Reporting negative Ames test results for Indian Acorus calamus L., rhizome, extracts, and beta asarone

Abstract

OBJECTIVE:

Acorus calamus L. (Sweet Flag), known in India as “Vacha,” is widely used in traditional medicine, particularly for cognitive enhancement in infants. While traditionally considered safe, β-asarone – a key constituent – has shown potential genotoxicity in some in vitro studies. This study aimed to evaluate the mutagenic potential of Indian A. calamus rhizome, its extracts, and pure β-asarone using the Ames test in accordance with Organization for Economic Co-operation and Development guidelines.

MATERIALS AND METHODS:

Samples tested included pure β-asarone, dried rhizome powder, and extracts prepared in acidic and alkaline aqueous media. Marker compounds (β-asarone, α-asarone, and shyobunone) were quantified. The Ames test was conducted under GLP using Salmonella typhimurium strains TA97a, TA98, TA100, TA102, and TA1535 (Moltox Inc., USA). Each strain was tested with and without metabolic activation using Aroclor 1254-induced rat liver S9 homogenate. Positive controls included 2-aminoanthracene, sodium azide, mitomycin C, 9-aminoacridine, and 4-nitro-o-phenylenediamine. The vehicle was used as a negative control. Doses tested ranged from 39.06 to 5000 µg/plate.

RESULTS:

No mutagenic response was observed in any of the tested samples across all five strains under either condition (±S9). Positive controls showed expected increases in revertant colonies. Revertant counts for test samples did not reach the threshold for a positive response (≥3 × control for TA1535, TA97a; ≥2× for TA98, TA100, and TA102).

CONCLUSIONS:

Under the test conditions, A. calamus rhizome and β-asarone did not exhibit mutagenicity. Further studies, including chronic toxicity and carcinogenicity evaluations, are recommended to confirm safety across formulations.

Introduction

Acorus calamus L. (Sweet Flag) is an important herb known globally. In India, its rhizome (Indian Name: Vacha) is well known in traditional knowledge and is gaining attention in drug development for brain functions. Vacha is described in Bhavprakash Nighantu.[1] It is known for its use as appetizer, digestive, nootropic, works in brain disorders, useful in treating hysteria, respiratory disorders, throat problems, pain relief including headache, migraine, Parkinson’s, and other brain disorders. A. calamus dried rhizome is used extensively in children and infants as a cultural practice by rubbing a dried clean rhizome on a grinding stone a few times with few drops of milk (either mother’s milk or cow’s milk) and the paste so obtained (will be few milligrams) is made to be licked by the child. It is believed that children who consume this paste experience enhanced brain development, improved learning abilities, and earlier speech development. In addition, with A. calamus rhizome, one black pepper, one dried Haritaki (Terminalia chebula, myrobalan), and Saunth (dried ginger, Zingiber officinale) is also rubbed along as illustrated in Figure 1a and b. Normally, 5 or maximum 10 circular movements are done on the grinding stone. One traditional medicine expert had told this author of a verse (in the Oriental Language Sanskrit) which reads as – “Vaachayati iti vacha ha” – meaning that which promotes speaking ability is called Vacha, an example of signature nomenclature adopted in tradition. So much so, if an infant or a child is speaking fluently or speaks a lot, questions are asked – did the child take too much of Vacha? This demonstrated acceptance of the safety profile of this rhizome. However, some old publications (referenced elsewhere in the paper) reported that pure β-asarone demonstrated in vitro genotoxicity/carcinogenicity, causing a concern.

Figure 1.

(a) Grinding stone with Acorus calamus rhizome. See in the center a small quantity of “paste of calamus ground with milk”, (b) Grinding stone with A. calamus Rhizome. See in the center a small quantity “paste of calamus ground with milk”. Shown on Right-hand - one black pepper, one myrobalan, and one dry ginger

A. calamus is not only cultivated and recognized in India but is also renowned worldwide. It is one of the few herbs that are known to be present with differing chromosome numbers, known as “ploidy status.” Depending on the ploidy nature, their chemistry also differs. Most commonly, 3 marker compounds – β-asarone, α-asarone, and shyobunone are estimated in A. calamus. Table 1 provides a summary of the data of these three markers in A. calamus of different chromosome numbers.

Table 1.

Ploidy status, occurrence, and some chemistry of Acorus calamus[2]

Taxonomic nature	Occurs in	β-asarone content	α-asarone content	Shyobunone content
Type I-diploid (2n=24)*	Primarily in North America and to some extent in Siberia	Free	Low levels or nil	13%–45%
Type II-triploid (2n=36)*	Mainly in Europe and to some extent in the Himalayan Region of India and Asia	About 0.3% on average	Not known	3.6%–32%
Type III- tetra ploidy (2n=48)*	Sub-tropical/tropical South and Southeast Asia, and in Temperate Far East Asia (including former USSR and Korea)	48%–84% (and in some locations only 2%–3%) (USSR and Korea)	2.7%–3.7%	1.4%–6.2%
Type IV-tetra ploidy (2n=48)*	Some parts of India, Pakistan, and Philippines	4%–8%	2.7%–3.7%	Not reported
Type IV-tetra ploidy (2n=48)*	Japan	20%–76%	Not reported	Low levels. not reported

*Detailed chemical composition of these Acorus calamus cytotypes is not being given for brevity

Environmental factors are responsible for determining the variety of A. calamus and the ploidy status. It is believed that Indian A. calamus may have undergone some changes in this regard. Recently, the Institute of Himalayan Bioresource Technology (IHBT – a National Laboratory of Council of Scientific and Industrial Research – CSIR) studied a few samples from the Himalayan region for their ploidy status and chemical composition. Their findings revealed that Indian A. calamus is not a tetraploid.[3] In addition, Sharma et al., 2020, discussed a detailed review of the Role of Vacha (A. calamus Linn.) in neurological and metabolic disorders, highlighting its importance through evidence from ethnopharmacology, phytochemistry, pharmacology, and clinical studies.[4]

Only one in vivo study has reported long-term administration of Vacha at doses higher than human dose showed toxicities, including carcinogenicity.[5] In general, the research conducted on rats has shown that β-asarone could induce liver cancer in high doses over prolonged periods of exposure. However, there are no recent studies have been conducted on this topic, except during 1980–1990. Uebel et al., 2021, reviewed the current status of toxicological evaluation of the three asarone isomers.[6] No human studies on the safety or mutagenicity/carcinogenicity of β-asarone or A. calamus are found in the literature by the authors. The same study documented a few animal studies reporting potential carcinogenicity to the liver at doses high compared to the human dose of A. calamus rhizome, following repeated administration for 23 months.[6] The World Health Organization (WHO) has published a monograph no. 498, β-asarone (WHO Food Additives Series16), which provides detailed information on the assessments mostly covering A. calamus oil and extracts.[5] No specific data or assessment are seen in this document specifically stating on Indian A. calamus.

In India, A. calamus is widely used in many traditional ayurvedic recipes and is considered safe to consume under medical advice. According to Bhavaprakash Nighantu (page 42), the recommended dose ranges from 25 to 500 mig. However, at a dosage of 1–2 g, it acts as a Vamak (emetic), inducing vomiting.[1]

A nonexhaustive list and some information about the content of the A. calamus rhizome in these recipes are provided in Table 2.

Table 2.

Traditional Ayurvedic recipes containing Acorus calamus as an ingredient[7]

Name of the preparation and format	Text reference#	Dosage	Quantity of Vacha as per text reference	Quantity of Vacha/dose
Aravindasava (A liquid)	Bhaishjyaratnavali, Balarogadhikara 185	3–12 ml	48 g in 35 kg	4 mg–16 mg
Asvagandhadyarista (A liquid)	Bhaishjyaratnavali, Murcchadhikara 13-17	12–24 ml	384 g in 39 kg	1.18 g–2.36 g
Brahma Rasayan (Semi solid jam like)	Astangahrudaya Uttarasthana, Adhyaya 39; 15-19	12 g	192 g in 110 kg	21 mg
Brhanmanjisthadi Kvatha Curna (powder)	Sarangdharasamhita, Madhyamakhanda, Adhyaya 2; 137-141	48 g	1 part of 45	1.06 g
Rasnadi Kvatha Curna (Maha) (powder)	Sarangdharasamhita, Madhyamakhanda, Adhyaya 2; 89-91	48 g	1 part of 75	63.84 mg
Maha Yogaraja Guggulu (Tablets/pills)	Sarangdharasamhita, Madhyamakhanda, Adhyaya 7; 56-60	½–1 g	3 g in 696 g	2.15 mg–4.3 mg
Dadhika Ghrta (Ghee Preparation-Clarified butter)	Ashtangahrudaya, Cikitsasthana, Adhyaya 14, 13-19	12 g	2.4 g in 16 kg	1.8 mg
Dhanvantara Ghrta (Ghee Preparation-Clarified butter)	Ashtangahrudaya, Cikitsasthana, Adhyaya 12, 19	48 g	11.63 g in 43.23 kg	13 mg
Narayana Curna (Powder)	Ashtangahrudaya, Cikitsasthana, Adhyaya 15, 14-16	1–3 g	1 part of 40	25 mg–75 g
Nimbadi Curna (Powder)	Bhaisajyaratnavali, Vataraktadhikara; 31-33	1–3 g	12 g of 432 g	27.7 mg–83.3 mg
Sudarsana Curna (Powder)	Bhaishjyaratnavali, Jvaradhikara, 308-312	1–2 g	1 part of 44	45.4 mg–90.8 mg
Kunkumadi Taila (Medicated oil)	Yogratnakar, Ksudrarogadhikara; 740	External	12 g in 8 kg	0.14%
Kottamcukkadi Taila (Medicated oil)	Sahasrayoga, Tailaprakarana; 12	External	21 g in 6.5 kg	0.32%
Citrakadi Taila (medicated oil)	Susrutsamhita, Bhagandra cikitsa; 50	External	16 g in 4 kg	0.40%
Rasnadi Curna/(Powder) Rasnadi Lepa (Semi solid for external application)	Sahasrayoga, Curnaprakarana; 61	External	1 part of 24	NMT 3 g
Grhadhumadi Lepa (Semi solid for external application)	Astangahrudaya, Cikitsasthana, Adhyaya 22; 36	External	1 part of 6	NMT 3 g
Sanjivani Vati (Pills/tablets)	Sarangdharasamhita, Madhyamakhanda, Adhyaya 7; 18-19	125 mg	1 part of 10	12.5 mg
Pradarantaka Lauha (Powder)	Bhaishjyaratnavali, Striogadhikara 75-76	500 mg	1 part of 30	16.65 mg
Candrodaya Vartti (powder)	Bhaishjyaratnavali, Netrarogadhikara 105	External	1 part of 9	NMT 3 g
Tamradi gutika (pills/tablets)	Sahasrayoga, Urdhvangarogacikitsa (netraroga); 51	External	10 parts of 61	15%
Ratnagiri Rasa (powder)	Bhaishjyaratnavali, Jvaradhikara; 433-436	125 mg	Q.S. (for Bhavana) 3 times	NMT 3 g
Kasturyadi (Vayu) Gutika (Pills/tablets)	Sahasrayoga, Gutikaprakarana; 8	125 mg	1 part of 41	3.03 mg
Kankayana Gutika (Pills/tablets)	Bhaishjyaratnavali, Gulmadhikara; 56-58	2 g	48 g in 816 g	118 mg
Khadiradi Gutika (Mukharoga) (Pills/tablets)	Carakasamhita, Cikitsasthana, Adhyaya 26; 201-206	2 g	12 g in 27.45 kg	0.87 mg

^#Brief reference to the authoritative texts of Ayurveda is given for each of the preparation cited in the table. Caution: These preparations are not for self-medication. Consult an Ayurvedic Vaidya for advice and prescription. NMT=Not more than; Q.S.=Quantity sufficient

No animal or human studies have been conducted in India specifically on the Indian A. calamus rhizome or β-asarone pure compound. Therefore, the authors decided to conduct a study on samples of pure β-asarone, dried rhizome powder, and extracts of dried rhizome prepared in both acidic and alkaline water mediums, using the Ames test in accordance with Organisation for Economic Co-operation and Development (OECD) guidelines to assess potential carcinogenicity. Details are reported in the following section.

Materials and Methods

Pure marker compounds of β-asarone (Lot No. T23E004, vials of 25 mg and 600 mg) from M/S Natural Remedies, Bangalore, α-asarone (Lot No. CFN93217, 20 mg), and shyobunone (Lot No. CFN98079, 20 mg) from M/S Chemfaces, China, along with their analytical data, were obtained and used. These markers had a peak purity above 90%. Dried rhizomes of A. calamus were sourced from the Authentic Botanical Council, which also supplied a certificate confirming their botanical identity. Samples have been retained for future reference.

While conventional botanical studies often employ aqueous or hydroalcoholic extracts, the present study aimed to simulate physiological conditions encountered during oral ingestion. Since herbal constituents are exposed to the acidic environment of the stomach (pH ~ 1–2) and the alkaline environment of the intestine (pH ~ 7–8), two aqueous extracts were prepared accordingly. This approach allowed for the evaluation of the mutagenic potential of not only the native compounds but also any degradation or transformation products generated under gastrointestinal-like conditions.

Importantly, β-asarone is known to be sparingly soluble in water and chemically unstable under stress. A recent study reported that β-asarone undergoes significant degradation under various conditions: 16.4% in acid, 23.9% in alkaline media, 19.6% under oxidative, 21.8% thermal, and 25.2% under photolytic stress. In aqueous solutions, β-asarone may also isomerize to α-asarone due to their cis-trans relationship. These findings underscore the importance of testing extracts prepared in pH-adjusted media to reflect chemical instability that may occur upon oral consumption.[8]

For the acidic extract, 500 g of coarsely powdered A. calamus rhizome was boiled in 2000 mL of 0.1 N hydrochloric acid (adjusted to pH 1.0) until the volume was reduced by half. The mixture was filtered, concentrated under vacuum, and dried to yield approximately 77.5 g of powder (yield: 15.5%).

For the alkaline extract, 500 g of the same rhizome powder was boiled in 2000 mL of sodium hydroxide solution (adjusted to pH 8.0). The extract was similarly reduced in volume, filtered, concentrated, and dried to yield approximately 82.5 g of powder (yield: 16.5%).

Due to budgetary constraints, enzyme-mediated digestion and other solvent-based extractions were not pursued. The acidic and alkaline aqueous extractions were considered relevant surrogates for gastrointestinal exposure in evaluating chemical stability and safety.

All samples were analyzed for β-asarone, α-asarone, and shyobunone content using validated high-performance liquid chromatography (HPLC) methods before subjecting them to the Ames test.

Analytical methods

Chromatographic conditions for α-asarone and β-asarone

HPLC system: make: Shimadzu HPLC System Model: Quaternary pump (model-LC 20AD), Auto sampler (model-SIL-20AC HT), diode array detector (model-SPDM20A), Column oven (CTD-10AS VP) Software: “Lab solution software version 6.12 SPI.” Stationary phase: Kromasil C18 Column (Dim: 4.6 mm × 150 mm) Mobile phase: 0.05% ortho phosphoric acid (OPA) in Water: Acetonitrile (55:45). Wavelength: 254 nm Sample injection volume: 10 µl Flow rate: 1 ml/min Column temperature: 25°C. Elution mode: Isocratic Run time: 20 min Retention time: β-asarone - 12 min 5 s. α-asarone - 14 min 4 s. Further details on the method validation and quantification of α-asarone and β-asarone in A. calamus extract samples are provided in a separate publication.[7]

Chromatographic conditions for shyobunone

HPLC system: Make: Shimadzu HPLC System Model: Quaternary pump (model-LC 20AD), Auto sampler (Model-SIL-20AC HT), Diode array detector (model-SPDM20A), Column oven (CTD-10AS VP) Software: “Lab solution software version 6.12 SPI (Shimadzu Corporation [Asia Pacific] PTE LTD, Kyoto, Japan).” Stationary phase: Kromasil C18 Column (Dim: 4.6 mm × 150 mm) Mobile phase: 0.05% OPA in Water: Acetonitrile: Methanol (15:40:45) wavelength: 210 nm Sample injection volume: 10 µl flow rate: 1 ml/min Column temperature: 25°C. Elution mode: Isocratic Run time: 10 min Retention time: 6 min 5 s. Further details on method validation and quantification of shyobunone in A. calamus extract samples are reported in a separate publication.[7]

Table 3 outlines the concentrations of the markers β-asarone, α-asarone, and shyobunone in the samples prepared for the study.

Table 3.

Contents of the markers in the samples prepared for the study

Sample description	Content of β-asarone (% w/w)	Content of α-asarone (% w/w)	Content of shyobunone (% w/w)
Dried rhizome powder (dissolved in ethanol for testing)	12.70	0.10	0.13
Water soluble extract	2.03	Not determined	Not determined
Ethanol+water extract (70:30)	12.73	Not determined	Not determined
Methanol extract	17.93	Not determined	Not determined
Extract in water in acidic medium (0.1N HCl)	1.20	0.030	Not detected
Extract in water in an alkaline medium (NaOH at about 7.2 to maximum 8 pH)	0.10	0.003	Not detected

Ames testing

Samples of A. calamus dried rhizome, its extracts in acidic medium and in alkaline medium, and pure β-asarone were subjected to the Ames test as per the OECD guidelines. The Ames testing was conducted by the subject experts at the Indian Institute of Toxicology Research (a National Laboratory of Council of Scientific and Industrial Research-CSIR) under Good Laboratory Practices. Necessary cultures were validated and used, and standard methods were employed.

Testing of β-asarone pure compound

The preliminary cytotoxicity study was performed at concentrations of 39.06, 78.125, 156.25, 312.5, 625, 1250, 2500, and 5000 µg/plate (90 mm petriplates), in the absence of a metabolic activation system based on precipitation test results. Preliminary cytotoxicity study of the test item and vehicle control was conducted using TA-98 and TA-100 tester strains by the plate incorporation method, in the absence of metabolic activation. Each concentration of test items, including the vehicle control, was tested three times in triplicate. In test strains TA-98 and TA-100, no precipitation was observed till the highest concentration (5000 µg/plate), and bacterial growth was observed on MGA plate. However, no precipitation and no cytotoxicity of the test item at concentrations 39.06, 78.125, 156.25, 312.5, 625, 1250, 2500, and 5000 µg/plate in the medium plate were observed. Therefore, in the mutagenicity study, subsequent highest concentrations of 312.5, 625, 1250, 2500, and 5000 µg/plate were chosen. Based on the findings of the mutagenicity test, no positive mutagenic response was observed with any of the tester strains (i.e., TA97a, TA98, TA100, TA102, and TA1535), in the absence of S9 activation or in the presence of metabolic activation at concentrations of 312.5, 625, 1250, 2500, and 5000 µg/plate. The preincubation method using Salmonella typhimurium test strains on the aforementioned concentrations was performed for mutagenicity study. The mean number of revertant colonies up to 5000 μg/plate did not show a significant increase relative to the respective control plates. Respective positive control exposed plates displayed a significant increase in the mean number of reverting colonies in all strains, demonstrating the sensitivity of the test system and the suitability of the conditions used in this analysis.

Same negative Ames test results were obtained for A. calamus dried rhizome and its extracts in both acidic and alkaline media. The four test reports conclude as under the test conditions, the test item “β-asarone pure marker, dried rhizome powder of A. calamus, and its extracts in both acidic and alkaline media” did not show any potential for mutagenic response with any of the tester strains either in the absence of metabolic activation (−S9) or in the presence of metabolic activation (+S9) (reports numbers-CSIR-IITR/GLP/326 [09.01.2024], 327 [09.01.2024], 328 [06.02.2024] and 329 [09.01.2024), available on file with the author).

Results

It was interesting to note that all the four samples, namely, dried calamus rhizome powder, its extract in acidic medium, alkaline medium, and pure β-asarone showed similar results, which were all reported as negative to the Ames test. None of the samples showed any potential carcinogen-related precipitation or activity when tested on the respective microbial cultures specified in OECD guidelines. Further to the common understanding that if a pure marker compound present in a botanical is found to exhibit potential toxicity, it is generally extrapolated that the botanical may also show a similar property. This study observed that conventional extraction methods utilizing water, or ethanol and water (or methanol) yielded higher percentages of the β-asarone content. However, when the same herb was extracted in acidic or alkaline media, the β-asarone content was significantly reduced, ranging from approximately 12.73% to 1.2%. Similarly, the α-asarone content decreased to 0.10%, whereas shyobunone was not detected. This reduction can be attributed to the instability of β-asarone and α-asarone in acidic and alkaline conditions, leading to their degradation during the extraction process, which involved boiling in these media. In addition, lower solubility in these media has some impacts. In many traditional preparations, A. calamus rhizome is purified by “shodhan” process involving the extraction of vacha in a solution of Chuna (quicklime) which is nothing but alkaline solution.

Discussion

The use of Vacha as described in this paper in small quantities to infants, and the presence of this herb in so many of the traditional preparations as described in this paper, some of which are under medical supervision for long periods, point to its safety profile when used properly. In these traditional preparations, vacha also undergoes “processing specifically” perhaps for enhancing its “safety profile or efficacy.” Authors have not heard of any traditional physicians (to whom they spoke) expressing concern about carcinogenicity due to such consumption, though the herb and its preparations do need to be used under medical supervision. Authors have also not come across any report of such an adverse event due to the consumption of vacha-containing products. Hence, this study and its report present interesting data.

This single study, which solely presents results from the Ames test, highlights the need for further investigation, including a battery of carcinogenicity/mutagenicity testing as well as chronic toxicity studies. Authors will explore funding agencies to undertake these studies. It is not possible to determine the chromosomal numbers with the dried rhizome sample. In a separate study, authors are involved in the collection of fresh A. calamus plants from different locations of India, and evaluating them for their chromosome numbers (ploidy status), which is not being reported here as the work in still underway.

Conclusions

The study demonstrated that A. calamus and its constituents, including β-asarone, as traditionally utilized in India, do not exhibit mutagenic properties under in vitro conditions. It is advised that further investigations be conducted to perform extensive toxicological assessments, including chronic toxicity and carcinogenicity studies, to gain deeper insights into the safety profile of A. calamus across various formulations and traditional applications.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.