2.4. Flavone and Isoflavone Derivatives
The 63 flavone (30‒92) and five isoflavone (93‒97) derivatives isolated by using SLH as the last separation stage illustrated that this technique plays an effective role in extraction of these compounds.
The simple flavone (
30) and its derivative 4′-hydroxy-5-methoxyflavone (
33) have been isolated from
Imperata cylindrica, whilst ethyl acetate extracts of rhizome were finally chromatographed via SLH with dichloromethane‒methanol (1:1) as eluent system [
28]. Ethyl acetate and ethanolic extracts gained from stem bark and aerial parts of
Albizzia julibrissin and
Athrixia phylicoides were extracted to isolate two aglycone flavones of 3′,4′,7-trihydroxyflavone (
31) [
29] and 5-hydroxy-6,7,8,3′,4′,5′-hexamethoxyflavon-3-ol (
32) [
30], respectively, through a separation procedure with SLH (eluent: methanol).
A well-known flavone luteolin (
34) (3,4,5,7-tetrahydroxy flavone) possessing several health benefits, such as anti-cancer [
31], cardio-protective [
32], anti-inflammation, and anti-allergy [
33] effects, has been isolated and purified from 11 plant species applying SLH as the final step: from hydroethanolic (70%) extract of
Brachychiton acerifolius leaf (eluent: methanol‒water 1:1) [
34], ethyl acetate extracts of
Thymus praecox aerial part [
35],
Ginko biloba leaf (eluent: methanol) [
36],
Rosmarinus officinalis sprig (eluent: methanol‒water 1:1) [
37],
Chamaemelum nobile flower (eluent: methanol‒dichloromethane 1:1) [
38],
Populus davidiana wood (eluent: methanol‒water 3:1, 1:1, 1:3) [
15], and
Solenostemon monostachys aerial part (eluent:
n-hexane‒ethyl acetate 3:7, 2:8, 1:9; ethyl acetate; ethyl acetate‒methanol 1:9, 2:8, 4:6, 5:5) [
39], aqueous fraction of
Phlomis bruguieri aerial part (eluent:
n-hexane‒MeOH‒acetone 30:60:10) [
40], methanolic extracts of
Taraxacum mongolicum aerial part (eluent: methanol) [
22] and
Dendrobium ellipsophyllum whole plant part (eluent: acetone) [
23], and
n-butanol extract of xylem part of
Populus tomentosa (eluent: methanol‒water 1:1, 1:3) [
41].
Moreover, 7-methoxy luteolin (
35) has been isolated from ethyl acetate extract of
Onopordum alexandrinum seeds via SLH as the final step with methanol‒water (9:1) as eluting solvent [
42]. Overall, five glycosylated luteolin (
36‒
40) have been purified applying gel filtration chromatography. Orientin (
36) which is luteolin 8-
C-glucoside was finally isolated by SLH (eluent: methanol) from petroleum ether extract of
Indocalamus latifolius leaf [
43].
Cynaroside (
37) as luteolin 7-
O-β-
d-glucoside have been previously isolated from six plant species: ethyl acetate extracts of
Tridax procumbens whole part [
44] and
Salvia macrosiphon aerial part (eluent: methanol) [
45], hydro-methanolic (80%) portion of
Tilia rubra leaf (eluent: methanol‒water 8:2), hydroethanolic extracts of leaf of
Olea europaea (eluent: ethanol 0–50% in water) [
46] and
Brachychiton acerifolius (eluent: methanol‒water 1:1) [
34], and chloroform extract obtained from
Citrus unshiu peel (eluent: methanol‒water 1:1) [
47].
From the methanolic extract of
Taraxacum mongolicum aerial part eluting with methanol through SLH, luteolin-7-
O-β-
d-galactopyranoside (
38) and luteolin-7-
O-β-
d-glucopyranoside (
39) [
22], and luteolin-4′-
O-β-glucoside (
40) from hydroethanolic (50%) extract of
Olea europaea leaf (eluent: ethanol 0‒50% in water) [
46] have been furtherly isolated.
Apigenin (
41), characterized as 4′,5,7,-trihydroxyflavone is considered as a natural flavone, and rich in several fruits, vegetables and medicinal plants possessing numerous pharmacological potencies, such as anti-inflammatory, antioxidant, antibacterial, antiviral, antidiabetic, antidepressant, and anticancer activities, and the treatment of amnesia and Alzheimer’s disease, and insomnia [
48,
49,
50,
51,
52]. SLH has been capable to isolate this natural product from hydroethanolic extracts of
Brachychiton acerifolius leaf (eluent: methanol‒water 1:1) [
34] and
Saccharum officinarum sugarcane top (eluent: chloroform‒methanol 1:1) [
53], ethyl acetate fractions of
Chamaemelum nobile flowers (eluent: methanol‒dichloromethane 1:1) [
38], and
Solenostemon monostachys aerial part (eluent:
n-hexane‒ethyl acetate 3:7, 2:8, 1:9; ethyl acetate; ethyl acetate‒methanol 1:9, 2:8, 4:6, 5:5) [
39],
n-butanol extract of xylem of
Populus tomentosa (eluent: methanol‒water 1:1, 1:3) [
41], and aqueous extract of
Phlomis bruguieri aerial part (eluent:
n-hexane‒methanol‒acetone 30:60:10) [
40].
From leaf hydroethanolic (70%) extract of
Brachychiton acerifolius, apigenin-7-
O-α-rhamnosyl (1→2)-β-D-glucuronide (
42), apigenin-7-
O-β-
d-glucoside (
43), and apigenin-7-
O-β-
d-glucuronide (
44) have been isolated eluting with methanol‒water (1:1) [
34]. Nonetheless, apigenin-7-
O-β-
d-glucoside (
43) were isolated from ethyl acetate extracts of aerial parts of
Thymus praecox [
35] and
Salvia macrosiphon (eluent: methanol) [
45] as two Lamiaceae species; moreover, apigenin-7-
O-β-
d-glucuronide (
44) was purified from
n-butanol fraction of
Erigeron multiradiatus whole part (eluent: chloroform‒methanol 1:1) [
54].
SLH was applied as the last chromatographic step in isolation of vitexin (
45) (apigenin 8-
C-glucoside) from hydroethanolic (60%) and petroleum ether extracts obtained from
Desmodium adscendens [
55] and
Indocalamus latifolius [
43] leaves, where methanol (20 to 100%) in water and pure methanol were used as eluting solvents, respectively.
Vitexin 2”-
O-xyloside (
46) and its iso-derivative namely isovitexin 2”-
O-xyloside (
48) have been formerly isolated from
Desmodium adscendens leaf hydroethanolic (60%) extract utilizing methanol (20 to 100%) in water as eluent [
55]; however, isovitexin (
47) is apigenin-6-
C-glucoside that has been isolated from ethanolic extract of
Croton zambesicus leaf with ethyl acetate in methanol (10 to 100%) as eluting solvent in SLH [
56].
Gohari et al. [
45] isolated apigenin-7,4′-dimethyl ether (
49) by finally exploiting SLH (eluent: methanol) from ethyl acetate extract fractionated from
Salvia macrosiphon aerial part. From ethyl acetate extract of
Aquilaria sinensis seeds, 7,4′-dimethylapigenin-5-
O-xylosylglucoside (
50) and 7,4′-dimethyl-5-
O-glucosideflavonoide (
55) eluting with methanol‒water (7:3), along with hydroxylgenkwanin (
51), lethedoside A (
52), 5,7-dihydroxyl-4′-methoxyflavone (
53), and 7,3′-dimethyl-4′-hydroxyl-5-
O-glucosideflavonoide (
54) using methanol as eluent have been isolated and purified via SLH [
57].
In another investigation, amentoflavone (
56) was isolated from
Ginko biloba leaf ethyl acetate extract by application of methanol as eluent [
36]. Hispidulin (
57) has been isolated from ethyl acetate extracts of sprig and flower of
Rosmarinus officinalis [
37] and
Chamaemelum nobile [
38] utilizing methanol‒water (2:1) and methanol‒dichloromethane (1:1), respectively. Root bark of
Morus alba has been previously partitioned and its ethyl acetate extract was subjected to separation of their phytoconstituents, finally through methanol‒water (8:2) as eluent in SLH, 2 known flavones kuwanon T (
58) and sanggenon J (
59), in addition, two novel secondary metabolites sanggenon V (
60) and sanggenon W (
61) have been isolated accordingly [
18].
Several other flavone derivatives have been also isolated and purified from different soluble-extracts of the species by SLH: hypoletin-7-
O-β-
d-xylopyranoside (
62) from leaf ethyl acetate extract of
Thuja orientalis (eluent: methanol) [
58], galangin (
63) from herb chloroform fraction of
Dalbergia cochinchinensis (eluent: methanol‒dichloromethane 1:1) [
12], 3′-geranyl-3-prenyl-2′,4′,5,7-tetrahydroxyflavone (
64) from ethyl acetate extract of
Morus alba root bark (eluent: methanol‒water 1:1) [
19], pectolinarigenin (
65) from chloroform fraction of
Cirsium Japonicum aerial part [
59], scutellarein-7-
O-β-glucuronide (
66) from
Erigeron multiradiatus n-butanol aerial part extract [
54], cirsimaritin (
67), cirsilinelol (
68), and eupatilin (
69) from chloroform extract of aerial part of
Centaurea bruguierana [
60], eluting with chloroform‒methanol (1:1), and eupafolin (
70) from ethyl acetate extract of
Chamaemelum nobile aerial part (eluent: methanol‒dichloromethane 1:1) [
38].
Tricin (
71) is 5,7,4′-trihydroxy-3′,5′-dimethoxyflavone, comprising many valuable bio- and pharmacological properties [
61], and it has been isolated from leaf ethyl acetate extract of
Sasa senanensis (eluent: methanol‒water 6:4) [
62], bract hydroethanolic (95%) fraction of
Zea mays [
63], and aqueous extract of
Phlomis bruguieri aerial part (eluent:
n-hexane‒methanol‒acetone 3:6:1) [
40].
The application of SLH on hydroethanolic (95%) extract of
Zea mayes bract has led to isolation of three tricin glucosides including tricin-5-
O-β-
d-glucopyranoside (
72), tricin-7-
O-β-
d-glucopyranoside (
73), and novel flavone namely tricin-7-
O-[β-
d-apifuranosyl (1→2)]-β-
d-glucopyranoside (
74) [
63]. Tricin-7-
O-β-
d-glucopyranoside (
73) has been furtherly isolated from two other Poaceae species
Avena sativa [
64] and
Indocalamus latifolius [
43], while a hydroethanolic (95%) fraction of bran and methanolic extract of leaf have been eluted by methanol in SLH column, respectively.
A new secondary metabolite 4′-methoxy-luteolin-7-phosphate (
75) has been formerly isolated by hiring SLH (eluent:
n-hexane‒methanol‒acetone 3:6:1) from aerial part aqueous extract of
Phlomis bruguieri [
40]. From an Asteraceae species
Santolina chamaecyparissus nepetin (
76) (eluent: methanol) was purified, where the dichloromethane extract of its aerial part was subjected to chromatographic procedure [
65].
Isoetin (
77) and its glycosylated analogous including isoetin-7-
O-β-
d-glucopyranosyl-2′-
O-α-
l-arabinopyranoside (
79), isoetin-7-
O-β-
d-glucopyranosyl-2′-
O-α-
d-arabinopyranoside (
80), and isoetin-7-
O-β-
d-glucopyranosyl-2′-
O-α-
d-xyloypyranoside (
81), along with genkwanin (
82) and genkwanin-4′-
O-β-
d-lutinoside (
83), have been isolated and purified by applying SLH as the last separation stage (eluent: methanol) from methanol extract of
Taraxacum mongolicum aerial part [
22]. Notably, a novel flavone isoetin 2′-methyl ether (
78) (5,7,4′,5′-tetrahydroxy-2′-methoxyflavone) has been isolated from
Bauhinia galpinii, where the ethyl acetate extract of the leaf were applied by using acetone‒methanol (1:1) as eluting solvent via SLH [
66].
By subjecting hydroethanolic (50%) extract of sugarcane top part of
Saccharum officinarum to various chromatographic methods, albanin A (
84), australone A (
85), and 5′-geranyl-5,7,2′,4′-tetrahydroxy-flavone (
86) have been finally isolated by exploiting chloroform‒methanol (1:1) and pure methanol as eluting solvents in SLH column [
53]. In another study, methanolic extract obtained from whole part of
Dendrobium ellipsophyllum were subjected to SLH (eluent: acetone) and chrysoeriol (
87) was consequently isolated [
23]. Xuan et al. [
28] isolated 4′-methoxyflavone-6-
O-β-
d-glucopyranoside (
88) for the first time in the nature from rhizome ethyl acetate extract of
Imperata cylindrica by SLH (eluent: methanol), whereas 5-hydroxyflavone (
89) was furtherly isolated from its petroleum ether extract by using dichloromethane‒methanol (1:1) as eluting mixture.
Three other flavones have also been isolated by SLH: texasin 7-
O-β-
d-glucopyranoside (
90) from ethyl acetate extract of
Leptadenia pyrotechnica aerial part [
67], tilianin (
91) from hydroethanolic (95%) extract of
Avena sativa bran (eluent: methanol) [
64], and 5-hydroxy-6,7,3′,4′-tetramethoxyflavone (
92) from flower chloroform extract of
Citrus aurantium (eluent: chloroform‒methanol 1:1) [
68].
Moreover, 5 isoflavone derivatives have been isolated by SLH as the last separation procedure. Formononetin-7-
O-β-
d-glucosy1 [
1,
2,
3,
4,
5,
6] glucoside (
94) and tectoridin (
95) have been purified from ethyl acetate extract of
Maackia amurensis bark (eluent: methanol‒water 6:4) [
69], whilst formononetin (
93) was extracted from
Aquilaria sinensis stem ethyl acetate extract (eluent: methanol) [
57]. Sphaerobioside (
96) and a well-known isoflavone genistein (
97) have been previously isolated by SLH eluting with methanol‒water (1:1) and methanol, respectively, from aqueous root fractions of
Cudrania tricuspidata [
25].
2.5. Flavonol Derivatives
SLH played a key role in isolation or purification of flavonoids specifically flavonol derivatives. The performed studies reported that SLH has been applied for isolation or purification of 79 different flavonol derivatives (98–176), while quercetin (98) with its analogous (99‒129), and kaempferol (130) and its analogous (131–152) were the most identified compound.
Quercetin (
98) (3,3′,4′,5,7-pentahydroxyflavone, C
15H
10O
7) is considered as one of the most beneficial flavonols and renowned for its antioxidant, anticancer, anti-inflammatory, and antiviral properties and endothelium-dependent vasodilation, and blood lipid-lowering effects [
70,
71,
72,
73]. SLH gel filtration chromatography has been able to isolate and purify quercetin (
98). Nineteen studies reported the successful isolation of this compound from 19 diverse species by using SLH. According to the literature, it seems the ethyl acetate fractions of various plant species are the richest extracts in case of quercetin (
98) content.
The calix part of
Fragaria ananassa was solvent-solvent partitioned and finally by utilizing SLH (eluent: methanol‒water 6:4), quercetin (
98) was isolated from the ethyl acetate extract [
74]. The ethyl acetate extract of
Gynura divaricate leaf has been furtherly subjected to isolate their major secondary metabolites, and the abovementioned compound was isolated by chloroform‒methanol (1:1) as an eluent system [
75]. By eluting methanol through SLH column, quercetin (
98) has been isolated from
Sarcopyramis bodinieri ethyl acetate extract [
76]. Quercetin (
98) has also been isolated from ethyl acetate extracts of
Chionanthus retusus flower (eluent: methanol‒water 8:2) [
24],
Tamarix hohenackeri aerial parts (eluent: methanol) [
77], whole part of
Pteris vittata (eluent: chloroform‒methanol 1:1) [
78],
Populus davidiana wood eluting with methanol‒water (3:1, 1:1, 1:3) [
15], and from aerial part of
Halimodendron halodendron (eluent: chloroform‒methanol 1:1) [
79].
Several researchers isolated quercetin (
98) from alcoholic extracts of different species via SLH as the last chromatographic step. Abou Zeid et al. [
34] isolated this flavonol from hydroethanolic (70%) extract of
Brachychiton acerifolius leaf eluting by methanol‒water (1:1) as eluent. In other phytochemical studies on
Byrsocarpus coccineus (Connaraceae family) [
80],
Juniperus chinensis (Cupressaceae family) [
81], and
Paulownia tomentosa (Scrophulariaceae family) [
14], this compound has been isolated from
n-butanol fraction of the leaf, herb, and bark, while methanol, chloroform‒methanol (4:1), and methanol‒water (1:1) were applied as eluents, respectively. The methanolic extracts of
Cheilanthes tenuifolia whole part [
82] and
Taraxacum mongolicum aerial part [
22] have been exploited to isolate this phytochemical eluting with methanol (0 to 60%) in water and pure methanol, respectively.
Hydroalcoholic fractions of some species have been previously applied for isolation and purification of quercetin (
98): hydro-methanolic (70%) extracts of leaf and aerial part of
Albizia amara [
83] and
Allium porrum [
84] eluting via methanol and methanol‒water (6:4), respectively, and hydro-ethanolic (50%) extract of sugarcane top part of
Saccharum officinarum (eluent: chloroform‒methanol 1:1) [
53]. Furthermore, this aglycone flavonol has been isolated from stem aqueous extract of
Bauhinia strychnifolia using methanol as eluent in SLH gel filtration method [
85]. Among all isolated quercetin derivatives (
99‒
129) by applying SLH, two aglycones, including 3-
O-methylquercetin (
99) and 3,3′-di-
O-methylquercetin (
100) have been isolated from the ethyl acetate extract of
Halimodendron halodendron (Fabaceae) aerial part with mixture eluting solvents of chloroform‒methanol (1:1) [
79].
Rutin (
101) (syn. quercetin-3-
O-
α-rhamnosyl (1→6)-
β-
d-glucoside or 3′,4′,5,7-tetrahydroxy-flavone-3-rutinoside), as a well-renown dietary flavonoid, has been reported to possess several remarkable pharmacological benefits, such as in the treatment of Parkinson’s, and Alzheimer’s diseases, and myocardial infraction, along with anti-depressant, antihypertensive, anti-allergic, antioxidant, and anticancer properties [
86,
87,
88]. However, this compound has been isolated by different methods, specifically solid-phase extraction and counter-current chromatography, and the size exclusion technique has also been applied to isolate this compound [
86]. By utilization of SLH as the final purification phase, rutin (
101) has been isolated from hydroethanolic (70%) and aqueous extracts of leaf and fruit of
Brachychiton acerifolius [
34] and
Cinnamomum zeylanicum [
89], respectively, by using methanol‒water (1:1), and from whole part methanolic fraction of
Cheilanthes tenuifolia eluting with methanol (0 to 60%) in water [
82].
Application of SLH has led to isolation of quercetin-3-
O-β-6′’-(
p-coumaroyl) glucopyranoside-3′-methyl ether (
102) (syn. helichrysoside-3′-methyl ether) from ethanolic leaf extract of
Croton zambesicus with chloroform (10 to 60%) in methanol as eluent [
56]. Two glycosylated quercetin analogous quercetin 3-β-
d-glucoside (
103) and quercetin 3-
O-α-arabinoside (
104) have been isolated using
n-butanol and ethyl acetate extracts of
Byrsocarpus coccineus leaf, respectively, in which methanol was as eluting solvent [
80].
From leaf ethyl acetate fractions of
Bauhinia galpinii and
Dryopteris filix-mas have been finally isolated quercetin-3-
O-β-galactopyranoside (
105) [
66] and quercetin-3-
O-α-
l-rhamnopyranoside (
106) [
90] exploiting acetone‒methanol (1:1) and pure methanol as eluting solvent systems, respectively. Quercetin-3-
O-α-
l-rhamnopyranoside (
106) has been furtherly isolated from aqueous and
n-butanol extracts of flower and leaf of
Cinnamomum zeylanicum [
89] and
Curcuma longa [
91], respectively, by using methanol‒water as eluent mixture in SLH gel filtration column. The leaf
n-butanol extract of
Ficus exasperate was extracted and quercetin-3-
O-β-rhamnoside (
107) accordingly isolated via SLH (eluent: toluene‒ethanol 7:3) [
92]. By utilization of methanol as eluting solvent through SLH column, quercetin-3-
O-glucopyranoside (
108) has been isolated from leaf methanolic and
n-butanol extracts of
Indocalamus latifolius [
43] and
Sambucus ebulus [
93], respectively. Another glycosylated quercetin derivative namely quercetin-3-
O-β-
d-glucuronide (
109) has been obtained by SLH from
n-butanol, ethanol, and ethyl acetate extracts of leaf, leaf, and stem parts of
Curcuma longa [
91],
Eugenia jambos [
94], and
Nelumbo nucifera [
95], while methanol‒water (8:2), ethanol‒water (7:3), and methanol were applied as eluents, respectively.
In similar studies, quercetin-3-
O-sambubioside (
110) and quercetin-3-
O-sophoroside (
112) have been isolated from
n-butanol and hydroethanolic (70%) extracts of
Eriobotrya japonica [
96] and
Poacynum hendersonii [
97] leaves, respectively, using methanol as solvent in SLH. Moreover, quercetin 3-
O-gentiobioside (
111) has been finally extracted by application of SLH from hydro-methanolic (70%) fraction of
Albizia amara leaf [
83] and
n-butanol extract of
Oryza sativa grain [
98] eluting with methanol‒water.
Hydro-methanolic (70%) extracts have been previously obtained from
Albizia amara leaf [
83] and aerial part of
Allium porrum [
84], then by using methanol‒water as eluents, quercetin 3-
O-
α-rhamnopyranoside (
113) has been isolated and identified. Three flavonol glucosides consist of quercetin-3-
O-α-
l-rhap-(1→2)-[α-
l-rhap-(1→6)]-β-
d-galactopyranoside (
114), quercetin-3-
O-α-
l-rhap-(1→6)-β-
d-galactopyranoside (
115), and quercetin-3-
O-α-
l-rhap-(1→2)-α-
l-rhamnopyranoside (
116) have been isolated from
Curcuma longa leaf
n-butanol extracts, eluting with methanol‒water (1:1) [
91]. Moreover, phytochemical analysis of hydro-methanolic (70%) extract of
Allium porrum aerial part was finally led to isolation of quercetin-3-
O-β-glucopyranosyl-7-
O-α-rhamnopyranoside (
117) and quercetin-4′-
O-β-glucopyranoside (
118), by using methanol‒water as eluting solvents with ratios of 2:8 and 4:6, respectively [
84].
Shi et al. [
22] isolated quercetin-3,7-di-
O-β-
d-di-glucopyranoside (
119), quercetin-3′,4′,7-trimethyl ether (
120), and quercetin-7-
O-[β-
d-glucopyranosyl(1→6)-β-
d-glucopyranoside] (
121) from methanolic extract of aerial part of
Taraxacum mongolicum (eluent: methanol). From ethyl acetate extracts of two plant species belonging to Asteraceae family including
Onopordum alexandrinum seed and
Tridax procumbens whole part, quercimeritrin (syn. quercetin-7-
O-glucoside) (
122) [
42] and quercetin-7-
O-β-
d-glucopyranosyl-(2→1)-α-
l-rhamnose (
123) [
44] have been isolated, respectively; moreover, quercimeritrin (
122) was isolated from aqueous extract of
Cudrania tricuspidata bark eluting with methanol‒water (1:1) [
25].
SLH gel filtration method has also been used for isolation and purification of other glycosylated quercetin derivatives: dihydroquercetin 7-
O-β-
d-glucoside (
124) from leaf
n-butanol extract of
Curcuma longa (eluent: methanol‒water 1:1) [
91], quercetrin (syn. quercetin 3-
O-rhamnoside) (
125) from leaf butanol extract of
Camellia japonica eluting with chloroform‒methanol (1:1) [
99], and isoquercetin (syn. quercetin 3-β-
O-glucoside) (
126) from ethyl acetate fraction of
Dorema glabrum aerial part (eluent: methanol‒water 8:2) [
100].
Quercitrin (syn. quercetin-3-rhamnoside) (
127) has been formerly isolated from
Thuja orientalis leaf ethyl acetate extract [
58], bran hydroethanolic (95%) extract of
Avena sativa [
64], and
Eriobotrya japonica leaf
n-butanol extract [
96], whilst methanol was used as eluting solvent. Isoquercitrin (syn. quercetin 3-
O-β-
d-glucopyranoside) (
128) has been previously isolated and identified from
n-butanol extracts of
Phyllanthus reticulatus leaf [
101] and
Juniperus chinensis herb [
81], using methanol‒water (1:1) and methanol for eluting, respectively. This compound has also been isolated from hydroethanolic (70%) and ethyl acetate fractions yielded from leaves of
Poacynum hendersonii [
97] and
Thuja orientalis [
58], respectively, eluting with methanol. Hiring SLH eluting with toluene‒ethanol (7:3) has been concluded to isolate isoquercitrin-6-
O-4-hydroxybenzoate (
129) from
n-butanol extract of
Ficus exasperate leaf [
92].
Kaempferol (
130) (3,4′,5,1-tetrahydroxyflavoune) is an aglycone flavonol which is naturally occurred in many plants’ parts through the phenylpropanoid pathway [
102,
103]. Pharmacological and biological activities of this nutraceutical compound have been extensively studied and reported to possess significant antiproliferative, cytotoxicity, anti-inflammatory, antioxidant, and antidiabetic activities [
104,
105,
106,
107,
108,
109].
This valuable compound has been isolated from 11 different plant species by employing SLH as the last chromatographic step. Ethyl acetate extracts might be considered as the richest fractions in kaempferol (
130) content: from
Fragaria ananassa calyx (eluent: acetone‒water 2:1) [
74],
Gynura divaricate leaf (eluent: chloroform‒methanol 1:1) [
75],
Gingko biloba leaf (eluent: methanol) [
36],
Chionanthus retusus flower (eluent: methanol‒water 7:3) [
24],
Populus davidiana wood (methanol‒water 3:1, 1:1, 1:3) [
15], and
Leptadenia pyrotechnica aerial parts [
67]. Kaempferol (
130) has been furtherly isolated from hydro-methanolic (70%) extracts of
Albizia amara leaf [
83] and
Allium porrum aerial part [
84] eluting with methanol and methanol‒water (8:2), respectively. From leaf hydroethanolic (70%) extract of
Brachychiton acerifolius applying methanol‒water (1:1) as eluting solvent [
34], and aqueous fractions of
Zygophyllum dumosum shoot [
110] and
Cudrania tricuspidata bark [
25] with methanol through SLH, kaempferol (
130) have been also isolated.
Among 23 kaempferol derivatives (
131–
152), only 7,4′-dimethoxykaempferol (
131) has been isolated as aglycone analogue from aerial part ethyl acetate extract of
Tamarix hohenackeri (Tamaricaceae family) using methanol as eluent in SLH column [
77]. The leaf ethanolic and ethyl acetate extracts of
Croton zambesicus and
Gingko biloba have been previously subjected to various chromatographic methods, and tiliroside (syn. kaempferol-3-
O-β-6′’(
p-coumaroyl)-glucopyranoside) (
132) [
56] and kaempferol 3-
O-rhamnopyranoside (
133) [
36] have been accordingly isolated via chloroform‒methanol (9:1) and methanol as SLH eluent, respectively.
Among all the isolated secondary metabolites from leaf
n-butanol extract of
Curcuma longa, kaempferol-3-
O-α-
l-rhamnopyranoside (
134) has been identified as a glycosylated flavonol exploiting methanol‒water (8:2) for eluting of samples in SLH [
91]. Kaempferin (syn. afzelin, Kaempferol-3-rhamnoside) (
135) has been previously isolated from two plant species of
Eriobotrya japonica [
96] and
Thuja orientalis [
58], whereas their leaves
n-butanol and ethyl acetate extracts were chromatographed by SLH with methanol, respectively. Methanol has been used as eluting solvent in isolation and purification of Kaempferol-3-rutinoside (
136) from
Sideroxylon foetidissimum leaf petroleum ether extract [
111] and kaempferol 3-
O-α-arabinoside (
137) from ethanolic fraction of
Opuntia dilleniid flower [
112].
Kaouadji et al. [
113] isolated kaempferol 3-
O-α-
l-(2-
E-
p-coumaroyl rhamnopyranoside) (
138) and kaempferol 3-
O-α-
l-(2-
Z-
p-coumaroyl rhamnopyranoside) (
139) from ethyl acetate extract of buds of
Platanus acerifolia by SLH (eluent: methanol). In a phytochemical investigation carried out on
Nelumbo nucifera, the ethyl acetate extract of stem by utilization of methanol in SLH gel filtration column kaempferol 3-
O-α-
l-rhamnopyranosyl-(1→6)-β-
d-glucopyranoside (
140), kaempferol 3-
O-β-(2″-
O-α-rhamnosyl)-glucuronide (
141), and kaempferol 3-
O-α-
l-rhamnopyranosyl-(1→2)-β-
d-glucopyranoside (
142), and kaempferol 3-
O-β-
d-glucuronopyranoside (
143) have been isolated and purified [
95].
SLH has been able to isolate astragalin (syn. kaempferol 3-
O-β-
d-glucopyranoside) (
144) from five plant species. Hydro-methanolic (70%) and hydroethanolic (95%) extracts of
Allium porrum aerial part [
84] and bran part of
Avena sativa [
64] have been applied to isolate astragalin (
144) applying methanol‒water (6:4) and methanol as eluting solvent, respectively. Furthermore, aerial parts ethyl acetate extracts of
Leptadenia pyrotechnica [
67] and
Dorema glabrum [
100], and
Fragaria ananassa calyx (eluent: acetone‒water 7:3) [
74] comprised the aforementioned compound.
From aerial part ethyl acetate extract of
Leptadenia pyrotechnica kaempferol-3-
O-α-
l-rhamnopyranosyl (1″′→6″)-
O-β-
d-glucopyranoside (
145) and kaempferol-3-
O-β-
d-glucopyranosyl (1″′→6″)-
O-β-
d-glucopyranoside (
146) [
67], whereas kaempferol 3-
O-(3″-
E-p-coumaroyl)-α-
l-rhamnopyranoside (
147) and kaempferol 3-
O-(2″
-O-E-p-coumaroyl)-β-
d-glucopyranoside (
148) were also isolated and identified from bran hydroethanolic (95%) extract of
Avena sativa (eluent: methanol) [
64].
Rayyan et al. [
16] reported isolation of a novel kaempferol glucoside, namely 8-methoxykaempferol 3-
O-(6″-malonyl-β-glucopyranoside) (
149) from hydro-methanolic (80%) extract of leaf and flower parts of
Crataegus spp. (Hawthorn) by increasing ratio of methanol (40 to 70%) in water using SLH.
According to previously performed phytochemical studies, three other glycosylated kaempferol derivatives have been furtherly isolated and purified by SLH gel filtration: kaempferol 7-
O-glucoside (
150) from seed ethyl acetate extract of
Onopordum alexandrinum (eluent: methanol‒water 9:1) [
42], kaempferol 7-
O-β-glucopyranoside (
151) from hydro-methanolic (70%) fraction of
Allium porrum aerial part (eluent: methanol‒water 6:4) [
84], and kaempferol 7-
O-α-
l-rhamnopyranoside (
152) from bran hydro-ethanolic (95%) extract of
Avena sativa (eluent: methanol) [
64].
Isorhamnetin (
153) has been isolated by using SLH eluting with methanol‒water (8:2) from hydro-methanolic (70%) extract of
Allium porrum aerial parts [
84]. Three isorhamnetin glucosides consist of isorhamnetin 3-
O-β-
d-rutinoside (
154) from aerial part ethyl acetate extract of
Halimodendron halodendron (eluent: chloroform‒methanol 1:1) [
79] and flower ethanolic fraction of
Opuntia dillenii (eluent: methanol) [
112], isorhamnetin 3-
O-monoglucoside (
155) from
n-butanol extract of
Sambucus ebulus leaf (eluent: methanol) [
93], along with isorhamnetin 3-
O-β-
d-glucopyranoside (
156) from
Dorema glabrum aerial part ethyl acetate extract (eluent: methanol‒water 8:2) [
100].
Exploiting SLH by eluting acetone‒methanol (1:1), myricetin (syn. 3,5,7,3′,4′,5′-hexahydroxyflavone) (
157) and myricetin-3-
O-β-galactopyranoside (
160) have been isolated from ethyl acetate extract of
Bauhinia galpinii leaf [
66]. Moreover, from stamen ethyl acetate, a fraction of
Nelumbo nucifera, myricetin 3′,5′-dimethylether 3-
O-β-
d-glucopyranoside (
158) (eluent: methanol) [
114], and a novel secondary metabolite myricetin 7-methylether 3-
O-xylopyranosylsyl-(1→2)-α-rhamnopyranoside (
159) have been previously isolated and identified from
Eugenia jambos ethanolic extract of the leaf (eluent: ethanol‒water 3:7) [
94].
By eluting methanol‒water and pure methanol through SLH column, myricitrin (syn. myricetin 3-
O-α-rhamnopyranoside) (
161) has been isolated from hydro-methanolic (70%) and ethyl acetate extracts of
Albizia amara [
83] and
Thuja orientalis [
58], respectively. Another study reported SLH was able to isolate penduletin (
162) and chrysosplenol D (
163) from aerial part methanolic extract of
Plectranthus cylindraceus [
115].
More flavonol derivatives have also been isolated and purified from ethyl acetate extracts of diverse plant species: sexangularetin (
164) from calyx of
Fragaria ananassa eluting with methanol‒water (4:1) [
74], a new natural product brassicin-4′-
O-β-
d-glucopyranoside (
165) via increasing acetone ratio (33 to 100%) in water from
Oryza sativa spp.
japonica grain [
116], 5,7,3′-trimethyl-4′-methoxyl-3-
O-β-
d-flavonoid glucoside (
166) and 8,3′-dihydroxyl-3,7,4′-trimethoxy-6-
O-β-
d-flavonoid glucoside (
167) from whole part of
Tridax procumbens [
44], a novel phytochemical ptevon-3-
d-glucoside (
168) from
Pterocarpus indicus leaf (eluent: dichloromethane‒methanol 1:1) [
117], leonurusoide E (
170) from
Leonurus japonicus eluting with methanol‒water (4:6) [
118], dillenetin (
172) from
Tamarix hohenackeri aerial part (eluent: methanol‒water) [
77], and tamarixetin 3-
O-rhamnopyranoside (
175) from
Firmiana simplex stem bark (eluent: methanol) [
119].
Furthermore, sophoflavescenol (
169) from root dichloromethane extract of
Sophora flavescens (eluent: dichloromethane‒methanol) [
120], 5,4′-dihydroxyflavone-3,6-di-
O-β-
d-glucoside-7-
O-β-
d-glucuronide (
171) from
Carthamus tinctorius aqueous flower fraction (eluent: water) [
121], 7-hydroxy-6-methoxyflavone (
173) from herb chloroform extract of
Dalbergia cochinchinensis (dichloromethane‒methanol 1:1) [
12], 3-
O-demethyldigicitrin (
174) from ethanolic extract of
Athrixia phylicoides aerial part (eluent: methanol), and artemitin (
176) from methanolic fraction of
Taraxacum mongolicum [
22] have been formerly isolated and purified by application of SLH as the final separation step.