Heavy metal contamination and genotoxicity assessment of anthelmintic medicinal plants in Assam, India

Medicinal plants possess several metals obtained naturally or through pollution. The study evaluates the genotoxic potentials and the presence of essential elements and toxic heavy metals in Sesbania sesban var. bicolor (leaves), Cyperus compressus (roots) and Asparagus racemosus (roots), which are used as anthelmintics in the traditional medicine of the Santhals in India. Genotoxicity study was performed as per Organization for Economic Co-operation and Development (OECD) guidelines in rats. Element contents were evaluated using inductively coupled plasma-optical emission spectrometry (ICP-OES). Administration of S. sesban var. bicolor (400 mg/kg), C. compressus (700 mg/kg) and A. racemosus (500 mg/kg) did not cause any abnormalities in the chromosomes. Elemental analysis revealed the presence of lead (5.00 ± 0.05, 4.98 ± 0.01, 4.99 ± 0.07 ppm), cadmium (4.99 ± 0.04, 4.90 ± 0.03, 5.00 ± 0.08 ppm) and arsenic (0.98 ± 0.03, 0.41 ± 0.05, 0.92 ± 0.03 ppm), respectively. Although genotoxic potentials were lacking, the presence of toxic heavy metals in them is a cause of concern.


Introduction
In recent years a growing interest in herbal medicine has developed due to its long history of usage and belief that they are natural and hence safe (Woo et al. 2012). However, considering medicinal plants to be safe and e cacious because of their long history of traditional use is not only unreliable but also  (Yuan et al. 2011). This has encouraged researchers to investigate medicinal plants for the presence of toxic elements in them (Brima 2017). Elements such as Al, As, Cd, Cu, Pb, Hg, Tl, Sn, and Zn are common adulterants found in medicinal plants (Woo et al. 2012). Toxic potentials of herbal medicine are attributed to two major factors, intrinsic (direct) which include active chemical constituents present in the herbs and extrinsic (indirect) which include contamination, adulteration and misidenti cation of the medicinal plants (Drew and Myers 1997). A vast majority of herbal medicine still await proper clinical trials and the assessment of their e cacy and safety has become a growing concern. Owing to reports of toxicity caused by usage of traditional herbal medicines, a thorough investigation on safety pro les of medicinal plants seems necessary (Nath et al. 2017).
The leaves of S. sesban var. bicolor, (Fabaceae) and roots of Cyperus compressus (Cyperaceae) and Asparagus racemosus (Asparagaceae) are used in the traditional medicine system of the Santhals to treat helminth infections. A. racemosus has also been claimed to possess anti-mutagenic properties (Akram et al. 2020). Since these medicinal plants are used frequently, this study was conducted to evaluate their potential genotoxic effects, if any in rodent models and also to evaluate the presence of both essential and toxic elements in them.

Chemicals
The following chemicals were procured from the respective companies to carry out this study. Colchicine

Genotoxicity studies
Genotoxicity studies were conducted on Wistar rats following the protocol of Guo and Wu (2008) with minor modi cations. The animals were dosed according to the dosage administered by traditional healers to their clients. The recommended dose was taken as the median dose and two doses, one exponentially lower and the other higher that the median doses were selected for the study. Accordingly, animals were dosed 100, 200 and 400 mg/kg b.w. of S. sesban var. bicolor leaf extract, 175, 350 and 700 mg/kg b.w. of C. compressus root extract and 125, 250 and 500 mg/kg b.w. of A. racemosus root extract.
Animals were divided into three groups, with ve animals in each. Group 1 served as the control and received only the vehicle. Group 2 and 3 received highest and lowest dose respectively. All the animals were dosed for 14 days. At the end of the dosing period, the animals were subjected to mitotic arrest by injecting colchicine intraperitonially (4mg/kg). The animals were then sacri ced after 2 h, according to the prescribed guidelines of IEC (Animal Model) of NEHU.
The femurs were dissected out and ushed with pre warmed (37±1°C) 0.06 M KCl through a 40 μm cell strainer into a 50 ml centrifuge tube. The samples were kept in Carnoy's xative then centrifuged at 1500 rpm for 15 min (4 times) with KCl. After each centrifugation, the supernatant was discarded and the pellet was used to carry out further examinations. Finally, the cells were resuspended in 2 ml xative and stored at -20° C for further use.
To make the slides, a few drops of the cell suspension was dropped on grease free, methanol cooled slides from a height of about 1 m. The slides were then warmed in ame for 2 seconds to allow the cells to spread following which they were allowed to dry, and then stained in 5% buffered Giemsa stain, (pH -7) for 10 minutes. The slides were then washed in distilled water to remove excess stain and allowed to dry. The slides were mounted in DPX and observed in oil at 100 x for various chromosomal aberrations which included chromatid breaks (CB), isochromatid breaks (ICB), chromosomal fragments (CF), exchanges (E) and sister chromatid unions (SCU). To score various chromosomal aberrations, about 100 well spread metaphase plates were examined per animal (Nath et al., 2017).

Elemental analysis
To perform elemental analysis, the powdered plant material (1 g) was subjected to acid digestion using HCl and HNO 3 in the ratio 2:1. The acid mixture was made to evaporate in a hot plate following which distilled water was added and then ltered. The ltrate was made to a nal volume of 50 ml then subjected to elemental by inductively coupled plasma-optical emission spectrometry (ICP-OES) using Thermo Fischer iCAP 7600. Essential elements such as chromium (Cr), cobalt (Co), copper (Cu), iron (Fe), manganese (Mn) and nickel (Ni) were estimated. Toxic heavy metals arsenic (As), cadmium (Cd), and

Statistical analysis
The results obtained in the study were analysed using OriginPro 8. All data are represented as mean ± standard error of mean (SEM). P≤0.05 was considered to be signi cantly different.

Genotoxicity studies
Genotoxicity studies of the three test plants did not reveal any abnormalities or deviations from the control group. The photomicrographs of bone marrow metaphase chromosome spreads revealed intact and normal chromosomes (Fig. 1).

Elemental Analysis
Elemental analysis of the studied plants revealed that the levels of essential elements namely, Co, Cr and Ni were found to be above the permissible limits as set by the WHO/FAO in all the three studied plant extracts. Cu, Zn, Fe, and Mn were found to be present in safe concentrations. Fe was however not detected in C. compressus. Two of the estimated toxic elements namely, Cd and As were found to be above the permissible limits of WHO/FAO in all the three plant species. Although Pb was detected in all the three plant extracts, it was found to be below the permissible limits (Table 1).  Exposure to As occurs through ingestion, inhalation, dermal contact and parenteral route (Tchounwou et al. 2003) and is known to cause vitamin A de ciency resulting in night blindness (Saha et al. 1999). The exposure of these metals may affect different organ systems, including the cardiovascular, dermatologic, nervous, hepatobiliary, renal, gastro-intestinal, and respiratory systems (Tchounwou et al. 2003).
Cr exposure may cause oxidative stress in the cell resulting into damage of DNA and proteins (Stohs and Bagchi 1995). It possesses mutagenic properties and is categorized as a group 1 human carcinogen (Zhang et al. 2011 Conclusions This is the rst study in the region that has detected the presence of toxic elements in medicinal plants.
Although the studied plants did not reveal genotoxic effects, the presence of toxic heavy metals in them is a cause of concern since these medicinal plants are used widely. The accumulation of these toxic elements can be attributed to presence of these elements in the soil of the studied area. Their uncontrolled consumption can result in adverse toxic effects in their users and hence their usage in the area should be done with caution.

Declarations Funding
The study did not receive any funding from any organizations or individuals.

Competing interests
All the authors declare that they have no competing interests.

Availability of data and materials
All data generated pertaining to this study have been included in this article.

Code availability
Not applicable Authors' contribution AKY proposed, conceptualized and supervised the study and interpreted the genotoxicity results of the study. ADS performed the genotoxicity experiments, handled the animals, analysed the data and wrote the rst draft. All authors read and approved the nal manuscript.

Ethics approval
All animals were procured from the animal room of the Department of Zoology, North-Eastern Hill University (NEHU). Genotoxicity studies were performed after approval and written consent was obtained from the Department of Zoology and IEC (Animal models), NEHU (