Antimicrobial Activity of Ginsenosides - Wiley Online Library

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May 21, 1998 - Protopanaxadiol, a genuine sapogenin of Gin- seng saponins. Chem. Pharm. Bull. 14: 595-600. Sonnenborg, U. (1987). Ginseng. Dtsch. Apoth.
Pharin. Pharmacol. Comniun. 1998, 4: 41 1-413 Received May 5 , 1998 Accepted May 21, 1998

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1998 Phdrm. Pharmacol. Commun.

Antimicrobial Activity of Ginsenosides L. BATTINELLI, M. T. MASCELLINO*, M. C . MARTINO, M . LU AND G. MAZZANTI

Institute of Pharmacology and Pharmacognosy, *Department of Infectious Diseases, University of Rome “La Sapienza” P.le Aldo Moro 5 00185 Rome, Italy Abstract

The antimicrobial activity of pure ginsenosides (Rbl, Rb2, Rc, Rd, Re, Rgl), two chemical fractions, and the total extract of Panax ginseng root was investigated. The results showed strong antimicrobial activity (minimum inhibitory concentration < lpg mL-’) by some pure ginsenosides belonging to both protopanaxadiol and protopanaxatriol groups against representative Gram-positive and Gram-negative bacteria, and yeasts. Conversely, Panax ginseng chemical fractions and the total extract were inactive against the microorganisms tested. This lack of antimicrobial activity may be due to possible antagonist effects between ginsenosides and other Panax ginseng components.

Panax ginseng C. A. Meyer (Arialiaceae) is a typical drug in Chinese traditional medicine and is widely used in Western countries. Ginsenosides are the main active components of P. ginseng. These compounds are a mixture of glycosides and belong to protopanaxadiol (Rb, , Rb2, Rc, Rd ginsenoside (Shibata et a1 1966)) or protopanaxatriol (Re, Rf, Rgl, Rg2 (Shibata et a1 1965)) groups. A large number of pharmacological studies have been carried out on P. ginseng root in order to confirm its tonic, stimulant and antifatigue properties (Sonnenborg 1987). Saito et a1 (1974) reported that P. ginseng facilitated recovery from exhaustion caused by forced running. Moreover, Petkov & Masharoff (1987) reported that P. ginseng improved learning and memory in rats, and that these effects depended on different mechanisms involving the brain amine content and direct interaction with central neurotransmitters (Petkov 1978). In addition, P. ginseng inhibited prolactin release in a dose-dependent manner by interacting with dopaminergic receptors on pituitary cells (Mazzanti et a1 1996). A variety of depressive effects on the central nervous system (CNS) have been observed, and include suppression of exploratory and spontaneous movements (Takagi et a1 1972a, 1974), prolongation of hexorbital sleeping time (Takagi et a1 1972b) and inhibition of conditioned avoidance response (Saito et a1 1977). Kim et a1 (1987) reported that pure ginseng saponins inhibited the development of tolerance and Correspondence: G. Mazzanti, Institute of Pharmacology and Pharmacognosy, University of Rome “La Sapienza” P.le Aldo Moro 5 , 00 I85 Rome, Italy.

physical dependence of morphine. Effects on cardiovascular and immune systems have been also described for P. ginseng, as have its antitumoral and antidiabetic activities (Wang 1980). Conflicting pharmacological effects of ginseng may be explained by the effect of different saponins. Ginsenoside Rbl inhibits many functions of the CNS, while Rgl stimulates the CNS. Kang et a1 (1995) demonstrated that ginsenosides from the protopanaxatriol group, but not from the protopanaxadiol group, enhance the release of nitric oxide from endothelial cells and may contribute to the beneficial effect of ginseng on the cardiovascular system. In this study pure ginsenosides, P. ginseng total extract and two chemical fractions were evaluated for antimicrobial activity against the most common bacteria and yeasts in human pathology.

Materials and Methods Plant material Plant material was kindly supplied by D.co Ulrich S.p.A., Nichelino (Turin, Italy). Specimens were identified and stored under vacuum in plastic bags. P. ginseng dried total extract, containing 14% saponins expressed as ginsenoside Rgl , was obtained from Indena S.p.A. (Milan, Italy). Pure ginsenosides Rbl, Rb2, Rc, Rd, Rgl and Re were purchased from Extrasynthese SA (Lyon, France). Microorganisms Five Gram-positive bacteria, seven Gram-negative bacteria and four yeasts obtained by clinical isola-

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L. BATTINELLI ET AL

Determination of minimum inhibitory concentration (MIC) MIC values were determined by a standard twofold dilution technique. Inocula (1 O5 CFU mL-') were prepared in the same medium by diluting bacterial suspensions in broth after overnight incubation. The MIC was the lowest concentration that inhibited bacterial growth after 18-24 h incubation at 37°C (Wilkinson & Gentry 1981). In order to determine bactericidal or bacteriostatic action of samples, the cultures not presenting growth were used to inoculate a plate of solid medium. The plates were observed after incubation at 37°C for 24 h (or 48 h for yeasts) and lack of colonies denoted bactericidal action. Samples were tested in triplicate.

Table 1. Microorganisms tested. Microorganism

Source

Staphylococcus aureus 360 Staphylococcus aureus 32 Staphylococcus capitis 425 Staphylococcus epidermidis 40 Streptococcus sanguis 189 Klebsiella oxytoca 487 Klebsiella pneumoniae 52 Escherichia coli 26 Salmonella enteriditis 68 Proteus vulgaris 72 Proteus mirabilis 608 Pseudomonas aeruginosa 374 Candida krusei 47 Candida humicola 50 Candida albicans 533 Candida albicans 875

Pharyngeal swab Pharyngeal swab Blood Pharyngeal swab Blood Sputum Sputum Urine Faeces Cervical swab Urine Pharyngeal swab Vaginal swab Vaginal swab Pharyngeal swab Vaginal swab

Results and Discussion tion were used (Table 1). Microorganisms were grown in Mueller-Hinton Broth (Becton-Dickinson, Milan, Italy) at pH 7.4. Preparation of plant extracts P. ginseng roots were extracted according to Shibata et a1 (1966) obtaining two fractions-GNS (neutral saponins containing protopanaxadiol aglycone) and GN4 (protopanaxathriol aglycone). The presence of protopanaxadiol and protopanatatriol ginsenosides in GNS and GN4 fractions, respectively, were detected by thin-layer chromatography performed using chloroform-methanolwater (65: 50: 10) as solvent system and 10% phosphoric acid as detection system. Rb2, Rc, Rd and predominantly Rbl ginsenosides were present in GNS. GN4 contained mainly Rg,, Re and traces of Rc. The fraction yields were 10.4% and 2.6% for GNS and GN4, respectively. GNS and GN4 were solubilized in water for biological tests. All extracts were adjusted to pH 6.5-7.0 and sterilized by filtration with a 0.22-prn filter.

,,

Antimicrobial activity of pure ginsenosides (Rb Rb2, Rc, Rd, Rgl and Re) was tested on Staphylococcus capitis 425, Staphylococcus epidermidis 40, Staphylococcus aureus 360, Klebsiella pneumoniae 52, Pseudomonas aeruginosa 374 and Candida albicans 533. Rbl and Rc ginsenosides were inactive against all microorganisms tested. Rb2 had strong antimicrobial activity (MIC< 1 ,ug mL-') against S. capitis 425 and C. albicans 533 and had bacteriostatic action against S. epidermidis 40 and K. pneumoniae 52. Rd was active only against C. albicans 533 (MIC = 9 ,ug mL-'). Protopanaxatriol ginsenosides, Rg, and Re, had bacteriostatic activity against S. epidermidis 40 and K. pneumoniae 52. Ginsenoside Re also showed strong antimicrobial activity (MIC< 1 ,ug mL-') against S. capitis 425 and C. albicans 533. The results are reported in Table 2. Interestingly, active ginsenosides belong to both protopanaxadiol and protopanaxatriol groups. GNS (protopanaxadiol ginsenosides) and GN4 (mainly protopanaxatriol ginsenosides) fractions were inactive against all microorganisms tested;

Table 2. Antimicrobial activity of pure ginsenosides. Minimum inhibitory concentration (pg m L-') Ginsenoside

S. capitis 425

S. epidermidis 40

Rb I Rb2 Rc Rd Rg I Re

>78 < I (78

>78 tl (>78) 39 (78) 78 (>78) i l (39) t1 (>78)

>78

>78 78 >78 >78 >78 >78

K. pneurnoniae 52

>78 t1 (>78) >78 >78 t1 (>78) i l (178)

Minimum bactericidal concentration (pg mL-') is given in parentheses.

P. aeruginosa 374

>78 >78 >78 >78 >78 >78

C. albicans 533

>78 t l (tl)

>78 9 (9) >78 < l (2)

ANTIMICROBIAL GINSENOSIDES

Table 3. Ginsenoside content (%) in Panax ginseng root. Protopanaxadiol aglycone 0.02 0.03 0.005 0.4 0.2 0.01 0.15 0.15

Protopanaxatriol aglycone Re Rf Rg I Rg2 Rh I

0.2 0.05 0.2 0.02 0.002

MIC values were constantly higher than the maximum concentration tested (2.5 mg mL-‘). A dried total extract enriched in saponins was studied to establish whether the lack of antimicrobial activity was due to a low ginsenosides concentration. This extract was inactive against all microorganisms tested. This result seems to conflict with results for active pure ginsenosides which had potency comparable with that of some widely used antibiotics. The inactivity of P. ginseng fractions and total extract does not seem to be due to the low concentration of ginsenosides because of their potency in inhibiting microorganism growth and their content in the whole drug (Table 3) (Sanada et a1 1978; Besso et a1 1982; Kasai et a1 1983; Matsuura et a1 1984). The lack of antimicrobial activity may be due to possible antagonist effect between ginsenosides and other P. ginseng components. Medicinal plants are often considered to possess the same biological properties as their pure components. In the whole drug, however, some chemical constituents can interact with the active principles, synergistically or antagonistically, modifying their biological action. Hence, this deductive approach is misleading. Acknowledgement This work was supported by a grant from Enrico ed Enrica Sovena Foundation.

References Besso, H., Kaisai, R., Saruwatari, Y., Fuwa, T., Tanaka, 0. (1982) Ginsenoside Ral and ginsenoside Ra2, new dammarane-saponins of Ginseng root. Chem. Pharm. Bull. 30: 2380-2385

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Kang, S. Y., Schini-Kerth, V. B., Kim, N. D. (1995) Ginsenoside of protopanaxatriol group cause endothelium-dependent relaxtion in the rat aorta. Life Sci. 56: 1577-1586 Kaisai, R., Besso, H., Tanaka, O., Saruwatari, Y., Fuwa, T. (1983) Saponins of red Ginseng. Chem. Pharm. Bull 31: 21 20-21 25 Kim, H. S., Oh, K. W., Park, W. K., Yamano, S., Toki, S. (1987) Effects of Panax ginseng on the development of morphine tolerance and dependence. Korean J. Ginseng Sci. 1 1 : 182-190 Mazzanti, G., Braghiroli, L., Bolle, P. (1996) Effects of Panax ginseng and Ginkgo biloba on in vitro prolactin secretion. Phytother. Res. 10: S33-S35 Matsuura, H., Kasai, R., Tanaka, O., Saruwatari, Y., Kunihiro K., Fuwa, T. (1984) Further studies on dammarane-saponins of Ginseng root. Chem. Pharm. Bull. 32: 1188- 1192 Petkov, V. D. (1978) Effect of Ginseng on the brain biogenic monoamines and 3’,5’-AMP system. Arzneim. Forsch. 28: 388-393 Petkov, V. D., Masharoff, A. H. (1987) Age and individual related specificities in the effects of standardized Ginseng extract on learning and memory (experiments on rats). Phytother. Res. 1: 80-84 Saito, H., Yoshida, Y., Takagi, K. (1974) Effect of Panax ginseng root on exhaustive exercise in mice. Jpn. J. Pharmacol. 24: 119-124 Saito, H., Tsuchiya, M., Naka, S., Takagi, K. (1977) Effects of Panax ginseng root on conditioned avoidance response in rats. Jpn. J. Pharmacol. 27: 509-516 Sanada, S., Kondo, N., Shoji, J., Tanaka, O., Shibata, S. (1974) Studies on the saponins of Ginseng. I. Structure of ginsenoside Ro, Rbl, Rb2, Rc and Rd. Chem. Pharm. Bull. 22: 421 428 Shibata S., Tanaka, O., Soma K., Iida Y., Ando, T., Nakamura, H. (1965) Studies on saponins and sapogenins of Ginseng. The structure of panaxatriol. Tetrahedron Lett. 207-213 Shibata, S., Tanaka, O., Ando, T., Sado, M., Tsushima, S., Ohsawa, T. (1966) Chemical studies on the oriental plant drug. XIV. Protopanaxadiol, a genuine sapogenin of Ginseng saponins. Chem. Pharm. Bull. 14: 595-600 Sonnenborg, U. (1987). Ginseng. Dtsch. Apoth. Ztg. 127: 433441 Takagi, K., Saito, H., Tsuchiya, M. (1972a) Pharmacological studies of Panax ginseng root: pharmacological properties of a crude saponin fraction. Jpn. J. Pharmacol. 22: 339-346 Takagi, K., Saito, H., Nabata, H. (1972b) Pharmacological studies of Panax ginseng root: estimation of pharmacological actions of Panax ginseng root. Jpn. J. Pharmacol. 22: 245-259 Takagi, K., Saito, H., Tsuchiya, M. (1974) Effect of Panax ginseng root on spontaneous movement and exercise in mice. Jpn. J. Pharmacol. 24: 41-48 Wang, B. X. (1980) Recent progress of pharmacological studies of Panax ginseng. Acta Pharm. Sin. 15: 312-320 Wilkinson, I. D., Gentry, L. 0. (1981) In vitro comparison of ceftazidime and nine other antimicrobial agents against hospital strains of Gram-negative bacteria. J. Antimicrob. 8: 53-56