Abstract

   Agarwood, a highly valuable resin/wood combination with diverse
   pharmacological activities but scarce supply, has a long history of
   being used as a medicine in several medical systems. Grafted Kynam
   agarwood (GKA) has been cultivated successfully recently and has the
   qualities meeting the definition of premium Kynam agarwood. However,
   there are few comprehensive comparisons between GKA and normal agarwood
   in terms of traits, global composition, and activity, and some key
   issues for GKA to be adopted into the traditional Chinese medical (TCM)
   system have not been elaborated. The two types of agarwood samples were
   evaluated in terms of trait characteristics, physicochemical
   indicators, key component groups, and global compositional profile.
   Furthermore, a molecular docking was performed to investigate the
   active ingredients. In vitro activity assays were performed to evaluate
   the activation of adenosine 5’-monophosphate (AMP)-activated protein
   kinase (AMPK) by GKA and normal agarwood. The results revealed that,
   overall, the traits, microscopic characteristics, chemical composition
   types, and bioactivity between GKA and normal agarwood were similar.
   The main differences were the content of resin (ethanolic extract
   content), the content of key component groups, and the composition of
   the different parent structural groups of 2-(2-phenethyl) chromones
   (PECs). The contents of total PEC and ethanol extract content of GKA
   were significantly higher than those of normal agarwood. The MS-based
   high-throughput analysis revealed that GKA has higher concentrations of
   sesquiterpenes and flindersia-type 2-(2-phenylethyl) chromones (FTPECs)
   (m/z 250-312) than normal agarwood. Molecular docking revealed that
   parent structural groups of FTPECs activated multiple signaling
   pathways, including the AMPK pathway, suggesting that FTPECs are major
   active components in GKA. The aim of this paper is to describe the
   intrinsic reasons for GKA as a high-quality agarwood and a potential
   source for novel drug development. We combined high-throughput mass
   spectrometry and multivariate statistical analysis to infer the
   different components of the two types of agarwood. Then we combined
   virtual screening and in vitro activity to construct a
   component/pharmacodynamic relationship to explore the causes of the
   activity differences between agarwood with different levels of quality
   and to identify potentially valuable lead compounds. This strategy can
   also be used for the comprehensive study of other TCMs with different
   qualities.

   Keywords: Grafted Kynam agarwood, Aquilaria sinensis, MS-based
   high-throughput analysis, Molecular docking, Flindersia-type
   2-(2-phenylethyl) chromones, AMPK

1. Introduction

   Agarwood is a valuable resin/wood combination. Resin is produced by a
   variety of plants in two genera, Aquilaria spp. and Gyrinops spp., in
   response to stimuli from external damage to help the plant heal itself
   [[36]1,[37]2]. Those stimuli from natural or artificial sources (knife,
   fire, fungus, etc.) put the Aquilaria tree in a state of stress and
   cause it to produce a special defensive mechanism to secrete resin
   [[38]3]. The majority of available agarwood in the market is from four
   species, including Aquilaria malaccensis Lam, Aquilaria crassna Pierre
   ex Lecomte, Aquilaria sinensis (Lour.) Gilg, and Aquilaria filaria
   (Oken) Merr [[39]4]. Natural agarwood has been used as incense and
   medicine for spiritual healing and physical healing. The therapeutic
   effects of agarwood are closely related to its quality with high
   quality agarwood containing higher concentrations of resins
   [[40]3,[41]5]. Agarwood has been included in every edition of the
   Pharmacopoeia of the People’s Republic of China (Volume 1) (Ch.P)
   [[42]6]. Although both domestic agarwood (Aquilaria sinensis (Lour.)
   Gilg) and imported agarwood (Aquilaria agallocha Roxb.) were included
   in the previous edition of Ch.P (1963 edition), only Aquilaria sinensis
   (Lour.) Gilg has been included in subsequent editions of Ch.P as the
   designated medicinal agarwood resource. It is hard for plants forming
   agarwood under natural condition, which results in the low supply of
   agarwood. With the demand outstripping the supply, natural agarwood
   resources are in danger of being depleted. Both Aquilaria spp. and
   Gyrinops spp. have been listed in Appendix II of the Convention on
   International Trade in Endangered Species of Wild Fauna and Flora
   (CITES).

   Kynam agarwood, a rare high-quality agarwood, also known as Kanankoh,
   Kyara, Kynam, Chi-Nan, Qi-Nan, and so on, has consistent
   characteristics of high resin content, soft texture, and unique aroma
   [[43]7,[44]8]. Because of the depletion of natural Kynam agarwood,
   Kynam agarwood is expensive and out of reach of the general public.
   Fortunately, recently developed artificial cultivation techniques can
   produce Kynam agarwood with native Aquilaria sinensis, referred as
   grafted Kynam agarwood (GKA). Some studies including germplasm
   genetics, incense smoke, and chemical composition studies have shown
   that GKA has the potential to become a high-quality agarwood. Yong Kang
   et al. performed DNA barcoding analysis on 58 batches of GKA in the
   market and found that it was closely related to A. sinensis in the
   phylogenetic tree and therefore tentatively identified its source
   species as A. sinensis [[45]9]. Yuan Chen et al. concluded that
   phenylethyl chromones with no or few substituents and sesquiterpenes in
   GKA were the main factors contributing to its aroma by comparing
   incense smoke analysis from GKA and normal agarwood [[46]10]. Meng Yu
   et al. suggested that the main differences between the GKA and normal
   agarwood were 2-(2-phenylethyl) chromone and 2-[2-(4’-methoxybenzene)
   ethyl] chromone, both of which were higher in GKA than in common
   agarwood [[47]11]. Feng Jian et al. conducted a full inspection of ten
   batches of GKA collected according to the 2020 edition of Ch.P and
   found that agarotetrol content did not meet the requirement of 0.1% in
   pharmacopoeia, but the ethanol extract content of GKA was much higher
   than the requirement of 10% in the pharmacopoeia, and the
   characteristics were consistent with the profiles of high-quality
   agarwood [[48]12]. Wen-Yi Kao et al. analyzed the chemical composition
   of the incense smoke produced from four agarwood including Kynam by
   headspace gas chromatography/tandem mass spectrometry and found that
   2-(2-phenylethyl) chromone derivatives were only found in the incense
   smoke produced from Kynam agarwood [[49]13]. Chen xiqin et al.
   investigated the in vitro anti-inflammatory activity of the essential
   oil extracts of Kynam and normal agarwood based on the LPS-induced
   RAW264.7 cell model and found that the essential oil extracts of Kynam
   agarwood had better anti-inflammatory activity [[50]14]. Despite the
   great efforts of these studies on the multi-component analysis of GKA
   and normal agarwood, more evaluations of GKA are required to further
   understand its pharmacological and therapeutic effects. Moreover, there
   are few studies studying the effects and mechanism of GKA at the
   molecular level and the downstream signaling pathways that are
   influenced by GKA, which gives doctors and regulators limited
   references for regulations. Study of GKA characteristic components,