*Article* **Proximate Composition and Antioxidant Activity of Selected Morphological Parts of Herbs**

**Wioletta Biel <sup>1</sup> , Urszula Pomietło <sup>2</sup> , Robert Witkowicz <sup>3</sup> , Ewa Pi ˛atkowska <sup>2</sup> and Aneta Kope´c 2,\***


**Abstract:** The aim of the study was to provide an analytical evaluation of the proximate composition, the total content of polyphenolic compounds and the antioxidant activity, of 27 selected plant materials collected in Poland (West Pomeranian). The basic chemical composition was determined in the ground samples according to the Association of Official Analytical Chemists methods. Antioxidant activity was tested using free radical methods ABTS•<sup>+</sup> , DPPH•<sup>+</sup> and the FRAP method. The lowest concentration of dry matter (DM) was measured in black chokeberry (88.82 g/100 g) and the highest was found in milk thistle (94.65 g/100 g) as well as black cumin (95.09 g/100 g). The content of total polyphenols, assessed using the Folin–Ciocalteu method, ranged from 291.832–7565.426 mg of chlorogenic acid equivalent (CGA)/100 g of DM. Antioxidant activity measured sequentially against the radical ABTS•<sup>+</sup> , DPPH•<sup>+</sup> and using the FRAP method was 26.334–1912.016 µM Trolox/g DM, 9.475–1061.068 µM Trolox/g DM and 26.252–1769.766 µM Trolox/g DM, respectively. The methanolic extract from milk thistle fruit in most assays was characterized by the lowest antioxidant activity and the lowest total content of polyphenolic compounds. Methanol extracts prepared from garlic, stinging nettle and cleavers showed the highest content of total polyphenols and antioxidant activity among the tested plant materials. The parts of plants with the highest antioxidant potential can be a source of new bioactive compounds, but further research is required to describe the profile of compounds harmful to human health.

**Keywords:** antioxidant activity; botanicals; herbs; polyphenols; principal component analysis (PCA); proximate composition

## **1. Introduction**

In recent years, the interest in antioxidants derived from herbal raw materials has increased significantly due to their health-promoting properties. Their preventive effect is appreciated in the context of chronic non-communicable diseases such as obesity, type II diabetes, atherosclerosis or neurodegenerative diseases, the common risk factor of which is oxidative stress [1]. In biological systems, the formation of free radicals is the result of many metabolic changes, including aerobic respiration and inflammatory reactions. Free radicals have proven useful in the fight against pathogens. This is due to their bacteriostatic and bactericidal effects, but they are also capable of removing cancer cells [2]. Free radicals are responsible for controlling blood flow through blood vessels, removal of xenobiotics from the body, and they are also responsible for transmitting signals within the cell [3–5]. Under normal conditions, there is a balance between the formation of free radicals and their removal. However, in conditions of disturbed homeostasis, the amount of free radicals increases, beyond the possibility of their systematic and efficient removal by enzymatic and non-enzymatic mechanisms [5]. During aerobic respiration, some of the electrons leave

**Citation:** Biel, W.; Pomietło, U.; Witkowicz, R.; Pi ˛atkowska, E.; Kope´c, A. Proximate Composition and Antioxidant Activity of Selected Morphological Parts of Herbs. *Appl. Sci.* **2023**, *13*, 1413. https://doi.org/ 10.3390/app13031413

Academic Editors: Luca Mazzoni, Maria Teresa Ariza Fernández and Franco Capocasa

Received: 18 December 2022 Revised: 16 January 2023 Accepted: 18 January 2023 Published: 20 January 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

the complexes of the respiratory chain, reducing the oxygen molecule by a one-electron or two-electron redox reaction, which leads to the formation of reactive oxygen species (ROS) [6,7]. This causes disturbances at the metabolic level due to high reactivity, short lifetime and extraordinary ease of chemical reactions of free oxygen radicals with cell components [5,7]. Enzymatic systems located in the mitochondrial matrix and cytoplasm, i.e., superoxide dismutase (SOD), glutathione reductase (GR), glutathione peroxidase (GPx), catalase (CAT) are responsible for the removal of these compounds [5,8].

Apart from enzyme systems, there are also defense systems based on proteins located in the blood plasma (ceruloplasmin, ferritin, transferrin, or albumin) that remotely bind copper and iron ions, preventing free radical reactions. These proteins interact with uric acid, complementing each others antioxidant potential [8]. Non-enzymatic defense mechanisms against free radicals are some vitamins (C, E, A, including provitamin A–βcarotene), coenzyme Q10 or polyphenolic compounds found in plants [9]. The excess of free radicals, including reactive oxygen species, together with the inefficiency in their removal, leads to lipid peroxidation, damage to cell organelles, proteins, and DNA degradation and mutation, which results in chronic non-communicable diseases [10].

The main antioxidant compounds present in plant products are polyphenolic compounds, including flavonoids [11]. Antioxidant compounds contained in herbs have following effects: anti-inflammation, antibacterial, antifungal, antiviral and immunostimulation effects [12]. Their effect cannot be reproduced on the basis of individually isolated antioxidant compounds, because the health benefiting effect includes many compounds simultaneously appearing in the plant [13].

Herbal raw materials, long known in traditional folk medicine, are mostly well-tested in terms of antioxidant properties, but there are still raw materials that have not been widely distributed in the phytotherapeutic and pharmaceutical industries [14]. Such raw materials may turn out to be new nutraceuticals, helping pharmacological substances (medicines) in the fight against common diseases. Adequate dietary intake of a variety of antioxidants, such as polyphenols, vitamin C, vitamin E, carotenoids and selenium, is associated with a lower risk of developing chronic non-communicable diseases [14,15].

Increasing amount of research results suggest that antioxidants affecting cells may also trigger interactions with specific proteins crucial for intracellular signaling cascades, modulating their expression and activity [15]. These compounds also affect epigenetic mechanisms and modulate the intestinal microbiota [16].

In the available literature, many studies are devoted to the antioxidant properties of various plants and their morphological parts (e.g., fruits, seeds, leaves, roots) [17–20]. Usually, in this type of article, the assessment of antioxidant properties is most often performed using the methods of determining total polyphenols, ABTS, DPPH and FRAP [12,18,21,22].

It is very important that in the published literature there are studies on the antioxidant properties of herbs, but most often they concern popular herbs, e.g., sage, oregano and basil. In our publication, we decided to research less popular plants, which, however, are often used for their culinary and medicinal properties in many countries around the world.

The aim of the study was the analytical evaluation of the basic chemical composition, the total content of polyphenolic compounds (TPC) the antioxidant activity, of 27 selected morphological parts of plant materials collected in Poland.

#### **2. Materials and Methods**

#### *2.1. Materials*

Plant material was collected in 2019 from the collection of medicinal and useful plants of the Experimental Station in Lipnik, Poland (53◦2003500 N, 14◦5801000 E). The collection was conducted by a team of botanists and agrotechnicians from the West Pomeranian University of Technology in Szczecin (Poland).

Samples of tested plants weighed and dried at room temperature (18–22 ◦C) for 3–4 days were ground to 0.1 mm by use of a laboratory mill type KNIFETEC 1095 (Foss Tecator, Höganäs, Sweden) and placed in sterile containers, according to the list presented in Table 1.

**Table 1.** Plant material.


#### *2.2. Methods*

#### 2.2.1. Proximate Composition

Before conducting analyses by the weight-dryer method, the dry matter content was determined and afterwards components in the air-dry mass were analysed. The proximate composition of the samples was determined according to the Association of Official Analytical Chemists (AOAC) methods [23]. To determine dry matter, samples were dried at 105 ◦C to constant weight (method 945.15). Crude fat (as ether extract EE); method 2003.06) was determined using the Soxhlet extraction method with diethyl ether as solvent; crude ash (CA; method 920.153 by incineration in a muffle furnace at 580 ◦C for 8 h; crude protein (CP; method 945.18) (N × 6.25) by Kjeldahl method using a Büchi B-324 distillation unit (Büchi Labortechnik AG, Switzerland). Crude fiber (CF) was determined as the residue after sequential treatment with 1.25% H2SO<sup>4</sup> and with 1.25% NaOH using an ANKOM<sup>220</sup> Fibre Analyser (ANKOM Technology, New York, NY, USA). Total carbohydrates were calculated as: nitrogen free extract (NFE) (%) = 100 − % (moisture + crude protein + crude fat + crude ash + crude fiber).

#### 2.2.2. Extraction

Methanolic extracts were prepared by weighing a 1.0 g sample of the material and extracting it for 1.5 h with 40 mL of 70% methanol (analytical grade) in a water bath with a shaker at 31 ◦C ± 1 ◦C. Then, the cooled extract was filtered using Munktell filter paper (84 g/m<sup>2</sup> ) and funnels into 50 mL containers. These extracts were used for subsequent polyphenols content determination and antioxidant analysis. The extracts were stored in a freezer at −18 ◦C ± 1 ◦C.

#### 2.2.3. Total Polyphenols Content

The content of polyphenols was determined using the method described by Swain and Hillis using the Folin–Ciocalteu reagent [24]. The absorbance of the obtained colored solutions was then measured using a spectrophotometer (Specto 2000 RD, LaboMed, We Los Angeles, CA, USA) at λ = 760 nm against 70% methanol. Total polyphenol content is expressed as chlorogenic acid equivalents (mg CGA/100 g DM).

#### 2.2.4. Antioxidant Activity

Antioxidant activity was measured using the method of Re et al. [25] with the ABTS•<sup>+</sup> radical (2,20 -azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)). The absorbance of the colored solution was measured using a spectrophotometer (Specto 2000 RD, LaboMed, Inc., Los Angeles, CA, USA) at a wavelength of 734 nm in the presence of 70% methanol used to prepare the extracts. The result was expressed in µM Trolox/g DM.

Antioxidant activity was determined by the method of Brand-Williams et al. [26] using the free radical DPPH•<sup>+</sup> . To a 1.5 mL sample suitably diluted with methanol, 3 mL of the prepared DPPH•<sup>+</sup> solution (diluted to an absorbance between 0.900–1.000) was added and the contents of the tube were mixed. The samples were left for 10 min of incubation protected from light and at room temperature. After this time, the absorbance was measured with a spectrophotometer (Specto 2000 RD, LaboMed, Inc. Los Angeles, CA, USA) at 515 nm against 99% pure undiluted methanol. The result was expressed in µM Trolox/g DM.

Antioxidant activity was determined by the FRAP method according to Benzie and Strain [27]. The absorbance at 593 nm against 70% methanol was measured using a spectrophotometer (Specto 2000 RD, LaboMed, Inc., Los Angeles, CA, USA). The results obtained are expressed in µM Trolox/g DM.

#### *2.3. Statistical Analysis*

All analyses were carried out in duplicate (proximate composition) and triplicate (total polyphenols and antioxidant activity). One factorial analysis of variance (ANOVA) and principal component analysis (PCA) were carried out using the STATISTICA v13.3 software. The significance of differences between the means was assessed using the Tukey test at *p =* 0.05.

#### **3. Results**

#### *3.1. Proximate Composition*

Seeds of black cumin and milk thistle were characterized by the highest level of dry matter (respectively, 95.09 g/100 g, 94.65 g/100 g) while the lowest level was found in black chokeberry fruit (88.82 g/100 g). The highest content of protein was found in fruit of fenugreek (26.164 g/100 g DM). Herb of stinging nettle was the best source of fiber (42.661 g/100 g DM), and herb of common dandelion (24.506 g/100 g DM) was the best source of crude ash (Table 2).

#### *3.2. Total Polyphenols Content*

The tested herbs were characterized by a varied content of TPC (Table 3). The three herbs with the highest total polyphenolic content are marjoram (6956.584 mg CGA/100 g DM), chamomile (7240.002 mg CGA/100 g DM) and cleavers (7565.426 mg CGA/100 g DM). It is worth mentioning that birch (6585.825 mg CGA/100 g DM), field horsetail (6545.378 mg CGA/100 g DM) and garlic (6512.095 mg CGA/100 g DM), contained more than 6000 mg CGA/100 g DM phenolic compounds. The lowest content of total polyphenols, among the tested plants, was shown in the milk thistle (291.832 mg CGA/100 g DM) test, and the difference between the other tested samples was statistically significant at *p =* 0.05.

*Appl. Sci.* **2023**, *13*, 1413


Means with at least same letter not differ statistically at *p* = 0.05. \* dry matter (DM), crude protein (CP), crude fiber (CF), ether extract (EE), crude ash (CA), nitrogen free extract (NFE).

**Table 2.** Proximate composition of analysed plant materials.

*Appl. Sci.* **2023**, *13*, 1413


**Table 3.** Total polyphenol content and antioxidant activity of selected raw materials.

48

DPPH•+—2,20

