Acorus calamus L. is an emergent aquatic macrophyte that is used in several medicines and food supplements. A. calamus ecotypes with low concentrations of β-asarone can be used for medicinal purposes. We investigated 50 populations of this species, representing the entire natural distribution range in Indian Himalayan Region (IHR), to understand natural variations in β-asarone concentration, ploidy level and genetic divergence. Our results showed that: 1) all four cytotypes (diploid, triploid, tetraploid and hexaploid) of A. calamus exist in natural populations in the IHR; 2) the β-asarone concentration is highly correlated with the ploidy level (diploid and hexaploid populations exhibit low β-asarone concentrations, whereas triploids and tetraploids show high β-asarone concentrations); 3) there is a significant correlation between the β-asarone concentration and its distribution along latitudinal and longitudinal ranges, but not ecological factors (mean annual temperature and mean annual precipitation); and 4) geographic boundaries play an important role in the population divergence of A. calamus. This study provides a foundation for understanding the genetic basis of natural variation in complex traits of A. calamus and for devising conservation strategies for this medically important plant.

https://www.longdom.org/open-access/high-levels-of-diversity-in-the-phytochemistry-ploidy-and-genetics-of-themedicinal-plant-acorus-calamus-l-2167-0412-S1-002.pdf

High Levels of Diversity in the Phytochemistry, Ploidy and Genetics of theMedicinal Plant Acorus calamus L.

Introduction: Acorus calamus Linn. ( A. calamus ) has been found use in medicines to cure fevers, for asthma, bronchitis and as an all-round sedative. β-asarone is an important phytochemical compound present richly in the rhizomes of Acorus calamus that imparts several therapeutic properties to the plant by the virtue of which the plant has occupied a significant therapeutic acclaim in ancient Ayurvedic text and is employed as one of the key ingredients in several traditional and herbal formulations. Thus, the study aims to develop and validate an efficient Reverse Phase High Performance Liquid Chromatography (RP-HPLC) method for quantification of β-asarone from rhizomes of A. calamus of the wild and marketed variety and also intends to apply the validated methods for the estimation of the biomarker from different formulations containing the rhizome as one of its ingredients. Methods: Separation was carried out on Cosmosil C 18 column eluted with mobile phase of methanol: distilled water (50:50, v/v) at flow rate of 1 mL/min. Detection was carried out at 304 nm using a photodiode array detector (PDA) and the method was validated as per International Conference on Harmonization (ICH) guidelines. Rhizome was collected from Kerala and also procured from the market.  Commercial traditional and herbal formulations like Sarasvata Curna, Maanasmitra Vatakam, Khadiradi Vati, Chandra Prabha Vati, Sanjeevani Vati, Mahashankha Vati, Smritisagara Rasa, Abana, Vacadi Taila, and Ashwagandharishtha were further subjected to RP-HPLC for separation and estimation of β-asarone. Results: The limit of detection (LOD) and limit of quantitation (LOQ) levels were found to be 0.025 µg/mL and 0.1 µg/mL, respectively. The content of β-asarone was found to be maximum in the sample collected from Kerala which was 0.2946±0.0152 mg/g. Conclusion: The developed method can be recommended for marker-based standardization and quality assurance of A. calamus and its formulations. Key words: Acorus calamus , Formulations, Rhizome, RP-HPLC, Validation.

Estimation and quantitation of β-asarone from Acorus calamus rhizome and its formulations using validated RP-HPLC method

Morphological characterization of diploid and triploid Acorus calamus (Acoraceae) from southern Western Siberia, parthenocarpy in sterile plants and occurrence of aneuploidy

Sweet flag rhizome (Acorus calamus L.) which belongs to family Araceae and is a perennial, semi-aquatic plant found in both temperate and sub-temperate zones. Super critical fluid extracted sweet flag rhizome oil has good antimicrobial activity. The rhizome oil showed maximum antimicrobial response against Gram positive bacterium Staphylococcus aureus with a diameter of zone of inhibition about 9.2 mm. Whereas the zone of inhibition diameter for Escherichia coli and Pseudomonas aeruginosa was observed to be 7.5 mm and 6.9 mm respectively. Maximum activity of sweet flag rhizome oil was also reported against fungal pathogens. Sweet flag rhizome recorded zone of inhibition of 12.5 mm against Mucur species. Whereas zone of inhibition of 11.5 and 10.3 mm against Aspergillus species and Pencillium. The minimum inhibitory concentration of SC-CO, extracted sweet flag rhizome oil found maximum zone of inhibition for Psedomonas aeruginosa (9.3 mm) and for Staphylococcus aureus (8.1 mm) and these were more sensitive at concentration of 100 μl/ml for sweet flag rhizome oil, whereas Escherichia coli was not at all sensitive at the concentrations used.

https://www.phytojournal.com/archives/2018/vol7issue3/PartAL/7-3-257-275.pdf

Antimicrobial activity of supercritical fluid extracted Acorus calamus Oil against different microbes

Introduction: The aim of study was to develop a simple, sensitive and precise gas chromatographic method for the analysis of β-asaronein ethanolic, ethyl acetate, aqueous extracts from the rhizomes of Acorus calamus and validate according to current ICH guidelines. Followed by evaluation of antimicrobial activity of prepared extracts in comparison with penicillin disc. Methods: The β-asarone was one chief active constituent from the rhizomes of Acorus calamus. The ethanolic, ethyl acetate, aqueous extract of rhizomes was prepared and further evaluated for its antimicrobial activity against Streptococcus Mutants by agar well diffusion method. The GC method was used for the analytical determination of β-asarone. The sample was estimated using gas chromatography with flame ionization as a detector. Nitrogen at a flow rate of 1.18 mL/min was used as a carrier gas and total run time was 10 minutes. The injection port and detector temperature were set to 225˚C and 270˚C, respectively. The retention time of β-asarone was found to be 6.9 minutes. Results: The linearity of the developed method was tested in the range of 100 ng/mL-500 ng/mL for β-asarone, limit of detection and limit of quantification was found to be 22.78 and 69.05 ng/mL respectively and the percentage recovery was from 99.63- 100.64%. Conclusion: A simple, precise and accurate GC-FID method has been developed for the determination of β-asarone in ethanolic, ethyl acetate and aqueous extracts of rhizomes. Key words: Acoruscalamus , β-Asarone, Alcoholic, Aqueous, Ethyl acetate, Extracts, GC- FID, Microbiological evaluaiton, Stability studies.

Gas Chromatographic Method for Analysis β-Asarone in Rhizome extracts of Acorus calamus and Their Microbiological Evaluation

Acorus calamus L. is a well- known herb for its traditional medicinal and pharmaceutical potentials. The rhizome of the plant is mainly used for medicinal purposes because it yields an essential oil known as “Calamus Oil”. The main component of the essential oil is β-asarone. In our present study, 20 different species of Acorus calamus were collected from different parts of South India and North East India. An attempt has been made to characterize all the accessions on the basis of their ploidy level by cytogenetic studies, and determining β- asarone content by HPLC analysis. All the accessions were screened for their ploidy level by staining the root chromosomes at metaphase stage. The plants observed were either diploid or triploid ruling out any tetraploids. HPLC analysis of powdered rhizome extracts for the β- asarone, indicated that it ranged from 2.2% to 7.2%. Our results also have revealed that there is no correlation between the ploidy status and the content of β-asarone. Indian accessions were found to have low to moderate levels of β- asarone content.

/pdf/hplc-profiling-of-b-asarone-content-and-cytogenetic-studies-wf3l7vhzss.pdf

HPLC Profiling of B Asarone Content and Cytogenetic Studies of Medicinally Important Indian Acorus Calamus l., Accessions

Acorus calamus L. (Family: Acoraceae) is a well-known traditional, endangered, medicinal and aromatic plant mainly found in India and China. The plant is also widely used in industrial, pharmaceutical and food industries. In the present study, 20 different accessions of Indian A. calamus were subjected to the study of genetic diversity (RAPD), and cytogenetic and phytochemical (β-asarone) analysis. For RAPD analysis, 9 primers were chosen, which generated 107 DNA fragments. The average percentage of polymorphism was recorded to be 67.23%. The primer OPA 12 showed the highest (100%) polymorphism, whereas the lowest (38.2%) polymorphism was observed for the primer OPBB 6. The polymorphism information content (PIC) values ranged 0.44 (OPA 7) to 0.18 (OPA11), while marker index (MI) values ranged 4.74 (OPA 7) to 0.36 (OPA 11). A dendogram was constructed by UPGMA method and the robustness of the tree was confirmed by bootstrap analysis with 1000 pseudo samples. For cytogenetic analysis, the 20 A. calamus accessions were screened for their ploidy status. The accessions were found to be either diploid or triploids. The phytochemical analysis of β-asarone content was determined through by HPLC method. The β-asarone concentration varied in the range of 2.2 to 7.2 mg/100 mg. The results of present study indicated the presence of low level of polymorphism among the A. calamus accessions of South India and North-East India. The phytochemical and cytogenetic analysis revealed that both diploid and triploid have low concentration of β-asarone irrespective of their geographical location.

http://nopr.niscair.res.in/bitstream/123456789/41025/1/IJBT%2015%284%29%20560-567.pdf

Genetic diversity analysis and chemical profiling of Indian Acorus calamus accessions from South and North-East India

Acorus calamus is a well-known medicinal and aromatic plant used for many industrial and pharmaceutical purposes. In our current study, twenty different accessions of Acorus calamus collected from North-East India and South India have been studied and an attempt has been made to classify them according to their ploidy level by cytogenetic analysis and to correlate them via PCR-RFLP technique for the NonTranscribed Spacer (NTS) region of the 5S-rRNA gene. β-asarone which is the major phyto-chemical found in Acorus sp. when present in higher concentrations was found to be carcinogenic. The scope of this study is to identify diploid Acorus calamus accessions as they are reported to have less concentration of β-asarone from those of triploids. Cytoyping was performed by staining of root metaphase chromosomes. For PCR-RFLP analysis, the NTS region of 5S rRNA gene was amplified by using primers, designed from the coding regions of 5S rRNA gene. The PCR products were digested with EcoRI restriction enzyme. Triploid accessions showed restriction digestion at 586 bp resulting in an additional fragment of 127 bp, which is absent in diploids. Ten diploid accessions and 10 triploid accessions were identified by cytogenetic technique and PCR-RFLP method. The sequences of triploid and diploid were analyzed using bioinformatics tools.

/pdf/an-alternative-method-for-cytotype-determination-in-acorus-1h1k59lmvm.pdf

P.E. Rajasekharan

Papers

HPLC Profiling of B Asarone Content and Cytogenetic Studies of Medicinally Important Indian Acorus Calamus l., Accessions

Genetic diversity analysis and chemical profiling of Indian Acorus calamus accessions from South and North-East India

An Alternative Method for Cytotype Determination in Acorus calamus L., Accessions by PCR- RFLP Technique