Antibacterial Properties of Bark
Extract Prepared
From ‘Iliahi
(Santalum freycinetianum):
An Endemic Hawaiian Sandalwood
Kahale Pali
Presented in partial fulfillment
of Biology 493
Advisor: Phil Bruner
ABSTRACT
The purpose of this study was to determine if an extract from the bark of Santalum freycinetianum had antibacterial properties. Alcohol and aqueous extracts of S. freycinetianum bark were tested against Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus using the Kirby-Bauer method. No significant antimicrobial effects were found. Traditional knowledge of sandalwood in medicinal use does not designate the specific part of the plant that exhibited antibacterial properties, nor the details of the preparation methods. Similarities between traditional and modern preparation techniques may be coincidental and could explain the results of this study. The lack of antimicrobial evidence may also be due to the long-term isolation of this species and its subsequent loss of any antibacterial activity.
INTRODUCTION
Indigenous people have used sandalwood (Santalum spp.) medicinally to remedy many ailments (Duke 1985). In India, a paste made from the ground wood of Santalum album L. was applied to the skin to relieve itching and inflammation (Chopra et al. 1982). The Chinese used S. album mixed with mucilage as a cure for facial acne (Li 1973). Grated wood of S. insulare Bert. was employed in the Cook Islands for earaches or headaches and was sometimes applied to skin infections (Whistler 1992).
The essential oil of S. album has been shown to decrease the incidence and quantity of skin papillomas in mice (Dwivedi and Abu-Ghazaleh 1997). It has also been reported to exhibit indirect antioxidant properties on laboratory mice by increasing the levels of glutathione-S-transferase (GST) activity and the acid soluble sulphydryl (SH) level in the liver (Scartezzini and Speroni 2000). Experiments conducted on rats showed hypotensive activity to extracts of Santalum ellipticum Gaud. (Bourke et al. 1973). Santalbic acid, derived from the oil of Santalum acuminatum R. BR., restricted the growth of Staphylococcus aureus, Staphylococcus epidermidis, and certain pathogenic fungi (Jones et al. 1995).
Extracts from sandalwood were found to be effective against some microorganisms that cause infections of the urogenital system. A herbal cream containing aqueous extracts of S. album was effective in treating vaginal infections (Rani et al. 1995) and has also proven to be a growth inhibitor of the yeast-like fungus Candida albicans (Hammer et al. 1998). Sandalwood oil was also found to inhibit replication of Herpes simplex viruses-1 and –2 (Benencia and Courreges 1999).
Santalum freycinetianum L. is one of four endemic Hawaiian sandalwood species which occur in mesic to wet forest on Kaua‘i, Lana‘i, O‘ahu, Maui, and Moloka‘i (Wagner et al. 1999). Santalum freycinetianum is hemiparasitic and extracts nourishment from other nearby trees. Early Hawaiians used the leaves and bark of sandalwood in a remedy to remove dandruff and destroy lice (Nagata 1971, Akana 1972). Hawaiian kahuna la‘au lapa‘au, or medicinal healers, used the finely ground scrapings of the native sandalwood (Akana 1972) as one of the components in treating some sexually transmitted diseases (Chun 1998). Its Hawaiian name is ‘iliahi, ‘ili meaning “skin” and ahi meaning “fire” (Puku‘i and Elbert 1986), and can be transliterated as “fiery skin” (Mano‘i pers. comm.). Santalum freycinetianum does not cause contact dermatitis and so its name may be suggestive of its medicinal properties (Mano‘i pers. comm.).
Despite extensive research on the genus Santalum, little has been published on S. freycinetianum. The purpose of this study was to determine if an extract from the bark of S. freycinetianum had antibacterial properties.
Materials and Methods
A specimen of S. freycinetianum was located on the Kaunala Trail, Pupukea, O‘ahu. A voucher specimen was prepared and taken to the National Tropical Botanical Gardens at Lawa‘i, Kaua‘i where identification was confirmed (Flynn pers. comm.). A bark sample (inner bark, outer bark, and cork layers) three centimeters by ten centimeters was obtained from an area on the trunk (36.5 cm in diameter) 30 cm above the base of the tree.
The bark sample was immediately rinsed with distilled water and cut into two equal pieces. These were ground separately, using a mortar and pestle. While the samples were being ground, a solvent was added. One sample was prepared with an aqueous extraction using 30ml. of distilled water. The other sample was extracted using 30 ml. of a 50% dilution of ethyl alcohol as an organic solvent. Both samples were filtered through sterile cheesecloth and collected in a sterile beaker. The beakers were covered and put into a shaker for 48 hours.
Broth cultures of Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus were prepared. Bacteria selected were representative of endospore-forming Gram-positive rods, facultatively anaerobic Gram-negative rods, Gram-negative aerobic rods, and Gram-positive cocci, respectively.
Each of the four bacteria were tested on twenty-five plates of Mueller-Hinton agar using the Kirby-Bauer method. Every plate was divided into six sections each. Six millimeter blank discs were made by hole-punching 610 grade filter paper and sterilizing the discs in an autoclave. A six-millimeter blank sterile disc was placed in section one and an erythromycin disc was placed in section six. A disc with four microliters of ethyl alcohol extract, ethyl alcohol, distilled water extract, and distilled water were placed in sections two through five. The plates were incubated at 35ºC for 18 hours. The zones of inhibition were then measured.
Results
The ethyl alcohol extract produced an average zone of inhibition of 0.2 mm against B. subtilis, 2.3 mm against E. coli, 3.4 mm against P. aeruginosa, and 1.4 mm against S. aureus (Fig. 1). The ranges of zones of inhibition were 0 to 2 mm for B. subtilis, 0 to 5 mm for E. coli, 0 to 8 mm for P. aeruginosa, and 0 to 3 mm for S. aureus.
The aqueous extract showed an average zone of inhibition of0.1 mm against E. coli, and 1.2 mm against P. aeruginosa (Fig. 1). There were no zones of inhibition of B. subtilis and S. aureus. The ranges of zones of inhibition were 0 to 3 mm for both E. coli and P. aeruginosa.
An analysis of variance (ANOVA) showed no significance between the blank control and either the alcohol or aqueous extract for B. subtilis and E. coli (p>0.05). For P. aeruginosa and S. aureus, ANOVA results found significance between the blank control and both the alcohol extract and the alcohol control (p<0.05), but there was no significance between the alcohol extract and the alcohol control (p>0.05).
Fig. 1. The average zones of inhibition of ‘Iliahi bark extract and controls.
Discussion
Although other Santalum spp. have been shown to contain antimicrobial properties, the results of this study found that the bark of the selected specimen of S. freycinetianum apparently lacked these qualities. In those studies where antimicrobial properties were demonstrated, the part of the plant utilized for testing was the wood (secondary xylem). One function of wood (secondary xylem) is for storage of compounds produced by the plant. Bark functions in dissolved carbohydrate transport (secondary phloem) and in protection (periderm). The stored compounds in the wood may contain the antimicrobial agents.
In an effort to mimic traditional use, the bark was selected for testing in this study. According to Chun (1998), “The flesh of the ‘iliahi is scraped and the bits and pieces are mixed with manena (Pelea spp.) and ‘awa (Piper methysticum)” (Scientific names added). This study assumed that the “flesh” was the bark of the native sandalwood.
In indigenous Hawaiian culture, many of the remedies are mixtures of several ingredients. One remedy for venereal diseases used parts of ‘iliahi (sandalwood, Santalum spp.), manena (Pelea spp.), ‘awa (Piper methysticum), nīoi (Eugenia spp.), and kauila (Alphitonia ponderosa or Colubrina oppositifolia). Perhaps it was one of the other ingredients that possessed the antimicrobial property and not the Santalum spp. Another possibility is the combination of all these ingredients synergistically led to the formation of an antimicrobial compound or activated an otherwise inactive antimicrobial compound.
The mode of preparing remedies was an important factor in traditional Hawaiian medicine. The specific preparation method could be the factor influencing the success of a particular cure. Unfortunately, preparation methods haven’t been adequately documented for all remedies. Modern lab methods used in this study can only be coincidentally similar to traditional preparation methods.
Many plants produce chemicals as a defense against insects, bacterial infections, or in response to grazing vertebrates. Long-term isolation of a species increases the chances of endemism and subsequent evolutionary changes, such as a loss of natural defenses. Isolation and lack of herbivory and/or bacterial infectious agents may have caused S. freycinetianum to lose its chemical defenses, possibly explaining why there were no antimicrobial properties in the bark.
Possibilities for future studies may include testing different parts of S. freycinetianum for antimicrobial activity. Comparing data from studies of other Santalum spp. may be helpful in determining similar medicinal properties of plant extracts. These properties may have economic benefits involving S. freycinetianum in cultivation.
Santalum research could look into the function of storing antimicrobial compounds in sandalwood. Is there a function or is it a by-product that coincidentally has antimicrobial activity?
Finally, sandalwood oil, containing 90% santalol, was found to inhibit Herpes simplex viruses-1 and -2 replication (Benencia and Courreges 1999). According to Scartezzini and Speroni (2000), santalol is a major constituent (90%) of oil extracted from the bark of S. album. An antiviral study using bark extract from S. freycinetianum may yield similar results.
ACKNOWLEDGEMENTS
I would like to thank the biology faculty of BYU-Hawaii for their revisions to my paper. Special thanks to: my advisor, Dr. Paul, for her advice and suggestions; Dr. Oba, for assistance with materials and methods; Dr. Winget, for help with data analysis; and Phil Bruner, for additional revisions to my paper. I would also like to thank Tim Flynn, former herbarium curator of the National Tropical Botanical Garden for identifying my voucher specimen and Lorrin Mano‘i for passing on his traditional knowledge.
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Benencia, F. and M. C. Courreges. 1999. Antiviral activity of sandalwood oil against herpes simplex viruses-1 and –2. Phytomedicine 6(2): 119-123.
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