Optimization of Jirisan Mountain Cudrania tricuspidata leaf substance extraction across solvents and temperatures.

OBJECTIVE: The aim of this study is to optimize the extraction of beneficial substance from Cudrania tricuspidata leaves grown at Jirisan Mountain in South Korea by three different solvents depending on extraction time and at different temperature.
METHODS: The total phenolic contents were determined by the method reported by Sánchez-Moreno et al. The total flavonoid contents were analyzed by Slinkard and Singleton. The DPPH radical scavenging activity was determined according to the method reported by Blois.
RESULTS: The extraction yield for each solvent is 9.05-14.1%, 2.17-5.67%, and 2.3-3.9% for D.W., ethanol, and hexane, respectively. The overall results were maximized for the extract obtained with D.W. for 5 min at 100°C. The average phenol contents were 77.11, 45.64, and 0.343 mg/g at 100°C in water, 78°C in ethanol, and 68°C in hexane, respectively. The flavonoid contents were the highest in the materials extracted with D.W., and were increased with increasing temperature, regardless of the extraction solvents, whether water (green), polar organic ethanol, or nonpolar organic hexane. In the ethanol extract, the flavonoid contents are increased gradually from 5.66 mg/g to 7.73 mg/g. The total flavonoid contents were proportional to the concentrations of the water extracts, ranging from 4.14 mg/g to 48.89 mg/g. The antioxidative activities of the water-extracted compounds are generally increased with increasing temperature from 42.5% to 85.5%. Those of the hexane extracts are increased slowly from 3.79% to 8.8%, while those of ethanol extracts are increased from 29.8% to 47.4%.
CONCLUSION: The extraction yields were dependent upon solvents for extraction as well as extraction time and the temperature. The optimal extraction time was 5 min and the extraction yields were increased with increasing temperature excepted hexane. Of the three tested extraction solvents, the greenest solvent of water shows excellent results, suggesting that water is among the most effective solvents for natural sample extractions for general medicinal, pharmaceutical, and food applications.

1. Introduction

Cudrania tricuspidata is a plant of the Moraceae family native to Korea, Japan, and China that often grows at the foot of mountains. Approximately ten kinds of Cudrania tricuspidata are known. The fruit is used in jams and alcoholic beverages, bark and roots are used for medicine, and leaves are used in teas and soups as substitutes for mulberry leaves. Cudrania tricuspidata has traditionally been used as a medicinal plant for conditions including eczema, tuberculosis, and chronic back pain [1]. According to previous studies, the plant has many beneficial characteristics, including antioxidative [2], antimicrobial [3], antitumor [4], antiobesity [5], anti-osteoarthritis [6], antiaging [7], antiinflammatory, and neuroprotective [8] properties. It also shows efficacy against cardiovascular diseases [9] such as atherosclerosis and hypertension [10].

Although organic solvents must be used to extract some natural materials, despite their potential hazards and generation of toxic substances, environmentally and biologically friendly “green” extraction technologies for natural substances are developing rapidly in the medicinal, pharmaceutical, and food industries [11-13]. Water is the greenest solvent; it is nontoxic to the human body, environmentally friendly, and safe to work with, and is therefore considered among the most environmentally sustainable solvents [14]. Water extraction temperatures can be varied to increase the extracted quantity of effective beneficial substances from natural sources. Moreover, high-temperature water extraction (HTWE) can shorten the extraction time. However, a precise understanding of the stability of natural extracts at various temperatures and time conditions is necessary for implementing HTWE. This study is intended not to characterize the beneficial chemical substances of Cudrania tricuspidata leaves (CTL), but to determine the optimal extraction conditions for obtaining beneficial substances with optimized antioxidative effects, total phenolic contents, and flavonoid contents using the different extraction solvents of water, ethanol, and hexane.

2. Materials and Methods

Materials and Methods

Materials

Cudrania tricuspidata leaves (CTL) used were collected in April and May of 2017 by a medicinal plant supplier from Jirisan Mountain in Sancheong County, Gyeongnam, South Korea.

Extraction from CTL

10 g of dried and ground CTL were mixed with 100 mL of distilled water (D.W.), 100 mL ethanol, or 100 mL hexane, respectively. The extraction was performed for time intervals of 5 min, 15 min, 30 min, 45 min, and 1 h. The temperature of the water extraction was varied from room temperature (RT) to 40°C, 60°C, 80°C, and 100°C. Ethanol extractions were performed at RT, 40°C, 60°C, and 78°C, while hexane extractions were performed at RT, 40°C, and 68°C. Heat-reflux extraction using a water-bath was performed to maintain the extraction temperature. All extracts were filtered with Whatman® Grade 2 qualitative filter papers (GE Healthcare), evaporated, and dried using a freezing dryer (FD5508, Ilshin Lab Co., Ltd.) before use.

Functional bioassays of CTL

For the functional bioassays, 1-mL samples of the extracts were centrifuged at 8000 rpm for 5 min and then filtered with a syringe filter (Hyundae, 0.45 μm). The total phenolic contents were determined by the method reported by Sánchez-Moreno et al. [15,16] from the standard curve prepared using garlic acid as a standard. The total flavonoid contents were analyzed by the standard curve using catechin, as described by Slinkard and Singleton [17,18]. The DPPH radical scavenging activity was determined according to the method reported by Blois [16,19].

Statistical Analysis

All experiments were independently performed in triplicate (n = 3) to obtain statistic values. The data reported are expressed as the mean ± standard deviation. One-way analysis of variance was used for statistical significance (p<0.05).

3. Results and Discussion

Many beneficial substances supporting human health have been extracted and analyzed both chemically and structurally from natural sources. Scientific efforts to discover new substances remain active (Table 1 and Table S1).

Table 1

Total phenols, flavonoids concentration and antioxidant activity of plant extracts.

Compound Name	Effects	Structure	Solubility (1 L)	Reference
Deoxynojirimycin (DNJ)	Antioxidant		Water: 16.32 g	[5]
Rutin	Antioxidant, blood vessel protection		Water: 0.13 g	[5]
Kaempferol	Antioxidant, neuroprotective, antidiabetic, anticancer, cardiovascular disorders		Water: slightly solubleEthanol: very soluble	[23]
Quercetin	Neuroprotective		Water: Practically insoluble; soluble in aqueous alkaline solutions	[24]
Resveratrol	Neuroprotective, anticancer, antiaging, antidiabetic, antifungal		Water: 0.03 gEthanol: 50 gDMSOa: 16 g	[25]
GABA	Antianxiety, antidiabetic		Water: 0.130 g	[26]

DMSO: Dimethyl sulfoxide

The three different extraction solvents of distilled water (D.W.), ethanol, and hexane were analyzed in terms of yield rates. The extraction yield for each solvent is represented in Fig. 1a, at 9.05–14.1%, 2.17–5.67%, and 2.3–3.9% for D.W., ethanol, and hexane, respectively. The extraction yield of D.W. was increased as the temperature increased, maximizing at 100°C. Ethanol and hexane yields were also increased with temperature and time. For hexane, yield was increased at room temperature, but decreased at 40°C and 68°C (Table S2). The overall results were maximized for the extract obtained with D.W. for 5 min at 100°C. Functional bioassays according to concentration were also performed on extracts with D.W. for 5 min at 100°C. As the concentrations of the samples were increased, the bioactivities also increased (Table S3). Although the extraction yield with D.W. was decreased when 20 g of dried and ground CTL were extracted using 100 mL solvent, the bioactivities were slightly increased. From the preliminary optimization of extraction yield with each solvent, 10 g of dried and ground CTL in 100 mL solvent showed the best results; these quantities were thus maintained for all later extraction experiments (Fig. 1b).

Figure 1

The yields of CTL extract. a. Extraction yields by D.W., ethanol (EtOH), and hexane. b. The yield of D.W. extracts and functional assay by D.W.

The polyphenol compounds in plants are important because of their hydroxyl group-related scavenging abilities, which facilitate binding with various compounds and grant antioxidative, anticarcinogenic, and antiinflammatory properties [20]. The polyphenol contents were analyzed based on the extraction solvents, temperatures, and times (Fig. 2a). Regarding the solvent used, D.W. yielded significantly higher phenol contents than ethanol and hexane did. For extraction temperature, the amount of extracted phenol increased as the temperature was increased. The average phenol contents were 77.11, 45.64, and 0.343 mg/g at 100°C in water, 78°C in ethanol, and 68°C in hexane, respectively. As the extraction time was extended, the phenol contents slightly decreased from 80.21 mg/g to 73.97 mg/g in water, but this difference was insignificant. In ethanol and hexane, the phenol contents slightly increased with time extension, from 43.64 to 46.59 mg and from 0.288 to 0.4mg, respectively (Table S4). The total phenolic compounds gradually increased from 30.32 mg/g to 79.2 mg/g by increasing the quantities of extract in D.W.

Figure 2

Total polyphenol contents of CTL by three different extraction conditions. a. The estimation of total polyphenol contents in extracts by three different solvents. b. The estimation of total flavonoid contents in extracts by three different solvents. c. The effects of extraction conditions on the DPPH radical scavenging activities of the CTL extracts.

Flavonoids are a subset of polyphenols and are widely distributed in natural materials. They have high antioxidative activities, effectively eliminating reactive oxygen species, in addition to antiviral, antiinflammatory, and anticancer functions [21]. The flavonoid contents were the highest in the materials extracted with D.W., and were increased with increasing temperature, regardless of the extraction solvents, whether water (green), polar organic ethanol, or nonpolar organic hexane. In the ethanol extract, the flavonoid contents are increased gradually from 5.66 mg/g to 7.73 mg/g (Fig. 2b). The total flavonoid contents were proportional to the concentrations of the water extracts, ranging from 4.14 mg/g to 48.89 mg/g (Table S5).

1,1-Diphenyl-2-dipicryldihydrazyl (DPPH) is a stable free radical that is reduced by antioxidant compounds, and thus is used to measure antioxidative activity [22]. The antioxidative activities of the water-extracted compounds are generally increased with increasing temperature from 42.5% to 85.5% (Fig. 2c). Those of the hexane extracts are increased slowly from 3.79% to 8.8%, while those of ethanol extracts are increased from 29.8% to 47.4%. Despite the lower quantities, the hexane extracts may show different properties than the water or ethanol extracts because of the nonpolar nature of the solvent. The total antioxidative activities in the water–extracted compounds were much higher than those of ethanol and hexane. The antioxidant activities of the extracts were steadily increased with increasing sample mass, with the exception of samples exceeding 10 g (Table S6).

4. Conclusion

This study systematically investigated changes in functionality, such as the total contents of polyphenols and flavonoids and the antioxidant activities, of CTL extracts when obtained using different extraction solvents, temperatures, and times. Water extraction yielded compounds showing not only significantly higher total contents of polyphenols and flavonoids, but also greater antioxidant activities, than extraction by organic polar or nonpolar solvents. The optimal extraction time was 5 min and the extraction yields were increased with increasing temperature, however hexane was the exception. Of the three tested extraction solvents, the greenest solvent of water shows excellent results, suggesting that water is among the most effective solvents for natural sample extractions for general medicinal, pharmaceutical, and food applications.

Table 1

Total phenols, flavonoids concentration and antioxidant activity of plant extracts.

Compound name	Effects	Structure	Reference
Myricetin	Neuroprotective		[1]
Oxyresveratrol	Skin protection, hepatoprotective		[2]
Cudratricusxanthone A	Antiplatelet, anticoagulant		[3]
Macluraxanthone B	HIV-lnhibitory		[3]
Imperatorin	Anticonvulsant, anticancer		[4]
Isoimperatorin	Antiinflammatory, hepatoprotective effect, antimycobacterial activity		[4]
Syringaresinol	Antioxidant, anticancer		[4]
Umbelliferone	Antihyperglycemic effect, antiinflammatory		[4]
Achilleol A	Antioxidant		[4]
4-(Methoxymethyl)phenol	Antioxidant		[4]
Erysubin A	Antileukemia		[4]
Lupalbigenin	Antioxidant		[4]
Millewanin H	Antioxidant		[4]
Cycloartocarpin	Antioxidant		[5]
Licoflavone C	Antioxidant		[6]
Carthamidin	Antioxidant		[6]
Millewanin G	Antioxidant		[6]
Furowanin B	Antioxidant		[7]
Isolupalbigenin	Antioxidant		[7]

Table S2

The extraction yield by three different solvents

Solvent	min	The extraction yield (%)
		20 °C	40 °C	60 °C	80 °C	100 °C
D.W.	5	12.35±0.09	12.38±0.10	12.40±0.09	13.10±0.10	14.00±0.11
	15	11.90±0.07	12.05±0.08	11.75±0.10	12.99±0.09	13.70±0.10
	30	10.35±0.06	11.82±0.08	11.09±0.09	12.08±0.07	13.00±0.09
	45	9.82±0.03	10.70±0.04	10.82±0.05	11.76±0.03	12.70±0.04
	60	9.05±0.03	9.80±0.04	10.20±0.05	11.02±0.04	11.25±0.06
Solvent	min	20 °C	40 °C	60 °C	78 °C
EtOH	5	2.17±0.06	2.98±0.08	3.78±0.09	4.03±0.09
	15	2.27±0.04	3.02±0.08	3.89±0.07	4.11±0.07
	30	2.55±0.06	3.15±0.09	3.94±0.07	4.68±0.08
	45	2.69±0.07	3.22±0.08	4.03±0.07	5.08±0.05
	60	2.73±0.05	3.26±0.07	4.06±0.06	5.67±0.08
Solvent	min	20 °C	40 °C	68 °C
Hexane	5	2.30±0.02	2.90±0.03	3.90±0.05
	15	3.00±0.06	3.10±0.07	3.70±0.05
	30	3.90±0.07	3.30±0.06	3.30±0.06
	45	3.30±0.04	3.00±0.03	3.00±0.06
	60	3.00±0.03	2.90±0.06	2.70±0.07

Table S3

The yield of extracts and the functional assay by D.W. at 100°C, 5min

	100ml
	1g	2.5g	5g	10g	20 g
The yield (%)	2.88±0.02	6.30±0.04	11.10±0.03	14.10±0.05	6.52±0.03
Phenolic (mg/g)	30.32±0.09	36.00±0.08	54.00±0.06	69.60±0.03	79.2±0.07
Flavonoid (mg/g)	4.14±0.07	10.84±0.09	22.60±0.05	42.47±0.05	48.89±0.05
DPPH (%)	18.19±0.06	26.70±0.04	41.32±0.07	70.76±0.08	78.44±0.09

Table S4

The total phenolic by three different solvents

Solvent	min	phenolic (mg/g)
		20 °C	40 °C	60 °C	80 °C	100 °C
D.W.	5	31.12±0.09	46.41±0.10	75.38±0.08	74.58±0.09	80.21±0.12
	15	36.00±0.08	60.00±0.08	75.72±0.12	75.00±0.09	80.40±0.10
	30	47.62±0.11	69.95±0.10	76.79±0.11	76.39±0.09	76.59±0.12
	45	49.20±0.09	70.68±0.11	72.00±0.10	74.04±0.11	74.40±0.13
	60	53.85±0.08	71.36±0.10	69.95±0.09	72.56±0.12	73.97±0.11
Solvent	min	20 °C	40 °C	60 °C	78 °C
EtOH	5	8.23±0.07	14.94±0.09	28.62±0.10	43.64±0.08
	15	12.80±0.06	20.80±0.05	30.40±0.05	44.80±0.07
	30	16.28±0.05	26.74±0.08	36.94±0.07	46.59±0.06
	45	16.64±0.06	27.04±0.05	33.60±0.09	46.59±0.08
	60	17.09±0.04	28.08±0.06	32.91±0.07	46.59±0.05
Solvent	min	20 °C	40 °C	68 °C
Hexane	5	0.16±0.01	0.19±0.01	0.29±0.01
	15	0.18±0.01	0.21±0.01	0.30±0.01
	30	0.19±0.01	0.22±0.01	0.32±0.01
	45	0.19±0.01	0.21±0.01	0.37±0.01
	60	0.21±0.01	0.24±0.01	0.40±0.01

Table S5

The total flavonoid by three different solvents

Solvent	min	flavonoid(mg/g)
		20 °C	40 °C	60 °C	80 °C	100 °C
D.W.	5	3.36±0.02	4.55±0.03	5.97±0.03	8.96±0.04	9.41±0.02
	15	3.47±0.07	4.65±0.06	5.89±0.09	9.08±0.07	9.47±0.09
	30	3.82±0.04	4.77±0.03	5.79±0.05	8.85±0.06	9.52±0.03
	45	3.78±0.05	4.71±0.03	5.61±0.05	8.83±0.02	9.38±0.02
	60	3.75±0.01	4.64±0.04	5.45±0.06	8.79±0.07	9.44±0.09
Solvent	min	20 °C	40 °C	60 °C	78 °C
EtOH	5	5.00±0.03	5.35±0.02	5.64±0.05	5.66±0.06
	15	4.22±0.05	5.57±0.04	6.12±0.07	6.69±0.05
	30	3.54±0.11	5.86±0.13	6.69±0.08	7.32±0.03
	45	4.22±0.05	6.51±0.10	7.17±0.17	7.55±0.11
	60	4.62±0.09	6.71±0.09	7.55±0.21	7.73±0.13
Solvent	min	20 °C	40 °C	68 °C
Hexane	5	0.12±0.02	0.12±0.01	0.12±0.02
	15	0.14±0.03	0.14±0.01	0.14±0.02
	30	0.14±0.02	0.14±0.02	0.17±0.01
	45	0.14±0.01	0.14±0.01	0.17±0.01
	60	0.14±0.02	0.14±0.01	0.16±0.01

Table S6

The effect of extracts condition on DPPH radical scavenging activity by three different solvents

Solvent	min	DPPH (%)
		20 °C	40 °C	60 °C	80 °C	100 °C
D.W.	5	42.50±0.06	55.30±0.10	68.20±0.11	78.00±0.10	85.50±0.09
	15	48.00±0.10	56.20±0.10	69.80±0.07	79.00±0.06	85.00±0.10
	30	50.60±0.08	57.10±0.07	66.80±0.11	80.70±0.09	84.10±0.10
	45	50.90±0.09	55.00±0.08	64.00±0.10	78.00±0.07	83.90±0.06
	60	51.20±0.10	53.70±0.11	61.00±0.10	76.40±0.10	84.00±0.10
Solvent	min	20 °C	40 °C	60 °C	78 °C
EtOH	5	29.80±0.10	35.90±0.10	47.60±0.10	47.40±0.09
	15	30.00±0.08	39.00±0.06	48.00±0.10	48.00±0.09
	30	31.40±0.09	44.28±0.10	48.40±0.10	49.50±0.11
	45	33.00±0.09	46.00±0.12	49.50±0.10	53.50±0.10
	60	36.80±0.09	48.75±0.08	51.60±0.07	57.50±0.08
Solvent	min	20 °C	40 °C	68 °C
Hexane	5	3.79±0.03	7.42±0.05	8.80±0.02
	15	2.75±0.03	8.42±0.02	9.60±0.04
	30	2.21±0.02	8.22±0.04	10.15±0.03
	45	2.00±0.01	8.09±0.01	9.60±0.01
	60	1.97±0.02	8.02±0.03	9.20±0.02