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Keywords:

  • natural antibacterial agent;
  • Cordyceps militaris;
  • Paecilomyces japonica;
  • Bombyx mori;
  • intestinal bacteria;
  • clostridia;
  • structure-activity relationship

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. EXPERIMENTAL
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

The growth-inhibiting activities of materials derived from the fruiting body of Cordyceps militaris cultured on Bombyx mori pupae (CM-1) and pupae separated from the culture (CM-2), and the fruiting body of Paecilomyces japonica cultured on B. mori pupae (PJ-1) and pupae separated from the culture (PJ-2), fresh B. mori larvae (BML), fresh B. mori female pupae (BMP), and Morus alba leaves (MAL) towards eight lactic acid-producing bacteria and 11 harmful intestinal bacteria were examined using an impregnated paper disc bioassay. At 10 mg per disc, methanolic extracts from CM-1 and CM-2 strongly inhibited growth of Clostridium difficile ATCC 9689, C. paraputrificum ATCC 25780, and C. perfringens ATCC 13124 without adverse effects on the growth of eight lactic acid-producing bacteria and the other eight harmful bacteria. The methanolic extracts from PJ-1, PJ-2, BML, BMP, and MAL did not affect growth of all test bacteria. The growth-inhibiting principle of CM-1 and CM-2 towards test clostridia was characterized as cordycepin by spectroscopic analysis. The contents of cordycepin in dried CM-1 and CM-2 were 0.69% and 0.54%, respectively. These results suggest that cordycepin may be produced from the fruiting body of C. militaris cultured on B. mori pupae and then translocated to its host insect and accumulated. Structure-growth inhibition relationships of cordycepin and its eight derivatives against C. perfringens ATCC 13124 indicate that a deoxy form at either 3′ or 2′ position appears critical for the inhibitory activity. Natural cordycepin and its two analogues, 2′-deoxyadenosine and tubercidin, merit further study as a potential antibacterial agent against various diseases caused by harmful intestinal bacteria such as clostridia. Copyright © 2006 Society of Chemical Industry


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. EXPERIMENTAL
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

In humans, the gastrointestinal microbiota participate in normal physiological functions and also contribute to the genesis of various disease states by biotransforming a variety of ingested or endogenously formed compounds to potentially harmful agents such as N-nitroso compounds, or by protecting against diseases by generation of beneficial products.1–3 Gastrointestinal ecological investigations have indicated that there are some differences in intestinal bacterial composition between cancer patients and healthy control subjects, and between young and elderly subjects.1, 2, 4 Unlike the normal gastrointestinal microbiota, the microbiota of cancer patients or elderly subjects is known to be mainly composed of Clostridium with few lactic acid-producing bacteria. The composition of the biota may also be influenced by factors such as drugs, diet, foods, and stress.1, 2 Additionally, repeated use of antibiotics for eradication of clostridia infection has disrupted gastrointestinal microbiota and resulted in the development of resistance.5 Disturbance of the microbiota may cause a variety of disease or abnormal physiological states. These problems substantiate the need for selective antibacterial alternatives for harmful gastrointestinal bacteria.

Cordyceps is the dry complex composed of the sclerotium of Cordyceps sinensis (Berk.) Sacc. (Clavicipitaceae) and the larval corpses of insects of the family Hepialidae, particularly Hepialus armoricanus Oberthür, on which the fungus is parasitic.6, 7 It may be an alternative source of materials for antimicrobial agents and much effort has been focused on Cordyceps-derived materials as potential sources of commercial antimicrobial agents or as lead compounds. It contains a range of bioactive chemicals such as amino acids, cordycepic acid (D-mannitol), cordycepin, galactosaminoglycan, nucleic acids, polysaccharides, and steroids.6–8 In the Chinese Pharmacopoeia, C. sinensis has long been considered to have natural medicinal properties such as hemostatic, mycolytic, antiasthmatic, and expectorant in the treatment of respiratory diseases as well as a tonic.6, 7Cordyceps militaris Link has also been used as a substitute for C. sinensis.7 In Korea, C. militaris and Paecilomyces japonica Yasuda have been used as substitutes for C. sinensis. They have been described as parasites to a variety of insect species under humid ground in Korea.9 Liquid culture of C. militaris collected from Bombyx mori (L.) pupae and its constituent cordycepin had growth-inhibiting activity towards Clostridium paraputrificum ATCC 25 780 and Clostridium perfringens ATCC 13 124.10 Little information exists with respect to modulating intestinal microorganisms with C. militaris and P. japonica, although these species have been widely cultivated and consumed as health food and tea in Korea. In a preliminary experiment, methanolic extracts of C. militaris fruiting body cultured on pupae of B. mori and pupae separated from the culture had potent growth-inhibiting activity towards C. perfringens ATCC 13 124 without adverse effects on Bifidobacteriun adolescentis ATCC 15 706.

This paper describes a laboratory study aimed at assessing growth-inhibiting activity of the fruiting body of C. militaris cultured on pupae of B. mori and pupae separated from the culture, the fruiting body of P. japonica cultured on pupae of B. mori and pupae separated from the culture, fresh B. mori larvae and pupae and Morus alba L. leaves towards eight lactic acid-producing bacteria and 11 harmful bacteria and at isolating growth-inhibiting constituents from C. militaris- and P. japonica-derived materials active towards harmful bacteria. Structure–growth inhibition relationships of the test compounds in C. perfringens ATCC 13 124 were also studied.

EXPERIMENTAL

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. EXPERIMENTAL
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Chemicals

Adenosine, adenosine 5′-monophosphate (AMP), adenosine 5′-diphosphate (ADP), adenosine 5′-triphosphate (ATP), 8′-bromoadenosine, 2-chloro-2′-deoxyadenosine, cordycepic acid, 2′-deoxyadenosine, and 7-deazaadenosine (tubercidin) were purchased from Sigma (St Louis, MO, USA). All other chemicals were of reagent grade.

Bacterial strains and culture conditions

The eight lactic acid-producing bacteria used in this study were Bifidobacterium adolescentis ATCC 15 706, B. bifidum ATCC 29 521, B. breve ATCC 15 700, B. infantis ATCC 25 962, B. longum ATCC 15 707, Clostridium butyricum ATCC 25 779, Lactobacillus acidophilus ATCC 4356, and L. casei ATCC 393. The 11 harmful bacteria used were Bacteroides distasonis ATCC 8503, B. fragilis ATCC 25 285, B. thetaiotaomicron ATCC 29 741, Clostridium difficile ATCC 9689, C. paraputrificum ATCC 25 780, C. perfringens ATCC 13 124, Escherichia coli ATCC 11 775, Eubacterium limosum ATCC 8486, Klebsiella pneumoniae ATCC 25 306, Salmonella typhimurium ATCC 13 311, and Staphylococcus aureus ATCC 12 600. Stock cultures of these 19 strains were routinely stored on brain heart infusion (BHI) broth (pH 7.6) (Difco, Sparks, MD, USA) containing glycerol at −70 °C, and when required were subcultured on Eggerth–Gagnon (EG) agar (Eiken Chemical, Tokyo, Japan). The plates were incubated at 37 °C for 2 days in an atmosphere of 5% H2, 15% CO2, and 80% N2 in an anaerobic chamber (Hirayama, Tokyo), except for the plates of S. aureus and E. coli, which were incubated at 37 °C for 2 days under aerobic conditions. The bacteria were then grown in BHI broth. All cultures were checked by plating for contamination at the end of the growth cycle.

Sample preparation

Dried C. militaris and P. japonica cultured on pupae of B. mori were obtained from Cunuli (Yeoju, Gyeonggi Province, Korea) and Ewoo (Gapyeong, Gyeonggi Province), respectively. These materials were separated into C. militaris fruiting body (CM-1) and pupae from the culture (CM-2), and P. japonica fruiting body (PJ-1) and pupae from the culture (PJ-2). The fruiting bodies (each 1 kg) and separated pupae (each 1 kg) were finely powdered, extracted with methanol (10 L) four times in a water bath at 60 °C for 3 h and filtered. Each combined filtrate of CM-1, CM-2, PJ-1, and PJ-2 was concentrated to dryness by rotary evaporation at 40 °C to yield about 34, 31, 24, and 31% (based on the weight of the dried fruiting bodies and separated pupae), respectively.

Bombyx mori larvae were reared on M. alba leaves at 25 ± 1 °C and 40–60% RH under a 16:8 h light:dark cycle. Fresh third instar larvae (BML) and female pupae (BMP) of B. mori were freeze-dried. Each dried B. mori larva (400 g) and pupa (400 g) were finely powdered, extracted with methanol (2 L) three times at room temperature for 3 h and filtered. Each combined filtrate of larvae and pupae was concentrated under vacuum at 40 °C to yield about 2.3% and 3.6% (based on the weight of the dried larvae and pupae), respectively.

Fresh M. alba leaves (MAL) were collected from the College of Agriculture and Life Sciences, Seoul National University, Suwon, Gyeonggi Province. They were dried in an oven at 60 °C. One hundred grams of the dried leaves were finely powdered, extracted with 500 mL of methanol three times at room temperature for 3 h and filtered. The combined filtrate was concentrated to dryness by rotary evaporation at 40 °C to yield about 16% (based on the weight of the dried leaves).

Isolation and identification of active principles

The methanolic extracts (each 200 g) from CM-1 and CM-2 were sequentially partitioned into hexane (14 and 59 g), chloroform (18 and 20 g), ethyl acetate (5 and 4 g), butanol (25 and 18 g), and water (138 and 99 g) portions. The organic solvent portions were concentrated to dryness by rotary evaporation at 40 °C, and the water portions were freeze-dried. For isolation of the active principles, 10 mg per disc of each CM-1- and CM-2-derived fraction in methanol was applied using an impregnated paper disc bioassay described below.

The butanol fractions (each 12 and 9 g) from CM-1 and CM-2 were chromatographed on a silica gel (70–230 mesh, 600 g, Merck, Darmstadt, Germany) column (5.5 cm × 70 cm), and successively eluted with a stepwise gradient of chloroform/methanol (100:0, 75:25, 70:30, 65:35, 60:40, 50:50, 30:70, and 0:100 v/v). Column fractions were monitored by thin-layer chromatography (TLC) on silica gel plates (SILC/UV254, 0.25 mm, Macherey-Nagel, Darmstadt, Germany) with chloroform/methanol (3:2 v/v). Fractions with similar Rf values on the TLC plates were pooled. Spots were detected by spraying with 30% H2SO4 and then heating on a hotplate. Five fractions were obtained from both CM-1 and CM-2 and bioassayed. Further separation of the constituents of the bioactive BF2 fractions (3.35 g from CM-1 and 2.55 g from CM-2) was achieved using high-performance liquid chromatography (HPLC) (Spectra System P2000 high-performance liquid chromatograph, Thermo Separation Products, San Jose, CA, USA). The column was a 7.8 mm ID × 300 mmµBondapak C18 (Waters, Milford, CA, USA) and a mobile phase of methanol/water (3:7 v/v) at a flow rate of 3.0 mL min−1 was used. Chromatographic separations were monitored using a UV detector at 254 nm. Finally, active principles (each 1.14 and 1.53 g) were isolated from CM-1 and CM-2. Similarly, the ethyl acetate fractions (each 5 and 4 g) from CM-1 and CM-2 were chromatographed on a silica gel column, and successively eluted with a stepwise gradient of chloroform/methanol. Three fractions were obtained from both CM-1 and CM-2. HPLC was used for further separation of the constituents from the bioactive EF3 fractions (1.25 g from CM-1 and 1 g from CM-2). Active principles (each 0.51 and 0.4 g) were isolated from CM-1 and CM-2.

The structures of the active isolates were determined by spectroscopic analyses. 1H and 13C nuclear magnetic resonance (NMR) spectra were recorded in CD3OD on a JEOL JNM-LA 400F7 spectrometer (Tokyo, Japan) at 400 and 100 MHz (using TMS as an internal standard), respectively. Mass spectra (MS) were obtained on a JEOL JMS AX505 WA spectrometer (Tokyo, Japan).

Growth-inhibiting assay

An impregnated paper disc bioassay was used to determine the growth-inhibiting activities of the test materials towards test microorganisms.11 Briefly, one loopful of bacteria was suspended in 1 mL of sterile physiological saline. An aliquot (0.1 mL) of the bacterial suspension was seeded on EG agar. Amounts (0.001 to 10 mg per disc) of each test material and compound in methanol (0.1 mL) were applied to paper discs (Advantec, 8 mm diameter and 1 mm thickness, Toyo Roshi, Tokyo, Japan). Control discs received methanol (0.1 mL). After drying in a fume hood, the paper discs were placed on the agar surface inoculated with test bacteria. All plates were incubated under the same conditions mentioned above. All tests of growth inhibition were repeated three times.

The inhibitory responses were classified as follows: very strong response, ++++, zone diameter >30 mm; strong response, +++, zone diameter 21–30 mm; moderate response, ++, zone diameter 16–20 mm; weak response, +, zone diameter 10–15 mm; and little or no response, −, zone diameter <10 mm.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. EXPERIMENTAL
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Growth-inhibiting activity of test materials

The growth-inhibiting effects in the impregnated paper disc bioassay of CM-1, CM-2, PJ-1, PJ-2, BML, BMP, and MAL on 11 harmful intestinal bacteria were investigated (data are not shown). At 10 mg per disc, methanolic extract of CM-1 strongly and moderately inhibited growth of C. perfringens ATCC 13 124 and C. paraputrificum ATCC 25 780, respectively, but exhibited weak inhibitory activity towards C. difficile ATCC 9689. At 10 mg per disc, methanolic extract of CM-2 moderately inhibited growth of C. perfringens ATCC 13 124 but exhibited weak inhibitory activity towards C. difficile ATCC 9689 and C. paraputrificum ATCC 25 780. The CM-1 and CM-2 extracts showed little or no growth-inhibiting activity for B. distasonis ATCC 8503, B. fragilis ATCC 25 285, B. thetaiotaomicron ATCC 29 741, E. coli ATCC 11 775, E. limosum ATCC 8486, K. pneumoniae ATCC 25 306, S. typhimurium ATCC 13 311, and S. aureus ATCC 12 600. Methanolic extracts from PJ-1, PJ-2, BML, BMP, and MAL did not affect growth of all test harmful bacteria at 10 mg per disc.

The growth-inhibiting activities of the test materials towards eight lactic acid-producing bacteria were also examined (data are not shown). At 10 mg per disc, no growth inhibition of all test lactic acid-producing bacteria was observed with methanolic extracts of CM-1, CM-2, PJ-1, PJ-2, BML, BMP, and MAL.

Identification and contents of active principles from C. militaris-derived materials

When fractions obtained from methanolic extracts of CM-1 and CM-2 were bioassayed, substantial differences were observed in the growth-inhibiting activities towards test clostridia (Table 1). The ethyl acetate and butanol fractions strongly inhibited growth of C. difficile ATCC 9689, C. paraputrificum ATCC 25 780 and C. perfringens ATCC 13 124 at 1 mg per disc, whereas the other three fractions exhibited little or no growth-inhibiting activities towards these organisms at 5 mg per disc.

Table 1. Growth-inhibiting effects of each solvent fraction from methanolic extracts of the fruiting body of Cordyceps militaris cultured on pupae of Bombyx mori and pupae separated from the culture on three harmful clostridia using the impregnated paper disc bioassay
Test materialDose (mg per disc)Clostridial strain
Clostridium difficile ATCC 9689Clostridium paraputrificum ATCC 25 780Clostridium perfringens ATCC 13 124
  • a

    C. militaris fruiting body cultured on B. mori pupae.

  • b

    B. mori pupae separated from the C. militaris culture.

CM-1a
 Hexane fraction5
 Chloroform fraction5
 Ethyl acetate fraction5+++++++++
1++++++
0.5++++
 Butanol fraction5+++++++++
1+++++++++
0.5+++++
 Water fraction5
CM-2b
 Hexane fraction5
 Chloroform fraction5
 Ethyl acetate fraction5++++++++
1+++++++
0.5++++++
 Butanol fraction5+++++++++
1+++++++++
0.5+++++++
 Water fraction5

Impregnated paper disc bioassay-guided fractionation of CM-1 and CM-2 provided an active constituent. The structure of the four isolates was characterized as the nucleoside cordycepin (3′-deoxyadenosine) (Fig. 1) by spectroscopic analyses, including mass spectrometry and NMR, and by comparison of published literature.10 The contents of cordycepin in CM-1 and CM-2 were 0.69% and 0.54% (based on the weight of the dried fruiting body and separated pupae), respectively.

thumbnail image

Figure 1. Structure of cordycepin, the antibacterial constituent from the fruiting body of Cordyceps militaris, cultured on Bombyx mori pupae and pupae separated from the culture.

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Growth-inhibiting activity of cordycepic acid and cordycepin

Because cordycepic acid is found to be one of the major components of C. sinensis and C. barnesii,7 the growth-inhibiting activity of the compound towards three harmful clostridia was compared with that of cordycepin (Table 2). At 0.01 mg per disc, cordycepin exhibited strong or very strong growth inhibition of C. difficile ATCC 9689, C. paraputrificum ATCC 25 780, and C. perfringens ATCC 13 124. This compound strongly inhibited growth of C. paraputrificum ATCC 25 780 at 0.001 mg per disc, whereas weak and moderate activity was observed towards C. difficile ATCC 9689 and C. perfringens ATCC 13 124, respectively, at the same dose. Cordycepic acid at 10 mg per disc exhibited little or no growth-inhibiting activity towards these three clostridia.

Table 2. Growth-inhibiting effects of cordycepin and cordycepic acid on three harmful clostridia using the impregnated paper disc bioassay
CompoundDose (mg per disc)Clostridial strain
Clostridium difficile ATCC 9689Clostridium paraputrificum ATCC 25 780Clostridium perfringens ATCC 13 124
Cordycepin1+++++++++++
0.1+++++++++
0.01+++++++++
0.001++++++
Cordycepic acid10

Structure–activity relationships

Structure–growth inhibition relationships of cordycepin and its eight derivatives against C. perfringens ATCC 13 124 are given in Table 3. At 0.1 mg per disc, strong growth-inhibiting activity was observed with cordycepin, 2′-deoxyadenosine, and tubercidin. At 0.01 mg per disc, cordycepin strongly inhibited growth of C. perfringens ATCC 13 124, whereas 2′-deoxyadenosine and tubercidin showed weak and moderate inhibitory activity, respectively. Weak growth-inhibiting activity was observed with adenosine and 2-chloro-2′-deoxyadenosine at 1 and 0.1 mg per disc. 8-Bromoadenosine exhibited weak and no growth-inhibiting activity at 1 and 0.1 mg per disc, respectively. At 1 mg per disc, no growth inhibition was observed with AMP, ADP, and ATP.

Table 3. Growth-inhibiting effects of cordycepin and its derivatives on Clostridium perfringens ATCC 13 124 using the impregnated paper disc bioassay
CompoundDose (mg per disc)
10.10.01
Cordycepin++++++++++
Adenosine++
Adenosine 5′-monophosphate
Adenosine 5′-diphosphate
Adenosine 5′-triphosphate
8-Bromoadenosine+
2-Chloro − 2′-deoxyadenosine++
2′-Deoxyadenosine+++++++
Tubercidin+++++++++

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. EXPERIMENTAL
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Among the intestinal microorganisms, clostridia are the most important causative agents of a wide variety of human diseases such as sudden death, toxicity, mutagenesis, carcinogenesis or ageing by biotransforming a variety of ingested or endogenously formed compounds to harmful agents such as N-nitroso compounds or aromatic steroids within the gastrointestinal tract.3, 4, 12, 13 On the contrary, bifidobacteria are often taken as useful indicators of human health under most environmental conditions because they play important roles in metabolism such as amino acid and vitamin production, aid defense against infection, are associated with longevity, antitumor activity, pathogen inhibition, improvement of lactose tolerance of milk products and immunopotentiation.2, 4, 14–16 It would therefore be desirable to inhibit both the growth of potential pathogens and/or increase the numbers of bifidobacteria in human gut. Selective growth promoters for bifidobacteria or inhibitors for harmful bacteria are especially important for human health because intake of these materials can normalize disturbed physiological functions that result in the prevention of diseases caused by pathogens in the gastrointestinal tract.

In the present study, methanolic extracts from CM-1 and CM-2 selectively inhibited growth of C. difficile, C. paraputrificum, and C. perfringens without growth inhibition of eight lactic acid-producing bacteria as well as the other eight harmful intestinal bacteria. Little or no growth-inhibiting activity was observed with the methanolic extracts from PJ-1, PJ-2, BML, BMP, and MAL. These results indicate that the growth-inhibiting compounds may be produced by interactions of C. militaris and its host insect, regardless of silkworm as host insect of C. militaris and mulberry leaves as the host plant of silkworm. On the basis of our results, P. japonica cannot be used as a substitute for C. militaris. We reported in our previous paper that mycelial culture of C. militaris collected from B. mori pupa possessed growth-inhibiting activity against C. paraputrificum ATCC 25 780 and C. perfringens ATCC 13 124.10

Cordyceps species and their constituents have potential as natural products for the eradication of harmful intestinal bacteria. They can be applied to humans in the same way as other conventional antimicrobial agents such as antibiotics. Additionally, certain natural products have been found to be highly effective against fungicide-resistant microorganisms17, 18 and drug-resistant Helicobacter pylori.19 Much concern has therefore been focused on the determination of the distribution, nature and practical use of Cordyceps species-based substances that have antibacterial activity. In the present study, the growth-inhibiting principle from CM-1 and CM-2 was identified as cordycepin with species selective activity as in the case of liquid culture of C. militaris collected from B. mori pupa.10 Of test bacteria, this compound selectively inhibited growth of C. difficile, C. paraputrificum, and C. perfringens. However, the methanolic extract from BMP exhibited little or no growth-inhibiting activity. The contents of cordycepin in dried CM-1 and CM-2 were 0.69% and 0.54%, respectively. These results suggest that B. mori pupae are only a nutritional source of C. militaris: cordycepin might be produced from C. militaris fruiting body and then translocated to its host insect and accumulated. C. militaris-derived materials might be good candidates for naturally occurring selective antibacterial agents because antibiotics are found to strongly inhibit growth of various lactic acid-producing bacteria like bifidobacteria and lactobacilli at low concentrations.10 It has been reported that the cordycepin content of C. militaris cultured in China was 0.75%, although the content varied according to cultivated source.8 Submerged cultivated C. militaris is found to secrete cordycepin into the environment.20 Cordycepin is found to possess antitumor activity,21 inhibition of nuclear RNA synthesis in nerve and glial cells,22 and antiviral activity towards HIV-1 in vitro,23 as well as antiprotozoal effect24 and insecticidal activity against Plutella xylostella (L.).25

Structure–growth inhibition relationships in intestinal organisms have been studied. Ahn et al.26 studied the structure–activity relationship between the six polyphenols derived from Thea sinensis L. leaves and growth inhibition towards C. perfringens and C. difficile: the gallate moiety of polyphenols seems to be required, but their stereochemistries do not appear critical for the inhibitory activity. The growth inhibition of pinenes against C. perfringens is found to be much more pronounced in (1R)-(+)-α-pinene than (1S)-(−)-α-, (1R)-(+)-β- and (1S)-(−)-β-pinenes: (R)-(+)-α form appears to be required for growth-inhibiting activity towards C. perfringens.27 In the current study, there was substantial difference in growth-inhibiting activity towards C. perfringens ATCC 13 124 among cordycepin and its eight derivatives. Cordycepin and 2′-deoxyadenosine were more active than adenosine with hydroxy groups at both 3′ and 2′ positions. The inhibitory activity of adenosine was comparable to that of 8-bromoadenosine and 2-chloro-2′-deoxyadenosine. These results indicate that a deoxy form at either the 3′ or 2′ position appears critical for the growth-inhibiting activity.

Results of this study indicate that C. militaris-derived materials, particularly cordycepin, could be useful as antibacterial agents for harmful clostridia such as C. difficile, C. paraputrificum, and C. perfringens. The antibacterial action of cordycepin may be an indication of at least one of the pharmacological actions of C. militaris. For the practical use of cordycepin and its analogues as novel antibacterial agents to proceed, further research is necessary to establish whether this activity is exerted in vivo after consumption of cordycepin or its analogues by humans. Other areas requiring attention are antibacterial mode of action of these compounds and effective formulation to improve potency and stability in human gastrointestinal tract.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. EXPERIMENTAL
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

This work was supported by grants from NaturoBiotech Co., Ltd and the Ministry of Education and Human Resources Development for the Brain Korea 21 Project of the Korean Government to YJA.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. EXPERIMENTAL
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
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