Subcritical-Water Extraction of Natural Products

Subcritical water refers to high-temperature and high-pressure water, but below water's critical point of 374 °C and 218 atm. A unique and useful characteristic of subcritical water is that its polarity can be dramatically decreased with increasing temperature. Therefore, subcritical water can behave similarly to methanol or ethanol. This makes subcritical water a green extraction fluid used for a variety of organic species. 

natural products;subcritical water extraction;alkaloids;glycosides;flavonoids;essential oils;quinones;terpenes;lignans;organic acids;polyphenolics;steroids;carbohydrates

1. Introduction

Among the various new green extraction and separation technologies developed recently, subcritical water extraction (SBWE) is the most promising one. Subcritical water refers to the liquid water at temperature and pressure below its critical point (Tc = The pressure of the subcritical water must be higher than the vapor pressure at a given temperature to keep water in the liquid state. With the increase of temperature, the physical-chemical properties of subcritical water change drastically.

2. Compounds Extracted by Subcritical Water

2.1. Flavonoids

Flavonoids, also known as bioflavonoids, are widely found in plants and berries. They are important natural compounds in human diets. They have been used to prevent and treat cardiovascular diseases. In addition, they have strong antioxidant activities and antibacterial activities. When high-flavonoid apples were fed to healthy mice, decreases in some inflammation markers were reported [73].
Generally speaking, flavonoids belong to phenols. Since they are widely investigated, they can be presented separately from phenols. Flavonoids have the general structure of a 15-carbon skeleton by connecting two benzene rings with a heterocyclic ring. The basic nucleus is 2-phenylchromone. Flavonoid compounds are usually poorly soluble in ambient water and most organic solvents. Table 1 summarizes SBWE of flavonoids from plant materials.
Table 1. SBWE of flavonoids.
Samples Medicinal Parts Compounds Extracted Extracts Activity Extraction Conditions Analytical Methods Other Extraction Methods (Solvent, Ratios of Yields) Ref.
In general, organic acids in natural products are widely distributed in the leaves, roots, and fruits of the plants. The synthetic organic acids through chemical synthesis, enzymatic catalysis, and microbial fermentation are not discussed in this review. Organic acids are mostly soluble in water or ethanol and exhibit acidic properties, but they are difficult to dissolve in other organic solvents. It is generally believed that aliphatic organic acids have no special biological activity, but some natural organic acids such as citric acid, malic acid, tartaric acid, ascorbic acid, etc. have antibacterial, anti-inflammatory, hypoglycemic, antioxidant, and immune regulation effects. Depending on the organic acid in free state or in salt form, the extraction solvents could be water, dilute alkaline solution, diethyl ether, petroleum ether and cyclohexane, and other lipophilic organic solvents. A summary of recent studies on SBWE of organic acids are shown in Table 3.
Table 3. Subcritical water extraction of organic acids.
Samples Medicinal Parts Compounds Extracted Extracts Activity Extraction Conditions Analytical Methods Other Extraction Methods (Solvent, Ratios of Yields) Ref.
Subcritical water has also bee used to extract essential oils, alkaloids, quinones, terpenes, lignans, and steroids from plant and other materials. A summary of SBWE of essential oils, alkaloids, quinones, terpenes, lignans, and steroids can be found in Table 6.

Table 6. SBWE of essential oils, alkaloids, quinones, terpenes, lignans, and steroids.


Medicinal parts

Compounds Extracted

Extraction Conditions


Other Extraction Methods (Solvent, Ratios of Yields)



Essential oils


Thymbra spicata L.


α-thujene, α-pinene, terpinen-4-ol, p-cymene, γ-terpinene, 1-carvone, thymol, carvacrol, etc.

100–175 °C, 1–3 mL/min, 2–9 MPa, 30 min





Aquilaria malaccensis


butanal, cyclopentanone, acetoxyacetone, benzaldehyde, acetophenone, creosol,etc.

100–271 °C, 1–34 min, 0.08–0.22 g/mL


HD (95.4%)


Mentha piperita L.



TP, menthone, menthol, eriocitrin, etc.

40–160 °C, 10.3 MPa, 1–30 min


convention (methanol 53.2%)


Coriandrum sativum L.


coriander seeds

thujene, sabinene, pinene, myrcene, cymene, limonene, ocimene, terpinene, terpinolene, etc.

100–175 °C, 1–4 mL/min, 0.25–1 mm, 2 MPa, 20 min


HD (1.54-fold), Soxhlet (hexane 1.4-fold)


Coriandrum sativum L.


coriander seeds

3,4-dimethoxycinnamic acid, coumaric acid, sinapic acid,cis-and trans-linalooloxides, linalool, etc.

100–200°C, 10–30 min, 3–9 MPa




Kaempferia galangal L.


ethyl-p-methoxycinnamate, d-limonene, eucalyptol, tridecane, camphor, borneol, tetradecane, etc.

120 °C, 10 MPa, 30 min


HD (82.3%), UWE (100%)


Piper betle


4-allyl resorcinol, chavibetol

2 MPa, 10–90 min, 50–250 °C, 0.25–1 mm, 1–4 mL/min


convention (water 92.2–111%; methanol 96.6–110 %)


Aquilaria malaccensis


nonacosane, triacontane, pentadecanal, 9-octadecenal, (Z)-, tetradecanal, tetrapentacontane, guaiacol

100–271 °C, 1–34 min






α-phellandrene, β-pinene, 1,8-cineole, borneol, nona-3,7-dienol, isobornyl acetate, γ-terpineol, etc.

15 min, 50–200 °C, 1.5–15 MPa, 0.5–5.0 mL/min




Citrus hystrix


linalool, isopulegol, neoisopulegol, citronellal, 4-terpineol, citronellol, geraniol, menthoglycol, etc.

120–180 °C, 5–20 g/mL, 5–30 min


HD (28.2%)


Coriandrum sativum L.




α-pinene, β-pinene, camphor, methylchavicol, γ-terpinene, linalool, geraniol, carvacrol, etc.

100–200 °C, 1:10 g/mL, 2 MPa, 20 min


HD (27.0%), Soxhlet (DCM 6.5-fold), SCCO2 (4-fold)


Lavandula L.

lavender flowers

a-thujene, a-pinene, camphene, sabinene, pinene, myrcene, hexylacetate, terpinene, limonene, etc.

125 °C, 3 MPa, 30 min


HD (1.2-fold), US-HD (1.3-fold), NaCl-HD (1.3-fold)







cytisine, matrine, sophoridine, sophocarpine, oxymatrine

70–190 °C, 5–14 min, 4.0–13.8 MPa


ASE (ethanol 78.1%)


black tea brick


theophylline, epicatechin gallate, caffeine, etc.

120–180 °C, 7–42 min, 6–18 mL/min




Symphytum officinale L.


lycopsamine, echimidine, lasiocarpine, symviridine

60–120 °C, 40 min


Panax ginseng C.A. Meyer stem

TP and flavonoids antibacterial 110 and 165 °C, 15 min

190 °C, 10 min
TEM, UV heating (water 95.4%; ethanol 91.3%) [15]
Chamomilla matricaria L. flowers TP, TF, 18 polyphenolic compounds, apigenin antioxidant, enzyme inhibitory activity
]. Similar results were obtained by Nkurunziza et al. [81] and Zhang et al. [83] who investigated the extraction of four flavonoids from okara and from Puerariae lobata, respectively.

2.2. Polyphenols

Polyphenols, also known as polyhydroxyphenols, are a structural class that is mainly natural, by the presence of more than one phenolic unit and being deprived of nitrogen-based functions. Many fruits, vegetables, herbs, tea leaves, nuts, and algae contain high levels of naturally occurring phenols. It has been reported that polyphenols can resist oxidation [31]. As shown in Table 2, extractions of polyphenols can be carried out either using a sole solvent such as water, methanol, ethanol or a mixture of solvents such as ethanol-water and methanol-water-formic acid.
Table 2. Subcritical water extraction of polyphenols.
Samples Medicinal Parts Compounds Extracted Extracts Activity Extraction Conditions Analytical Methods Other Extraction Methods (Solvent, Ratios of Yields) Ref.

HRE (methanol 2.8-fold)


hydrastis canadensis


hydrastine, berberine

100–160 °C,1–10 MPa, 5–60 min, 0.5–1.5 mL/min


reflux (methanol 90.8%), UAE (methanol 106%)




TP, theobromine, theophylline, caffeine, epicatechin, etc.

120–220 °C, 15–75 min,

1:10–1:30 g/mL




Musaceae, Beta vulgaris


dopamine, total betacyanin, betaxanthin

150°C, 5 min, 3 MPa, 1:20 g/mL


infusion (100%), decoction (1.2-fold), maceration (97.4%), UAE (101%), MAE (50.3%)



arabica, C. arabica, C. canephora var. robusta,

C. canephora var. robusta

coffee silver skin

total sugar, reducing sugar, protein, TP, caffeine, HMF, etc.

180–270 °C, 10 min, 1.0–5.3 MPa


convention (0.1 M HCl 96.6%; 0.1 M NaOH 1.5-fold)




Rheum tanguticum



33–67 min, 100–200°C, 1.4–4.6 mL/min,




Garcinia mangostana Linn

mangosteen pericarps

TP, xanthone

120–160 °C, 1–10 MPa, 5–60 min, 10–30% DES

UV-vis, FT-IR, SEM



Phaleria macrocarpa

mahkota dewa fruits


4.0 MPa, 5 h, 50–150 °C




Lithospermum erythrorhizon



shikonin, acetylshikonin, β-dimethylacrylshikonin, etc.

40–60 mesh, 120 °C, 5 MPa


SCCO2 (86.3%), Soxhlet (ethyl acetate 95.4%), UWE (1.4-fold)


Morinda citrifolia



4 MPa, 150 and 220 °C, 1.6–4 mL/min




Morinda citrifolia


1,2-dihydroxyanthraquinone, alizarin

110–220 °C, 2–6 mL/min


ethanol (3 d)


Morinda citrifolia



4 MPa, 150–200 °C, 2–6 mL/min


convention (ethanol 81.16%), Soxhlet (ethanol 97.94%), UAE (ethanol 79.62%) SWBE (96.41%)




Hedyotis diffusa Willd.



ursolic acid


120–200 °C, 10–50 min,

20–40 mL/g, 0.6–3.0 MPa


maceration (ethanol 58.8%), HRE (ethanol 78.4%), UAE (ethanol 90.4%), MAE (ethanol 74.9%)


Centella asiatica



asiatic acid, asiaticoside

100–250°C, 10–40 MPa, 5h




basil, oregano


limonene, citronellol, etc.

100 and 150 °C, 10 min




Ganoderma lucidum


ganodermanon-triol, ganoderic acids, lucidumol

100–200 °C, 5–10 MPa, 5–60 min




Orostachys japonicus



triterpene, camellia, etc.

110–260 °C, 5–20 min, 10 MPa




Betula pendula

birch bark

betulinic acid

160–200 °C, 10–30min, 10 MPa




Inula racemose


igalan, soalantolactone, alantolactone

23.2–56.8 min, 1.3–4.7

 mL/min, 129.5–230.5 °C


Soxhlet (ethanol 100%), UAE (ethanol 70.36%), SCCO2 (76.06%)


Semen richonsanthis


3,29-dibenzoylkarounidiol, polysaccharides

80–160 °C, 5.0–30.0 min




Cucurbita pepo

pumpkin peel

14 carotenoid compounds

120 °C, 3 h, 5 MPa


SCCO2 (75.4%)


Betula pendula

birch bark

sesquiterpenes, steroids

10 min, 100–200 °C




S. rebaudiana

Bertoni leaves

steviol glycosides, tannins, chlorophyll A

100–160 °C, 5–10 min,

10.34 MPa, 1:3 g/mL






Linum usitatissimum L.


SDG lignan, phenolics, flavonoids

160–180 °C, 5–60 min, 10 MPa




Sesamum indicum L.

sesame seeds

lignans, TP, flavonoids, flavonols

140–220 °C, 8–14 MPa, 0–95% ethanol, 0–75 min




Linum usitatissimum L.


total fat content, SDG lignan

120–180 °C, 15–90 min, 10–13.8 MPa




Sinopodophyllum hexandrum



Allium ursinum L. leaves TP, TF, 5-HMF, catechin, p-cumaric, ferulic acids, etc. antioxidant (DPPH, ABTS), Millard products 120–200 °C, 10–30 min, 0–1.5% HCl, 1:10 g/mL HPLC-DAD   [31]

12 mL/g, 3 MPa, 2ml/min, 120–240 °C




Panax ginseng Meyer root TP, chlorogenic acid, caffeic acid, gallic acid, etc. antioxidant (DPPH, ABTS, FRAP, HRS)  100–240 °C, 15 min,

4–9 MPa, 200 rpm
65–210 °C, 5–60 min

323–423 K, 1–7 h,

0.7–4.0 MPa HPLC, LC-MS convention (water 69.6%; ethanol 34.1%; methanol 108%), HRE (water 85.7%; ethanol 60.8%; methanol 115%), Soxhlet (water 86.1%; ethanol 55.8%; methanol 113% methanol)[23]
[36] Terminalia chebula1:30–1:100 g/mL fruits TP, allic acid, corilagin ellagic acid antioxidant (ABTS) 120–220 °C, 2–4 mL/min, 4 MPaTLC, UV, HPLC-MS   [18]
TLC, UV, MS, NMR, HPLC Helicteres isora L.Soxhlet (water 74.5%; ethanol 46.3%), HWE (water 46.3%)  [ hexadecanoic acid, octadecnoic acid, heptadecen-8-carbonic acid etc. antibiofilm, antioxidant,

antimicrobial, antienzymatic
160 °C, 30 min, 1 MPa, 1: 30 g/mL GC-MS, UV
Punica granatum L. pomegranate seed TP, kaempferol

antioxidant (DPPH, ABTS) 80–280 °C, 5–120 min, 1:10–1:50 g/mL, 6.0 MPa  HPLC-DAD, UV, HPLC-ABTS+34] [27] leaching (water 40.6%; methanol 79.7%; ethanol 41.7%; acetone 45.5%), UAE (water11.3%; methanol 20.6%; ethanol 18.9%; acetone 15.2%), Soxhlet (methanol 71.4%; acetone 39.7%) [144] Allium cepa onion wastes quercetin-4′-glycoside, quercetin, etc.   40–160 °C, 5 min, 5 MPa,

1–10 mm, pH 3.0–7.0
LC-MS/MS HPLC-UV convention (methanol and hydrochloric acid 94.3%)
Lycium ruthenicum Murr.[19]
fruits total anthocyanin, seven anthocyanins antioxidant (ABTS, DPPH) 110–170 °C, 30–90 min, 1–3 min/L HPLC, UPLC-MS
Teucrium montanum L.UAE (water 59.8%; methanol 81.1%)
antiproliferation effect (MTT)
XiLan olive fruit olive dreg TP, chlorogenic acid, gallic acid, syringic acid, etc. antioxidant (ABTS, DPPH, reducing power) 100–180 °C, 5–60 min, 1:20–1:60 g/mL LC-MS-IT-TOF, UV convention (methanol 3.2%; ethanol 0.6%; DMK 0.9%) aerial parts[33] Crocus sativus L. stigmas TP, dodecane, γ-terpinene, tetradecane, etc. antioxidant (DPPH, FRAP), antibacterial 100–180 °C, 10–30 min,

1:10 g/mL
GC/MS, UV-vis   [21]
Punica granatum L. peels TP, TF, punicalin, etc.   100–220 °C, 5–30 min, 3.0 MPa UV-vis, HPLC MAE (water 121%; ethanol 146%) [109] Saururus chinensis, etc. skin, leave, peel, etc. quercetin, isorhamnetin, kaempferol, isoquercitrin, etc.
Castanea sativa shells tannins, phenolic acids, flavonoids, anthocyanins  10 MPa, 110–200 °C, 5–15 min HPLC   antioxidant (DPPH, FRAP, ABTS) 51–249 °C, 6–30 min UV-vis,

[110 Camellia sinensis leaves epigallocatechin gallate   80–120 °C, 3–7 min,

40–60 mL/g
HPLC convention (water 87.6%) [44]
Origanum vulgare L. leaves
110–150 °C, 1–10 min, 0.5–10 MPa
Camellia oleifera Abel. seeds free fatty acids (palmitic acid, stearate, oleic acid, etc.), tea saponin antioxidant (DPPH)
rutin, naringin, epicatechin, etc. antioxidant (DPPH, FRAP) 60–200 °C, 30 min,

1–10 MPa, 1:10 g/mL HPLC-PDA, UV   [145] sunflower seeds (Natura) dehulled seeds total proteins, total carbohydrates, TP TP, flavanone, flavone, flavanol antioxidant (DPPH, TEAC, ABTS)
convention (ethanol, 1.13-fold)
60–160 °C,2–7 MPa,

5–60 min, 1:3–1:25 g/mL GC-MS, FT-IR Soxhlet (petroleum ether 100%), cold pressed (100%) [37]antioxidant capacities 60–160 °C, 5–120 min,

3 MPa, 1:10–1:30 g/mL
GC-FID, UV-Vis, HPLC Soxhlet (hexane 67.3%) [38] 10.34 MPa, 30 or 15 min

25–200 °C
HPLC- 100–180 °C, 5–25 min, 1:20–1:60 g/mL, 1–5‰ NaHSO3
cottonseed (Egypt) cottonseed linoleic acid, palmatic acid, oleic acid, myristic acid   180–280 °C, 5–60 min, 1:2–2:1 g/mL GC-FID, heating (hexane 89.5%) [39]

orange peels reducing sugar, TP, pectin, hesperidin, narirutin antioxidant (DPPH, FRAP) 110–150 °C, 10–30 mL/min

10 MPa

Soxhlet (ethanol 79.2%), shaker (ethanol 250%), UAE (ethanol 114%) [50]
orange peels flavones, 7-hydroxyflavone   100–150 °C, 0.5 mL/min GC-FID UAE (methylene chloride) [70]
Citrus unshiuHPLC-ESI-MS, UV HWE (water 33.9%) [115]
German chamomile flowers 9 phenolic acids and derivatives antioxidant, cytotoxic, enzyme 100 °C, 1–9 MPa, 30 min UHPLC-DAD, MS/MS   [116]
Fagopyrum tataricum grains phenols, 13 phenolics, 4 flavonoids, 3 anthocyanins antioxidant (TEAC, CAA and FRAP), cytotoxicity 220 °C, 60 min, 5 MPa, 1:60 g/mL HPLC-MS, UV UAE (water 83.5%) [117]
A. uva–ursi herbal dust TP, TF antioxidant (DPPH, reducing power) 120–220 °C,3 MPa, 10–30 min, 0–1.5% HCl
green coffee (Robusta Uganda) beans chlorogenic acid   130–170 °C, 40–90 min, 0–30 % ethanol HPLC convention (ethanol 66.7%) [45]
Nannochloropsis gaditana   fatty acids, omega-3, omega-6, lipid   156.1–273.9 °C, 6.6–23.4 min, 33–117 g/L GC-FID, SEM Soxhlet (n-hexane 100%)
(20% methanol 18.7%) [150] Saccharina japonica   gallic, caffeic, vanillic, syringic, chlorogenic, p-hydroxybenzoic acids, etc. antioxidant (DPPH, ABTS, total antioxidant (FRAP) 100–250 °C, 5 min, 5 MPa, 0.25–1.00 M ILs HPLC, UV convention (DMK 0.2%; DCM 0.3%; Et2O 0.8%; IL 1.6%) [52 Markovich
Crocus sativus L.] peels rutin, naringin, hesperidin, naringenin   0.5–14 MPa, 5–15 min, 100–190 °C HPLC   [74]
Allium cepa L. peels UV maceration (water 38.5%; ethanol 69.5%) [118]
Hippophaë rhamnoides L. seed residue TP, TF, proanthocyanidins antioxidant (DPPH) Curcuma longa L.
stigmas picrocrocin, safranal, crocin   5–15 min, 105–125 °C GC-MS, UV, HPLC   [151] Haematococcus pluvialis   p-hydroxybenzoic acid, gallic acid, siringic acid, vanillic acid, etc. antioxidant (ABTS, TEAC), antimicrobial activity 50–200 °C, 20 min,

10 MPa
Momordica charantia
Glycyrrhiza uralensis Fisch licorice

TP, glycyrrhetic acid, glycyrrhizin, liquiritin antioxidant (DPPH, reducing power) 50–300 °C, 10–60 min,

0.002–5 MPa
HPLC, UV-Vis   [152] TP, TF, quercetin antioxidant (DPPH, TBA, FTC) 110 and 165 °C, 15 min, p < 3.4 MPa HPLC, UV heating (ethanol 153%; water 45.6%) [75]
fruits TP, gallic acid, gentisic acid, chlorogenic acid antioxidant (ABTS) 130–200 °C, 10 MPa, 2–5 mL/min Hippophae rhamnoides leaves TP, TF, isorhamnetin, kaempferol, quercetin antioxidant, cytotoxicity 25–200 °C, 15 min,

10.34 MPa
HPLC, UV, FM maceration (water 21.3%), Soxhlet (ethanol 64.6%) TP, chlorogenic acid, gallic acid, vanillic acid, etc.[ antioxidant, antifungal, antibacterial, cytotoxic76]
60–200 °C, 30 min, 1 MPa, 1:40g/mL HPLC-DADUV   [133] Allium cepa L. peels
Prunus avium L., Prunus cerasus L. stems 3 alcohols, 10 organic acids, etc. antioxidant, antiproliferative 150 °C, 30 min, 2 MPa GC-MS, UV   [134]
Castanea sativa nuts ellagic acid, feru lic acid, gallic acid, etc.
2.5. Carbohydrates
Carbohydrates is a very common term that include sugars, starch, and cellulose, which are an important class of organic compounds widely distributed in nature. The saccharides are divided into four groups: monosaccharides, disaccharides, oligosaccharides, and polysaccharides. As shown in Table 5, carbohydrates extracted by SBWE possess antioxidants [153], antimitotic [154], and growth inhibitory effects [55].
Table 5. Subcritical water extraction of carbohydrates.
Samples Medicinal Parts Compounds Extracted Activity/Mixtures Extraction Conditions Analytical Methods Other Extraction Methods (Solvent, Ratios of Yields) Ref.






, Amaranthaceae

ginseng root

sugar, fructooligosaccharides, beta-ecdysone

80–180 °C, 5–15 min, 2–12 MPa




Panax ginseng C.A. Meyer

ginseng root

TP, maltol, panaxadiol, panaxatriol

150–200 °C, 5–30 min, 100 MPa


convention (water 32.6%; methanol 24.1%; ethanol 18.7%)


Panax ginseng C.A. Meyer

ginseng root

total ginsenosides, total sugar,

1-oleanane ginsenosides, etc.

120–200 °C, 20 min, 1:20 g/mL, 6.0 MPa


heating (water, 30.9%; ethanol 94.4%)




wood, cane

E-piceid, E-piceatannol, E-resveratrol, E-parthenocissin, etc.

100–190 °C, 5–30 min, 10 MPa


ASE (116% for cane; 103% for wood; 1.5-fold for root)


Withania somnifera L

root leaves

TP, withanoside IV V, withaferin A, withanolide A, B

100–200 °C, 10–30 min, 10 MPa


maceration (water 31.7%), Soxhlet (ethanol 39.2%), MAE (methanol 45.8%)


Acanthophyllum glandulosum




121 °C, 0.15MPa,

15 min, pH 4–9




Vaccaria segetalis

cowcock seed

vaccarosides, segetosides

125–175 °C, 15–180 min


USE (methanol 46.8%; water 27.9%; ethanol 5.2%)


Lycium barbarum berries total sugar content antioxidant (FRAP, TEAC), immunomodulatory 1:30 g/mL, 110 °C, 5 MPa HPGPC HWE (water 71.5%), UAE (water 89.9%), UWE (water 132%) [11]
sunflower sunflower heads galacturonic acid, pectin   10–50 min, 2–8 mL/g, 100–140 °C, 0.2–1 MPa TG/TGA, DSC, UV−vis, FTIR, HPSEC, NMR   [40]
Aronia melanocarpa chokeberry stems 1 amino acid, 8 alcohols, 11 sugars, 2 fatty acids, etc. antioxidant (DPPH), enzyme inhibitory activity 130 °C, 3.5 MPa, 20 min, 1:20 g/mL GC-MS   [49]
Paeonia lactiflora root albiflorin, paeoniflorin   100–260 °C, 10–60 min,10–40 mL/g Lentinus edodes fruit bodies hetero–polysaccharides, xylose, mannose, etc.HPLC reflux (water 83.5%), UAE (ethanol 77.8%) [146]
TP, TF, kaempferol, quercetin
antioxidant (DPPH)
170–230 °C, 3 MPa, 30 min,

pH 2–10
HPLC, UV-vis heating (ethanol 26.7%) [77]
antioxidant 120–135 °C,

15–60 min HPLC   [135]
Solanum tuberosum potato peel TP, gallic acid, caffeic acid, chlorogenic acid, protocatechuic acid, etc.   100–240 °C,

30–120 min, 6 MPa
HPLC, UV convention (methanol 1.6%; ethanol 2.0%) [136]
antioxidant (OH·, DPPH, ABTS) 120–160 °C,30–50 min, 0.033–0.05 g/mL UV-vis, SEM, GC, GPC, FT-IR   [54] Morus nigra L. fruits TP, TF, cyanidin 3-glucoside, etc. 40–80 °C, 20–60 min, 2–6 mL/min, 15 MPa tyrosinase inhibitory activity UPLC-DAD-ESI-MS/MS shaker (ethanol:water 116%), UAE (ethanol:water:TFA 134%) [147
Lentinus edodes]
fruit bodies l-rhamnose, d-arabinose, d-xylose, d-mannose antioxidant (ABTS), growth inhibitory effect 100–150 °C, 10–30 min, 5 MPa FT-IR, UV-Vis, AFM, GC, HP SEC-MALLS   [55] Stevia rebaudiana leaves TP, stevioside,

rebaudioside A
antioxidants (DPPH) 100–150°C, 30–60 min, 23 MPa, 1:10 g/mL HPLC-UV, UV   [148]
Lentinus edodes fruit bodies polysaccharides, rhamnose, arabinose, xylose, etc. antioxidant (DPPH, reducing power) 140 °C, 40 min, 1:25 g/mL, 5 MPa GC, FT-IR, AFM, SEM   [56] Erigeron breviscapus whole parts scutellarin, 20 inorganic elements, etc.HPLC, UV Soxhlet (methanol 4.9%), UAE (methanol 4.0%) [132]
antioxidant (DPPH) 120–140 °C, 5–15 min, 150–420 um HPLC, HPLC-MS reflux (methanol 86.1%; ethanol 84.8%) [
Lentinula edodes fruit bodies TCC, total β-glucan, chitin HMGCR, immuno-

149] 200 °C, 11.7 MPa, 15–60 min GC-MS, HPSEC, NMR UAE (water 65.2%), HWE (water 32.3%), SPE (water 33.0%) [57]
Grifola frondosa fruit bodies total polysaccharide, total protein antioxidant (DPPH, reducing power) 100–230 °C, 2–4 min, 20–100 mesh, 5 MPa FT-IR, SEM HWE (water ~87.8%) [58]
Sagittaria sagittifolia L. fruit bodies polysaccharides antioxidant (DPPH, ABTS, reducing power) 150–190°C, 12–20 min, 1:20–1:40 g/mL, pH 7–9 FT-IR, 1H and 13C NMR, UV HWE (water 55.8%) [59]
Sagittaria sagittifolia L. fruit bodies l-rhamnose, d-arabinose, d-xylose, d-mannose antioxidant,

170°C, 16 min HPLC, GC, SEM, IR, AFM, HPSEC-MALLS HWE (water 75.6%); UAE (water 96.1%) [60]
Sagittaria sagittifolia L. fruit bodies α-pyranose polysaccharide, β-pyranose polysaccharide immuno-stimulatory 1 MPa, pH 7,170 °C, 16 min, 30:1 mL/g IR, GC-FID, UV, HPSEC, AFM   [61] Morus nigra L., Teucrium chamaedrys L., Geranium macrorrhizum L., Symphytum officinale L. leaves, flowers
Cordyceps militaris fruit bodies total sugars, protein and uronic acid   180 °C, 13 min, pH = 8,

21 mL/g
wheat bran monosaccharide, etc. antioxidants (DPPH) 160–180°C, 5–60 min HPAEC-PAD, SEC   [153] Achillea millefolium L.
Saccharina japonicaherbal dust TP, TF, HMF, chlorogenic acid antioxidant (DPPH, TEAC, ABTS)   fucoidan, fucose, glucose, galactose, mannose, etc.120–200 °C, 10–30 min

0–1.5% HCl, 3 MPa
HPLC, UV-vis   80–180 °C, 15–90 min, 1:10–1:50 g/mL, 6 MPa[ antioxidant, antimitotic

UV convention (water 19.6%; methanol 104%; ethanol 80.0%) anti-proliferative 100–180 °C, 5–15 min, 2–8 MPa FTIR, TGA, UV-Vis[119] convention (0.05 M HCl 100%) [154] Curculigo latifolia root TP, TF, pomiferin, etc. antioxidant (DPPH, ABTS, TEAC) 100–200 °C, 10 MPa

30–120 min, 0.5 mL/min
LC-MS, UV   [79
grape (Croatina)]
pomace TP, TF antioxidant (DPPH) 100–140 °C, 8–15 MPa, 1–2 mL/min UV convention (water 5.3%; ethanol 7.87%) [120]
Citrus grandis L. pomelo peel pectin   90–120 °C, 3–10 MPa HPSEC-MALLS   [155] Citrus unshiu peels hesperidin and narirutin
Matricaria chamomilla  L. flowers
Theobroma cacao L.110–190 °C

3–15 min polyphenolic compounds, etc. antioxidant, cytotoxic, enzyme inhibitory cacao pod husks65–210 °C, 30 min, 4.5 MPaHPLC   [80]
UHPLC-ESI-MS/MS, UV xylose, arabinose, etc.   121 °C, 30 min, 10.3 MPa  [121]
Actinidia deliciosa pomace TP, chlorogenic acid, protocatechuic acid, etc. antioxidant (DPPH, FRAP, ABTS) 170–225 °C

10–180 min, 5 MPa
UV, HPLC, pH   FT-IR, GC-FID, SEM convention (4% citric acid 76.1%)[ [156] Glycine max okara genistin, daidzin, genistein, daidzein   100–200 °C, 5 min, 2–5 MPa, 10–30 g/mL HPLC Soxhlet (methanol, 108%)
Kappaphycus alvarezii. A   ĸ-carrageenan, glucose, 3,6-anhydrogalactose, etc.[81 antioxidant (DPPH, ABTS)]
60–180°C, 5 MPa, 5 min FTIR, TGA, XRD convention (water 94.3%; water with IL 101%) [157] onion skins quercetin, quercetin-4′-glucoside   100–190°C, 5–30 min,

9–13 MPa
HPLC convention (methanol, 92.8%) [82]
Puerariae lobata root puerarin, daidzin, daidzein

  100–200 °C, 15–75 min

1:10–1:25 g/mL
HPLC reflux (ethanol 91.6%), UAE (water 95.9%) [83]
Coriandrum sativum seeds TP, TF antioxidant (DPPH) 100–200 °C, 10–30 min

3–9 MPa
UV   [84]
] convention (ethanol 56.4%; methanol 35.8%; water 6.2%) [91]
palatiferum Radlk.
Salvia officinalis L. by–products TP, TF antioxidant (DPPH, TEAC, reducing power) 120–220°C, 10–30 min, 3 MPa, 0–1.5% HCl UV maceration (water 59.9%) [111]
Pistacia vera L. hulls gallotannin, anacardic acid, etc. antioxidant (ABTS, FRAP) 110–190 °C, 6.9 MPa, 4 mL/min HPLC-ESI/MSn UAE (methanol 83.9%)) [112]
Zingiber officinale pulp and peel 6-gingerol, 6-shogaol antioxidant (FRAP) 10 MPa, 110–190 °C, 5–40 min HPLC convention (methanol 114%; water 77.1%) [113] Citrus unshiu peels
Sorfhum bicolor L. bran TP, oligomeric procyanidins, taxifolin, taxifolin hexoside antioxidant (DPPH, ABTS), antiproliferative 110–190 °C, 5–40 min, 1:10–1:50 g/mL HPLC, ESI-MS/MS heating (water 74.9%) [114] flavanones, polymethoxy-Flavones, etc. anticancer, cardioprotectives 120–180 °C, 1.0–2.0 mL/min, 5.0 ± 0.1 MPa GC, HPLC, convention (methanol 75.0%; ethanol 41.6%; acetone 17.2%)
Nelumbo nucifera[85]
seed epicarp TP, proanthocyanidin dimers, trimer, cyanidin, etc.137 Nelumbo nucifera seedpods TP, TF, proanthocyanidin dimer, isoquercetin, etc. antioxidant, antiproliferative (HepG2) 100–180 °C, 30–70 mL/g, 5–25 min, 1–6‰ NaHSO3 UV-Vis, HPLC, ESI-MSn HWE (water 91.4%) [122]
Vitis vinifera L. grape pomace catechins, flavonols, tannins, proanthocyanidins, etc. antioxidant (DPPH, ABTS) 40–120 °C, 10 min, 10.34 MPa, 10–40% NADES UV, HPLC-ESI-MS   [123]
hypnea musciformis   chlorogenic, protocatechuic, and gallic acids, TP, TF, etc. antioxidant (DPPH, ABTS), emulsify 120–270 °C, 10 min,

1:50–1:150 g/mL
pH, UV, HPLC   [138]
Carica papaya L. papaya

TP, 18 phenolic acids, 20 flavonoids, 1 stilbene, etc. antioxidant (DPPH, β-carotene bleaching) 70–150 °C, 10 MPa, 1–40 min, 4 mL/min LC-ESI-MS/MS, UV Soxhlet (water 37.1%) [139] sweet chestnut bark TP, tannins, ellagic and gallic acids, ellagitannins, etc. antioxidant (DPPH) 150–250 °C, 10–60 min, 10–30 mL/g, 4.5 MPa UV-Vis, HPLC  
Zingiber officinale ginger

Pseuderanthemum palatiferum leaves TCC, monosaccharides anticoagulant, antioxidant 150–200°C, 5–10 mL/min HPLC, GPC, NMR, UV convention (0.1 M NaOH 48.8%) [158] rhizome[ 12 sugars, 8 diols, 4 phenolic acids, etc. antimicrobial, cytotoxic 150 °C, 1 h, 1:10 g/mL HPLC-ESI-TOFMS heating (water)124]
wheat bran140 TCC, reducing sugar, arabinose, xylose, etc.] antioxidant, α-amylase inhibitory 140 °C, 5 MPa, 30 min SEC-MALLS, FT-IR, DLS, DSC, UV SBWE (water with citric acid 97.6%); UWE (water with citric acid 103%) [159] Symphytum officinale root TP, TF antioxidant (DPPH), enzyme inhibitory 120–200 °C,10–30 min, 0–1.5% HCl UV, ELISA
Chlorella sp. microalgae   TP, caffeic acid, ferulic acid, p-coumaric acidUAE (methanol 2.5%; ethanol 17.4%); maceration (methanol 4.4%; ethanol 29.8%) antioxidant (DPPH)[125]
100–250 °C,

5–20 min UV, SEM, HPLC   [141]
Lycium barbarum L. fruits polysaccharides antioxidant (O2·, OH·, DPPH) 5 MPa, 25 mL/g, 110 °C, 1 h UV HWE (water 86.2%); UAE (water74.9%); UWE (water 111%) [160] Pinot Nero grape skins
Vitis viniferaTP   80–120 °C, 2 h,10 MPa, 2–5 mL/min UV-Vis   [126] Phlomis umbrosa
vine-canes TP, flavonoids, phenolic acids, flavonols antioxidant, antiradical
Cocos nucifera L. defatted coconut mannose, galactosamine, xylose, rhamnose, etc. antioxidant,

hypoglycaemic, adsorption
125–250 °C, 50 min 1:10–1:50 g/mL, 10–50 min, HPLC, UV

120–200 °C, 20–100 mesh[ HPLC, XRD, TGA, DTGA, SEM, FT-IR142]   [161] whole part TP, TF, iridoids glycosides Coffea arabica L. spent coffee grounds TP, caffeoylquinic acid, feruloylquinic acid, etc.antioxidant (DPPH, ABTS) 110–200 °C, 10 MPa, 1–25 min antioxidant (DPPH, ABTS)
Cinnamomum Cassia BlumeHPLC,

ESI-MS convention (ethanol; methanol; water) [86]
160–180 °C, 35–55 min, 14.1–26.3 g/L cinnamonHPLC-ABTS+, MS, UV coumarin, cinnamic acid, cinnamaldehyde, cinnamyl alcohol, etc.   [127]
110–130°C, 20–60 min, 2–4 MPa, 1:10 g/mL HPLC  
okara   polysaccharides, TP, TF antioxidant (ABTS, DPPH) 1:30 g/mL, 160–230 °C, 10 min UV   [162]Actinidia deliciosa peels CurcumalongaTP, TF, L. rhizomesantioxidant (DPPH, ABTS, FRAP) 120–160 °C, 0–30 min, 3 MPa,

pH 2–5.5
UV-vis, pH curcumin, demethoxycurcuminconvention (ethanol 81.9%)  [87]
120–160 °C, 6–22 min, 1–2.5 MPa
Saccharina japonica  HPLC-UV, SEM   [128] polysaccharide, fucoidan, alginate antioxidant (ABTS, DPPH, FRAP) 100 –150 °C, 1–5 MPa, 1:30–1:50 g/mL IR, DSC, TGA, 1HNMR, HPLC, HPSEC-ELSD   [163] Scutellaria baicalensis root baicalin, baicalein, wogonin,

  110–160 °C, 10–90 min,

20–100 mesh
Curcuma longaHRE (ethanol 93.0%) L.[88]
rhizomes α-phellandrene, curcumin, β-caryophyllene, trans-β-farnesene, β-bisabolene, γ-curcumin, etc.
Passiflora edulis  fruit peel90–150 °C, 1–4 mL/min, 2 MPa, 0.5–1.5 mm pectic polysaccharide, mannose, glucose, etc. antioxidant (DPPH) 100–160 °C, 5.64–7.94 min, 10–30% ethanol HPLC, UV, viscometer   [164] Citrus unshiu pomaces TP, polymethoxylated flavones, sinensetin, etc. antioxidant (DPPH, TP) 25–250 °C, 10–60 min,

0.1–5.0 MPa
HPLC, UV   [89]
Chlorella vulgaris, Sargassum vulgare, Sargassum muticum, Porphyra spp., Cystoseira abies–marina, Undaria pinnatifida and Halopitys incurvus, Rosmarinus officinalis L., Thymus vulgaris, Verbena officinalis microalgae, algae, leavescitrus unshiu peels hesperidin, narirutin, prunin, naringenin, sinensetin, etc. antioxidants (DPPH, FRAP), enzyme 145–175 °C, 15 min

5 MPa, 0.75–2.2 mL/min
HPLC 2M HCl extraction 42.9%; 2 M NaOH extraction 38.9% [90]
citrus unshiu peels hesperidin and narirutin   110–200 °C, 5–20 min,

10 ± 1 MPa
HPLC, MS/MS leaves TP, TF, protein, saponin, sugar, apigenin, kaempferol antioxidants (DPPH, FRAP, ABTS), 110–270 °C, 15 min, 8 MPa

1:70 g/mL
HPLC, UV convention (water 77.7%; methanol 32.8%), Soxhlet (ethanol 43.7%) [92]
Glycyrrhiza uralensis Fisch. root TP, TF, liquiritin, flavanone, isoflavone antioxidants (DPPH, ABTS) 80–320°C, 2–100 min, 7.0 MPa, 1:30 g/mL, pH 3–11 HPLC,

UAE (water 20.6%; ethanol 44.9%), MAE (water 25.6%; ethanol 63.8%) [93]
Tagetes erecta L. flower residues TP, TF, 5-HMF, reducing sugar, free amino acids antioxidants (DPPH, ABTS) 80–260 °C, 15–90 min

1:20–1:60 g/mL,120 rpm
HPLC-DAD, UV leaching (water 9.4%; methanol 69.9%; ethanol 68.8%; acetone 94.0%), UAE (water 9.9%; methanol 69.8%; ethanol 64.3%; acetone 87.6%) [94]

GC -FID HD (80.7%), Soxhlet (n-hexane 1.2-fold) [129]
Curcuma longa L. rhizomes curcumin, demethoxycurcumin, bisdemethoxycurcumin   rhizomes curcumin, demethoxycurcumin, bisdemethoxycurcumin   90–250 °C, pH 1.0–5.5 5.0 MPa, 0.5 mL/min HPLC, UPLC, LC-MS Soxhlet (acetone, 1.17-fold) [ Daucus carota leaves polyphenols, luteolin   110–230 °C, 0–114 min, 4 MPa UV, PLC   [95]
Matricaria chamomilla L. flowers TP, TF, apigenin-7-O-glucoside, etc. antimicrobial, cytotoxic activity 200 °C, 40 min, 1:50 g/mL UHPLC, HESI-

Soxhlet (ethanol 129%), MAE (ethanol 117%), UAE (ethanol 104%) [96]
sugar, TP, melanoidins antioxidant (ABTS, O2¯) 100–200 °C, 20 min, 10 MPa UV   [165] 131]
rice bran bran protein, TCC, TP antioxidant (DPPH) 120–250 °C,0.5–5 mL/min UV, UV-Vis   [166]
Nizamuddinia zanardinii   TCC, rhamnose, xylose, arabinose, fucoidan, fucose antioxidant, anticancer, macrophage, etc. 425 rpm, 10–30 min, 90–150 °C, 0–40 mL/g, 0.75 MPa, 1500 W FT-IR, GC-MS, SEM, UV, HPSEC-MALLS-RI   [167]
Dendrobiumnobile Lindl. stems polysaccharide, arabinose, galactose, glucose, etc. antioxidant (OH·, ABTS) 0.5–1.5 MPa, 5–20 min 120–160 °C, 1:25 g/mL UV−vis, GPC, HPLC, HPAEC   [168]
Ecklonia maxima   TP, polysaccharide, sulphate, and alginate antioxidant (ABTS) 100–180 °C, 5–30 min, 10–50 mL/g, 4 MPa UV, elemental analysis, ICP-MS convention (70% ethanol 0%; 0.05 M HCl 20.1%) [169]
Vitis vinifera grape pomace glucose, fructose, galactose, arabinose, mannose, etc. antimicrobial, antioxidant (DPPH) 170–210 °C, 10 MPa, 5–10 mL/min HPLC, UV   [170]
green coffee beans spent coffee grounds carbohydrates, phenolics antioxidant, antibacterial 150–220 °C, 7 MPa, 10 mL/min, HPLC, UV,   [171]
Tamarindus indica seed TP, xyloglucan antioxidant (DPPH) 100–200°C, 1:20 g/mL SEC, UV convention (water 74.6%) [172] Silybum murianum L seeds taxifolin, silychristin, silydianin, and silybin   75–250 °C, 40–60 min, 12.5 MPa, 0.1–0.5 mm HPLC
Mentha arvensis leaves carbohydrates, apocynin antioxidant (DPPH) 180–260 °C, 1:20 g/mL, 5 min HPLC, GC-MS, UV,convention (ethanol 101%; water 43.6%) [97]
  [173] Echinacea purpurea L. flowers TP, TF antioxidant

103.4–216.56 °C, 3 MPa, 5.86–34.414 min UV-vis   [98]
Humulus lupulus pellets TP, desmetylxanthohumol, prenylflavonoids, etc. anti-inflammatory 50–200 °C, 30 min, 10 MPa HPLC,

convention (hexane 17.2%; ethanol 105%) [99]
Kunzea ericoides leaves TP, TF, 5-HMF, quercetin, catechin, syringic acid, etc. antioxidant

150–210 °C, 0–40 min

15–35 g/mL, 4 MPa
HPLC, UV convention (ethanol 37.5%) [100]
Pistacia atlantica subsp. mutica hull TP, kaffesaure, ethyl vanillin, flavanomarein, etc. antioxidant (DPPH), reducing power 110–200 °C, 30–60 min,

10–50 g/mL
HPLC-DAD, UV HWE (85 °C 42.8%) [101]
Satureja hortensis L. whole part TP, TF, rosmarinic acid, rutin, quercetin, etc. cytotoxic, antibacterial 140 °C, 30 min

4 MPa, 1:20 g/mL
HPLC-PDA, UV maceration (ethanol 57.2%), Soxhlet (ethanol 18.4%), UAE (ethanol 69.2%), MAE (ethanol 81.3%) [102]
Urtica dioica L. leaves TP, TF, twenty-seven compounds cytotoxic, antifungal, antimicrobial 125 °C, 30 min, 3.5 MPa,

1:30 g/mL
UHPLC-HESI-MS/MS UAE (water 48.5%), MAE (water 100%) [103]
Chamomilla recutita R. flowers 2 flavonoids, 4 esters, 1 amino acid, 11 phenols, etc.   150 or 200 °C, 5.0 ± 0.1 MPa,

1.7 mL/min, 40 min
UV, HPLC, GC-MS   [104]
Glycine max okara TP, gallic acid, syringic acid, ferruric acid, etc. antioxidant (ABTS, DPPH, FRAP) 150 °C, 4 MPa, 5–275 min

20 mg/mL
UV, HPLC   [105]
Carménère grape pomace flavanols, stilbenes, and phenolic acids   90–150 °C, 5 min, 10 MPa, 15–50% glycerol UPLC-MS   [106]
Zingiber officinale root TP, TF, four macro- and five microelements antioxidant (OH·, ABTS, TRP, etc.) 80–180 °C, 1 h, 5MPa,

1:10 g/mL
UV-vis, ICP-MS convention (water, 62.5%) [107]
Momordica foetida leaves quercetin, kaempferol, isorhamnetin   100–300 °C, 5 mL/s

6.9± 1.4 MPa psi
UHPLC-q-TOF-MS   [108]
Ko and coworkers have investigated the relationship between flavonoid structure and SBWE. They found that flavonoids with an OH side chain were optimally extracted at lower temperatures than O-CH3 and H side chains. The optimal temperatures of the glycoside forms are lower than that of the less polar aglycones [30]. Turner et al. found that different glycosidic compounds may be converted by their respective aglycones in less than 10 min reaction time in water from onion waste [20
2.3. Organic Acids
The use of SBWE was explored for the extraction of gallic acid, chlorogenic acid, caffeic acid, ferulic acid, vanillic acid, and coumaric acid from various matrices. Inevitably, some other active components such as phenolics [23], flavonoids [108], proteins [23], lipids, peptides, amino acids, and other organic compounds were often coextracted. Švarc-Gajić et al. [134] have used SBWE for the extraction of alcohols, organic acids, sugars, and other organic compounds from both sweet and sour cherry stems, finding the chemical compositions of the two samples similar. Harun et al. [51] reported lipid extraction with a relatively high content of eicosapentaenoic acid from Nannochloropsis gaditana, finding 237 °C and 14 min to be the optimum extraction conditions.

2.4. Glycosides

Glycosides are compounds in which sugars or sugar derivatives are bound to another type of non-sugar substance (also called aglycones, ligands or substituents). Glycosides are linked by an O- N-, S-, or C-glycosidic bond between a sugar and a non-sugar component, which are widely found in the root, stems, leaves, flowers, and fruits of plants. Most glycosides are colored crystals, and generally a little bitter.
Glycosides extracted by SBWE have been proven to have antioxidant activities and tyrosinase inhibitory activity, as shown in Table 4 [36,144,145,146,147,148,149,150,151,152]. Gao et al. [144] has performed SBWE of phenolic compounds from pomegranate seed residues at 80–280 °C. The results showed that TP increased with the rise of extraction temperature from 80 °C to 220 °C and decreased from 220 °C to 280 °C. At 80–220 °C, the breakage of the bonds led to the increase of TP, however, a higher temperature caused the phenolics to degrade. In addition, they compared SBWE with leaching and UAE using water (room temperature) and organic solvents namely methanol, ethanol, and acetone. TP and antioxidant capacities of SBWE (120 °C) were not as high as organic solvents; however, with respect to the extraction time (2 h for leaching vs. 30 min for SBWE) and toxicity, subcritical water is more acceptable. Meng and Cheng [149] have studied 13 phenolic compounds and 20 inorganic elements of Erigeron breviscapus. They also have found similar results, as the glycosides are not stable at a high temperature and with a long extraction time. For example, scutellarein and apigenine are the aglycones of corresponding acutellarin and apigenin 7-glucuronide, when at high temperature glycosidic bonds become unstable and begin to decompose to its glycone and aglycone. Haznedaroglu et al. [147] have optimized the parameters such as temperature, extraction time, and flow rate. Temperature and extraction time were found as the most effective parameters for TP and total flavonoids while extraction time and flow rate for anthocyanin contents. In addition, temperature and time were the leading parameters for the effectiveness of extracts on tyrosinase inhibition.
Table 4. Subcritical water extraction of glycosides.
Samples Medicinal Parts Compounds Extracted Extracts Activity Extraction Conditions Analytical Methods Other Extraction Methods (Solvent, Ratios of Yields) Ref.
Phaleria macrocarpa fruits mangiferin
Mangifera indica
quercetin3-d-glucoside, mangiferin antioxidant (DPPH) 100 °C, 4 MPa, 10 g/min, 3 h UV, HPLC
2.6. Essential oils, alkaloids, quinones, terpenes, lignans, and steroids2.6. Essential Oils, Alkaloids, Quinones, Terpenes, Lignans, and Steroids