INTRODUCTION:

The use of traditional medicine in both developing and developed countries is significantly increasing in recent times. According to World Health Organization (WHO), more than 80% of the world’s population relies on traditional medicine for their primary healthcare needs. The undesirable oxidation and growth of microorganism in food are always associated with some undesirable changes even causing harmful issues such as development of food spoilage, food-borne illness and off-flavours. The world trade in plant based drugs and its products are many fold expanding continuously; because the general awareness of the wide spread toxicity and harmful after effects associated with the long-term use of synthetic drugs and antibiotics^1-3. On the other hand, herbal medicine are considered as safe and effective alternative remedies and therapeutic agents for the good health in most developing countries^4,5.

Researches have been conducted on the antimicrobial and antioxidant activity of the various medicinal plants^6,7. The genus Acorus belongs to the family Araceae, comprises about 40 species. Acorus is derived from Acoron (coreon = the pupil of the eye). Acorus gramineus (Grassy-leaved sweet flag), which is native eastern Asia, is an aquatic or wetland plant. A. calamus (Sweet flag) is a native of central Asia and eastern Europe and grows worldwide widly along swamps, rivers and lakes and grows wild in marshy places up to 2000 m altitude in the Himalaya and in some parts of South India⁸. The two species are semi-evergreen perennial hairless herb^9,10. Acorus gramineus is renowned indigenous Korean and Chinese medicinal plant, officially listed in the Korean and Chinese Pharmacopoeia. The rhizomes of A. gramineus has been considered to possess natural medicinal value such as a digestant, expectorant and as a stimulant against digestive disorders, diarrhea and epilepsy¹¹. And the volatile oil of A. gramineus stimulating olfraction is effective for treatment of the Alzheimer’s disease rat¹². The major bioactive components of A. gramenius rhizomes are associated with various pharmacological activities including antibacterial, antifungal, anthelminthic and pesticidal activity¹³. The rhizomes of Acorus calamus was used by the ancient Greek and Chinese as traditional herbal medicine. In Eastern and Western herbal medicine, the rhizomes of A. calamus are used for the treatments of various ailments like diarrhea, headache, rheumatism and various disorders and as insecticide. And it has used for Ayurvedic medicinal practice in India¹⁴. There has been a tradition of washing hair and skin with water that is soaked the rhizomes of A. calamus and biological activity of the plant has been under way for cosmetic use in Korea¹³. Various bioactive phytoconstituents and biological activities of A. calamus have been reported over the years^15-17. A steadily increasing progress has been witnessed towards investigating chemical composition and bioactivities of this two Acorus species. The aim of present study is to compare the in vitro antimicrobial and antioxidant activity of leaves and rhizomes of A. gramineus and A. calamus.

MATERIALS AND METHODS:

Plant material:

The leaves and rhizomes of Acorus gramineus were collected from a farm in Hwasoon-gun, Jellanamdo Province of Korea (127°4′9.13″ E, 34°9′23.93″ N) and the leaves and rhizomes of Acorus calamus were collected from a farm in Wanju-gun, Jellabukdo Province of Korea (127°25′78.35″E, 35°9′6.25″N). The two provenances were collected in June 2014 and the leaves and rhizomes were air-dried at room temperature for 2 weeks. The air-dried leaves and rhizomes were pulverized using an electric mill.

Microbial strains:

The tested microorganisms included three Gram-positive bacteria (Bacillus cereus ATCC 11778, Bacillus subtilis ATCC 9327, Methicillin resistant-Staphylococcus aureus (MRSA) KCCM 40510 and Staphylococcus aureus ATCC 13301) and three Gram-negative bacteria (Escherichia coli ATCC 15489, Pseudomonas fluorescens KCCM 41443 and Salmonella typhimurium KCCM 11862).

Extract preparation for antimicrobial activity:

Two hundred gram of the powdered leaves and rhizomes was macerated with 1,000ml of ethanol for 24 hrs. The percolates were then filtered through Whatman No.2 filter paper. The crude ethanol extract was subsequently fractionated with 500ml of hexane, after which the top hexane layer (comprising the hexane fraction) was gathered. The remaining layer was successively partitioned with 500ml of diethyl ether, ethyl acetate and water in a separating funnel (forming the ether, ethyl acetate and water fraction). Each fraction was subsequently concentrated in vacuo to 30ml at 30°C and tested for antimicrobial activity. Antimicrobial activity was measured only with the ethyl acetate and water fraction.

Determination of antimicrobial activity:

The Gram-positive and Gram-negative bacteria were cultured on nutrient broth agar at 30℃ for 18~24hrs prior to testing, then subcultured three times for another 18~24hrs. The turbidity of bacterial cell suspensions was brought to 0.3 optical density (OD) at 660nm by adding sterile broth and was then used for the tests. Next, 200 µl of the bacterial cell suspensions was poured uniformly onto nutrient broth agar plates and then paper disks (8.0mm in diameter) containing the ethyl acetate, or water fraction were carefully placed on the inoculated Petri dishes. The diameters of inhibition zone of the tested bacteria were measured in mm after the cultures were incubated for 24hrs at 37℃. The minimal inhibition concentration (MIC) was determined as the lowest concentration that caused an inhibition zone¹⁸.

Extract preparation for antioxidant activity:

The powdered leaves and rhizomes of Acorus calamus and A. gramineus (50g) were extracted with 1,000ml heating water (80℃) and ethanol at room temperature for 3hrs. Each samples was run in triplicate. The extracts were filtered with 0.2µm syringe filter and concentrated in vacuoto 30ml at 30℃.

DPPH free radical scavenging activity:

The DPPH free radical scavenging activity was evaluated using the Blois method (Blois, 1958), with slight modification. Briefly,160µl of each sample extract was mixed with 40µl of 1.5×10^-4M DPPH solution (in methanol). The mixtures were gently mixed and allowed to stand at room temperature for 30min, and the absorbance at 520nm was measured using a microplate spectrophotometer reader (Molecular Devices). The antioxidant activity of each extract was expressed in terms of IC₅₀ values (the concentration required to inhibit DPPH radical formation by 50%). L-Ascorbic acid was used as a reference¹⁹.

Determination of total polyphenol content:

The total polyphenol content was determined using the Folin-Denis method, with slight modification. The extracts were centrifuged at 1,200rpm for 10 min, and the supernatant collected. The 0.5ml of clear supernatant of the samples were mixed with 8ml of distilled water, after which 0.5ml of Folin-Denis’ reagent was added. After 3min, 1ml of sodium carbonate (10% in distilled water) was added and the solution was allowed to stand for 2 hrs at 22°C in darkness. The absorbance was then measured at 700nm using an UV-vis. spectrophotometer (HP-8453, USA). A standard curve prepared from tannic acid (50-300mg/l) was used for quantification and the total polyphenol content was expressed as mg tannin/g dry weight¹⁹.

Statistical analysis:

All experiments were performed in triplicate. The data were recorded mean±standard deviation and analyzed using statistical SPSS software (SPSS Inc., Chicago, USA. Version 25.0).

RESULTS AND DISCUSSION:

Antimicrobial activity of fraction of leaves and rhizomes from Acorus gramineus and Acorus calamus:

The rhizomes of Acorus calamus possess strong, characteristic and aromatic odor and is found in the northern temperate and subtropical regions of Asia, North America and Europe, and the study on the plant is extensive. On the other hand, Acorus gramineus is distributed in Korea, Japan, China and Taiwan, and the research on the plant is limited. In order to evaluate the antimicrobial activity of A. gramenius and A. calamus, the minimal inhibition concentration (MIC) of ethyl acetate and water fraction was measured. The lower the MIC value indicated a higher antimicrobial activity. The MIC values against three Gram-positive bacteria are presented in Table 1. The ethyl acetate fraction exhibited comparable antimicrobial activity against tested Gram-positive bacteria, except for rhizomes of A. calamus have no MIC value against B. subtilis. In special, MIC value against MRSA was lower than that against the other two tested Gram-positive bacteria. On the other hand, the water fraction was not detect MIC value against the tested Gram-positive bacteria. In addition, the antimicrobial activity of A. gramineus was slightly higher in rhizomes fraction compared to leaves fraction against Gram-positive bacteria. The result published by Li and Wah, rhizomes extract of A. calamus has been shown higher antibacterial activity compared to the leaves extract³. Ether extract of A. calamus was shown that bigger inhibition zone diameter was detected on P. aeruginosa followed by S. aureus and E. coli and B. subtilis showed smaller inhibition zone than the former two tested bacteria¹. Another earlier study reported that extracts obtained with ethyl acetate from A. calamus were found to be highly effective against tested fungi and yeast and tested bacteria were resistant to both rhizomes and leaves extract except for E. coli¹. And the ethyl acetate fraction of leaves and rhizomes from A. gramineus, unlike A. calamus detected MIC against all the tested Gram-positive and Gram-negative bacteria. Escherichia coli was more sensitive to the tested fractions compared to the other two tested Gram-negative bacteria (Table 2). These finding is similar with the finding of Susanah et al.²⁰who reported that E. coli was more sensitive to the A. calamus extract compared to S. aureus and C. albicans. The results of this study show that not only the rhizomes of the two Acorus plants, which have been used for medicinal purposes, but also the leaves show the potential as materials for medicinal or functional products.

Table 1. Minimal inhibition concentration (MIC) of ethyl acetate and water fraction of ethanol extract from Acorus gramineus and Acorus calamus against 3 Gram-positive bacteria

Fraction	Plant	Part	Minimal inhitition concentration (MIC, mg/ml)
Fraction	Plant	Part	*B. subtilis*	MRSA	*S. aureus*
Ethly acetate	A. gramineus	Leaves	1,50	0.20	0.20
		Rhizomes	0.50	0.25	0.25
	A. calamus	Leaves	2.00	0.15	0.25
		Rhizomes	-	0.125	0.25
Water	A. gramineus	Leaves	-	-	-
		Rhizomes	1.00	-	-
	A. calamus	Leaves	-	-	-
		Rhizomes	-	-	-

Table 2. Minimal inhibition concentration (MIC) of ethyl acetate and water fraction of ethanol extract from Acorus gramineus and Acorus calamus against 3 Gram-negative bacteria

Fraction	Plant	Part	Minimal inhitition concentration (MIC, mg/ml)
Fraction	Plant	Part	*E. coli*	*P. fluorescens*	*S. typhimurium*
Ethly acetate	A. gramineus	Leaves	0.50	0.20	2.00
		Rhizomes	0.10	0.50	2.00
	A. calamus	Leaves	1.50	1.50	1.50
		Rhizomes	0.10	0.25	-
Water	A. gramineus	Leaves	-	-	2.00
		Rhizomes	1.00	-	1.00
	A. calamus	Leaves	-	-	2.00
		Rhizomes	1.00	-	-

Antioxidant activity of fraction of leaves and rhizomes from Acorus gramineus and Acorus calamus:

The yield of ethanol and water extract from Acorus gramineus and A. calamus is shown in Table 3. The yield of rhizomes is higher than that of leaves. The evaluation of antioxidant activity in plant is vital for discovery of natural antioxidant²¹. For their conceivable antioxidant activity, the ethanol and water extract was subject to screening. The ability of the two Acorus ethanol and water extracts for scavenging free radicals was evaluated through the use of the synthetic radical DPPH. The IC₅₀values of ascorbic acid and the ethanol and water extract from A. gramineus and A. calamus was shown in Table 4. The half inhibitory concentration (IC₅₀) of ethanol extract of A. gramineus rhizomes was found to be 38.11±5.78µg/ml, the value means the highest activity. On the other hand, the IC₅₀of ethanol extract of A. calamus leaves (69.97±7.27µg/ml) was lower than that of A. calamus rhizomes (376.63±21.55 µg/ml). There is a lot of difference between this finding and the results reported by Ho et al.²², which revealed that the IC₅₀value of methanol extract of A. gramineus whole plant was 1045.51±0.69µg/ml. The IC₅₀value of A. gramineus showed low radical scavenging activity in the DPPH assay. In DPPH assay conducted on the water extracts, the A. calamus rhizomes had the lowest IC₅₀value among the tested extract (79.46±12.21µg/ml). This is quite different with the results of the radical scavenging activity of the ethanol extracts. There is a growing interest in the correlation between phytochemicals and their pharmacological activity in medicinal plants, and the research is actively being conducted²³. Among the various antioxidants such as phenolic compounds, carotenoids, vitamins and terpenoids, etc., phenolic compounds are gaining importance due to their safety and multiple effects, including antioxidant activity^24,25. Phenolic compounds present in the plants are known for their ability of scavenging free radical. Tea and herbal materials contribute to the major source of phenolic compounds^26,27. The total polyphenol content is expressed as milligram of tannin equivalent per gram dry weight (Table 5). For the ethanol extract of A. gramineus rhizomes, the total polyphenol content was 5.33±0.44mg/g; as the rhizomes water extract of A. calamus, the total polyphenol content was 3.94±0.81 mg/g. It shows that no correlation between antioxidant activity and total polyphenol contents and the total phenolic content is lower compared to that of tea plant (201.51 to 265.48mg/g)²⁸.

Table 3. The yield of solvent extract from leaves and rhizomes of Acorus grmineus and Acorus calamus

Plant	Part	Yield (%)
Plant	Part	Ethanol	Water
A. gramineus	Leaves	7.25	13.92
	Rhizomes	12.65	6.58
A. calamus	Leaves	12.31	6.97
	Rhizomes	10.95	13.46

Table 4. DPPH free radical scavenging activity of extract from leaves and rhizomes of Acorus gramineus and Acorus calamus

Plant	Part	DPPH free radical scavenging activity (IC₅₀value, µg/ml)
Plant	Part	Ethanol	Water
A. grmineus	Leaves	284.57±5.46	309.71±14.44
	Rhizomes	38.11±5.78	231.24±7.13
A. calamus	Leaves	69.97±7.27	145.32±5.58
	Rhizomes	376.63±21.55	79.46±12.21
Ascorbic acid		6.74±0.13

All data were presented by mean±standard deviation

Table 5. The total polyphenol content in extract from leaves and rhizomes of Acorus gamineus and Acorus calamus

Plant	Part	Total polyphenol content (mg/g)
Plant	Part	Ethanol	Water
A. gramineus	Leaves	8.82±0.87	5.32±0.56
	Rhizomes	5.33±0.44	12.64±1.01
A. calamus	Leaves	17.42±3.43	13.68±2.23
	Rhizomes	4.94±1.33	3.94±0.81

All data were presented by mean±standard deviation

CONCLUSION:

This study revealed the in vitro antimicrobial and antioxidant activity of Acorus gramineus and A. calamus.

The activities of the two plants were different according to strain and extraction solvent. The characterization of the antimicrobial and antioxidant activity of the two Acorus plants could lead to more useful source in the pharmaceutical and health food industry.

CONFLICT OF INTEREST:

None of the authors have any conflict of interest to declare.

ACKNOWLEDGEMENT:

This work was supported by Research promotion program of SCNU.

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Received on 08.06.2021 Modified on 18.04.2022

Research J. Pharm. and Tech 2023; 16(1):13-17.

DOI: 10.52711/0974-360X.2023.00003