2.1. Basic Chemical Composition of Sprouts and Microgreens
The basic chemical composition of the tested sprouts and microgreens is presented in
Table 1. The sprouts and microgreens differed in their chemical composition (
p < 0.05).
Mean dry matter content ranged from 4.7 (mung bean) to 46.4% (lentil) for sprouts, and from 4.1 (kale) to 8.1% (green pea) in microgreens. This was overall similar to the values previously reported by Xiao et al. [
12]. Lower dry matter content probably reflected more advanced vegetative growth attained by the microgreens. High content of water in microgreens and some sprouts (black medick, sunflower, beetroot and mung bean) indicated high water storage capacity and adaptations preventing water loss.
The tested sprouts and microgreens showed low content of soluble solids (SSC). This was especially true for microgreens represented by kale, radish, beetroot, and amaranths, and for sprouts represented by black medick, sunflower or leek. To date, there have been no reports on SSC in sprouts and microgreens, however, their content seems to be lower than in artichoke [
14] or other vegetables. The content of sugar was only examined in sprouts of lentil, broccoli and radish (up to 1.8 g/100 g fresh weight fw). The main identified saccharide was fructose, with glucose and saccharose taking the second and third place, respectively. Sorbitol, mannose and rhamnose occurred only in leek, broccoli and lentil, respectively. The lowest sugar content was detected in black medick and mung bean (>0.2 g/100 g fw) sprouts, while in beetroot sprouts, similarly to microgreens, no sugars were identified. Our results corroborated those obtained for sea buckthorn fruits [
15] and artichoke [
14], but differed from those for microgreens analyzed by Choe et al. [
10]. Apart from their growth-related functions, sugars also serve as signaling molecules that modulate light signaling pathways. Moreover, intense sucrose accumulation in the leaves is associated with a reduction in nitrates or decreased photosynthetic rate [
16].
Sprouts and microgreens were characterized by high pH (>5.3), and titratable acidity lower than 0.7 and 0.4 g/100 fw, respectively. The content of organic acid was significantly higher for microgreens than for sprouts. In our study, oxalic acid predominated over the other organic acids, but its levels were 10 times higher in microgreens than in sprouts. The remaining organic acids, i.e., citric, malic or quinic and succinic acids, were at similar levels in both materials (
Table 1). These trends were in agreement with those reported by Silva et al. [
3]. Low amounts of phytic acid were detected only in black medick, sunflower and beetroot. Apart from different aromatic compounds, sugars, organic acids and pH are crucial for organoleptic characteristics as well as acceptability, quality and nutritional values and microbial stability. Organic acids are known for their both beneficial and adverse effects on human health. For example, citric and malic acids support the action of antioxidants due to their ability to chelate metal ions, i.e., copper and iron that catalyze adverse oxidation processes. Phytic acid affects the uptake of micronutrients in the digestive tract [
17]. On the other hand, high content of oxalic acid salts is unfavorable, because they can form hardly soluble crystals of calcium oxalate in various organs, including atherosclerotic plaques in the coronary arteries. Oxalic acid is known to reduce mineral bioavailability from food. High content of oxalic acid in microgreens is the effect of postharvest treatment with this chemical as an effective method for preserving the food against bacteria and pathogens.
Additionally, plant treatment with oxalic acid significantly delays quality deterioration during storage. The acid specifically reduces yellowing of the leaves by stopping chlorophyll degradation, slowing down the respiration rate, electrolyte leakage, and ammonia production [
18]. In recent years, many authors have reported on the beneficial effects of applying oxalic acid on artichokes [
19] or rocket and baby spinach leaves [
18].
Pectins are among the most complex components of plant cell walls. Depending on their molecular composition and chemical configuration, pectin matrices can change mechanical behavior of plants [
20] in terms of their elasticity, hardness or fibrousness. Unfortunately, the analyzed sprouts and microgreens featured low content of pectins, except for lentil sprouts. From the human perspective, pectins play an important health-promoting role by impacting metabolic and physiological processes of the organism [
20].
Mineral elements are important for human health, and their adequate daily intake is necessary for a good condition and wellness of the body [
9]. Some authors [
9,
10] suggested that microgreens are a perfect source of minerals. No significant differences between sprouts and microgreens were detected, but the content of ash was higher in microgreens, especially for amaranths and kale (
Table 1). According to previous studies, sprouts, microgreens or “baby” vegetables at early growth stages are better sources of nutrients than their mature counterparts [
21,
22]. The content of ash depends on the agrotechnical techniques and growing conditions, such as soil and climatic factors, exposure to light, and the type of species or varieties [
22].
2.2. L-ascorbic Acid Content in Sprouts and Microgreens
Significant differences were found between the content of L-ascorbic acid in microgreens and sprouts, as shown in
Table 1. Sprouts of broccoli (114.0 mg/100 g fw) and radish (94.0 mg/100 g fw) accumulated the greatest amounts of L-ascorbic acid, while its level in all microgreens fell below 32 mg/100 g fw. According to literature data [
23], L-ascorbic acid is absent in unsprouted seeds of soybean, buckwheat, and mung bean, but its level increases significantly during germination and sprouting. The content of L-ascorbic acid in sprouts and microgreens was similar to that reported by Marton et al. [
17] and Xiao et al. [
12], respectively. Its levels in coriander (13.09 mg/100 g), cress and purple basil (73.27 and 98.04 mg/100 g fw) [
4] were similar to those in the investigated microgreens.
Literature reports show that the content of L-ascorbic acid in sprouts and microgreens is not very high [
12]. It may depend on multiple factors, such as cultivar, cultivation method, biotic and abiotic stress, harvest date and storage, access to light, water and nitrogen fertilizers [
4]. Another essential factor is the time of germination, as the most intense synthesis of L-ascorbic acid occurs within a few days after sowing, and then its content is maintained at a constant level [
12,
17]. In some sprouted seeds (soybean and buckwheat), the initial peak was followed by a decrease in L-ascorbic acid content until reaching its original values [
23]. This may explain why microgreens exhibited low levels of this compound. It should be remembered that regular consumption of vitamin C helps in diminishing oxidative stress, limiting undesirable reactions of enzymatic oxidation, and is necessary for collagen synthesis in humans [
4,
12,
23].
2.3. Chlorophyll and Carotenoid Contents in Sprouts and Microgreens
The color of sprouts and microgreens is one of the first attributes that affects consumers’ choice and acceptability of the product, and, thus, it is considered an important parameter determining their quality. The results regarding identification and quantification of chlorophylls and carotenoids (carotene and xanthophylls) analyzed by UPLC-PDA-Qtof-ESI-MS system are summarized in
Table 2. Fourteen major compounds, of which eight belong to chlorophylls, were identified in sprouts and microgreens. In general, microgreens were richer in chlorophylls and carotenoids than sprouts.
The content of chlorophylls in microgreens ranged from 195.6 to 638.5 μg/g fw and decreased in the following order: amaranth > green peas >> beetroot > radish > kale. The content of chlorophylls in sprouts oscillated from 6.0 to 108.5, with the highest value recorded for lentil. Previous studies [
4] reported similar levels of chlorophylls (118.0–715.1 μg/g fw) in variable microgreens, such as mustard, pack choi, radish, or green and purple basil. The major compounds in the analyzed samples were chlorophylls (
a, a’, and
b), but some compounds, e.g., pheophytin (
a’,
b and
b’), were not evaluated in sprouts. Both chlorophyll
a and
b and chlorophyll isomers
a′ and
b′, and pheophytin
a′ are the major chlorophylls present in green plants [
24]. The primary photosynthetic pigment is chlorophyll
a. Chlorophyll
b is not necessary for photosynthesis to occur, and, therefore, not all cells that perform photosynthesis contain chlorophyll
b. An increase in the content of chlorophyll
b results from adaption to shade, as it allows a plant to absorb a broader range of light wavelengths. Light is not only crucial for plant growth and development, but it also plays an important role in enhancing cellular metabolism and synthesis of defense-related secondary metabolites [
25]. Sprouts and microgreens are very often grown in special, controlled environment growth chambers with artificial light sources [
26]. This may explain why in all the analyzed samples, chlorophyll
a predominated over chlorophyll
b. The chlorophyll
a:
b ratio reflects the conditions in which sprouts and microgreens were cultivated. A high ratio indicates that a sample was exposed to the sun or intense light, as the high level of chlorophyll
b is responsible for the more yellow color. In microgreens, this ratio was at a similar level (1.8–1.9), but in sprouts, chlorophyll
a was up to 8.8 times more abundant than chlorophyll
b, which meant than sprouts were richer in chlorophyll
a than
b, and the green color was more intense. Kyriacou et al. [
4] determined the chlorophyll
a:b ratio in mibuna and radish microgreens, which reached 2.85 and 4.20, respectively.
Yellow-orange fruits and green leafy vegetables are generally considered the richest source of carotenoids. Our study demonstrated an increase in carotenoid levels at germination and sprouting time, which makes sprouts and microgreens moderately good sources of these compounds. The content of carotenoids followed the same pattern as the content of chlorophyll (
Table 2). In microgreens, it ranged from 1510.1 to 4073.5 μg/g fw and decreased in the following order: amaranth > kale > green pea > beetroot > radish. The content of carotenoids in sprouts was more variable and ranged from 22.5 (mung bean) to 948.8 (radish) μg/g fw. Major compounds in the analyzed sample included oxygenated carotenoids, such as xanthophylls (lutein, neo- and zeo-xanthin) and carotenes
α+β. The analyzed microgreens were richer in (
α+β)-carotene, lutein, zeo- and neo-xanthin and neochrome than sprouts. According to the literature, plant parts exposed to sun or light generally accumulate significantly greater amounts of xanthophyll and
β-carotene and trace amounts of
α-carotene.
α- and
β-carotene and cryptoxanthins are precursors of provitamin A [
27], and, therefore, their levels were calculated. The content of provitamin A in sprouts was approximately 21 times lower (
p < 0.05) than in microgreens. The content of provitamin A in microgreens ranged from 281.6 to 698.6 μg RAE (recommended dietary allowance) [
27], which means that a diet containing radish or kale helps in meeting daily dietary recommendations for this compound. Daily carotenoid intake in adults in European countries, e.g., Spain, reaches 9.54, but in France, it is up to 16.06 mg [
27].
Carotenoids play a major role in the protection of plants against photooxidative processes [
25] and in the protection of humans against cancers of, e.g., liver, colon, lung, pancreas or prostate [
10,
28]. They are also efficient antioxidants [
22,
28]. Consumption of carotenoid-rich products has been demonstrated to bring health benefits in the form of ameliorating degenerative and cardiovascular diseases [
7,
10,
29].
2.4. Polyphenol Content in Sprouts and Microgreens
UPLC-PDA-FL analysis of the methanolic extracts of sprouts and microgreens revealed their phenolic composition (
Table 2). Polyphenol profiles differed significantly across sprout and microgreen samples, and variations were observed with respect to major phenolic components. All samples contained some flavan-3-ols (catechins and polymeric procyanidins), phenolic acids (mainly caffeoylquinic, ferulic, sinapic, chlorogenic and their derivatives), flavonols (mainly quercetin, kaempferol, isorhamnetin), and flavones (daidzein and genistein). Some samples (radish, beetroot, amaranth) comprised also anthocyanins, mainly cyanidin derivatives. Phenolic acids, such as caffeoylquinic, ferulic, sinapic, or chlorogenic acid and their derivatives, were common in all investigated samples. Dominant plant flavonols include derivatives of quercetin, kaempferol, and isorhamnetin [
2,
8]. Sprouts were richer in total polyphenols (26.7–191.1 mg/100 g fw; especially radish, lentil, broccoli, and sunflower) than microgreens (23.3–132.9 mg/100 g fw; especially amaranth and green pea).
Phenolic acids were a dominant group of polyphenols only in sprouts of radish, broccoli and sunflower (55, 60, 68% of total phenolic compounds, respectively) and in microgreens of green pea and amaranth (23 and 32% of total phenolic compounds, respectively). Lentil sprouts were rich in polymeric procyanidins and isoflavones, and broccoli sprouts additionally in monomeric flavan-3-ols and polymeric procyanidins. Anthocyanins were quantified in radish and beetroot sprouts and microgreens, and in amaranth microgreens, they accounted for 35% of total phenolic compounds. As mentioned above [
2,
4], microgreens contained only scant amounts of flavonols and their glycosides that are common in mature green vegetables, which suggests significant phenolic transformation during plant ontogenesis. L-phenylalanine is converted into
trans-cinnamic acid by phenylalanine ammonia lyase during synthesis of phenolic compounds. It can be therefore expected that sprouts and microgreens are rich in phenolic acid and flavan-3-ols that are primary compounds synthesized via the phenylpropanoid metabolic pathway. The sprouts of cress are particularly rich in flavonoids, and of radish in phenolic acids, while lentil sprouts are less abundant in these compounds [
17].
Samotyja et al. [
1] determined polyphenol content in the sprouts of five species (mung bean, radish, sunflower, wheat and lentil) and found their highest levels in sunflower and radish. The content of polyphenolic compounds doubles in germinated seeds of quinoa, buckwheat and wheat as compared with their seed levels. Most of these compounds were found in buckwheat seeds and sprouts. Kyriacou et al. [
4] examined 13 microgreens belonging to
Lamiaceae,
Brassicaceae,
Malvaceae,
Chenopodiaceae, and
Apiaceae species, and found green and purple basil microgreens (
Lamiaceae) to be the richest in phenolic compounds. Additionally, they identified alternative phenolic-rich microgreens from the
Apiaceae family. Cevallos-Casals and Cisneros-Zevallos [
5] compared polyphenolic content in the seeds and sprouts of 13 different crops (broad bean, sunflower, soybean, mung bean, radish, fenugreek, broccoli, lentil, wheat, kale, mustard, alfalfa and onion), and found the sprouts of mustard, sunflower and broccoli the most abundant in these compounds. A study by Silva et al. [
3] demonstrated that the sprouts of alfalfa, soybean and mung bean are a rich source of numerous bioactive compounds with antioxidant properties (phenolic acids, flavones, flavonols, isoflavones, monoterpenes) that may inhibit or delay cell degeneration and damage caused by oxidative stress.
2.5. Amino Acid Content in Sprouts and Microgreens
This work presents a qualitative and quantitative analysis of free amino acids in the sprouts and microgreens performed using a derivatization method with AQC and LC-PDA-QTof-ESI-MS operated in negative ionization mode. We analyzed 24 amino acids of variable abundance as present in
Table 3. Two the most abundant free amino acids in sprouts and microgreens included L-asparagine (particularly high content in lentil, black medick, and leek sprouts and radish and green bean microgreens), and L-glutamine (particularly high content in radish, broccoli, sunflower, leek, and beetroot sprouts, and in kale and amaranth microgreens). The content of L-ornithine (only characteristic of lentil sprouts and kale microgreens), L-proline, L-methionine (not detected in beetroot sprouts and microgreens and amaranth microgreens), L-homocysteine (only characteristic of black medick sprouts and kale microgreens) in sprouts was always very low (<1%). Additionally, L-cysteine was detected in microgreens, but L-proline was absent in these samples.
The sum of free amino acids was generally higher in sprouts and ranged from approximately 162.3 (radish) to 455.0 (beetroot) mg/100 g fw for microgreens, and from 321.4 (beetroot) to 1292.1 (lentil) mg/100g fw for sprouts (
Table 3).
The concentration of amino acids in radish or beetroot sprouts and microgreens varied significantly, and might, as suggested by Silva et al. [
30], result from metabolic changes during growth and ripening. According to Tarasevičiene et al. [
31], amino acid levels in broccoli were growing for up to 72 h of germination and then stabilized to remain very similar after 120 h. These authors [
31] indicated that changes in amino acid content during germination could be related to protein hydrolysis, synthesis, and re-arrangement that involved mobilization of protein reserves in cotyledons and synthesis of new proteins necessary for the sprout growth. Furthermore, the amino acids produced by hydrolysis of the protein reserves are not used solely for the synthesis of new components but may also be used as energy sources, especially at early stages of germination.
Generally, even though the content of amino acids in sprouts and microgreens is low, they are still important sources of these compounds, as some amino acids cannot be produced by the human body and need to be supplied with food (e.g., L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-threonine, L-tryptophan, and L-valine). The content of these amino acids ranged from 56.3 (beetroot) to 248.2 (broccoli) mg/100g fw in sprouts and from 24.6 (beetroot) to 83.7 (radish) mg/100 g fw in microgreens (
Table 3). Apart from nutritional properties, amino acids also affect taste and flavor, as a number of them have a distinctively bitter taste (e.g., tyrosine, arginine, leucine, valine, methionine, and histidine). Additionally, methionine and L-homocysteine, as amino acid thiols, play an important role in abiotic stress tolerance in plants, and they are the most important antioxidants that protect cells from oxidative damage [
32].
2.6. Biological Properties of Sprouts and Microgreens and Their Anti-Oxidant, Anti-Diabetic, Anti-Obesity and Anti-Cholinergic Activity
Sprouts and microgreens are rich in natural antioxidants, including phenolic compounds, carotenoids, chlorophylls, ascorbic acid, etc., that determine their considerable antioxidant capacity. Antioxidant properties were assessed using (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), ferric reducing ability of plasma (FRAP), and oxygen radical absorbance capacity (ORAC)) assays. As shown in
Table 4, sprouts accumulate significantly higher amounts of oxygen radical scavengers than microgreens. ORAC capacity in sprouts was the highest in beetroot and descended in the following order: beetroot, broccoli > radish, lentil >> black medick, sunflower > leek, mung bean, while in microgreens, this order was as follows: beetroot >> kale > radish > amaranth > green pea.
A similar tendency was observed for scavenging radical ABTS and reduced ferrous ion (Fe
+3) in the FRAP assay. Overall, the antioxidant capacity was higher in sprouts than in microgreens, with a notable exception of mung bean sprouts. The antioxidant capacity measured for mung bean sprouts was below 0.01 mmol Trolox/100 g fw. Mung bean sprouts had low content of polyphenols, chlorophylls and L-ascorbic acid. Previous works [
5,
15,
33] reported that antioxidant potential can be related to the content of phenolics and other bioactive compounds. Antioxidant capacity measured by ABTS and FRAP assays correlated with the content of L-ascorbic acid (r
2 = 0.671 and 0.872); polyphenols (r
2 = 0.682 and 0.538), particularly flavan-3-ols (r
2 = 0.742 and 0.649); and polymeric procyanidins for the ABTS assay (r
2 = 0.758). For the ORAC test, the strongest correlations were determined for polyphenols (r
2 = 0.327), flavan-3-ols (r
2 = 0.481) and L-ascorbic acid (r
2 = 0.438). Other bioactive compounds, such as volatiles and organic acids found in the sample, should not be ignored, as they may contribute to the obtained results. Silva et al. [
3] mentioned that volatile compounds or organic acids present in the sprouts were reported as antioxidants, and the antioxidant capacity of the sprouts resulted from their interactions. As suggested by Cevallos-Casals and Cisneros-Zevallo [
5], consumption of fresh sprouts could provide similar antioxidant benefits to those offered by commonly consumed fruits.
The anti-diabetic (α-amylase and α-glucosidase) and anti-obesity (pancreatic lipase) assays were used to determine biological activity of the samples (as IC
50).
Table 4 presents differences in inhibitory activity of α-amylase and α-glucosidase in the tested sprouts and microgreens. The strongest inhibition of α-amylase and α-glucosidase, constituting an alternative approach to preventing diabetes mellitus, was found in the sprouts of black medick, mung bean, beetroot, and broccoli, and in beetroot microgreens. Considerable inhibition of α-glucosidase was also determined for radish microgreens and leek sprouts. High inhibitory activity towards pancreatic lipase was noted for all investigated sprouts and microgreens (IC
50 < 0.6), except for lentil sprouts (
Table 4). Our data indicate that sprout and microgreen extracts inhibit pancreatic lipase with similar intensity and are considerably less efficient in inhibiting α-amylase and α-glucosidase. Polyphenols are well known for their capacity of binding proteins by hydrogen bonding or hydrophobic effects, leading to their complexation and precipitation. This means that extracts of different plants could potentially inhibit enzymes by aggregation.
The hypoglycemic effect of polyphenolic compounds is also due to their antioxidant potential involved in restoring the insulin-secreting machinery in pancreatic cells, or their ability to inhibit the activity of carbohydrate-hydrolyzing enzymes (α-amylase and α-glucosidase) [
10,
11,
34].
Additionally, bioactive free amino acids are produced during germination and then in sprouts and microgreens [
35]. Free amino acids, such as leucine, arginine, alanine, phenylalanine, isoleucine, lysine, and methionine, are known stimulators of insulin production and release from pancreatic cells, as they participate in the synthesis of phosphatidylinositol-3-kinase, which is an important component of insulin receptor/tyrosine kinase. Therefore, soybean sprouts may be, for example, used to reduce diabetes prevalence in Indonesia [
35]. Our study confirmed a strong correlation between amino acid content and anti-diabetic (r
2 = 0.716 and r
2 = 0.502 for α-amylase and α-glucosidase, respectively) and anti-obesity activity (r
2 = 0.839). For comparison, a correlation between polyphenols and anti-diabetic and anti-obesity activity was lower than r
2 = 0.630, except for polymeric procyanidins (r
2 > 0.940) and flavonols (r
2 > 0.710). The beneficial health effects of sprouts of many plant species were confirmed in human studies. A randomized clinical trial [
36] involving patients with type 2 diabetes showed that a four-week diet enriched in pulverized broccoli sprouts significantly lowered blood levels of triglycerides, the oxidized low density lipoprotein (LDL) to LDL ratio, the atherogenic index of plasma (API), insulin and the insulin resistance index (HOMA-IR) as compared with the control.
Inhibition of cholinesterase activity is of key importance in symptomatic treatment of Alzheimer’s disease [
14]. It is crucial that the patients’ diet contain potential inhibitors that block the enzyme and thus increase the content of acetylcholine in cholinergic synapses and improve neurotransmission. Some sprouts and microgreens were characterized in terms of their capacity to inhibit the activity of acetylcholinesterase (AChE) and butylcholinesterase (BuChE), as shown in
Table 4. Generally, inhibition of AChE and BuChE was species-dependent, as it was impossible to indicate which group (sprouts or microgreens) provided more effective inhibition. The strongest inhibition of AChE and BuChE was noted for radish and kale sprouts and for green bean and amaranth microgreens. Lentil and black medick sprouts and radish and beetroot microgreens were at the opposite site of the spectrum. The inhibitory effect on AChE and BuChE was stimulated by polyphenols, carotenoids and L-ascorbic acid, resulting in average correlation coefficients of r
2 = 0.431–0.412, r
2 = 0.386–0.387, and r
2 = 0.577–0.507, respectively. Previous works [
33,
37] indicated that the inhibitory activity depended on the number and position of -OH (especially in position
orto) and/or -OCH
3 groups substituted in the phenyl ring. The same authors [
33] reported on a strong inhibition of AChE and BuChE by chlorogenic acid and flavonol derivatives. According to Silva et al. [
3], who analyzed
Glycine max L. Merr.,
Vigna radiata L. and
Medicago sativa L. sprout extracts, no AChE inhibitory activity was found at the tested concentrations.
2.7. Principal Component Analysis (PCA)
PCA was employed to better understand the relationship between biological activity and bioactive compounds and chemical composition of sprouts and microgreens.
Figure 1 is a bidimensional representation of all of the variables and samples of sprouts and microgreens defined by the two first PCs as PC1 vs. PC2. Four PCs explained the cumulative percentage of total variation, while PC1 and PC2 explained 64.28% of total variation. PC1 (right side of the figure) explained 43.21% of the total variation and accounted mainly for bioactive compounds (sum of polyphenols, amino acids, and L-ascorbic acid) and biological activity (as ORAC, ABTS, FRAP, α-amylase and α-glucosidase, pancreatic lipase, AChE, and BuChE). However, antioxidant capacity might also be conferred by polyphenolic compounds and L-ascorbic acid. Broccoli, radish, and lentil sprouts were plotted among positive PC1 values, whereas the other sprout and microgreen samples were plotted in the negative half of the chart. PC2 explained 21.07% of total variation and accounted mainly for antioxidant capacity (ORAC, ABTS, FRAP), anticholinergic activity (AChE, BuChE), α-amylase, sum of polyphenols and carotenoids, chlorophylls, organic acids, L-ascorbic acid, ash, sugars, and titratable acidity. Kale, amaranth, and green pea microgreens exhibited a high total content of carotenoids and chlorophylls, contrary to lentil sprouts which were rich in amino acids. The other samples, including microgreens of beetroot and radish and sprouts of beetroot, sunflower, leek, black medick, and mung bean, were plotted mainly at the bottom of the chart due to their lowest biological activity and low content of selected bioactive compounds.
