Antiyeast activities of crude saponin fractions (MIC μg/ml)
Saponins are a class of natural products which are structurally constructed of aglycone (triterpene or steroid) and sugars (hexose and/or uronic acid). The name ‘saponin’ comes from soap as its containing plants agitated in water form soapy lather. Saponins are widely distributed in many plants and are relatively widespread in our foodstuffs and herbal preparations. Saponins traditionally used as a natural detergent. In addition to this physical property, plant-derived triterpenoid and steroidal saponins have historically received a number of industrial and commercial applications ranging from their use as sources of raw materials for the production of steroid hormones in the pharmaceutical industry, to their use as food additives and as ingredients in photographic emulsions, fire extinguishers and other industrial applications which take advantage of their generally non-ionic surfactant properties [1-3]. They also exhibit a variety of biological activities, and have been investigated toward the development of new natural medicines and prove the efficacy of traditional herbal medicines . Other interesting biological applications for various specific saponins include their uses as anti-inflammatory , hypocholesterolemic  and immune-stimulating  whose properties are widely recognized and commercially utilized.
As to the application of saponins to foods and cosmetics, it is indispensable that sufficient amounts of plant resources are available, and that the content of saponins must be high. Furthermore, a plant must have a long history of human use as foodstuffs or ingredients of cosmetics, and their safety should be officially guaranteed.
The saponins of Quillaja bark and licorice root are widely utilized in the world. The Quillaja saponaria (Rosaceae) tree has remained of special interest, because of its bark containing 9-10 % saponins. A large amount of Quillaja saponin is utilized in photosensitized film as a surfactant. It is used also in beverages, food ingredients, shampoos, liquid detergents, toothpastes and extinguishers as an emulsifier and long-lasting foaming agent. Recently, the saponin mixture possesses the immunoadjuvant property and has pharmaceutical application as suspension stabilizer .
Nearly 50,000 tons of licorice roots (Glycyrrhiza spp., Leguminosae) are consumed on a year basis. Licorice extract and its major saponin, glycyrrhizin (yield: more than 2.5%), are used as a medicine and as a sweetener and flavor enhancer in foods and cigarettes .
It is known that the deterioration of cooked foods is caused mainly by yeast, and that many skin diseases are due to infection by dermatophytic fungi and yeasts. In an expansion of utilization of saponins in foods and cosmetics, we have examined antifungal and antiyeast saponins.
2. Screening of antiyeast saponins
Crude saponin fractions from several plants were subjected to an antiyeast screening test using Candida albicans and/or Saccharomyces cerevisiae. Preparation of saponin fraction for screening test was following methods. Each plant material was extracted with hot 50% of MeOH. A suspension of the MeOH-extract in H2O was chromatographed on a column of Diaion HP-20 eluting with 40%-, and MeOH. The MeOH eluate (crude saponin fraction) was subjected to the screening test.
Inhibitory activity against each yeast was determined using agar dilution method. The inhibitory activity of the samples was assessed as the minimum inhibitory concentration (MIC), the lowest concentration tested at which no growth was observed.
Table 1 shows the screening results of antiyeast activity tests of crude saponin mixtures from several plants. The saponin fraction from licorice root, quillaja bark, gypsophila root and soy bean seed showed no activity (MIC:>1000μg/ml) and that of hedera leaf, marronier seed, ginseng root, camellia seed, saponaria rhizome and tea seed showed a weak activity (MIC:500∼1000μg/ml), wheras crude saponin fraction from pericarps of Sapindus mukurossi and the stems of Mohave yucca exhibited significant activity, the active principles of both these materials were further investigated in detail.
|Licorice root||>1000||>1000||Ginseng root||1000||1000|
|Quillaja bark||>1000||>1000||Camellia seed||1000||1000|
|Gypsophila root||>1000||>1000||Saponaria rhizome||NT||1000|
|Hedera leaf||1000||1000||Tea seed||500||500|
|Soy been seed||NT||>1000||Yucca stem||NT||250|
|Marronier seed||1000||1000||Sapindus pericarp||250||250|
3. Sapindus pericarps
Addition of an antifungal and antiyeast ingredient to cosmetics is desirable for the protection of skin against, and prevention of, dandruff generation, dermatomycosis and cutaneous candidiasis.
Significant antiyeast activity was observed for the crude saponin fraction from the pericarps of Sapindus mukurossi (Sapindaceae), a tall tree that grows abundantly in China and Japan. Pericaps of this plant have been used as a natural detergent, and are utilized as foaming-stabilizing agents in chemical fire extinguishers in Japan. The pericarps have also been used as an antitussive, anti-inflammatory and anthelmintic agent as well as for treatment of dermatomycosis. In Japan, the pericarps is called “enmei-hi”, which means “life prolonging pericarps”, and in China, it has been called “wu-huan-zi”, which means “non-illness fruit”.
4. Antifungal and antiyeast oleanane-saponins of Sapindus pericarps
The percarps were extracted with hot 90% MeOH. A suspension of the MeOH-extract in H2O was chromatographed on a column of highly porous polymer (Diaion H-20) eluting with H2O and 50%- and 85%-MeOH, successively. 85%-MeOH eluate gave a saponin-mixture (mono- and bis-desmosides, SP-mix). Hederagenin (1) was obtained from SP-mix by usual acid hydrolysis. Saponins 2-7 were isolated from SP-mix, such as monodesmosides: saponin A (2), sapindoside B (3), saponin C (4), sapindoside A (5), mukurozi-saponin E1 (6) etc. and bisdesmosides: mukurozi-saponin Y1 (7) etc. . The structures of these saponins are shown in Figure 1.
Antidermatophytic activities of these saponins are shown in Table 2. All the monodesmosides exhibited strong growth inhibition. It is noteworthy that activity of sapindoside A is almost as strong as that of griseofulvin, the well-known antidermatophytic antibiotic. Griseofulvin does not show inhibitory activity against a pathogenic yeast, Candida albicans, while these monodesmosides ehhibited significant inhibition. The bisdesmosides, mukurozi-saponin Y1 showed no activity.
It was found that while purified monodesmosides of pericarps are sparingly soluble in water, their solubility was greatly increased in the presence of bisdesmosides . These phenomena are important for the biological activities of the pericarps.
5. Structure-antifungal activity relationship
Figure 1 showed antidermatophytic activity against Tricophyton rubrum was investigated for a variety of oleanane saponins. Saponins 8-10 were separated from roots of Anemone rivularis . Saponins 11-13 were isolated from bupleurum roots , and saponins 14 and 15 were prepared from 11 and 12, respectively by the reference . Saponin 16 was isolated from roots of Kalopanax septemlobus . Saponin 17-20 were isolated from brans of Chenopodium quinoa [15, 16], and saponin 21 from rhizome of Thladiantha hookeri var. pentadactyla , derivative 1 (22) was prepared from 21, and derivative 2 (23) from 22 .
It was disclosed that for growth inhibition, the presence of free 28-COOH, 23-OH and 3-O-glycosyl groups is essential (Figure 2). A sugar moiety was prerequisite for the antifungal activity of oleanane saponin. All the bisdesmosides of hederagein, such as kalopanaxsaponin B (16), the 28-COOH of which is glycosylated, showed no activity. Mono- and bisdesmosides of oleanolic acid, such as saponin CP4 (8), which lack a 23-OH, also showed no growth inhibition. Saikosaponins, the active principles of Bupleurum radix, lack a 28-COOH, exhibiting no activity. Thalandioside H1 (21), a bisdesmoside which was isolated from Thandiantha hookeri var. pentaphyla in yield of 10% without any chromatography (Nie et al., 1989), showed no activity, while a monodesmoside of hederagein derived from this bisdesmoside, exhibited activity. Activity was also obserbed for hederagenin-3-O-α-L-arabinoside (24) which was prepared from 17 .
|Trichophyton mentagrophytes||T. rubrum||Epidermophyton floccosum||Sabouraudites canis||Candida albicans|
|2. saponin A||6.25||6.25||6.25||3.13||12.5|
|3. sapindoside B||6.25||6.25||3.13||3.13||12.5|
|4. saponin C||6.25||6.25||6.25||3.13||25|
|5. sapindoside A||3.13||1.56||3.13||1.56||12.5|
|6. mukurozi-saponin E1||6.25||6.25||6.25||3.13||12.5|
|7. Mukurozi-saponin Y1||>100||>100||>100||>100||>100|
|* positive control|
6. Antimicrobial activity of the saponin fraction of Sapindus pericarps
For commercial utilization as ingredient in cosmetics, the saponin fraction was prepared as follows. The methanolic extract was subjected to chromatography on Diaion HP-20. After removal of other water-soluble constituents by elution with water and then 50% of MeOH, the saponin fraction was obtained by elution with 80% MeOH.
The saponin fraction showed moderate antibacterial activity against Gram-positive bacteria, while no activity was obserbed against Gram-negative bacteria (Table 3).
A summarized in Table 4, the saponin fraction exhibited growth inhibition against food deteriorating yeasts, Pichia nakazawae, Debaryomyces hansenii and Hansenula anomala, as well as against Malassezia furfur which is associated with dandruff generation. The activity of saponin fraction against common fungi was not so strong, while it exhibited remarkable growth-inhibitory effects against the following dermatophytic fungi and pathogenic yeast, Tricophyton rubrum, T. mentagrophytes, Sabouraudites canis, and Epidermophyton floccosum (which are known as dermatophytic fungi) and against Candida albicans, a pathogenic yeast which causes cutaneous candidiasis.
|Gram-positive, MIC:μg/ml||Gram-negative, MIC:μg/ml|
|aureus IID 671||400||Escherichia coli HUT 215||>400|
|epidermidis IID 866||400||Pseudomonas aeruginosa JCM 2776||>400|
|Streptococcus mutans IFO 13955||400||Alcaligenes faecalis IFO 13111||>400|
|Bacillus subtilis IFO 3007||400||Proteus vulgaris IFO 3851||>400|
|Saccharomyces cerevisiae IFO 0203||100||Candida utilis IFO 0396||100|
|Pichia nakazawae HUT 1688||50||Hansenula anomala HUT 7083||50|
|Malassezia furfur IFO 0656||200||Debaryomyces hansenii IFO 0018||>400|
|Aspergillus niger IFO 4343||>400||Rhizopus nigricans IFO 4731||>400|
|Mucor pusillus HUT 1185||100||Penicillium citrinum IFO 4631||>400|
7. Sapindus saponin fraction as an antidermatophytic ingredient in cosmetics
It is difficult to use Sapindus saponin fraction as a food ingredient without long-term toxicity test, because we have no history of this fraction or Sapindus extract as a foodstuff. Furthrmore, it tastes very bitter, changing the taste of foods. On the other hand, the extract has been used as a folk detergent,and is listed in the Japanese Cosmeic Ingredient Codex (JCIC), being authorized as an ingredient in cosmetics by the Ministry of Health and Welfare in Japan. We reconfirmed the safety of the saponin fraction by dermal toxicity tests. It did not show primary dermal irritant, sentitization, phototoxicity or photosensitization effects. The present study strongly suggests that the saponins of the pericarps as an ingredient in toiletries, are valuable not only as detergents, but also for the prevention of dermatomycosis, cutaneous candidiasis as well as for dandruff generation.
8. Mohave Yucca (Yucca schidigera)
Yucca species (Agavaceae), grows widely in North and Central America. Mohave yucca, Y. schidigera, has been used as a foodstuff and folk medicine by Native Americans as well as early California settlers to treat a variety of ailments including arthritis and inflammation , and is approved for use in food and beverages by the U.S. Food and Drug Administration (FDA) under Title 21 CFR 172.510, FEMA number 3121. Yucca products are currently used in a number of applications. Yucca powder and yucca extract are used as animal feed additives, as in reference . Other applications include the use of the extract of this plant is now utilized as a long-lasting foaming agent in carbonated beverages, root beer, regular and low-alcohol beers, and in shampoos and foaming cosmetics. Recently, the potential of biological activities of saponins and phenolics from this plant was reviewed .
9. Antiyeast and antifungal spirostanoid saponins from Mohave yucca
The presence of steroidal saponins in this plant has been reported previously [21,22]. As to the saponin constituents of this plant, a monodesmoside named YS-1 is isolated and identified as in . We have conducted the isolation and identification of individual saponins that had not been achieved prior to this study [24,25].
The EtOH extract of this plant was subjected to colomn chromatography on highly porous polymer, Diaion HP-20, which is styrene-divinylbenzene polymer. After successive elution with water and 60% and 80% MeOH, a saponin fraction which showed significant antiyeast activity against Saccharomyces cerevisiae was obtained by elution with 90% MeOH. This fraction was subjected to successive chromatography on silica gel and then octadesysilylated silica gel (ODS) and was finaly separated by HPLC on ODS to give fourteen yucca saponins 25-38.
Figure 3 shows the structure of all of these saponins and their sapogenins. The antiyeast activities of each saponin from Y. schidigera against six kinds of yeast, Saccharomyces cerevisiae (brewers yeast), Candida albicans (a pathogenic yeast) and Hansenula anomala, Pichia nakazawae, Kloeckera apiculata and Debaryomyces hansenii (food-deteriorating yeasts) were determined and are summarized in Table 5.
Those saponins having a branched-chain trisaccharide moiety without any oxygen functionalities at C-2 and –12 exhibited potent antiyeast activities, while saponins with 2β-hydroxyl (5,6,13, and 14) or 12-keto (4 and 12) groups showed very weak or no activity. A saponin (11) with a disaccharide moiety exhibited relatively low activities. The aglycons showed no antiyeast activity.
10. Antimicrobial activity of the saponin fraction
For the commercial utilization of Mohave yucca, the antimicrobial activity of the saponin fraction which was obtained by column chromatography of the extract on Diaion HP-20 (vide supra) was investigated. It showed no or only weak growth inhibition against both Gram-positive and Gram-negative bacteria (Table 6).
|S.c.a||C.a. b||H.a. c||P.a. d||K.a. e||D.h. f|
|Gram-positive bacteria, MIC (μg/ml)|
|Staphylococcus||Bacillus circulans IFO 3329||>1,000|
|aureus IID 671||1,000||Lactobacillus|
|aureus IFO 3060||1,000||plantarum IFO 3070||>1,000|
|epidermidis IID 866||>1,000||rhamnosus IFO 12521||>1,000|
|Bacillus||Enterococcus faecalis IFO 3971||>1,000|
|subtilis IFO 3007||1,000||Streptococcus mutans IFO 13955||>1,000|
|licheniformis IFO 12200||1,000|
|Gram-negative bacteria, MIC (μg/ml)|
|Escherichia coli HUT 215||>1,000||Pseudomonas|
|Alcaligenes faecalis IFO 13111||1,000||aeruginosa JCM 2776||>1,000|
|Proteus vulgaris IFO 3851||1,000||fluorescens JCM 2779||>1,000|
|Klebsiella pneumoniae IFO14940||1,000|
|Yeast, MIC (μg/ml)|
|Saccharomyces||Kloeckera apiculata IFO 154||62.5|
|cerevisiae IMO 293||62.5||Debaryomyces|
|cerevisiae HUT 2075||31.3||hanenii IFO 18||31.3|
|cerevisiae JCM 2223||62.5||hanenii IFO 27||62.5|
|Hansenulla sp.||31.3||hanenii IFO 47||31.3|
|anomala HUT 7083||31.3||hanenii IFO 7011||125|
|laurentii IFO 609||125||rouxii IFO 845||31.3|
|Pichia||rouxii IFO 1130||31.3|
|nalazawae HUT 1688||31.3||Candida famata IFO 664||31.3|
|carsonii IFO 946||31.3|
|Fungi, MIC (μg/ml)|
|niger IFO 4343||>1,000||awamoi HUT 2014||>1,000|
|oryzae HUT 2065||>1,000||awamoi HUT 2015||>1,000|
|oryzae HUT 2175||125||Mucor pusillus HUT 1185||15.6|
|oryzae HUT 2188||>1,000||Rhizopus|
|oryzae HUT 2192||>1,000||formosaensis IFO 4756||>1,000|
|sydowii HUT 4097||>1,000||nigricans IFO 4731||>1,000|
|Penicillium expansum IFO 5453||>1,000|
|Dermatophytic yeast and fungi, MIC (μg/ml)|
|Tricophyton||Sabouraudites canis IFO 7863||31.3|
|rubrum IFO 5807||15.6||Epidermophyton floccosum IFO 9045||31.3|
|mentagrophytes IFO 5809||31.3||Candida albicans TIMM 0134||62.5|
The antiyeast and antifungal activities are summarized in Table 7. The saponin fraction exhibited potent antiyeast activity. Infection of boiled rice such as “sushi” and “musubi” with Hansenula anomala and Kloeckera apiculata results in odor smelling like an organic solvent. Infection of cooked beans and processed fish meat with Candida famata and Pichia carsonii causes oders smelling like kerosene. Pichia nakazawae, Debaryomyces hansenii and Zygosaccharomyces rouxii are film-forming yeasts, damaging “soy sauce” and “miso”, oriental fermented seasonings. The saponin fraction exhibited strong growth inhibition against these food-deteriorating yeasts.
The saponin fraction showed less activity against common fungi, while it significantly inhibited the growth of dermatophytic yeast and fungi.
Potassium sorbate has been utilized in foods as a preservative. Its antiyeast activity depends upon pH. Between pH 5.0 – 3.0, potassium sorbate completely inhibited the growth of yeast at the concentration of 0.05%, while at less acidic pH (near neutral), the activity decreased remarkably. In contrast to this, such pH dependence was not observed for the yucca saponin fraction. In the range of pH 6.3 – 3.0, it entirely inhibited the growth of yeasts at the concentration of 0.03%.
11. Effects of several culture conditions against antimicrobial activity of yucca extract
The inhibitory effects of yucca extract on the growth of the yeasts isolated from ume-zuke, a salted Japanese apricot fruit product were investigated with (2% or 5%) or without sodium chloride (Table 8). From the results of MICs of yucca extract without sodium chloride, the genera Debaryomyces, Kloeckera, Pichia, Saccharomyces and Zygosaccharomyces are sensitive to yucca extract, while the genera Cryptococcus, Rhodotorula and Sporobolomyces are tolerate to yucca extract. For the difference between these yeasts, latter yeast belong anamorphic basidiomycetous genera.
The inhibitory effect was enhanced and showed a broad antiyeast spectrum when yucca extract was used in combination with sodium chloride.
Table 9 shows the effects of several cultural conditions against antiyeast activity of yucca extract. The antiyeast activity of yucca extract was strengthened under the condition of chemical and physical conditions, low pH, alcohol, heating and high OP. While the high-polymer substances, such as polysaccharides and protein reduced antiyeast activity of yucca extract. It is interested that antiyeast activity of yucca extract was inhibited by free unsaturated fatty acids, palmitoleic acid, oleic acid and linoleic acid. On the other hand, saturated fatty acids, palmitic acid and stearic acid and oils composed of unsaturated fatty acids, olive oil, soybean oil and egg lecithin had no effect on the antiyeast activity of yucca extract.
|Yeast||MIC (μg/ml)||Yeast||MIC (μg/ml)|
|C. albicans 221||1000||500||250||P.anomala 201||500||250||250|
|C. guilliermondii 212||>2000||1000||500||P.anomala 202||250||250||250|
|C. guilliermondii 213||>2000||2000||500||P.anomala 203||250||125||125|
|C. guilliermondii 222||1000||500||250||P.anomala 204||500||250||250|
|C. guilliermondii 224||1000||250||250||P.anomala 206||250||125||125|
|C. guilliermondii 227||>2000||>2000||2000||P.anomala 211||500||250||250|
|C. krusei 222||>2000||>2000||1000||P.anomala 216||500||250||125|
|C. lipolytica 223||62.5||62.5||62.5||P.anomala 219||500||250||250|
|C. parapsilosis 224||1000||500||500||P.anomala 223||500||250||250|
|C. tropicalis 225||>2000||>2000||1000||P.anomala 256||500||250||250|
|C. valida 226||1000||500||125||P.anomala 260||250||250||250|
|C. versatilis 228||500||250||250||P.anomala 261||500||250||250|
|C. zeylanoides 229||250||250||125||P.anomala 262||500||250||250|
|C. neoformans 231||>2000||>2000||1000||P. farinosa 207||250||250||125|
|D.hansenii 201||1000||125||62.5||R. rubra 233||>2000||1000||500|
|D.hansenii 214||1000||1000||1000||S. cerevisiae 203||500||250||62.5|
|D.hansenii 215||1000||1000||2000||S. cerevisiae 208||250||250||62.5|
|D.hansenii 220||2000||2000||>2000||S. farmentati 209||500||250||62.5|
|D.hansenii 225||1000||2000||2000||S. fibullgera 211||2000||1000||1000|
|D.hansenii 263||>2000||2000||1000||S. servazzii 210||2000||1000||1000|
|G. candidum 218||500||125||NG||S. pombe 212||62.5||NG||NG|
|G. capitatum 219||2000||1000||NG||Sporobolomyces|
|Hansenula||S. albo-rubescens 234||>2000||>2000||2000|
|H. saturnus 202||1000||500||250||Torulaspora|
|Issatchenkia||T. delbrieckii 4188||500||125||62.5|
|I. orientalis 237||125||125||62.5||T. delbrieckii 4952||500||500||250|
|K. apiculata 203||1000||1000||500||Z. bailii 213||2000||250||NG|
|K. apiculata 208||1000||500||500||Z. rouxii 214||500||250||250|
|K. apiculata 258||1000||1000||500||Z. rouxii 215||250||250||250|
|K. apiculata 266||>2000||>2000||2000||Z. rouxii 216||125||125||250|
|K. apiculata 4631||2000||1000||62.5|
|K. apiculata 12219||2000||2000||500|
|K. corticis 217||1000||1000||NG|
|K. corticis 236||2000||250||NG|
|K. corticis 12828||500||250||NG|
|K. japonica 12220||500||500||250|
|NG : No growth recognized without yucca extract|
|low pH||heating||alcohol||polysaccharide||protein||lipid||high OP***|
12. Utilization of the yucca extract as an anti-food deteriorating agents
Yucca extract is non-toxic and non-mutagenic. It is recognized as safe for human food use by U.S.FDA (listed in 21 CFR 172.510). The extract is tasteless and odourless, exerting no influence on the taste of foods. It is readily soluble in water and stable on heating. Based on the present study, commercial application of the extract for extending the shelf life of cooked foods and fermented seasonings is now under development .
Figure 4 shows the application of yucca extract to sponge cake. Addition of 0.2% of yucca extract to sponge cake had effective on the growth of fungi and yeasts stored in room for one week.
The application of yucca extract to strawberry jam was showed in Figure 5. The jam mixed 0.02% and 0.04% of yucca extract and stored in room for one week shows no change, whereas control jam was contaminated by fungi.
The microbial safety of foods and cosmetics continues to be a major concern to consumers, regulatory agencies and food industries throughout the world. Although synthetic antimicrobials are approved in many countries, the recent trend has been for use of natural preservatives, which necessitates the exploration of alternative sources of safe, effective and acceptable natural preservatives. Many plant extracts possess antimicrobial activity against a range of bacteria, yeast and fungi, but the variations in quality and quantity of their bioactive constituents is major disadvantage to their industrial uses.
Based on the present study, mukurozi extract and yucca extract are considered to be effective for the preservation of foods and cosmetics. Both mukurozi and yucca plants have been consumed by humans for a long time. These plants also have wide application due to little pH or food component interaction.
Thus our works demonstrate that the saponin fraction from Sapindus pericarps and Mohave yucca stems can be recommended as alternative preservations for foods and cosmetics.