT, Kim DJ, Kim CH, et al. A prospective nationwide study of drug-induced liver injury in Korea. Am J Gastroenterol. 2012;107: 1380–7. Hillman L, Gottfried M, Whitsett M, et al. Clinical features and outcomes of complementary and alternative medicine induced acute liver failure and injury. Am J Gastroenterol. 2016;111:958– 65. Zhu Y, Niu M, Chen J, et al. Hepatobiliary and pancreatic: comparison between Chinese herbal medicine and Western medicineinduced liver injury of 1985 patients. J Gastroenterol Hepatol. 2016;31:1476–82. Philips CA, Paramaguru R, Joy AK, Antony KL, Augustine P. Clinical outcomes, histopathologic patterns and chemical analysis of ayurveda and herbal medicine associated with severe liver injury—a single center experience from South India. Indian J Gastroenterol. 2018; 37: https://doi.org/10.1007/s12664-0170815-8. Devarbhavi H, Choudhury A, Reddy V, et al. Acute on chronic liver failure secondary to drugs: causes, outcome and predictors of mortality. J Hepatol. 2016;64:232A. Saper RB, Phillips RS, Sehgal A, et al. Lead, mercury, and arsenic in US- and Indian-manufactured Ayurvedic medicines sold via the Internet. JAMA. 2008;300:915–23. Navarro VJ, Khan IA, Avula B, Verma M. The frequency of herbal and dietary supplement mislabelling: experience of the Drug Induced Liver Injury Network. Hepatology. 2017;66:149A.

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