ALPS Biotech Co., Ltd., National Biotechnology Research Park, Nangang, Taipei City 11571, Taiwan.

*Corresponding authors:

Shui-Tein Chen ALPS Biotech Co., Ltd., National Biotechnology Research Park, Nangang, Taipei City 11571, Taiwan. Tel: +886-2-27887626; Email: [email protected]

Yerra Koteswara Rao ALPS Biotech Co., Ltd., National Biotechnology Research Park, Nangang, Taipei City 11571, Taiwan. Email: [email protected]

Citation: Chen ST, Rao YK. (2022). An Overview of Agarwood, Phytochemical Constituents, Pharmacological Activities, and Analyses. Traditional Medicine. 3(1):8.

Received: April 21, 2022

Published: July 17, 2022

ABSTRACT

Agarwood is a resin-impregnated heartwood obtained from the plants belongs to the genera, Aquilaria, Daphne, Gonystylus, Gyrinops and Wikstroemia. It is traditionally used for the production of perfume and incense stick, and pharmaceutical applications. Agarwood usually induced by the natural (traditional), conventional, and non-conventional methods. The major groups of phytochemicals identified in agarwood extracts are sesquiterpenes, 2-(2-phenylethyl)-4H-chromen-4-one derivatives (PECs), and aromatic compounds. These phytochemicals are showed various pharmalogical properties such as anti-inflammatory, cytotoxic, neuroprotective, anti-diabetic, anti-bacterial, etc. Several analytical techniques are applied to analyze the agarwood phytochemicals including sesquiterpenes, which exists mostly in the form of essential oils, and the fragrance constituents of PECs. The present review summarize the agarwood traditional uses, induction methods, phytochemical constituents, potential pharmacological activities, along with analyses methods. This review was carried out by searching various scientific databases, including Google Scholar, PubMed, Elsevier, ACS publications, Taylor and Francis, Wiley Online Library, MDPI, Springer, Thieme, and ProQuest. The present review provides a scientific basis for future studies and necessary information for the development of agarwood based therapeutic agents.

Keywords: Agarwood; Traditional uses; Induction methods; Chemical constituents; Biological activities; Analyses

INTRODUCTION

Agarwood, known as aloeswood or eaglewood, is an aromatic dark resin-impregnated heartwood obtained from wounded tree species of the Thymelaeaceae family [1]. The plant family Thymelaeaceae contains 54 genera, including Aquilaria, Daphne, Gonystylus, Gyrinops and Wikstroemia [1]. The species of the genus Aquilaria, Gonystylus, and Gyrinops produce agarwood [1]. In particular, the genus Aquilaria contains 57 species, among these 21 are accepted in the plant list [1]. So far, fifteen species of Aquilaria and nine species of Gyrinops are reported as agarwood producing plants [2]. Agarwood (resin)-producing species are found from the forests of Southeast Asia including, Bangladesh, Bhutan, China, India, Indonesia, Laos, Malaysia, Myanmar, Singapore, Taiwan, Thailand, and Vietnam [1,2]. They are usually found in lowland tropical forests with optimal sunlight, shade and moisture. Agarwood-producing species have a small flower similar to that of ‘jasmine’, and the fruit is bitter [3].

Healthy Aquilaria tree does not produce agarwood [2]. The healthy wood is white, soft, even-grained and not having a perfumed smell, as compared with the dark, hard and heavy scented characterictics resin-impregnated agarwood [2]. The agarwood resin developed through pathological, wounding and non-pathological mechanisms [4]. The formation of agarwood occurs naturally in response to natural injuries such as lightning, insects and mold attacks [4]. The deposited resin around the wounds over the years accumulate and eventually forms agarwood [4]. Therefore, Agarwood is termed as the resin-impregnated pieces of wood [4], and its formation is related to the self-defense mechanism of Aquilaria trees in response to biotic and abiotic stresses [1,2]. Stresses trigger the defense responses of Aquilaria species, which in turn initiate the secondary metabolite biosynthesis and the accumulation of agarwood resin [1,2].

The prominent species of agarwood producing Aquilaria species are, A. beccariana Tiegh., A. crassna Pierre ex Lecomte, A. filaria (Oken) Merr., A. hirta Ridl., A. khasiana Hallier f., A. malaccensis Lamk., A. microcarpa Baill., A. rostrata Ridl., A. sinensis (Lour.) Spreng., and A. agallocha [5]. Among these A. agallocha, A. crassna, A. malaccensis, and A. sinensis gain significant attention due to their therapeutic uses in traditional Southeast Asian medical systems [5]. Accordingly, these species appear frequently in the literature, particularly A. crassna, A. malaccensis and A. sinensis [1,5]. The agarwood producing Aquilaria species and their native place are presented in Table 1. With the increasing demand for agarwood, the population of agarwood species is declining rapidly in the wild. Currently, the genus Aquilaria is listed as endangered species and protected under Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) regulation [1]. The index of CITES species listed A. agallocha Roxb. as a synonym of A. malaccensis Lamk [1]. A. malaccensis Lamk., is also synonym to Aquilariella malaccensis (Lam.) Tiegh., and Agallochum malaccense (Lam.) Kuntze [6]. The International Union for Conservation of Nature and Natural Resources (IUCN) Red List of Threatened Species is listed A. crassna as critically endangered, and A. malaccensis and A. sinensis as vulnerable [7].

Table 1: Agarwood producing Aquilaria species and their place of origin.

Species	Place of origin (Native)
Aquilaria apiculata Merr	Philippines
Aquilaria baillonii Pierre ex Lecomte	Cambodia, Thailand, Laos, Vietnam
Aquilaria banaensis P.H.H6	Vietnam
Aquilaria beccariana Tiegh	Indonesia
Aquilaria citrinicarpa (Elmer) Hallier f.	Philippines
Aquilaria crassna Pierre ex Lecomte	Thailand, Cambodia, Vietnam
Aquilaria cumingiana (Decne) Ridl	Malaysia
Aquilaria khasiana Hallier f.	India
Aquilaria malaccensis Lam.	India, Myanmar, Malaysia, Indonesia, Philippines
Aquilaria microcarpa Baill	Indonesia
Aquilaria parvifolia (Quisumb) Ding Hon	Philippines
Aquilaria rostrata Ridl	Malaysia
Aquilaria rugosa Kiet Kessler	Vietnam
Aquilaria sinensis (Lour.) Gilg	China
Aquilaria subintegra Ding Hon	Thailand
Aquilaria urdanetensis (Elmer) Hallier f.	Philippines
Aquilarla yunnanensis S.C. Huang	China

1.1. Traditional uses

Agarwood is known as “wood of God” because of religious practices [4]. The word “aloes” which means agarwood is found in the Sanskrit poet, Kâlidâsa, dated back to c. 4^th–5^th century CE [4]. Agarwood is considered the finest natural incense and has been used in many cultures, such as the Arabian, Chinese, Indian, and Japanese cultures [8]. Agarwood also associated with religious history, rituals and ceremonies in Buddhism, Christianity, Hinduism, and Islam [8]. It is known as gaharu in the Indonesia and Malaysia, jin-koh in Japan, chen xiang (沉香) in Chinese, agar in India, chim-hyuang in Korea, kritsana noi in Thailand, tram huong in Vietnam, and oud in the Middle East [8]. Agarwood is widely used as therapeutic perfumes, traditional medicine, religious purposes and aromatic food ingredient (Table 3) [3]. In the traditional Chinese and Ayurvedic medicines as an aphrodisiac, sedative, cardiotonic and carminative, as well as to treat gastric problems, coughs, rheumatism and high fever [9]. Agarwood is a traditional Chinese medicine included in the 2020 edition of Chinese Pharmacopoeia [10]. In traditional Arabian medicine, agarwood essential oil is used for aromatherapy [3]. In Thailand, agarwood has been used for a long time as a traditional treatment for infectious diseases such as diarrhea and skin diseases [3]. Additionally, A. crassna extract has been using as the ingredient of Ya-hom, a traditional Thai herbal formulation for the treatment of fainting by targeting the cardiovascular system.

1.2. Grading system

The market price of agarwood has commercial attention. However, the grading process of agarwood is largely depends on the human experience from the age-old practices of each country [1]. In general, the classification of agarwood oil quality is based on wood physical properties, long lasting aroma when burnt, color, resin content, high fixative properties and consumer perception, etc [1]. The higher the grade of agarwood, the richer the layers of aroma [1]. The best agarwood fragrance is mellow and sweet, full of penetration and persistence, and the powdery waxy material on the surface can be scraped off and kneaded it into a ball [1]. Its aroma is regarded as a symbol of high quality. Complexity and variability in agarwood composition are major challenges associated with its grading process. The morphological grading system of agarwood is shown in Table 2.

Table 2: Assesment of agarwood quality using grading system.

Observational Feature	Grading Categary
	A	B	C
Sense of oiliness	Strong	Strong	Mild
Aroma	Strong, feel sweet and cool	Less potent odor, feel sweet and slightly spicy	Mild aroma, feel slightly sweet, salty
Resin density	High dense, compact, sink in water when soaked	Dense, less compact, half-sinkage in water	Light and not dense, full-floating on water
Weight	Hard texture, brittle, and not hollowed	Texture little hard, little brittle, slightly hollow	Loose texture, not brittle, and hollow

1.3. Economical value

Agarwood is a valuable, non-timber forest product used different societies for medicinal, aromatic, cultural and religious purposes [8]. As the wealth of the consumer countries are gradually increased in recent decades, the market’s demand for agarwood started to exceed its supply [1]. The market value of agarwood derivative products is dependent on the classification or grading of agarwood, which is determined by a cumulative factor of the fragrance strength and longevity, resin content, geographical origin and purity (for oil) [8]. Global agarwood prices can be ranging from US$20 – 6,000 per/kg for the wood chips depending on its quality or US$ 10,000 per/kg for the wood itself [1]. High quality wood is used as incense in Arabian households and for the ‘koh-doh’ incense ceremony in Japan [8]. High-quality agarwood products can reach prices as high as US$100,000/kg. In the form of oud oil which is distilled from agarwood for perfumery, can be sold for US $1500 per 11.7 g [1,9]. The annual global market for agarwood has been estimated to be in the range of US$ 6 – 8 billion [1,9]. Agarwood has commercial importance in three categories i.e. perfume production, incense stick, and pharmaceuticals as described below [8].

1.3.1. Perfume

Agarwood oil is an essential oil obtained by water and steam distillation of agarwood [1]. Agarwood oil is a yellow to dark amber, viscous liquid with a characteristic balsamic and woody odour [9,11]. It is used in luxury perfumery for application. Agarwood perfumes are commonly prepared in both alcoholic and non-alcoholic carriers [9,11]. Agarwood perfume has a unique smell obtained from fragrance essential oil and aromatic compound [11]. The oil is also used as a fragrance in the production of cosmetics and personal care products, such as soaps and shampoos [9,11]. Agarwood resin is a key ingredient in old and new Arabic perfume products, and used as an element within high-quality perfumes in Arabic, Japanese and Indian cultures [4]. Traditionally in the Middle East, agarwood oil is used as a scent, and Minyak attar (water-based) [4]. “Attar” is an example of a water-based perfume containing agarwood oil, which is traditionally used by Muslims to lace prayer clothes [4]. Agarwood oil is one of the most important ingredients in Chinese perfume industry; additionally it became a prominent in the modern western perfume and fragrance industry.

1.3.2. Incense stick

Burning agarwood produces fragrance, which is used as incense for ceremonial purposes in Buddhism, Confucianism and Hinduism [11]. The incense also functions as an insect repellent [4,8]. The aromatic compounds are the main chemical components in agarwood smoke and create an atmosphere of peace and serenity [1,4]. It scent heavenly, woody nuance, balsamic and warm aura of bittersweet when the chromones break into low molecular weight at high temperature [1,4]. In Taiwan, the agarwood stick is used in traditional festivals or ceremonies to bring safety and good luck to the believer [11]. The agarwood incense stick is used in the bathroom as a customary sense, during Ramdan prayer by the Muslim, and Puja celebration by the Hindu religious practice [11].

1.3.3. Pharmaceutical use

Agarwood plays a vital role in the field of medicine, contains various chemical components, including several sesquiterpenes, 2-(2-phenylethyl)chromens (PECs), and aromatic compounds, etc [3,6,12]. These compounds display various biological properties such as anticancer, anti-inflammatory, antioxidant, antibacterial, antifungal, antidiabetic, and so on [3,6,12]. Traditionally agarwood is prescribed to treat pleurisy by the Sahih Muslim, relieve pain, arrest vomiting, and asthma [6,12]. A. malaccensis products are an essential source in the field of Ayurveda for treating various diseases such as appetizer, analgesic, antipyretic, antihistaminic, styptic, carminative, cytotoxic, insecticidal, general tonic, etc [6,11,12]. Agarwood materials have also been formulated into a balm (muscle rub) and candle wax [11]. The pharmaceutical and traditional use of agarwood in different countries/locations are presented as in Table 3.

Table 3: The pharmaceutical and traditional use of agarwood in different countries/locations.

Place	Traditional use	Preparations/route of intake
Bangladesh	Treatment of rheumatism	Agarwood taken orally
China	Treatment of circulatory disorders, abdominal pain, vomiting, dyspnea, asthma	Heartwood in Chinese medicines, and Heartwood decoction
India	Treatment of diarrhoea, dysentery, vomiting, anorexia, mouth and teeth diseases, facial paralysis, shivering, sprains, bone fracture	Heartwood in Ayurvedic formulation such as Chawanprash, Arimedadi Taila and Mahanarin Taila
Indonesia	Treatment of joint pain, Sedation, detoxification, treatment of stomachaches, incense sticks	Wood burned and smoke held over the affected area
Japan	Stomachic and sedative agent	Infusion or decotion
Korea	Treatment of cough, acroparalysis, croup, asthma, stomachic agent, tonic, sedative and expectorant	Infusion or decotion
Malaysia	Tonic, stimulant and carminative agent after childbirth	Heartwood mixed with coconut oil
	Treatment of rheumatism and body pains	Heartwood decoction (mixed with other types of woods)
	Treatment of small pox	Heartwood prepared into Ointment
Philippines	Stop bleeding of the wounds	Bark and roots
	Treatment of malaria (substitute for quinine)	Bark, wood and fruits
Thailand	Treatment for diarrhoea, dysentery and skin diseases, antispasmodic and cardiovascular function enhancer in fainted patient Treatment of fainting, nausea and vomiting	Traditional medicinal preparation ‘Krisanaglun Folk medicine ‘Ya–Hom’
Tibet	Treatment of nervous and emotional disorders Cardioprotective agents	Infusion or decotion

1.3.4. Other uses

The uses of agarwood is not restricted to incense and perfumery. Solid pieces of agarwood are carved into natural art sculptures, beads, bracelets and boxes [4,11]. The wood of A. agallocha is used as decorative ornaments (China), ‘joss sticks’ (China and India), and flea and louse repellents (India), whereas the bark has been used to manufacture paper (China) [1,4]. In India, the wood of A. malaccensis used as fuel for fumigation, and the bark has been used to make cloth and rope [11].

1.4. Agarwood induction methods

Agarwood is a valuable non-timber product, and its demand is much greater than its supply. The agarwood (resin) induction mechanism is not fully understood or elucidated. High demand of quality agarwood in conjunction with the depletion of the wild Aquilaria trees, leads to the artificial induction of agarwood resin formation. Modern artificial agarwood formation techniques are mainly biochemical methods, such as chemical reagent invasion and bacteria inoculation (Figure 1) [2].

1.4.1. Natural (Traditional) methods

Naturally, agarwood formation is often linked to the physical wounding or damage of Aquilaria trees caused by thunder strike, animal grazing, pest and disease infestations [13]. These events expose the inner part of the trees toward pathogenic microbes, which evoke the defense mechanism of Aquilaria to initiate the resin production [13]. This natural formation process of agarwood has greatly inspired the development of diverse artificial induction methods (Figure 1). For example, the microbial species of Actinobacteria sp., Acidobacteria sp., Aspergillus sp., Alcaligenes sp., Bacillus sp., Chaetomium sp., Curvularia sp. Fusarium sp. Lasiodiploidia sp., Penicillium sp., Proteobacteria sp., Pseudomonas sp., and Trichoderma sp. are involved in the agarwood formation [1,13]. For more details, please refer the recent review article [221].

1.4.2. Conventional methods

Various conventional methods are applied to initiate agarwood resin formation. The techniques often involved the physical penetration into the trunk (wounding), mechanical wounding, axe chopping, nailing, holing, burning, insertion of a microbial (mainly fungal) concoction (pathology) and response of the tree towards the administered stress (non-pathological) [2,5]. Many pure-culture strains of fungi such as Aspergillus niger, A. fijiensis, Chaetomium sp., Fusarium solani, Lasiodiplodia sp. (L. hormozganensis), Gongronella butleri, Saitozyma podzolica, Cladorrhinum bulbillosum, Humicola grisea, Penicillium sp., Trichoderma lentiforme, Phaeoacremonium rubrigenum, and Tetracladium marchalianum are isolated from natural agarwood are found to be effective biological agents to induce agarwood formation in healthy Aquilaria trees [14‒16]. For more details, please refer the recent review article [221]. Therefore, fungal-interaction induction methods coupled with the application of biological inoculum are developed for agarwood induction [2,5]. The advantage of using fungal inoculum is that it is generally believed to be safe for handling and eco-friendly. However, fungal inoculation will normally give rise to localized and inconsistent quality of agarwood due to the different fungal consortium used [2,5]. As a solution, laborious holing process and long incubation time is required to maximize the colonized surface area on the tree to produce better quality of agarwood [17]. The fungal infected Aquilaria trees are reported to deposit agarwood resin around the infected sites as barrier to prevent further fungal intrusion [13,14]. Agarwood resin deposition accompanied with color changes of internal tissues occured within a year by injuring the trees [8]. Although it is cost effective and requires only personnel with little or no scientific knowledge on agarwood, but these conventional induction methods usually result in inferior quality and uncertain yield of agarwood. Mass cultivation and large plantation of Aquilaria trees using these conventional methods have greatly resolved the shortage of agarwood supply in the global market.

1.4.3. Non-conventional methods

Artificial induction of agarwood formation is the use of chemical, insect and pathogen-inducing techniques is increasingly common in agarwood induction [18]. Chemical inducers normally comprise of phytohormones, salts, minerals and biological-derived substances [2,18,19]. Various chemical induction approaches are developed, including cultivated agarwood kit (CA-kit), the whole-tree agarwood inducing technique (Agar-Wit) and biologically agarwood-inducing technique (Agar-bit). CA-kit is a combined method based on physical wounding and chemical induction, where the inducing agent is applied into the Aquilaria tree via an aeration device inserted into the wound [2]. Agar-Wit is a transpiration-assisted chemical treatment to form an overall wound in the tree, where the preloaded inducer in a transfusion set is distributed via plant transpiration [18]. Similarly, Agar-bit method adopts the idea of distributing the inducing reagent by plant transpiration, except that the reagents are injected directly into the stems of the tree [20]. Chemical inducers are suitable for mass production of agarwood with easier quality control than biological inoculum. However, in spite of the fast results and high yields, the application of chemical inducers still poses skepticism of toxicity on both human and environment. All of these induction techniques in any case mimic the natural processes of agarwood formation, which have their own strengths and weaknesses. The agarwood induction methods are presented in Figure 1. On the other hand, in vitro culture of various parts of Aquilaria spp. and Gyrinops spp. are studied at various tissue culture laboratories [2]. The tissue culture techniques identified the key regulator genes of Aquilaria spp. and Gyrinops spp. involved in the agarwood production [2].

Figure 1: Schematic presentation of Agarwood induction techniques.

2. PHYTOCHEMICAL CONSTITUENTS OF AGARWOOD

The chemical constituents of healthy Aquilaria trees without resin-formation differ from the resin-impregnated portions of the plants [6,12]. The phytochemical analysis of agarwood resin has been the subject of many studies [1,6,12]. The types and derivatives of chemical constituents in agarwood are extremely wide and diverse, indicating the different types of fragrance properties of agarwood from different species and regional sources [1,6,12]. Agarwood resin constituents were isolated using solvent extraction, with subsequent purification via column chromatography and structural elucidation using spectroscopic techniques, including NMR [1,6,12]. Essential oils are produced by the hydrodistillation of resin followed by GC-MS or the newer technique of supercritical fluid extraction (SFE) [21]. The chemical constituents in agarwood may vary considerably in terms of quality, source plant origin, extraction methods, agarwood induction method, or agarwood-formation process, collection time, analytical approach etc [1,6,12]. The agarwood chemical constituents produced by Aquilaria species including A. sinensis, A. malaccensis (syn. A. agallocha), A. crassna, A. filaria, and Gyrinops salicifolia, as well as an unidentified Aquilaria spp [1,6,12]. Previous chemical investigations of agarwood species resulted in the isolation and structure characterization of several sesquiterpenes, 2-(2-phenylethyl)-4H-chromen-4-one derivatives (PECs), and aromatic compounds are the main characteristic chemical constituents [6,12,22]. The types of agarwood chemical constituents are described below.

2.1. Sesquiterpenoids

Sesquiterpenes are composed of three isoprene units. They are mainly distributed in plants existing mostly in the form of volatile constituents present in essential oils. The constituents of agarwood essential oil is mainly composed of sesquiterpenoids, and low abundant of volatile aromatic metabolites, which gives an unique and fragrant-smelling property of agarwood [1]. The sesquiterpenes isolated from agarwood exhibit various types (Figure 2), including acoranes (A), agarospiranes (B), cadinanes (C), eudesmanes (D), eremophilanes (E), guaianes (F), humulanes (G) and prezizaanes (H), zizaanes (I).

Figure 2: Types of sesquiterpenes from agarwood.

2.1.1. A. Acoranes

The spiro sesquiterpenes, acoranes (A1‒A3), are reported from the agarwood of A. sinensis (Figure 3). The compounds A2 and A3 are a pair of stereoisomers.

Figure 3: Chemical structures of acorane-type sesquiterpenes from agarwood.

2.1.2. B. Agarospiranes (vetispiranes)

The spirocyclic sesquiterpenes, agarospiranes are reported in agarwood from A. sinensis, A. malaccensis and A. agallocha (Figure 4, and Table 4). The first agarospirane sesquiterpene discovered in agarwood is agarospirol (B1) from the agarwood of A. agallocha [23]. The allyl ether 2,14-epoxy-vetispir-6-ene (B10) and enol ether 2,14-epoxy-vetispira-6(14),7-diene (B11) are reported from the essential oil of A. agallocha [24]. Vetispira-2(11),6(14)-dien-7-ol (B8) and vetispira-2(11),6-dien-14-al (B9) might be artefacts [25]. The sesquiterpenes, agarospiranes have limited distribution and are mainly found in the agarwood species of A. agallocha, A. malaccensis, and A. sinensis (Figure 4, Table 4). Phytochemical examination of 95% ethanol extract of A. agallocha agarwood, resulted in the isolation of agarospirane-type sesquiterpenes (agarospiranic aldehyde A, and B, B13, B14) [26].

Figure 4: Chemical structures of agarospirane-type sesquiterpenes from agarwood.

Table 4: Agarospirane-type sesquiterpenes from agarwood.

No.	Name	Source	Ref.
B1	Agarospirol	A. agallocha A. malaccensis A. sinensis	27,23 28 29
B2	Baimuxinol	A. sinensis	30
B3	Baimuxinic acid	A. sinensis	30
B4	Baimuxinal [Oxoagarospirol]	A. sinensis A. malaccensis A. agallocha	29, 31,32 33,34, 35
B5	(4R,5R,7R)-1(10)-spirovetiven-11-ol-2-one	Kyara-Vietnam	36
B6	2-Oxo-12-hydroxy-hinesol	A. sinensis	37
B7	Isoagarospirol		25
B8	Vetispira-2(11),6(14)-dien-7-ol	A. agallocha	24
B9	Vetispira-2(11),6-dien-14-al	A. agallocha	24
B10	2,14-Epoxy-vetispir-6-ene	A. agallocha	24
B11	2,14-Epoxy-vetispira-6(14),7-diene	A. agallocha	24
B12	rel-(2R,5R,10S)-6(7)-Spirovetiven-11,12,13-triol	Aquilaria spp.	38