Chemical Compound Characterizations of Patchouli Leaf Extract via GC-MS, LC-QTOF-MS, FTIR, and 1H NMR

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Chemical Compound Characterizations of Patchouli Leaf Extract via GC-MS, LC-QTOF-MS, FTIR, and 1H NMR

Sangeethavani Sundarajan*

Faculty of Chemical Engineering and Natural Resources University Malaysia Pahang,

Kuantan, Pahang

Nour Hamid Abdurahman

Faculty of Chemical Engineering and Natural Resources University Malaysia Pahang,

Kuantan, Pahang

Abstract The demand for patchouli (Pogostemon cablin) essential oil is increasing globally due to its diverse importance in the nutraceutical and pharmaceutical industries. The oil from this plant is an embodiment of different varieties of chemical compounds. It is then important to identify the bioactive compounds in the extracted oil to identify the embedded potential uses of the oil. Hence, this study focused on the characterization of extracted patchouli oil through microwave- assisted hydrodistillation (MAHD) technique using gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry quadrupole time of flight (LC-Q-TOF-MS), nuclear magnetic resonance (NMR), and Fourier transform infrared transmission (FTIR). The obtained results reflected that patchouli leaf oil is endowed with appreciable quantities of non-oxygenated and oxygenated compounds (from GC-MS analysis); 18 tentatively identified phenolic compounds from LC-Q-TOF-MS analysis; several peaks showing the presence of O-H stretching in alcohol, C=O stretching vibration of carbonyl aldehyde, C-H bending stretching of alkane, and C=C stretching from aromatic rings were identified from the FTIR analysis. Additionally, the NMR results reflected the abundance of patchouli alcohol in the oil which can contribute to its aroma. Thus, patchouli leaf oil is an important plant endowed with different bioactive compounds.

Keywords Bioactive compounds; Characterization; Microwave-assisted hydrodistillation; Patchouli; Gas- chromatography mass spectrometry; Nuclear magnetic resonance

  1. INTRODUCTION

    Patchouli leaves (Pogostemon cablin), a plant belonging to the Lamiaceae family is endowed with essential oil [1]. This plant is usually cultivated in a tropical area particularly southeast Asia [2]. It is an aromatic plant that possesses higher content of essential oil that emanates from its young twigs and leaves. Patchouli oil is a dark orange or brownish colored liquid with a woody, earthy and camphoraceous odour [3]; [1]; [2]. The oil plays an important role in the production of perfumery products such as soaps, cosmetics products and detergents because it possesses a long-lasting odour with fixative properties [4]. Moreover, the patchouli plant is used in traditional Asian medicine as an anti-stress, antiseptic, relieve headaches and fever [5]. Several therapeutic activities of patchouli oil had previously been reported, including antifungal, anti-depressive, anti-bacterial, anti- inflammatory, sedative, febrifuge, and diuretic [4]; [1].

    The composition of patchouli essential oil is complex and unique in relation to other essential oils [4]; however, it is distinct due to the presence of sesquiterpenes. Patchoulol is

    one of the main constituents of this oil and a primary compound accountable for its fragrance [6]. Patchoulol acts as fragrance binder to give long-lasting characteristic for the fragrance as compared to patchoulol, -himachalene, -/-/- patchoulenes, seychellene, and -guaiene [4]; [5]. Moreover, a report had illustrated that patchoulol and -patchoulene are responsible for the oil aroma [4]. Hence, this can increase the trading values of this oil if patchoulol and -patchoulene are present in larger amounts. Conventionally, the essential oil is obtained from a plant matrix through hydrodistillation technique. However, this technique had been reported to take a longer period of extraction, consumed a larger amount of energy with a potential of degenerating the bioactive compounds in the oil. Thus, employing a modern technique of extraction is important to fill up the shortcomings of hydrodistillation technique. The microwave-assisted hydrodistillation extraction technique is being employed due to its fast start-up, efficient heating and faster energy transfer [7]. In addition, characterization of plant extract/oil is essential to tentatively identify the embedded bioactive compounds. Several characterization techniques are being employed to tentatively identify the chemical compounds, these include gas chromatography-mass spectrometry (GC-MS), gas chromatography (GC), liquid chromatography-mass spectrometry quadrupole time of flight (LC-Q-TOF-MS), Fourier transform infrared transmission (FTIR), nuclear magnetic resonance (NMR), and among others.

    Although, the bioactive compounds in the patchouli oil had previously been reported [5]; [8], however, comprehensive tentative characterization using gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry quadrupole time of flight (LC-Q-TOF-MS), nuclear magnetic resonance (NMR), and Fourier transform infrared transmission (FTIR) is yet to be reported. Thus, this study focuses on the characterization of oil from patchouli leaves oil to disclose the embedded bioactive compounds.

  2. MATERIALS AND METHODS

    1. Raw material and chemicals

      Dried samples (patchouli leaves) were obtained from Gaya Naturals company, Tawau, Sabah. The dry leaves were separated from stems and further dried at room temperature for several days. Then, the patchouli leaves were blended into powder form. Anhydrous sodium sulphate and

      dichloromethane utilized in this study were obtained Faculty of Chemical and Natural Resources Engineering laboratory.

    2. The procedure of essential oil extraction using MAHD

      A modified domestic microwave extractor comprising power and time control was used to extract oil from patchouli leaves. Fig. 1 shows the set up for the MAHD extractor for this study. A 30 g of patchouli powder was mixed with 180 mL of distilled water in a round-bottomed glass chamber. The microwave was operated at 400 W for 1 h. After 1 h, the hydrosol (water + essential oil) was collected from the Clevenger. Then, the hydrosol was placed in a separating funnel by adding few drops of dichloromethane to separate the essential oil from water. Thereafter, anhydrous sodium sulphate was added to eliminate any trace of water in the extracted oil. The obtained oil was then stored in a vial at -4 C for further analysis. The experiment was repeated for 240, 300, 360, and 420 mL of distilled water.

    3. Liquid chromatography-mass spectrometry (LC-Q-TOF- MS) analysis

      The chemical constituents in the patchouli essential oil were identified using LC-Q-TOF- MS (Waters, USA). LC-Q- TOF-MS has higher sensitivity and selectivity in characterizing and identifying the chemical compounds in the extracted oil of patchouli leaf when compared with other characterization methods. The patchouli oil was diluted using analytical grade ethanol to prepare a concentration of 100 mg/mL. Then, the concentration of the oil is further re-diluted to obtain 20 ppm prior to injection into the mass spectrometer. The QTOF-MS instrument was operated under the following conditions viz; desolvation flow rate (800 L/h), desolvation temperature (550 °C), operation mode (+ve and ve mode), scan time (0.200 s to 4.00 min), ms mode (high definition), collision energy interval (4.00-45.00 eV), and scanning range (100-1000 m/z).

      Fig. 1. MAHD set-up

    4. Fourier transform infrared transmission (FTIR) analysis

      Fourier transform infrared transmission was utilized to recognize functional groups in the patchouli leaf oil. This nalysis was carried out to determine the bonding structures present in the essential oil by studying the position of peaks in the IR spectra. The IR spectra were acquired by utilizing an FTIR spectrometer (Nicolet iS5 iD7 ATR; Thermo Scientific, Germany) equipped with OMNIC software. The wavenumber ranging from 4000-500 cm-1 was used to analyze the oil.

    5. Nuclear magnetic resonance (NMR)

    1H NMR spectra were recorded on a Bruker AMX500 spectrometer operating at 500 MHz for the proton nucleus at room temperature. The patchouli oil samples were used to obtain 1H NMR spectra with the following acquisition parameters: Acquisition time 3.75 s, 16 scans, 10 s D1, spectral width 4370.63 Hz, and FID resolution 0.133Hz. Phase correction and baseline correction were manually performed.

  3. RESULTS AND DISCUSSIONS

    1. Identified chemical compounds through GC-MS analysis

      Patchouli essential oil was analyzed using gas chromatography-mass spectrometry (GC-MS) to identify the chemical compounds in the oil (Table 1). A total number of twenty-nine chemical compounds were identified. The oil reflected about 60.25% non-oxygenated and 39.49%

      Table 1 List of chemical compounds in patchouli essential oil using GC-MS analysis

      Number

      Compounds

      Molecular Formula

      Mass percentage of chemical compounds in crude extracted (%)

      Oxygenated Terpenes

      1

      Patchouli alcohol

      C15H26O

      26.25

      2

      3-Ethylphenol

      C8H10O

      6.69

      3

      Aristol-9-en-8-one

      C15H22O

      1.61

      4

      Cashmeran

      C14H22O

      1.49

      5

      Ethyl chrysanthemate

      C12H20O2

      0.83

      6

      -Pinone

      C9H14O

      0.6

      7

      Turmerone

      C15H20O

      0.48

      8

      5-Isopropenyl-1,2-dimethylcyclohex-2-enol

      C11H18O

      0.44

      9

      2(1H) Naphthalenone, 3,5,6,7,8,8a-hexahydro-4,8a-dimethyl-6-(1-methylethenyl)-

      C15H22O

      0.42

      10

      2-(2-Methylpropylidene)-1H-indene-1,3(2H)-dione

      C13H12O2

      0.29

      11

      Corymbolone

      C15H24O2

      0.28

      12

      2-(2-Furyl)-1,3-thiazolidine

      C7H9NOS

      0.11

      13

      2-Ethylphenol

      C8H10O

      nd

      14

      3,4-Dimethyl-3-cyclohexene-1-carboxaldehyde

      C9H14O

      nd

      15

      4-Fluoro-3-nitrotoluene

      C7H6FNO2

      nd

      16

      2-Propenoic acid, 6-methylheptyl ester

      C11H20O2

      nd

      Sesquiterpenes

      17

      Valencene

      C15H24

      13.29

      18

      Aromadendr-1-ene

      C15H24

      11.86

      19

      Azulene

      C15H24

      11.76

      20

      -Patchoulene

      C15H24

      9.57

      21

      Aromandendrene

      C15H24

      3.61

      22

      Patchoulene

      C15H24

      2.96

      23

      Dehydroaromadendrene

      C15H22

      1.65

      24

      Caryophyllene

      C15H24

      1.43

      25

      Longifolene

      C15H24

      1.23

      26

      alpha-guaiene

      C15H24

      0.61

      27

      Guaiazulene

      C15H18

      0.47

      28

      o-Xylene

      C8H10

      0.39

      29

      m-Xylene

      C8H10

      0.36

      30

      Cyclopentane-3'-spirotricyclo [3.1.0.0(2,4)] hexane-6'-spirocyclopentane

      C14H20

      0.33

      31

      3,4-Dimethylstyrene

      C10H12

      0.16

      32

      Naphthalene, 6-butyl-1,2,3,4-tetrahydro-

      C14H20

      0.05

      33

      -Gurjunene

      C15H24

      nd

      34

      Guaia-3,9-diene

      C15H24

      nd

      35

      Cyclohexane

      C15H24

      nd

      36

      Benzene, (2-methyl-1-propenyl)-

      C10H12

      nd

      37

      5-(3-Fluorophenyl)-2H-tetrazole

      C7H5FN4

      nd

      38

      (-)-Tricyclo [6.2.1.0(4,11)] undec -5-ene,1,5,9,9 tetramethyl (isocaryophyllene-I1)

      C15H26

      nd

      39

      Bicyclo [4.2.0] oct-1-ene, exo-7-(1-cyclohexen-1-yl)-

      C14H20

      nd

      40

      Clovene

      C15H24

      nd

      41

      -Selinene

      C15H24

      nd

      42

      alpha-Himachalene

      C15H24

      nd

      43

      1,4-Dimethyladamantane

      C12H20

      0.52

      Total non-oxygenated compounds (%)

      60.25

      Total oxygenated compounds (%)

      39.49

      Total identified (%)

      99.74

      Table 2 Phenolic compounds in patchouli leaf oil

      Terchebin

      No.

      Observed RT (min)

      Component name

      Chemical formula

      Observed (m/z)

      Response

      Adducts

      Total fragment found

      1

      5.42

      Tellimagrandin

      C41H30O26

      983.1007

      4645

      +HCOO

      23

      2

      5.53

      Pedunculagin

      C34H24O22

      783.0700

      3582

      -H

      41

      3

      7.29

      Furosin

      C27H22O19

      695.0756

      4773

      +HCOO

      71

      4

      5.82

      Kukoamine A

      C28H42N4O6

      529.3018

      4237

      -H

      11

      5

      6.02

      1,2,3,4,6-Penta-O'Galloyl- -D'Glucopyranoside

      C41H32O26

      985.1175

      7074

      +HCOO

      21

      6

      6.57

      Geraniin

      C41H28O27

      951.0737

      5234

      -H

      55

      7

      6.64

      Casuarinin

      C41H28O26

      981.0840

      4895

      +HCOO

      28

      8

      6.84

      C41H30O27

      953.0916

      4723

      -H

      64

      9

      7.69

      Mallotinic acid

      C34H26O22

      831.0899

      3684

      +HCOO

      46

      10

      0.51

      Schizonepetoside E

      C16H28O8

      349.1838

      428455

      +H

      7

      11

      1.45

      Xanthumin

      C17H22O5

      307.1520

      5614

      +H

      0

      12

      3.89

      Sonchuside A

      C21H32O8

      435.1989

      176863

      +Na, +K

      34

      13

      4.41

      Hookeroside C

      C38H62O15

      781.3982

      110716

      +Na

      16

      14

      5.45

      2-Hydroxyesculentic acid

      C30H46O7

      541.3127

      290124

      +Na

      48

      15

      6.13

      11-Oxo-kansenonol

      C30H46O4

      493.3288

      813628

      +Na

      30

      16

      7.02

      Akebonoic acid

      C29H44O3

      463.3179

      420054

      +Na

      30

      17

      7.51

      3,30-Dihydroxylup-20 (29)-en-27-oic acid

      C30H48O4

      495.3443

      73459

      +Na

      19

      18

      3.28

      2-Hydroxyilicic acid

      C15H24O4

      291.1569

      77053

      +Na

      5

      oxygenated compounds. This is because the heat transfer in MAHD is generated from the oil gland to the surrounding solvent [9]. Hence, the maximum quantity of essential oil can be extracted from patchouli oil gland using MAHD. Patchoulol is the main oxygenated compound extracted from patchouli leaves. Moreover, the higher the number of oxygenated compounds, the higher the quality of essential oil. MAHD method shows higher probability in the production of natural aroma of the patchouli oil.

    2. Identified chemical compounds through LC-Q-TOF-MS analysis

      The phenolic compounds in the patchouli leaf oil were analyzed using LC-Q-TOF-MS. A total of 18 phenolic compounds were identified as shown in Table 2. Tannins, phenolic alkaloids, flavonoids, sesquiterpenoids lactones, and triterpenoids are the phenolic compounds found in the patchouli oil. From the identified compounds, eight chemical compounds are tannins. Tannins are the complex mixture of polyphenol which can act as an antioxidant [10], anti-inflammatory [11] and anti-microbial [12]. However, 1,2,3,4,6-Penta-O' galloyl–D'glucopyranoside is a compound reported to possess anti-cancer [13] and anti- tumor effects [14]. Geraniin has good radical scavenging activity [15] while Casuarinin possesses anti-oxidant properties that could inhibit the growth of T24 bladder cancer cells [15]. Moreover, Kukoamine A is a phenolic alkaloid reported to possesses natural anti-oxidant properties with mechanisms involving free radical scavenging [15]. Other pharmaceutical effects such as anti- hypertension, anti-analgesic, anti-inflammatory, antisepsis, and enhancing autoimmune still abound [16].

    3. Identified chemical compounds through FTIR analysis

      Fig. 2 illustrated the FTIR spectra for patchouli oil obtained from MAHD method. The infrared spectroscopy (IR) characteristics fingerprint peaks for patchouli oil falls within the range of 3400-800 cm-1. These spectra show that there is an overlap of the absorption spectrum of different components in the oil because patchouli essential oil is a

      complex mixture of volatile oils [17]. There are some observable peaks from MAHD spectrum. The peak at 3313 cm-1 represents O-H stretching in alcohol [18] which shows the abundance of patchoulol, an important material in perfumery. Another observable peak at 1635 cm-1 corresponds to C=O stretching vibration of carbonyl aldehyde [17]. It proves that patchouli oil contains higher amounts of aldehyde compounds. The peak at 1445 cm-1 can be attributed to C-H bending stretching in alkane [19] and C=C stretching from aromatic rings while the peak at 1373 cm-1 is a characteristic of O-H bending in a carboxylic acid [18]. The peak at 886 cm-1 is a representation of C-H bending vibration [17]. Summary of major peaks and their representation are listed in Table 3. From Table 3, five functional groups were identified from the MAHD spectrum.

      Fig. 2. FTIR spectrum

      Table 3 Summary of important peaks and their representation

      Functional group representation

      Position of bands (cm-1)

      C-H bending vibration

      886

      O-H bending

      1373

      C-H bending stretching alkane and C=C stretching aromatic

      1445

      C=O stretching vibration and N-H bending

      1635

      O-H stretching

      3313

    4. Identified chemical compounds through NMR analysis

    The representative one-dimensional 1H NMR spectra of patchouli leaf oil are shown in Fig. 3. The vertical scale B was magnified for better visibility. Table 4 shows the chemical shifts from both NMR spectrum A and B with their protons. The chemical shift at 0 ppm is the reference point. The highest peak in the NMR spectrum A which is at

    Fig. 3. 1H-NMR spectrum of ethanolwater extract of Patchouli essential oil. Vertical scale in B is magnified with respect to A.

    Table 4 The chemical shifts with their protons

    Chemical Shift, (ppm)

    Protons

    0.7011

    R-CH3

    1.7136

    R3C-CH

    1.8677

    =C-CH

    2.9922

    -C C-H

    4.4543

    HCOH

    8.0

    ArH

    Chemical Shift, (ppm)

    Protons

    0.7011

    R-CH3

    1.7136

    R3C-CH

    1.8677

    =C-CH

    2.9922

    -C C-H

    4.4543

    HCOH

    8.0

    ArH

    4.4543 ppm represents the alcohol group [20]. This proves the abundance of patchouli alcohol in patchouli leaves

    extract which contributes to the aroma of patchouli essential oil [21]. The peak of 8.0 ppm is characterized by the signals of aryl group (patchoulene, azulene and valencene) which play an important role in aroma [20]; [22] and act as anti-

    inflammatory [23], anti-ulcer [23]; [24], and anti-diabetic

    [24].

  4. CONCLUSION

This study has successfully characterized the oil obtained from patchouli leaves through MAHD using GC- MS, LC-QTOF-MS, FTIR, and NMR. The obtained results have clearly indicated that the oil possessed about 60.25% non-oxygenated and 39.49% oxygenated compounds through GC-MS analysis; the LC-Q-TOF-MS results showed that the oil comprised 18 phenolic compounds which are majorly tannins, phenolic alkaloids, flavonoids, sesquiterpenoids lactones, and triterpenoids. Additionally, there was the presence of O-H stretching in alcohol indicating the abundance of patchoulol, C=O stretching vibration of carbonyl aldehyde showing that the patchouli oil contains higer amounts of aldehyde compounds, C-H bending stretching of alkane, and C=C stretching from aromatic rings. The NMR results indicated that the abundance of patchouli alcohol in the oil which can be contributed to the aroma. Thus, patchouli leaf oil is an important plant that is endowed with varieties of chemical compounds.

REFERENCES

  1. Kusuma HS, Mahfud M (2017) Microwave-assisted hydrodistillation for extraction of essential oil from patchouli (Pogostemon cablin) leaves. Periodica Polytech Chem Eng 61:82- 92.

  2. Sundaresan V, Singh SP, Mishra AN, Shasany AK, Darokar MP, Kalra A, Naqvi AA (2009) Composition and comparison of essential oils of Pogostemon cablin (Blanco) Benth. (Patchouli) and Pogostemon travancoricus Bedd. var. travancoricus. J Essent Oil Res 21:220-222.

  3. Buré CM, Sellier NM (2004) Analysis of the essential oil of Indonesian patchouli (Pogostemon cabin Benth.) using GC/MS (EI/CI). J Essent Oil Res 16:17-19.

  4. Donelian A, Carlson LHC, Lopes TJ, Machado RAF (2009) Comparison of extraction of patchouli (Pogostemon cablin) essential oil with supercritical CO2 and by steam distillation. J Supercrit Fluid 48:15-20.

  5. Kusuma HS, Altway A, Mahfud M (2018) Solvent-free microwave extraction of essential oil from dried patchouli (Pogostemon cablin Benth) leaves. J Ind Eng chem 58:343-348.

  6. Deguerry F, Pastore L, Wu S, Clark A, Chappell J, Schalk M (2006) The diverse sesquiterpene profile of patchouli, Pogostemon cablin, is correlated with a limited number of sesquiterpene synthases. Arch Biochem Biophys 454:123-136.

  7. Alara OR Abdurahman NH (2018) Kinetics studies on effects of extraction techniques on bioactive compounds from Vernonia cinerea leaf. J Food Sci Tech.

  8. Tsubaki N, Nishimura K, Hirose Y (1967). Hydrocarbons in patchouli oil. Bull Chem Soc Jpn 40:597-600.

  9. Jeyaratanam N, Nour AH, Kanthasamy R, Nour AH, Yuvaraj AR, Akindoyo JO (2016) Essential oil from Cinnamomum cassia bark through hydrodistillation and advanced microwave assisted hydrodistillation. Ind Crop Prod 92:57-66.

  10. Owolabi OO, James DB, Sani I, Andongma BT, Fasanya OO, Kure B (2018) Phytochemical analysis, antioxidant and anti-inflammatory potential of Feretia apodanthera root bark extracts. BMC Complem Altern M 18:12.

  11. Minhas AM, Khan AU, Miana GA (2018) Anti-inflammatory actions of Berberis lycium (whole plant) in acute and chronic models of inflammation in mice. J Anim Plant Sci 28:754-760.

  12. Abubaka HMG, Usman H, Karumi Y (2018) Evaluation of antimicrobial activity and phytochemical composition of some fractions of methanol stem bark extract of Diospyros mespiliformis. Bayero J Pure Appl Sci 11:47-51.

  13. Kawk SH, Kang YR, Kim YH (2018) 1, 2, 3, 4, 6-Penta-O-galloyl- -d-glucose suppresses colon cancer through induction of tumor suppressor. Bioorganic med chem lett 28:2117-2123.

  14. Sekowski S, Bitiucki M, Ionov M, Zdeb M, Abdulladjanova N, Rakhimov R, Zamaraeva M (2018) Influence of valoneoyl groups on the interactions between Euphorbia tannins and human serum albumin. J Lumin 194:170-178.

  15. Li K, Zeng M, Li Q, Zhou B (2018) Identification of polyphenolic composition in the fruits of Rubus chingii Hu and its antioxidant and antiproliferative activity on human bladder cancer T24 cells. J Food Meas Charact 1-10.

  16. Liu J, Jiang X, Zhang Q, Lin S, Zhu J, Zhang Y, Zhao Q (2017) Neuroprotective effects of Kukoamine A against cerebral ischemia via antioxidant and inactivation of apoptosis pathway. Neurochem Int 107:191-197.

  17. Li YQ, Kong DX, Wu H (2013) Analysis and evaluation of essential oil components of cinnamon barks using GCMS and FTIR spectroscopy. Ind Crop Product 41:269-278.

  18. Hosseini SF, Zandi M, Rezaei M, Farahmandghavi F (2013) Two- step method for encapsulation of oregano essential oil in chitosan nanoparticles: Preparation, characterization and in vitro release study. Carbohydr Polym 95:50-56.

  19. Wen P, Zhu DH, Wu H, Zong MH, Jing YR, Han SY (2016) Encapsulation of cinnamon essential oil in electrospun nanofibrous film for active food packaging. Food Control 59:366-376.

  20. Mullen CA, Strahan GD, Boateng AA (2009) Characterization of various fast-pyrolysis bio-oils by NMR spectroscopy. Energy Fuels 23:2707-271.

  21. Fan L, Jin R, Liu Y, An M, Chen S (2011) Enhanced extraction of patchouli alcohol from Pogostemon cablin by microwave radiation- accelerated ionic liquid pretreatment. J Chrom B, 879:3653-3657.

  22. Sales A, Paulino BN, Pastore GM, Bicas JL (2018) Biogeneration of aroma compounds. Curr Opin Food Sci 19:77-84.

  23. Wu JZ, Liu YH, Liang JL, Huang QH, Dou YX, Nie J, Wu QD (2018) Protective role of – patchoulene from Pogostemon cablin against indomethacin-induced gastric ulcer in rats: Involvement of anti-inflammation and angiogenesis. Phytomedicine 39:111-118.

  24. Leino TO, Turku A, Yli-Kauhaluoma J, Kukkonen JP, Xhaard H, Wallén EA (2018) Azulene- based compounds for targeting orexin receptors. Eur J Med Chem 157:88-100.

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