Investigation of Chemical Constituents and Antioxidant Activity of Biologically Active Plant-Derived Natural Products

The aim of this publication is to present rapid screening methods (visual/colorimetric) that will enable quick identification of the presence of biologically active compounds in aqueous solutions. For this reason, 26 plant extracts obtained by ultrasound-assisted extraction were analysed for the content of these compounds. Higher plants, used as a raw material for extraction, are common in Europe and are easily available. The article proposes a comparison of various protocols for the identification of various compounds, e.g., phenolic compounds (phenols, tannins, anthocyanins, coumarins, flavones, flavonoids), vitamin C, quinones, quinines, resins, glycosides, sugars. Initial characterisation of the composition of plant extracts using fast and inexpensive methods allows you to avoid the use of time-consuming analyses with the use of advanced research equipment. In addition, the antioxidant activity of plant extracts using spectrophotometric methods (DPPH, ABTS, FRAP assay) and quantitative analysis of plant hormones such as abscisic acid, benzoic acid, gibberellic acid, indole acetic acid, jasmonic acid, salicylic acid, zeatin, zeatin riboside, and isipentenyl adenine was performed. The obtained results prove that the applied visual methods show different sensitivity in detecting the sought chemical compounds. Therefore, it is necessary to confirm the presence or absence of bioactive substances and their concentration using modern analytical methods.

The impressive contribution of plant-based extracts to virtually all aspects of human life has promoted their use to an increasing extent. For this reason, it is crucial to accelerate and reduce the cost of the production of new and innovative bioproducts and solutions. In view of the fact that the extraction process is a crucial first step in the development of new formulations, the research within this article has been designed to present the methods that could be used as a primary screening when no data are available on the chemical composition of examined extracts to evaluate the efficiency of the extraction techniques, to ensure that the active ingredients were not destroyed during preparation, and thus to reduce the time and costs of further purification of the obtained natural products. The choice of our examined plants was based on the ease and economic acquisition of raw materials (plants commonly found in the natural environment) and richness of active compounds that may be found in them. A total of 26 different extracts were tested for the content of phenolic compounds (phenols, tannins, anthocyanins, coumarins, flavones, flavonoids), vitamin C, quinones, quinines, resins, glycosides, sugars, antioxidant activity, and plant hormones. In most of the analyses, basic qualitative methods were used to provide a quick answer regarding the content of specific active compounds. The extracts were produced by means of ultrasound-assisted extraction, which is considered as a more environmentally friendly technique while allowing the extraction of bioactive compounds on a larger scale.
The aim of the publication was a comprehensive characterisation of plant extracts obtained from a number of higher plants. A given compound was determined using a series of methods, due to which it was possible to select visual protocols, the most sensitive ones indicating the presence of the given compound in the extract.

Results
The tested methods allowed rapid identification of the presence or absence of bioactives in the extract; however, in order to determine the exact amount of tested compounds, it is necessary to use more sophisticated analytical methods.
Throughout the paper, the following abbreviations were used for the particular extract: Alv L (solution/extract prepared based on aloe leaves), Am Fr (black chokeberry fruits), Arv H (common mugwort herb), Bv R (beetroot roots), Co F (common marigold flowers), Ea H (field horsetail herb), Ep F (purple coneflower flowers), Ep L (purple coneflower leaves), Hp H (St. John's wort herb), Hr Fr (sea-buckthorn fruits), Lc S (red lentil seeds), Mc F (chamomile flowers), Ob H (basil herb), Pm H (broadleaf plantain herb), Poa H (common knotgrass herb), Ps S (pea seeds), Pta L (common bracken leaves), Sg L (giant goldenrod leaves), So R (comfrey roots), To F (common dandelion flowers), To L (common dandelion leaves), To R (common dandelion roots), Tp F (red clover flowers), Ur L (nettle leaves), Ur R (nettle roots), Vo R (valerian roots). In order to better visualise the obtained effects, the tables also show the tube with the extract before treatment (always the first on the left). The changes were usually observable immediately (up to 5 min) after following the appropriate procedures.

Phenolic Compounds (Total Phenolic Compounds, Tannins, Anthocyanins, Coumarins, Flavones, Flavonoids)
Several protocols for rapid phytochemical screening can be used to determine the presence of bioactive compounds in the examined samples. According to the literature, to assess the prevalence of phenolic compounds the ferric chloride test can be implemented. Authors who used this method found that these compounds were present after the appearance of a dark green [30][31][32], deep blue [31], violet [33], bluish black [34][35][36], or bluish-green [36] colour. Similar results were presented by other researchers who stated that violet [37], blue or green [38], or deep blue or black colour [39] indicates the presence of phenols. The second method, the lead acetate test, is also widely applied to detect these compounds in samples. Their presence can be confirmed when white precipitate is developed [31,40]. However, it is worth mentioning that the lead acetate test reveals very little helpful information and has the drawback of involving the heavy metal, lead, which creates environmental disposal problems. As a third method, the zinc hydrochloride test can be deployed-the appearance of yellow or orange colour after a few minutes proves the presence of phenols [37]. In another method, the Shinoda test, a yellow or orange colour demonstrates their existence [37]. The total phenolic content can be quantified using the Folin-Ciocalteu test, and when the bluish colour occurs it confirms the presence of phenolic compounds and their concentrations are verified by measuring the absorbance of the solutions [41].
The formation of green-blue [39], violet or blackish red [33,37] colouration in the ferric chloride test [39]; the yellow precipitate in the lead acetate test [31,37,39,42,43]; a red or magenta colour in the zinc hydrochloride test [37]; or a pink scarlet, green to blue, or crimson red colour emerging within minutes in the Shinoda test indicates flavonoids [31,33,36,37,43]. In the alkaline reagent test, the addition of sodium hydroxide solution causes an intense yellow colour which changes to colourless after the addition of hydrochloric acid, which may also suggest their presence [30,34,37]. In the aluminium chloride test, if the addition of aluminium chloride solution induces the light-yellow colour, the existence of flavonoid is observed. The addition of sodium hydroxide and hydrochloric acid makes the solution colourless, which also confirms their presence [32,39]. Among other methods used to identify these compounds, the ammonium test (a yellow colour at the ammonia layer [37,39]), the ammonia and sulphuric acid test (a yellow colour [30]), and the Millon's test (a white precipitate which turns to red after gentle heating [37]) can be mentioned. Photos of the Millon's test are presented in our previous article, where we conducted the analyses of proteins [44].
The ferric chloride test is likewise used for the analysis of tannins. Their presence can be confirmed when the formation of a greenish black precipitate [38][39][40]42,43,45] or a green, violet [37], or dark blue [33,38] colour is observed. Other authors have stated that the greater addition of ferric chloride changes the blue or greenish black colour to olive green [46]. The occurrence of a blackish blue colour indicates the presence of gallic tannins and a green-blackish colour shows the presence of catechol tannins [47]. The yellow [34,37,45,48] or white coloured precipitate in the lead acetate test [42,43] or a yellow to red precipitate in the alkaline reagent test may indicate the presence of tannins in the solution [37]. These compounds can also be detected in samples using other tests, among others: gelatin test (the white precipitate [37]), potassium dichromate test (the yellowish brown colour precipitate) [45], HCl test (the red coloured precipitate-phlobatannins [34,38,39,47]), and bromine water test (the buff coloured precipitate-condensed tannins; no precipitatehydrolysable tannins [49]).
The sodium hydroxide test is employed in the analysis of anthocyanins (a blue-green colour [39]) and coumarins and flavones (a yellow colour [33,34,38,42]). In the sulphuric acid test, the yellowish orange colour indicates flavones [40] or anthocyanins, orange to crimson indicates flavonones, and yellow to orange colour indicates flavones [37].
The bromine water test can be used to detect the presence of glycosides (a yellow precipitate develops [37]) and carbohydrates (the solution discolours (by aldose)) [37]).
In the case of our results, the ferric chloride test clearly identified the presence of phenolic compounds in the following extracts: Am Fr, Ea H, Ep F, Ep L, Hr Fr, Pm H, Poa H, To F, To L, Tp F, Ur L. The colour of the extracts changed into a uniform dark green colour, without precipitation or turbidity of the solution (Table 1). Quantitative analysis of total polyphenol content, with the use of the Folin-Ciocalteu test, confirmed that qualitative methods show different sensitivities-indicating the presence of TPC in extracts that contain a great as well as a low amount of them but do not show their content even although these compounds are present. The highest levels of TPC could be found in Ep F, Pta L, and Ep L (3.2-2.2 mg·mL −1 ) and the lowest in Ps S, Ur R, Ur L, Lc S, and To R (0.07-0.18 mg·mL −1 ) ( Table 1). The appearance of a white precipitate in the lead acetate test indicates the presence of phenols- Table 2. This was observed with the following extracts: Alv L, Bv R, Mc F, Ob H, Pm H, Ur R. White precipitation can also indicate the presence of tannins. Evident yellow/orange colour of the solution, which is typical for the presence of phenols in the zinc hydrochloride test, was observed in the extract Bv R, Co F, Ep F, Ep L, Sg L, Tp F, Ur L, Ur R, Vo R ( Table 2). The results for the Shinoda test in most cases coincide with the results for the zinc hydrochloride test, used to detect phenols in plant extracts ( Table 2).

Arv H
The appearance of a dark green colour and the precipitation of a fine precipitate were observed + + − 1.00 ± 0.12

Bv R
The appearance of a brown colour and the formation of a precipitate were observed − − − 0.53 ± 0.02

Co F
A change in colour of the solution to dark green and a jelly-like consistency was observed

Ea H
The appearance of a dark green colour was observed + + − 0.42 ± 0.02

Ep F
The appearance of a dark green colour was observed + + − 3.17 ± 0.03

Ep L
The appearance of a dark green colour was observed

Hp H
A dark green colour change and precipitation were observed

Hr Fr
The appearance of a dark green colour was observed + + − 0.52 ± 0.02 Poa H-common knotgrass herb; Ps S-pea seeds; Pta L-common bracken leaves; Sg L-giant goldenrod leaves; So R-comfrey roots; To F-common dandelion flowers; To L-common dandelion leaves; To R-common dandelion roots; Tp F-red clover flowers; Ur L-nettle leaves; Ur R-nettle roots; Vo R-valerian roots.      The ferric chloride test was inconclusive in the determination of flavonoids in the extracts (Table 1). According to literature data, the appearance of a green-blue colour may indicate the presence of flavonoids [39]. No such change was observed for any of the tested extracts. The potential presence of flavonoids in the extract should be confirmed by another method. The Millon's test (vide Table 4 in the work of Godlewska et al. [44]) revealed that the formation of precipitation, which could be considered as a positive result for the presence of flavonoids, occurred only in tubes with Co F (before boiling (1) an orange-brown precipitation formed, while after boiling (2) a white precipitation formed), EP F ((1) a brown precipitate, (2) a red precipitate), Lc S ((1) a white precipitate, (2) a white precipitate), Ps S ((1) a yellowish precipitate, (2) a white precipitate), So R ((1) a brown precipitate, (2) a brick-red precipitate). Yellow precipitation in the lead acetate test typical to tannins and flavonoids present in extracts was detected for Arv H, Co F, Ea H, Poa H, Pta L, To F, To L, To R, Vo R ( Table 2). The colour change of the extract in the zinc hydrochloride test to red/magenta, indicating the presence of flavonoids, performed with the same test, was observed only for the extract Am Fr ( Table 2). The presence of flavonoids detected by the Shinoda test was in the following extracts: Am Fr, Bv R, Ep F, Hr Fr, Lc S, Ps S, Tp F. The Shinoda test was more effective in detecting flavonoids in plant extracts than the zinc hydrochloride test ( Table 2). The use of the alkaline reagent test did not allow the detection of flavonoids in plant extracts ( Table 3). The ammonium test did not give a clear answer as to the content of flavonoids ( Table 6). The unequivocal yellow colour, which indicates the presence of flavonoids in plant extracts, was observed only for Hr Fr, Poa H, and To R. A yellow colour, which indicates the presence of flavonoids in extracts using the ammonia and H 2 SO 4 test, was observed for Co F, Ea H, Hr Fr, Mc F, To F, To R, and Tp F. After applying the ammonium chloride test, discoloration of the solution to some degree could be observed in most cases (with the exception of Am Fr).          In the case of tannin identification using the ferric chloride test, in addition to the greenish-black colour, which is typical for phenolic compounds, a precipitate was also observed, especially in the following extracts: Arv H, Bv R, Hp H, Mc F, Ob H, Pta L, Sg L, So R, Vo R (Table 1). In the case of the determination of tannins by the gelatin test, a change in the colour of the extract was mainly observed, and not the formation of a characteristic white precipitate (Table 3). This has been seen with the following extracts: Hp H, Lc S, and Ps S. A yellow to red precipitate indicating the presence of tannins in the plant extracts (alkaline reagent test) was present only in a few extracts: Ea H, Lc S, Pta L, To F, and Ur L ( Table 3). Using the bromine water test, no tannins were detected in most botanical extracts ( Table 3). The use of the potassium dichromate test did not allow the detection of tannins in plant extracts (Table 4). For this reason, the dichromate test for identifying tannins is not recommended, as it has given all negative results, and additionally dichromate poses a disposal issues. Furthermore, the bromine water test very rarely gave positive outcome for any class of compound and could easily be recommended not to be used. The characteristic yellowish-brown precipitate was not observed. A similar situation occurred in the case of detecting tannins (phlobatannins) with the HCl test. Dark (red) colour precipitate was observed only in the following extracts: So R and Vo R (Table 4).      Using the NaOH test, the presence of anthocyanins was not detected in the botanical extracts (Table 5). In none of the cases was the colour of the extract blue-green. The appearance of a yellow colour in the extract during this test indicates the presence of coumarins and flavones. Such a colour was unequivocally observed in the extracts Hr Fr, Mc F, Poa H, and To R. The H 2 SO 4 test in many cases did not give a clear answer as to the presence of anthocyanins and flavones in plant extracts. A stable yellowish-orange colour that indicated the presence of flavones and anthocyanins was observed for Alv L, Am Fr, Hp H, Poa H, Pta L, Tp F, Ur L.     Poa H-common knotgrass herb; Ps S-pea seeds; Pta L-common bracken leaves; Sg L-giant goldenrod leaves; So R-comfrey roots; To F-common dandelion flowers; To L-common dandelion leaves; To R-common dandelion roots; Tp F-red clover flowers; Ur L-nettle leaves; Ur R-nettle roots; Vo R-valerian roots.

Vitamin C
In the DNPH test (2,4-dinitrophenylhydrazine), the formation of yellow precipitate indicates the presence of vitamin C [34]. The presence of vitamin C, using the DNPH test, was observed only for Lc S and Ps S extracts (Table 6).

Quinones, Quinines, Resin
The literature shows that the sulphuric acid test (the appearance of red colour) [38,42,43], the hydrochloric acid test (the formation of yellow precipitation) [34,37], and the ammonia test (a pink coloured precipitate) [38] can be applied to detect the presence of quinones/anthraquinones. In the sodium hydroxide test, a deep colouration (e.g., purple, red) can be attributed to the presence of quinine [33]. Furthermore, in the acetone test, a turbid solution implies the presence of resin [33].
The application of the H 2 SO 4 test, HCl test, ammonia test, and NaOH test did not allow the detection of quinones and quinines in plant extracts (Table 7).                         The acetone test was used to detect resins in plant extracts. Their presence (turbidity of the solution) was confirmed in the following extracts: Alv L, Am Fr, Arv H, Co F, Ea H, Lc S, Ob H, Pm H, Ps S, Pta L, Sg L, So R, To F, Ur R, and Vo R.

Glycosides
Glycosides can be found in samples using a number of rapid approaches. Authors who used the Keller-Killiani test showed that the presence of brown [36,38,42,43] or a reddish-brown ring at the junction of two layers [45] indicates the appearance of cardiac glycosides. Other authors stated that cardiac glycosides are present in sample when the colour of the acidic layer above the ring changes to bluish green [37,45] or greenish [36] and the lower layer to reddish brown [37] or violet [36]. In the Baljet test, the yellow to orange colour exhibits the occurrence of cardiac glycosides [37]. In the Borntrager's tests (1), the anthraquinone glycosides can be found in samples when the ammoniacal (lower) layer shows a rose, pink, or red colour [37,39,42,43,50]. In the modified Borntrager's tests (2), the pink colour indicates the presence of glycosides [32,38]. In the sulphuric acid test, the appearance of reddish precipitate indicates the presence of glycosides [40]. Photos are available in our previous article, in analyses of protein content (vide Table 4 in the work of Godlewska et al. [44]). The Molisch test can also be used as another method. In this protocol, the formation of a reddish-violet ring at the junction of two layers confirms the presence of glycosides [40]. The next method is Liebermann's test, in which the appearance of a colour from violet through blue to green suggests the presence of glycosides [34]. Photos are presented in our previous article (vide Table 5 in the work of Godlewska et al. [44]).
No glycosides were detected in most botanical extracts using the bromine water test ( Table 3). The use of the Baljet test did not show the presence of cardiac glycosides in most of the extracts tested (Table 8). Molisch's test can be used to quickly screen extracts for the content of glycosides and sugars. The appearance of a reddish-violet ring at the junction of two liquids was easily visible in many botanical extracts ( Table 9). The Borntrager test (2) was not effective in the detection of glycosides as well as sugars, and neither was the Borntrager test (1) in the detection of cyanogenic glycosides in the tested plant extracts. In all tubes subjected to the Liebermann's test, no violet or blue colour was observed, which could likewise indicate the presence of these compounds. Extracts that may be considered to contain glycosides to some extent due to the greenish colour are Ep L and Mc F.
The Keller-Killiani test (Table 9), like the Baljet test (Table 8), did not provide full clarity on the presence of cardiac glycosides in plant extracts.

Sugars
Various protocols can be used to detect the presence of sugars. One of them is the Fehling's test. The simple (reducing) sugars are present in samples when first a yellow, then a brick red precipitate is noted [31,33,37,39,42,43,45,47]. The next one is Benedict's testwhen the solution turns green [42,43] or red [31,40], or if the reddish-brown precipitate forms [33] it might suggest the presence of carbohydrates/reducing sugars. In the Molisch's test, the appearance of a purple or reddish colour [38,47] or purple [30,34,37,40,45] or red brown [31,40,45] coloured ring at the junction of the two liquids shows the occurrence of carbohydrates. Additionally, the Borntrager's test can also be applied, and when a change in colour of the ammonia layer is observed it indicates the presence of carbohydrates [37]. In the Selwinoff's test, a red colouration implies fructose content in the solution [37], while in the Barfoed's test, the formation of red precipitation reveals the presence of monosaccharaides [47].
The deployment of the bromine water test did not allow the determination of sugars in most botanical extracts (Table 3). No yellow/red precipitate was observed after using Fehling's test, indicating the presence of sugars in the extracts (Table 10). Benedict's test showed a clear change in the colour of the extract to green and the formation of a red-brown precipitate, which indicated the presence of sugars (reducing sugars) in almost all extracts tested. Selwinoff's test gives a red coloured compound when linked with resorcinol. The colour of the extracts changed to red for Am Fr, Bv R, and Hp H. The red precipitate is the result of the Barfoed test, which indicates the presence of simple sugars and was observed in the following extracts: Arv H, Pta L, and To R.

Antioxidant Activity
Plant-derived extracts possessed varied antioxidant activity (Table 11). The analysis conducted using the DPPH assay showed that the highest radical scavenging potential demonstrated the following extracts: Pta L, Hp H, Ep F, Am Fr, Sg L, To L, and Ob H (9.57-2.48 µM Trolox·mL −1 ) and the lowest: Lc S, Ur L, Ur R, and Ps S (0.14-0.15 µM Trolox·mL −1 ). The greatest DPPH inhibition ratio showed extracts based on Pm H, Hr Fr, and Arv H (31.58-28.12%), while the smallest were based on Lc S, Ur L, Ps S, Ur R, and Ep L (2.00-2.37%). On the other hand, the relative ability of the antioxidants present in bioproducts to scavenge the ABTS free radicals was the strongest in Ep L, Ep F, Hp H, Am Fr, To L, Poa H, and Pta L (19.00-6.33 µM Trolox·mL −1 ), and the weakest in To R, Alv L, Ur L, Ea H, Hr Fr, and Lc S (0.81-1.90 µM Trolox·mL −1 ). The ABTS inhibition ratio was the highest for Poa H (5.37%) and So R (4.47%) and the lowest for Ob H, Sg L, and Pta L (0.34-0.54%). The most effective scavenging of the FRAP radical exhibited compounds present in extracts Pta L, Ep L, Ep F, Ob H, Sg L, Hp H, and Am Fr (20.25-8.73 µM Trolox·mL −1 ), while the least were in Lc S, Ps S, Ur R, To R, Ur L, and Alv L (0.40-1.38 µM Trolox·mL −1 ).

Plant Hormones
Of the seven plant hormones analysed (Table 12)
Among the rapid, qualitative methods used to assess the presence of phenolic  SO 4 test. By comparing these results with quantitative analysis data obtained with the use of the Folin-Ciocalteu test, it can be noted that qualitative tests vary significantly in sensitivity in detecting the targeted bioactive compounds. This assay is widely used to assess TPC in foods; however, it is not specific for their determinations and is highly dependent on the composition of the matrix, which can vary in terms of the types phenolics and the amount of particular compounds. For instance, reducing sugars or vitamin C may hamper the accuracy of this assay [58,59]. The Folin-Ciocalteu test showed that all extracts contained phenolic compounds in the range of 0.07 mg·mL −1 (Ps S) to 3.17 mg·mL −1 (Ep F). It can also be seen that the extracts prepared from Lc S and Ps S contained one of the lowest TPC contents despite the content of the vitamin C (the content of reducing sugars was not found). In contrast, the content of reducing sugars in extracts containing the highest amount of TPC, namely Ep L and Pta L, was confirmed in only one or two cases, respectively (the presence of vitamin C was not found). The point-biserial Correlation results for the comparison of methods used to detect phenolic compounds (PC) are included in Table S1. The analysis takes into account quantitative variable (Folin-Ciocalteu test results) and nominal variable (presence and absence of PC marked by plus or minus sign). There are two cases considered, depending on how to define the "−/+" sign: (a) treated as "+" (rpb+), (b) as "−" (rpb−). The values of the point-biserial correlation coefficient rpb+ show that there is a positive, medium strength correlation for the Ferric chloride test, and a positive, low strength correlation for the Zinc hydrochloride test. When the rpb− coefficient is investigated, the findings indicate a similar pattern, with the difference that the Shinoda test is characterised by a positive, low strength correlation.
The ferric chloride test allowed detection of the presence of PC only in four extracts (Ep F, Ep L, To L, Am Fr) out of nine, with the highest concentration ranging from 3.17 mg·mL −1 to 1.0 mg·mL −1 . Meanwhile, this test confirmed their presence in extracts that contained lower levels of them; for example, Ur L (0.13 mg·mL −1 ), Poa H (0.36 mg·mL −1 ), and Ea H (0.42 mg·mL −1 ). This assay was also appropriate for the determination of tannins in the following extracts: Arv H, Bv R, Hp H, Mc F, Ob H, Pta L, Sg L, So R, andVo R, but was ambiguous in the determination of flavonoids. The Acetate test allowed detection of phenols in Alv L, Bv R, Mc F, Ob H, Pm H, andUr R, as well as tannins and flavonoids in Arv H, Co F, Ea H, Poa H, Pta L, To F, To L, To R, and Vo R. The zinc hydrochloride test confirmed the presence of phenols in Bv R, Co F, Ep F, Ep L, Sg L, Tp F, Ur L, Ur R, and Vo R, and flavonoids in Am Fr. The results of the presence of phenols with the use of the Shinoda test in most cases coincide with the results for the zinc hydrochloride test, while the presence of flavonoids was verified in Am Fr, Bv R, Ep F, Hr Fr, Lc S, Ps S, and Tp F. However, the alkaline reagent test did not detect flavonoids in plant extracts. The Millon's test can also be used to determine flavonoids, and in our extracts they were detected in Co F, EP F, Lc S, Ps S, and So R. The presence of tannins can be indicated using the gelatin test (positive for Hp H, Lc S, and Ps S), the alkaline reagent test (positive for Ea H, Lc S, Pta L, To F, and Ur L), and the HCl test (phlobatannins) (positive for So R and Vo R). However, the use of the bromine water test and the potassium dichromate test did not allow the detection of these compounds.
The NaOH test did not prove to be effective in the determination of anthocyanins, but it enabled the identification of coumarins and flavones (positive for Hr Fr, Mc F, Poa H, and To R). The H 2 SO 4 test in many cases did not give a clear answer as to the presence of anthocyanins and flavones in plant extracts (positive for Alv L, Am Fr, Hp H, Poa H, Pta L, Tp F, and Ur L). Comparing both NaOH and H 2 SO 4 tests for detecting anthocyanins and flavones, the latter seems to be more sensitive, but the presence of these active compounds in plant extracts was confirmed in most cases by both tests. The ammonium test did not give a clear answer as to the content of flavonoids (positive for Hr Fr, Poa H, and To R). The ammonia and H 2 SO 4 test seems to be more precise in the detection of flavonoids in plant extracts than the ammonium test. The ammonia and H 2 SO 4 test indicated the presence of flavonoids in Co F, Ea H, Hr Fr, Mc F, To F, To R, and Tp F. The ammonium chloride test showed that most extracts contained flavonoids (with the exception of Am Fr). The comparison of sensitivity of applied methods for the detection of polyphenolic compounds has been included in Supplementary Materials (Tables S1-S4). Among the examined tests for the presence of phenolic compounds in plant extracts, the most sensitive test was the ferric chloride test. The visual results largely coincide with the total polyphenol content, determined by the Folin-Ciocalteu test (Table S1). Failure to detect phenolic compounds with the ferric chloride test coincided with a very low concentration of these compounds in the extract using the spectrophotometric technique (Folin-Ciocalteu reagent). Phenolic compounds are common in plants and are easily extracted using water as a solvent. Based on the studies carried out, the ferric chloride test can also be recommended for the detection of tannins in plant extracts (Table S2). For the detection of flavonoids in plant extracts, many tests (aluminium chloride test, ammonium test, ammonia and H 2 SO 4 test) gave inconclusive results. To the greatest extent, the results obtained for these tests coincided with the detection of flavonoids using the lead acetate test, which can be used as the first to screen plant extracts for the presence of flavonoids (Table S3). In the case of detecting anthocyanins in plant extracts, the NaOH test turned out to be useless-these compounds were not detected in any of the extracts tested. However, for their initial detection in extracts, the H 2 SO 4 test can be used. The same applies to the screening of extracts for the presence of flavones. The H 2 SO 4 test was more sensitive than the NaOH test (Table S4).
Vitamin C, an omnipresent plant and animal metabolite [60], exhibits multifarious biological and pharmaceutical functions [61]. It is crucial in the prevention of scurvy [60]; helps to lower blood cholesterol [62]; and is necessary for collagen, carnitine, and neurotransmitters biosynthesis [63,64]. It supports detoxification, assists the adequate function of the immune system, and is involved in the primary prevention of commonly encountered diseases, including diabetes, eye diseases, atherosclerosis [63] cardiovascular disease, and cancer [60]. In view of the fact that this vitamin is not synthesized by the human body, it has to be provided with diet [62]. Vitamin C is extensively utilised in the feed, food, and pharmaceutical industry as a nutritional supplement and preservative [61,65]. In our analysis, the DNPH test allowed detection of its presence only in Lc S and Ps S extracts. The study of this molecule is greatly handicapped by its oxidation under exposure to air, light, and heat.
Quinine, a cinchona alkaloid, belongs to the aryl amino alcohol group of drugs. It has played an invaluable role in the treatment of malaria since the 18th century and still plays a key role in the treatment of this disease. In turn, quinones, a class of compounds containing a benzene ring with a carbonyl group [66], are used in industry as oxidants, dehydrating agents [67], and dyes [68]. The analysis using the H 2 SO 4 test, HCl test, ammonia test, and NaOH test did not confirm the presence of the tested compounds in any of the obtained extracts. The comparison of methods used to detect of quinones are presented in Supplementary Materials (Table S5). Among the tests for the detection of quinones in plant extracts (H 2 SO 4 test, HCl test, ammonia test), the HCl test was the most sensitive.
Plant resins are a complex mixture of specialised metabolites; for example, alkaloids, phenols, and terpenes [69][70][71][72] as well as alcohols, aldehydes, esters, and amorphous neutral substances [69]. Due to their diverse biological activities (e.g., antimicrobial, antiinflammatory, antioxidant, anticancer, antiulcer, haemostatic, immunostimulant) [70,[72][73][74][75][76], resins are used as a raw material in the medical and pharmaceutical industry [70,73] but also as fuel additives, paint thinners, rosin, and varnishes as well as components in polishes [69]. One of the fast tests to verify the presence of resins is the Acetone test. This assay confirmed their existence in the following extracts: Alv L, Am Fr, Arv H, Co F, Ea H, Lc S, Ob H, Pm H, Ps S, Pta L, Sg L, So R, To F, Ur R, and Vo R.
Another group of compounds examined as a part of this study were glycosides, which can be sourced from plant or animal origin [77,78]. Various types of glycosides can be distinguished: among others, triterpene, β-sitosterol, flavonoid, iridoid, phenylpropanoid, anthraquinone, kaempferol, and saponin. The biological activity is strongly related to their stereochemistry [77,79]. Glycosides have been recognized and utilised as alternative drugs in the treatment of various cancers and have other notable therapeutic potential and clinical utility [77,79,80]. For instance, flavonoid glycosides possess antioxidant, antiinflammatory, anti-allergic, anti-microbial, and anti-cancer activities and thus find use in the prevention and management of diseases [78,79]. Cardiac glycosides are used for the treatment of cardiac arrhythmia, congestive heart failure, and atrial fibrillation; exhibit strong anticancer activity; and evoke cell proliferation or activation of cell death by apoptosis or autophagy [77,78,81,82]. Visualisation of the presence of glycosides can be conducted with the use of various methods. The Molisch's test proved to be the most sensitive in detecting these compounds (positive for Alv L, Arv H, Co F, Ea H, Lc S, Mc F, Ob H, Poa H, Ps S, Sg L, So R, Tp F, Ur L, and Ur R). However, the Borntrager test (1), the Borntrager test (2), the Keller-Killiani test, the Baljet test, and the bromine water test did not provide reliable confirmation of the presence of glycosides in plant extracts. The use of the Liebermann's test also did not assure a full clarity of their appearance. The extracts which could be to some extent considered as a glycoside containing are Ep L and Mc F. The summary of protocols used for the confirmation of the presence of glycosides can be found in Supplementary Materials (Table S6). For the detection of glycosides in plant extracts, Molisch's test is undoubtedly recommended.
The principal source of sugars, the main products of photosynthesis [83][84][85], are beet and cane sugar, while other sources may include honey, corn syrup, fruits, and vegetables [86]. The most abundant free sugars found in plants are disaccharides (sucrose and maltose) and monosaccharides (glucose and fructose) [83,87]. These compounds are used in food products to provide sweetness and energy, but also play a key role in preservation, fermentation, colour, flavour, and texture [86,88,89]. The highest sensitivity in determining the presence of sugars showed the Benedict's test (all extracts with the exception of Lc S and Ps S) and Molisch's test (positive for Alv L, Arv H, Co F, Ea H, Lc S, Mc F, Ob H, Poa H, Ps S, Sg L, So R, Tp F, Ur L, and Ur R). Selwinoff's test (positive for Am Fr, Bv R, and Hp H) and the Barfoed test (positive for Arv H, Pta L, and To R) proved to be less effective in the identification of carbohydrates. The use of Fehling's test, the Borntrager test (2), and the bromine water test were not sensitive in the detection of sugars. The comparison of methods used for the detection of sugars has been included in Supplementary Materials (Table S7). Both Molisch's test and Benedict's test were effective in detecting sugars in the tested plant extracts.
Antioxidants, compounds able to prevent/inhibit/reduce oxidation processes [90,91], can be sourced from microorganisms, plants, and animal tissues [92]. The industry has utilised them to prevent metal corrosion and oxidative degradation of polymers (e.g., rubbers, plastics, and adhesives), but they have also found use as food preservatives (enrichment and inhibition of disruption, sourness, and colour change) [90][91][92][93], and as stabilisers in fuels and lubricants [91,93], but also in pharmacology, cosmetics, and medicine [92] (in the prevention of degenerative illnesses, e.g., cancers, cardiovascular, and neurological diseases, cataracts and oxidative stress dysfunctions) [93]. In recent years, due to their numerous biological activities (e.g., anti-aging and anti-inflammatory), the interest in the utilisation of antioxidants is rapidly growing [92]. The measurements of antioxidant activity with the use of three examined assays (DPPH, ABTS, and FRAP assays) revealed that Pta L, Hp H, Ep F, and Am Fr had the highest reducing power. Additionally, the greatest antioxidant activity was also noted for Sg L, Ob H (DPPH assay and FRAP assay), To L (DPPH assay and ABTS assay), and Ep L (ABTS assay and FRAP assay). The extract Poa H was characterised by one of the highest activities in the ABTS test, while in the DPPH test and FRAP test it was characterised by one of the lowest. The lowest reducing power was observed for Vo R, Ea H, Poa H, To R, Alv L, Ur R, Ps S, Ur L, and Lc S (all three assays) as well as for Ur R and Ps S (DPPH assay and FRAP assay). Therefore, it can be seen that despite the differences between these tests, the results obtained are relatively comparable.
Plant hormones, which can be found in plants, algae, and plant-associated bacteria and fungi, play a vital role in plant growth and development (e.g., promote fruit ripening and leaf drop, stimulate seed germination and gemmation, increase yield and resistance to adverse environmental conditions) [94][95][96][97]. The use of these compounds in agriculture and horticulture is of great importance, and since their first discovery and commercial availability, farmers have incorporated them into the crop production to improve numerous aspects of the cultivation processes [96,98,99]. The conducted studies proved that the obtained extracts could constitute a source of plant hormones, especially gibberellic acid (e.g., Ep F, Pm H, Sg L, To R, Ur L, Ur R).
Bromine water test-to the extract solution (2 mL) 0.2 mL of bromine water [37] was added.
Barfoed's test-extracts (2 mL) were mixed with Barfoed's reagent (1 mL) and heated (water bath, 2 min) [47]. Barfoed's solution was prepared by dissolving 13.3 g of copper acetate in 200 mL of water and then 1.8 mL of glacial acetic acid was added.
The percentage of DPPH and ABTS scavenging effects were calculated by the following equation: where A control is the absorbance of the addition of ethanol and A sample is the absorbance of tested extracts.

Plant Hormones
HPLC-qualitative and quantitative HPLC chromatographic analysis of plant hormones were performed in the reverse phase system, using a LaChrom-Merck liquid chromatograph with a DAD diode detector (L-7450), a pump (L-7100), a degasser (L-7612), a 20 µL dosing loop with a thermostat (L-7360), a Rheodyne dispenser, and a steel column LiChrocart C18 250 mm × 4.6 mm filled with a stationary phase with a grain diameter of dp = 5 µm. The samples were analysed at 30 • C. Separation of standard substances was performed using an isocratic elution in 1% aqueous solution of acetic acid and acetonitrile (75:25, v/v) at pH 4.0. Mobile phases for the determination of hormones in the plant samples consisted of 40% acetonitrile-0.1% acetic acid in water (eluent A) and 0.1% acetic acid in methanol (eluent B). The following gradient was used: 0-18 min, 100% A; 18-25 min, linear gradient up to 100% B; 25-35 min 100% B; 35-40 min, linear gradient to 100% A. Post-run time was 15 min. Elution was performed with a solvent flow rate of 0.8 mL·min −1 and an injection size of 20 µL. Detection was carried out at a wavelength of λ = 230 to 287 nm. Hormones were identified by comparing their retention times (tR) with the standards. Abscisic acid (ABA), benzoic acid (BA), gibberellic acid (GA3), indole acetic acid (IAA), jasmonic acid (JA), salicylic acid (SA), zeatin (Z), zeatin riboside (RZ), and isipentenyl adenine (IP) in the tested extracts was calculated on the basis of a calibration curve determined for each identified hormone. All samples were filtered through 0.22 µm membrane filters before injection into HPLC [101,102].

Conclusions
The current study represents the systematic screening of bioactive compounds extracted from twenty-six biomasses. The detailed phytochemical study of the content of phenolic compounds (phenols, tannins, anthocyanins, coumarins, flavones, flavonoids), vitamin C, quinones, quinines, resins, glycosides, and sugars, as well as antioxidant activity and the content of plant hormones, have been reported. The applied protocols are accessible, inexpensive, and provide a quick answer regarding the presence or absence of bioactive compounds. Several methods could be used for rapid screening, while modern analytical methods are necessary for the final confirmation of the concentration of bioactive compounds.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/molecules28145572/s1, Table S1: The comparison of methods used to detect phenolic compounds (PC); Table S2: The comparison of methods used to detect tannins (TN); Table S3: The comparison of methods used to detect flavonoids (FD); Table S4: The comparison of methods used to detect anthocyanins (AC), and flavones (FL); Table S5: The comparison of methods used to detect quinones (QNO); Table S6: The comparison of methods used to detect glycosides; Table S7: The comparison of methods used to detect sugars.