INTRODUCTION:

The lipophilic profile of plant organisms is represented by lipids (fatty acids), terpenoids, alcohols, aldehydes, hydrocarbons, vitamins, etc. Fatty acids in living organisms are structural components of membrane lipids and a source of reserve energy. In addition to their structural and energetic function, they are involved in the formation of an immune response in plants. In the human body, fatty acids are involved in the synthesis of eicosanoids (prostaglandins, thromboxanes, leukotrienes), which regulate the work of the cardiovascular system. Fatty acids are subdivided into saturated (no double bonds), unsaturated (one double bond), and polyunsaturated (from two or more double bonds), depending on the location and number of double bonds in the hydrocarbon chain. Essential fatty acids are polyunsaturated fatty acids that are not synthesized in the body and are supplied only with food.^1-7

Essential oils are secondary metabolites, predominantly of a terpenoid structure, that perform a protective function in plant organisms and exhibit antimicrobial activity. They are used as an antiseptic, anti-inflammatory, diuretic, antispasmodic, tonic, and for therapeutic purposes. In the food industry, essential oils are used as food preservatives.^8-13

Pectorales species No. 2 (PS No. 2) is a multi-component herbal medicine. The composition of PS No. 2 includes leaves of coltsfoot (40%), leaves of plantain (30%), licorice roots (30%). The infusion of the collection is used to treat inflammatory diseases of the respiratory tract, accompanied by a cough with difficult sputum secretion (including bronchitis and tracheitis). Studies of the PS No. 2 lipophilic profile are not presented in the scientific literature. There is information about the lipophilic composition of the components of PS No. 2. Fatty acids n-hexadecanoic (palmitic) acid (up to 38.47%), 9,12-octadecadienic (linoleic) (up to 25.78%), eicosotrienic acid (2.4%) were detected by GC-MS in plantain extracts.¹⁴ There is evidence of the presence of arachidonic and behenic acids.¹⁵

Aromatic compounds (odorants) acetic acid (223000 μg/kg), hexanoic acid (63100 μg/kg), hexanal (4750 μg/kg), anethole (2840 μg/kg), butanoic acid (1570 μg/kg), γ-nonalactone (1110 μg/kg), linalool (1060 μg/kg), pentanoic acid (580 μg/kg), estragol (473 μg/kg), (421 μg/kg), phenylacetic acid (376 μg/kg) , eugenol (320 μg/kg), carvacrol (211 μg/kg), 3-methylbutanoic acid (190 μg/kg), 2-methylbutanoic acid (174 μg/kg), 1,8-cineole (96.9 μg/kg) kg), benzaldehyde (91.2 μg/kg), thymol (55.5 μg/kg), decanal (53.4 μg/kg), 3-ethylphenol (51.8 μg/kg), 2,3-butanedione (44.9 μg/kg), octanal (37.2 μg/kg), 2,4-nonadienal (35.5 μg/kg), 2-methylbutanal (33.9 μg/kg), 2,6-nonadienal (30.7 μg/kg) were identified in licorice roots.¹⁶

Gyawali R. et identified 2-ethoxy-1-propanol (23.38 mg/kg), 4-terpiniol (7.77 mg/kg), ethyl acetate (7.65 mg/kg), hexanal (5.83 mg/kg), hexanol (4.9 mg/kg), γ-nonalactone (2.56 mg/kg), para-cymene-8-ol (2.45 mg/kg), acetic acid (2.15 mg/kg ), solavetivone (1.97 mg/kg), indole (1.85 mg/kg), tetramethylpyrazine (1.78 mg/kg), myrtenal (1.72 mg/kg), α-terpineol (1.7 mg/kg), hexanoic acid (1.53 mg/kg), pulegone (1.32 mg/kg), methyl linoleidate (1.29 mg/kg) in the roots of Ural licorice.¹⁷14 fatty acids were identified by GC in petroleum ether extract of licorice roots, among which about 30% were saturated and 70% unsaturated. Palmitic acid prevailed among saturated fatty acids and among unsaturated ones – linoleic acid (52%) and oleic acid (12%).¹⁸

Iranian researchers using GC-FID found 4,4-dimethyltetracyclodecane (up to 21.9%), 1-undecene (up to 21.9%), 14-hydroxy-cis-caryophyllene (up to 21.1%), humulene epoxide II (up to 21.1%), caryophyllene oxide (up to 12.1%), α-cadinol (up to 11.1%), nerolidol (up to 11, 1%) 2-phenylethylanthranilate (up to 10.2%), β-selenene (up to 6.8%), spatulenol (up to 5.4%), α-copaen (up to 4.3%), (E) -caryophyllene (up to 4.1%) germacrene D (up to 3.3%), 1-tridecene (up to 3.1%), thymol methyl ester (up to 2.3%), α-humulene (up to 1.5%).¹⁹

Katsuba et. investigated the fatty acid composition of the leaves of coltsfoot. The amount of unsaturated fatty acids was 67.32%, saturated – 31.23%. Unsaturated fatty acids are represented mainly by linolenic (30.45%), linoleic (26.65%), oleic (6.2%), gondoic (1.92%). The main saturated acids are palmitic (24.42%), stearic (2.3%).²⁰

The objective of this work is to determine the fatty acid composition and the profile of volatile lipophilic compounds in PS No. 2.

MATERIALS AND METHODS:

Fatty acid profile:

Sample Preparation:

1-2 g of the sample was placed in a 50 ml test tube, 20 ml of a chloroform/methanol mixture (2:1 v/v) was added, tightly closed with a stopper and shaken for 1.5 hours on a laboratory shaker. Then 6.6 ml of distilled water was added, mixed and centrifuged for 5 min at 3000 rpm. The lower chloroform layer of the lipophilic fraction of each sample was placed into 50 ml round-bottom flasks, previously brought to constant weight and weighed on an analytical balance. Chloroform was distilled off from the flasks on a vacuum rotary evaporator. The remaining lipophilic fraction was dried to constant weight (10-20 min) at a temperature of 75-80° C. Then it was cooled in a desiccator and the flask was weighed on an analytical balance to constant weight. Approximately 10 mg of the lipophilic fraction of each sample was added to screw-capped tubes with weight-recorded gaskets. An 850 μlof previously prepared standard mixturesolution (undecanoic acid methyl ester, C=0.564 mg/mL, glyceryltritridecanoate, C=0.500 mg/mL and butylhydroxytolueneas antioxidant,C=0.011 mg/mL) in methanol were added. 1 mL of methanol, 20 µL of hexane, and 20 µL of acetyl chloride were added to the samples. Tubes were tightly sealed with caps and after a short intensive shaking were placed in a desiccator for 1 hour at 80 ° C for methylation. 2.5 mL of hexane and 100 µl of water were added after cooling the samples to room temperature and stirred vigorously on a laboratory shaker for about 10 seconds. 1 mL of the upper hexane layer with methyl esters was transferred to vials for GC analysis after stratification.

Conditions for GC-FID Analysis:

Sample injection volume – 1 μl, split mode – 30:1, carrier gas – nitrogen, flow rate – 0.9 ml/min. Injector temperature– 260 ° C, detector temperature– 240 °C. Separation conditions: initial temperature– 140°C (isotherm for 5 min.), then increasing at 4°C/min up to 220°C (isotherm 25 min). Data were collected and processed using Agilent ChemStation Rev.B.04.03 and Microsoft Excel 2007 software. The content of fatty acids was calculated using undecanoic acid methyl ester as an internal standard. The completeness of transesterification and extraction of methyl esters into hexane was controlled using the glyceryltritridecanoate. Conversion factors of methyl esters to free forms were used to calculate totalFAs content according to previous works.^21,22

Volatile compounds:

Sample Preparation:

3 g of homogenized sample was placed in a 20 ml vial for vapor phase analysis. Then 7 ml of highly purified water was added, stirred for 20 seconds on a vortex, and sealed. A SPME fiber was placed in the space above the sample and incubated for 30 min on a tile heated to 110°C. Next, the fiber was extracted and GC analysis was performed.

Conditions for GC-MS Analysis:

The fiber coated with divinylbenzene/carboxene/ polydimethylsiloxane 50/30 μm (Supelco) was used in the experiment; before the analysis, the fiber was conditioned according to the manufacturer's recommendations. Agilent Technologies 7890A gas chromatograph (USA) with an Agilent Technologies 5975C mass detector (USA) and a Supelcowax 10 chromatographic column 60 m ×0.53 mm ×1 μm were used. The following temperature program was used: 35°C for 5 min, heating up to 220°C at a rate of 4°C/min, isotherm 40 min. Carrier gas–helium, splitless mode, injector temperature–225 °C. Mass detector parameters: scanning range–35-400 m/z, ionization source temperature–230°C, quadrupole temperature– 150°C, electron impact ionization with energy 70 eV. The results were processed using “MSD ChemStation E02.02.1431”. The software “The NIST Mass Spectral Search Program for the NIST/EPA/NIH Mass Spectral Library Ver. 2.0 gm, build May 19 2011” with a set of commercially available mass spectra libraries was used to identify the spectra. Components that havethe value of coincidence coefficient (analyzed compound spectrum to library spectrum) more than 700 were taken into account and identified. Subsequent processing of the obtained data was performed using the Microsoft Office Excel 2007 SP3 MSO software package.

RESULTS AND DISCUSSION:

Fatty acid profile

The results of the study of the PS No. 2 fatty acid composition are presented in Fig. 1 and Table 1.

Table 1: Ratio of the content of FA methyl esters, % of the total FA

No.	Name	Index	Content,%
Saturatedfattyacids
1.	lauric	12:0	1,51
2.	myristic	14:0	1,66
3.	pentadecylic	15:0	0,35
4.	palmitic	16:0	24,81
5.	margaric	17:0	0,56
6.	stearic	18:0	4,40
7.	arachidic	20:0	1,71
8.	behenic	22:0	1,18
9.	lignoceric	24:0	1,14
Total Saturated Fatty Acids			37,32
Unsaturatedfattyacids
1.	hexadecenoic	16:1	0,80
2.	palmitoleic	16:1 9-cis	0,43
3.	heptadecenoic	17:1	0,23
4.	elaidic	18:1 9-trans	0,40
5.	oleic	18:1 9-cis	9,15
6.	vaccenic	18:1 11-trans	1,01
7.	cys, trans-linoleic	18:2 9-cys, 12-trans	1,17
8.	trans, cys - linoleic	18:2 9-trans, 12-cys	0,12
9.	linoleic	18:2	18,39
10.	γ - linoleic	18:3 ω-6 6c,9c,12c	0,87
11.	α - linoleic	18:3 ω-3 9c,12c,15c	26,86
12.	gondoic	20:1	0,42
13.	eicosadienoic	20:2	0,43
14.	eicosatriene n6	20:3 8,11,14-cys	1,82
15.	arachidonic	20:4 n6	0,16
Total Unsaturated Fatty Acids			62,26

Thus, the main compounds in the fatty acid composition of PS 2 are palmitic acid, linoleic acid, and α-linolenic acid, which confirms the literature's data.

Volatile compounds:

91 compounds were detected in the volatile fraction of the PS 2. The data are presented in Table 2.

Table 2: Profile of volatile compounds in PS No. 2

1.	tR	Components content in the mixture,%	Name
2.	8.082	0,017	isoprene
3.	14.216	0,023	butyraldehyde
4.	14.436	0,252	methacrylaldehyde
5.	15.176	0,033	butanone
6.	15.859	0,538	isovaleraldehyde
7.	16.426	0,299	non-1-ene
8.	17.92	0,065	1,3-cyclopentadiene, 5-(1,1-dimethylethyl)-
9.	18.499	0,568	valeraldehyde
10.	19.634	0,035	methylpentanone
11.	20.185	0,416	α -pin-2(10)-en
12.	22.037	0,072	camphene
13.	22.785	5,653	caproicaldehyde
14.	23.813	0,089	β-pin-2(3)-en
15.	23.981	0,055	iso-hexanol
16.	24.299	0,123	thujone
17.	25.009	0,171	pent-4-enal
18.	25.822	0,143	myrcene
19.	26.174	0,336	phellandrene
20.	26.884	0,443	methyl n-amylketone
21.	27.012	0,287	heptanaldehyde
22.	27.591	1,176	limonen
23.	27.809	0,141	methylcrotonaldehyde
24.	28.065	1,187	cineol
25.	28.543	1,213	hexenal
26.	28.657	0,287	amylfuran
27.	29.408	0,323	1,4-terpinene
28.	29.636	0,169	3-octanone
29.	30.463	1,901	4-isopropylmethylbenzene
30.	30.79	0,088	methylhexylketone
31.	30.969	0,111	caprylicaldehyde
32.	31.048	0,103	1,2,4-trimethylbenzene
33.	31.46	0,680	iso-heptylalcohol
34.	31.881	0,117	3- methylamylalcohol
35.	32.504	0,880	trans-heptenal
36.	32.753	3,368	1-hexanol
37.	33.211	0,083	1,2,3-trimethylbenzene
38.	34.034	2,748	cis-hex-3-enyl alcohol
39.	34.322	0,017	methylcaprylate
40.	34.48	0,061	methylheptylketone
41.	35.238	0,791	3-octen-2-one
42.	35.435	0,473	fenchone
43.	36.021	5,850	iso-octenylalcohol
44.	36.235	2,347	α-thujone
45.	36.879	1,795	β-thujone
46.	37.512	0,088	tetramethylpyrazine
47.	37.669	7,561	isomentone
48.	38.139	1,614	(s)-3-ethyl-4-methylpentanol-1
49.	38.623	2,965	mentanone
50.	39.027	0,682	3,5-octadien-2-one
51.	39.187	2,526	linalool
52.	39.354	0,358	dillether
53.	39.541	0,446	octylalcohol
54.	39.696	2,701	benzaldehyde
55.	40.734	0,482	octadienone
56.	41.23	0,619	neoisomenthol
57.	41.639	1,174	1-terpinene-4-ol
58.	42.179	0,081	cis-4-(isopropyl)-1-methylcyclohex-2-en-1-ol
59.	42.314	1,023	dihydrocarvone
60.	42.519	1,431	menthol
61.	42.936	0,461	dihydrocarvone
62.	43.53	2,544	pulegone
63.	43.848	1,104	estragole
64.	44.291	0,444	α-terpineol
65.	45.111	0,266	p-menth-4-en-3-one
66.	46.064	23,439	carvon
67.	46.376	0,141	δ-cadinene
68.	46.872	0,101	nerol
69.	47.157	0,117	myrtenol
70.	47.352	0,291	methylsalicylate
71.	47.435	0,255	cuminaldehyde
72.	47.783	0,033	decadienaldehyde
73.	48.04	0,202	geraniol
74.	48.222	0,048	β-damascenone
75.	48.355	4,388	anethol
76.	48.48	0,152	p-cimenol-8
77.	48.61	0,272	5,9-undecadien-2-one, 6,10-dimethyl-
78.	48.834	1,253	caproicacid
79.	49.149	0,195	methoxyphenol
80.	49.43	0,354	benzylalcohol
81.	50.438	0,468	phenylethylalcohol
82.	51.04	0,317	α-calacorene
83.	51.377	0,535	β-ionone
84.	53.181	0,910	caryophylleneepoxide
85.	54.12	0,430	anisaldehyde
86.	54.538	0,413	caprylicacid
87.	56.626	0,331	spathulenol
88.	57.805	0,439	thymol
89.	59.046	0,256	carvacrol
90.	62.363	0,181	2,4-di-tert-butylphenol
91.	66.745	0,113	diethylphthalate
92.	67.732	0,265	dihydroactinidiolide

The volatile compounds of PS No 2 are presented by abundant amounts of carvone (23,439%), isomentone (7,561%), iso-octenyl alcohol (5,850%), anethol (4,388%), hexyl alcohol (3,368%), mentanone (2,965%), cis-Hex-3-enyl alcohol (2,748%), benzaldehyde (2,701%), pulegone (2,544%), linalool (2,526%), α-thujone (2,347%). Hexanoic acid, anethol, estragol, carvacrol, 1,8-cineol, benzaldehyde, thymol, octanal, hexanol, para-cymene-8-ol, α-terpineol, hexanoic acid, pulegon, which, according to literature data, were identified in licorice roots. The presence of spatulenol, α- phellandrene, and heptanal compounds detected in the volatile fraction of PS2 detected in the leaves of coltsfoot also confirms the literature data.

CONCLUSION:

The composition and content of fatty acids in PS No 2 were determined. 24 fatty acids were identified with a predominance of unsaturated (62.26%) over saturated (37.32%). Palmitic acid (24.81%), linoleic acid (18.39%), and α-linolenic acid (26.86%) were the most abundant. The composition and content of 91 volatile compounds of the lipophilic fraction PS No 2 were determined by GC-MS. The predominant compounds were carvone (23,439%), isomentone (7,561%), iso-octenyl alcohol (5,850%), anethol (4,388%), hexyl alcohol (3,368%), mentanone (2,965%), cis-Hex-3-enyl alcohol (2,748%), benzaldehyde (2,701%), pulegone (2,544%), linalool (2,526%), α-thujone (2,347%). Hexanoic acid, anethol, estragol, carvacrol, 1,8-cineol, benzaldehyde, thymol, octanal, hexanol, para-cymene-8-ol, α-terpineol, hexanoic acid, pulegon, spatulenol, α- phellandrene, heptanal can be considered as marker compounds for standardization purposes.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

REFERENCES:

1. Kachroo A. Kachroo P. Fatty acid–derived signals in plant defense. Annualreviewofphytopathology 2009; 47: 153–176.doi: 10.1146/annurev-phyto-080508-081820

2. Gladyshev M I. Essential Polyunsaturated Fatty Acids and their Dietary Sources for Man. Journal of Siberian Federal University. Biology. 2012; 4: 352–386 [in Russian]

3. Joshi H, Arun B Joshi, Sati H, Gururaja MP, Shetty PR, Subrahmanyam EVS, Satyanaryana D. Fatty Acids from Memecylonumbellatum (Burm.). Asian J. Research Chem. 2009; 2(2): 178–180

4. Vaidya N.Choure R. Electrochemical analysis of fatty acids obtained from the natural resource seed of Perillafrutescens. Asian J. Research Chem. 2011; 4(5): 705–707.

5. Bubenchikov RA.Korableva TV.Pozdnyakova TA.Kuleshova ES. The study of the fatty acid composition of compass lettuce (Lactucaserriola L.). Research J. Pharm. and Tech. 2020; 13(12): 6105–6108.doi: 10.5958/0974-360X.2020.01064.1

6. Kurkin VA.Mubinov AR.Avdeeva HV.Ryazanova TK. Comparative research of fatty acid composition and volatile components of fatty oils from seeds of Nigella sativa and Arganiaspinosa. Research J. Pharm. and Tech. 2021; 14(3): 1586–1590. doi:10.5958/0974-360X.2021.00280.8

7. Ponugoti M. Panda SP.Kulandaivelu U. Rao GK.Alavala RR. Varma NJ. Isolation and evaluation of anti-inflammatory activity of epigallocatechin from Senegaliarugata along with PUFAs. Research Journal of Pharmacy and Technology. 2021; 14(11): 5739–4. doi:10.52711/0974-360X.2021.00998

8. Mishra A. P. et al. Combination of essential oils in dairy products: A review of their functions and potential benefits. Lwt. 2020; 133: 110116. doi: 10.1016/j.lwt.2020.110116

9. Suganya S. Bharathidasan R. Senthilkumar G. Madhanraj P. Panneerselvam A. Antibacterial activity of essential oil extracted from Coriandrumsativam (L.) and GC-MS analysis. Research J. Science and Tech. 2012; 4(5): 203–207

10. Malathy BR. Ajitha PS. Sangeetha KS. Thampy S. Kamala G. Antimicrobial activity of commercial essential oils on human pathogens. Research Journal of Pharmacy and Technology. 2021; 14(8): 4440–4. doi: 10.52711/0974-360X.2021.00771

11. Joicy CM. Sivaraj C. Arumugam P. In-vitro antioxidant, antidiabetic, antibacterial and cytotoxic activities of essential oil extracted from flowers of Illiciumverum L. Research Journal of Pharmacy and Technology. 2021; 14(5): 2452–8. doi:10.52711/0974-360X.2021.00431

12. Ridwan RD, Sidarningsih, Wijayanti U. The anti-bacterial activity of gingival mucoadhesive patch from Thymus vulgaris essential oil towards Aggregatibacter actinomycetemcomitans and Fusobacteriumnucleatum. Research J. Pharm. and Tech. 2021; 14(2): 645–649. doi:10.5958/0974-360X.2021.00115.3

13. Labiad H.Aljaiyash A.Ghanmi M.Satrani B.Ettahir A.Aouane M.Fadli M.Chaouch A. Exploring the provenance effect on chemical composition and pharmacological bioactivity of the Moroccan essential oils of Laurusnobilis. Research J. Pharm. and Tech. 2020; 13(9): 4067–4076. doi:10.5958/0974-360X.2020.00719.2

14. Mazzutti S. et al. Green-based methods to obtain bioactive extracts from Plantagomajor and Plantagolanceolata. The Journal of Supercritical Fluids. 2017; 119: 211–220. doi: 10.1016/j.supflu.2016.09.018

15. Adom MB. et al. Chemical constituents and medical benefits of Plantago major. Biomedicine and Pharmacotherapy. 2017; 96: 348–360. doi: 10.1016/j.biopha.2017.09.152

16. Wagner J. Granvogl M.Schieberle P. Characterization of the key aroma compounds in raw licorice (Glycyrrhiza glabra L.) by means of molecular sensory science. Journal of Agricultural and Food Chemistry. 2016; 64(44): 8388–8396. doi: 10.1021/acs.jafc.6b03676

17. Gyawali R. et al. Effect of γ-irradiaton on the volatile compounds of licorice (Glycyrrhizauralensis Fischer). European Food Research and Technology. 2008; 226(3): 577–582. doi: 10.1007/s00217-007-0591-2

18. Yunusova S. G. et al. Lipids of Glycyrrhizaglabra roots. Russian Chemical Bulletin. 1995; 44(2): 359–362. doi: 10.1007/BF00702152 [in Russian]

19. Norani M. Ebadi MT.Ayyari M. Volatile constituents and antioxidant capacity of seven Tussilagofarfara L. populations in Iran. Scientia Horticulturae. 2019; 257: 108635. doi: 10.1016/j.scienta.2019.108635

20. Kacuba IK. Kislichenko VS. Novosel EN. Study of the fatty acid composition of leaves, flowers and roots of coltsfoot. Aktual'nyeproblemymediciny. 2013; 23(18): 161. [in Russian]

21. Golay PA. Moulin J. Determination of labeled fatty acids content in milk products, infant formula, and adult/pediatric nutritional formula by capillary gas chromatography: Collaborative study, final action 2012.13. Journal of AOAC International. 2012; 99(1): 210–222.

22. Golay PA. Dong Y. Determination of labeled fatty acids content in milk products, infant formula, and adult/pediatric nutritional formula by capillary gas chromatography: Single-Laboratory Validation, First Action 2012.13. Journal of AOAC International. 2015; 98 (6): 1679–1696.

Received on 22.02.2024 Modified on 17.06.2024

Research J. Pharm. and Tech 2024; 17(11):5412-5416.

DOI: 10.52711/0974-360X.2024.00827

Volatile compounds and fatty acids of mixture herbal product Pectorales Species No 2

MATERIALS AND METHODS:

RESULTS AND DISCUSSION:

CONCLUSION:

CONFLICT OF INTEREST:

REFERENCES: