Total Phenolic Content,Flavonoid Content,and Antioxidant Potential of Hydrosols From Six Aromatic Plants

Abstract

Objective

The objective of this study was to evaluate the total flavonoid content (TFC), total phenolic content (TPC), and antioxidant activity of hydrosols derived from six aromatic plant species cultivated in Guangxi, China (Eucalyptus grandis × E. urophylla, Pogostemon cablin (Blanco) Benth., Cymbopogon citratus (DC.) Stapf, Rosmarinus officinalis L., Plumeria rubra L., and Baeckea frutescens L.). Furthermore, the correlations among these parameters were analyzed.

Methods

The volatile components of the hydrosols were analyzed by gas chromatography - mass spectrometry (GC - MS). The total flavonoid content of the hydrosols was determined by the sodium nitrite - aluminum nitrate colorimetric method. The total phenolic content of the hydrosols was measured by the Folin - Ciocalteu colorimetric method. The antioxidant activity of the hydrosols was assessed by the 2,2-Diphenyl-1-picrylhydrazyl (DPPH) scavenging method and the 2,2′-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS⁺) radical scavenging method.

Results

According to the GC - MS analysis results, among the three hydrosols in which phenolic compounds were detected, the Plumeria rubra hydrosol had the highest phenolic compound content. In the results of total flavonoid and total phenolic content measurements, the Plumeria rubra hydrosol had the highest total flavonoid and total phenolic contents, which were 74.88 ± 3.28 μg/ml and 117.80 ± 2.03 μg/ml, respectively. In the antioxidant activity measurement results, Plumeria rubra hydrosol showed the highest DPPH and ABTS⁺ radical scavenging activities, with IC₅₀ values of 8.45 mg/mL and 53.65 mg/mL, respectively.

Conclusion

The relationship plots between the total flavonoid and total phenolic contents of the hydrosols and the IC₅₀ values of the hydrosols for DPPH and ABTS⁺ radical scavenging capacity show that there are negative correlations between them. This study demonstrates a significant correlation between the total flavonoid, total phenolic content and antioxidant activity in six aromatic plant hydrosols.

Keywords

hydrosols volatile chemical compositions flavonoid content phenolic content antioxidant activity

Introduction

Antioxidants comprise a class of stable compounds capable of mitigating oxidative damage through multiple mechanisms, including free radical scavenging, metal ion chelation, antioxidant enzyme activation, and suppression of oxidative pathways.^1,2 Antioxidants find extensive applications in both the food and pharmaceutical industries, playing crucial roles in preventing diseases induced by reactive oxygen species (ROS) - metabolic byproducts of cellular redox reactions, as well as food deterioration.^3,4 In the food industry, antioxidants serve as preservatives to extend shelf life. However, growing concerns about the toxicity of synthetic antioxidants have led to an increasing demand for natural alternatives, particularly plant-derived antioxidants.

In recent years, the safety of synthetic chemicals has been questioned by the general public. Studies have shown that these substances not only have negative impacts on human health but also contribute to environmental pollution. As a result, there is a growing body of research on plants and their derivatives, such as essential oils and hydrosols.^5,6 Hydrosols are by-products of plant steam distillation. They consist of distilled water and water-soluble components of essential oils.⁷ Similar to essential oils, they possess a wide range of extensive biological activities, such as antimicrobial, skin whitening, complexion improvement, moisturizing, antioxidant, and anti-inflammatory effects.^8,9 However, hydrosols are less harmful to the human body, have low production costs, and are easy to obtain, but research on hydrosols is still limited.¹⁰ Considering its unique advantages and great application potential, it is particularly necessary to conduct in-depth research on hydrosol, which is also the starting point and main motivation of this study. Eucalyptus grandis × E. urophylla, Pogostemon cablin (Blanco) Benth., Cymbopogon citratus (DC.) Stapf, Rosmarinus officinalis L., Plumeria rubra L., and Baeckea frutescens L. are commonly used medicinal plants in Guangxi with a long history of folk application. However, research on the biological activities of these species remains insufficient. Therefore, this paper investigates the hydrosols of these six plants to address the research gap and promote the sustainable utilization of Guangxi’s characteristic medicinal resources.

Eucalyptus grandis × E. urophylla is originally native to Australia. It is an artificially pollinated hybrid variety belonging to the Myrtaceae family and Eucalyptus genus. This hybrid shows significant heterosis, with fast growth and relatively strong stress resistance. It has been successfully introduced to southern China and other subtropical regions, where it has become the dominant species in Chinese eucalyptus plantations.^11,12 Eucalyptus grandis × E. urophylla has wide applications. Its trunk is commonly used for papermaking, production of composite boards, medicine and other fields. Furthermore, it is also an important landscaping plant in China, mainly used for urban, mountain, and roadside greening.^13,14 Research has found that the essential oil from Eucalyptus grandis × E. urophylla leaves and its components possess excellent biological activities, including antibacterial, herbicidal, insecticidal, and acaricidal effects.¹⁵

Pogostemon cablin (Blanco) Benth. is mainly distributed in subtropical regions. It is a famous medicinal material in China and occupies an important position among aromatic plants, because its uses are relatively wide and its fragrance is very unique.^16,17 This plant can be used as a traditional Chinese medicine to treat gastric discomfort, headaches, vomiting, diarrhea, and nasal congestion. In addition, it also possesses multiple beneficial biological activities, including antioxidant, anti-inflammatory, antiviral, and antibacterial effects.^18,19 Additionally, it holds significant value in the cosmetics and perfume industries, where it is commonly used to produce personal care products such as body lotions, creams, perfumes, soaps, and other similar items.²⁰

Lemon grass (Cymbopogon citratus (DC.) Stapf), a perennial plant of the Poaceae family, possesses a lemon-like fragrance and is widely distributed in tropical regions including India, Indonesia, and Malaysia. Because it contains compounds including alkaloids, flavonoids, saponins, reducing sugars, steroids, quinones, tannins, and carbopol, it possesses biological activities such as antibacterial and antioxidant effects, and can be used to treat cough, pneumonia, influenza, and headaches. Additionally, it may also be used to treat HIV side effects, including secondary bacterial infections.^21-23 The essential oil exhibits bactericidal, insecticidal, and anticancer properties, making it can be applied in industries including food industry, pharmaceutical formulations, perfumery, and cosmetic products.^24,25

Rosmarinus officinalis L. is a perennial medicinal plant, belonging to the Lamiaceae family. It possesses antibacterial, antioxidant, antidepressant and anticholinesterase activities, and can also be used to prevent or treat dementia.^26,27 Studies have shown that its extracts and essential oils possess antioxidant, anticancer, anti-inflammatory and antibacterial activities, and are therefore widely used in food, pharmaceutical and pesticide industries.^28,29

Plumeria rubra L.(Apocynaceae family), originally from tropical America, is widely planted in China’s Fujian, Guangxi, Guangdong, Hainan and other regions. Studies have demonstrated that the essential oil of P. rubra contains chemical constituents such as iridoids, triterpenoids, and flavonoids.^30,31 Medicinally, it can be employed in the treatment of leprosy, inflammatory disorders, diabetes mellitus, ulcers, acne, asthma, and infertility.^32,33

Baeckea frutescens L. (Myrtaceae) is a medicinal plant species native to the Malay Peninsula and Sumatra. It is widely distributed throughout southern China and Southeast Asia. Studies have revealed that this species exhibits diverse biological activities, including antimicrobial, anti-inflammatory, antioxidant, and insecticidal properties.^34,35 The leaves of B. frutescens can be processed to yield essential oil and hydrosol, both demonstrating significant antimicrobial efficacy. The essential oil exhibits multiple pharmacological properties, including anti-inflammatory, insecticidal, antipruritic, febrifugic, and analgesic effects, along with the ability to eliminate dampness.³⁶ B. frutescens is also utilized in aromatherapy and therapeutic massage for managing various conditions, including headache, rheumatism, menstrual disorders, and dyspepsia.³⁷

This study aimed to extract hydrosols from six aromatic plants (Eucalyptus grandis, Pogostemon cablin, Cymbopogon citratus, Rosmarinus officinalis, Plumeria rubra, and Baeckea frutescens), analyze their chemical constituents, determine total flavonoids/phenolics and antioxidant activity, and investigate their correlations.

Materials and Methods

Chemicals and Drugs

Rutin standard (Sigma-Aldrich, USA). Sodium nitrite (NaNO₂, analytical grade); Aluminum nitrate (Al(NO₃)₃, analytical grade); Sodium hydroxide (NaOH, analytical grade); Gallic acid (Sigma-Aldrich, USA); Anhydrous sodium carbonate (Na₂CO₃, analytical grade); Folin-Ciocalteu reagent (Sigma-Aldrich, USA); DPPH (Sigma-Aldrich, USA); ABTS⁺ (Adamas, USA); Ethanol (Yueqiao Reagent Plastics Co., Ltd., Taishan, China).

Plants and Hydrosols Preparation

The leaves of Eucalyptus grandis × E. urophylla utilized in this research were collected in Yongfu County, Guilin, Guangxi, China, in March 2022. The plant material was authenticated by Senior Engineer Yuhua Deng from Huangmian Forestry Farm. The aerial parts of Pogostemon cablin (Blanco) Benth. were purchased from Guilin Dingkang Traditional Chinese Medicine Decoction Co., Ltd., Guangxi, China, in October 2021. The plant material was identified by Guilin Dingkang Chinese Herbal Medicine Co., Ltd. and confirmed to comply with the standards for Pogostemon cablin (Blanco) Benth in the Chinese Pharmacopoeia 2015 Edition, Volume I of the Chinese Pharmacopoeia. Cymbopogon citratus (DC.) Stapf was purchased from Guangxi Dongxing Liangjiayou Agriculture and Forestry Technology Co., Ltd., in July 2018, and was identified by Associate Professor Lu Yuting from Guangxi University of Science and Technology. Rosmarinus officinalis L., Plumeria rubra L., and Baeckea frutescens L. were purchased from Yulin Shishuyuansheng Chinese Herbal Medicine Purchasing and Sales Department, Yuzhou District, Guangxi, China, in November 2020, October 2023, and June 2018 respectively, and were identified by Associate Professor Lu Yuting from Guangxi University of Science and Technology. All plant material samples used in this study are deposited at the Laboratory of the College of Biochemical Engineering, Guangxi University of Science and Technology, China.

Exactly 100 g of dried plant material was weighed into a 3000 mL round-bottom flask, mixed with 2000 mL distilled water, and subjected to hydrodistillation for 6 h using a steam distillation apparatus. After cooling and phase separation, the hydrosol fraction was collected from the lower layer of the extraction tube.^38,39

GC - MS Analysis

GC - MS analysis was conducted to identify the chemical composition of six botanical hydrosols. GC - MS analysis was performed using an HP-5 capillary column (30 m×0.25 mm×0.25 μm) for the determination of volatile components in six plant hydrosols. The temperature program was set as follows:

The volatile constituents of Eucalyptus grandis × E. urophylla,Cymbopogon citratus (DC.) Stapf, Rosmarinus officinalis L. and Baeckea frutescens L. hydrosol were analyzed via direct injection of the neat sample (split ratio 1:15). The temperature program was set as follows: 50°C held for 2 min, increased to 120°C at 4°C/min and maintained for 3 min, then raised to 280°C at 8°C/min and held for 5 min.

The volatile constituents of Pogostemon cablin (Blanco) Benth. hydrosol were analyzed through direct injection of the neat sample (split ratio 1:5). The temperature program was set as follows: 80°C held for 3 min, then increased to 250°C at 5°C/min.

The volatile compounds of Plumeria rubra L. hydrosol were analyzed by direct injection of the extracted aromatic fraction in splitless mode. The temperature program was set as follows: 60°C held for 5 min, then increased to 260°C at 2°C/min, and maintained at 260°C for 5 min, with a solvent delay of 7 min.

The mass spectra of each peak were searched against the NIST17 standard spectral library using computer software. Only compounds with both SI (Similarity Index) and RSI (Reverse Similarity Index) values greater than 900 when compared with the NIST17 library were discussed. The chemical components were determined by referring to standard spectra and mass spectral fragmentation patterns. The relative percentage content of each component was calculated using the peak area normalization method.

MS conditions: EI ion source, electron energy 70 eV, electron multiplier voltage 1.5 kV, mass scan range 50–550 amu, full scan mode.^40,41

Antioxidant Activity

Total Flavonoid Content

The total flavonoid content of the hydrosols was determined using the aluminum nitrate-sodium nitrite method. An accurately measured 2.0 mL aliquot of the diluted sample extract was transferred into a 10 mL stoppered test tube, then 3.0 mL of 60% ethanol solution, 0.3 mL of 10% sodium nitrite, 0.3 mL of 10% aluminum nitrate, and finally 4.0 mL of 4% sodium hydroxide were added, diluted to 10 mL with 60% ethanol solution, mixed evenly, and allowed to stand for 12 minutes.⁴² The absorbance was measured at a wavelength of 510 nm, and a standard curve (0–40 μg/mL) was plotted using rutin as the standard. The total flavonoid content was then determined by correlating the measured absorbance with the established standard curve. The experiment was repeated three times, and the average value was taken.

Total Phenolic Content

The total polyphenol concentration was measured using the Folin-Ciocalteu colorimetric method. Specifically, 1.00 mL of the appropriately diluted sample extract was transferred to a 10 mL stoppered test tube. Then, 1 mL of Folin-Ciocalteu reagent and 2 mL of 12% (w/v) sodium carbonate solution were added sequentially. The mixture was brought to a final volume of 10 mL with distilled water, thoroughly mixed, and allowed to react for 1 hour at room temperature under light-protected conditions. Absorbance measurements were performed at 765 nm using a reagent blank (prepared with extraction solvent in place of sample extract) as reference. The total polyphenol content was quantified based on the established standard curve.^43,44 The experiment was repeated three times, and the average value was taken.

Determination of DPPH Radical Scavenging Activity

The DPPH radical working solution (0.02 mg/mL) was prepared using anhydrous ethanol as solvent and stored under light-protected conditions. In the test system, equal volumes (2 mL each) of DPPH radical solution and essential oil samples at varying concentrations were combined and mixed. The reaction kinetics were monitored by recording absorbance at 517 nm wavelength at 10-minute intervals over a 180-minute duration, with all determinations performed in triplicate. The control absorbance (A_c) was measured by replacing the essential oil sample with anhydrous ethanol, while the blank absorbance (A_j) was determined using anhydrous ethanol instead of the DPPH radical solution. Both measurements were performed in triplicate and averaged.⁴⁵ The DPPH radical scavenging activity was calculated as follows:

S % = [1 - (A_{i} - A_{j}) / A_{c}] \times 100 %

(1)

Determination of ABTS+ Radical Scavenging Activity

A 7 mmol/L ABTS+ solution was mixed with a 2.45 mmol/L potassium persulfate solution in equal volumes. The mixture was then stored at room temperature under light-protected conditions for 24 h before use. The ABTS⁺ radical working solution was prepared by adjusting its concentration with absolute ethanol to achieve an absorbance of 0.70 (±0.002) at 734 nm. For ABTS⁺ radical scavenging activity assessment, 2 mL of each essential oil sample at varying concentrations was mixed with 2 mL of ABTS⁺ radical working solution. After incubation in the dark for 6 min, the absorbance (A_i) was measured at 734 nm. The control absorbance (A₀) was determined by replacing the essential oil with absolute ethanol.⁴⁶ All measurements were performed in triplicate, and the ABTS⁺ radical scavenging rate was calculated using the following equation:

S % = [(A_{0} - A_{i}) / A_{0}] \times 100 %

(2)

Statistical Analysis

The results were expressed as mean values with their corresponding standard deviations. All analyses were performed in triplicate, with each analysis considering the mean value.

Results

Analysis of Volatile Components in Hydrosol

GC - MS analysis revealed 24, 12, 17, 13, 18, and 17 volatile compounds in the hydrosols of Eucalyptus grandis, Pogostemon cablin, Cymbopogon citratus, Rosmarinus officinalis, Plumeria rubra, and Baeckea frutescens, respectively. Table 1 demonstrates that the major volatile components in Eucalyptus grandis hydrosol were eucalyptol (39.03%), α-terpineol (17.58%), endo-borneol (10.02%), and trans-pinocarveol (9.14%); the main volatile components identified in Pogostemon cablin hydrosol were patchouli alcohol (29.10%), furfural (3.20%), norpatchoulenol (3.14%), benzeneacetaldehyde (3.13%), and pogostone (3.06%); the main volatile components of Cymbopogon citratus hydrosol were geranial (26.22%), neral (14.66%), 2-methyl-3-buten-2-ol (11.42%), cis-linalool oxide (furanoid) (7.12%), trans-linalool oxide (furanoid) (6.14%), and geraniol (5.81%); the main volatile components of Rosmarinus officinalis hydrosol were verbenone (60.82%), D-(+)-camphor (14.50%), 1,8-cineole (7.79%), and endo-borneol (5.80%). ; the volatile components of Plumeria rubra hydrosol were predominantly composed of eugenol (36.62%), followed by linalool (11.16%), cis-linalool oxide (8.61%), geraniol (8.08%), and α-terpineol (7.04%); the volatile profile of Baeckea frutescens hydrosol was characterized by the following major components: terpinen-4-ol (18.95%), α-terpineol (16.45%), 1,8-cineole (16.08%), linalool (12.69%), cis-linalool oxide (7.20%), endo-borneol (5.45%), and trans-linalool oxide (furanoid) (5.34%).

Table 1.

Volatile Composition Analysis of Hydrosols From Six Aromatic Plants

Chemical compound	CAS	RI_ref	RI_exp	Relative percentage content/%
Chemical compound	CAS	RI_ref	RI_exp	E. grandis×E. urophylla hydrosol	P. cablin hydrosol	C. citratus hydrosol	R. officinalis hydrosol	P. rubra hydrosol	B. frutescens hydrosol
2-methyl-3-buten-2-ol	115-18-4	614^a	546	0.16	-	11.42	0.47	-	-
1-pentanol	71-41-0	765^a	n^c	-	2.58	-	-	-	-
3-methyl-butanal	590-86-3	652^a	635	0.76	-	-	-	-	-
1-penten-3-ol	616-25-1	684^a	700	0.25	-	-	-	-	-
3-methyl-1-butanol	123-51-3	736^a	742	0.17	-	-	-	-	-
(Z)-2-penten-1-ol	1576-95-0	767^a	783	0.53	-	-	-	-	-
Prenol	556-82-1	775^a	790	-	-	1.10	-	-	-
3-methyl-2-butenal	107-86-8	782^a	800	0.24	-	0.70	-	-	-
Benzaldehyde	100-52-7	962^a	957	-	1.30	-	-	2.78	0.44
Furfural	98-01-1	833^a	839	1.88	3.20	-	0.46	-	0.63
2-hexenal	505-57-7	851^a	859	0.61	-	-	-	-	-
3-hexen-1-ol	544-12-7	856^a	864	-	-	-	0.33	-	-
6-methyl-5-heptene-2-one	110-93-0	986^a	1021	-	-	0.79	-	-	-
Dihydrocarvone	7764-50-3	1192	1217	-	0.25	-	-	-	-
p-cymene	99-87-6	1025^a	1024	-	-	-	-	-	0.24
Verbenone	80-57-9	1205^a	1224	-	0.64	-	-	-	-
Eucalyptol	470-82-6	1032^a	1031	39.03	-	-	7.79	-	16.08
Benzeneacetaldehyde	122-78-1	1045^a	1041	0.48	3.13	0.60	-	-	-
Benzyl alcohol	100-51-6	1036^a	1028	-	-	-	-	0.67	-
cis-Linalool oxide	5989-33-3	1074^a	1075	2.46	0.68	7.12	-	8.61	7.20
trans-Linalool oxide	34995-77-2	1086^a	1088	1.96	-	6.14	-	5.23	5.34
Fenchol	2217-02-9	n^b	1122	1.68	-	-	-	-	1.92
(E)-p-menth-2-en-1-ol	29803-81-4	1140^a	1129	-	-	-	0.20	-	-
Chrysanthenone	473-06-3	1123^a	1132	-	-	-	0.36	-	-
α-campholenal	4501-58-0	1125^a	1134	0.70	-	-	-	-	-
.(+)-Nopinone	38651-65-9	1152^a	1137	-	-	-	-	-	0.77
trans-pinocarveol	547-61-5	1139^a	1147	9.14	-	-	-	-	-
Camphene hydrate	465-31-6	1148^a	1149	-	-	-	-	-	0.29
D-(+)-camphor	464-49-3	1143^a	1151	-	-	-	14.50	-	-
linalool	78-70-6	1099^a	1100	-	-	2.66	0.19	11.16	12.69
Pinocarvone	30460-92-5	1164^a	1171	1.87	-	-	-	-	-
(3R,6S)-2,2,6-Trimethyl-6-vinyltetrahydro-2H-pyran-3-ol	39028-58-5	1173^a	1175	-	-	-	-	-	0.42
Endo-borneol	507-70-0	1167^a	1166	10.02	-	-	5.86	-	5.45
3,7-dimethyl-1,5,7-octatriene-3-ol	29957-43-5	1107^a	1094	-	-	-	-	0.99	-
p-Cymen-8-ol	1197-01-9	1183^a	1185	-	-	-	-	-	2.30
Terpinen-4-ol	562-74-3	1177^a	1178	0.59	-	-	1.59	-	18.95
trans-p-mentha-2,8-dienol	7212-40-0	1123^a	1208	-	-	0.58	-	-	-
trans-p-mentha-1(7),8-dien-2-ol	21391-84-4	n^b	1197	2.32	-	-	-	-	-
α-terpineol	98-55-5	1189^a	1191	17.58	0.72	0.78	4.56	7.04	16.45
(-)-myrtenol	19894-97-4	1213^a	1210	1.01	1.24	-	-	-	-
cis-p-mentha-2,8-dien-1-ol	3886-78-0	1122^a	1209	-	-	0.88	-	-	-
levoverbenone	1196-01-6	1204^a	1218	-	-	-	60.82	-	-
trans-carveol	1197-07-5	1217^a	1229	1.95	-	-	-	-	-
cis-p-mentha-1(7),8-dien-2-ol	22626-43-3	n^b	1237	2.73	-	-	-	-	-
2-(5-methyl-5-vinyltetrahydro-2-furanyl)propanal	53447-46-4	1145^a	1136	-	-	-	-	0.27	-
Norpatchoulenol	41429-52-1	1553	1575	-	3.14	-	-	-	-
α-phellandren-8-ol	1686-20-0	1167^a	1154	-	-	-	-	0.22	-
Bornyl acetate	76-49-3	1285^a	1293	-	-	-	0.16	-	-
1,4-dimethyl-7-(prop-1-en-2-yl)decahydroazulen-4-ol pogostole	21698-41-9	1655^a	1672	-	3.06	-	-	-	-
Neral	106-26-3	1240^a	1228	-	-	14.66	-	0.75	-
patchouli alcohol	5986-55-0	1660^a	1679	-	29.10	-	-	-	-
exo-2-hydroxycineole acetate	57709-95-2	1344^a	1352	0.68	-	-	-	-	-
Geraniol	106-24-1	1255^a	1247	-	-	5.81	-	8.08	-
Geranial	141-27-5	1270^a	1258	-	-	26.22	-	1.26	-
Nerol	106-25-2	1228^a	1217	-	-	-	-	1.47	-
3,7-dimethyl-2-octen-1-ol	40607-48-5	1463^a	1219	-	-	-	-	1.34	-
Eugenol	97-53-0	1357^a	1360	-	-	3.87	-	36.62	0.84
Humulenol-II	19888-00-7	1632	1642	-	-	-	-	-	0.37
Geranic acid	4698-08-2	1355^a	1370	-	-	-	-	1.44	-
Methyleugenol	93-15-2	1402^a	1393	-	-	-	-	0.98	-
(E)-Nerolidol	40716-66-3	1564^a	1549	-	-	-	-	0.31	-

Note: “-” indicate not detected; RI_ref: Literature retention indices; RI_exp: Experimental retention indices relative to C₇–C₄₀ n-alkanes

^aData from the NIST Chemistry WebBook Retention indices

[n]^bnot found

[n]^cnot detected

The volatile components of the six aromatic plant hydrosols were primarily terpenoids, with the following relative contents: Eucalyptus grandis (90.09%), Pogostemon cablin (38.83%), Cymbopogon citratus (64.85%), Rosmarinus officinalis (96.03%), Plumeria rubra (23.21%), and Baeckea frutescens (70.09%). As shown in Table 2, Plumeria rubra hydrosol contained the highest phenolic content (36.62%), followed by Cymbopogon citratus (3.87%) and Baeckea frutescens (0.84%) hydrosols. However, no phenolic compounds were detected in the other aromatic plant hydrosols analyzed, flavonoid compounds were not detected in any of the six tested hydrosol samples. Therefore, the total flavonoid content and total phenolic content of hydrosols derived from six aromatic plant species cultivated were determined by sodium nitrite-aluminum nitrate colorimetric method and Folin - Ciocalteu method, respectively.

Table 2.

Volatile Composition Classification and Relative Percentage Content of Hydrosols From Six Aromatic Plants

Volatile component classification	Relative percentage content(%)
Volatile component classification	E. grandis×E. urophylla hydrosol	P. cablin hydrosol	C. citratus hydrosol	R. officinalis hydrosol	P. rubra hydrosol	B. frutescens hydrosol
Phenolic Compounds	-	-	3.87	-	36.62	0.84
Aldehyde Compounds	4.67	7.63	42.18	0.46	5.06	1.07
Alcohol Compounds	40.58	37.46	36.49	7.34	45.12	81.72
Ester Compounds	0.68	-	-	0.16	-	-
Acid Compounds	-	3.95	-	-	1.44	-
Ketone Compounds	1.87	3.95	0.79	61.18	-	0.77
Epoxide Compounds	43.45	-	13.26	7.79	14.11	12.96
Heterocyclic Compounds	1.88	3.2	-	0.46	-	1.65
Hydrocarbon Compounds	-	-	-	-	-	0.24
Monoterpene Hydrocarbons	-	-	-	-	-	0.24
Oxygenated Monoterpenes	90.09	2.89	64.85	96.03	21.20	69.48
Sesquiterpenes	-	-	-	-	-	-
Oxygenated Sesquiterpenes	-	35.94	-	-	2.01	0.37

Note. “-” indicate not detected.

Antioxidant Activity

Total Flavonoid and Total Phenolic Content

Using rutin as the standard, the total flavonoid content in the six aromatic plant hydrosols was determined by the sodium nitrite-aluminum nitrate colorimetric method. Similarly, using gallic acid as the standard, the total phenolic content was measured by the Folin-Ciocalteu method. The total flavonoid content of the six aromatic plant hydrosols ranged from 4.97 to 74.88 μg/mL (Table 3). Among these, Plumeria rubra hydrosol exhibited the highest flavonoid concentration. The ranking of flavonoid content was as follows: Eucalyptus grandis hydrosol < Pogostemon cablin hydrosol < Rosmarinus officinalis hydrosol < Baeckea frutescens hydrosol < Cymbopogon citratus hydrosol < Plumeria rubra hydrosol; The total phenolic content of the hydrosols ranged from 10.86 to 117.80 μg/mL (Table 3), with Plumeria rubra hydrosol showing the highest concentration. The phenolic content ranking was as follows: Pogostemon cablin hydrosol < Eucalyptus grandis hydrosol < Rosmarinus officinalis hydrosol< Baeckea frutescens hydrosol < Cymbopogon citratus hydrosol< Plumeria rubra hydrosol.

Table 3.

Total Flavonoid and Phenolic Contents in Six Aromatic Plant Hydrosols

Hydrosol	Total flavonoid contents(μg/mL)	Total phenolic contents(μg/mL)	Combined TFC and TPC (μg/mL)
E. grandis×E. urophylla hydrosol	4.97±1.01	16.45±0.93	21.42
P. cablin hydrosol	5.18±0.37	10.86±0.36	16.04
C. citratus hydrosol	47.40±0.16	49.08±0.40	96.48
R. officinalis hydrosol	17.42±0.09	16.74±0.81	34.16
P. rubra hydrosol	74.88±3.28	117.80±2.03	192.68
B. frutescens hydrosol	39.81±4.41	42.05±1.10	81.86

DPPH and ABTS+ Radical Scavenging Activitiy

The IC₅₀ values for antioxidant capacity ranged from 53.65 to 627.75 mg/mL across the six aromatic plant hydrosols (Table 3). Notably, Plumeria rubra hydrosol demonstrated the lowest IC₅₀ value (53.65 mg/mL), indicating its superior DPPH radical scavenging capacity among the tested samples. The six plant hydrosols demonstrated the following descending order of DPPH radical scavenging capacity: Plumeria rubra hydrosol > Baeckea frutescens hydrosol > Cymbopogon citratus hydrosol > E. grandis×E. urophylla hydrosol > Rosmarinus officinalis hydrosol > Pogostemon cablin hydrosol.

The IC₅₀ values of the six aromatic plant hydrosols against ABTS⁺ ranged from 8.45 to 223.72 mg/mL (Table 4). Among them, Plumeria rubra hydrosol demonstrated the strongest ABTS⁺ scavenging ability, with an IC₅₀ of 8.45 mg/mL, whereas Pogostemon cablin hydrosol showed the weakest activity, yielding an IC₅₀ of 223.72 mg/mL (Table 4). Similar to the total phenolic content, the six aromatic plant hydrosols exhibited the following descending order of ABTS⁺ radical scavenging activity: Plumeria rubra hydrosol > Cymbopogon citratus hydrosol > Baeckea frutescens hydrosol > E. grandis×E. urophylla hydrosol > Rosmarinus officinalis hydrosol > Pogostemon cablin hydrosol.

Table 4.

Antioxidant Capacity (DPPH, ABTS) of Six Aromatic Plant Hydrosols

Hydrosol	IC₅₀ values(mg/mL)
Hydrosol	DPPH·	ABTS⁺·
E. grandis×E. urophylla hydrosol	374.27	157.86
P. cablin hydrosol	627.75	223.72
C. citratus hydrosol	146.90	28.51
R. officinalis hydrosol	567.87	161.94
P. rubra hydrosol	53.65	8.45
B. frutescens hydrosol	131.95	53.60

The results demonstrated that all tested hydrosols exhibited strong free radical scavenging capacities against both DPPH and ABTS⁺ radicals. Notably, Plumeria rubra hydrosol exhibited the highest total flavonoid content, total phenolic content, and showed the most potent antioxidant activity. Based on these findings, this study further investigated the correlation between total flavonoid content, total phenolic content and the antioxidant activities of the six aromatic plant hydrosols.

To determine the relationship between the total phenolic content, total flavonoid content, and the sum of total phenolic and total flavonoid contents in the hydrosols and their IC₅₀ values for antioxidant activity, we created a relationship diagram for them (Figure 1). It can be intuitively seen from the figure that there is a negative correlation between the two, meaning that the higher the total flavonoid and total phenolic contents in the hydrosols, the stronger their antioxidant activity.

Figure 1.

Relationship between total flavonoid, total phenolic contents and antioxidant IC₅₀ values in six aromatic plant hydrosols. (a) Relationship between total flavonoid content and DPPH IC₅₀ values; (b) Relationship between total phenolic content and DPPH IC₅₀ values; (c) Correlation between the combined content of total flavonoids and phenolics with DPPH IC₅₀ values. (d) Relationship between total flavonoid content and ABTS⁺ IC₅₀ values; (e) Relationship between total phenolic content and ABTS⁺ IC₅₀ values; (f) Correlation between the combined content of total flavonoids and phenolics with ABTS⁺ IC₅₀ values

Discussion

The volatile composition of hydrosols is influenced by multiple factors including plant species, harvesting time, extraction processes, and analytical methodologies.^47,48 The observed differences in either the types or concentrations of volatile components between the present study and previous research may be attributed to these variable factors.

Previous studies on the water-soluble volatile constituents of Eucalyptus grandis× E. urophylla have reported different major components, including isovaleric acid (15.03%), eucalyptol (10.75%), α-terpineol (9.85%), 2-methylbutyric acid (7.93%), benzyl alcohol (7.08%), 1,3,3-trimethyl-2-oxabicyclo[2.2.2]octan-6-ol (5.90%), and borneol(5.26%),⁴⁹ the current findings are generally consistent with previous results, but showing some differences in component concentrations, this may be because they extracted, dried, and concentrated the distilled hydrosol before determining the volatile components, which is different from the extraction method used in this paper. The volatile components of Eucalyptus grandis × Eucalyptus urophylla essential oil are somewhat different from those of its hydrosol. Zhou et al⁵⁰ analyzed the essential oil components of Eucalyptus grandis × Eucalyptus urophylla, and the results showed that the main volatile components of the essential oil were α-pinene (17.02%), α-terpineol (13.63%), aromadendrene (11.08%), d-limonene (8.47%), and endo-borneol (7.77%). The essential oil is mainly composed of monoterpene hydrocarbons (67.34%), while the hydrosol is mainly composed of oxygenated monoterpenes (90.09%). The highest content volatile component in both patchouli essential oil and hydrosol is patchouli alcohol, and the other components are different.^51,52 Yu et al⁴⁷ reported different major components in Cymbopogon citratus hydrosol: (E)-3,7-dimethylocta-2,6-dienal(33.0%), (Z)-3,7-dimethylocta-2,6-dienal (19.3%), geraniol (9.3%), and linalool (6.8%), the results show partial similarity in composition but differ in specific components and their relative abundances. The main volatile components of Cymbopogon citratus essential oil are similar to those of its hydrosol, but the contents are different.^53-55 Politi et al.⁵⁶ analyzed the volatile composition of rosemary hydrosol obtained through hydrodistillation, reporting the main volatile components: 1,8-cineole (47.10%), D-(+)-camphor (5.40%), borneol (3.70%), verbenone (2.80%), and linalool (2.00%), while both studies identified similar major components, significant differences were observed in their relative concentrations. Taglienti et al ‘s research results on the volatile components of rosemary essential oil showed that its main component was α-pinene,⁵⁷ which is quite different from the volatile component content of its hydrosol. Quoc Toan et al⁵⁸ ‘s research on the volatile components of Baeckea frutescens oil showed that its main components were tasmanone (21.46%), β-pinene (15.64%), 1,8-cineole (11.32%), α-thujene (8.74%), α-pinene (7.18%), linalool (7.44%), terpinen-4-ol (5.11%), and α-terpineol (4.46%). Similar to the above results, the volatile components of Baeckea frutescens oil hydrosol and essential oil are similar, but there are some differences in content. To our knowledge, no previous studies have reported on the volatile compositions of Pogostemon cablin, Plumeria rubra or Baeckea frutescens hydrosols.

From the classification and relative percentage content of volatile components in the six aromatic plant hydrosols, it can be seen that the content of oxygenated terpenes in the hydrosols is the highest. This may be because oxygen-containing terpenoids have higher water solubility compared to hydrocarbon terpenoids.^59,60 Terpenoids are the most abundant class of compounds among natural products, functioning as plant secondary metabolites with broad-spectrum biological activities, including antioxidant, antimicrobial, anti-inflammatory, and anticancer properties, and are widely applied in pharmaceutical, food, and cosmetic industries.^61-63 These hydrosols contain high content of terpenoid compounds, so they have high utilization value.

From the six aromatic plant hydrosols total phenolics and total flavonoids content results, the rankings of the two substances are not parallel. For example, in the total flavonoids content, Eucalyptus grandis hydrosol has the lowest content, while in the total phenolics content, Pogostemon cablin hydrosol has the lowest content. This may be because flavonoids and phenolic compounds have different biosynthetic pathways in different species.⁶⁴ This situation has also been observed before, similar to this paper, the rankings of total phenolics and total flavonoids content were not consistent in different species.^65,66

Except for the fact that the antioxidant activity of Plumeria rubra essential oil has not been reported, compared with the other five plant essential oils, the hydrosols all exhibited mild but statistically significant antioxidant activity.^4,50,67,68 This can be attributed to the abundance of oxygen-containing derivatives in hydrosols,⁶⁹ which have stronger polarity and water solubility compared to essential oils, so hydrosols have a unique advantage in enriching hydrophilic antioxidants. In the food and cosmetic fields, hydrosols can serve as a natural source of low-irritation,⁷⁰ hydrophilic antioxidants, complementing the lipophilic antioxidants of essential oils.

In addition, the ranking of antioxidant activity is different between essential oils and hydrosols. For example, the IC₅₀ value of Pogostemon cablin essential oil for DPPH· scavenging activity is lower than that of Cymbopogon citratus essential oil, while the opposite is observed in hydrosols. This suggests that Cymbopogon citratus hydrosol may contain certain highly active hydrophilic phenolic compounds, and the specific mechanism of action requires further investigation.

The experimental results on the correlation between the total flavonoid and total phenolic contents in the hydrosols and their antioxidant activity indicate that there is a positive correlation between the two. The results of this study are consistent with the experimental results of (Ahmed et al),⁷¹ whose results stated that the antioxidant activity of basil (Ocimum basilicum L.) extract showed a high correlation with the contents of total flavonoids and total phenolics. The higher the contents of total flavonoids and total phenolics, the stronger the antioxidant activity. Asem et al⁷² examined the relationship between the total phenolic and total flavonoid contents of Malaysian stingless bee propolis extracts and their antioxidant activities. Pearson correlation coefficient analysis was conducted to examine the relationship between the antioxidant activity of propolis extracts and their total flavonoid and total phenolic contents. The results showed that both total flavonoid content and total phenolic content were positively correlated with antioxidant activity in the propolis extracts. Notably, the ABTS⁺ radical scavenging assay revealed a strong association with the total phenolic and total flavonoid contents, with R² values of 0.763 and 0.773, respectively. Furthermore, studies on the correlation between total flavonoid, total phenolic contents and antioxidant activity in plant extracts have also confirmed these results.^73,74

Overall, these consistent findings across different studies further confirm that total flavonoid and total phenolic contents can serve as indicators of the antioxidant potential of hydrosols, which is consistent with the general principles of the field of phytochemistry.

This study fills a research gap regarding hydrosols that have been overlooked as byproducts of essential oil distillation, and provides a theoretical basis for hydrosols to be recognized as natural antioxidants with significant development potential and for their application in the food, cosmetic, and pharmaceutical industries. However, this study also has limitations, as it only used GC-MS to analyze the volatile components of the six plant hydrosols, HPLC-MS was not used to qualitatively and quantitatively analyze non-volatile components such as total flavonoids and total phenolic compounds;only the DPPH and ABTS⁺ radical scavenging methods were used to determine the in vitro antioxidant activity of the six plant hydrosols, which cannot reflect in vivo absorption, metabolism, and actual antioxidant effects. Therefore, the mechanism of action of the antioxidant activity needs to be further studied.

Conclusions

This study determined the volatile components, total flavonoid content, total phenolic content, and antioxidant activity of hydrosols derived from six aromatic plants cultivated in Guangxi. Using GC - MS analysis, we identified 24, 12, 17, 13, 18, and 17 chemical constituents in Eucalyptus grandis × E. urophylla, Pogostemon cablin, Cymbopogon citratus, Rosmarinus officinalis, Plumeria rubra, and Baeckea frutescens hydrosols, respectively, among all hydrosols, terpenoids were the most abundant compounds. Among the three hydrosols in which phenolic compounds were detected, the Plumeria rubra hydrosol had the highest phenolic compound content. The total flavonoid and total phenolic content measurements showed that: the Plumeria rubra hydrosol had the highest total flavonoid and total phenolic contents, the Eucalyptus grandis × E. urophylla hydrosol had the lowest total flavonoid content, and the Pogostemon cablin hydrosol had the lowest total phenolic content. Among the antioxidant activities of the six aromatic plant hydrosols, the Plumeria rubra hydrosol showed the highest scavenging rates against DPPH· and ABTS⁺·, with IC₅₀ values of 53.65 mg/mL and 8.45 mg/mL, respectively. In contrast, Pogostemon cablin hydrosol displayed the weakest antioxidant activity, with IC₅₀ values of 627.75 mg/mL (DPPH) and 223.72 mg/mL (ABTS⁺). The correlation analysis revealed a negative relationship between the total flavonoid, total phenolic contents of the hydrosols and their antioxidant IC₅₀ values, indicating that higher total flavonoid and phenolic contents were associated with stronger antioxidant activity. In the future, the interaction mechanisms among the compounds responsible for the antioxidant activity of hydrosols can be studied and elucidated, and hydrosol products with enhanced effects can be developed.

Footnotes

Acknowledgement

The authors are indebted to researchers from Guilin Dingkang Traditional Chinese Medicine Decoction Co., Ltd., Yulin Institute for Food and Drug Control, Guangxi Zhuang Autonomous Region, as well as Associate Professor Yuting Lu from the Faculty of Medical Science, Guangxi University of Science and Technology, and Senior Engineer Yuhua Deng for the identification of the plant.

ORCID iD

Mengdan Li

Author Contributions

Y.T., M.L., Q.Z., F.C. and N.L. conceived and designed the project; M.L., F.H. and N.L. conduced the experiment; Y.T. and M.L. analyzed the data;Y.T., M.L. wrote and edited this paper. All authors have read and agreed to the published version of the manuscript.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was funded by National Natural Science Foundation of China (22268012) and Guangxi Natural Science Foundation Project (2021GXNSFAA220067).

Declaration of Conflicting Interests

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

Data Availability Statement

Data is contained within the article.*

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