Aquilaria Identification Methods and Phytochemicals

Aquilaria Identification Methods and Phytochemicals: Comparison

Please note this is a comparison between Version 1 by Mubasher Hussain and Version 2 by Peter Tang.

Aquilaria sinensis (Lour.) Gilg is the resin-containing wood of the Aquilaria. Agarwood is a traditional Chinese medicine included in the 2020 edition of Chinese Pharmacopoeia. The main phytochemicals of agarwood include terpenoids, dominated by sesquiterpenes. For centuries, terpenoids have been used in traditional Chinese medicine and have been shown to possess various pharmacological properties, including bacteriostatic, antibacterial, sedation, analgesia, anti-inflammation, anti-asthmatic, hypoglycemic, antidepressant, and many others. Alongside biological activity screening, phytochemical advances and pharmacological research have also made certain progress.

agarwood
chemistry
medicinal plant
pharmacological effects
phytochemicals

1. Introduction

Aquilaria sinensis (Lour.) Gilg is the resin-containing wood of the Aquilaria. Agarwood is a traditional Chinese medicine included in the 2020 edition of Chinese Pharmacopoeia ^[1]. There are about 17 species of agarwood in the world, most of which are distributed in China, India, and Southeast Asian countries. In recent years, the majority of agarwood, headed by Aquilaria sinensis (Lour.) Spreng, has continuously attracted attention. Agarwood plants are likely to secrete a variety of secondary metabolites when they are damaged, including by lightning, chopping, burning, moth-eating, or microbial invasion of natural factors ^[2]. The traditional artificial agarwood formation techniques are based on physical methods such as cutting and digging holes. Modern artificial agarwood formation techniques are mainly biochemical methods, such as chemical reagent invasion and bacteria inoculation, which are deposited in the xylem after a complex agarwood formation process after wound formation ^[3][4]. There are few reports about artificial agarwood, mostly focused on drilling and chemical reagent dripping, but insect infestation is the best method for producing natural agarwood (Figure 1). In recent years, there are many kinds of agarwood commodities and crafts in the market, and the phenomenon of counterfeit mixing is serious. At present, there are many ITS (Internal Transcribed Spacer) intergenic regions for the identification of agarwood species, and the research on agarwood has made some progress, but its identification method is not yet mature. At present, a variety of methods have been established to control the quality of medicinal materials, including the coloration of chemical reagents, the content of alcohol extracts, the content of agarwood tetraol, the content of chromone, the HPLC fingerprint of alcohol extract, etc. Most of the effective components of agarwood are focused on the study of volatile oil, chromone, and sesquiterpene. Agarwood is a valuable traditional Chinese medicine and natural high-grade spice in the world. It has the effects of relieving pain, asthma, and vomiting. It has significant effects when combined with other traditional Chinese medicines such as Bawei Chenxiang Powder and Chenxiang Tongbian Powder. This study focused on pharmacological and clinical research of antibacterial, bacteriostatic, anti-tumor, antidepressant, and antioxidation, as well as the application for cardiovascular and cerebrovascular diseases ^[5]. There are 17 Aquilaria spp distributed around the world, as shown in Table 1.

Figure 1.

Wound tissues and agarwood sample collection from

Aquilaria sinensis

Table 1. The species of Aquiliria and its distribution.

Species	Origin	References
Aquilaria malaccensis Lam.	India, Myanmar, Malaysia, Indonesia, Philippines

used a variety of different extraction methods for identifying different agarwood species by the ITS2 sequence analysis method, using its DNA. The results showed that the DNA barcode sequence based on ITS2 can accurately identify the real and fake species, and the species can be identified by sequence comparison. The similarity of the comparison result with the original agarwood is up to 100%, which proves that this method can effectively distinguish true and fake products and the relationship between species and genera. Meanwhile, Niu et al. ^[18] identified the sequence-specific regions (ITS1 and ITS2) using PCR amplification. The results showed that there were various sequence-specific regions in the rDNA sequence of agarwood. Shen et al. ^[19] analyzed the sequence-specific regions (ITS sequence of DNA), and the different regions were distinguished by sequence alignment.

3.2. Phytochemicals

The major chemical constituents from Aquilaria plants are sesquiterpenoids and chromones (Table 2, Table 3 and Table 4). These are divided into two categories: (A) Volatile compounds of agarwood and (B) Non-volatile compounds of agarwood.

Table 2. Aromatic compounds from agarwood.

Name	Contents (%)	Chemical Structure	Reference

Chemical structure of 2-(2-phenylethyl) chromones in agarwood.

Name	Chemical Structure	Reference


^[	⁶
Benzylacetone	0.95
^]	^[	⁷^]^[8]
			^[20]

2-[2-(4-Hydroxyphenyl)ethyl]chromone

Rel-(1AR,2R,3R,7bS)-1a,2,3,7b-Tetrahydro-2,3-hidydroxy-5[2-(4-methoxyphenyl)ethy]-7H-oxireno[f]^[1]benzophran-7-one
Oxidoagarchromones A
^[
²⁷
^]

3.3. Pharmacological Uses

A variety of phytochemicals from agarwood has obvious pharmacological uses and has always been an interesting topic among researchers and scientists (Table 5). The majority of phytochemicals from Aquilaria plants are sesquiterpenoids and chromones, which have been widely used in the pharmacological industry.

Table 5. A summary of pharmacological effects of agarwood.

Pharmacological Action	Active Substance	Action Mechanism

^[29] extracted and separated the volatile oil of Malaysia agarwood. After analysis and screening, the content of γ-eucalyptus oleol was determined as the quality standard of agarwood volatile oil. Chen et al. ^[30] detected the incense in different fragrance processes by GC-MS, analyzed the different chemical components, distinguished the incense in different ways by the method of grey relational analysis, and, finally, reflected the quality of incense by ranking. The several chemical components of agarwood were isolated by high-performance liquid chromatography (HPLC). After evaluation and analysis, the content of agaropiric acid in incense was selected as the index to establish a standard method for evaluating the quality of artificial incense, with the content of agaropiric acid as the evaluation standard ^[31].

Table 6. Quality grading system of agarwood.

Morphological Feature	Grade
Morphological Feature	A	B	C

Antibacterial and bacteriostatic	Sesquiterpenes	^[21]	Its antibacterial mechanism can induce cell apoptosis through the process of nuclear condensation and cleavage.
Aquilaria sinensis (Lour.) Gilg
2,4-Di-tert-butylphenol
	China		^[7]^[

Anti-tumor6,8′-Dihydroxy-2-2′-bis(2-phenylethyl)-4H,4′H-5,5′-bichromone-4,4′-dione
Agarotetrol
Color	Dark brown or khaki with a small number of white spots	Yellowish or reddish-brown with more white spots	Khaki or yellowish-brown with a large number of white spots
	4.15
Density of resin	Sesquiterpene, chromone, and triterpene	⁸	Very dense and compact, sink in water when soaked
^]
		Its mechanism may be related to the induction of apoptosis through nuclear condensation and breakage, including the destruction of mitochondrial membrane potential.	full-floating on water

	Dense and less compact, half-sinkage in water
Aquilaria microcarpa Baill	Indonesia
Sedation, analgesia, and anti-inflammation
	^[	^6]^[7]^[
	Sesquiterpene and chromone
⁸	^]	^[⁹

	Light and not dense,		The mechanism of analgesia and sedation may be related to the regulation of gene expression of GABAA receptor, enhancement of GABAA receptor function, and promotion of Cl⁻¹^]

Weight	Hard texture, brittle, and not hollowed	influx, and the anti-inflammatory mechanism may be related to the inhibition of granulocyte respiratory burst, inhibition of pro-inflammatory cytokines (IL-1 β, IL-6, and TNF- α), and decrease in lipid peroxidation (MDA).	The texture is a little hard, a little brittle, and slightly hollow	Loose texture, not brittle, and hollow
Aquilaria apiculata Merr
Relieving cough and relieving asthma
	Philippines
	Terpenoid/chromone
^[	⁸	^]
	It is speculated that the mechanism by which agarwood relieves asthma may be related to anti-inflammation, anti-apoptosis, improvement of pathological changes of lung and an intestinal tract, abnormal function, and balance of immunity.
Aroma	Strong odor, feel sweet and cool.	Less potent odor, feel sweet and slightly spicy	The aroma is light, feel slightly sweet and salty
Aquilaria baillonii Pierre ex Lecomte
Antidepressant
	Cambodia, Thailand, Laos, Vietnam	^[6]^[8]
	Diterpene
Sense of oiliness		The mechanism may be related to the inhibition of the corticotropin-releasing factor (CRF) gene expression and the hyperactivity of the hypothalamus–pituitary–adrenal (HPA) axis, as well as the inhibition of corticotropin receptor gene transcription and protein expression in the cerebral cortex and hippocampus.	Have a strong sense of oiliness
Aquilaria banaensis P.H.H6

		Have a strong sense of oiliness	The sense of oiliness is weak	Anti-oxidation and anti-aging
	Vietnam

	^[	^6]^[7]
^[	²²	^]^[23]
				Flavonoids and sesquiterpenes	The mechanism may be related to the regulation of reactive oxygen clusters and proinflammatory cytokines by microglia and the release of stress hormones.
			Aquilaria beccariana Tiegh	Indonesia
			Isoagarotetrol
^[	⁶	^]^[7]

Aquilaria citrinicarpa (Elmer) Hallier f.	Philippines	^[6]^[8]^[

2-(2-phenylethyl) chromone
¹⁰	^]
Aquilaria cumingiana (Decne) Ridl	Malaysia	^[8]

2-[2-(4-methoxyphenyl) ethyl] chromone
Aquilaria khasiana Hallier f.	India	^[6]^[7]^[8]
Aquilaria apiculata Merr	Philippines	^[8]^[10]
Aquilaria parvifolia (Quisumb) Ding Hon	Philippines	^[6]^[10]
Aquilaria rostrata Ridl	Malaysia	^[6]^[7]^[9]
Aquilaria rugosa Kiet Kessler	Vietnam	^[8]
Aquilaria subintegra Ding Hon	Thailand	^[8]^[9]
Aquilaria urdanetensis (Elmer) Hallier f.	Philippines	^[8]^[10]
Aquilarla yunnanensis S.C. Huang	China	^[7]^[8]

2. Economic Value and Aquilaria Species Distribution

Economic Value

High-quality agarwood can be sold at US $100,000 per kg, while those of poor quality can only be sold at US $100 per kg. As a high-quality and high-grade agarwood essential oil, it can even be sold for US $1500 per 11.7 g ^[11][12][13]. The higher the grade of agarwood, the richer the layers of aroma. 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. Its aroma is regarded as a symbol of high quality. There are various kinds of volatile components, each with a unique aroma ^[12][14][15]. Aquiliria spp. Asiatic distribution map is shown in Figure 2.

Figure 2.

Aquiliria

spp. Asiatic distribution map. Red spot showing the distribution in different countries.

3. Aquilaria Identification Methods and Phytochemicals

3.1. Identification Methods

There are many kinds of agarwood trees, and their morphological characteristics are very similar, which increases the difficulty in the identification of agarwood. Even if there are different batches of agarwood from the same producing area and different producing areas, the chemical constituents measured by the same experimental method are very different ^[16]. It is necessary to establish a set of systematic and accurate identification methods. At present, the differences in intragenic regions of Internal Transcribed Spacer (ITS), ITS1, and ITS2 are often used to distinguish different species and genera. For example, Zou et al. ^[17]

5,6,7,8,-Tetrahydroxy-5,6,7,8-tetrahydro-2-[2-(4-methoxyphenyl)ethy]-chromone
3,5-Di-tert- butylphenol	2.70
	4-Methoxyphenylacetone	0.95

Table 3.

Table 4. Chemical structure of terpenoids from agarwood.

Cardiovascular system


Chromone

	The mechanism may inhibit cardiomyocyte apoptosis after ischemia/reperfusion by regulating B lymphocyte tumor-2 gene (Bcl-2) and to down-regulating rabbit anti-human monoclonal antibody (Bax).

Name	Chemical Structure	Reference
7α,15-Dihydroxydehydroabietic acid		^[24]
Methyl 7-oxodehydroabietate
7α-hydroxypodocarpen-8(14)-en-13-on-18-oic acid
18-norpimara-8(14),15-dien-4αα-ol
18-norpimara-8(14), and 15-dien-4α-ol
7α, 12α, 13α-trihydroxyabiet-8(14)-en-18-oic acid acetonide		^[25]
6α, 13α, 14α-trihydroxyabiet-7-en-18-oic acid
13α, 14α, 15-trihydroxy-7-oxoabiet-8-en-18-oic acid
13β, 14β-epoxyabiet-7-en-18, 6α-olide
7α, 12β, 13β-trihydroxyabiet-8(14)-en-18-oic acid
7α-hydroxyabieta-15-methoxy-8,11,13-trien18-oic acid
7α-hydroxyabieta-15-methoxy-8,11,13-trien18-oic acid
12α-ethoxyabieta-7,13-dien-18-oic acid
7,13-dioxopodocarpan-18-oic acid
α-Agarofuran		^[26]
(5S,7S,10S)-(-)Selina-3,11-dien-9-one
(+)-(4S,5R)-Dihydrokaranone
α-Guaiene
Agarospirol
8-β-H-Dihydrogmelofuran
(-)-bornyl ferulate

3.4. Grading System for Agarwood Identification

The morphological grading system of agarwood is shown in Table 6. At present, the incense sold in the market comes from a wide range of sources, which needs to be processed before it can be used in medicine. It is difficult to distinguish its quality by observing its morphological characteristics, and the handicrafts of incense are even more difficult to identify. It is almost impossible to identify through the naked eye, the circulation in the market is more chaotic, and the phenomenon of adulteration and fraud is more common. The Chinese Pharmacopoeia (2020 edition) indicates that the content of the ethanol extract of resin shall not be less than 10%, and the content of agarotetrol (C₁₇H₁₈O₆₎ shall not be less than 0.1% ^[1]. Xie et al. ^[28] digitized the chemical composition extracted from HPLC-Q-TOF-MS fingerprints, and the quality was evaluated according to the proportion of components. Ismail et al.

3.5. Agarwood Induction Technique

3.5.1. Natural Induction

With the rapid development of medicine, the demand for incense resources is increasing day by day, and it is even more valuable as medicinal material. Most of the agarwood resources mainly come from the wild, and agarwood needs to be damaged and can be harvested for a long time, and the number of agarwood resources that can be harvested is decreasing day by day.

3.5.2. Artificial Induction

The molecular mechanism of agarwood induction is shown in Figure 3. Artificial incense will replace wild incense to become the main source. The artificial incense-forming technology mainly includes the physical injury method, chemical reagent method, biological planting method, and many more ^[2]. With the development of incense forming technology, most of the artificial incense is mainly composed of chemical techniques. Zhao and Fang ^[32] evaluated the quality of agarwood produced by the whole-tree agarwood-inducing technology, and preliminarily considered that the quality of incense produced by this technology conformed to the standard of the Chinese Pharmacopoeia. However, the quality of artificial incense is still different from that of natural incense, it is far from the ratio of high-quality insects in natural incense, and there are few reports on how to use insect microorganisms to cause damage to the incense to form high-quality insect incense. The method of using artificial intervention to make the fragrance quality close to that achieved using natural insects is still a blank, so it is necessary to combine incense with insect microorganisms. To produce the incense using high-quality insect leakage is an area that still requires considerable effort. At present, the studies on artificial incense technology are limited, a problem needs to be solved. Therefore, there is a need to establish a standardized incense base and explore the method of combining animals and plants to improve incense technology and provide high-quality medicinal resources.

Figure 3.

A schematic diagram showing the molecular mechanism of agarwood induction.

References

Chinese Pharmacopoeia. National Pharmacopoeia Committee. 2020. Available online: http://wp.chp.org.cn/front/chpint/en/ (accessed on 19 December 2021).
Liu, Y.Y.; Wei, J.H.; Gao, Z.H.; Zhang, Z. A review of quality assessment and grading for agarwood. Chin. Herb. Med. 2017, 9, 22–30.
Kumeta, Y.; Ito, M. Characterization of α-humulene synthases responsible for the production of sesquiterpenes induced by methyl jasmonate in Aquilaria cell culture. J. Nat. Med. 2016, 70, 452–459.
Liu, Y.Y.; Chen, D.L.; Wei, J.H.; Feng, J.; Zhang, Z.; Yang, Y.; Zheng, W. Four new 2-(2-phenylethyl) chromone derivatives from Chinese agarwood produced via the whole-tree agarwood-inducing technique. Molecules 2016, 21, 1433.
Yumi, Z.; Has-Yun, H.; Kerr, P.G.; Phirdaous, A.; Hamzah, M.S. Aquilaria spp. (agarwood) as source of health beneficial compounds: A review of traditional use, phytochemistry and pharmacology. J. Ethnopharmacol. 2016, 189.
Barden, A.; Anak, N.A.; Mulliken, T.; Song, M. Heart of the matter: Agarwood use and trade and CITES implementation for Aquilaria malaccensis. Traffic Network Rep. 2000, 46, 17–18.
Gärdenfors, U.; Hilton Taylor, C.; Mace, G.M.; Rodriguez, J. The application of IUCN red list criteria at regional levels. Conserv. Biol. 2001, 15, 1206–1212.
Germplasm Resources Information Network Taxonomy for Plant. Available online: https://trade.cites.org/# (accessed on 12 December 2021).
Hsuan, K. The Concise Flora of Singapore. Gymnosperms and Dicotyledons; University Press: Singapore, 1990.
Parris, B.S. Flora of Peninsular Malaysia; Forest Research Institute: Kuala Lumpur, Malaysia, 2010.
Wong, Y.F.; Chin, S.T.; Perlmutter, P.; Marriott, P.J. Evaluation of comprehensive two-dimensional gas chromatography with accurate mass time-of-flight mass spectrometry for the metabolic profiling of plant–fungus interaction in Aquilaria malaccensis. J. Chromatogr. 2015, 1387, 104–115.
Ismail, N.; Ali, N.A.M.; Jamil, M.; Rahiman, M.H.F.; Tajuddin, S.N.; Taib, M.N. A review study of agarwood oil and its quality analysis. J. Teknol. 2014, 68, 37–42.
Sen, S.; Talukdar, N.C.; Khan, M. A simple metabolite profiling approach reveals critical biomolecular linkages in fragrant agarwood oil production from Aquilaria malaccensis–a traditional agro-based industry in North East India. Curr. Sci. 2015, 108, 63–71.
Ishihara, M.; Tsuneya, T.; Uneyama, K. Components of the agarwood smoke on heating. J Essent. Oil Res. 1993, 5, 419–423.
Ishihara, M.; Tsuneya, T.; Uneyama, K. Components of the volatile concentrate of agarwood. J. Essent. Oil Res. 1993, 5, 283–289.
Du, H.f.; Xiong, L.y.; Lin, L.; Sshuai, O.; Qi, L.k. Simultaneous determination of five kinds of chromone in Aguilariae lignum resinatum by HPLC. CTMFJ 2015, 20, 87–90.
Zou, M.M.; Xia, Z.Q.; Lu, C.; Wang, H.; Ji, J.; Wang, W.J. Genetic diversity and differentiation of Aquilaria sinensis (Lour.) Gilg revealed by ISSR and SRAP markers. Crop Sci. 2012, 52, 2304–2313.
Niu, X.; Ji, K.; Lu, G. Preliminary studies on identification of Aquilaria sinensis (L.) Gilg by the PCR product of rDNA ITS sequencing. Gd. Agric. Sci. 2010, 37, 167–169.
Shen, Y.; Tan, X.; Zhao, X.; Pang, Q.; Zhao, S.J. Pharmacy, Ribosomal DNA ITS sequence analysis of Aquilaria sinensis from different geographical origin in China. Chin. J. Trad. Chin. Med. Pharm. 2009, 24, 539–541.
Mei, W.L.; Zeng, Y.B.; Liu, J.; Dai, F. GC-MS analysis of volatile constituents from five different kinds of Chinese eaglewood. J. Chin. Med. Mat. 2007, 30, 551–555.
Yang, J.J. Study on quality evaluation method of agarwood from China. Master’s Thesis, Hainan University, Haikou, China, 2015. Available online: https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CMFD&dbname=CMFD201702&filename=1015592278.nh&uniplatform=NZKPT&v=UxkWd_b6Efird7uOwyv1LxhWn2np-jaCL5kGrHJG0gB9s3ToeGtfVufTgjYZpRhf (accessed on 19 December 2021).
Shimada, Y.; Tominaga, T.; Konishi, T.; Kiyosawa, S. Studies on the agarwood (Jinko). I. Structures of 2-(2-phenylethyl) chromone derivatives. Chem. Pharm. Bull. 1982, 30, 3791–3795.
Shimada, Y.; Konishi, T.; Kiyosawa, S.; Nishi, M.; Miyahara, K.; Kawasaki, T. Studies on the agalwood (Jinko). IV.: Structures of 2-(2-Phenylethyl) chromone derivatives, agarotetrol and isoagarotetrol. Chem. Pharm. Bull. 1986, 34, 2766–2773.
Yang, L.; Qiao, L.R.; Xie, D.; Dai, J.G.; Guo, S.X. Diterpenoids from Chinese eaglewood. Chin. J. Chin. Mat. Med. 2011, 36, 2088–2091.
Yang, L.; Qiao, L.R.; Ji, C.X.; Xie, D.; Gong, N.B.; Lu, Y.; Zhang, J.J.; Dai, J.G.; Guo, S.X. Antidepressant abietane diterpenoids from Chinese eaglewood. J. Nat. Prod. 2013, 76, 216–222.
Sukkaew, S.; Pripdeevech, P.; Thongpoon, C.; Machan, T.; Wongchuphan, R. Volatile constituents of murraya koenigii fresh leaves using headspace solid phase microextraction–gas chromatography–mass spectrometry. Nat. Prod. Commun. 2014, 9, 1783–1786.
Chen, H.Q.; Wei, J.H.; Yang, J.S.; Zhang, Z.; Yang, Y.; Gao, Z.H.; Sui, C.; Gong, B. Chemical constituents of agarwood originating from the endemic genus Aquilaria plants. Chem. Biodivers. 2012, 9, 236–250.
Xie, B.; Luo, H.; Huang, X.; Huang, F.; Zhang, Q.; Wu, X.; Zhou, X.; Wu, H. Pharmacokinetic studies of six major 2-(2-phenylethyl) chromones in rat plasma using ultra high performance liquid chromatography with tandem mass spectrometry after oral administration of agarwood ethanol extract. J. Separ. Sci. 2021, 44, 2418–2426.
Ismail, N.; Ali, N.A.M.; Jamil, M.; Rahiman, M.H.F.; Taib, M.N.; Tajuddin, S.N. Major volatile chemical compounds of agarwood oils from Malaysia based on z-score technique. J. Chem. Nat. Compd. 2015, 51, 776–779.
Chen, X.Y.; Huang, Y.Q. Grey relational analysis of volatile components and anti-tumor activity of agarwood. Proprietary Chin. Med. 2018, 40, 224–227.
Zhou, X.; Fan, Y.; Lei, Z.; Pan, Q.; Zhong, Z.; Liu, D.; Zhang, W.; Gao, X. Correlation between agaropiric acid and extract contents in artificial Aquilariae lignum resinatum. Chin. J. Exp. Tradit. Med. Form. 2016, 22, 55–59.
Zhao, Y.; Fang, Z.J. Quality assessment of Chinese eaglewood by the technique of Aquilaria sinensis quintana making incense and the determination of benzyl acetone. J. Gd. Pham. Univ. 2013, 29, 253–257.