This study was conducted during 2018 to Kinshasa in DR Congo. The bulk-marketed black teas were collected from two markets of Kinshasa (La Liberté and Zikida) (Table 1). They were finely ground in a high-speed mill (Retsch ZM 100 Model) and sieved at 180 µm particle size.

All solvents used were of analytical and HPLC grade and purchased from Merck VWR (Leuven, Belgium). 2-Aminoethyldiphenylborat and vanillin were purchased from Sigma (Bornem, Belgium). L-012 (8-amino-5-chloro-7-phenylpyrido

(*) LEBEST is a packaged black tea (2 g/tea bag) from Ceylon used such as reference tea or standard tea.

[3,4–d]pyridazine-1,4(2H,3H)dione) was purchased from Wako Chemicals GmbH (Neuss, Germany).

Horseradish peroxidase (HRP) was obtained from Roche (Mannheim, Germany) and human Myeloperoxidase was from Calbiochem, EMD Millipore (Bellirica, MA, USA). Caffeic acid (purity 96%), gallic acid (purity: 97%) was purchased from Sigma-Aldrich. Rutin (purity ≥ 99%), isoquercitrin (purity ≥ 99%) and, hyperoside (purity ≥ 98%) were HPLC grade and purchased from Extrasynthese. Water was treated using a Milli-Q water ultra-purification system before use.

Powder observations were made using lactic acid reagent (European Pharmacopeia reagent) [7]. For the microscopic analysis, two drops of lactic acid reagent dropped on the slide were mixed with a small quantity of powder and then covered with a cover glass. The obtained microscopic preparation was warmed up to boiling. Observations were made with a Zeiss Primo Star 200 microscope and pictures were taken with Smart Phone Samsung E7.

Aqueous extracts were prepared by infusion of 10 g of sample powders with 100 mL of water for 10 minutes. The infusion was cooled to room temperature before filtration and the evaporation of the solvent was performed by lyophilisation (a Christ Alpha 1-4 LSC® apparatus). The extracts were then weighed and kept in dark hermetic flasks at 4˚C.

The extracts were solubilized in water, thus their effect was compared to a control test performed with water alone.

Each concentration was tested in triplicate in each assay, and at least three different assays were performed. All results were expressed as mean values ± standard deviation (SD). The statistical analysis was performed with GraphPad 7.0 (GraphPad Software, San Diego California, USA). Two-way ANOVA was used; multiple comparisons of all data were performed using the “Tukey” Multiple Comparisons Test and the level of statistical significance was set at p < 0.05. The IC₅₀ values were calculated with GraphPad Prism 7.0 under application of the function “log (inhibitor) vs. normalized response-variable slope” after converting the concentrations into their decimal logarithm.

Examined under a microscope using European Pharmacopeia reagent (Lactic acid reagent), the powders of different samples showed the following diagnostic characters: numerous asterosclerites (Figure 2), numerous fragments of unicellular trichomes; fragments of abaxial epidermis consisting of cells with slightly wavy walls, numerous anomocytic stomata, slightly thickened walls, accompanied by cells of the underlying palisade parenchyma; numerous isolated and grouped sclereids (Figure 3), palisade parenchyma usually arranged in 2 layers, spongy parenchyma including cells containing cluster crystals of calcium oxalate; fragments of spiral vessels accompanied by lignified fibers. Numerous starch grains, drop oils were also identified. Microscopy analysis showed equally foreign elements such as insect pests (mites) and grain of sand in three samples (Figure 4).

The detection, prevention and eradication of mites and insects infestation is an important hygienic and economic consideration for natural products. The presence of mites or insects in studied black teas is critical for quality of teas consumed by the Congolese population. Bulk-marketed black teas are conserved in the woven sacks and are sold outdoors in the large plastic basin. Storage conditions affect the quality of tea products. The main factors influencing tea quality and polyphenol stability during storage are light, oxygen, and temperature [6]. Weather conditions to Kinshasa (DR Congo), are characterized by high temperatures and high humidity. Effective preventive measures of natural products involve good hygiene in the warehouse (removal of spillages, old debris and packaging materials, elimination of sources of infections such as floor cracks and crevices), effective stock control (regular inspection, rotation of stock, early recognition of infestation), optimum storage conditions (maintenance of cool, dry environment) and good packaging (woven sacks and bags, multiply paper sacks stitched at the seams, paper, polythene film, flimsy cardboard are all penetrable by insects and mites) [13]. High temperatures and high humidity could also lead to proliferation of Aspergillus, Fusarium, and Penicillium, the natural fungal flora usually found in the food chain that produce mycotoxins [14]. Mycotoxins are reported such as the major chemical contaminants found in tea products together with pesticides residues and heavy metals. Few reports focused on the mycotoxin detection in tea samples, however aflatoxins, ochratoxin A, and fumonisins were found in black tea samples. Acting such as carcinogenic and teratogenic agents, mycotoxins are recognized as a serious threat to human health [14].

Awareness-raising campaigns among sellers and food quality control authorities should be conducted to improve the quality of food for the Congolese people.

Phytochemical analysis revealed the presence of anthocyanidins, phenolic acids, flavonoids, iridoids, terpenes. TLC and HPLC fingerprints of all black tea samples were nearly similar and showed the presence of glycosylated flavonoids and phenolic acids as main compounds. By comparison with standards, they showed that extracts of teas contain chlorogenic acid, gallic acid, ferulic acid such as phenolic acids and astragalin, hyperoside, kaempferol, quercetin-3-O-rutinoside (rutin) such as flavonoids (Figure 5). HPLC analysis revealed that hyperoside, rutin and kaempferol 3-O-rutinoside was the main glycosylated flavonoids in accordance with previous studies [15]. Hyperoside was the major pic in the all tea HPLC chromatograms (Figure 6).

Previous phytochemical analysis showed that the chemical components of tea leaves include polyphenols, alkaloids (caffeine, theobromine, theophylline, etc.), volatile oils, polysaccharides, amino acids, lipids, vitamins (e.g., vitamin C), inorganic elements (e.g., aluminium, fluorine and manganese), etc. However, the polyphenols are primarily responsible for the beneficial healthful properties of tea [5] [16] [17].

Polyphenol composition of tea varied with the types of teas [16]. The major polyphenolic compounds in green tea are catechins, and in black tea are flavonoids predominantly polymeric of the type theaflavins and thearubigins. Black tea is produced by fermentation, which is in fact an enzymatic oxidation evoked by polyphenol oxidases native to tea leaves. There are two possible ways of processing in order to obtain black tea using “orthodox rollers” or “crush-tear-curl” (CTC) machines. During black tea manufacturing the most considerable changes occur in the content of catechins. A profound loss in catechin content is observed and at the same time new compounds (theaflavins and thearubigins) are formed as a result of enzymatic oxidation [3] [6] [15] [18]. Black tea retains a high number of the antioxidants polyphenols such as flavonoids. Black tea was reported to contain more gallic acid compared to green tea. Green tea contains six primary catechin compounds, namely catechin, gallocatechin, epicatechin, epigallocatechin, epicatechin-3-gallate and epigallocatechin-3-gallate (EGCG) [6] [19]. Black teas consumed in DR Congo seem to be CTC tea such as the cases of many Africa’s countries [15].

Results from the quantitative determination of anthocyanins, flavonoids and total phenolic content are summarized in Table 2. Anthocyanin contents were determined as catechin equivalents in grams per 100 grams of dry weight (g CE/100g DW), while total polyphenol contents were calculated as gallic acid equivalents in grams per 100 grams of dry weight (g GAE/100g DW). Flavonoid content was determined as quercetin equivalents in grams per 100 grams of dry weight (g QE/100g DW). The anthocyanin, flavonoid and total phenolic contents varied significantly (p < 0.05) between the studied teas. The tea 1 showed the highest value of total phenol content and anthocyanins, the tea 7 the highest value of flavonoid content.

Radical scavenging activity of tested infusion extracts of teas, determined by two biochemical in vitro methods namely ABTS and DPPH assays is presented in Table 3 and is expressed as IC₅₀ values. IC₅₀ is the amount of antioxidant necessary to decrease the initial concentration of radical by 50%.

Lower IC₅₀ value indicates a higher antioxidant activity. All the extracts had significant scavenging effects with antiradical activities connected to their ability to scavenge free radicals according to their IC₅₀ values. IC₅₀ for infusion extracts showed that Tea 8 and TeaTB are the most active in ABTS assay; while in DPPH assay Tea 3 is the most active, but there is not significative difference with Tea 8 and TeaTB. Our results obtained from ABTS and DPPH assays show that all the infusion extracts of bulk-marketed black teas possessed antioxidant properties comparable to the reference packaged tea (TeaTB). The tea “LEBEST” is the main packaged black tea marketed in DRC. The two-way Anova test showed that there are no statistical differences between TeaTB (The reference tea) and Tea 4, 6, 8 and 9 in ABTS assay and Tea 1, 2, 3, 4, 6 and 8 in DPPH assay (p < 0.05) (Figure 7 and Figure 8).

Our results were correlated between the two chemical methods used. The differences observed between the two methods could be attributed to the fact that DPPH assay only detect hydrophilic antioxidants in the contrary of ABTS assay. The highest radical-scavenging capacities of infusion extracts of studied teas are not in adequacy with their anthocyanin, flavonoid and total phenolic contents. The basic bioactive compounds in tea are polyphenols. Antioxidant potential of studied black teas varied related to their quantity of polyphenol compounds. The antioxidant activity of tea polyphenols is not only due to their ability to scavenge superoxide but also due to increased activity of some detoxifying enzymes such as glutathione peroxidase, glutathione reductase, glutathione-S-transferase, catalase [2] [16].

Peroxidases represent a family of isoenzymes actively involved in oxidizing reactive oxygen species, innate immunity, hormone biosynthesis and pathogenesis of several diseases. Even though peroxidases perform a great role in protective mechanisms but still some peroxidases can also lead to some deleterious reactions like co-oxidation of endogenous substrates, drugs and xenobiotics which lead to lipoprotein oxidation, carcinogenesis and liver necrosis. So nowadays even variety of inhibitors are also used against different types of tissue specific peroxidases to treat various types of diseases [20]. Peroxidases generally use H₂O₂ as one of the substrates. The MPO is, a pro-oxidant enzyme involved in secondary cell damage. MPO and its metabolites are biomarkers for infectious diseases and a wide array of non-infectious and neurodegenerative disorders [20] [21]. In MPO direct technique, all infusion extracts of teas exhibited a dose-dependent inhibitory effect on MPO activity in the range concentrations of 1 to 20 µg·mL⁻¹. This inhibitory effect was significantly higher (p < 0.0001) and showed a better interaction of molecules of tea extracts with the enzyme (Figure 9).

Altogether the results showed that the extracts tested showed the highest acellular and the highest inhibition on MPO activity. Phenolic compounds such as catechins, glycosylated flavonoids of tea extracts were reported to be excellent inhibitors of MPO and the pyrogallol group in the B ring, the 3, 4’, and 5-OHs, and of the C2-C3 double bond are essential for both binding and reactivity with peroxidase such as MPO [22] [23].

The inhibition of HRP-catalyzed oxidation reactions was studied by using L-012 chemiluminescence method. L-012 is a luminol-based molecule that has been reported to produce much stronger chemiluminescence (CL) than other CL probes [21]. Tea extracts showed a less effective inhibition of HRP oxidant activity and the effect appears at the concentration of 80 µg·mL⁻¹. At concentration of 100 μg·mL⁻¹, the percentage of inhibition effect was approximatively 50% for Tea 5, Tea 6, Tea 7, Tea 8 and TeaTB.

Horseradish peroxidase (HRP) was used for the investigation of inhibitor activity of anti-inflammatory drugs [24]. The results of this assay showed that tea extracts exhibited a moderate capacity to inhibit HRP catalytic activity related to their polyphenolic contents. Phenolic compounds contained in tea extracts, should be responsible of exhibited inhibitory properties on HRP. Mahfoudi et al. (2017) reported that flavonoids exhibited a great capacity to inhibit HRP and the presence of 4’-OH groups on the B ring (catechol structure) is most important for this potent peroxidase inhibition [25].

In some acute and chronic pathologies peroxidases generate a battery of highly diffusible reactive oxidants, which instigate oxidative damage in the host tissues at the inflammatory sites exacerbating tissue damage [26]. Our results demonstrated that all extracts from bulk-marketed black teas exerted a noticeable inhibitory effect on the MPO and on HRP catalytic activity comparable to tea “LEBEST”, the packaged black tea used such as reference. Obtained results showed that unpackaged black tea marketed, largely consumed by Congolese people would procure the health benefits under different conditions. However, storage conditions must be improved to preserve the nutritional and therapeutic values of tea.