A Comprehensive Review on Chemotaxonomic and Phytochemical Aspects of Homoisoflavonoids, as Rare Flavonoid Derivatives

Homoisoflavonoids (3-benzylidene-4-chromanones) are considered as an infrequent flavonoid class, possessing multi-beneficial bioactivities. The present study gives an overview on phytochemical aspects of homoisoflavonoids, including utilized plant species, parts, extracts, and separation techniques. Overall, these compounds have mainly been isolated and identified from bulbs and rhizomes of the plants belonging to Asparagaceae and Fabaceae families, particularly the genera of Ophiopogon, Dracaena, Scilla, Polygonatum, and Caesalpinia.


Introduction
Homoisoflavonoids (HIFs), a small, rare, and unique class of the flavonoids, are naturally occurring oxygen heterocyclic compounds possessing two aromatic rings, and an additional carbon between the B and C rings on the isoflavonoid skeleton ( Figure 1) [1,2]. As demonstrated in Figure 1, the most updated classification of HIFs is categorized into five groups: sappanin, scillascillin, brazilin, caesalpin, and protosappanin types [3,4].
The major aim of this paper is to gather the most updated information on chemosystematic and phytochemical features of HIFs in order to facilitate future scientific works. Contrary to the papers published [1,17,18] which deal with the phytochemistry and biological potencies of these compounds, the emphasis of this review is on separation techniques implemented for isolation and purification of different homoisoflavonoid (HIF) derivatives from diverse plant species in detail.
The keyword of "homoisoflavonoid" was applied to search the associated published data through databases covering PubMed, Web of Science, and SciFinder since 1980 (last search: 31 January 2021).
The keyword of "homoisoflavonoid" was applied to search the associated published data through databases covering PubMed, Web of Science, and SciFinder since 1980 (last search: 31 January 2021).

Separation Techniques Used for the Isolation and Purification of Homoisoflavonoids
Given that HIFs are mostly polar compounds, polar organic solvents have been used for the isolation and purification. Besides crystallization methods, the following chromatographic techniques were predominantly applied: semi-preparative and preparative column chromatography (CC) using reverse phase material as stationary phase and methanol (MeOH), water (H2O), and acetonitrile (MeCN) as mobile phase solvents; normal phase CC on silica gel using MeOH, chloroform (CHCl3), dichloromethane (CH2Cl2), acetone (Me2CO), etc. as eluent; Sephadex ® LH-20 (SLH) with MeOH, ethanol (EtOH), and CH2Cl2 as a solvent system; flash chromatography (FC) and preparative thin layer chromatography on normal (PTLC) and reverse phases (RP-PTLC). These methods are discussed in detail in the following sections.

Chemotaxonomy of Homoisoflavonoids
Based on the literature, HIFs have been isolated mainly from Asparagaceae, Fabaceae (syn. Leguminosae), and Portulacaceae families.

Homoisoflavonoids Isolated from Asparagaceae Family
The Asparagaceae family, belonging to the Asparagales order, comprises over 153 genera and 2500 species of flowering plants. This large family is distributed nearly all around the world [19].
Overall, HIFs have been isolated and identified from 23 genera and 49 species within to this family. Most of the identified compounds were isolated from Ophiopogon japonicus, Polygonatum spp. (mainly P. odoratum), Scilla spp., Dracaena spp., and Bellevalia spp.

Separation Techniques Used for the Isolation and Purification of Homoisoflavonoids
Given that HIFs are mostly polar compounds, polar organic solvents have been used for the isolation and purification. Besides crystallization methods, the following chromatographic techniques were predominantly applied: semi-preparative and preparative column chromatography (CC) using reverse phase material as stationary phase and methanol (MeOH), water (H 2 O), and acetonitrile (MeCN) as mobile phase solvents; normal phase CC on silica gel using MeOH, chloroform (CHCl 3 ), dichloromethane (CH 2 Cl 2 ), acetone (Me 2 CO), etc. as eluent; Sephadex ® LH-20 (SLH) with MeOH, ethanol (EtOH), and CH 2 Cl 2 as a solvent system; flash chromatography (FC) and preparative thin layer chromatography on normal (PTLC) and reverse phases (RP-PTLC). These methods are discussed in detail in the following sections.

Chemotaxonomy of Homoisoflavonoids
Based on the literature, HIFs have been isolated mainly from Asparagaceae, Fabaceae (syn. Leguminosae), and Portulacaceae families.

Homoisoflavonoids Isolated from Asparagaceae Family
The Asparagaceae family, belonging to the Asparagales order, comprises over 153 genera and 2500 species of flowering plants. This large family is distributed nearly all around the world [19].
Overall, HIFs have been isolated and identified from 23 genera and 49 species within to this family. Most of the identified compounds were isolated from Ophiopogon japonicus, Polygonatum spp. (mainly P. odoratum), Scilla spp., Dracaena spp., and Bellevalia spp. Among them, bulbs of Ophiopogon japonicus, determined to contain almost 60 HIFs, are deemed as the richest natural source of this compound class.
Details about single species, investigated plant parts and soluble fractions, applied chromatographic techniques, and names of the isolated compounds are listed in Table S1. Figure 2 exhibits the abundance variation of HIFs isolated from different species of Asparagaceae.
Among them, bulbs of Ophiopogon japonicus, determined to contain almost 60 HIFs, are deemed as the richest natural source of this compound class.
Details about single species, investigated plant parts and soluble fractions, applied chromatographic techniques, and names of the isolated compounds are listed in Table S1. Figure 2 exhibits the abundance variation of HIFs isolated from different species of Asparagaceae.

Ophiopogon japonicus
The species of Ophiopogon japonicus is known as "Maidong" in China and has diverse folk medicinal applications such as fever treatment in consumptive ailments or general debility, dehydration of febrile disease, and dry mouth [20]. This species is considered as the major source of HIF compounds. Its tuberous roots are rich in these constituents, explicitly, ethyl acetate (EtOAc) and hydro-alcoholic soluble fractions. From the ether extract using CC on silica gel (eluent: benzene (Bz)), followed by recrystallization, methylophiopogonone A, methylophiopogonanone A, and a novel derivative, namely, 5hydroxy-7,8-dimethoxy-6-methyl-3-(3′,4′-dihydroxybenzyl)chroman-4-one have been
Reverse-phase CC (RP-CC) on silica gel has been carried out to isolate four new HIF derivatives from the CHCl 3 -soluble partition of the tuber part; a homoisoflavanone homoisopogon A and one homoisoflavan homoisopogon C, and two homoisoflavans named homoisopogon B and D were consequently purified by eluting gradient mobile phase H 2 O-Me 2 CO (2:3 and 1:1), respectively [23]. The EtOAc-soluble fraction of the roots also contained ophiopogonone D and ophiopogonanone G, isolated as the new secondary metabolites. In this study, the extract was initially subjected to CC on silica gel by increasing the ratio of Me 2 CO in petroleum ether (PET) as mobile phase, then chromatographed with CC on polyamide, where H 2 O-MeOH (10:0 to 0:10) was applied as mobile phase, and, finally, the aforementioned compounds were purified by SLH (eluent: MeOH) and repeated HPLC via an isocratic mobile phase MeOH in H 2 O (7:3) [24].
In a similar investigation, novel constituents dracaeconolide A and B, together with four known derivatives, were isolated from the EtOAc fraction of D. cochinchinensis resin, using C18 RP silica gel as stationary phase and H 2 O-MeOH (6:4 to 0:1) and H 2 O-MeOH (1:1 to 0:1) as mobile phases [59]. The MeOH-soluble part of its reddish resin has further been subjected to SLH (eluent: MeOH) and HPLC (mobile phase:  [62]. Ten previously identified HIFs were isolated from the Me 2 CO soluble part of the resin of D. draco [63]. Furthermore, from the D. loureiri stems, four known derivatives were isolated using silica gel CC and PTLC as separation tools [64].
Whole Urginea depressa parts have been extracted using different solvents and, finally, CH 2 Cl 2 and NHEX fractions were exploited for the isolation of their predominant components. Six undescribed natural products have been isolated and identified as HIF

Homoisoflavonoids Isolated from Fabaceae Family
A total of 39 novel and 57 previously identified HIFs have been isolated from 13 species belonging to the Fabaceae (syn. Leguminosae) family. Most of the isolated HIFs belong to Heamatoxylon campechianum and Caesalpinia spp. (Table S2, Figure 3).

Caesalpinia millettii
8-Methoxyisobonducellin has been isolated and characterized for the first time from the Me 2 CO fraction of C. millettii stems; SLH CC (eluent: MeOH) was used as the final separation step. Moreover, four known HIFs consisting of 8-methoxybonducellin, eucomin, bonducellin, and intricatinol were isolated by means of CC on silica gel and SLH [95].
In the course of a newer study, the methanolic extract of the heartwood of H. campechianum has been subjected to silica gel (mobile phases:

Portulacaceae Family
Portulaca oleracea, belonging to Portulacaceae family, is extensively distributed all over the world. In folk medicine, it has been acknowledged to be a natural antispasmodic, diuretic, antiseptic, antiscorbutic, analgesic, etc. agent [119][120][121][122]. Besides all the identified phytoconstituents from this species, several HIFs were isolated and described (Table S3).

Structural Identification of Homoisoflavonoids
The structural characterization of HIFs has relied on various spectroscopic techniques, mainly nuclear magnetic resonance (NMR), mass spectrometry (MS), spectrophotometric ultraviolet (UV), and infrared (IR). Both proton ( 1 H) and carbon-13 ( 13 C) NMR spectra in one-dimensional (1D) or 2D experiments such as 1 H-1 H correlation spectroscopy (COSY), nuclear Overhauser effect spectroscopy (NOESY), heteronuclear multiple bond correlation (HMBC), and heteronuclear single quantum coherence (HSQC) have been applied to the structure elucidation of these compounds. Since homoisoflavonones possess a five-proton spin system on carbons 2, 3, and 9, they are able to be characterized according to their protons' chemical shifts in those positions. The homoisoflavans are usually identified by the analysis of the seven-proton spin system spread over carbons 2, 3, 4, and 9; ring A of HIFs has two or three substituents, while ring B can be recognized for its oxygen substituent at position 4 ( Figure 1). The determination of physical properties, such as melting point, plays a role. Moreover, in several studies, circular dichroism (CD) and optical rotatory power ([α]D) were utilized to ascertain the absolute stereochemistry of the HIF structures.

Conclusions and Perspectives
HIFs are a small class of flavonoids with privileged biological activities. According to the literature, these bioactive phenolics are mainly present in Asparagaceae, Fabaceae, Portulacaceae, Cucurbitaceae, and Polygonaceae families. The tuberous root of Ophiopogon japonicus (Asparagaceae) is the richest source of HIFs, specifically its EtOAc-soluble fraction. The EtOAc partition of the Polygonatum odoratum rhizome is known for its diverse HIF derivatives. From the plants belonging to the Fabaceae family, the heartwood parts of Heamatoxylon campechianum and Caesalpinia spp., particularly C. sappan, are considered as a striking natural source of these compounds. To date, over 300 HIFs have been reported in the literature [1]. The preponderance of information documented herein was to overview the chemotaxonomic standpoint of HIFs and separation techniques used for their isolation. They do not only display a broad structural diversity but are also known for a variety of biological properties and some of these compounds stand out for their unique pharmacological properties. More phytochemical and pharmacological studies will be required to fully exploit the potential of these interesting compound class substances.