Phytochemistry of Schizandra Chinensis (Turcz.) Baill Cultivated in

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Phytochemistry of Schizandra Chinensis (Turcz.) Baill Cultivated in Bulgaria P. Merdzhanov,*,1 N. Delchev,2 O. Teneva,3 V. Gochev,4 and A. Stoyanova1 1Department

Essential Oils, University of Food Technology, 26 Maritza Boulevard, Plovdiv 4002, Bulgaria 2Department Analytic Chemistry, University of Food Technology, 26 Maritza Boulevard, Plovdiv 4002, Bulgaria 3Department Chemical Technology, Paisii Hilendarski University of Plovdiv, 24 Tzar Asen Strasse, Plovdiv 4000, Bulgaria 4Department Biochemistry and Microbiology, Paisii Hilendarski University of Plovdiv, 24 Tzar Asen Strasse, Plovdiv 4000, Bulgaria *E-mail: [email protected].

The chemical composition of the oil fruits from of Schizandra chinensis (Turcz.) Baill, cultivated in Bulgaria were analyzed using GC and GC/MS. The major constituents (over 3%) of the essential oil (1.65%) were found to be α-himachalene (10.83%), α-ylangene (10.50%), β-himachalene (9.02%), schisandrin (8.23%), β-chamigrene (6.44%) and γ-muurolene (5.52%). The essential oil studied demonstrated antimicrobial activity against Gram-positive and Gram-negative bacteria and yeasts. Oleic acid (80.8%), stearic acid (14.5%) and palmitic acid (4.7%) were the main components in the triacylglycerol fraction (24.6%). In the tocopherol fraction α-tocopherol (96.7 %) predominated, and in sterol fraction: β-sitosterol (91.0%) and campesterol (5.1%). Keywords: Schizandra chinensis (Turcz.) Baill ; essential oil ; antimicrobial activity ; lipid fraction

© 2016 American Chemical Society Jeliazkov (Zheljazkov) and Cantrell; Medicinal and Aromatic Crops: Production, Phytochemistry, and Utilization ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Introduction The genus Schizandra (also known as Schizandra) includes 25 species of deciduous vines belonging to the Schizandraceae family (Magnolia vine family). All but one is native to the forests of Northern China, the Russian Far East, Korea and Japan. The Chinese name for Schizandra is wu-wei-zi, which means “five taste-fruits” or “five flavor herb” and alludes to the fact that the fruits contain all five flavors: sweet, sour, bitter, pungent and salty. Sucking on a dry fruit is an interesting experience because of its various flavors. Schizandra chinensis (Turcz.) Baill vines prefer some shade and well-drained, deeply cultivated sandy soil with plenty of moisture, rich compost and cold temperatures. Like most vines they need to grow on an arbor, wall or fence. They usually begin bearing fruit after the second or third year. Schizandra is propagated by seed, cuttings or layering (1). Schizandra is being used by western herbalists as an overall tonic and recovery herb for various deficiencies and weak body conditions. It can be used in combination with other herbs for chronic stress, chronic fatigue, insomnia, poor memory. Some clinical studies have shown that the berries improve brain efficiency while at the same time calming the central nervous system. When taken over several weeks as an adaptogen (a substance that helps the body adapt to stresses), Schizandra helps improve energy levels, reduces tiredness, and improves the immune system’s response, thus being a valuable tonic for many people (2, 3). Pharmacological studies on animals have shown that Schizandra increases physical working capacity and delivers a stress-protective effect against a broad spectrum of harmful factors including heat shock, skin burn, cooling, frostbite, immobilisation, swimming under load in an atmosphere with decreased air pressure, aseptic inflammation, irradiation, and heavy metal intoxication. The phytoadaptogen provides a beneficial effect on the central nervous, sympathetic, endocrine, immune, respiratory, cardiovascular and gastrointestinal systems, on the development of experimental atherosclerosis, on blood sugar and acid-base balance, and on uterus myotonic activity (4). The main active principles present in all parts of the plant are dibenzo cyclooctadiene lignans (5, 6). Krotova and Efremov (7) investigated the chemical composition of Schizandra fruits. They established that the content of essential oil was 1.65 % and lipids were 40.3%. The volatile components of various species of the plant were studied by many authors (6, 8–10). The major compounds determined by Deng et al. (11) through steam distillation of the Schizandra chinensis fruits from China were: α-santalene (28.51%), 2,4aα, 5,6,7,8-hexahydro-3,5,5,9-tetramethyl-1H-Benzocycloheptene (13.31%), ζ-cadinene (13.25 %), 4-isopropylidene-1-vinylmenth-8-ene (7.71%); by Wang et al. (12): (-)-1,7-dimethyl-7-(4-methyl-3-pentenyl)-tricyclo [2.2.1.0(2,6)] heptane (18.06%), (1 alpha, 4 beta, 8 alpha)-1,2,3,4,4α,5,6,8αoctahydro-7-menthyl-4-methylene-1-(1-methylethyl)-naphtalene (15.58%), 2,4α,5,6,7,8-hexahydro-3,5,5,9-tetramethyl-1H-benzocycloheptene (11.45%), 1-methyl-4-(1-methylethyl)-1,3-cyclohexadiene (9.90%); 1-methyl-4-(1,2,2trimethylcyclopenthyl)-benzene (9.13%); while the fruits from Russia examined by Krotova and Evremov showed the following composition (7): α- and β-chamigrenal (26.5%), α- and β-chamigrene (19.5%), sesquicarene (10.5%). The 236 Jeliazkov (Zheljazkov) and Cantrell; Medicinal and Aromatic Crops: Production, Phytochemistry, and Utilization ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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essential oil had antimicrobial activity against Gram-positive and Gram-negative bacteria and proved to be a weaker antioxidant compared to BHT (12). The essential oil, ethanol extract and various fractions (petroleum, ethyl acetate and n-butanol of ethanol extract) demonstrated a strong antioxidant activity (10). Bulgaria is a relatively small country in South-East Europe, which occupies 111000 km2 and is located in the centre of the Balkan Peninsula, sharing borders with Romania, Serbia, the Republic of Macedonia, Greece, Turkey and the Black Sea. Besides being very picturesque and rich in history and culture, Bulgaria possesses a highly varied topography and a range of micro-climatic areas: mountains, plains, rivers and seacoast valleys. The climate ranges from moderate continental in the northern part to Mediterranean/subtropical in southwest and southeast regions. As a result of these favorable climatic conditions, soils and other natural factors, Bulgaria has been the perfect place for growing of medicinal plants and therefore a producer of essential oils for over 400 years. Bulgaria has been long renowned as one of the two major global suppliers of rose oil and, also, as a source of many other essential oils, including lavender, dill, pine, clary sage, basil, bigroot geranium and milfoil. Shizandra is a new plant for Bulgaria introduced as a crop in the late 90s of the twentieth century. Nowadays it is grown in the region of Pleven - Northern part. The harvested fruits are used in the food industry for preparing various products - jams, juices and more, as well as in traditional medicine. The aim of present study is the production of the essential and vegetable oils from Schizandra fruits grown in Bulgaria and determination of their chemical composition and characteristics for possible application in natural cosmetics, pharmaceuticals and food products.

Materials and Methods Sample. The fruits were harvested in 2014 in the vicinity of the town of Pleven, Bulgaria. Pleven’s climate is temperate continental. The average annual temperature is around 13°C. The region is characterized by the fertile black earth soils known as chernozems. The moisture of the aerial fruits (12.5%) was determined by drying to 105°C, according to Russian Pharmacopoeia (13). Isolation of essential oil: The air-dried fruits were ground in a laboratory mill to a size of 0.7-1cm. The oil was prepared through hydro distillation for 5h in a laboratory glass apparatus as per British Pharmacopoeia, modified by Balinova and Diakov (14). The oil obtained was dried over anhydrous sodium sulfate and stored in tightly closed dark vials at 4°C until analyzed. Isolation of fruit oil: The ground fruits were extracted with n-hexane in Soxhlet apparatus for 18h. The solvent was partly removed in a rotary vacuum evaporator, the residue was transferred to a pre-weighed glass vessel and the remaining solvent was removed under stream of nitrogen to a constant weight, in order to determine the oil content (15). 237 Jeliazkov (Zheljazkov) and Cantrell; Medicinal and Aromatic Crops: Production, Phytochemistry, and Utilization ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Chemical composition of the essential oil: GC analysis was performed using Agilent 7890A gas chromatograph; column HP-5 ms (30m x 250μm x 0.25μm); temperature: 35°C/3 min, 5°C/min to 250°C for 3min, total 49min; carrier gas: helium 1ml/min constant speed; split ratio: 30:1. GC/MS analysis was carried out on a mass Agilent 5975C spectometer, carrier gas: helium, column and temperature the same as of the GC analysis. The identification of chemical compounds was performed by comparing their relative retention time to library data. The components identified were arranged in accordance with their retention time and the percent. Fatty acids: The total fatty acid composition of the oil was determined by GC after transmethylation of the respective sample with 2N methanolic KOH at 50°C according to Christie (16). Fatty acid methyl esters (FAME) were purified by TLC on 20cm x 20cm glass plates covered with 0.2mm Silica gel 60 G layer (Merck, Darmstadt, Germany) with mobile phase n-hexane:acetone, 100:8 (by volume). Determination was performed on a gas chromatograph equipped with a 30m x 0.25mm x 25μm (I.D.) capillary EC 30-Wax column (Hewlett Packard GmbH, Vienna, Austria) and a flame ionization detector. The column temperature was programmed from 130°C (hold 4min), at 15оC/min to 240°C (hold 5min); injector and detector temperatures were 250°C. Hydrogen was the carrier gas at a flow rate 0.8ml/min; split was 50:1. Identification was performed by comparison of the retention times with those of a standard mixture of FAME subjected to GC under identical experimental conditions (17). Sterols: Unsaponifiables were determined by weight after saponification of the lipid fraction and extraction with hexane (18). The unsaponifiable matters (100 mg, precisely measured) were applied on 20cm x 20cm glass plates (ca. 1 mm thick Silica gel G layer) and developed with n-hexane:acetone, 100:8 (by volume). Free sterols (Rf = 0.4) were detected under UV light by spraying the edges of each plate with 2´,7´-dichlorofluorescein, they were then scraped, transferred to small glass columns and eluted with diethyl ether. The solvent was evaporated under a stream of nitrogen and the residue was weighed in small glass containers to a constant weight. Sterol composition was determined by GC using HP 5890 gas chromatograph (Hewlett Packard GmbH, Vienna, Austria) equipped with a 25m x 0.25mm DB – 5 capillary column (Agilent Technologies, Santa Clara CA, USA) and a flame ionization detector. Temperature gradient was from 90°C (hold 2min) up to 290°C at a rate 15°C/min and then up to 310°C at a rate of 4°C/min (hold 10min); the injector temperature was 300°C and the detector temperature was 320°C. Hydrogen was used as carrier gas at a flow rate of 0.8ml/min; split 50:1. Identification was confirmed by comparison of retention times with those of a standard mixture of sterols (19). Tocopherols: Tocopherols were determined directly in the oil by high performance liquid chromatography (HPLC) by a Merck-Hitachi (Merck, Darmstadt, Germany) unit equipped with a 250mm x 4mm Nucleosil Si 50-5 column (Merck, Darmstadt, Germany) and a fluorescent detector Merck-Hitachi F 1000. The operating conditions were as follows: mobile phase n-hexane:dioxan, 96:4 (by volume), flow rate 1.0 ml/min, excitation 295nm, emission 330nm. 20μl 1% solution of crude oil were injected. Tocopherols were identified by comparing the retention times to those of authentic individual pure tocopherols. 238 Jeliazkov (Zheljazkov) and Cantrell; Medicinal and Aromatic Crops: Production, Phytochemistry, and Utilization ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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The tocopherol content was calculated on the base of tocopherol peak areas in the sample vs. tocopherol peak area of the standard tocopherol solution (20). Determination of antimicrobial activity: The antimicrobial effect of the essential oil was tested against Gram-positive bacteria Bacillus cereus (food spoilage isolate), two strains of Staphylococcus aureus (ATCC 6538 and one food spoilage isolate) and Listeria monocytogenes (food spoilage isolate), as well as the following Gram-negative bacteria: two strains of Escherichia coli (ATCC 25922 and one clinical isolate), two strains of Salmonella abony (ATCC 6017 and one clinical isolate), three strains of Pseudomonas aeruginosa (ATCC 27853, one clinical isolate and one food spoilage isolate) and Pseudomonas fluorescens (food spoilage isolate), sources given in Table 2. Additionally antimicrobial testing against two strains of Candida albicans (ATCC 10231 and one clinical isolate) was performed. All strains were deposited in the Microbial Culture Collection of the Department of Biochemistry and Microbiology (University of Plovdiv, Bulgaria). The bacterial strains were stored on Nutrient Agar (NA, HiMedia Ltd., India) and the yeasts strains were stored on Sabouraud Dextrose Agar with chloramphenicol (SDA, HiMedia Ltd.). Stock solutions of the samples for antimicrobial testing were prepared by dissolving the respective compound in 2% DMSO (Sigma-Aldrich Co.). The antibacterial activity evaluation of the studied compounds was performed according to Clinical Laboratory Standard Institute (21) reference method for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. The anticandidal activity of the studied compounds was tested according to CSLI (22) reference method for broth dilution antifungal susceptibility testing of yeasts. Briefly stock solutions were added to the RPMI1640 broth medium buffered to pH 7.0 with 0.165mol/L MOPS buffer (3-N-morpholinopropanesulfonic acid, Sigma-Aldrcih,Co) to reach dilutions with final sample concentrations, after inoculation with microbial test suspension, between 1024μg/mL and 4μg/mL. Controls consisting of inoculated medium without tested sample and without DMSO, as well as with DMSO were also prepared. The DMSO concentration in the broth dilution assay was low to keep the effect on microbial growth to a minimum. Antimicrobial activity determined by broth microdilution tests was expressed as Minimal Inhibitory Concentration (MIC) in µg/mL. MIC was defined as the lowest concentration of the tested compound at which total inhibition of microbial growth was detected. All tests were performed in triplicate.

Results and Discussions The content of the essential oil was 1.65% and was comparable with the data from the literature (7). The essential oil was light yellow, with a specific odor. The chemical composition of the essential oil was given in Table 1. Thirty two components representing 91.81% of the total content were identified. Twenty two of them were in concentrations over 1% and the rest 11 constituents were in concentrations under 1%.The major constituents (over 3%) of the oil were as follows: α-himachalene (10.83%), α-ylangene (10.50%), β-himachalene (9.02%), schisandrin (8.23%), β-chamigrene (6.44%) and γ-muurolene (5.52%). 239 Jeliazkov (Zheljazkov) and Cantrell; Medicinal and Aromatic Crops: Production, Phytochemistry, and Utilization ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

The difference in the quantities of the chemical composition of the essential oil extracted in our laboratories and the reported data may be due to the environmental conditions under which the plant has grown as well as to the variation in conditions of analysis.

Table 1. Chemical Composition of Essential Oil from Schizandra chinensis

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No.

RI

Compounds

%

1.

α-Pinene

939

1.13

2.

Camphene

954

1.66

3.

β-Pinene

979

0.34

4.

Myrcene

990

0.39

5.

α-Phellandrene

1003

0.10

6.

α-Terpinene

1018

0.58

7.

p-Cymene

1025

1.08

8.

Limonene

1029

0.91

9.

γ-Terpinene

1060

2.50

10.

α-Terpinolene

1088

0.33

11.

Terpinene-4-ol

1179

0.61

12.

3-Methoxy-p-cymene

1215

1.35

13.

Bornyl acetate

1288

2.41

14.

α-Ylangene

1375

10.50

15.

β-Bourbonene

1388

0.26

16.

β-Elemene

1396

1.13

17.

β-Caryophyllene

1419

2.69

18.

(Z)-β-Farnesene

1448

1.21

19.

α-Humulene

1455

0.43

20.

β-Chamigrene

1468

6.44

21.

β-Eudesmene

1477

0.45

22.

β-Himachalene

1486

9.02

23.

α-Himachalene

1491

10.83

24.

γ-Muurolene

1496

5.52

25.

Alloaromadendrene

1499

1.50

26.

β-Humulene

1512

2.27 Continued on next page.

240 Jeliazkov (Zheljazkov) and Cantrell; Medicinal and Aromatic Crops: Production, Phytochemistry, and Utilization ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

Table 1. (Continued). Chemical Composition of Essential Oil from Schizandra chinensis

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No.

RI

Compounds

%

27.

α-Cubebene

1525

2.87

28.

Dehydroaromadendrene

1577

0.74

29.

Longipinocarvone

1748

3.72

30.

Gomisin F

2380

1.69

31.

Schisandrin

2420

8.23

32.

Gomisin A

2480

1.22

The distribution of major groups of aroma substances in the essential oil was shown in Figure 1. Sesquiterpene hydrocarbons were the dominant group in the oil (69.67%), followed by monoterpene hydrocarbons (8.54%) and oxygenated sesquiterpenes (4.05%). The results were similar to those in the literature.

Figure 1. Groups of components in the oil,%. 1 - Monoterpene hydrocarbons; 2 – Oxygenated monoterpenes; 3 – Sesquiterpene hydrocarbons; 4 – Oxygenated sesquiterpenes ; 5 - Phenyl propanoid hydrocarbons; 6 – Oxygenated phenyl propanoids ; 7 – Other compounds. 241 Jeliazkov (Zheljazkov) and Cantrell; Medicinal and Aromatic Crops: Production, Phytochemistry, and Utilization ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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The results of antimicrobial testing were shown in Table 2. The summery results of the oil studied demonstrated its antimicrobial activity against the tested microbial strains. Gram-positive bacteria were the most sensitive microbes, followed by yeasts and Gram-negative bacteria. The less sensitive were three strains of P. aeruginosa. The ability of P. aeruginosa to grow in a form of biofilm and the production of extracellular polysaccharide increased antimicrobial resistance of these bacteria mainly through permeability barrier. These strains also produced two types of soluble pigments, pyoverdin and pyocyanin, which probably participate in cell defense against antimicrobials. The oil demonstrated equal antimicrobial activity against Gram-negative bacteria P. fluorescens and both strains of yeasts belonging to species C. albicans. The results obtained are comparable with the results published for the same essential oil by other authors.

Table 2. Аntimicrobial Activity of the Essential Oil Test microorganisms

Origin

MIC,%

B. cereus

Minced meat

50

S. aureus

ATCC 6538

128

S. aureus

Pork fillet

128

L. monocytogenes

Chicken breasts

128

E. coli

ATCC 25922

256

E. coli

Clinical isolate

256

S. abony

ATCC 6017

256

S. abony

Clinical isolate

256

P. aeruginosa

ATCC 27853

1024

P. aeruginosa

Clinical isolate

1024

P. aeruginosa

Minced meat

1024

P. fluorescens

Chicken breasts

512

C. albicans

ATCC10231

512

C. albicans

Clinical isolate

512

The triacylglycerol fraction was 24.6%, and not corresponding with the data found in the literature (7). The fatty acid composition was presented in Table 3. Data show that three fatty acids were determined, constituting 100% of the total oil content. The fatty acids found in the triacylglycerol fraction were oleic acid (80.8%), stearic acid (14.5%) and palmitic acid (4.7%). Regarding the individual presence of oleic acid, the oil was similar to the oils from other nontraditional 242 Jeliazkov (Zheljazkov) and Cantrell; Medicinal and Aromatic Crops: Production, Phytochemistry, and Utilization ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

materials such as grape seeds, watermelon, tobacco and poppy seeds (23, 24). Schizandra fruits oil was found to contain very high amounts of the saturated stearic and palmitic acids, which came close to the levels in other oils, like olive oil and corn oil (25).

Table 3. Fatty Acid Composition

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No.

Fatty acids

Content,% (w/w)

1

C 16:0 Palmitic

4.7

2

C 18:0 Stearic

14.5

3

C 18:1 Oleic

80.8

Phytosterols, more commonly known as plant sterols are currently approved by the U.S.Food and Drug Administration for use as a food additive; however, there is some concern that they may block absorption not only of cholesterol but of other important nutrients as well. Sterols were present in the so called non-saponificated part from the lipid fraction. The total content in the oil was found to be 0.8%. The individual sterol composition was given in Table 4. β-sitosterol (91.0%) and campesterol (5.1%) predominated in the sterol fraction. The data obtained made obvious that regarding its sterol content and composition, Schizandra fruits results were similar to the findings for cotton seed oil (26).

Table 4. Sterol Composition No.

Sterols

Content,% (w/w)

1.

Cholesterol

0.2

2.

Campesterol

5.1

3.

Stigmasterol

0.3

4.

β- Sitosterol

91.0

5.

Δ5-

Avenasterol

2.2

6.

Δ7-

Stigmasterol

0.9

7.

Δ7,25-

0.3

Stigmastendiol

243 Jeliazkov (Zheljazkov) and Cantrell; Medicinal and Aromatic Crops: Production, Phytochemistry, and Utilization ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

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Tocopherols are a class of organic chemical compounds, mostly with vitamin E activity. α-Tocopherol is the main source found in supplements and in European diet, where the main dietary sources are olive and sunflower oils, while γ-tocopherol is the most common form in the American diet due to a higher intake of soybean and corn oil. The total content of tocopherols in the lipid fraction was comparatively higher – 660mg/kg. The tocopherol composition was presented in Table 5. The α-tocopherol predominated in the oil, followed by β-tocopherol. The oil with higher content of α-tocopherol proved superior to a number of common food oils, thus showing similarity to some non-traditional oils (23, 24).

Table 5. Tocopherol Composition No.

Tocopherols

Content,% (w/w)

1.

α-Tocopherol

96.7

2.

β-Tocopherol

3.3

Conclusion Schizandra fruits (Schizandra chinensis (Turcz.) Baill) can be used as a nontraditional material for production of oil rich in biologically active substances as essential oil, lipid fraction (sterols and tocopherols) for nutritive purposes, as well as for an additive in fodder mixtures to enrich them with valuable nutrients.

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