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Abstract

Background/Objective: Volatile compounds present in green Ginkgo biloba L. leaves extracts contain compounds that can undertake free radical scavenging. The assumption was that volatile compounds present in yellow Ginkgo biloba leaves extracts have a different composition compared to green Ginkgo biloba leaves extracts. The investigation was conducted in order to determine the compounds in yellow Ginkgo biloba leaves’ extracts that can undertake free radical scavenging. Methods: The volatile compounds present in yellow Ginkgo biloba leaf extracts were obtained by hydrodistillation and supercritical carbon dioxide (CO₂) extraction. The obtained essential oils were analyzed by gas chromatography–mass spectrometry. Results: The essential oil obtained by hydrodistillation consisted of monoterpenes (3.1%), oxygenated monoterpenes (15.3%), sesquiterpenes (6.2%), oxygenated sesquiterpenes (51.4%), esters (1.3%), benzopyrans (7.1%), an oxolane (1.5%), and a rose ketone (0.8%). Caryophylene-4(12),8(13)-dien-5α-ol (7.1%) was the most abundant compound, followed by 14-hydroxy-(Z)-caryophyllene (5.8%), caryophyllene oxide (5.8%), and hydroxydihydroedulan (5.6%). The supercritical fluid extraction was done under the pressure of 30 MPa, at the temperature of 40 °C and the CO₂ flow rate was 2 kg·h⁻¹. The obtained chromatogram revealed the presence of 12 compounds. The main constituents were squalene (110.5 mg per 100 g of plant material) and α-tocopherol (99.3 mg per 100 g of plant material). Conclusion: The well-known compound that has a free radical scavenging activity α-tocopherol was determined in yellow Ginkgo biloba leaves extract obtained by supercritical CO₂ extraction.

Keywords

yellow leaves hydrodistillation

Ginkgo biloba L. is the only extant species within the division Ginkgophyta.^1-4 G. biloba extracts are herbal remedies used for many centuries.⁵ G. biloba herbal remedies have maintained historical and cultural alternatives for pharmaceutically made medicaments.^6,7 G. biloba is a medicinal plant and most special extracts are made from its leaves.^8,9 The leaves are green in spring and summer and turn to grey-yellow, yellow, brown, or blackish in autumn. Ginkgo leaves major constituents are alkanes, lipids, sterols, terpenoids, carotenoids, carboxylic acids, benzenoids, phenylpropanoids, flavonoids, biflavonoids, catechins, proanthocyanidins, and carbohydrates.^10-13 Characteristic constituents are terpenetrilactones with the trivial name ginkgolides, while sesquiterpene lactones are bilobalides.^12,14

Thin-layer chromatographic analysis is used for ginkgolides and bilobalide identification.¹⁴ High-performance liquid chromatographic analyses for determination of flavonoids, ginkgolides, and bilobalide have been developed.^10,15 Chemical assays have been developed for qualitative and quantitative flavonoid glycoside determination and are carried out after hydrolysis of the aglycones.¹⁶ The qualitative biflavones identification can be determined by high-performance liquid chromatography.¹⁷ The qualitative and quantitative diterpene ginkgolides and sesquiterpene bilobalide determination can be done by high-performance liquid chromatography.¹²

The changes in G. biloba leaf lipids through the early stages of degradation were done in order to identify biomarkers of the Ginkgoale.¹⁸ The fresh, senescent, and litter leaves were extracted with CH₂Cl₂:MeOH (2:1, v/v) and obtained were internal lipids.¹⁸ It was found that α-tocopherol is present in G. biloba leaves and can be a useful biomarker for the Ginkgoale.¹⁸

The G. biloba leaves, collected in September, where the essential oil was obtained by hydrodistillation, contained hydrocarbons (31.14%), alcohols (5.61%), ketones (46.17%), aldehydes (1.78%), acids (5.41%), and esters (1.1%).¹⁹ The essential oil obtained by hydrodistillation, when the leaves were collected in June, consisted of sesquiterpenes (42.11%), diterpenes (14.08%), hydrocarbons (5.89%), esters (6.12%), aldehydes (2.59%), alcohols (2.36%), monoterpenes (1.94%), acids (0.57%), and a ketone (0.11%).²⁰ Essential oils, obtained after hydrodistillation of leaves, collected from May to late November from three different trees, two young and one old tree, composition varied from month to month. The constituents were hydrocarbons, esters, aldehydes, ketones, and acids.²¹ The ionones were detected only in one young tree and limonene only in the old one.²¹

G. biloba green leaves have been used for the extraction of biologically active compounds using various extraction techniques.^22-25 The carbon dioxide (CO₂) in its supercritical state is a solvent enabling the extraction of lipophilic compounds. To increase the polarity of CO₂ fluid, in the process, cosolvents were added to improve extraction efficiency.²⁵ For the extraction of flavonoids and terpenoids from G. biloba green leaves, after the primary extraction with 70% ethanol, followed the extraction with the supercritical CO₂ (SC CO₂) using the CO₂ containing 5% ethanol as a cosolvent.²⁵ The most favorable experimental conditions were 30 MPa, 60 °C, and CO₂ containing 5% ethanol as a cosolvent.²⁵ The influence of pressure, temperature, and time of the SC CO₂ extraction on the yield, using the cosolvents, was investigated.²³

It is well known that the leaf's development stage influences the phytochemical profile. Leaf senescence is characterized by chloroplast degeneration and catabolism of phytochemicals. Phytochemicals disassembled from senescent leaves, which were not relocated to phloem tissues, were extracted by hydrodistillation and SC CO₂ extraction in order to gain insight into G. biloba yellow leaves essential oils. Data on the SC CO₂ extraction of yellow G. biloba leaves are not available. The SC CO₂ was the method of choice to study the lipophilic phytochemicals in G. biloba yellow leaves under the most favorable experimental condition.

The aim of the study was to determine the phytochemical profile of G. biloba yellow leaves essential oils obtained by hydrodistillation and SC CO₂ extraction.

The essential oil obtained by hydrodistillation yielded 0.2% and after the SC CO₂ extraction, the yield was 1.4%. In the essential oil, obtained by hydrodistillation, 50 compounds were identified (Table 1). In the essential oil, obtained by SC CO₂ extraction, 12 compounds were identified. The yield of the phytochemicals in the G. biloba yellow leaves essential oil, obtained by SC CO₂ extraction, is expressed in milligrams of α-tocopherol equivalents per 100 g of dry leaves (Table 2).

Table 1.

Phytochemical Profile of Ginkgo biloba Yellow Leaves Essential Oil Obtained by Hydrodistillation.

No.	Compound	RI exp^a	RI lit^b	%
1	α-Pinene	939	936	1.0
2	Camphene	946	946	1.6
3	p-Cymene	1021	1020	0.5
4	cis-Linalool oxide (furanoid)	1067	1067	0.3
5	Linalool	1093	1096	0.6
6	Camphor	1136	1141	0.4
7	Borneol	1160	1165	2.3
8	Terpinen-4-ol	1174	1174	0.8
9	α-Terpineol	1190	1186	1.3
10	Verbenone	1206	1204	0.9
11	β-Cyclocitral	1216	1217	0.3
12	Vitispirane	1272	1279	1.5
13	Bornyl acetate	1281	1287	3.0
14	Dihydrocarveol acetate	1293	1295	0.1
15	Chrysanthenone epoxide	1298	1290	1.3
16	Edulan I	1310	1313	1.5
17	Silphiperfol-4,7(14)-diene	1352	1358	0.2
18	2-Ethyl-3-hydroxyhexyl 2-methylpropanoate	1374	1373	0.4
19	cis-β-Caryophyllene	1404	1408	0.2
20	trans-α-Ionone	1430	1428	0.8
21	Hydroxydihydroedulan	1437	1453	5.6
22	α-Humulene	1453	1453	1.7
23	Geranyl acetone	1458	1453	4.0
24	Ledene	1478	1492	1.4
25	ar-Curcumene	1486	1479	0.5
26	α-Selinene	1494	1498	0.4
27	α-Muurolene	1500	1500	0.2
28	Cyclobazzanene	1507	1514	0.1
29	iso-Italicene epoxide	1514	1514	4.8
30	trans-Calamenene	1523	1521	1.5
31	Spathulenol	1551	1572	4.4
32	Italicene epoxide	1558	1547	4.3
33	trans-Nerolidol	1568	1561	0.6
34	Caryophyllene oxide	1580	1582	5.8
35	Clovenol	1584	1581	4.6
36	Cubebane-11-ol	1589	1584	1.2
37	Salvial-4(14)-en-1-one	1592	1594	0.2
38	Humulene epoxide II	1606	1608	0.6
39	Isospathulenol	1612	1625	0.6
40	Caryophylla-3(15),7(14)-dien-6-ol	1630	1635	1.0
41	Caryophylla-4(12),8(13)-dien-5α-ol	1634	1639	7.1
42	α-Muurolol (=Torreyol)	1640	1644	1.8
43	β-Eudesmol	1647	1649	3.5
44	14-Hydroxy-(Z)-caryophyllene	1655	1666	5.8
45	4-Hydroxy-9-epi-(E)-caryophyllene	1670	1668	3.4
46	Eudesma-4(15),7-dien-1β-ol	1684	1687	0.6
47	Hexahydrofarnesyl acetone	1839	1838	1.1
48	Ethyl hexadecanoate	1996	1992	0.4
49	Ethyl oleate	2172	2171	0.4
50	Ethyl octadecanoate	2198	2196	0.1

^aRI exp: retention indices on HP-5MS column relative to C₈-C₄₀ n-alkanes.

^bRI lit: retention indices obtained from the literature and NIST Webbook.

Table 2.

The Phytochemical Profile of Ginkgo biloba Yellow Leaves Essential Oil Obtained by Supercritical CO₂ (SC CO₂) Extraction.

No.	Compound	RI exp	Yield [in milligram α-tocopherol equivalents per 100 g of dry leaves]
1.	α-Cadinol	1657	0.5
2.	Hexahydrofarnesyl acetone	1839	0.5
3.	Neophytadiene	1840	38.9
4.	Ethyl hexadecanoate	1996	14.6
5.	1-Octadecanol	2081	3.0
6.	Phytol	2119	36.6
7.	Ethyl oleate	2171	4.0
8.	Ethyl octadecanoate	2198	5.8
9.	1-Eicosanol	2282	3.7
10.	Squalene	2832	110.5
11.	α-Tocopherol	3112	99.3
12.	β-Sitosterol	3197	0.8

The essential oil obtained by hydrodistillation consisted of monoterpenes (3.1%), oxygenated monoterpenes (15.3%), sesquiterpenes (6.2%), oxygenated sesquiterpenes (51.4%), esters (1.3%), the oxolane vitispirane (1.5%), two benzopyrans (7.1%), and a rose ketone trans-α-ionone (0.8%). The essential oil obtained by hydrodistillation of leaves collected in September, contained 1.1% of esters while the essential oil, from the leaves collected in November, had 1.32% of esters.¹⁹ Ketones dominated in the essential oil from the leaves collected in September, while in the essential oil, from the leaves collected in November, the geranyl acetone presented 3.95% and hexahydrofarnesyl acetone was present in 1.09%.¹⁹ The rose ketone was identified previously only in essential oil obtained from young tree leaves while in the essential oil obtained from leaves collected in November, it presented 0.85%.²¹ The most abundant compounds in the essential oil obtained from the leaves collected in November were: caryophyllene-4 (12),8(13)-dien-5α-ol (7.1%), 14-hydroxy-(Z)-caryophyllene (5.8%), caryophyllene oxide (5.8%), hydroxydihydroedulan (5.6%), iso-italicene epoxide (4.8%), clovenol (4.6%), spathulenol (4.4%), geranyl acetone (4.0%), and 4-hydroxy-9-epi-(E)-caryophyllene (3.4%) (Table 1). Oxygenated sesquiterterpenes were the most abundant compounds present in the essential oil (Table 1).

The essential oil obtained by SC CO₂ extraction was characterized by high amounts of squalene and α-tocopherol, followed by neophytadiene and phytol (Table 2). The α-cadinol, hexahydrofarnesyl acetone, and β-sitosterol were present in small amounts. The SC CO₂ extraction of green G. biloba leaves revealed the presence of flavonoids and terpenoids.²⁵ The phytochemical analysis of the essential oil of G. biloba yellow leaves collected in November, revealed the presence of one sesquiterpenoid alcohol α-cadinol; a diterpene neophytadiene; a ketone hexahydrofarnesyl acetone; three esters: ethyl hexadecanoate, ethyl oleate, and ethyl octadecanoate; alcohols: 1-octadecanol, phytol, and 1-eicosanol; a triterpenoid squalene, α-tocopherol, and one phytosterol β-sitosterol (Table 2). The hexahydrofarnesyl acetone and three esters were present in both essential oils. The operating parameters applied for the SC CO₂ extraction yielded the presence of α-cadinol, neophytadiene, phytol, 1-octadecanol, 1-eicosanol, squalene, α-tocopherol, and β-sitosterol. The presence of α-tocopherol can be attributed to the applied operating temperature of 40 °C during the SC CO₂ extraction. The essential oil obtained by hydrodistillation did not contain α-tocopherol due to the high temperatures applied during the hydrodistillation. The essential oil obtained by SC CO₂ extraction had low terpenoid content due to the low operating temperature during the SC CO₂ extraction process. The differences in chemical composition of essential oils obtained by two different extraction techniques can be attributed to the temperature applied. The novelty of this study is the presence of α-tocopherol in the SC CO₂ essential oil of G. biloba yellow leaves. The quantity of α-tocopherol is high and yellow leaves can be a reliable source for obtaining α-tocopherol from the SC CO₂ extract.

This study reports the phytochemical profile of G. biloba yellow leaves essential oils obtained by hydrodistillation and SC CO₂ extraction. It can be concluded that the essential oil obtained by hydrodistillation is a valuable source of oxygenated sesquiterpenes. The essential oil obtained by SC CO₂ extraction is rich in squalene and α-tocopherol. The identification of α-tocopherol is a starting point for further investigations in order to find the optimal conditions for its isolation from the G. biloba yellow leaves.

Experimental

Plant Material

G biloba yellow leaves were collected in November 2021 in Zrenjanin, Serbia (45°22′51″ N, 20°23′20″ E). Yellow leaves were air-dried for several days. Dried G. biloba yellow leaves were grounded in a rotating blade coffee grinder Bosch MKM 6003. After drying, the water content was determined according to AOAC Official Method 925.40 and was 9.3 ± 0.1%. The grounded plant material was sieved for 20 min using the vertical vibratory sieve shaker (Labortechnik GmbH). The average particle size distribution was determined using a nest of 3 sieves of aperture sizes 0.5, 0.4, and 0.315 mm. The mass of fragments remaining on each sieve was used to calculate the distribution of fragments by the Rosin-Rammler distribution function and was normalized concerning the total mass.²⁶ The distribution revealed that the average particle size was 0.37 ± 0.08 mm.

Chemicals

The CO₂ used for extraction was 99.97% (Messer Tehnogas AD). The standard compound α-tocopherol was from Dr. Ehrenstorfer (Augsburg, Germany). All other chemicals were of analytical reagent grade.

Hydrodistillation

Air-dried grounded plant material was hydrodistilled for 3 h using Clevenger-type apparatus. The essential oil obtained was dried over anhydrous sodium sulfate.

SC CO₂ Extraction Procedure

Grinded yellow leaves (50 g), in a powder form, were poured into the extraction vessel made from stainless steel bar (AISI 304) 100 mm outside diameter and height 500 mm, with the center hole of Ø 40 mm. The volume of the extraction vessel was 500 mL. The cap of the extraction vessel held a plug connected to the extraction vessel through the thread. The plug had O-ring seals in two places. The plug had a filter element with the ability to filter particles of 2 to 10 µm (Norman Ultraporous 4202T-6T-2M) to prevent the withdrawal of the plant material. The extract was collected in a separator with the glass tube with previously determined glass tube mass. The temperature was controlled by a controller. The input CO₂ line leading towards the extraction vessel was preheated by a heat exchanger supported by the water heating system. Liquid CO₂ was precooled through the stainless steel coil by an ethylene glycol/ethanol cooling bath. Before the extraction vessel, CO₂ was preheated through the stainless steel coil at the extraction temperature. The CO₂ flow rate was controlled by a flow meter. The mass of the plant material in the extraction vessel, grounded plant material particle size, and the extraction time (90 min) were kept constant during the experiment. The extraction lasted for 90 min since a longer extraction time did not increase the extraction yield significantly.

Gas Chromatography–Mass Spectrometry Analysis

The extract sample analyses were carried out on Agilent 7890A GC equipped with 5975 MSD. The capillary column was HP-5MS (5% phenyl-methyl polysiloxane, 30 m × 250 μm × 0.25 μm). The injection port temperature was 250 °C, split-less injection. The transfer line temperature was 280 °C. The initial oven temperature was set at 200 °C for 3 min, and programmed with 8 °C·min⁻¹ to 280 °C. The carrier gas was He. Mass spectrometry (MS) conditions were: scan (45 to 450 amu) and the MS source temperature was 250 °C. Injected sample volume was 1 μL. The phytochemicals present in the essential oils were identified on the basis of their mass spectra, retention times, and retention indices (RIs). The identification of phytochemicals was carried out based on computer matching with NIST 2008 MS library. Quantification of compounds was provided using calibration curves. The standard compound was dissolved in n-hexane to prepare six different concentrations of α-tocopherol. The R² for the calibration curve was 0.999. All analyses were performed in triplicate.

Footnotes

Declaration of Conflicting Interests

The author declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Nina Djapic

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Essential Oils of Ginkgo biloba Yellow Leaves Obtained by Hydrodistillation and Supercritical Carbon Dioxide Extraction

Abstract

Keywords

Experimental

Plant Material

Chemicals

Hydrodistillation

SC CO2 Extraction Procedure

Gas Chromatography–Mass Spectrometry Analysis

Footnotes

Declaration of Conflicting Interests

Funding

ORCID iD

References

SC CO₂ Extraction Procedure