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Journal of Applied Science and , 10(3) Special 2015, Pages: 21-39

AENSI Journals Journal of Applied Science and Agriculture ISSN 1816-9112

Journal home page: www.aensiweb.com/JASA

Review: Tea ( Alternifolia) As A New Material For Biocomposites

1,2Rodney, J., 2Sahari, J., 3Mohd Kamal Mohd Shah

1Faculty of Science and Natural Resources, Universiti , 88400 Kota Kinabalu, Sabah, Malaysia. 2Knowledge and Management Division, Sabah Economic Development & Investment Authority (SEDIA), 88873, Kota Kinabalu, Sabah, Malaysia. 3Faculty of , Universiti Malaysia Sabah 88400, Kota Kinabalu, Malaysia

ARTICLE INFO ABSTRACT Article history: Melaleuca alternifolia or commonly known as tea tree is a tall or small tree in the Received 25 November 2014 genus Melaleuca. It is popular for its oil, which is where it has been Received in revised form 26 December employed largely in various industries of its antimicrobial properties. Research works 2014 are still ongoing mainly focusing on the tea tree oil properties, ultimately almost none Accepted 1 January 2015 of them investigating on the residue which is the leaves. Environmental issues become Available online 10 January 2015 the world major concern, which create awareness among industrial player to turn back to natural fibre in producing products. In recent time, productions of composites from Keywords: agro waste have received considerable attention. This paper aims to rationalize the Tea tree Melaleuca alternifolia potential of tea tree (Melaleuca alternifolia) leaves as a new source of natural fibres or Biocomposites Agricultural waste material in order to become the potential or reinforcer in the development of a new biocomposite.

© 2015 AENSI Publisher All rights reserved. To Cite This Article: Rodney, J., Sahari, J., Mohd Kamal Mohd Shah., Review: Tea Tree (Melaleuca Alternifolia) As A New Material For Biocomposites. J. Appl. Sci. & Agric., 10(3): 21-39, 2015

INTRODUCTION

Agricultural waste, or agro-wastes are by-products from agricultural activities, which can be husk, straw, cobs or fiber (Abba et al., 2013). In recent time, industrial players have move towards green and sustainable , as global warming and depletion of resources occurs every now and then, which lead to the productions of composites from agro waste. Malaysia, a tropical country, blessed with varieties of and , produces a massive amount of agricultural waste, which are 47,402 dry kilotonne/year (Goh et al., 2010) and most of it contributed from industries (Sumathi et al., 2008). Natural fibres from non- materials in the form of fibres and/or particles have received attention by different wood-based industries, as this can reduce the large amount of wood used as feedstock by wood-based industries and would preserve and maintain the environment without much destruction (Alwani et al.,2014). Table 1 shows the examples of agro waste. As we are moving forward, every potential natural fibre, which derived from the agricultural wastes, will be explored in order to have more options of natural fibres compared to ready existing natural fibres.

2.0 Tea tree (Melaleuca alternifolia): 2.1 History of tea tree (Melaleuca alternifolia): Tea tree (Fig. 1 and Fig. 2) is native to , where it is found from to north-east . The Australian abrorigines have long using the tea tree leaves to treat cuts and wounds. Crushed leaves were applied directly to an injury, then held in place with a mud pack. This poultice helped fight infection in the wound. The widespread usage of the tea tree oil started with the finding of Australian government chemist named Arthur R. Penfold in 1922. He found out that the oil is an extremely powerful antiseptic, 12-times more effective than phenol (carbolic acid) without doing any damage to the skin. He later presented his findings to the Royal Society of New South Wales and England (Tenny, 1996).

Table 1: Examples of agro waste (Abba et al., 2013). Type Examples References Wheat Straw, Husk Wang and Sun, 2002 Rice Husk, Hull, Straw and Stalk Panthapulakkal et al., 2005 Sorghum Husk, Straws, Cobs, Stover, Leaves McGee, 2006 Millet Husk, Straws, Cobs, Stover, Leaves Choi et al.,2006 Fronds, Husk, Shells Maniruzzaman et al.,2012

Corresponding Author: Rodney, J., Faculty of Science and Natural Resources, Universiti Malaysia Sabah, 88400 Kota Kinabalu, Sabah, Malaysia. 22 Rodney, J. et al, 2015 Journal of Applied Science and Agriculture, 10(3) Special 2015, Pages: 21-39

Coffee Hull, Husk, Ground Azim, 2012 Cotton Stalks Aziz and Ansell, 2004 Peanut Shells Mwaikambo and Ansell, 2003 Sugarcane Bagasse Ismail et al., 2009 Nuts Hull, Shells Ndazi et al., 2006

Fig. 1: Tea tree (Melaleuca alternifolia).

Fig. 2: Melaleuca alternifolia (Peter, 1991).

Although Australians use the name ‗tea tree‘ to refer to many Australian native from the genera of Leptospermum, Melaleuca and Neofabricia (family ) (Craven, 1999), it should be noted that the only source of Australian tea tree oil is in the genus Melaleuca, particularly from the species Melaleuca alternifolia (Maiden and Betche) Cheel. The term ―tea‖ should not be mistaken with the tea (). Southwell and Lowe (1999) explained that the name tea tree arose when Captain James Cook, on his exploratory voyage of Australia in 1770, encountered a myrtaceous shrub (possibly a Leptospermum) with leaves that were used in his sailors as a substitute for tea (Camellia sinensis). Subsequently, these myrtaceous , now known as the genera Leptospermum, Melaleuca, Kunzea and Baekea, were collectively known as ―tea tree‖. Weiss (1997) however notes that this should not to be confused with the Maori or Samoan derived ―titree‖ or ―ti-palm‖, a name to plants of the Cordyline genus.

2.2 Botanical Features and Description: Melaleuca alternifolia or commonly known as tea tree is a tall shrub or small tree in the plant genus Melaleuca (which contain of 230 species native to Australia (Craven, 1999)) and is in the family of Myrtaceae. 23 Rodney, J. et al, 2015 Journal of Applied Science and Agriculture, 10(3) Special 2015, Pages: 21-39

It is an evergreen tall shrub up to 7 meter high with a bushy crown and whitish papery bark. The leaves are narrow, scattered to whorled and are 10-35mm long and 1 mm wide. The leaves are very rich in . The flowers are white, solitary, each within a bract, and have petals 2-3 mm long. The stamens appear in bundle of 30-60 and female part known as style in 3-4 mm long. The fruit has many seeds, is woody, cup-shaped capsule and 2-3 mm in diameter (Peter, 1991). Tea tree oil is extracted from the tree Melaleuca alternifolia that grows naturally in Australia, and has been shown to have many beneficial medicinal uses as an antiseptic, antifungal and antibacterial agent (Carson et al., 1995). Tea tree oil is considered to be a general treatment for acne, eczema, skin infections like herpes, wounds, burns, insect bites and nail mycosis. Practically, the commercial tea tree oil is produced from Melaleuca alternifolia (Maiden and Betche) Cheel. The Melaleuca genus belongs to Myrtaceae family and roughly Melaleuca alternifolia can reach height typically 5-8 metres if it is left to grow (Colton and Murtagh, 1999).

2.3 Requirements: Melaleuca alternifolia is adaptable to a wide range of soil types, yet it requires specific climate and soil conditions to produce a consistently high yield (Colton and Murtagh, 1999). A highly productive commercial can be achieved when it is planted on a site which mimics its natural conditions of damp soil in humid, sub-tropical areas of northern New South Wales(Colton et al., 2000). The productivity of depends on the yield and oil concentration in the leaves. The total production of biomass of Melaleuca alternifolia is determined by various factors including temperature, water availability, plant density and month of harvest (Murtagh, 1996). SEDIA (2007) had demonstrated that the propagation of tea tree can be done by seedlings, cuttings and tissue culture. a) Climate and rainfall: Native to the wet sub-tropical area, the plant responds well to warm temperatures and a continuous supply of moisture. According to Lowe and Murtagh (1997), high humidity is necessary as it is proven from research that concentration of oil increases with humidity. Although tea tree needs ample moisture to grow, it can survive in very dry conditions. In extreme drought, it will shed its leaves and will re-shoot after rain (Small, 1981b). The best temperature for tea tree growth is between 18-34°C. The temperature can directly affect the growth rate and oil concentration of tea tree. Tea tree requires abundant rainfall ranging from 1000mm-1600mm per year. b) Soil management: The plant grows well in deep sandy loam or friable loam due to its high water holding capacity. The optimum soil pH for tea tree is between the ranges of 4.5-5.5 (Weiss, 1997). Tea tree does not grow well on shallow light sandy soils, heavy clay soils and acid sulfate soils. Some tea tree growers have reported growth responses from regular applications of commercial foliar nutrient preparations as well. However, only the minimum quantities of fertilizers promoting high yields of quality oil may be used. To limit groundwater contamination with nitrates and other nutrients, additions of fertilizer must consider the soil texture and organic matter content of the soil. From studies done by MARDI, the rate of 150-200kg N/ha, 50kg P2O3, 70kg K2O/ha is recommended (Nordin, 2005). This rate is equivalent to 1.125 t/ha of NPK fertilizer applied split into four applications per year. The usage of chemical can be reduced substantially as soil organic matter content increases. c) Plant spacing and population: Studies in Australia shows that the highest yield is being obtained at the densest planting with optimum population exceeding 27,000 trees/ha (Small, 1981b) and these high plant populations achieve full grown cover more quickly after harvest and compete better with weeds by shading. A study done by Prastyono (2008) discovered that trees planted in narrower spacing, 33,333 trees/ha had higher oil concentration than those at the widest spacing, 16,667 trees/ha. SEDIA (2007) reported that in Sabah, Malaysia, they had successfully planted approximately 10,000 trees/ha with planting density of 0.8m between rows and 0.6m within rows, where they investigated that the higher planting density in the tea tree plantation resulted in less weeding and ultimately to higher biomass per hectare.

2.4 Crop protection: a) Weed control: Weeds can reduce tea tree growth during both the initial establishment phase and in the annual regrowth cycles. Newly planted tea tree seedlings have a very restricted system of only 2.5cm diameter, and a shoot height of 10-15cm. Their small size makes them poor competitors against rapidly growing weeds. Tea tree shoot growth also declines quickly with increased shading. Weed shading is most likely in the first three months after 24 Rodney, J. et al, 2015 Journal of Applied Science and Agriculture, 10(3) Special 2015, Pages: 21-39 planting, when tea tree seedling and weeds are of similar height. Older tea tree seedlings of approximately 0.5cm in height are still at risk of shading by erect, broadleaf weeds and climbing weeds. Tea tree will be prone to weed competition for water where the root systems interact where soil and water is insufficient to meet both tree and weed needs. This is particularly likely in soils with low water holding capacities (e.g. sandy loams) and/or which receive low rainfall. The majority of tree and weed occur in the surface 30cm of soils (Bowen, 1985), giving the weeds advantage for water uptake. Thus young tea tree seedlings with their shadow root system are very susceptible to moisture competition, and water stress results. Such a mechanism of weed interface is very important for tree seedling and extreme water stress can cause deaths (Nambiar and Zed, 1980). b) Pest and disease: Damage by pest to tea tree is still relatively low and the need to control is an unnecessary added cost. In addition the use of chemicals at inappropriate rates can cause residues which affect the oil quality and price. Nonetheless, the need to understand the biological cycle and development of pest outbreaks allow the generation of a model that accounts for field behavior and action thresholds. Table 2 shows the insect damaging seedling of tea tree, Table 3 shows the insect damaging foliar of establish plants and Table 4 shows the insect feeding on wood and bark.

Table 2: Insect damaging seedling of tea tree (Campbell and Maddox, 1999). Pest Activity African black beetles Ring barks tea tree seedling at or below ground level which causes dehydration and death Mole crickets Burrows and forages in horizontal tunnels in top 5cm of soil causing damage to newly set of tea tree transplants by severing tea tree roots Cut worms Attack seedlings, cutting near ground level and feeding on the felled plants

2.5 Harvest: Joseph et al. (2009) reported the plants can be harvested in about 9 months when they reach a height about 2m. It is cut at about 15 - 30cm above the ground level so that the stumps can regrow and can be subsequently harvested. The best time to harvest the plant is in the dry season when the yield of oil is generally higher. The oil is extracted from the twig and leaves. It has a pale green colour with the smell of champor. Tea tree crop is ready to harvest once its canopy is fully developed and tea trees have reached maximum leaf yield. Once trees have reached this stage they tend to start losing lower leaves and stems begin to thicken. The harvested trees should be chopped into smaller portion to facilitate distillation of tea tree oil. Timing is crucial from point of harvest to distillation as oil can vaporize and reduce yield. To minimize loss, the time frame given is less than 24 hours. It is best to harvest the trees during drier periods to minimize the loss of trees from fungal infection to the cut stems. The choice of harvesting machineries or equipment will depend on farm size. Small scale farms can use circular saw blade brush cutter or machete. Larger scale can opt for harvesting to reduce harvesting time.

Table 3: Insect damaging foliar of establish plants (Campbell and Maddox, 1999). Pest Activity Chrysomelids Adult beetle feeds and lay eggs on expending flush growth Scarabaeids (Pasture Scarabs) Causes extensive localized defoliation of tea tree. The beetles feed and mate on trees then return to the soil to lay their eggs Psyllids Causes pitting and some distortion of the leaves and shoots Eriophyid Mites Distort new foliage and cause the leaf margins on the ventral surface to bend; cells on the leaf surface within the distorted area bubble and become hairy. The mites live within the distorted area Aphids Sucks on shoots of new flush encouraging black sooty mould grows in the wounded area Scale insects Attacks plants weakend by defoliating or -sucking insects Leaf hopper Suck sap from the expanding shoots on flush growth. Feeding causes the witting of the shoot which develops into a distinct purple coloration Sawflies Wasps insert eggs in rows into the leaf tissue of young follage. Once hatched the larvae feeds on the leaves causing major defoliation Moth larvae (leaf roller) Feed on exposed leaves and shelters itself by forming pockets by weaving few leaves together

25 Rodney, J. et al, 2015 Journal of Applied Science and Agriculture, 10(3) Special 2015, Pages: 21-39

Table 4: Insect feeding on wood and bar (Campbell and Maddox, 1999). Pest Activity Cerambycidae (Longicorn beetles) Feed on wooding parts of the tea tree plant weevil Feeds on bark of the stems within plantation causing death if population is dense Termites Enters the plant through the root system and continue to feed within the stems Stem-boring Lepidoptera Larvae chew the wood in the shoot down to the cambial layer

2.6 Postharvest: a) Distillation: Tea tree oil is obtained by passing steam through the oil-bearing parts of the plant which are the leaves and terminal branchlets. According to Southwell and Lowe(1999), the main woody part of the plant does not contain oil. Leaf oil content can range from 0.5-3 %, but yield from traditional design water distillation is 1%. SEDIA (2007) reported that they had successfully gain 0.8% of tea tree oil from tea tree planted in Sabah, Malaysia. Steam distillation is the appropriate method of extraction for tea tree oil, not only because it causes minimum change to the composition of the oil extraction, but also because steam is readily available, cheap, not hazardous (chemically), can be used at low pressure and can be recycled (Southwell and Lowe, 1999). Steam distillation enables the aromatic oil to be extracted at a temperature which is constant and low enough as not to cause any damage to the oil. Tea tree oil from Melaleuca alternofilia is a mixture of various , and their alcohols. The monoterpenes terpinen-4-ol, γ-, 1,8-cineol, p-cymen, α-terpineol, α-, terpinolenes, and account for 80-90% of the oil. The chemical composition of tea tree oil is defined by international standard ISO 4730 and the identical Australia Standard AS 2782-2009. The standard specifies that the acceptable level of terpinen-4-ol is a minimum 30%, and a maximum content of 15% 1,8-cineole (International Standards Organisation, 1996 ).

2.7 Tea tree (Melaleuca alternifolia) oil – TTO: 2.7.1 Composition and chemistry: Carson et al. (2005) have reviewed all literature related to tea tree (Melaleuca alternifolia) up to 2005. According to them, tea tree oil (TTO) is consisted of hydrocarbons, mainly monoterpenes, sesquiterpenes and their associated alcohols. Sharp (1983) discovered the are volatile, aromatic hydrocarbons and may be considered as of which has the formula C5H8.Series of finding on the composition of tea tree oil have been reported. As early as Guenther (1968) claimed there are 12 components, later on Laakso (1965) cited in Altman (1988) said it contained 21 components, and 48 components was discovered by Swords and Hunter (1978). Then, Brophy et al. (1989) found out that there are almost 100 components and their range concentrations after tested over 800 tea tree oil samples using gas chromatography and gas chromatography . Finally, international standard has regulated a standard for TTO, which is ―Oil of Melaleuca –terpinen-4-ol type‖ which sets maxima and/or minima for 14 components of the oil (International Organisation for Standardisation, 1996).(See Table 5). Terpinen-4-ol (Fig. 3) and 1,8-cineole (Fig. 4) are both most crucial compound in tea tree oil (Carson et al., 2005; Joseph et al.,2009). As shown in the Table 5, Terpinen-4-ol is the major compound of tea tree oil, which has long been considered as the main antimicrobial properties to TTO. This is the reason why to optimize the antimicrobial activity; a lower limit of 30% has been set and no upper limit. However, for 1,8-cineole, there is an upper limit, which is 15% and no lower limit has been set. This is due to cineole is considered as the skin and mucous membrane irritant causes and has disinfectant effects. Subsequently, by this limitation of both terpinen- 4-ol and 1,8-cineole, this harmony of combination can give the optimise effects in treatments (Joseph et al., 2009).

Fig. 3: Terpinene

26 Rodney, J. et al, 2015 Journal of Applied Science and Agriculture, 10(3) Special 2015, Pages: 21-39

Fig. 4: Cineole

Table 5: Composition of tea tree (Melaleuca alternifolia) oil (Carson et al.,2005) Component Compositon (%) ISO 4730 Range1 Typical composition2 terpinen-4-ol ≥ 303 40.1 γ-terpinene 10 - 28 23.0 α-terpinene 5 – 13 10.4 1,8-cineole ≤ 153 5.1 terpinolene 1.5 - 5 3.1 ρ-cymene 0.5 - 12 2.9 α-pinene 1 – 6 2.6 α-terpineol 1.5 - 8 2.4 aromadendrene traces - 7 1.5 δ-cadinene traces - 8 1.3 limonene 0.5 - 4 1.0 sabinene traces – 3.5 0.2 globulol traces - 3 0.2 viridiflorol traces – 1.5 0.1

1 (International Standards Organisation. 1996), 2 (Brophy et al.,1989), 3 no upper or lower limit set

2.7.2 Commercial production: The revolution of tea tree (Melaleuca alternifolia) industry started when Penfold reported its medical properties in 1920s and 1930s. Then TTO has attracted attention from stakeholders due to its value in economic potential. At the early stage, the plant material was always hand-cut and usually distilled on the spot. During World War II, tea tree oil was supplied as medical kits for the soldiers, as those who involved in the bush-cutters can be exempted from national services (Carson and Riley, 1994). Productions are declining after World War II as demands are not so strong and the development of antibiotics and other natural products. Eventually, the industry of TTO starting to spark again in the 1970s and 1980s as natural products started to get attention, while commercial plantation establishment in those years turn the industry to merchanise and make big and consistent quantities of products (Joseph et al.,2009; Johns et al.,1992). TTO was one of the success stories in Australian essential oil production during the early 90‘s with prices topping AUD 65/kg. This attracted large numbers of new growers entering the industry until production exceeded demand and prices tumbled to less than AUD 50/kg, especially in the late 90‘s (Davis, 1999). This has shaken out the industry and the largest producers have all ceased production, leaving small and medium sized producers to supply the market. Prices have been on the increase again and are breaching the AUD 50/kg mark and are still increasing. However, many large customers including some of the major European and US chain who carried the product have discontinued demand, due to lack of confidence in future supply. Likewise, many major personal care companies have also switched to other natural additive in their product ranges, leaving the task ahead for tea tree producers to convince the cosmetic and industries to support the product. This unstable period has opened the door for procedures in countries like China and other Asian countries to step up production and compete with Australian producers. Therefore, TTO is famous to be used in a wide range of products, either as formulated or pure oil into many kinds of value-added products as a preservative, antiseptic, antibacterial, antifungal and even anti-pest agent. Those are include shampoos, conditioners, soaps, bath oils, mouthwashes, toothpastes, deodorants, moiturisers, face cleansings and washes, foot sprays and powders, shaving products, antiseptic creams, body lotions, sun blocks, lip balms, post-waxing treatments, acne creams and many other health products and dog shampoos and other veterinary care products (Southwell and Lowe, 1999; Colton et al., 2000; Wrigley and Fagg, 1993). 27 Rodney, J. et al, 2015 Journal of Applied Science and Agriculture, 10(3) Special 2015, Pages: 21-39

2.7.3 Antimicrobial activity of TTO: Antimicrobial is the synonym word to describe TTO. The ethno botanical usage of TTO was first discovered from Bundjalung Abrogines of Northern New South Wales. Tea tree leafs were crushed before inhaled to treat coughs and colds, or was sprinkled on any wounds after applying poultice (Shemesh and Mayo, 1991). Apart from that, soaking tea tree leafs in order to make it as an infusion were made as reported by Shemesh and Mayo (1991), and Low (1990), to treat skin ailments or sore throats. In 1925, Penfold and Grant (1925) compared TTO and other oils with disinfectant carbolic acid or phenol, the gold standard of the day, in a test known as the Rideal-Walker (RW) coefficient. TTO‘s activity was directly compared with that of phenol and rated at 11 times as active. At that point, TTO was then promoted as a therapeutic agent (Anon, 1930; Anon ,1933; Anon, 1933b). The answers of which component of TTO to act as the antimicrobial activities have yet to be found. Early signs from RW coefficients were that much of activity can be attributed to terpinen-4-ol and α-terpineol (Penfold and Grant, 1925), as supported by recent studies (Carson et al.,1995a; Raman et al.,1995; Hammer et al., 2003). Of all components of TTO, they concluded that some possess some degree of antimcobial activity, some considered less active and none are inactive. Cox et al. (2001a) demonstrated by in vitro that components of TTO may experience synergistic or antagonistic effects on the overall antimicrobial activity, as it may has interactions with other essential oil such as lavender (Cassella et al., 2002), and other essential oil such as β- triketones from manuka oil (Christoph et al., 2001a; Christoph et al., 2001b). As such, more works need to be done to answer this puzzles.

2.7.4 Antibacterial activity of TTO: In 1955, Atkinson and Brice (1955) assessed plants of Myrtaceae family for antibacterial activity of TTO by both agar and broth dilution assays. Antibacterial titres (% v/v) as determined by agar and broth dilution assays were 0.63 and 0.31 respectively for Staphylococcus aureus, 1.25 and 0.24 for Salmonella typhi and 0.31 and 0.10 for Mycobacterium phlei. Low et al., (1974) were then examined the same agar dilution method by Atkinson and Brice, and discovered MICs (% v/v) of 0.062for S. aureus and 0.031 for S. typhi. With TTO, S. aureus could not be recovered whereas viable Pseudomonas aeruginosa were recovered (Low et al.,1974). The susceptibility data for bacteria tested against TTO (% v/v) can be seen in Table 6. The range of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) are between 00.6% - 1.0%.

Table 6: Susceptibility data for bacteria tested against M. alternifolia oil (% v/v) (Carson et al, 2005). Bacterial species MIC, MICrange or MIC90 MBC, MBCrange or MBC90 Acinetobacter baumannii 1.08 1.08 Actinomyces viscosus 0.66 0.66 Actinomyces spp. 1.014 1.014 Bacillus cereus 0.32 Bacteroides spp. 0.061, 0.51 0.06-0.121 Corynebacterium sp. 0.2-0.32, 2.08 2.08 Enterococcus faecalis 0.5-0.752 Enterococcus faecalis (vancomycin R) 0.5-14, >810 0.5-14, >810 Escherichia coli 0.253, 7, 0.0811 0.253, 7 Fusobacterium nucleatum >0.66 Klebsiella pneumoniae 0.258, 0.32 0.258 Lactobacillus spp. 1.014, 2.01 2.01, 14 Micrococcus luteus 0.06-0.58 0.25-6.08 Peptostreptococcus anaerobius 0.26, 0.251 0.03-0.121 Porphyromonas endodentalis 0.025-0.114 0.025-0.114 Porphyromonas gingivalis 0.116 Prevotella spp. 0.031, 0.251 0.031 Prevotella intermedia 0.003-0.114 0.003-0.114 Propionibacterium acnes 0.052, 0.31-0.635 0.513 Proteus vulgaris 0.0811, 0.32, 2.010 4.010 Pseudomonas aeruginosa 1->2.02 ,1-810, 3.08 2->810, 3.08 Staphylococcus aureus 0.63-1.255, 0.57, 10 1.010, 2.07 Staphylococcus aureus (methicillin R) 0.0411, 0.254, 9 0.54, 0.59 Staphylococcus epidermidis 0.63-1.255, 1.08 4.08 Staphylococcus hominis 0.58 4.08 Streptococcus pyogenes 0.1212 0.2512 Veillonella spp. 0.016-1.014 0.03-1.014

1 Hammer et al., 1999a; 2 Griffin et al., 2000; 3 Gustafson et al., 1998; 4 Nelson, 1997; 5 Raman et al., 1995; 6 Shapiro et al., 1994; 7 Carson et al., 1995b; 8 Hammer et al., 1996; 9 Carson et al., 1995a; 10 Banes- Marshall et al., 2001; 11 Mann & Markham, 1998; 12 Carson et al., 1996; 13 Carson & Riley, 1994; 14 Hammer et al., 2003. 28 Rodney, J. et al, 2015 Journal of Applied Science and Agriculture, 10(3) Special 2015, Pages: 21-39

The mechanism of action of TTO against bacteria has been partly explained. Assumptions about its mechanism of action were made on the basis of its hydrocarbon and lipophilicity. Carson et al. (2005) once again were reviewing studies that had been done. They summarize that from the treatment of S. aureus against TTO precipitates the leakage of potassium ions (Cox et al.,2000; Hada et al.,2003), and 260 nm-absorbing materials (Carson et al.,2002), and it inhibits respiration (Cox et al.,2000). Carson et al. (2005) also reviewed that TTO also produces morphological changes apparent under electron microscopy (Reichling et al.,2002) and sensitizes previously tolerant cells to sodium chloride (Carson et al.,2002). But, there is no significant lysis of whole cells was observed by spectrophotometrically (Carson et al.,2002) or electron microscopy (Reichling et al.,2002), only modest uptake of propidium iodide was observed (Cox et al.,2001b) after treatment with TTO, and no cytoplasmic membrane damage as evidenced by lactate dehydrogenase release could be detected (Reichling et al.,2002). In E. coli, a modest loss of 280 nm-absorbing materials has been reported (Cox et al.,2001b). The detrimental effects on potassium homeostasis (Cox et al.,1998) , morphology (Gustafson et al.,1998), glucose- dependent respiration (Cox et al.,1998) and ability to exclude propidium iodide have been discovered. In contrast to the absence of whole cell lysis seen in S. aureus treated with TTO, lysis occurs in E. coli treated with TTO (Gustafson et al.,1998). All in all, these observations confirm that TTO compromises the structural and functional integrity of bacterial membranes.

2.7.5 Antifungal activity of TTO: For C. albicans, by either the broth or agar dilution assay, the Individual MICs and MIC90s that have been reported were 0.04% (Beylier, 1979), 0.2% (Griffin et al.,2000), 0.25% (Vazquez et al.,2000), 0.3% (Christoph et al.,2000) and 0.44% (Nenoff et al.,1996). Studies done to investigate activity of TTO against filamentous fungi found out that these fungi are susceptible, with a few exceptions. All isolates of Aspergillus niger, Rhizopus oligosporus and Penicillium spp. showed zones of inhibition to either 20 μl or 35 μl oil on a paper disc (Concha et al.,1998; Chao et al.,2000). MICs for the filamentous fungi, mostly obtained by the agar dilution method, were in the range of 0.2 – 1.0% for isolates of A. flavus, A. niger, Penicillium spp., Rhizopus spp. and Scopulariopsis spp. (Beylier, 1979; Bassett et al.,1990; Southwell,1993; Rushton et al.,1997; Christoph et al.,2000; Griffin et al.,2000).

2.7.6 Antiviral activity of TTO: Bishop (1995) was first shown the antiviral activity of TTO using tobacco mosaic virus and tobacco plants with agricultural applications in mind. A field trial was conducted in which Nicotiniana glutinosa plants were sprayed with 100, 250 or 500 ppm TTO or control solutions, and all plants were then experimentally infected with tobacco mosaic virus. After10 days, there were significantly fewer lesions per cm2 of leaf of plants treated with TTO as compared to controls. In 2001, Schnitzler et al. (2001) investigated the activity of TTO and eucalyptus oils against herpes simplex virus (HSV). Briefly, the activity of TTO was determined by incubating virus with varying concentrations of TTO, and then using these treated viruses to infect cell monolayers. After 4days, the numbers of plaques formed by virus treated with TTO, or untreated control virus, were determined and compared. The concentration of TTO inhibiting 50% of plaque formation, as compared to controls, was 0.0009% for HSV1 and 0.0008% for HSV2. These studies also showed that at the higher concentration of 0.003%, TTO reduced HSV1 titres by 98.2% and HSV2 titres by 93.0%. Also, by applying TTO at different stages in the virus replicative cycle, TTO was shown to have the greatest effect on free virus (prior to infecting cells) although when TTO was applied during the adsorption period a reduction in plaque formation was seen also.

2.7.7 Antiprotozoal activity of TTO: Carson et al. (2005) mentioned that TTO caused a 50% reduction in growth (as compared to controls) of the protozoa Leishmania major and Trypanosoma brucei at concentrations of 403 μg/ml and 0.5 μg/ml, respectively (Mikus et al.,2000). Further investigation showed that terpinen-4-ol contributed significantly to this activity. In a different study, TTO at 300 μg/ml killed all cells of Trichomonas vaginalis (Viollon et al.,1996). This combined with anecdotal in vivo evidence that Trichomonas vaginalis infections may be successfully treated with TTO suggested by Peña (1962) that further work is warranted.

2.7.8 Anti-inflammatory activity of TTO: Over the last decade, Hart et al., (2000) has demonstrated that through in vitro work terpinen-4-ol can inhibit the production of several inflammatory mediators (such as interleukins) by human peripheral blood monocytes. This suggests a mechanism by which TTO may reduce the normal inflammatory response. Terpinen-4-ol also suppresses superoxide production by agonist-stimulated monocytes, but not neutrophils (Brand et al.,2001). By in vivo, the oedema associated with the efferent phase of a contact hypersensitivity response has been modulated by applied TTO (Brand et al.,2002a). This activity was attributed primarily to 29 Rodney, J. et al, 2015 Journal of Applied Science and Agriculture, 10(3) Special 2015, Pages: 21-39 terpinen-4-ol and α-terpineol. In addition, topical TTO reduced histamine-induced skin oedema of the type that is often associated with immediate type allergic hypersensitivities (Brand et al.,2002b). This kind of activity also seems to be due mainly to terpinen-4-ol.

2.7.9 Anti-insect activity of TTO: Essential oils (Fig. 5) merely related to insecticidal agents, even though only few investigated scientifically. TTO has been evaluated in vitro against Pediculus humanus capitis (head lice) by Veal (1996) and Downs et al., (2000). In a pilot study of lice treatment by McCage et al., (2002) a shampoo containing several plants extracts including TTO performed well. Scabies sarcopti (scabies) has also been tested with TTO in vitro and found to be susceptible to the oil (Walton et al., 2000). However, in contrast, a natural mosquito repellent product containing TTO provided almost no protection against Aedes aegypti by in vitro (Chou et al.,1997).

Fig. 5. Chemical structures of selected components of essential oils.

McDonald and Tovey (1993) demonstrated the activity of several essential oils (including TTO) against house dust mites was compared to that of benzyl benzoate, a standard treatment. Oils of citronella and TTO were as effective as 0.5% benzyl benzoate and TTO at a concentration of 0.8% killed 79% of mites after a 10 min exposure time. In the second study, TTO was the most effective at killing the house dust mite Dermatophagoides pteronyssinus, when compared to lemon essential oils and lavender (Priestley et al.,1998). TTO at a concentration of 10% caused 100% immobility after 30 min and100% mortality after 2 h. According to Callander and James (2012), tea tree oil has insecticidal action against sheep blowfly (L. cuprina) eggs and larvae, stimulating larvae to leave the wound and through antimicrobial andanti-inflammatory properties that aid in wound healing. The study of Benelli et al. (2013) extends the number of effective essential oils against the Mediterranean fruit fly and provides useful information for the development of new tephritid control tools. They have shown tea tree oil cultivated in Italy to be toxic against the Mediterranean fruit fly, C. capitata and its parasitoid, P. concolor. Through contact and fumigation assays, TTO showed lower LC50values towards C. capitata over P. concolor. In ingestion formulation, the LD50 value was lower in C. capitata than in P. concolor. James and Callander (2012a) suggested that tea tree oil has potential for the development of tea tree oil- based ovine lousicides, where they demonstrated that immersion of wool for 60s in formulations containing concentrations of 1% tea tree oil and above can caused 100% mortality of adult lice and eggs. They both then conducted another experiment at the same year which they discovered that immersion dipping of sheep shorn in both 1% and 2%formulations had reduced lice to almost zero after 20 weeks of treatment. Second treatment of sheep with 6 months wool by jetting (high pressure spraying into the fleece) also reduced louse numbers to 91%-94% with 1% tea tree oil formulations and 78%-84% with 2% tea tree oil formulations. Tea tree oil has insecticidal effects as recorded by Williamson et al. (2007) and has repellent effects (Canyon and Speare , 2007; Eamsobhana et al.,2009; Maguranyi et al.,2009). According to Callander and James (2012), tea tree oil has insecticidal action against sheep blowfly (L. cuprina) eggs and larvae, stimulating larvae to leave the wound and through antimicrobial and anti-inflammatory properties that aid in wound healing. 30 Rodney, J. et al, 2015 Journal of Applied Science and Agriculture, 10(3) Special 2015, Pages: 21-39

3.0 Under-utilize parts of tea tree (melaleuca alternifolia) as the potential natural fibre source:

Fig. 6: Tea tree leaf before and after grinded.

The plants (Fig. 6) can be harvested in about 9 months when they reach a height about 2m (Joseph et al.,2009). According to Small (1981b), the first harvest of tea tree can reach 20t/ha (approximate 27,000 trees/hectare) of fresh biomass weight. In the second and third harvest, the trees are capable of producing fresh biomass up to 25 t/ha and 30 t/ha respectively under good growing condition and management. Here, we can simply calculate that in every hectare we can get grossly 20,000kg of harvested tea tree ready for distillation. A study done by Prastyono (2008) demonstrated two types of spacing: 33,333 trees/ha (narrow) and 16,667 trees/ha (wide). He discovered that wide spacing give higher leafiness score, and lower oil concentration compared to narrow spacing. Meanwhile, SEDIA (2007) had planted 10,000 trees/ha in their tea tree plantation in Kimanis, Papar, Sabah. This evidence shows that tea tree can be planted almost everywhere in the world. Tea tree oil is produced by steam distillation of the leaf, and the yield of oil is typically 0.5-3% of the wet weight of the plant (Southwell and Lowe, 1999; Carson et al., 2005). Murtagh (1996) showed that the leaf yield of tea tree was strongly correlated with the total yield of biomass (with a correlation coefficient of 0.94), yet, the proportion of leaf in twig and proportion of twig in the total biomass also influenced the total yield of biomass. Small stems also included in distillation as this plant is shrubs and not trees. Roy et al. (1990) stated that the leaves and the small branches are about 35% dry weights. Eventually, there are a lot of left-over residue or waste, which are the leaves after the distillation. In normal practice, after distillation, the tea tree leaf will be burned or will be composted. Richard (2003) mentioned that in Australia, there are some companies dry the residue leaf after distillation, and then universally return it to the plantation as mulch. He also added that some companies also employ the dried residue leaf as a boiler fuel, and only 15-30% of it required to fuel the boiler furnace for another distillation and this shows that it is a fuel positive process. However, burning can cause environment pollution and the burning activities are monitored by local authorities. Ahmed et al. (2002;2004) mentioned that decomposing and burning the leaves in-situ will not contribute in improving plantation yield and other uses of these beneficial agricultural wastes must therefore be found (Mohamed et al.,2009) in order to solve this problem. In addition, the residue which is the leaf, may contain some remaining tea tree oil, as the consequences from the distillation process. Tea tree oil, which contains antimicrobial properties in general, will make the leaves to take more time to be composted, as the main agent in composting process are microbes it-self (Zhang et al.,2013). Based on these facts, it gives a good consideration to use these left-over leaves which contain natural fibre as the reinforce of filler in biocomposite. As we are moving towards zero-waste and green technology nowadays, scientists and technologists tend to enjoy discovering the future potential development of every parts of plant. Sahari et al. (2012) had investigated the physical and chemical properties on every part of palm tree, from the bunch, frond, trunk and black sugar palm fibre or ijuk. Among all of the parts, the sugar palm frond exceeds others in term of tensile strength which is 421.4 N/mm2, while tensile strength for sugar palm bunch (SPB), ijuk and sugar palm trunk (SPT) is 365.1, 276.6 and 198.3 N/mm2, respectively. The highest cellulose content was obtained from SPF (66.5%), followed by SPB (61.8%), ijuk (52.3%), and SPT (40.6%). These discoveries were then extended to few findings where they developed biocomposite with every different part of the sugar palm tree, with different percentages of fibres and different tests in order to improvise the composites (Sahari et al., 2011a; Sahari et al., 2011b; Sahari et al. 2013). Sugar palm tree parts such as bunch, frond, trunk, and ijuk, are considered wastes, under-utilized and in an abundant source. However, these rubbishes have been converted into high value added new green composite products. The same thing can be applied for the residue of tea tree leaf, which is emphasized in this paper on its potential as the source of fibres. It is good to take into consideration regarding the practicality and economic value of tea tree, from planting to the process of getting the yield. Furthermore, it is advised to compare with other available and well known natural fibres which have existed and survived long in global market. However, this paper aims to stress on the potential of tea tree waste, the leaf, and to some extent, the branches and trunk too, in which most of the industrial player have forgotten. As in Malaysia, which recognized as the largest producer in palm oil, rubber, 31 Rodney, J. et al, 2015 Journal of Applied Science and Agriculture, 10(3) Special 2015, Pages: 21-39 sugar, cocoa and other commodities, the issues of, can tea tree compete with those industry in the market, will rise.

4.0 Agro waste: Fibre source in biocomposite: There is a huge demand in using biofibre as reinforcer and/or biofibre in composite. Their highly specific stiffness, flexibility during processing and low cost (on a volumetric basis) make them attractive to manufacturers. The awareness regarding non-renewable resources is becoming insufficient for the demand has increased as our unavoidable dependence on renewable resources has arisen. The main target of developing natural fibre is to create a new type of composite that can replace the available composite derived from glass fibre (Sain et al, 2005). The role of natural fibre reinforced composite is to become the alternative to replace the material derived from metal, and wood. There are two categories of fibre, primary and secondary, depending on their utilization (Faruk et al.,2012). Primary plants are those grown for their fibre content; such as jute, kenaf etc. while secondary plants are plants in which the fibres are produced as a by-product; such as palm oil, pineapple etc. Six basic types of natural fibres are: bastfibres (jute, flax, hemp, ramie and kenaf), leaf fibres (abaca, sisal, and pineapple), seed fibres (coir, cotton and kapok), core fibres (kenaf, hemp and jute), grass and reed fibres (wheat, corn and rice) and all other types (wood and roots).

Fig. 7: Classifications scheme for natural fibres (Mohanty et al., 2005b; Mueller and Krobjilowski,2003)

According to Mohanty et al. (2005b) natural fibres are derived from plants, animals, and inorganic material as shown in Fig. 7 (Mueller and Krobjilowski,2003). Among the natural fibres, plant fibres are the main sources of fibres that can be found in a large quantity. Rowell (1995) reported that plant fibres are classified according to what part of the plant they come from. Different types of fibres provide different properties (see Table 7). One of the key to know the plant fibre is by understanding the chemical composition, in which it is really closely related and affecting its performance when applying it as composite material. Rowell (1998) reported that plant fibre also referred as lignocellulosics, which comprise of cellulose, hemicellulose and lignin. It is also comprises of minor amounts of starch, , sugar and other organic compounds. Reddy and Yang (2005) mention that cellulose, hemicellulose, and lignin are the three main components of any plant fibres and the proportion of these component in a fibre depends the age, source of fibre and extraction (see Table 8). There are many types of fibres can be taken from various types of plant. In this paper, we are focusing on natural fibres which are derived from leaves.

Table 7: Some properties of plant and synthetic fibres (Bismarck et al.,2005). Fibre Density Diameter Tensile Strength Young‘s Elongation at g/cm3 (μm) (MPa) Modulus Break (%) (GPa) Flax 1.5 40-600 345-1500 27.6 2.7-3.2 Hemp 1.47 25-500 690 70 1.6 Jute 1.3-1.49 25-200 393-800 13-26.5 1.16-1.5 Kenaf - - 930 53 1.6 Ramie 1.55 - 400-938 61.4-128 1.2-3.8 Sisal 1.45 50-200 468-700 9.4-22 3-7 PALF - 20-80 413-1627 34.5-82.5 1.6 Abaca - - 430-760 - - Oil palm EFB 0.7-1.55 150-500 248 3.2 25 Cotton 1.5-1.6 12-38 287-800 5.5-12.6 7-8 Coir 1.15-1.46 100-460 131-220 4-6 15-40

32 Rodney, J. et al, 2015 Journal of Applied Science and Agriculture, 10(3) Special 2015, Pages: 21-39

Table 8: Chemical composition and moisture content of plant fibres (Bismarck et al.,2005). Fibre Cellulose Hemicellulose Lignin (wt%) Pectin (wt%) Moisture (wt%) (wt%) (wt%) Content (wt%) Flax 71 18.6-20.6 2.2 2.3 8-12 1.7 Hemp 70-74 17.9-22.4 3.7-5.7 0.9 6.2-12 0.8 Jute 61-71.5 13.6-20.4 12-13 0.2 12.5-13.7 0.5 Kenaf 45-57 21.5 8-13 3-5 - - Ramie 68.6-76.2 13.1-16.7 0.6-0.7 1.9 7.5-17 0.3 Sisal 66-78 10-14 10-14 10 10-22 2 PALF 70-82 - 5-12.7 - 11.8 - Abaca 56-63 - 12-13 1 5-10 - Oil palm EFB 65 - 19 - - - Cotton 85-90 5.7 - 0-1 7.85-8.5 0.6 Coir 32-43 0.15-0.25 40-45 3-4 8 -

4.1 Sisal as biocomposite: To name a few, there are many researches have been done by using leaf fibres as filler or reinforce in biocomposite. Suppakarn and Jarukumjorn (2009) had added magnesium hydroxide and zinc borate into sisal/PP composites as flame retardants. They had discovered that the addition of the flame retardants reduced the burning rate and increased the thermal stability of the composite. Zhang et al. (2005) discovered sisal/plasticized wood flour composites were fully biodegradable. Towo et al. (2008b) had prepared the treated sisal fibre composites with an epoxy and matrix, and performed tests on fatigue evaluation and dynamic thermal analysis.composites containing alkali treated fibre had better mechanical properties than the untreated. Sisal fibres also investigated with other matrices such as rubber (Jacob et al.,2007; Wongsorat et al.,2010), bio polyurethane (Bakare et al.,2010), cellulose acetate (Peres de Paula et al.,2008), phenol formaldehyde (Zhong et al.,2007) and polyethelene (Favaro et al.,2010) regarding their mechanical, morphological, chemical and cure characteristic.

4.2 Abaca as biocomposite: Bledzki et al. (2007; 2008) examined the mechanical properties of abaca fiber reinforced PP composites regarding different fiber lengths (5, 25 and 40 mm) and different compounding processes (mixer-injection molding, mixer and direct compression molding process). It was observed that, with increasing fiber length (5–40 mm), the tensile and flexural properties showed an increasing tendency though not a significant one. Among the three different compounding processes compared, the mixer-injection molding process displayed a better mechanical performance (tensile strength is around 90%higher) than the other processes. When abaca fiber PP composites were compared with jute and flax fiber PP composites, abaca fiber composites had the best notched Charpy and falling weight impact properties. Abaca fiber composites also showed higher odor concentration compared to jute and flax fiber composites. Abaca fibers have been investigated with cement (SavastanoJr et al.,2009), polyurethane (El-Meligy et al.,2010), aliphatic polyester resin (Teramoto et al.,2004), PP (Paul et al.,2008), urea formaldehyde (El-Meligy et al.,2004), PE (Ibrahim et al.,2010), polyester (Gohil and Shaikh,2010), and polyvinyl alcohol (Sathasivam et al.,2010) as matrices, in order to evaluate the composites properties.

4.3 Pineapple leaf fibre: Pineapple leaf fiber was reinforced with polycarbonate to produce functional composites (Threepopnatkul et al.,2008). The silane treated modified pineapple leaf fibers composite exhibited the highest tensile and impact strengths. The thermo gravimetric analysis showed that the thermal stability of the composites is lower than that of neat polycarbonate resin. In addition, the thermal stability decreased with increasing pineapple leaf fiber content. On the other hand, Kengkhetkit and Amornsakchai (2014) had demonstrated whole ground pineapple leaf (WGL), pineapple leaf fiber (PALF) and non-fibrous material (NFM) are examined as fillers for polypropylene reinforcement. It was found that PALF provided the highest improvement in all mechanical properties tested (tensile, flexural and impact tests) and also heat distortion temperature, followed by WGL and NFM, respectively. NFM, although it provided only slightly improved tensile and flexural properties, could maintain or even improve impact strength.

4.4 Research works related in Malaysia: Various studies related to development of biocomposites reinforced by natural fibres had been done in Malaysia. Apart from being a tropical country where natural fibres are available in abundant resource, in fact Malaysia is also focusing on agriculture activities, and as a consequence, more and more agricultural wastes are available too. From oil palm empty fruit brunch (EFB) (Abu Bakar and Hassan, 2009; Khalina et al.,2009), oil palm stem plywood (Paridah et al.,2009), (Nur Yuziah. 2009) and (Sahrim, and Hazleen. 2009), (Anuar et al.,2009), sugar palm tree (Sahari et al., 2011a; Sahari et al., 33 Rodney, J. et al, 2015 Journal of Applied Science and Agriculture, 10(3) Special 2015, Pages: 21-39

2011b; Sahari et al. 2012; Sahari et al. 2013), and many more including pseudo-stem, coconut shell, kenaf, rice husk, coir, sugarcane bagasse, roselle, and wood (Mohamad Awang et al., 2009). As mentioned before, it is good to investigate more on tea tree as the source of fibres, and see how far it can compete with existing natural fibre which some of them has been well established long ago.

Conclusion: Melaleuca alternifolia is popular for its oil, which has been employed largely in various industries of its antimicrobial properties. Research are still ongoing mainly focusing on the tea tree oil properties, however almost none of them investigating on the residue which is the leaf. Though there are research works that exploring on leaf as the source of fibres reported before, however studies on the tea tree as a new source of natural fibres or material in order to become the potential filler or reinforcer have yet been reported elsewhere. Furthermore, there is still no exploration on the development of any parts of tea tree, including its branch and trunk, as value added products. The combination of tea tree leaves with any matrix to become a new biocomposite is interesting from the view of both aspects of economy and environment, as what have been reported before. Thus, since tea tree leaf remains largely unknown by many people and very little information is available elsewhere, more studies need to be done to unveil its significance and to promote its usefulness for a better tomorrow.

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