Studies on the chemical composition of plants used in traditional medicine in Congo

2.1 Preparation of samples prior to analysis

Medicinal plants compiled in Table 1 were collected and identified by Dr Edmond Sylvestre Miabiangana in the area of Brazzaville, Congo. After drying the medicinal plant samples were transported to the Department of Analytical Chemistry at the Medical University of Gdansk, Poland, then ground using a sample preparation mill (Knifetec 1095, Foss Tecator, Sweden), and stored in plastic containers in a dry and dark place prior to analysis.

About 0.5 g of samples was then digested using a microwave unit (Jupiter, Sineo, China) applying a four-stage digestion program: step I – 150°C (10 min), step II – 160°C (5 min), step III – 180°C (5 min), and step IV – 190°C (15 min). The obtained digests were transferred into 50 mL volumetric flasks and the volume was done with distilled water.

To measure the contents of phenolic acids, flavonoids, polyphenolic compounds, and ascorbic acid, as well as determine the antioxidant activity, aqueous extracts were prepared by pouring 0.5 g of plant material in 50 mL of boiling redistilled water. After filtration, the extracts were transferred to 50 mL volumetric flasks, and the volume was made with redistilled water. Redistilled water was obtained using a distillation apparatus (Heraeus Quarzglas, Germany).

2.2 Determination of metallic elements

The contents of essential elements, Fe, Mn, Zn, Cu, Cd, and Pb, were determined by the flame atomic absorption technique (SpectrAA 250 Plus, Varian, Australia) using standard analytical procedures and an external calibration method. An air–acetylene mixture was used during the measurements along with the following analytical wavelengths (nm) for the particular metallic elements: 248.3 (Fe), 279.5 (Mn), 213.9 (Zn), 324.8 (Cu), 228.8 (Cd), and 217.0 (Pb). Chromium, Co, Al, and Se were assayed by the inductively coupled plasma-optical emission spectroscopy (ICP-OES) technique using a PerkinElmer Optima 7000DV spectrometer (Perkin-Elmer, Waltham, MA, USA).

Phosphorus was determined in the digests of medicinal plants by a spectrophotometric technique using a molybdenum blue reaction [11] at 650 nm as the analytical wavelength and an external calibration curve.

2.3 Total phenolic content (TPC)

The TPC of the Congo plant extracts was determined using the Folin–Ciocalteu method, as previously described by Singleton [12]. The absorbances of the extracts from Congo folk medicines were measured at 760 nm using an SP-870 Metertech UV/Vis spectrophotometer (South Korea). The gallic acid calibration curve (0.1–0.154 mg/mL) was used to express the results as milligrams of gallic acid equivalents per gram of dry mass (mg GAE/g dm).

2.4 Total flavonoid content (TFC)

The TFC of the Congo plant extracts was determined based on the method of the European Pharmacopeia [13] with some modifications. An appropriate amount of the extract was mixed with 0.1 mL of 5% (w/v) AlCl₃ solution. The mixture was incubated for 30 min in the dark at room temperature and the absorbance was then measured at 430 nm using an SP-870 Metertech UV/Vis spectrophotometer (South Korea). The TFC was expressed in milligrams of quercetin equivalents (QE) per gram of dry mass (mg QE/g dm) using a calibration curve obtained from quercetin standard solutions (5–50 μg/mL).

2.5 Total phenolic acid content (TPAC)

The procedure described in the Polish Pharmacopeia VI [14] was applied for TPAC determination using Arnov’s reagent. An appropriate amount of the extract was mixed with 0.2 mL of 0.5 M HCl, 0.2 mL of Arnov’s reagent and 0.2 mL of 1 M NaOH. The absorbance was measured at 490 nm. The results are expressed in milligrams of caffeic acid equivalents per gram of dry weight (mg CAE/g DW) based on a calibration curve registered for caffeic acid (5–40 μg/mL).

2.6 Ascorbic acid determination and antioxidant activity

The ascorbic acid content in extracts of medicinal plants from Congo was assayed based on the analytical procedure described elsewhere [15]. Antioxidant activity was determined in the extracts of plants using FRAP, DPPH, and ABTS methods, also described previously [15].

2.7 Statistical evaluation of the results

All measurements were carried out in triplicate, and the results were presented as the arithmetic mean ± standard deviation (SD). Table 2 presents the statistical evaluation of the results of element’s determination in terms of their precision and recovery obtained for selected certified reference materials (CRMs) using FAAS. Validation parameters for ICP-OES were published in our previous article [23]. For statistical evaluation of experimental data using principal component analysis (PCA), the Statistica 7.1 program (Tulsa, USA) was used.

Validation of methods applied for TPAC, TPC, and TFC was performed by evaluating the following parameters: linearity, limit of detection (LOD), and limit of quantification (LOQ), intra- and inter-day precision, recovery, and stability. Good linearity was found over the determined ranges for all analytes, with correlation coefficient values significantly higher than 0.980. The LOD and LOQ were calculated in accordance with the following equations: LOD = 3.3Sxy/b and LOQ = 10Sxy/b, where Sxy is the SD of the response and b is the slope of the calibration curve. The values of LODs and LOQs were less than 3.5 and 13.2 μg/mL, respectively. These results show that the analytical method had excellent resolution and sensitivity. Intra-day precision was validated with a standard solution of assayed phenolic compounds three times within 1 day, while inter-day precision was validated with the same standard solution over three consecutive days. Consequently, the precision was acceptable, and the coefficient of variation values ranged from 0.6 to 1.4% and 1.2 to 2.5% for intra- and inter-day variations, respectively.

The mean recovery was found to be in the satisfactory range, 92.6–97.4%, with a relative SD of less than 4.5%. The peak areas and retention times of the determined phenolic compounds were analyzed every 8 h within 48 h for the stability test, and they were found to be quite stable, while retention CV was lower than 1.6% for peak area and 0.5% for retention time. All validation values described above were obtained for phenolic compound analysis.

3.1 Contents of metallic elements, phosphorus, and selenium

Results of element determination in ten analyzed medicinal plants from Congo are presented in Table 3. The characteristic feature of the presented data is the fact that the arithmetic mean and the SD represent a similar range of concentrations. This is the effect that these values were obtained after measuring the elements in all analyzed samples of folk medicines from Congo, and their level was largely different. As for results obtained for P, the only non-metal element analyzed, it was found in the wide range from about 0.14 to 2.96 mg/g dm. An especially high level of P was determined in the leaf of Alchornea cordifolia, almost 3 mg/g dm. On the other hand, the lowest P concentrations were noticed in two plant samples – Ochna afzelii and Syzygium brazzavillense, at about 135.00 and 151.00 mg/kg dm, respectively. Other research on the phosphorus level has shown that in plants originating from the DR Congo, it was found in the range from 1258.00 to 1567.00 mg/kg dm [16], which is rather close to the highest results obtained in our study. The P level in the plant material from Congo is lower than those reported in European plants used in medicine [17,18]. The reason for this can be the different P levels in soils of Africa and Europe but also anthropogenic factor cannot be neglected. The latter effect is mainly due to the use of fertilizers containing phosphorus, which can elevate the concentration of this element in plants [19].

The concentrations of Ca, Mg, Na, and K, also shown in Table 3, indicated several plants with characteristic levels of them. Calcium concentration was found to be the highest in the bark of Zanthoxylum gilletii, 40.49 mg/g dm, and in the leaf of A. cordifolia, 39.74 mg/g dm. The lowest Ca level was determined in Q. africana, 8.55 mg/g dm. In general, the Ca level in our study was higher than those determined by other researchers, both in plants from Africa [16] and Iran [20]. As for Mg, the richest plant material from Congo with these elements was the bark of S. brazzavillense, about 12 mg/g dm, as well as the bark of Mitragyna stipulosa, which contained almost 11 mg/g dm. Lower than the values given above, the Mg concentration was determined in plants from DR Congo, about 1.70–1.90 mg/g dm [16], but a similar Mg level was found in South African medicinal plants [8]. Sodium content was the highest in the sample of A. cordifolia, about 32.00 mg/g dm, and in the bark of P. macrophylla, 31.00 mg/g dm. Relating these values to Na levels determined by other researchers [16,20], they are higher. The contents of K varied in different samples depending on plant species. The highest K level was found in the bark of M. stipulosa, about 27.00 mg/g dm, and the lowest in the bark of S. brazzavillense, about 4.80 mg/g dm. The former value is quite similar to the concentration of K found in plants from South Africa [8] and DR Congo [16].

The contents of microelements in medicinal plants from Congo are presented in Table 3, too. Among microelements essential for human life, the order in which their concentrations (in mg/kg dm) were obtained is as follows: Fe > Mn > Zn > Cu. The plant materials with relatively high levels of these metals, such as samples Q. africana, Garcinia huillensis, M. stipulosa, and A. cordifolia, can be listed. When comparing the obtained levels of essential elements with those in the literature, it can be stated that these levels strongly depend on plant species and analyzed plant organs. For example, the level of Cu in medicinal plants in DR Congo [16] was determined in a similar range of concentrations, about 10.00–11.00 mg/kg dm. The same can be noticed for Zn, Fe, and Mn [8,16,21]. The results of essential element assays determined for plants from Congo in this study also remain in agreement with their levels in medicinal plants originating from Europe [17,18].

Table 3 also presents the results of the determination of harmful elements for humans, such as Cd, Pb, Cr, Co, and Al. Cadmium and lead were assayed by the FAAS technique, and the other elements by ICP-OES. The highest level of Cd was found in the sample of Zanthoxylum giletii, 4.5 mg/kg dm, and in Q. afzelii, 5.0 mg/kg dm. On the other hand, for plants such as Q. africana, M. stipulosa and G. huillensis, Cd was below the LOD for the FAAS technique. The level of Cd detected in the plants from Congo was however higher than that found in other African plants, for example, in Nigeria [22]. Perhaps the reason for this can be higher Cd contamination of soils near Brazzaville in Congo. Lead was determined in the highest concentration in the sample of bark from M. stipulosa, about 14.20 mg/kg dm. In other investigated plants, the Pb level was much lower or below the LOD. This is typical for African plants since the Pb level can vary depending on local contamination and/or anthropogenic factor. In South African plants [8], the level of Pb was determined in a lower amount in comparison with the Pb concentration in the bark of M. stipulosa, and in other research studies performed on plants from DR Congo [16] its level was about 1.00 mg/kg dm or lower.

Elements determined by ICP-OES represented a low range of concentrations in comparison with those assayed by FAAS. Particularly rich in Se were plants such as M. stipulosa (42.30 mg/kg dm), Pausinystalia johimbe (15.90 mg/kg dm), and Q. africana (15.80 mg/kg dm). The aluminum level was the highest in the sample of Q. africana – 678.00 mg/kg dm, about ten times higher than that detected in other medicinal plants. Chromium and Co levels were determined as the highest in the sample of M. stipulosa, 15.00 and 0.29 mg/kg dm, respectively.

3.2 Phenolic compound analysis

3.3 PCA

PCA was performed in order to find factors responsible for the differentiation of the results of the chemical composition of Congo plants. To construct the experimental database, all results for metallic and non-metallic elements were taken into consideration. The same was done for the results of TPAC, TPC, TFC, ascorbic acid, and antioxidant activity assayed by FRAP, DPPH, and ABTS methods. In this way, the experimental database was obtained with the dimensions of 20 × 10. PCA calculations revealed that the first three principal components described together 77% of the variability among the investigated samples. The eigenvalues of PC1, PC2 and PC3 were 8.0, 4.8, and 2.7, respectively. Figure 1 shows the distribution of the studied African medicinal plants samples in three-dimensional plots PC1, PC2, and PC3. There are several characteristic plant samples in this plot. For example, in the right part of Figure 1, one can see sample No. 2 (A. cordifolia), and sample No. 6 (M. stipulosa), which is located in front of the plot in the left area. Their location is characteristic, since they are far away from the others, which is caused by specific values of elements and other parameters determined. On the other hand, sample No. 10 (G. huillensis) can be found in the central area of the plot, and it is characterized by a low value of PC3. To explain the loading values for particular principal components, it is necessary to study Figure 2. It is clear that PC1 is correlated with several element concentrations, such as Na (positively), Cu, Fe, and Al (negatively), as well as TPC, ascorbic acid, and antioxidant activity. On the other hand, PC2 is negatively correlated with the Mn level in the studied plant samples, and PC3 is negatively correlated with the TPACs. As it was found in the recent application of PCA in the interpretation of experimental data for Chinese medicinal plants [23], this statistical method is also well suited for complex evaluation of results of Congo medicinal plants and finding the factors responsible for the differentiation of investigated materials.

Figure 1

Distribution of the studied African medicinal plants samples in 3D plot of PC1, PC2, and PC3.

Figure 2

Loading plot obtained for PCA results of African medicinal plants.