Characterization of Mulberry Root Bark Extracts ( L.) Based on the Extraction Temperature and Solvent

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IJIE

ISSN 1598-3579, http://dx.doi.org/10.7852/ijie.2020.41.2.36

Received : 16 Nov 2020 Revised : 27 Nov 2020 Accepted : 2 Dec 2020 Keywords:

mulberry root bark, extraction, structural analysis, polyphenol, flavonoid, antioxidant activity Mulberry root bark is one of potential plant sources for antioxidant materials which can be used

for the relief of oxidative stress. To explore the effects of solvent type and temperature on the structural characteristics and antioxidant activity of the root bark extracts, we prepared various extracts of mulberry root bark (Morus alba L.) using 0 – 100 % ethanol (EtOH) at RT – 100^oC.

EtOH concentration and temperature critically affected the extraction yields, the content of bioactive components, and antioxidant activity of the extracts. Use of high content of EtOH solvent and low temperature resulted in the low extraction yield. Meanwhile, it was revealed that the extract prepared using absolute EtOH at room temperature contained polyphenols and flavonoids with the highest contents among other extracts. Interestingly, the temperature differently affected the polyphenol and flavonoid contents according to the solvent types. In the case of 30% EtOH solvent, polyphenol and flavonoid contents increased with an increase in temperature, whereas in the case of 70 and 100 % EtOH, these contents decreased. Using the radical scavenging assay, it was confirmed that the 100% EtOH extracts had higher antioxidant activity compared to distilled water (DW) extracts regardless of temperature. Also, heating might extract more antioxidant components from the root bark. Especially, the extract prepared using 30% EtOH solvent at 100^oC showed the highest antioxidant activity. Taken together, these experimental results imply that the extraction parameters should be designed carefully considering the productivity, the extracted bioactive components, and antioxidant activity.

Introduction

Oxidative stress occurs when overproduced reactive oxygen and nitrogen species (RONS) are not neutralized by antioxidant systems of the body (Liguori et al., 2018). The excessive RONS can damage cellular structure and biomolecules such as lipid,

protein, and DNA (Weidinger and Kozlov, 2015), causing various diseases and age-associated functional losses (i.e.

cancer, diabetes, cardiovascular and neurodegenerative diseases, chronic obstructive pulmonary and kidney disease, and other degenerative diseases in humans) (Liguori et al., 2018; Uttara et al., 2009). Therefore, these days, developments of food or

† Sora Lee and Soo Hyun Kim contributed equally this work.

*Corresponding author.

Wan-Taek Ju, PhD

Sericultural and Apicultural Division, National Institute of Agricultural Sciences, RDA, Wanju-gun, 55365, Republic of Korea.

Tel: +82-63-238-2855 E-mail: wantaek@korea.kr

Characterization of Mulberry Root Bark Extracts ( Morus alba L.) Based on the Extraction Temperature and Solvent

Sora Lee

^1†

, Soo Hyun Kim

^1†

, You-Young Jo

²

, Seong-Wan Kim

¹

, Hyun-Bok Kim

¹

, HaeYong Kweon

¹

, and Wan-Taek Ju

¹

*

1Sericultural and Apicultural Materials Division, National Institute of Agricultural Sciences, RDA, Wanju-gun, 55365, Republic of Korea.

2R&D Performance Evaluation & Management Division, Rural Development Administration, Jeonju-si, 54875, Republic of Korea

Abstract

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factors more than one and the optimum extraction conditions are insufficient. Therefore, in this study, we investigated the effects of solvent type and temperature on the extraction yield, polyphenol content, and antioxidant activity and discussed the extraction conditions for obtaining high amount of antioxidant substances.

Materials and methods

Preparation of mulberry root bark extract

The crude powder of fresh Morus alba L. root bark was obtained from Uljin plantation (Uljin, Gyeongsangbuk-do, Korea). The powder (50 g) was soaked in various solvents (1 L) containing 0 – 100 %v/v of ethanol (EtOH; Fisher Scientific, Pittsburgh, PA, USA) and incubated for 2 h for extraction.

Extraction was carried out at room temperature (RT) and controlled temperature (50, 70, and 100°C) using heating mantle and condenser. The extraction mixture heated up to 87 – 95°C when the setting temperature was 100°C. After the extraction, the extract was filtered. The final products were lyophilized and stored at -80°C. The production yield was calculated using the mass ratio of the finally obtained powder to the input mulberry root bark.

Characterization of structural properties of each extract

Attenuated Total Reflection Fourier Transform Infrared (ATR- FTIR) spectroscopy measurement were performed and the spectra of the extract were recorded with Fourier transform spectrometer (S100, Perkin Elmer, USA) in the range of 1000 to 4000 cm^-1. Thirty-two scans were averaged with resolution 4 cm^-1. The circular dichroism (CD) spectra of each extract (0.01 %) were acquired using a Jasco J-1500 circular dichroism spectropolarimeter (Jasco, Easton, MD, USA) using 10 mm path-length quartz high precision cell (Jasco). All spectra were recorded as an average of 3 scans in the range of 190–300 nm with a bandwidth of 4 nm at a scan rate of 500 nm/min. The background CD spectrum of ultrapure water was subtracted from all sample spectra. The x-axis represents the wavelength (190-300 nm). The y-axis indicates the ellipticity signals (mdeg), where positive value means greater absorbance of left-handed than right-handed light. All spectra were smoothed using a Savitzky- Golay filter using Spectra Analysis software (Jasco) (Jastrzebska et al., 2016).

medicine that supplement antioxidants have drawn attention.

Plants have biosynthesis systems to produce various antioxidant substances capable of neutralizing RONS (Kasote et al., 2015).

Phytochemical analyses have confirmed antioxidant components in various plant extracts and evaluated their antioxidant activities.

Especially, mulberry (Morus alba L.) is a good plant source of natural antioxidants. Mulberry is widely cultivated throughout the world traditionally for raising silkworms Bombyx mori.

In recent years, the use of mulberry has been also appreciated as medicines, fruits, vegetables, and animal feed since it was revealed that mulberry contains various bioactive components for each part (i.e. fruit, leaf, twig, and root bark). Especially, the root bark of mulberry is documented in the Pharmacopoeia of various Asian countries including Korea, China, and Japan and it has been used for the treatment of lung heat, cough, edema, and oliguria (Wei et al., 2016). Triterpenoids, flavonoids, coumarins, and alkaloids contained in the root bark are known to play critical roles in antioxidant, anti-inflammatory, and hypoglycemic effects, etc. (Chan et al, 2016)

Bioactive substances can be obtained from the plant matrix by extraction processes under specific conditions. Because the composition of functional components and their bioactivities can be influenced by extraction conditions (Kobus-Cisowska et al., 2020), the extraction parameters such as solvent type, temperature, time, or physical treatments should be carefully controlled. Choi et al. (2015) characterized the compositions of phenolic compounds and biological activities of water and ethanol extracts of various parts of mulberry tree such as fruit, leaf, twig, and root bark. The experimental results demonstrated the ethanol extracts showed higher bioactive components and higher radical scavenging activity. In addition, using response surface methodology (RSM), Radojković et al.

(2012) systematically investigated the effects of the extraction parameters such as temperature (40 – 80°C), solvent-solid ratio (10 – 30 mL/g), and solvent types (40 – 80 %v/v of ethanol content) on the phenol and flavonoid contents and antioxidant activity of black mulberry (Morus nigra L.) extracts and the authors concluded 59.92°C, solvent-solid ratio of 20.73 mL/

g, and 59.47 % ethanol content were the optimal extraction conditions.

Although the researches on phytochemistry have identified the antioxidant activities of various extracts obtained from mulberry root bark of Morus alba L., investigation on the collaborated actions of extraction

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/ absorbance of blank] × 100 (%). IC⁵⁰ was determined as the concentration where radical scavenging rates reach 50%.

Results and discussion

Effect of extraction solvent and temperature on product yield

The extraction conditions are important factors to affect not only extraction yield but also structural and bioactive properties of the extract which determine the functionality and applicability to various industries. In this study, to explore the effects of the EtOH content in the extraction solvent and temperature on the characteristics of the extracts, the extraction process was carried out using the solvents containing 0 – 100 % EtOH at different temperature from room temperature to 100°C.

Table 1 shows the extraction yields of different extraction conditions. The yields were in the range from 9.9 to 27.44 %, indicating that large amount of mulberry root bark components (72.56 – 90.1 %) were insoluble in both water and ethanol. The insoluble products might be cellulose, lignin, or their derivatives.

The extraction yields slightly decreased around 0.1 – 3.8 %point compared to distilled water (DW) groups with the increase of EtOH content from 0 to 70 % showing the similar value to the previously reported one (i.e. 26 % for water extract and 23

% for 80% ethanol extract prepared in the presence of sonic treatment for 2 h.) (Choi et al., 2015). In addition, compared to DW groups, the yields remarkably declined around 11.2 – 12.8

%point when 100% EtOH solvent was used, indicating that most components of root bark are insoluble in the absolute EtOH.

Solvent polarity is the critical factor determining the solubility of molecules (Nawaz et al., 2020). According to the literature, the polarity index (PI) of water is 1 and that of ethanol is 5.2, indicating that ethanol is less polar than water (Abarca-Vargas et al., 2016). Therefore, we could expect that the solvent containing

Determination of total polyphenol and flavonoid

Total polyphenol content in the extract was determined by applying Folin-Denis’s method with a slight modification (Folin and Denis, 1912). Twenty microliters of the extract (25 mg/mL) were added into the mixture of 1.58 mL water and 100 μL of Folin Denis’s phenol reagent. The reaction was allowed for 1 min at room temperature. Three hundred microliters of 20% Na²CO³ was added into the reaction mixture. After incubation for 2 h at room temperature, the absorbance at 750 nm was recorded with a Multiskan GO plate reader (Thermo Scientific, USA). Total flavonoid content was determined according to the established protocol (Lister et al., 1994). One hundred microliters of the extract (25 mg/mL) were mixed with 890 μL of diethylene glycol and 10 μL of NaOH (1 N). The mixture was incubated in 37°C water bath for 1 h. The absorbance at 420 nm was measured with a Multiskan GO plate reader (Thermo Scientific). Gallic acid and naringin were used as the standards for determination of total polyphenol content and flavonoid content, respectively.

Measurement of radical scavenging activity of each extract

Radical scavenging activity was determined using 2-diphenyl- 1-picrylhydrazyl (DPPH) assay according to an established protocol with minimum modification (Marsden, 1958). One hundred microliters of the extracts with various concentrations (1 – 5000 μg/mL) were added into 100 uL of DPPH (Sigma- Adrich, St. Louis, MO, USA) dissolved in methanol (Carlo Erba, Sabadell, Barcelona, Spain) at 60 μM. The mixture was incubated for 30 min at room temperature in dark. The absorbance was read at 540 nm using a Multiskan GO plate reader (Thermo Scientific). The percentage of DPPH radical scavenging activity was expressed as [1-absorbance of sample

Table 1. Extraction yield (%) of mulberry root bark prepared under different extraction conditions. The liquor ratio and the extraction time were fixed to 1:20 and 2 h, respectively.

Extraction condition DW 30% EtOH 50% EtOH 70% EtOH 100% EtOH

RT 21.14 21.02 19.16 18.36 9.90

50°C 24.88 21.96 23.60 21.90 12.10

70°C 26.32 24.48 24.96 24.20 13.64

100°C 27.48 25.24 25.88 23.64 14.86

* RT: room temperature; DW: distilled water.

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Effect of extraction conditions on structural characteristics

FT-IR and CD spectroscopy are powerful tools for obtaining the information about structures and component of extracts.

According to the FT-IR spectra depicted in Fig. 1A-D, absorbance peaks at 3300 cm ^-1 indicating hydroxyl group of phenolic compound and carbohydrate were shown (Santos et al., 2019). The peaks at 2924 and 2854 cm^-1 indicate C-H bonds of methylene groups (Santos et al., 2019). Various marker peaks were exhibited in the range of 1800 – 800 cm^-1. In fact, in Fig.

1E-H, we could observe the peaks at 1607, 1513, and 850 cm^-1 corresponding to the conjugated C-C=C bond and C-H bond of aromatic groups (Nandiyanto et al., 2019; Oliveira et al., 2016). The complex peaks around 1200 – 900 cm^-1might be originated from the glycosidic bond of carbohydrates (Natalello et al., 2005). Therefore, we expected the phenolic compounds, alkaloids, hydrocarbons, and carbohydrates might be contained in the extracts. Phytochemical researches identified that various less amount of EtOH is relatively suitable to obtain the polar

products.

Meanwhile, there was positive correlation between the extraction temperature and yield, suggesting that heating helps the extraction of substances from the root bark matrices regardless of solvent types. Especially, the extraction condition using DW at 100°C resulted in the highest extraction yield (i.e.

27.48 %). Generally, for many solids, the solubility is known to increase at higher temperature. The increase in the kinetic energy with high temperature allows the solvent molecules to more effectively enter the root bark matrices and dissociate the molecular interactions between the components (Lu et al., 2020). On the other hand, excess heat applied to samples can make negative effects on the structural stability of extracted components (Huaman-Castilla et al., 2019). Therefore, the extraction solvent and temperature should be controlled with careful consideration about the extraction yield of desired component, degradation of extracts and economic advantages.

Fig. 1. FT-IR spectra of mulberry root bark extracts prepared under different conditions. Mulberry root bark was extracted in the solvents with different EtOH content (0 – 100%) at various temperature (RT – 100°C). The spectra are categorized by extraction temperature. A-D:

full spectra; E-H: zoomed spectra. (A, E) RT, (B, F) 50°C, (C, G) 70°C, (D, H) 100°C. RT and DW indicate the room temperature and distilled water, respectively.

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except for the slight growth of the absorbance around 1200 – 900 cm^-1of the extracts prepared at higher temperature. This result suggested that more carbohydrate polymers could be extracted by increasing the temperature.

In addition to FT-IR measurement, we performed CD spectroscopic analysis to confirm the presence of the optically active molecules in the extracts. The optically active molecules differently absorb the left and right circularly polarized light, making different ellipticity spectrum. As depicted in Fig. 2, the spectra of each extract are different according to the extraction condition. Especially, negative bands around 210, 240, and 288 nm were observed in most extract samples. Those negative bands were grown strongly with the increase of EtOH content and the intensities of the bands decreased with the increase of temperature. According to the literatures reporting the negative bands at 210 nm corresponds to the n-π* transition of carboxyl group (Chen et al., 2019), we thought the growth of bands at 210 nm in the case of high content of EtOH was attributed to the increase of phenolic acid content in the extracts. However, we could not find strong bands at 210 nm in the DW extracts types of phenols (resveratrol, oxyresveratrol, chlorogenic acid,

mulberroside, maclurin, and moracins), non-anthocyanins flavonoids (rutin, quercetin, and kaempferol-3-rutinoside), polyhydroxylated alkaloids (1-deoxynojirimycin and fagonime), and polysaccharides are contained in Morus alba L. root extracts (Wen et al., 2019; Wei et al., 2016; Kumar and Chauhan, 2011).

Especially, with the increase of EtOH content, the growth of peaks at 2924, 2854, 1607, 1513, and 850 cm^-1 was observed, suggesting that large quantity of hydrocarbons and phenolic compounds were extracted from the root bark. On the other hand, the absorbance around 1200 – 900 cm^-1 significantly decreased when 100% EtOH was used for the extraction. We thought water-soluble polar carbohydrates did not prefer to be dissolved in 100% EtOH which has lower polarity than water.

Considering that carbohydrates account for high fraction of plant tissue (Pettolino et al., 2012), the decrease of extraction yield (Table 1) was due to the lower solubility of carbohydrates in the higher content of EtOH. In contrast to the notable effects of EtOH content on the components extracted from the root bark, the extraction temperature did not significantly affect them

Fig. 2. CD spectra of mulberry root bark extracts prepared under different conditions. Mulberry root bark was extracted in the solvents with different EtOH content (0 – 100%) at various temperature (RT – 100°C). The spectra are categorized by extraction temperature. (A) RT, (B) 50°C, (C) 70°C, (D) 100°C. RT and DW indicate the room temperature and distilled water, respectively.

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that alkaloids and phenolic compounds including flavonoids are extracted more especially when the solvent containing high content of EtOH was used for the extraction at low temperature.

Effect of extraction conditions on total polyphenol and flavonoid contents

Next, we quantitatively evaluated the contents of bioactive components contained in the root bark extracts. Total polyphenol contents were recorded in range from 12.13 to 20.37 mg/g and flavonoids contents were in range from 4.36 to 18.87 mg/g (Fig.

3). The extraction conditions similarly affected both contents.

When the EtOH content increased, more polyphenols and flavonoids were extracted from the root bark. Especially, the contents in 100% EtOH extracts increased around 62.43 – 69.72

% for polyphenol content (Fig. 3A) and 112.58 – 257.54 % for flavonoid content (Fig. 3B) compared to DW extracts. Because polyphenols including flavonoids have hydrophobic moieties in their chemical structure, the solubility was higher in high regardless of temperature. This means that there are few phenolic

acids in DW extracts. γ-Aminobutyric acid (GABA), a non- protein natural amino acid, is one of potent phenolic acids which can influence the ellipticity at 210 nm. Choi et al. demonstrated that GABA content in the root bark was highest (~223.9 mg/100g) among various parts of mulberry tree including fruit, leaf, and twig (Choi et al., 2015). Also, GABA can be obtained more in EtOH solvent (Le et al., 2020) and the extraction temperature has negative correlation with the extraction yields of GABA (Tiaokun et al., 2017). The negative bands around 240 and 288 nm observed in all spectra might be attributed from the flavonoids in the extract. According to the previous literature, the isomers of 2,3-trans-3,4-Flavan-3,4-diols (2R, 3S, 4R) and 2,3-cis-3,4-trans-Flavan-3,4-diols (2R, 3R, 4S) showed the strong negative bands around both 240 and 290 nm (Ferreira et al., 2004; Slade et al., 2005). Flavan-3,4-diol has another name as leucoanthocyanidin which is found in whole plants (Husain, 1992). Taken together, FT-IR and CD spectra demonstrated

Fig. 3. Total polyphenol (A) and flavonoid (B) contents of mulberry root bark extracts prepared under different conditions. Mulberry root bark was extracted in the solvents with different EtOH content (0 – 100%) at various temperature (RT – 100°C). RT and DW indicate the room temperature and distilled water, respectively.

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compounds. These combinational negative effects of heat and EtOH on phenolic components and flavonoids were consistent with CD spectroscopic results (Fig. 2).

Effect of extraction conditions on radical scavenging activity

Given the fact that the structural and componential analyses showed the different contents of bioactive components in each extract, we could hypothesize that the extraction solvent and temperature might affect the antioxidant activities of the extract. Therefore, the antioxidant activity of each extract was investigated using DPPH radical scavenging assay. Fig. 4 shows the concentration which presents 50% radical scavenging activity (IC⁵⁰) of each extract. IC⁵⁰ values of each extract were different according to the extraction conditions, indicating that EtOH and temperature can affect the extraction of antioxidative components from the root bark. In all temperature groups, IC⁵⁰ values of DW extracts (16.75, 14.48, 11.46, and 12.78 μg/mL for RT, 50, 70, and 100°C, respectively) were higher than those of 100% EtOH extracts (15.08, 6.97, 10.03, and 8.31 μg/mL for RT, 50, 70, and 100°C, respectively), indicating that 100% EtOH dissolved more antioxidative components. These lower IC⁵⁰ values were thought to be due to the higher polyphenol and flavonoid contents of 100% EtOH extract than those of DW extracts as exhibited in Fig. 3. We could also observe that all extracts prepared under heat exposure conditions have low IC⁵⁰ values compared to the extracts prepared without heating, regardless of solvent types.

Especially when 30 and 50% EtOH were used, IC⁵⁰ values more dramatically decreased with increase of temperature. However, concentration of EtOH solvent which has lower polarity than

DW (Abarca-Vargas et al., 2016). These increasing rates were lowest when the extraction was performed at 100°C (i.e. 62.43%

for polyphenol contents and 112.58% for flavonoid contents).

However, the contents were not significantly different between 30-70% solvent when the extraction temperature was 50, 70, and 100°C.

Interestingly, the extraction temperature affected the contents differently according to the solvent type. When DW and 50%

EtOH were used, the polyphenol and flavonoid contents were not affected by the extraction temperature, showing the similar values around 10 and 5 mg/mL, respectively. In case of 30%

EtOH solvent, however, the contents slightly increase from 12.98 to 14.62 mg/mL for total polyphenol and from 6.81 to 9.36 mg/mL for flavonoid when the temperature increased from RT to 100°C. In contrast, when 70 and 100% EtOH were used, polyphenol and flavonoid contents have negative correlation with the temperature. In fact, with increase of extraction temperature from RT to 100°C in case of 70% EtOH solvent, polyphenol content decreased from 16.87 mg/mL to 13.6 mg/mL (Fig. 3A).

Similarly, the content decreased from 20.37 mg/mL (at RT) to 17.07 mg/mL (at 100°C) when 100% EtOH was used as the extraction solvent. These collaborated effects of EtOH content and temperature were also shown in flavonoid content results (Fig. 3B). It was reported that polyphenols such as kaempferol, quercetin, catechin, epicatechin and procyanidins could be degraded at high temperature during the extraction process (Huaman-Castilla et al., 2019). Therefore, we expected that heat and EtOH might combinedly make the damages in the phenolic

Fig. 4. DPPH radical scavenging activity of mulberry root bark extracts prepared under different conditions. Mulberry root bark was extracted in the solvents with different EtOH content (0 – 100%) at various temperature (RT – 100°C). IC⁵⁰ concentrations were plotted according to the extraction conditions. RT and DW indicate the room temperature and distilled water, respectively.

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critical roles in antioxidant activity, further studies on various components including polysaccharides, amino acids, and minerals in extracts prepared under different conditions need to be performed.

Acknowledgement

This work was carried out with the support of “Research Program for Agriculture Science and Technology Development (Project No. PJ015107012020)” Rural Development Administration, Republic of Korea.

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Conclusion

In conclusion, we investigated the effects of extraction parameters such as solvent type and temperature on several characteristics of mulberry root bark extracts. The experimental results showed that the extraction yields, structural characteristics, the content of phenolic components, and antioxidant activities in the extract were critically affected by EtOH content and temperature.

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