Antimicrobial botanicals

J. Wille

Microbiology Department, Bioderm Technologies, Inc., Chesterfield, New Jersey 08515

We describe the successful preparation and antibacterial activity of many different botanical extracts that are effective antibacterial agents against both the gram-positive bacterium, Staphylococcus epidermis, isolated from human armpit skin, yeast cells (Saccharomyces cerevisae), and gram-negative bacteria Pseudomonas aeruginosa. The botanical extracts were prepared as water or alcohol extracts, which were diluted in to saline solutions to ascertain their antibacterial activity. The microbiological methods and results confirm that all of the cited examples of botanical extracts including: Allspice (Pimenta diocia), Applephenon®, a commercially available green apple extract, ripe autumn olive berry (Eleagnus umbelifera), dried “Baes pepper korns from Guadalupe, cloves (Syzgium aromaticum), corn tassels (Zea mays), curly dock seeds (Rumex crispus), dried hops leaves (Humulus lupulus), dried Lavender flowers (Lavendula provence), Linden tree catkin (Tilia Americana ), dried oak tree catkin (Quercus alba), dried Oregano leaves (Origanum vulgare), Chinese Prickly Ash (Pericarpium zanthoxylli), commercially available dried green tea leaves (Camellia sinensis) have proven antibacterial activity against underarm derived S. epidermis. Several botanical extracts including Citricidal®, and HIDROX®, a commercial water-soluble extract of green olive pulp, and White Willow Bark extract were included as positive controls in the microbiological assays as they are known antioxidants and antimicrobial agents. The demonstrated bactericidal activity of all of the cited botanical extracts has a strong correlation to the polyphenolic content of the extracts.

Keywords antibacterial, botanicals, medicinal herbs, plant extracts, polyphenolic compounds

1. Introduction

Historically, botanicals have been an important source of both new pharmaceuticals and new cosmetic ingredients. Even today plants have yield more new medicinal compounds and cosmetic ingredients than the chemical synthesis approaches exemplified by the recent reliance on combinatorial chemistry methods [1]. Recent interest has shifted to the use of safer and natural botanical ingredients to preserve personal care products and or to kill pathogenic bacteria in dermatological preparations. The worldwide search for novel antibacterial natural products from plants has led to exotic plants in tropical rainforests of the Amazon and to the ocean depths. Yet, it must be noted, that the screening of higher plants for bioactive ingredients has barely scratched the surface of the more than 250,000 species of flowering plants (Angiosperms), and very few of the 50,000 species of monocots (grasses and ornamentals) relatives to the 200,000 species of dicot herbs, shrubs, trees, and ornamentals). Thus, it fair to conclude that a systematic and rationale approach to this task has yet to be formulated. Among the antimicrobials medicinal plants found in the desert and canyons of the American southwest are: Acacia, Algerita, Camphor Weed, Chaparral, Condalia, Cypress, Dessert Willow, Epizote, Eucalyptus, Juniper, Mesquite, Pinon, Sage and Yerba Mansa [2]. A partial list of antiseptic extracts from common medical herbs includes Arnica, Anise seeds, Bearberry, Blueberry Buchu, Burdick, Fenugreek seeds, Lemongrass Marigolds, Marshmallow, Plantain, Privet, Rosemary, Sage, Silver Birch leaves, and Wild Pansy, Wild Strawberry, and Yarrow [3, 4]. Several medicinal herbs have reached the market as common antiseptic remedies including Goldenseal with berberine as it active ingredient, Thyme with its active ingredient thymol, the fungicide, Tea Tree oil, and Witch Hazel, a distillate for antisepsis and treatment of insect bites [4]. A cursory review of the patent literature discloses antimicrobial botanicals derived essential oils, e.g., limonine, geraniol, and other terpenoids [5]. A polyphenolic derivative obtained from licorice extracts was reported to inhibit the growth of oral microbials [6]. Certain metals complexed with the essential oil, Hinokitiol, act as a bactericide against vancomycin-resistant enterococcus bacteria [7]. A method for inhibiting the production of bacteria which constitutively express PBP2 in combination with the antibiotic beta-lactam using tea extracts is active against methacillin-resistant S. aureus (MRSA) [8]. It is well known, that tea extracts yield antibacterial catechins such as trihydroxybenzoic acid. Among the bioactive antibacterials derived from indigo plant are kaemerferol, gallic acid, caffeic acid, indirubin, pheophenorbide and trptanrthrin [9]. Lastly, an antibacterial protein extracts have been reported from seeds of marigolds and paprika [10]. Here we take a different approach to the search for novel antibacterial botanicals, which narrows the search to plants that grow in the wild, or are cultivated primarily as a foodstuff but for which there is no present commercial medicinal or cosmetic uses. This has led to the discovery of many new antimicrobial botanical sources.

Microbial pathogens and strategies for combating them: science, technology and education (A. Méndez-Vilas, Ed.)

© FORMATEX 2013

____________________________________________________________________________________________

883

2. Materials and Methods

2.1. Source of botanicals

a) Applephenon® was obtained from Ashai Breweries, Tokyo, Japan. A method for the preparation of an apple extract enriched in polyphenols was reported [11], the main one being oligomeric procyanidins. Applephenon® is a commercial preparation of this apple extract; b) HIDROX ™ , derived from pulp of olives, was purchased from CreAgri, Inc, Hayward,CA; c) White Willow bark extract was obtained from Active Concepts, Middlesex, NJ); d) Citricidal® a grapefruit seed oil was purchased from Samuel Allen Associates, Ripton, VT. All of the remained botanicals were collected from the wild or were from herbs grown from seed.

2.2. Extraction procedures

Botanical extracts were prepared as follows: approximately 10 grams leaves, berries seeds, etc, were blended with 90 grams of water or 90 grams of 50% alcohol (ethanol) in a Hamilton Beach blender for 2 minutes (liquefy setting) at room temperature. The blended material was centrifuged for 10 minutes at 300 rpm at 50 C., and the clarified supernatant harvested by decantation into sterile glass flasks. The clarified extract was stored at 40 C for further study. In the case of commercial powders, powders were weighed out and 10 grams dissolved directly into 100 mL of distilled water or 50% ethanol [12].

2.3. Polyphenol Test: Assay conditions

Fifty microliters of each botanical extract was deposited in one well of a 96-well culture dish (see figure 1) and reacted with 50 microliters of an aqueous solution of ferric chloride (10%) deposited in the same well. An instantaneous color change occurs giving a green-black color of varying intensity correlated with the amount of polyphenols present in the extract. As a control for color change 100microliters if un-reacted ferric chloride was deposited in wells C1, C7 and C12. All extracts were tested for their polyphenol content by titrating them against a 10% aqueous ferric chloride solution. Polyphenols will react with ferric chloride (oxidant) and turn from an orange color to a dark green to black color if the botanical polyphenol content has strong antioxidant activity. The antioxidant activities of botanical polyphenols were measured colorimetrically by the intensity of green-black reaction product formed in the test.

2.4. Microbiological tests

Three different microbiological assays were employed: a) the self-sterilization assay, in which extracts were inoculated directly into bacterial culture medium to determine if there were any bacteria that would grow out upon incubation at 37oC. A negative response would indicate that the extract has endogenous bactericidal activity. The extracts were assessed for self-sterilization by inoculating small aliquots of a 10% (w/v) botanical extract into sterile beef heart infusion (BHI) medium and incubated at 37o C for 48 hours; b) the challenge assay, in which various dilutions of each self-sterilizing extract were challenged by the adding a 10 microliter aliquot containing 1x105 Staphylococcus epidermis bacteria to 1.0 ml of sterile saline and also continuing various concentrations of the test botanical extracts. The challenge assay tubes were incubated at 37o C for 48 hours. The assay included a positive control in which no botanical extract is added to the test. Positive controls become slightly turbid indicating the growth of the bacteria, while assays containing botanical extracts remain clear, indicating the bactericidal activity; and c) the post-challenge-test. This test is done to show that the botanical extracts had actually killed the bacteria rather merely preventing their growth. Small aliquots of the challenge media were further re-inoculated into fresh sterile BHI bacterial culture medium for an additional 48 hours. Bacterial growth is expected from the positive control tubes, while the absence of any bacterial growth in the post-challenge botanical extract tests assures that the extract has killed all 105 bacteria that were initially added in the challenge assay.

3. Results

3.1. Polyphenolic content of botanicals

Figure 1 presents photographic results for 24 different botanical test substances (rows A and B) and the unreacted (row C, yellow) controls.

Microbial pathogens and strategies for combating them: science, technology and education (A. Méndez-Vilas, Ed.)

© FORMATEX 2013

____________________________________________________________________________________________

884

Fig. 1 Ferric Chloride (0.5%) reaction for detection of polyphenols present in botanical extracts (see text below for details). Row A: 1. Corn Tassel extract in 50% ethanol (2X concentrated) (+++); 2. Green Tea Leaf extract (5%) in 50% ethanol (++++); 3. Pericarpium zanthoxyli (2%)-water-soluble extract (++); 4. Hydrox powder (5%) in 50% ethanol (0/+); 5. Autumn Olive berry extract (Prep II) in 50% ethanol -hexane fraction- (0); 6. Autumn Olive berry (Prep I) in50% ethanol extract (++); 7. Corn Tassel-70% ethanol extract (+/++); 8. Powdered Tassel pellet of 70% ethanol extract (+); 9. White Willow bark (5%) extract in 50% ethanol (+++); 10. ApplepehonSH extract (5%) in 50% ethanol (+++/++++); 11. Cocoa powder extract (5%) in 50% ethanol (++); 12. Corn Tassel extract (7%) in 50% ethanol ((+++). Row B: 1. Caffeic acid (1%) in 70% ethanol ((+++); 2. Gallic acid (1%) in 70% ethanol (+++); 3. Cinnamon bark powder (5%) in 50% ethanol (++); 4.Tomato paste (5%) extract in 50% ethanol (0/+); 5.French Lavender dried flower extract (5%) in 50% ethanol (+/++); 6.Tilia tree dried catkin (5%) in 50% ethanol (+++); 7. Corn Tassel (7%) in 70% ethanol (+/++); 8. Autumn Olive berry (Prep II) in 50% ethanol ((+/++); 9. Curly Dock dried tassel extract (10%) in 50% ethanol (++); 10. Red Pepper fresh vegetable extract in 50% ethanol (0); 11. Red Grapes fresh fruit extract in 50% ethanol ((0/+); 12. Baes dried pepper korn spice extract in 50% ethanol ((++/+++). Row C: numbers: 1, 7 and 12 10% Ferric Chloride solution The most reactive botanicals are: 1) a proanthocyanidin–rich extract prepared from unripe apples and available commercially (Applephenon SH®, (Figure 1, A10), 2) a hydroxytyrosol-enriched extract derived from hydrolyzedaqueous olive pulp (HIDROX ™, (Figure 1, A4), 3) a hydroalcoholic extracts of Autumn Olive (Eleagnus umbelifera) ripe berries, (Figure 1, A6), 4) water and hydroalcoholic extracts from green tea leaves Camellia sinensis, (Figure1 A2), 5) White Willow bark extract-enriched in polyphenols, , (Figure 1, A9), 6) a hydroalcoholic extract of Linden tree catkins, (Figure 1, B6), 7) a hydroalcolholic extract of seed pods of Curly Dock, (Figure 1, B9), 8) water extracts of the dried fruit of prickly ash (Pericarpium zanthoxyli)(Figure 1, A3). In addition, the following botanical extracts were found to be positive in the ferric chloride test: a water extract of powdered Tasselin, hydroalcoholic extracts of dried herb leaves of Oregano, Hops, Lavender flowers, dried “Baes” seeds (from island of Guadelope), Cocoa powder, Cinnamon bark, and dried catkins of the Oak tree. In support of these results, a diphenylpicrylhydrazine (DPPH) assay (data not shown) confirmed the antioxidant activity determinations made on all of the above extracts.

3.2. Self-sterility assays

Figure 2 is a photograph showing typical results of self-sterilization assays for Tasselin (corn tassel extract) tested for sterility as a water extract (A), and methanol (B) and ethanol (C) extracts produced by sequential extraction of the water extract by methanol followed by ethanol.

Fig. 2 Tasselin extracts prepared from corn tassels and tested for self-sterility. A- Water extract, B- methanol extract, and C-70% ethanol extract The three dishes labeled A, B, and C clearly indicates that no endogenous bacteria in the extracts survived the action of the antibacterial agents in Tasselin. The positive dish had heavy bacterial growth is not shown. Self-sterility was found to be an intrinsic property of extracts from Autumn olive berry, green tea, prickly ash, Applephenon, dried

Microbial pathogens and strategies for combating them: science, technology and education (A. Méndez-Vilas, Ed.)

© FORMATEX 2013

____________________________________________________________________________________________

885

Curly dock flowers, dried Oregano leaves, dried Hops leaves, dried Lavender flowers, dried Baes “pepper korns”, cocoa powder, cinnamon bark, and Linden tree catkins (data not shown).

3.3. Challenge assay.

A typical example of a positive challenge test type data is presented in Figure 3 for green tea extracts.

Fig. 3 Color photograph of 6-well cluster dish showing the results of testing green tea botanical extract for antibacterial activity in the challenge assay test.(see text for details).

The above figure presents a photograph of a test 6-well cluster dish. The top three wells are control wells. They were inoculated with S. epidermis in the absence of green tea extract. They appear cloudy indicating growth of S. epidermis bacteria even though they were also grown in the presence of different concentrations of ethanol (0, 10 and 5%). The bottom left well was left deliberately left empty for color contrast. The bottom middle and right test wells were inoculated with both S. epidermis and 0.5% (w/v) green tea extracts and as with the above controls also with10% and 5% ethanol. The test wells are clear indicating that the green tea extract completely inhibited all bacterial growth. This is an all-or-none result meaning that no bacteria can grow at the fixed concentration of test botanical in the presence of a standard fixed concentration of bacteria. It must be pointed out that the challenge assay does not rule out that the test botanical was merely bacteriostatic. To rule this possibility out, we perform a post-challenge assay. In which a small inoculum (10 uL) is removed aseptically from the test challenge well and reinoculated in to sterile growth medium. If nothing grows out after 48-72 hours, the test botanical is said to have killed all the bacteria exposed to it. Since nothing did grow out in the post challenge test, green tea hydroalcoholic extract was effective in killing five-logs of S. epidermis bacteria. With this caveat in mind, only the results of the post-challenge assays are presented below.

3.4. Post-challenge assay

The following ten botanical preparations were tested first in the challenge assay and then the negative growth result was retested in the post challenge assay to assure the antibacterial activity was a microbiocidal effect. a) Autumn Olive berry extract. Duplicate culture dishes were tested in post-challenge assay, bearing a 1/5X dilution of a hydroalcoholic extract (10% ethanol) of Autumn Olive ripe berries. There was a 7-log killing of S. epidermis. The positive control cultured in the absence of the botanical extract displayed heavy growth of the bacteria as expected. b) Hydroxytyrosol (HIDROX) versus Apple Extract (Applephenon®). We compared a commercial preparation of water-soluble olive pulp extract enriched in the antioxidant hydroxytyrosol for its antibacterial activity at 1% (w/v) compared with a commercial preparation of an green apple extract (Applephenon) at 1% (w/v) that is enriched in antioxidant catechins. Both were effective in fully killing all of the spiked S. epidermis bacteria. As expected, control saline spiked with S. epidermis was contaminated with spiked bacteria.

Microbial pathogens and strategies for combating them: science, technology and education (A. Méndez-Vilas, Ed.)

© FORMATEX 2013

____________________________________________________________________________________________

886

c) Prickly Ash (P. zanthoxyll) water-soluble extract. Post-Challenge-testing exhibited excellent antibacterial activity as a 1% of water-soluble extract. The saline control grew S.epidermis bacteria in sterile BHI culture medium, while no bacteria grew in the 1% Prickly Ash extract. d) Corn Tassel Powder water-soluble extract (Tasselin). Post-challenge-testing showed luxuriant S. epidermis bacterial growth in the duplicate non-treatment dishes, and no growth in the duplicate dishes containing the water-soluble Tasselin extract. In a separate experiment a hydroalcoholic extract of corn tassels (Tasselin) was bactericidal at 1% against the gram -negative bacteria, Pseudomonas aeruginosa. e) Allspice (Pimenta) extracts. Post-challenge-testing showed that in both the 10% ethanol and water controls there was luxuriant S. epidermis bacterial growth, while both the water-soluble and hydroalcoholic extracts of Allspice (Pimenta) displayed no growth at 20% of full extract strength. There was no bacterial growth in the 10% of full strength water extract, but the 10% of full strength hydro alcoholic extract was not sufficiently potent to kill all of the bacteria. f) Extracts of Oregano leaves, Oak catkins, and Hops leaves. Post-challenge-testing showed strong bactericidal activity against S. epidermis using a 20% of full strength hydroalcoholic extracts of Oregano leaves, for Oak (Quercus) dried catkins, and for dried Hops flowers. By contrast, a saline control with no botanical extract exhibited luxuriant bacterial growth. g) Extracts of Curly Dock, Lavender and Baes “pepper korns” A 20% dilution of the full strength of each botanical extract displayed antibacterial activity in both the challenge and post-challenge assay indicating that they were biocidal against S. epidermis. h) Minimal Bactericidal Concentration of Applephenon®. Three different dilutions of Applephenon (0.25, 0.5 and 1% aqueous solutions prepared from dried powders were first tested in the challenge assay. All were inhibitory of S. epidermis growth. In further aqueous serial dilutions the minimal inhibitory concentration was determined to be less than 0.063% (w/v). In a separate study (data not shown), Applephenon® (1% aqueous solution) killed 9-logs of Candida albicans yeast cells in the challenge assay. No yeast or bacteria grew out in the post-challenge assays. i) Applephenon® is a broad-acting bactericide. Applephenon® was also shown to be bactericidal for the gram-negative bacterium, P. aeruginosa. In this instance, the minimal inhibitory bactericidal concentration was less than 0.125% of an aqueous solution (w/v). j). Whole Clove Extracts and Anise Seed Extracts. Whole cloves and Anise were first tested and found to be antibacterial in the challenge assay against S. epidermis when tested at 20% of full-strength of these botanical extracts prepared either in water or in 50% ethanol. They were also bactericidal as determined in the post-challenge assay.

4. Discussion

A simplified cold alcohol extraction procedure was developed and employed to obtain bioactive antimicrobial activities from many different common wild plants and garden herbs. This method was first applied to corn tassels and extracts thereof shown to possess anti-inflammatory properties [12]. In a later study a crude hydroalcoholic extract of corn tassels was chemically fractionated and a phenolic compound purified and identified as 4- hydroxyl-1-oxindole-3-acetic acid with good antioxidant and antityrosinase activity [13]. We have now extended this methodology to the search for novel antimicrobial botanicals. Three different microbiological assays were developed to assess their antibacterial activity, In summary, self-sterility, challenge and post challenge assays established the antimicrobial activity of three commercially available botanical extracts powders including Applephenon®, prepared from green apples [11], and Hidrox ®, an extract from olive pulp, and white willow bark. A commercial preparation of grapefruit seed oil sold under the name Citricidal® as a preservative when tested at 0.5% was a good biocide in our microbiological assays. Although linden tree catkin extracts gave a positive response in the ferric chloride reactivity it failed in the microbiological assays. All the rest eight tested botanicals prepared by our hydroalcoholic procedure possess antimicrobial activity. For example, hydroalcoholic extracts of green tea leaves with known antioxidant activity and strong phenolic reactivity proved to have excellent antibacterial properties. This proved also to be the case with each of the other botanical extracts tested for both phenolic ferric chloride reactivity and antimicrobial activity.

5. References

[1] Rouhi AM. (2003). Rediscovering natural products. Chem Eng News, 13 October, pp 77-91. [2] Moore M. (1989). Medicinal Plants of the Desert and Canyon West. Museum of New Mexico Press. [3] Stary F. (1996). The Natural Guide to Medicinal Herbs and Plants. Barnes & Noble Books, New York by arrangement with

Aventinum Publishers, Prague, Czech Republic. [4] Foster S and Johnson RL. (2006). National Geographic Desk Reference to Nature’s Medicine, National Geographic Society

Washington, D.C. [5] Chastain DE et al. Method for killing yeast and fungi with Carveol, U.S. Patent No. 5,308,873 issued May 3, 1994. [6] Zhou JH. Composition and method for inhibiting oral bacterial. U.S. Patent No.: 6,139,523, issued November 20, 2001. [7] Iwai K. Bactericide against vancomycin resistant enterococcus. U.S. Patent No. 6,387,417 issued May 14, 2002 [8] Hamilton-Miller J.M.C. Antibacterial agent containing teat extract or active fraction thereof and beta.-lactam antibiotic. U.S.

Patent No.: 5,879,683 issued March 9, 1999. [9] Aga H et al. Physiologically active extract obtained from indigo plant polygonum. U.S. Patent No. 6,524,625 issued February

25, 2003.

Microbial pathogens and strategies for combating them: science, technology and education (A. Méndez-Vilas, Ed.)

© FORMATEX 2013

____________________________________________________________________________________________

887

[10] Ziegenfuss et al. Antibacterial protein extracts from seeds of marigold and paprika. U.S. Patent No. 6,086,885 issued July 11, 2000.

[11] Akazome Y. Characteristics and physiological function of polyphenols. BioFactors2004; 22 (1-4):311-314. [12] Wille J.J. Anti-irritant botanicals. U.S. Patent No.:7,399,493B2 issued July 15, 2008. [13] Wille J.J. and Berhow MA. (2011). Bioactives derived from ripe corn tassels: a possible new natural skin whitener, 4-hydroxy-

1-oxindole-3-acetic acid. Current Bioactive Compounds, 2011; 7(2): 126-134.

Microbial pathogens and strategies for combating them: science, technology and education (A. Méndez-Vilas, Ed.)

© FORMATEX 2013

____________________________________________________________________________________________

888