Streszczenie

Wprowadzenie: Wolne rodniki i reaktywne formy tlenu są połączeniami zwykle obecnymi w zdrowym organi­zmie, będąc naturalnymi produktami wielu szlaków metabolicznych, są one istotne dla procesów sygnałowych w komórce oraz utrzymania homeostazy. Jako źródła reaktywnych form tlenu moż­na wymienić komórki fagocytujące lub enzymy, takie jak oksydaza ksantynowa. Czasem poziom reaktywnych form tlenu znacznie wzrasta. Taki stan może prowadzić do niszczenia bardzo waż­nych struktur komórkowych, takich jak kwasy nukleinowe, białka lub lipidy. W takich sytuacjach można dostarczać organizmowi silnych przeciwutleniaczy w postaci leków lub składników die­ty. Bogatym źródłem przeciwutleniaczy np. związków fenolowych są surowce roślinne, które są przedmiotem naszych badań.
Materiał/Metody: Potencjał przeciwwolnorodnikowy wyciągów mierzono za pomocą rodnika DPPH (rodnik 2,2-di­fenylo-1-pikrylohydrazylowy) i wyrażono jako liczbę jednostek przeciwwolnorodnikowych na mg wyciągu (TAU_515/mg) i na g surowca (TAU_515/g). Ilość związków fenolowych oznaczono kolo­rymetrycznie za pomocą odczynnika fenolowego Folin-Ciocalteu (3H₂O · P₂O₅ · 13WO₃ · 5MoO₃ · 10H₂O).
Wyniki: Największą aktywność przeciwwolnorodnikową zaobserwowano dla wyciągów otrzymanych z Cinnamomi cortex; liczba jednostek aktywności przeciwwolnorodnikowej na mg wyciągu (TAU515/mg) była równa 10,31±1,052. Najsłabsze właściwości przeciwwolnorodnikowe wykazywał wyciąg z Zingiberis rhizoma (0,28±0,174) i wyciąg z Cichorii radix (0,38±0,669). Największą liczbę związków fenolowych zmierzono dla wyciągów z Bistortae rhizoma z wartością (w pro­centach) 78,6±13,5. Współczynnik korelacji pomiędzy liczbą jednostek aktywności przeciwwol­norodnikowej w wyciągach i liczbą związków fenolowych wynosił 0,7273. Gdy liczbę jednostek aktywności przeciwwolnorodnikowej obliczono na g surowca (TAU_515/g) najsilniejsze właściwości przeciwwolnorodnikowe wykazywał Bistortae rhizoma (1406±274,9), najsłabsze Cichorii radix (122±158,3).

Słowa kluczowe:surowce roślinne • wyciągi roślinne • związki fenolowe • właściwości przeciwwolnorodnikowe

Summary

Introduction: Free radicals and reactive oxygen species are compounds usually present in healthy organisms as natural products of many metabolic pathways, and they are important in cell signaling and ho­meostasis. As a source of reactive oxygen species one can mention phagocytic cells and enzy­mes such as xanthine oxidase. Sometimes the level of reactive oxygen species strongly increases. This may lead to damage of very important cell structures such as nucleic acids, proteins or li­pids. In this situation one should provide the organism with powerful antioxidants as a medicine or in the diet. A rich source of strong antioxidants such as phenolic compounds is plant raw ma­terials, which are the subject of our study.
Material/Methods: Antiradical potential of extracts was measured with DPPH radical (2,2-diphenyl-1-picrylhydra­zyl) and was expressed as the number of units per mg of extracts (TAU_515/mg) and per g of raw material (TAU_515/g). The amount of phenolic compounds was determined colorimetrically using Folin-Ciocalteu phenol reagent (3H₂O · P₂O₅ · 13WO₃ · 5MoO₃ · 10H₂O).
Results: The strongest antiradical activity was noted for extracts obtained from Cinnamomi cortex; the number of antiradical units per mg of extract (TAU_515/mg) was 10.31±1.052. The lowest antiradical features were exhibited by extract from Zingiberis rhizoma (0.28±0.174) and extract from Cichorii radix (0.38±0.669). The highest amount of phenolic compounds was measured for extracts from Bistortae rhizoma, with a value (in percentage) of 78.6±13.5. The correlation coefficient between the number of antiradical units in extracts and amount of phenolic compounds in these extracts was 0.7273. When the number of antiradical units was calculated per g of raw material (TAU_515/g) the strongest antiradical properties were noted for Bistortae rhizoma (1406±274.9), the weakest for Cichorii radix (122±158.3).

Key words:Plant raw materials • Plant extracts • Phenolic compounds • Antiradical properties

Abbreviations:

TAU_515/mg – the number of total antiradical units calculated per mg of extract using DPPH;
TAU_515/g – the number of total antiradical units calculated per g of raw material using DPPH.

Introduction

Plant raw materials are known to be a very rich source of natural antioxidants such as phenolic compounds [11,28], carotenoids [22] and tocopherols [14]. The most effective are polyphenolic compounds. Among them one can men­tion, in decreasing order of antiradical activity: green tea phenols such as epigallocatechin gallate (very strong antio­xidants) [7]; tannins (also strong antioxidants) [27]; pheno­lic acids (the antioxidant activity strongly depends on the number of hydroxyl groups in the molecule and the posi­tion of these groups) [13]; and flavonoids (flavonols seems to be the strongest among them, and their activity strongly depends on the number and position of the hydroxyl gro­up in the B ring) [17,23]. Green and black tea polyphenols, as well as tannins, always exhibit strong antioxidative fe­atures [26]. The activity of flavonoids and phenolic acids varies from strong, such as myricetin [9], gallic [18] and caffeic acids [12], to the almost completely inactive dio­smin [19] and salicylic acids [18].

Free radicals are formed in animal and human organisms as a result of normal metabolic processes, for example as a result of action on xanthine oxidase [1], or action of cells of the immunological system, for example stimulated pha­gocytes produce bulks of superoxide radical anion and hy­drogen peroxide [5]. However, sometimes the overproduc­tion of free radicals takes place, for instance during chronic or acute inflammatory states [16]. Then the natural antioxi­dant strength of the organism may not be enough, and pro­viding the organism with antioxidants in the form of me­dicine or dietary components could be advantageous [4].

Such a source of antioxidants could be plant extracts rich in phenolic compounds, especially those with strong an­tioxidant and antiradical activity.

In this study, different extracts were obtained from 6 raw materials: Bistortae radix (root of bistort), Dioscoreae rhizoma (root of wild yam), Curcumae radix (root of te­mulawak), Cichorii radix (root of common chicory), Zingiberis rhizoma (rhizomes of ginger), and Cinnamomi cortex (bark of Ceylon cinnamon). The antiradical activi­ty of each extract was measured and the general antiradi­cal potential of raw materials was evaluated.

Material and Methods

Preparation of extracts

Each raw material, i.e. Bistortae rhizoma (50 g), Dioscoreae rhizoma (50 g), Curcumae radix (50 g), Cichorii radix (49.9 g), Zingiberis rhizoma (49.4 g), and Cinnamomi cortex (50 g), was extracted with 900 ml of methanol for 4 days at 50°C. 180 ml of methanol solution was poured off and condensed to dryness under reduced pressure to obtain extract WA. The remaining 780 ml of methanol so­lution was condensed to dryness under reduced pressure and the dry extract was dissolved in hot water. After co­oling, the aqueous solution was stored at 4°C for 24 hours for the precipitate to form. Then the precipitate was separa­ted (extract WD). After precipitate separation, the aqueous solution was extracted with ethyl acetate (1000 ml). The ethyl acetate and remaining aqueous solution were conden­sed to dryness to obtain extracts WB and WC respectively.

The extracts of each raw material were marked with the following additional letters: b – Bistortae rhizoma, d – Dioscoreae rhizoma, cu – Curcumae radix, cy – Cichorii radix, i – Zingiberis rhizoma, ci – Cinnamomi cortex. For example, extracts from Bistortae rhizoma were marked as WAb, WBb, WCb and WDb.

Colorimetric measurement of total phenols

Total phenolic compounds in extracts were measured ac­cording to Singleton and Rossi et al. [15].

A 20% aqueous solution of sodium carbonate (Na₂CO₃) was prepared. 0.5 ml of Folin-Ciocalteu phenol reagent (3H₂O · P₂O₅ · 13WO3 · 5MoO₃ · 10H₂O) was added to 7 ml of water. 0.5 ml of methanol solution of extract (2.9 mg/ml) was added to the reaction mixture. After 3 min, 2 ml of aqueous solution of sodium carbonate was added and the sample was heated in a boiling water bath for 1 min. The absorbance was measured at 685 nm in a glass cuvette with 1-cm optical path against blank (without extract). The amo­unt of phenolic compounds was expressed in percentage per mg of extract.

Measurement of antiradical activity with DPPH radical

Antiradical activity of extracts was measured according to the method of Brand-Williams et al. [2].

50 µl of methanol solution of extract at the concentra­tion 2.9 mg/ml was added to 2 ml of methanol solution of DPPH (2,2-diphenyl-1-picrylhydrazyl radical) (0.037 mg/ml) in a cuvette, mixed and absorbance was measured at 515 nm in a glass cuvette with optical path 1 cm at the time of 0 min (start time) and after 1 minute. The control sample was prepared by the addition of 50 µl of methanol instead of extract solution and the absorbance was measu­red at 0 and after 1 minute. The measurement was repe­ated five times and the standard deviation was calculated.

Antiradical unit definition and calculation of number of units per mg of extract and g of raw material

Antiradical unit definition and number of units per mg of extracts and g of raw material were calculated accor­ding to Sroka et al. [20]. One unit of antiradical activi­ty is the amount of substance which causes a decrease of absorbance of the reaction mixture of 1 after 1 mi­nute at 20°C.

The amount of antiradical units per mg of extract was me­asured according to the equation:

where TAU_515/mg is the number of antiradical units per mg of extract; A₀ is the absorbance of the sample at 0 min; A₁ is the absorbance of the sample after 1 minute of reaction; m is the amount of extract [mg] in 1 ml of reaction mixture.

The maximal error ΔTAU_515/mg was calculated according to the total differential method.

The number of antiradical units per g of raw material was calculated according to the equation:

where TAU_515/g is the number of antiradical units per g of raw material (approximate value); TAU_515/mg/met is the num­ber of antiradical units per mg of methanol extract; me is the weight of extract [mg]; m^_R is the weight of raw mate­rial [g] taken for extraction.

The maximal error ΔTAU_515/g was calculated according to the total differential method.

Results and Discussion

Antiradical activity of investigated extracts expressed as the number of antiradical units per mg of extract is de­monstrated in Figure 1 and Table 1. The strongest anti­radical features were noted for extracts WBci and WDci from Cinnamomi cortex; the number of antiradical units (TAU_515/mg) was 10.3±0.868 and 10.31±1.052 respectively. The lowest number of antiradical units was calculated for extract WCi (0.28±0.174) obtained from Zingiberis rhizoma and extract WCcy (0.39±0.669) from Cichorii radix.

Figure 1. The number of antiradical units per mg of extracts (TAU_515/mg). WAb, WBb, WCb and WDb are methanol, ethyl acetate, aqueous extracts and precipitate (see preparation of extracts) from Bistortae rhizoma, WAd, WBd, WCd, WDd are respective extracts from Dioscoreae rhizoma, WAcu, WBcu, WCcu, WDcu are extracts from Curcumae radix, WAcy, WBcy, WCcy, WDcy – extracts from Cichorii radix, WAi, WBi, WCi, WDi – extracts from Zingiberis rhizoma, WAci, WBci, WCci, WDci – extracts from Cinnamomi cortex.

Table 1. The weight of extracts [mg], number of antiradical units per mg of extracts (TAU_515/mg), number of antiradical units per g of raw material (TAU_515/g), amount of phenolic compounds [%]. WAb, WBb, WCb and WDb are methanol, ethyl acetate, aqueous extracts and precipitate (see preparation of extracts) from Bistortae rhizoma, WAd, WBd, WCd, WDd are respective extracts from Dioscoreae rhizoma, WAcu, WBcu, WCcu, WDcu are extracts from Curcumae radix, WAcy, WBcy, WCcy, WDcy – extracts from Cichorii radix, WAi, WBi, WCi, WDi – extracts from Zingiberis rhizoma, WAci, WBci, WCci, WDci – extracts from Cinnamomi cortex

Amounts of phenolic compounds measured by the colori­metric method of Singleton et al. [15] are shown in Figure 2 and Table 1. The highest amount of phenols was noted for extracts WBb, WAb and WDb, with the value of 78.6±13.5, 45.2±7.68 and 45.1±7.24 respectively.

Figure 2. Amount of phenolic compounds in extracts expressed in percentage. WAb, WBb, WCb and WDb are methanol, ethyl acetate, aqueous extracts and precipitate (see preparation of extracts) from Bistortae rhizoma, WAd, WBd, WCd, WDd are respective extracts from Dioscoreae rhizoma, WAcu, WBcu, WCcu, WDcu are extracts from Curcumae radix, WAcy, WBcy, WCcy, WDcy – extracts from Cichorii radix, WAi, WBi, WCi, WDi – extracts from Zingiberis rhizoma, WAci, WBci, WCci, WDci – extracts from Cinnamomi cortex.

Pearson’s correlation coefficient between the number of antiradical units in extracts and the amount of phenolic compounds was 0.7273 (Fig. 3).

Figure 3. Correlation coefficient between amount of phenolic compounds in extracts and number of antiradical units (TAU_515/mg) per mg of extracts.

The antiradical features of raw materials (TAU_515/g) are demonstrated in Figure 4 and Table 1. When the number of antiradical units was calculated per g of raw materials, the highest value was calculated for Bistortae rhizoma (1406±274.9), the lowest for Cichorii radix (122±158.3).

Figure 4. Number of antiradical units in g of raw materials (TAU_515/g). TAU_515/gb is the number of antiradical units per g of Bistortae rhizoma, TAU_515/gd – number of antiradical units per g of Dioscoreae rhizoma, TAU_515/gcu – per g of Curcumae radix, TAU_515/gcy – per g of Cichorii radix, TAU_515/gi – per g of Zingiberis rhizoma, TAU_515/gci – per g of Cinnamomi cortex.

We chose for the study roots, bark and rhizomes because these parts of plants are always rich in antioxidant pheno­lic compounds, first of all tannins.

Root of common bistort exhibited the highest antiradical features (1406±274.9) among raw material investigated in this study. This result is much lower in comparison to the antiradical potential of green tea leaves (7601±92) which we demonstrated in our previous study [26]. The relati­vely strong antiradical activity well correlated positively with the highest phenolic compounds in bistort extracts. According to the literature, root of bistort contains 15-20% tannins [3], which are known to be powerful antiradical compounds [27].

The second in terms of antiradical activity appeared to be bark of cinnamon. Our investigation showed that the amount of phenolic compounds was lower than in root of bistort. According to the literature [24], among phenolic compounds, bark of cinnamon contains phenolic carboxy­lic acids (hydroxycinnamic acid derivatives, dihydroxyben­zoic acid), tannins, and especially oligomeric proanthocy­anidins, in amounts of no more than 2%. We demonstrated higher amounts of phenols than reported in the literature for this raw material. Our results could be caused by the presence of additive of primary bark. The cinnamon bark antiradical activity was lower than root of common bistort when calculated per g of raw material (TAU_515/g) but the extracts exhibited stronger antioxidant features (TAU_515/mg).

Antiradical potential of rhizome of ginger was average in comparison to raw materials investigated in this study but low in comparison to the above-cited tea leaves. The main therapeutic component of rhizome of ginger is essential oil, but some phenolic compounds were identified in this raw material, such as quercetin, rutin, catechin, and phe­nolic acids (gallic, vanillic ferulic, tannic acid) [6]. The gallic and tannic acids are known to have strong antiradi­cal properties [10,27].

Rhizomes of yams did not show strong antiradical activi­ty in our investigation. There is some information in the literature showing certain antioxidant properties of yam rhizome [21].

Root of temulawak and root of common chicory appe­ared to exhibit the lowest antiradical properties. Root of temulawak contains 1-2% dicinnamoyl methane deriva­tives (curcumin), which are not very effective antiradical compounds [25].

Root of common chicory is mainly used as a bitter medici­ne. Among the phenolics, there were identified monocaf­feoyl tartaric acid, chicoric acid, chlorogenic acid, some cyanidin and delphinidin derivatives, flavonoids such as quercetin and luteolin derivatives [8]. The amount of total phenols appeared to be very small, which correlated with low antiradical features.

One can conclude that:
a) Common bistort appeared to have the strongest antiradi­cal features, probably due to the presence of high amo­unts of total phenols and especially tannins – strong an­tiradical agents;
b) Antiradical activity positively correlated well with the total phenol amounts.

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The authors have no potential conflicts of interest to declare.

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