Evaluation of proximate composition , bioactive lignans and volatile composition of Schisandra chinensis fruits from Inje and Mungyeong , Republic of Korea

Article history: Received on: 08/08/2016 Revised on: 06/09/2016 Accepted on: 11/10/2016 Available online: 29/11/2016 The present study aimed to determine the proximate composition, mineral content, active ingredients (schisandrin, schisandrin A, gomisin A and gomisin N) and volatile composition of Schinsandra chinensis fruits collected from Inje and Mungyeong, two major cultivating areas in Republic of Korea. The bioactive ingredients were determined by high performance liquid chromatography (HPLC). The volatile composition of the supercritical carbon dioxide extracts (SFE) from the fruits of S. chinensis was determined by solid phase microextraction (SPME) gas chromatography/mass spectrometry (GC/MS). The proximate composition was found to be higher in Mungyeong than Inje samples. The minerals such as Mn, Ca and P were higher in the samples obtained from Inje when compared with Mungyeong. The active ingredients such as schisandrin (5018.67 μg/g), gomisin N (4772.87 μg/g) and schisandrin A (717.18 μg/g) were found to be higher level in Mungyeong samples with the exception of gomisin A. The SPME–GC/MS analysis revealed the identification of 40 components from each place, representing 97.22% (Inje) and 96.81% (Mungyeong) of the SFE. Ylangane, α-himachalene, longipinene and italicene were detected as the major components in the SFE. In conclusion, Inje and Mungyeong are the suitable places to collect S. chinensis fruits with higher level of nutrient and chemical contents.


INTRODUCTION
The genus Schisandra belongs to the family of Schisandraceae and contains 23 deciduous vine species that are widely distributed in East Asia.In the genus, Schisandra chinensis (Turcz.)Baill. is an important traditional medicinal plant and mainly cultivated in northeastern regions of China, Japan, Korea and Russia (Sun et al., 2010).This cash crop has been cultivated in alpine areas of Republic of Korea and the quality has been known to be determined by the climate of cultivating areas.The fruits of S. chinensis (Korean nameomija) are traditionally used for the treatment of various .disorders such as cough, spontaneous sweating, palpitation, spermatorrhea, dyspnea, kidney disorders, mouth dryness, dysentery and amnesia (Wang et al., 2008;Teng and Lee, 2014).The fruits contain various bioactive components including essential oil, organic acids, vitamins, lignans, terpenes, amino acids, polysaccharides, etc (Huang et al., 2008;Stacchiotti et al., 2009;Gao et al., 2009;Lu and Chen, 2009).Among them, lignans and the essential oil with terpenes are the most bioactive ingredients in the fruits of S. chinensis (Wang et al., 2008).Pharmacological studies have showed that the lignans exhibited various biological activities, including hepatoprotective, anticancer, antioxidant, cardioprotective, adaptogenic and central nervous system protecting activities etc. (Jiang et al., 2005;Huang et al., 2007;Hu et al., 2012;2013).Owing to the wide range of bioactive properties, the consumption of S. chinensis fruits has gained increasing popularity as dietary supplements.
In addition to lignans, the volatile components from the fruit of S. chinensis were widely used in pharmaceuticals and cosmetic industries.The steam distillation or hydrodistillation is a conventional method widely used to isolate the volatile components from various medicinal and aromatic plant materials (Herrero et al., 2010).
However, the conventional techniques have many disadvantages such as low extraction efficiency, longer extraction time and toxic solvent residue.Further, the essential oil quality is also highly affected due to the effect of the high temperatures (Fornari et al., 2012).
Previously, some authors have reported the volatile composition of S. chinensis fruits obtained from steam distillation, supercritical fluid extraction (SFE), Soxhlet, microwave assisted extraction and headspace solid-phase micro-extraction (SPME) methods (Deng et al., 2003;Li et al., 2003;Wang et al., 2008;Chen et al., 2012;Teng and Lee, 2014).In recent times, SFE has attracted significant attention as an effective and environmentally friendly extraction technique to replace conventional methods (Capuzzo et al., 2013).The SFE method has been developed significantly in the extraction of S. chinensis fruits, but most of the studies are related to the extraction of lignans (Wang et al., 2008).Furthermore, SPME combined with GC/MS is a novel technique, solvent-free, rapid and very simple to extract and detect the volatile components (Delgado et al., 2010).Hence, SPME-GC/MS was used in the present study to analyze the volatile composition of SFE from the fruits of S. chinensis .
According to the previous results, there were considerable qualitative and quantitative variations in the volatile composition from the fruits of S. chinensis and these variations might be influenced by various biotic and abiotic factors (Dhouioui et al., 2016).In the previous studies, the samples were mainly collected from China in relation to volatile composition of S. chinensis .
In addition, Inje and Mungyeong are the two major cultivating areas of S. chinensis in Republic of Korea, but there has been no comparative study on the nutritional and chemical composition of this fruit.Further, there is no report on volatile composition of SFE of S. chinensis fruits using SPME-GC/MS.Therefore, the present study was designed with the aim of determining and comparing the proximate composition, mineral content, active ingredients and volatile composition of S.chinensis fruits collected from Inje (Gangwon-do) and Mungyeong (Gyeongsangbuk-do) in Republic of Korea.

Materials
The matured and dried fruits of S. chinensis were procured from different locations of Inje (10 places) and Mungyeong (10 places) in Republic of Korea during February 2014 (Fig. 1).The lignans such as schisandrin, schisandrin A, gomisin A and gomisin N were purchased from Sigma-Aldrich Chemicals (St. Louis, MO, USA).All other chemicals and solvents used in the study were of analytical grade.

Analysis of proximate composition
In the proximate composition, moisture, crude protein, crude lipid, crude fiber and ash contents of S. chinensis fruit samples were determined by standard methods (AOAC, 2000).Moisture content of sample was determined using the direct drying method.The sample was dried in a hot air-oven set at 105ºC until constant weight of the sample was obtained.Protein content of the sample was determined according to the principle of Kjeldahl method.A conversion factor of 6.25 was used to convert the measured nitrogen content to protein content.Lipid content of sample was determined by using a Soxhlet extractor with diethyl ether as solvent.The crude fiber was determined by alternately digesting the dried, defatted sample in 1.25% HCl and 1.25% NaOH.Ash content of sample was determined using the dry ashing method.The sample was incinerated in a cold muffle furnace set at 550ºC until whitish/greyish ash was obtained.

Determination of mineral composition
The minerals such as iron (Fe), manganese (Mn), copper (Cu), calcium (Ca), potassium (K), magnesium (Mg), sodium (Na), and phosphorus (P) were determined in the fruit samples from Inje and Mungyeong.Ten milliliters of concentrated HNO 3 (70%) were added to 0.2 g of the fruit samples in a test tube.The dispersion was digested in a digestion block at 100ºC for 3 h.After cooling, the digested products were diluted to 40 ml with deionized water, and centrifuged at 5000 rpm for 10 min.The clear solution was used for mineral determination by an inductively coupled plasma-optical emission spectrometer(Integra XL, GBC Scientific, Australia).For P, absorbance was measured in a UV-visible spectrophotometer (HP 8453E, Hewlett-Packard

Inje Mungyeong
Co., USA) at 420 nm.The results are expressed as g/100 g dry weight of sample for each mineral element.

Quantitative determination of active components by HPLC
The dried and pulverized fruits of S. chinensis (0.3 g) were extracted with 25 ml of methanol in an ultrasonic bath for 30 min at room temperature and centrifuged for 10 min at 5,000 rpm.Then the supernatant was filtered through 0.45 µm membrane syringe filter and the filtrate was used for HPLC analysis.
The active components (lignans) were quantified by Shiseido NANOSPACE SI-2 HPLC system (Shiseido, Tokyo, Japan).The standard stock solutions of schisandrin, schisandrin A, gomisin A and gomisin N were prepared in methanol.Chromatographic separation was performed using a reversedphase column Unison UK-C18 (4.6 mm x 150 mm x 3 μm) at a column temperature of 30ºC.The mobile phases used to elute were distilled water (A) and acetonitrile (B), with an isocratic condition (A, 40% and B, 60%) for 30 min.The injection volume of the sample was set at 10 μL, with a fixed flow rate of 1 mL/min, and the detector wavelength was set to 254 nm (Accela PDA, Thermo Fischer Scientific, Bremen, Germany).

Supercritical CO 2 extraction (SFE)
The SFE was performed by ISA-SCCO-S-050-500 (ILSHIN Autoclave Co. Ltd., Daejeon, Republic of Korea).A hundred grams of dried fruits (not-pulverized) were loaded into a stainless steel extraction vessel.And the CO 2 was pressurized with a high-pressure pump and then charged into the extraction column to desired pressure.Back pressure regulators are used to set the system pressure (in extractor and separator).To optimize the SFE conditions for S. chinensis fruits, the extraction was conducted at different pressures (200, 300 and 400 bar) and temperatures (40, 50, 60 and 70ºC).The CO 2 flow rate was maintained at 30 mL/min.Each extraction process was performed for 60 min and the yield of extract obtained from the different extraction conditions was expressed as percent of the dry weight of fruits.

Solid phase microextraction (SPME) conditions
SFE (1 g) obtained from the fruits of S. chinensis was introduced into SPME vial (20 mL).The SPME device coated (fused-silica fiber) with a 100 µm layer of polydimethylsiloxane (Supelco, Bellefonte, PA, USA) was used for extraction of the volatiles and the vial was sealed with a silicone septum.The fiber was exposed in the SPME vial at 60ºC for 30 min and immediately introduced in the gas chromatography injector.

Gas chromatography/mass spectrometry (GC/MS) analysis
GC-MS analysis was performed with a Varian CP 3800 gas chromatography equipped with a VF-5 MS polydimethylsiloxane capillary column (30 × 0.25 mm x 0.25 µm) and a Varian 1200 L mass detector (Varian, CA, USA).Helium was used as a carrier gas at the rate of 1 mL/min.Oven temperature was kept at 50ºC for 5 min initially, and then raised with rate of 5ºC/min to 250ºC/min.The injected volume of essential oil was 10 μL with a split ratio of 1:10.The injector temperature was set at 250ºC.The mass spectra were recorded in the electrospray ionization mode at 70 eV in a scan range of 50 -600 m/z.The components of essential oils were identified by comparing the retention indices of the GC peaks obtained using homologous series of n-alkanes (C 8 -C 20 ) with those reported in literature (Adams, 2007).The mass spectra of the peaks were also matched with standards reported in literature and National Institute of Standards and Technology (NIST, 3.0) library.

Proximate composition and mineral content of Schisandra chinensis fruits
In the proximate composition analysis, moisture, crude protein, lipid and fiber and ash contents of the fruit samples obtained from Inje and Mungyeong were determined.The mean value of proximate composition for the fruits of S. chinensis is shown in Table 1.The moisture value of the fruits from Inje and Mungyeong were 3.88% and 3.81%, respectively.The major proximate components in the fruit samples of Inje and Mungyeong were crude lipid (13.51% and 14.73%, respectively) and crude fiber (13.77% and 14.19%, respectively) followed by crude protein (9.50% and 10.58%, respectively).The fruit samples also registered considerable amount of ash content (Inje -5.41% and Mungyeong -6.11%).From the results, the fruit samples from Mungyeong possess higher level of proximate composition than Inje samples.In general, a significant amount of ash content specified the presence of appreciable amounts of inorganic nutrients in the plant materials.The presence of substantial amount of lipids reveals the potential of this fruits to have dietary purposes with good nutritional qualities (Iqbal et al., 2012).Further, the fruits also possess considerable amount of crude fiber and crude protein contents.The results of proximate composition indicated the fruits of S. chinensis possess a high nutritional value.Previously, Kim and Choi (2008) investigated the physicochemical and antioxidative properties of S. chinensis fruits.The fruits contained 57.5% of moisture, 18.8% of crude fat, 12.6% of carbohydrate, 11.1% of crude protein, 4.9% of ash and 5.4% of crude fiber.The authors also reported the amino acid contents of the fruits with the largest portion of glutamic acid 131.7 mg/100 g followed by 51.5% aspartic acid.In addition, the fruits contained 7 types of free sugar contents with the glucose and fructose were registered as the dominant sugars.
It is essential to understand the chemical composition of the fruits, especially the composition of minerals and other trace elements, because the mineral composition of foods has a fundamental role in the diet of human.Table 1 shows the mineral contents of S. chinensis fruit samples from Inje and Mungyeong.The mineral composition of S. chinensis fruit samples varied considerably between two different places.Among the macrominerals, K was the most concentrated mineral in the fruits from both the places, Inje and Mungyeong (1085.82 and 1246.53 mg/100 g, respectively) followed by Mg (124.71 and 129.05 mg/100 g, respectively) and Ca (82.92 and 78.46 mg/100 g, respectively).Fe was the most concentrated micro-mineral in the fruits of Inje and Mungyeong (13.43 and 15.03 mg/100 g, respectively) followed by Mn (6.49 and 5.61 mg/100 g, respectively).Among the eight minerals analyzed, Fe, Cu, Mg and Na contents were found to be higher in the Mungyeong samples than Inje samples.These minerals are essential for the correct functioning of the human body.
The composition of minerals and other nutrients in the plant materialis mainly influenced by various biotic and abiotic factors.Hwang et al. (2015) studied the mineral contents of S. chinensis fruits from Korea and China.The mineral contents of S. chinensis fruits from China (100 g) were K (923.31mg), Mg (83.91 mg), Ca (14.80 mg), Mn (6.19 mg), Fe (4.30 mg), Zn (1.12 mg), Na (2.20 mg) and Cu (0.30 mg).In addition, the authors reported that the contents of K and Zn were found to be significantly higher in Korean fruits than the Chinese fruits.Kim and Choi (2008) reported the presence of 10 minerals in the fruits with the highest levels of K (912.6 mg/100 g) and Ca (613.8 mg/100 g), followed by Al, Mg, Na and Mn.The present study also confirmed that the fruit samples comprised higher levels of K, Mg and Ca minerals.Further, these nutrients were found to be higher in the current study when compared to that of previous reports.Potassium is an essential nutrient and has an important role in the synthesis of amino acids and proteins.Nour et al.
(2014) stated that the nutrition with a high ratio of K/Na have been associated with a lower incidence of hypertension.

Active components
Previously, a number of studies have reported that the lignans from S. chinensis possess various pharmacological properties, such as hepatoprotective, antioxidant and anticarcinogenic activities, and strong inhibitory effect on human immunodeficiency virus (Wang et al., 2008;Choi et al., 2006;Ip et al., 1996;Lee and Kim et al., 2010;Xie et al., 2010).Further, those studies suggest that the bioactive lignans mainly comprised of dibenzocyclooctadiene skeletons with (S)-or (R)-biphenyl configurations.Until now, more than 40 lignans have been characterized from the different organs of S. chinensis (Kim et al., 2010;Takimoto et al., 2013).
In the present study, the most bioactive lignans such as schisandrin, schisandrin A, gomisin A and gomisin N were quantified by HPLC (Fig. 2).The fruits contained the highest amount of schisandrin followed by gomisin N, gomisin A and schisandrin A. When compared to the fruit samples of Inje, the contents of schisandrin (5018.67 µg/g), gomisin N (4772.87µg/g) and schisandrin A (717.18 µg/g) were higher in the samples of Mungyeong.The level of Gomisin A was higher in Inje samples (1383.03µg/g) than Mungyeong samples (1202.60 µg/g).

Fig. 2:
Active ingredients in the fruits of Schisandra chinensis from two different places (Inje and Mungyeong).Values are mean of three replicate determinations (n=10) ± standard deviation.Zhang et al. (2009) established a rapid and specific HPLC method for simultaneous determination of six major lignans in S.chinensis such as schisandrin, schisandrol B, schisantherin A, deoxyschisandrin, γ-schisandrin and schisandrin C. The six lignans were successfully separated on a C18 column and the mobile phase consisted of acetonitrile and water with the detection wavelength of 225 nm.In addition, Hu et al. (2013) successfully validated the method to quantify 11 lignans (schisandrin, gomisin J, schisandrol B, angeloylgomisin H, gomisin G, schisantherin A, schisantherin B, deoxyschisandrin, γ-schisandrin, schisandrin B and schisandrin C) in S. chinensis by HPLC.Recently, a simplified sample extraction method using matrix solid phase dispersion followed by HPLC determination was established for the determination of five most abundant lignans, schisandrin, schisandrol B, schisantherin A, deoxyschisandrin and γschisandrin in S. chinensis (Zhang et al., 2016).In the current study, schisandrin was the most abundant component in the fruits followed by gomisin N, gomisin A and schisandrin A. The contents of schisandrin, gomisin N and schisandrin A were found to be higher in Mungyeong samples than Inje samples.However, the concentration and distribution of these lignans in S. chinensis are mainly influenced by plant origins and harvest seasons.Among the various lignans, schisandrin is a prominent lignin with multiple pharmacological properties including antioxidant, antiinflammatory, antitumor, sedative and hepatoprotective effects (Park et al., 2011;Kang et al., 2012;Zhang et al., 2016).Previous studies have shown that the lignans, gomisin N and gomisin A, significantly inhibited the liver damage from toxic chemicals.Gomisin A has been shown to inhibit acetylcholinesterase activity and anti-hypertensive effect and improve scopolamine-induced memory impairment in mice.
Gomisin A showed antiinflammatory properties by potentially inhibiting the proinflammatory mediators through the down-regulation of receptorinteracting protein 2 and activation of nuclear factor-kappa B (Park et al., 2012;Jeong et al., 2014).Schisandrin B (gomisin N) enhanced the cytotoxic and pro-apoptotic potentials of doxorubicin (Li et al., 2006;Kim et al., 2010).Gomisin N remarkably inhibited the nitric oxide production in lipopolysaccharide-induced RAW 264.7 cells and reduced the mRNA expression and the secretion of pro-inflammatory mediators (Oh et al., 2010).In addition, gomisin N exhibited antiproliferative properties against various cancer cell lines with the IC 50 values of 10-70 μM (Min et al., 2008).Furthermore, Giridharan et al. (2011;2012) reported that the lignan, schisandrin B effectively prevent scopolamine-induced dementia and cisplatin-induced memory deficits in animal model.Schisandrin A is also an important bioactive lignan and is a strong antioxidant which possesses hepatoprotective and antitumor properties.It also showed positive effects on preventing memory impairment in mice (Hu et al., 2012;Cheng et al., 2013;Lu et al., 2014).

Volatile composition of SFE
The SFE yield of S. chinensis fruits was significantly influenced by the extraction temperature and pressure.In the different pressures performed for the optimization of extraction condition, there were no yield at 200 and 300 bar.Further, the yield was increased with increase of temperature [50ºC (0.01%), 60 ºC (0.02%) and 70ºC (0.45%)]at constant pressure of 400 bar.At the constant temperature (70 ºC) and pressure (400 bar), the SFE yields of Inje and Mungyoeng samples were 0.42±0.13%and 0.45±0.13%,respectively.The results revealed that the pressure plays a vital role in the extraction yield of S. chinensis fruits when compared with the temperature.Previously, several studies have reported that the temperature and pressure played an important role on the yield of essential oils from various plant materials by SFE method (Ansari and Goodarznia, 2012;Ahmed et al., 2012).Wang et al. (2008) obtained the SFE yield of 185.6 mg/g from the fruits of S. chinensis with the operating conditions of pressure, 25 MPa; temperature, 50ºC; carbon dioxide flow rate, 25 L/h; and extraction time, 3 h.In the present study, the SFE yield was very low, because the fruits samples were not pulverized as well as the extraction time was 1 hour.The volatile composition of the SFE from S. chinensis fruits was determined by SPME-GC/MS and the result is presented in Table 2.In order of elution on VF-5ms.b Components identified based on mass spectra and retention indices.c RI, Retention indices reported in the literature.The SFE of fruits obtained from 10 sites of each place were pooled together and used for the SPME-GC/MS analysis.
According to the previous reports, sesquiterpene hydrocarbons and oxygenated sesquiterpenes are the main volatile components from the fruits of S. chinensis and responsible for its specific fragrance.The results of the present study also clearly showed that the fruits mainly composed of sesquiterpenes group of components with ylangene, α-himachalene, longipinene as the major components.In the previous report, 40 components were identified in the essential oil of S. chinensis fruits and the main components were ylangene (37.72%), β-himachalene (10.46%) and α-bergamotene (8.57%) (Chen et al., 2012).In another study, Teng and Lee (2014) compared the simultaneous distillation extraction with Soxhlet and microwave assisted extraction methods.The results revealed that the major ingredients in the oil extracted by simultaneous distillation extraction were ylangene (15.01%), α-phellandrene (8.23%), β-himachalene (6.95%), and cuparene (6.74%).However, the oils obtained by other extraction methods mainly contained aromatics such as schisandrins and gomisin A. Deng et al. (2003) compared the volatile composition of S. chinensis obtained from steam distillation and headspace SPME and identified 33 and 35 volatile compounds, respectively.Further, Li et al. (2003)investigated the essential oil composition of S. chinensis obtained from steam distillation and characterized 48 different volatile components from the oils.Wang et al. (2008) investigated the comparison of volatile composition from the fruits of S. chinensis obtained by SFE, steam distillation, Soxhlet extraction and ultrasound-assisted extraction and identified 37, 45, 27 and 37 compounds in the samples, respectively.Further, the authors stated that the SFE method shared 32 compounds in common with the other three methods.Among them, schisandrin (16.4%), methostenol (16.2%), and β-tocopherol (11.0%),αylangene (7.94%) 2-methyl-2-bornene (7.64%), 1-butyl-1,3,5,7cyclooctatetraene (6.50%)and α-farnesene (5.58%)were the major components in the SFE.In the current study, totally 42 components (40 components from each place) were identified in the SFE of fruits.Ylangene, α-himachalene, longipinene, italicene, bornyl acetate and aromadendrene epoxide were the most abundant components in the SFE.
The composition of SFE of S. chinensis fruits is totally different from previous report and analysis methods might be responsible for these variations (Wang et al., 2008).Moreover, this is the first report on the volatile composition of SFE of S. chinensis using SPME-GC/MS.The findings of the previous and the present studies clearly suggested that the volatile composition of S. chinensis fruits is mainly influenced by the extraction techniques, place of sample collection and environmental conditions (biotic and abiotic factors).

CONCLUSION
In the present investigation, the fruits of S. chinensis collected from both the places, Inje and Mungyoeng possess almost similar and appreciable levels of proximate composition, mineral contents, bioactive lignans and various volatile components.The considerable amount of nutrients and bioactive components in the S. chinensis fruits suggest that the fruits may have the potential for enriching ingredient for locally processed foods and can contribute to certain nutritional requirements in the human diet.

Fig. 1 :
Fig. 1: Map of Inje and Mungyeong, the two major cultivating areas of Schinsandra chinensis fruits in Republic of Korea.

Fig. 3 :
Fig. 3:Percentage composition of different chemical groups in the supercritical carbon dioxide extract of Schisandra chinensis fruits from two different places (Inje and Mungyeong).

Financial
support and sponsorship: This research was supported by the Ministry of Trade, Industry & Energy (MOTIE), Korea Institute for Advancement of Technology (KIAT) through the 4

Table 1 :
Proximate composition and mineral content of fruits of Schisandra chinensis from two different places (Inje and Mungyeong).

Table 2 :
Volatile composition of supercritical carbon dioxide extract of Schisandra chinensis fruits from two different places (Inje and Mungyeong)