Herb-induced Liver Injury-A Guide to Approach. Lessons from the Tinospora cordifolia (Giloy) Case Series Story.

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 high performance thin layer chromatography 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 the 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 the serological/histopathological identification of active herb/metabolites in human tissues.

Simultaneous Quantification of Pharmacological Markers Quercetin and Berberine Using High-Performance Thin-Layer Chromatography (HPTLC) and High-Performance Liquid Chromatography (HPLC) from a Polyherbal Formulation Pushyanuga Churna.

Background: Ayurvedic medicines show great promise due to their holistic approach in the treatment of diseases. But proper standardization is necessary for their integration into mainstream medicine. One such well-known formulation is Pushyanuga Churna. As safety and efficacy of a multiherbal formulation is dependent on the authenticity of the ingredients used, chromatographic techniques play a significant role in the quality control of complex herbal medicines. Objective: In the current research, marker-based standardization of Pushyanuga Churna using validated HPTLC and HPLC methods have been established. Methods: Pushyanuga Churna was prepared in-house using authentic ingredients. Pharmacologically active biomarkers quercetin and berberine were simultaneously estimated using validated HPTLC and HPLC methods. Chromatographic fingerprints were developed for the formulation along with all the ingredients that can be used as tools to identify Pushyanuga Churna. Results: In-house formulation and Patanjali (marketed formulation) were observed to be rich in both markers. Quercetin was found to be more abundant than berberine in the ingredients. The deviation of marker content in marketed formulations can be attributed to variation in the ingredients used in the preparation. Conclusions: The routine use of such scientifically accepted methods will help in establishing the quality of the formulation and help in building confidence in traditional medicine. Highlights: The study highlights evaluation of biomarkers from the formulation and its ingredients as an important method to ensure the batch to batch consistency in quality of the formulation.

Activity based evaluation of a traditional Ayurvedic medicinal plant: Saraca asoca (Roxb.) de Wilde flowers as estrogenic agents using ovariectomized rat model.

ETHNOPHARMACOLOGICAL RELEVANCE: Saraca asoca (Roxb.) de Wilde, Ashok, is a popular traditional plant used for gynecological disorders. In India, the juice of Ashok flowers is traditionally consumed as a tonic by women in case of uterine disorders. But despite the use, its estrogenic potency is not yet evaluated and thus lacks the scientific recognition and acclaim. AIM OF THE STUDY: This study is designed to investigate the estrogenic potential of standardized ethanolic extract of Saraca asoca flowers (SAF) using ovariectomized (OVX) female albino Wistar rat model. MATERIALS AND METHODS: Saraca asoca flowers were extracted in ethanol using hot maceration technique and the extract was standardized in terms of content of four phytoestrogens like quercetin, kaempferol, ß-sitosterol and luteolin using HPTLC technique. Safety of the extract was evaluated at a dose of 2000mg/kg body weight in female albino Wistar rats as per the OECD guidelines. Bilateral ovariectomy surgery was performed for the excision of both the ovaries. The OVX animals were treated with the ethanolic extract of SAF at three dose levels- 100mg/kg, 200mg/kg and 400mg/kg body weight in distilled water as a vehicle, orally once a day for two weeks. Estradiol valerate was employed as a modern drug for comparative evaluation of the results. Estrogenic potency was studied by assaying the activities of serum and plasma marker enzymes and hormones viz. G6PDH, LDH, 17ß-estradiol, progesterone along with cholesterol, triglycerides and HDL, and vaginal cornification. The uterotrophic effect was evaluated by studying the histoarchitecture of the uterus, effect on uterine weight and changes in the levels of uterine glycogen content. RESULTS: HPTLC revealed the presence of markers like quercetin, kaempferol, ß-sitosterol and luteolin from the ethanolic extract of SAF. The content of the four markers was found to be 1.543mg/g, 0.924mg/g, 4.481mg/g and 2.349mg/g, respectively. SAF extract was found to be safe at an oral dose of 2000mg/kg body weight in rats. Among the three doses administered to ovariectomized rats, treatment with high dose was found to be more efficacious when compared with ovariectomized rats. CONCLUSION: The findings of this study firmly support the estrogenic potency of ethanolic extract of SAF which may be by the reason of phytoestrogens.

A validated HPLC-ESI-MS/MS method for quantification of 2-hydroxy-4-methoxy benzoic acid from rat plasma and its application to pharmacokinetic study using sparse sampling methodology.

The phenolic compound, 2-hydroxy-4-methoxy benzoic acid (HMBA), is one of the major phytoconstituents of Decalepis arayalpathra (Joseph & Chandra.) Venter, a rare and endemic medicinal plant found in the Western Ghats of India. HMBA has been attributed to possess several biological effects including anti-inflammatory, anti-pyretic, anti-oxidant and anti-diabetic. The present article describes a rapid and sensitive liquid chromatography-tandem mass spectrometric method (HPLC-MS/MS) for the determination of HMBA in rat plasma. In brief, the developed assay involves pre-treatment of the plasma samples by an optimized solid phase extraction method (recoveries for HMBA greater than 90%) followed by chromatographic separation on a Cosmosil C18 (150mm×4.6mm i.d.; 5µm particle size) analytical column with mobile phase of methanol and 10mM ammonium formate (95:5 v/v; 0.2% formic acid) delivered at a constant flow rate of 1.0mL/min. The detection and quantification was performed using an Applied Biosystems Hybrid Q-Trap API 2000 mass spectrometer equipped with electrospray ionization source (ESI) functioning in negative mode. The developed assay was validated as per the US FDA bioanalytical guidelines with the calibration curve linear over the concentration range of 5.05-2019.60ng/mL (r(2)≥0.9936) for HMBA from rat plasma. Further, the validated HPLC-MS/MS method was successfully applied to pharmacokinetic study of HMBA after oral administration of D. arayalpathra tuber extracts to female albino Wistar rats using sparse sampling methodology. Following oral administration, the maximum mean concentration in rat plasma (Cmax -1301.57±128.22ng/mL) was achieved at 1.5h (Tmax) and the area under the curve (AUC0-48h) was 8985.02±229.54ngh/mL. The elimination half-life (t1/2) and terminal elimination rate constant (Kel) were 2.48h and 0.28 L/h, respectively.

Standardization of Shadbindu Taila: An Ayurvedic oil based medicine.

Shadbindu Taila (ST) is an Ayurvedic formulation used as a remedy for loosening of tooth, weakness of the eyesight, loss of hair, diseases of head, etc., Present study is an attempt to develop some newer approaches for the quality control and standardization of ST. Standardized operating procedure for the preparation of ST was developed in accordance with Ayurvedic Formulary of India. Preliminary phytochemical, physicochemical, and chromatographic evaluation of ST was carried out. Safety of ST was evaluated in terms of skin irritation test and presence of heavy metals. Chemical characterization of ST was done on the basis of kaempferol using validated -High Performance Thin Layer Chromatographic (HPTLC) method. ST did not show presence of any of the heavy metals analyzed and was found non-irritant on rabbit skin. The quality control parameters resulted after scientific evaluation of ST can be used as reference standard for quality control/assurance laboratory of a pharmaceutical firm in order to have a proper quality check over its preparation and processing.

A rapid HPLC-ESI-MS/MS method for determination of ß-asarone, a potential anti-epileptic agent, in plasma after oral administration of Acorus calamus extract to rats.

ß-Asarone (BAS), a phenylpropanoid from Acorus calamus Linn., has shown biological effects in the management of cognitive impairment conditions such as Alzheimer's disease. The present paper describes a selective and sensitive liquid chromatography-tandem mass spectrometric method (HPLC-MS/MS) using electrospray ionization source (ESI) for quantification of BAS in rat plasma. Briefly, the plasma samples were pre-treated using a simple solid-phase extraction method. The separation of BAS and the internal standard, caffeine, was achieved on an Agilent Zorbax XDB C(18) column (50 × 2.1 mm i.d., 5 µm) using 0.2 mL/min isocratic mobile phase flow. The detection was performed using an Applied Biosystems Hybrid Q-Trap API 2000 mass spectrometer equipped with an ESI source operated in positive mode. Also, the developed bioanalytical method was validated as per the US FDA bioanalytical guidelines over the concentration range of 9.79-4892.50 ng/mL (r(2) ≥ 0.9951) for BAS from rat plasma. The mean percentage recovery (n = 3) for the low, middle and high quality control samples was 86.92 ± 3.89, 85.30 ± 1.09 and 87.24 ± 4.03%, respectively. The applicability of the validated HPLC-MS/MS method was demonstrated by successful measurement of BAS from plasma following oral administration of Acorus calamus rhizome extracts to three female albino Wistar rats.

Evaluation of quality and efficacy of an ethnomedicinal plant Ageratum conyzoides L. in the management of pediculosis.

BACKGROUND: Infestation with the head louse, Pediculus humanus capitis, is one of the most common parasitic infestations of human worldwide. Traditionally, the main treatment for control of head lice is chemical control that includes wide variety of neurotoxic synthetic insecticides. The main difficulty posed in controlling the head louse infestation is increasing lice resistance to synthetic pediculicidal drugs. Plant-based drugs; especially essential oil components and standardized extracts have been suggested as an alternative source of materials for insect control. Ageratum conyzoides L. (Asteraceae) has been reported to possess antifungal and insecticidal properties. In the present research work, an attempt has been made to evaluate in vitro pediculicidal activity of A. conyzoides. METHODS: A filter paper diffusion bioassay was carried out in order to determine the pediculicidal activity of different extracts of A. conyzoides. RESULTS: The study elucidates the active plant part and suitable extract responsible for the therapeutic efficacy of this plant in the management of pediculosis. CONCLUSION: Findings of the present study indicate the potential of A. conyzoides extract to be included in the formulations as a pediculicidal agent.

A liquid chromatography/electrospray ionization tandem mass spectrometric method for quantification of asiatic acid from plasma: application to pharmacokinetic study in rats.

RATIONALE: Asiatic acid (AA), a pentacyclic triterpene from Centella asiatica (L.) Urban, has shown numerous therapeutic activities. However, none of the published works to date has used high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) for determination of AA from biological fluids. Therefore, the present paper describes a sensitive HPLC/electrospray ionization (ESI)-MS/MS method for quantification of AA in rat plasma. METHODS: Ammonium adduct formation of AA was essential in the development of a sensitive method with the rat plasma samples being pre-treated by a simple solid-phase extraction method. The separation was achieved on a Cosmosil C(18) column using a gradient mobile phase flow. Detection was performed using an Applied Biosystems API Q-Trap 2000 mass spectrometer equipped with an ESI source operated in positive mode with colchicine used as internal standard. RESULTS: An eight-point calibration curve over the concentration range of 1.02-407.88 ng/mL for AA from rat plasma provided an optimum linear detector response (with r(2) >0.9983). The mean percentage recovery (n = 3) for the low, middle and high quality control samples was 91.23 ± 1.88%, 90.36 ± 0.55% and 89.71 ± 0.21%, respectively. The intra-day and inter-day precision and accuracy of the quality control samples were within ≤5% and ±7% correspondingly. CONCLUSIONS: The developed method was validated as per US FDA guidelines and applicability demonstrated by successful measurement of AA from plasma following oral administration of C. asiatica extracts to Wistar rats. The results suggest that the method could be applied to therapeutic monitoring of AA and pharmacokinetic studies in human volunteers.

Wound healing efficacy of Jatyadi Taila: in vivo evaluation in rat using excision wound model.

ETHNOPHARMACOLOGICAL RELEVANCE: In traditional Indian medicinal treatise there are several Ayurvedic formulations mentioned which have been claimed as potential wound healing agents like Madhu Ghrita and Jatyadi Taila. Jatyadi Taila (JT) is a medicated oil formulation (Taila) popularly used in the treatment of various topical wounds. AIM OF THE STUDY: Though JT has its composition recorded in ancient Ayurvedic texts, there have been minimal attempts to standardize its use in the management of wound. The current work evaluates the wound healing efficacy of JT and also provides evidence of the dermal absorption kinetics of Karanjin from JT. MATERIALS AND METHODS: JT was subjected to preliminary phytochemical evaluation. Therapeutically active marker components ß-sitosterol, lupeol and karanjin were detected and separated using HPTLC. As a part of safety evaluation, skin irritation potential of JT was evaluated on rabbit skin. Excision wound model in rats were used to evaluate the wound healing efficacy of JT. Histopathological and biochemical evaluations of excised skin tissues at wound sites were carried out. The HPTLC method developed was also validated to evaluate the pharmacokinetics of Karanjin from JT after topical application on pinna of rabbit. RESULTS: Preliminary phytochemical evaluation of JT revealed presence of flavonoids, essential oils, tannins, glycosides, steroids and alkaloids while resins were found to be absent. HPTLC confirmed the presence of karanjin, lupeol and ß-sitosterol in JT. JT was found to be non-irritant when applied to the skin of rabbits. Topical application of JT on excision wounds caused significantly faster reduction in wound area as compared to the application of modern topical formulation (Neosporin(®)) and untreated control wounds. Animals treated with JT showed significant increase in protein, hydroxyproline and hexosamine content in the granulation tissue when compared with the untreated controls. Wound healing potential of JT was found to be dose dependant. HPTLC method was successfully used to evaluate the pharmacokinetics of Karanjin after topical application of JT on rabbit pinna. CONCLUSIONS: Current work demonstrates a modern approach towards standardization of the use of traditional topical formulation JT. The results justify the traditional claim of JT for its use in the management of wounds.

A validated RP-HPLC method for quantitation of trigonelline from herbal formulations containing Trigonella foenum-graecum (L.) seeds.

BACKGROUND: Trigonella foenum-graecum (L.) (Fabaceae, Fenugreek) is an important ingredient of Ayurvedic and other marketed herbal formulations. Fenugreek seeds are employed in many traditional systems as an antibacterial and antidiabetic agent, gastric stimulant and galactogogue. Trigonelline, a major phytoconstituent found in fenugreek seeds, shows estrogenic, anti-diabetic and anti-invasive activity. Therefore, it is a suitable bioactive marker to establish the quality of crude drug and its formulations. OBJECTIVE: To develop an efficient and effective RP-HPLC method for estimation of trigonelline from Trigonella foenum-graecum seeds and its marketed herbal formulations. MATERIALS AND METHODS: Separation and detection of trigonelline was carried out on a Cosmosil CN-MS column eluted with methanol:distilled water [95:5, v/v; pH 3.5 using hydrochloric acid]. Detection was carried out at 267 nm using a Photo Diode Array detector. Fenugreek seeds and two marketed herbal formulations were subjected for HPLC analysis of Trigonelline. RESULTS: The RP-HPLC method was validated as per ICH guidelines and the content of trigonelline in marketed polyherbal formulations such as Dibet powder and Amyron syrup was determined. The LOD and LOQ were found to be 5.00 ng/mL and 50.00 ng/mL, respectively. Detector response was linear from 100.00 to 8000.00 ng/mL. The method was found to be simple, sensitive, accurate, reproducible and rugged. CONCLUSION: This work can be recommended for quality assurance and marker-based standardization of formulations containing fenugreek seeds.

Effect of Asteracantha longifolia Nees. against CCl4 induced liver dysfunction in rat.

Significant recovery after treatment with the whole plant slurry of A.longifolia Nees. was observed in plasma AST, ALT and cholesterol levels in CCl4 induced hepatotoxic rats. This was amply supported by electron micrographs, which indicated normalization of cytoarchitecture of mitochondria and endoplasmic reticulum. The results suggest that the slurry of the plant is useful as a liver tonic.