Advertisement

Herb-induced liver injury (HILI) – A guide to approach. Lessons from the Tinospora cordifolia (Giloy) case series story

Published:November 06, 2022DOI:https://doi.org/10.1016/j.jceh.2022.11.001

      Abstract

      Background

      Tinospora cordifolia (TC) is being increasingly consumed in India for its health and suggested immune-enhancing benefits in preventing and countering COVID-19. We previously published our experience of hepatotoxicity with self-medication of TC in six individuals. Since herb-induced liver injury (HILI) has been described with Tinospora crispa (TCR) consumption, it was contested that our patients may have mistakenly self-medicated with TCR which is similar in appearance to TC.

      Methods

      We collected the four plant samples and two commercial preparations that were consumed by our patients for further analysis. The six samples underwent HPTLC phytochemical analysis and DNA barcoding studies for the confirmation of the genus and species. The four plant part samples which included stems and leaves were also analysed by a botanist for characteristic morphological and microscopic features.

      Results

      Based on morphological, microscopic, phytochemical, and DNA studies, the four plant part samples were identified as TC. The two commercial preparations could not be analysed on phytochemical analysis or DNA barcoding studies due to other ingredients that most likely interfered with the analysis. The herb consumed by our study subjects was confirmed to be Tinospora cordifolia.

      Conclusion

      We have highlighted the key morphological and phytochemical differences between these two species. We propose an algorithmic approach to accurately identify the implicated herb in cases of HILI. Future studies on causality need to focus on serological/histopathological identification of active herb/metabolites in human tissues.

      Abbreviations:

      TC (Tinospora Cordifolia), HILI (Herb-Induced Liver Injury), TCR (Tinospora Crispa), HPTLC (High Performance Thin Layer Chromatography), DNA (Deoxyribonucleic Acid), COVID-19 (Coronavirus Disease 2019), RUCAM (Roussel Uclaf Causality Assessment Method), DILI (Drug Induced Liver Injury)

      Introduction

      Tinospora Cordifolia (TC) has been a popular over-the-counter herbal immune boosting supplement for a vast proportion of Indians. Tinospora Cordifolia (TC), popularly known as Guduchi, Moonseed, Giloy and Amrita in Sanskrit, is a popular herb used in many traditional medicinal practices.

      Belwal T, Nabavi S, Nabavi S, Dehpour A, Shirooie S. Naturally Occurring Chemicals against Alzheimer’s Disease London, UK: Academic Press; 2020. Chapter 3.2.13, Tinospora cordifolia; p. 351-358.

      With the start of the COVID-19 pandemic in March of 2020, the self-medicated use of Giloy increased. In July of 2021, we published our experience in patients presenting with liver injury following the consumption of TC.
      • Nagral A.
      • Adhyaru K.
      • Rudra O.S.
      • Gharat A.
      • Bhandare S.
      Herbal Immune Booster-Induced Liver Injury in the COVID-19 Pandemic - A Case Series.
      We presented 6 cases of patients with acute hepatitis and a history of consumption of TC. After thorough history-taking and acquisition of routine serological parameters, the patients were subjected to a liver biopsy. The details of patient profiles and histological features are as in Table 1. We had concluded that the herb TC due to its immune booster properties, caused an auto-immune like hepatitis or unmasking of a latent auto-immune liver disease. In a previous paper, Karousatos et al
      • Karousatos C.M.
      • Lee J.K.
      • Braxton D.R.
      • Fong T.-L.
      Case series and review of Ayurvedic medication induced liver injury.
      had described a single patient with giloy related possible liver injury toxicity although the patient was not biopsied. Subsequently, others have described their experience with TC-induced hepatotoxicity.
      • Kulkarni A.V.
      • Hanchanale P.
      • Prakash V.
      • Kalal C.
      • Sharma M.
      • Kumar K.
      • et al.
      Tinospora Cordifolia (Giloy)-Induced Liver Injury During the COVID-19 Pandemic-Multicenter Nationwide Study From India.
      • Sahney A.
      • Wadhawan M.
      • Kumar A.
      Tinospora cordifolia—A Double Edge Sword?.
      • Gupta D.
      • Sonawane A.
      Heart-leaved Moonseed- Innocuous or Baneful.
      • Rastogi M.
      • Jagdish R.
      • Vij V.
      • Bansal N.
      Herbal Immune Booster–Induced Liver Injury in the COVID-19 Pandemic.
      • Gupta H.
      • Nigam N.
      • Singh S.
      • Roy A.
      • Dhiman R.
      Immune Boosting Gone Wrong? A COVID-Concoction-Conundrum.
      • Parikh P.
      Knowledge of Herbal Medicines – Is a Reverse Bridge Course an Urgent Necessity?.
      • Gupta S.
      • Dhankhar Y.
      • Har B.
      • Agarwal S.
      • Singh S.
      • Gupta A.
      • et al.
      Probable Drug-Induced Liver Injury Caused by Tinospora species: A Case Report.
      In an editorial by Bjornsson et al. on our publication of the potential hepatoxicity of TC, the editors analysed the data and concluded that the liver injury of TC was suggestive of a drug induced autoimmune hepatitis.
      • Björnsson E.S.
      • Navarro V.J.
      • Chalasani N.
      Liver Injury Following Tinospora Cordifolia Consumption: Drug-Induced AIH, or de novo AIH?.
      Table 1Biochemical,Serological,Histopathological, HPTLC and DNA Profile of Six Patients Consuming Tinospora Cordifolia.
      Patient 1Patient 2Patient 3Patient 4Patient 5Patient 6
      Age/sex40/M54/F38/M62/F56/F56/F
      TC Preparation consumedStemStemStemCommercial preparation (Syrup)StemCommercial preparation(Tablet)
      Laboratory parameters Hb (g/dl)13.710.9912.411.511.8
      TLC (cells/cmm)1600062009200840085506690
      Platelets (cells/cmm)388000187000371000103000241000140000
      Presentation total bilirubin (direct bilirubin)

      (mg/dl)
      7.9 (6.7)15.3 (10.9)7.4 (3.9)9.1 (5.7)12.2 (7.5)9.13 (4.41)
      Peak total bilirubin (direct bilirubin) (mg/dl)45.1 (25)24.9 (21)20 (10.7)15.1 (12.5)12.2 (7.5)9.1 (4.4)
      Peak AST (IU/L)17731195150422221099455
      On presentation ALT (IU/L)2894768560202256207
      Peak ALT (IU/L)31147681482855256472
      Time to normalisation of LFT (in days)95164781113853
      Autoimmune profile (pre/post)a ANANegative/-1:100/-ve1:100/-ve1:320/1:320Negative/-Negative/-
      Anti-SMANegative/-Negative/-Positive/NegativeNegative/-Weakly positive/Negative
      Serum IgG (700–1600 mg/dl)Normal/-Normal/-Normal/-2570/17212045/1680
      Revised AIH score b (pre/post when steroids given)1219/211519/2218/2118
      Liver injury pattern on biopsyHepatocellularHepatocellular + CholestaticHepatocellularHepatocellularHepatocellularHepatocellular
      Updated RUCAM score refc c454774
      HPTLC (Band at Rf 0.61)SeenSeenSeenNot seenSeenNot seen
      DNA BarcodingTCTCTCNRTCNR
      Abbreviations - M: Male, F: Female, Hb: Haemoglobin, TLC: Total leucocyte count, AST: Aspartate aminotransferase, ALT: Alanine aminotransferase, LFT: Liver function tests, ANA: Anti-nuclear antibody, Anti-SMA: Anti-smooth muscle antibody, IgG: Immunoglobulin G, AIH: Autoimmune hepatitis, HPTLC: High Performance Thin Layer Chromatography, DNA: Deoxyribo Nucleic Acid, TC: Tinospora Cordifolia+++: Prominent/Abundant ++: Moderate +: Mild/Occasional -: Not seen/done. aPre-stopping the drug and post-stopping the drug, - implies not done as previous ANA/ASMA were negative or IgG was normal. bRevised AIH score: Pre-treatment: Definite AIH >15 Probable AIH l0-15 Post-treatment: Definite AIH >17 12 Probable AIH 12–17. cUpdated RUCAM score and resulting causality grading: #0, excluded; 1–2, unlikely; 3–5, possible; 6–8, probable; and 9 highly probable.
      In India, herbal products have been widely used for centuries.
      • Jaiswal Y.S.
      • Williams L.L.
      A glimpse of Ayurveda - The forgotten history and principles of Indian traditional medicine.
      However, the drugs have not gone through rigorous phased trials to establish their efficacy and safety. Besides, most of the commercial preparations are mixtures of several herbs and their interactions as well as toxicities have not been well researched. Since Tinospora crispa (TCR), a closely related plant has been described to be potentially hepatotoxic,
      • Kadir F.A.
      • Othman F.
      • Abdulla M.A.
      • Hussan F.
      • Hassandarvish P.
      Effect of Tinospora crispa on thioacetamide-induced liver cirrhosis in rats.
      ,
      • Langrand J.
      • Regnault H.
      • Cachet X.
      • Bouzidi C.
      • Villa A.F.
      • Serfaty L.
      • et al.
      Toxic hepatitis induced by a herbal medicine: Tinospora crispa.
      the Ayurveda community queried whether our patients mistakenly consumed the wrong herb and whether that could be an explanation for the reported Herb-Induced Liver Injury (HILI).
      The aim of our study was to correctly identify the herbs consumed by our patients. To that end, we have been able to provide an algorithmic approach to a patient who consumes an unknown herb that causes liver injury, and which serves as a guide to identify the herb and further management.

      Methods

      An institutional ethical clearance (EC/1087/2021 dated 7-6-2021) was obtained. A written informed consent was taken from all patients who participated in our study
      • Nagral A.
      • Adhyaru K.
      • Rudra O.S.
      • Gharat A.
      • Bhandare S.
      Herbal Immune Booster-Induced Liver Injury in the COVID-19 Pandemic - A Case Series.
      , and samples of the plant parts consumed by them were collected. There were six patients described to have HILI with TC, four of them had consumed extracts of the stem (n=4), while two consumed commercially available preparations containing TC (n=2).
      The samples of the plant parts (stem and leaves) were preserved by wrapping them in dry paper, which were then examined by a botanist for morphological characteristics. Plant samples (stem pieces) provided by patients were sectioned by hand to obtain transverse sections of stem. The sections were stained with toluidine blue and safranin for microscopic evaluation of anatomy (both gross anatomy and microscopic tissue anatomy).
      All 6 samples, including the 2 commercial preparations were processed for phytochemical analysis by High Performance Thin Layer Chromatography (HPTLC). After being identified by the botanists, controls for the plant parts (TC and TCR) were obtained from plant nurseries maintained at Mahim Nature Park and Sanjay Gandhi National Park, Mumbai respectively. Methanolic extracts of the specimen were prepared by overnight extraction and were spotted on 20 × 10 cm TLC plate, precoated with silica gel 60 F254 (0.2 mm thickness; Cat. No. 1.05554.0001; Merck, Darmstadt, Germany). Samples were spotted using the CAMAG Linomat V Automatic Sample Spotter (Muttenz, Switzerland) fitted with a syringe (100 μL; Hamilton). The plates were developed in a CAMAG glass twin trough chamber (20 × 10 × 4 cm) with mobile phase of chloroform:ethylacetate:ethanol:formic acid (10:15:5:0.5 v/v/v/v). After drying, the plates were derivatized with methanolic sulphuric acid and visualized under 366 nm. Densitometric scanning was performed at 366 nm (mercury lamp) using CAMAG TLC Scanner 4 linked to WINCATS software. Distinctive bands obtained for authenticated specimen were compared with that obtained from the patient samples.
      The 6 samples were also processed for DNA analysis for confirmation of the species.
      • 1.
        DNA was isolated from the plant sample provided by the patient while the quality was initially evaluated on 1.0 % agarose gel. A single band of high-molecular-weight DNA was observed indicating the purity of the sample.
      • 2.
        Mitochondria, chloroplast, and nuclear genes were amplified using universal markers such as maturase K (matK), ribulose 1,5-biphosphate carboxylase (rbcL) and internal transcribed spacer (ITS) and the amplified products were purified.
      • 3.
        Sanger sequencing of amplicons was carried out using BDT v3.1 Cycle sequencing kit on ABI 3730xl Genetic Analyzer.
      • 4.
        The amplicon gene sequences were used to carry out BLAST with the ‘nr’ database of NCBI GenBank database and Barcode of Life Data (BOLD) system database.

      Results

      • (i)
        Morphological and micro-anatomical studies
      The fresh plant specimens of stem and leaf were evaluated for gross morphological and micro-anatomical features. The observations made are in concurrence with those reported earlier.
      • Begum N.
      • Raj P.
      • Ravikumar K.
      Comparative pharmacognostical and histochemical studies on the three different species of Tinospora on stem and leaf.
      ,
      • Parveen A.
      • Adams J.S.
      • Raman V.
      • Budel J.M.
      • Zhao J.
      • Babu G.N.M.
      • et al.
      Comparative Morpho-Anatomical and HPTLC Profiling of Tinospora Species and Dietary Supplements.
      Tinospora species appear similar in gross observation, especially in their external morphologies. They all have broadly cordate leaves [heart-shaped leaves are attached at the tip of a stalk (petiole) within a notch] and cylindrical stems often with lenticels (raised pores or openings present on the outer surface of the stem, responsible for gaseous exchange). On closer evaluation, distinctive morpho-anatomical features of stem and leaves can be observed. The broad differences have been elaborated in Table 2. Leaves are smooth in both T. cordifolia and T. crispa. The stem of TC is mainly used as the crude drug. The young stems in both TC and TCR are green with smooth surfaces but swollen at nodes (points on the stem from where leaves or branching twigs arise). Older stems are light brown in colour and their surface shows wart like outgrowths that clearly protrude out. These protuberances are due to circular lenticels. A transverse section of the stem taken by hand shows a single-layered epidermis in both TC and TCR as seen in Figure 1.
      • (ii)
        High Performance Thin Layer Chromatography
      Table 2Comparative list of characteristic differences between TC and TCR.
      Tinospora Cordifolia
      • Björnsson E.S.
      • Navarro V.J.
      • Chalasani N.
      Liver Injury Following Tinospora Cordifolia Consumption: Drug-Induced AIH, or de novo AIH?.
      ,
      • Jaiswal Y.S.
      • Williams L.L.
      A glimpse of Ayurveda - The forgotten history and principles of Indian traditional medicine.
      Tinospora Crispa
      • Björnsson E.S.
      • Navarro V.J.
      • Chalasani N.
      Liver Injury Following Tinospora Cordifolia Consumption: Drug-Induced AIH, or de novo AIH?.
      ,
      • Jaiswal Y.S.
      • Williams L.L.
      A glimpse of Ayurveda - The forgotten history and principles of Indian traditional medicine.
      ,
      • Upadhyay A.K.
      • Kumar K.
      • Kumar A.
      • Mishra H.S.
      Tinospora cordifolia (Willd.) Hook. f. and Thoms. (Guduchi) - validation of the Ayurvedic pharmacology through experimental and clinical studies.
      Distribution in IndiaArunachal Pradesh, Assam, Bihar, Delhi, Gujarat, Goa, Karnataka, Kerala, Maharashtra, Odisha, Sikkim, Tamil Nadu, Uttar Pradesh, West Bengal
      • Singh B.
      • Nathawat S.
      • Sharma R.A.
      Ethnopharmacological and phytochemical attributes of Indian Tinospora species: A comprehensive review.
      West Bengal, Odisha, Arunachal Pradesh and Assam,
      • Nagarkar B.
      • Kulkarni R.
      • Bhondave P.
      • Kasote D.
      • Kulkarni O.
      • Harsulkar A.
      • et al.
      Comparative Hepatoprotective Potential of Tinospora cordifolia, Tinospora sinensis and Neem-guduchi.
      extending to Southeast Asia
      Stem Morphology
      • ● Less prominent tubercules
      • ● Deciduous climber with seriate stems
      • ● More prominent blunt tubercles
      • ● Fleshy old stems - striate and often with aerial roots
      Micromorphology of Stem
      • ● Transverse section is wedge-shaped
      • ● More layers of cork cambium with distinct walls
      • ● Protoxylem is not clearly distinguishable in pith
      • ● Less prominent pericyclic layer that continues forming a sclerenchymatous cap of lignified cells
      • ● Fewer vascular bundles
      • ● Transverse section is circular
      • ● Few layers of lenticels with wavy walls.
      • ● Pith shows protoxylem
      • ● Larger number of vascular bundles
      • ● More prominent pericyclic layer
      Leaf Morphology
      • ● Heart shaped, 10-20 cm long with a 3-4 cm petiole broadly ovate-cordate, sinuate at base, abruptly cuspidate-acuminate at apex
      • ● Leaf surface has characteristic epicuticular waxy coating
      • ● Heart shaped 6-12 cm long with a 5-15 cm petiole, broadly ovate to oblong-orbicular, shortly or deeply cordate at base and caudate at apex
      • ● Epicuticular waxy layer is absent on leaf surface
      Flowers
      • ● Inflorescences are usually 5-15cm and solitary, being pseudo-racemose, axillary, or leafless branches
      • ● Male inflorescences are 5-10 cm or more; female inflorescence are 2-6 cm long with yellowish-green flowers
      Botanical terms explained in order of appearance: tubercules - protuberances, seriate - arranged in rows, striate - with parallel lines or grooves, cork cambium - inner layer of the bark/epidermis, Protoxylem - the first formed vascular tissue seen in the central part of the stem, pericyclic layer - cells that surround the vascular bundles, lignified cells - cells with thick cell walls that help water conduction, petiole -stalk, sinuate - wavy margin, cuspidate-acuminate - tapering tip with a sharp point, epicuticular - outermost layer, ovate -egg shaped, oblong-orbicular - rounded, caudate -attenuated to tail-like.
      Table 3Published data on liver injury related to Tinospora cordifolia (Giloy
      Giloy is one of the local names for Tinospora cordifolia in India.
      ).
      S.noStudyNo. of patientsHerbFormMax. Bilirubin (mg/dl)Max. AST/ALT (IU/L)Max. INRBiopsyTreatmentOutcome
      1.Karousatos CM et al
      • Karousatos C.M.
      • Lee J.K.
      • Braxton D.R.
      • Fong T.-L.
      Case series and review of Ayurvedic medication induced liver injury.
      1GiloyCommercial preparation3.71086/14511.0NoSupportive treatmentComplete resolution
      2.Kulkarni AV et al
      • Kulkarni A.V.
      • Hanchanale P.
      • Prakash V.
      • Kalal C.
      • Sharma M.
      • Kumar K.
      • et al.
      Tinospora Cordifolia (Giloy)-Induced Liver Injury During the COVID-19 Pandemic-Multicenter Nationwide Study From India.
      43Giloy, giloy containing formulationsPlant parts / local chemists’ preparations / commercial preparations46.61637/12696Yes (22/43)One – liver transplant; Others Supportive treatment2 mortalities; Others had Complete resolution
      3.Sahney A et al
      • Sahney A.
      • Wadhawan M.
      • Kumar A.
      Tinospora cordifolia—A Double Edge Sword?.
      3GiloyPlant part (twig)232300/21502.3YesSupportive +

      Steroids (2)
      Complete resolution
      4.Gupta D et al
      • Gupta D.
      • Sonawane A.
      Heart-leaved Moonseed- Innocuous or Baneful.
      3GiloyCommercial preparation (tablet):1

      Plant part (twig): 2
      24.991198/1044Not specifiedNoSupportive treatmentComplete resolution
      5.Rastogi M et al
      • Rastogi M.
      • Jagdish R.
      • Vij V.
      • Bansal N.
      Herbal Immune Booster–Induced Liver Injury in the COVID-19 Pandemic.
      2GiloyPlant part8.9679/4361.29YesSupportive +

      Steroids
      Complete resolution
      6.Gupta H et al
      • Gupta H.
      • Nigam N.
      • Singh S.
      • Roy A.
      • Dhiman R.
      Immune Boosting Gone Wrong? A COVID-Concoction-Conundrum.
      2Tinospora cordifolia (TC)Commercial preparation (juice):1

      Plant part (twig): 1
      20.1697/6452.8YesSupportive +

      Steroids
      Complete resolution
      7.Parikh P
      • Parikh P.
      Knowledge of Herbal Medicines – Is a Reverse Bridge Course an Urgent Necessity?.
      2Tinospora cordifoliaCommercial preparation (capsule)9460/4001.34YesSupportive +

      Steroids
      Complete resolution
      8.Gupta S et al
      • Gupta S.
      • Dhankhar Y.
      • Har B.
      • Agarwal S.
      • Singh S.
      • Gupta A.
      • et al.
      Probable Drug-Induced Liver Injury Caused by Tinospora species: A Case Report.
      2Tinospora Species not specifiedPlant part (stem): 1

      Commercial preparation (pellets):1
      Not specifiedNot specifiedNot specifiedYesSupportive-1

      Liver transplant-1
      Complete resolution
      # Giloy is one of the local names for Tinospora cordifolia in India.
      Figure 1
      Figure 1Hand cut transverse sections of stem of Tinospora cordifolia (A) and Tinospora crispa (B) stained with Toluidine Blue and Saffranine showing distinctive vascular bundles [the conducting vessels in the stem (indicated by yellow arrows) that help in transport of minerals and water] in each species. The sections are viewed under a light microscope at 40X.
      The 4 specimens and 2 commercial preparations were evaluated chromatographically using HPTLC at Ramnarain Ruia College Laboratory and phytochemical fingerprints obtained were compared with those of authenticated plant samples. The distinctive band seen in the Tinospora Cordifolia at Rf 0.61 (Figure 2) is absent in the Tinospora Crispa specimen (Figure 3), where Rf is Resolution Front, which is the ratio of distance travelled by the analyte to the distance travelled by the solvent. The distinctive band at Rf 0.61 of Tinospora Cordifolia shown in Figure 2 is seen in all plant material reportedly consumed by the patients. The 2 marketed formulations of Tinospora Cordifolia consumed by the patients (syrup and capsule) did not show the distinctive HPTLC band at Rf 0.61, although the labels on these commercial preparations mentioned only Giloy as the content and with no other constituent. The findings for each patient have been mentioned in Table 1.
      • (iii)
        DNA Barcoding
      Figure 2
      Figure 2HPTLC fingerprint of various Tinospora samples along with sample of Tinospora crispa, visualized at 366nm after derivatization with 10% methanolic sulphuric acid
      Track Details: Track 1 - Tinospora cordifolia leaf (control sample); Track 2 – Patient 1 sample stem; Track 3 - Patient 2 sample leaf; Track 4 – Patient 2 sample stem; Track 5 – Patient 3 sample capsule; Track 6 – Tinospora crispa leaf (control sample); Track 7 – Patient 4 sample leaf; Track 8 - Patient 4 sample stem; Track 9 – Patient 5 sample syrup; Track 10 - Patient 6 sample stem; Track 11 – Tinospora cordifolia stem (control sample); Track 12 - Tinospora cordifolia leaf (control sample)
      Note the distinct band at Rf 0·61(arrows) seen in all Tinospora cordifolia samples and in patient samples (as seen in earlier ).Note the absence of the band at Rf 0·61 in Tinospora crispa (Track no. 6). The general pattern of bands seen in T. crispa is different from those in T. cordifolia (Tracks 1,6, 11 and 12).
      Figure 3
      Figure 3The Thin Layer Chromatography (TLC) fingerprint of phytochemicals from Tinospora cordifolia and Tinospora crispa. The TLC chromatograms are shown along with the densitometric scan at 366nm and the Rf values of respective bands in a table. The distinct absence of bright band at Rf 0·61 is absent in the Tinospora crispa.
      All six samples underwent DNA barcoding for identification of the species at Barcode Biosciences. As observed in HPTLC, the two commercial preparations of the tablet and syrup did not show up as TC on the DNA analysis also.
      According to our analysis, all four samples of stems provided for plant DNA analysis were of Tinospora cordifolia. This can be interpreted from the sequence similarity table, phylogenetic tree, and distance matrix. The sequence similarity table shows us the length of query coverage (which should be 100% for identifying a species). The percentage identity for the 100% query covered sequence when is more than 98%, can be concluded as same species. Always the highest percentage of matched species is concluded as the species.
      Similarly, the distance matrix and phylogenetic tree also can show the closest species. The sample shares one branch with the nearest species in the phylogenetic tree whereas in the case of the distance matrix, the distance among the species should be less than 0·002. The lesser the distance, the closest the species. Further, the control samples of T. Cordifolia and T. Crispa were also confirmed on DNA analysis. The DNA findings of the 6 patients have been stated in Table 1.

      Discussion

      The COVID pandemic saw an increase in the use of Ayurvedic products, unsupervised use of plant parts, and renewed interest in wellness products.

      Why Ayurveda gained a lot of popularity since Covid-19 - Times of India [Internet]. The Times of India. Available from: https://m.timesofindia.com/life-style/health-fitness/health-news/why-ayurveda-gained-a-lot-of-popularity-since-covid-19/amp_articleshow/83058289.cms

      It has been suggested that a closely related species, Tinospora crispa (TCR), which is morphologically similar to Tinospora Cordifolia (TC) but known for its hepatotoxicity, could have been mistakenly consumed by our patients.
      • Sharma R.
      • Prajapati P.K.
      Remarks on "Herbal Immune Booster-Induced Liver Injury in the COVID-19 Pandemic - A Case Series.
      Hence a study to provide further insights into the characterization of these plant species was planned. It is not only important for knowing the purity of original plant species in the preparations consumed by patients but also for providing a platform for biochemical and phylogenetic relationship studies in similar looking herbs.
      Owing to the time in the pandemic when laboratories were not functioning at full capacity, analysis of the plant and plant parts was not readily possible. The samples, provided by the patients, have now been analysed morphologically, phytochemically, and by DNA barcoding. The results prove beyond doubt that the patients did in fact, consume TC. The fact that we analysed pure plant parts in four of our patients helped in the identification of the species as opposed to the commercial preparations (capsule and syrup) which could not be reliably analysed. It has been estimated that over 35% of websites have wrongly identified TC and TCR.
      • Huang W.-T.
      • Tu C.-Y.
      • Wang F.-Y.
      • Huang S.-T.
      Literature review of liver injury induced by Tinospora crispa associated with two cases of acute fulminant hepatitis.
      This experience convinced us that accurate species identification is imperative for implicating a causative herb in HILI.
      Tinospora Cordifolia (TC) is a large, glabrous, perennial, deciduous, climbing shrub, of the genus Tinospora, belonging to the family Menispermaceae.
      • Upadhyay A.K.
      • Kumar K.
      • Kumar A.
      • Mishra H.S.
      Tinospora cordifolia (Willd.) Hook. f. and Thoms. (Guduchi) - validation of the Ayurvedic pharmacology through experimental and clinical studies.
      The genus Tinospora has 34 species, of which nine are found in various parts of India.
      • Singh B.
      • Nathawat S.
      • Sharma R.A.
      Ethnopharmacological and phytochemical attributes of Indian Tinospora species: A comprehensive review.
      Of these, T. Cordifolia and T. Sinensis have therapeutic applications.
      • Nagarkar B.
      • Kulkarni R.
      • Bhondave P.
      • Kasote D.
      • Kulkarni O.
      • Harsulkar A.
      • et al.
      Comparative Hepatoprotective Potential of Tinospora cordifolia, Tinospora sinensis and Neem-guduchi.
      T. crispa, is a similar-looking plant of the family Menispermaceae, and of known hepatotoxic potential.
      • Langrand J.
      • Regnault H.
      • Cachet X.
      • Bouzidi C.
      • Villa A.F.
      • Serfaty L.
      • et al.
      Toxic hepatitis induced by a herbal medicine: Tinospora crispa.
      ,
      • Huang W.-T.
      • Tu C.-Y.
      • Wang F.-Y.
      • Huang S.-T.
      Literature review of liver injury induced by Tinospora crispa associated with two cases of acute fulminant hepatitis.
      The differences in morphology can be appreciated in Figure 4 and Figure 5.
      Figure 4
      Figure 4External morphology of leaf and stem of Tinospora cordifolia and Tinospora crispa. Note the prominent warty protuberances on the stem surface in T. crispa.
      Figure 5
      Figure 5Illustrations of Tinospora cordifolia (A) and Tinospora crispa (B) showing morphological differences in the plant species.
      In High Performance Thin Layer Chromatography (HPTLC), a technique of separation of the phytochemicals, both TC and TCR showed distinct bands that help in differentiating one from the other. T. cordifolia showed a light blue fluorescent band at Rf 0·61 which was characteristically absent in T. crispa. HTPLC performed on commercial samples labelled giloy did not yield any result possibly due to the low concentration of the extract or the interference of other additives in the commercial preparation. DNA barcoding is a method of species identification using a DNA sequence from a specific gene. Individual sequences (bar code loci) can be used to discriminate the closely related species and identify new cryptic species also
      • Kress W.J.
      • Erickson D.L.
      A two-locus global DNA barcode for land plants: the coding rbcL gene complements the non-coding trnH-psbA spacer region.
      . Depending on the taxon and complexity of the species, different barcode loci are used for the purpose. For example, Tinospora cordifolia belongs to the Menispermaceae family which includes more than 400 plant species and various subspecies that may have similar appearances. The simple flow chart in Figure 6 shows the procedure and use of DNA barcoding in plants. Figure 7 demonstrates the phylogenetic tree of a plant species. As the original i.e. ancestral plant evolves and diversifies into various species (A to E), it is important to differentiate these species from each other as they may look similar or have very closely related properties. This is important in validating purity of medicinal herb species in a formulation. Hence along with phytochemical study, we also undertook DNA studies.
      Figure 6
      Figure 6Flow chart depicting simple procedure and use of DNA barcoding in medicinal plants.
      Figure 7
      Figure 7Simplified phylogenetic tree to show how present species evolve from original plant species.
      Tinospora cordifolia has been reported to contain phytoconstituents like terpenoids, alkaloids, lignans, carbohydrates, bitters, steroids, glycosides, sesquiterpenoids, aliphatic compounds, essential oils, fatty acids, and polysaccharides.

      Council of Scientific & Industrial Research, Govt of India. The Wealth of India, Raw Materials; New Delhi, India: The Publication & Information Directorate. 1982;10:252.

      It is a Rasayana drug, widely used in the Ayurvedic system of medicine as an immune modulator for improving the body's resistance to infections.

      Sivarajan VV, Balachandran I. Ayurvedic drugs and their plant sources. New Delhi, India: CBS Publishers & Distributors Pvt Ltd. 1999;527–544.

      The stems of T. cordifolia, contains various constituents like phenyl propanoid glycosides like cordifolioside A and B, syringin, Phytosterol; 20-β-hydroxyecdysone, diterpenoids like tinosporaside, tinosporin, tinosporidine, tinocordifolioside, and alkaloids like columbin, isocolumbin, berberine and magnoflorine.

      Council of Scientific & Industrial Research, Govt of India. The Wealth of India, Raw Materials; New Delhi, India: The Publication & Information Directorate. 1982;10:252.

      ,
      • Qudrat-I-Khuda M.
      • Khaleque A.
      • Bashir A.
      • Roufk M.D.A.
      • Ray N.
      Tinospora cordifolia—isolation of tinosporon, tinosporic acid and tinosporol from fresh creeper.
      • Phan V.K.
      • Chau V.M.
      • Nguyen T.D.
      • La V.K.
      • Dan T.H.
      • Nguyen H.N.
      • et al.
      Aporphine alkaloids, clerodane diterpenes, and other constituents from Tinospora cordifolia.

      Patel, U., Girme, A., Patel, K. et al. A validated HPTLC method for quantification of cordifolioside A, 20-β-hydroxyecdysone and columbin with HPTLC–ESI–MS/MS characterization in stems of Tinospora cordifolia. JPC-J Planar Chromat.

      Cordifolioside A has been reported to have significant immunostimulant with cardio and radioprotective activities.
      • Maurya R.
      • Wazir V.
      • Kapil A.
      • Kapil R.S.
      Cordifoliosides A and B, two new phenylpropene disaccharides from Tinospora cordifolia possessing immunostimulant activity.
      20-β-hydroxyecdysone has been reported to be a potent immunoprotective wound-healing agent.
      • Sharma U.
      • Bala M.
      • Kumar N.
      • Singh B.
      • Munshi R.K.
      • Bhalerao S.
      Immunomodulatory active compounds from Tinospora cordifolia.
      Columbin has anti-inflammatory, anti-cancer, and antioxidant properties.
      • Sharma U.
      • Bala M.
      • Kumar N.
      • Singh B.
      • Munshi R.K.
      • Bhalerao S.
      Immunomodulatory active compounds from Tinospora cordifolia.
      Based on the published potent immunomodulatory properties of phytochemicals, TC could augment the immune response in patients with autoimmune problems, leading to clinically significant complications as was experienced in our patients.
      • Maurya R.
      • Wazir V.
      • Kapil A.
      • Kapil R.S.
      Cordifoliosides A and B, two new phenylpropene disaccharides from Tinospora cordifolia possessing immunostimulant activity.
      Studies of T. crispa extracts and isolated compounds of T. crispa have shown a broad range of pharmacological activities such as anti-inflammatory, antioxidant, immunomodulatory, cytotoxic, antimalarial, cardioprotective, and anti-diabetic effects.
      • Ahmad W.
      • Jantan I.
      • Bukhari S.N.
      Tinospora crispa (L.) Hook. f. & Thomson: A Review of Its Ethnobotanical, Phytochemical, and Pharmacological Aspects.
      However, studies in rats and humans have indicated hepatotoxic effects of ethanolic extracts of T. crispa.
      • Kadir F.A.
      • Othman F.
      • Abdulla M.A.
      • Hussan F.
      • Hassandarvish P.
      Effect of Tinospora crispa on thioacetamide-induced liver cirrhosis in rats.
      ,
      • Langrand J.
      • Regnault H.
      • Cachet X.
      • Bouzidi C.
      • Villa A.F.
      • Serfaty L.
      • et al.
      Toxic hepatitis induced by a herbal medicine: Tinospora crispa.
      A multicentre study from India further reiterated the findings of our study by highlighting the liver injury associated with the use of giloy in commercial pure and multiherbal preparations.
      • Kulkarni A.V.
      • Hanchanale P.
      • Prakash V.
      • Kalal C.
      • Sharma M.
      • Kumar K.
      • et al.
      Tinospora Cordifolia (Giloy)-Induced Liver Injury During the COVID-19 Pandemic-Multicenter Nationwide Study From India.
      In their study, in patients with a history of consumption of commercial preparations of giloy, they analysed the product for heavy metal contaminants, potential hepatotoxic organic compounds and residual pesticides.
      • Kulkarni A.V.
      • Hanchanale P.
      • Prakash V.
      • Kalal C.
      • Sharma M.
      • Kumar K.
      • et al.
      Tinospora Cordifolia (Giloy)-Induced Liver Injury During the COVID-19 Pandemic-Multicenter Nationwide Study From India.
      Twenty-five patients consumed giloy in a pure or extracted form, while eighteen consumed it as a part of a multi-herbal formulation. Heavy metal contamination with mercury, arsenic, and lead above prescribed limits was found in two samples, while others noted multiple phytochemicals with potential immunomodulatory effects and hepatotoxic potential.
      • Karousatos C.M.
      • Lee J.K.
      • Braxton D.R.
      • Fong T.-L.
      Case series and review of Ayurvedic medication induced liver injury.
      We did not perform the analysis for any heavy metals in the two commercial preparations as the main aim of our study was to identify the correct herb. However, in the commercial preparations analysed by us, there seemed to be multiple constituents as the main constituent on the label ‘Giloy’ could not be identified in both HPTLC and DNA studies.
      Herbs are easily consumed without medical supervision with the impression that they are completely safe. In a patient with documented liver injury and a history of consumption of an herb, we propose an algorithm to accurately identify the herb (Figure 8). Documentation of such observations will aid in establishing the hepatotoxic potential of herbs. Further, a positive autoimmune profile or raised IgG does not rule out HILI and a detailed drug/herb history needs to be taken. Liver biopsy although optional, would be prudent to rule out alternate etiologies or suspected dual etiologies eg. NAFLD and HILI or AIH and HILI.
      Figure 8
      Figure 8Algorithm to detect herb induced liver injury (HILI) in patients with history of herb consumption and liver injury.
      We did not estimate the heavy metals in the commercial preparations of the herbs and this could be considered a limitation of the study although the patients took “pure” TC. Further, HPTLC and DNA barcoding are expensive and not available in commercial labs, making its utilization difficult in the identification of the herb implicated in HILI.
      In conclusion, we have described our methods – botanical, HPTLC and DNA barcoding to identify a herb and suggested an algorithmic approach. Using these methods, we have been able to confirm the self-medicated herbs by our patients as being that of Tinospora cordifolia.Patients presenting with HILI, represent only a fraction of patients with this problem. CAMs are widely consumed by the Indian population but not all develop HILI. Data regarding the denominator of patients consuming herbs and the numerator i.e., those developing HILI is unknown. Besides data on patient characteristics, dosages at which one develops HILI, interactions with other drugs and herbs is also lacking and may be an area of study in the future. Besides, identifying specific phytochemicals in the blood samples of the patient for direct identification of the implicated herb may be an area of future research and potentially become an inexpensive, serum-based test for identification of HILI.

      Role of Funding Source

      No funding source.

      Author Contributions

      A Nagral contributed to the conception and design of the article, editing and critical revision of the manuscript. O S Rudra contributed to the design of the article, acquisition of the data and writing of the manuscript. S Menezes contributed to the design of the article and editing of the manuscript. S Menon contributed to drafting the manuscript and analysis and interpretation of the HPTLC data. S Shailajan contributed to acquiring and editing the botanical data. S Mallakmir was responsible for interpreting the DNA studies. R Reddy contributed to the critical revision of the manuscript. All authors have read, edited, and approved the final document prior to submission.

      Declaration of Competing Interest

      There are no conflicts of interest.

      Acknowledgements

      Mausume Mohanty, Sukhada S Thorat, Swati Singh, Yash Gangal, Mitali M. Kelkar, Vimal Temkar “Given his role as Editor in Chief, Dr A.C. Anand was not involved in the peer-review of this article and has no access to information regarding its peer-review. Full responsibility for the editorial process for this article was delegated to Dr Madhumita Premkumar’’

      References

      1. Belwal T, Nabavi S, Nabavi S, Dehpour A, Shirooie S. Naturally Occurring Chemicals against Alzheimer’s Disease London, UK: Academic Press; 2020. Chapter 3.2.13, Tinospora cordifolia; p. 351-358.

        • Nagral A.
        • Adhyaru K.
        • Rudra O.S.
        • Gharat A.
        • Bhandare S.
        Herbal Immune Booster-Induced Liver Injury in the COVID-19 Pandemic - A Case Series.
        J Clin Exp Hepatol. 2021; 11: 732-738
        • Karousatos C.M.
        • Lee J.K.
        • Braxton D.R.
        • Fong T.-L.
        Case series and review of Ayurvedic medication induced liver injury.
        BMC Complement Med Ther. 2021; 21: 91
        • Kulkarni A.V.
        • Hanchanale P.
        • Prakash V.
        • Kalal C.
        • Sharma M.
        • Kumar K.
        • et al.
        Tinospora Cordifolia (Giloy)-Induced Liver Injury During the COVID-19 Pandemic-Multicenter Nationwide Study From India.
        Hepatol Commun. 2022; 6: 1289-1300https://doi.org/10.1002/hep4.1904
        • Sahney A.
        • Wadhawan M.
        • Kumar A.
        Tinospora cordifolia—A Double Edge Sword?.
        Journal of Clinical and Experimental Hepatology. 2022; 12: 230-231
        • Gupta D.
        • Sonawane A.
        Heart-leaved Moonseed- Innocuous or Baneful.
        Journal of Clinical and Experimental Hepatology. 2022; 12: 254-255
        • Rastogi M.
        • Jagdish R.
        • Vij V.
        • Bansal N.
        Herbal Immune Booster–Induced Liver Injury in the COVID-19 Pandemic.
        Journal of Clinical and Experimental Hepatology. 2022; 12: 258-259
        • Gupta H.
        • Nigam N.
        • Singh S.
        • Roy A.
        • Dhiman R.
        Immune Boosting Gone Wrong? A COVID-Concoction-Conundrum.
        Journal of Clinical and Experimental Hepatology. 2022; 12: 235-236
        • Parikh P.
        Knowledge of Herbal Medicines – Is a Reverse Bridge Course an Urgent Necessity?.
        Journal of Clinical and Experimental Hepatology. 2022; 12: 249-251
        • Gupta S.
        • Dhankhar Y.
        • Har B.
        • Agarwal S.
        • Singh S.
        • Gupta A.
        • et al.
        Probable Drug-Induced Liver Injury Caused by Tinospora species: A Case Report.
        Journal of Clinical and Experimental Hepatology. 2022; 12: 232-234
        • Björnsson E.S.
        • Navarro V.J.
        • Chalasani N.
        Liver Injury Following Tinospora Cordifolia Consumption: Drug-Induced AIH, or de novo AIH?.
        J Clin Exp Hepatol. 2022; 12: 6-9
        • Jaiswal Y.S.
        • Williams L.L.
        A glimpse of Ayurveda - The forgotten history and principles of Indian traditional medicine.
        J Tradit Complement Med. 2016; 7: 50-53
        • Kadir F.A.
        • Othman F.
        • Abdulla M.A.
        • Hussan F.
        • Hassandarvish P.
        Effect of Tinospora crispa on thioacetamide-induced liver cirrhosis in rats.
        Indian J Pharmacol. 2011; 43: 64-68
        • Langrand J.
        • Regnault H.
        • Cachet X.
        • Bouzidi C.
        • Villa A.F.
        • Serfaty L.
        • et al.
        Toxic hepatitis induced by a herbal medicine: Tinospora crispa.
        Phytomedicine. 2014; 21: 1120-1123
        • Begum N.
        • Raj P.
        • Ravikumar K.
        Comparative pharmacognostical and histochemical studies on the three different species of Tinospora on stem and leaf.
        Journal of Pharmacognosy and Phytochemistry. 2019; 8: 650-655
        • Parveen A.
        • Adams J.S.
        • Raman V.
        • Budel J.M.
        • Zhao J.
        • Babu G.N.M.
        • et al.
        Comparative Morpho-Anatomical and HPTLC Profiling of Tinospora Species and Dietary Supplements.
        Planta Med. 2020; 86: 470-478
      2. Why Ayurveda gained a lot of popularity since Covid-19 - Times of India [Internet]. The Times of India. Available from: https://m.timesofindia.com/life-style/health-fitness/health-news/why-ayurveda-gained-a-lot-of-popularity-since-covid-19/amp_articleshow/83058289.cms

        • Sharma R.
        • Prajapati P.K.
        Remarks on "Herbal Immune Booster-Induced Liver Injury in the COVID-19 Pandemic - A Case Series.
        J Clin Exp Hepatol. 2022; 12: 247-248
        • Huang W.-T.
        • Tu C.-Y.
        • Wang F.-Y.
        • Huang S.-T.
        Literature review of liver injury induced by Tinospora crispa associated with two cases of acute fulminant hepatitis.
        Complement Ther Med. 2019; 42: 286-291
        • Upadhyay A.K.
        • Kumar K.
        • Kumar A.
        • Mishra H.S.
        Tinospora cordifolia (Willd.) Hook. f. and Thoms. (Guduchi) - validation of the Ayurvedic pharmacology through experimental and clinical studies.
        Int J Ayurveda Res. 2010; 1: 112-121
        • Singh B.
        • Nathawat S.
        • Sharma R.A.
        Ethnopharmacological and phytochemical attributes of Indian Tinospora species: A comprehensive review.
        Arab J Chem. 2021; 14103381
        • Nagarkar B.
        • Kulkarni R.
        • Bhondave P.
        • Kasote D.
        • Kulkarni O.
        • Harsulkar A.
        • et al.
        Comparative Hepatoprotective Potential of Tinospora cordifolia, Tinospora sinensis and Neem-guduchi.
        J Pharm Res Int. 2013; 3: 906-916
        • Kress W.J.
        • Erickson D.L.
        A two-locus global DNA barcode for land plants: the coding rbcL gene complements the non-coding trnH-psbA spacer region.
        PLoS One. 2007; 2: 508
      3. Council of Scientific & Industrial Research, Govt of India. The Wealth of India, Raw Materials; New Delhi, India: The Publication & Information Directorate. 1982;10:252.

      4. Sivarajan VV, Balachandran I. Ayurvedic drugs and their plant sources. New Delhi, India: CBS Publishers & Distributors Pvt Ltd. 1999;527–544.

        • Qudrat-I-Khuda M.
        • Khaleque A.
        • Bashir A.
        • Roufk M.D.A.
        • Ray N.
        Tinospora cordifolia—isolation of tinosporon, tinosporic acid and tinosporol from fresh creeper.
        Sci Res. 1966; 3: 9-12
        • Phan V.K.
        • Chau V.M.
        • Nguyen T.D.
        • La V.K.
        • Dan T.H.
        • Nguyen H.N.
        • et al.
        Aporphine alkaloids, clerodane diterpenes, and other constituents from Tinospora cordifolia.
        Fitoterapia. 2010; 81: 485-489
      5. Patel, U., Girme, A., Patel, K. et al. A validated HPTLC method for quantification of cordifolioside A, 20-β-hydroxyecdysone and columbin with HPTLC–ESI–MS/MS characterization in stems of Tinospora cordifolia. JPC-J Planar Chromat.

        • Maurya R.
        • Wazir V.
        • Kapil A.
        • Kapil R.S.
        Cordifoliosides A and B, two new phenylpropene disaccharides from Tinospora cordifolia possessing immunostimulant activity.
        Nat Prod Lett. 1996; 8: 7-10
        • Sharma U.
        • Bala M.
        • Kumar N.
        • Singh B.
        • Munshi R.K.
        • Bhalerao S.
        Immunomodulatory active compounds from Tinospora cordifolia.
        J Ethnopharmacol. 2012; 141: 918-926
        • Ahmad W.
        • Jantan I.
        • Bukhari S.N.
        Tinospora crispa (L.) Hook. f. & Thomson: A Review of Its Ethnobotanical, Phytochemical, and Pharmacological Aspects.
        Front Pharmacol. 2016; 7: 59