Introduction

Dalbergia odorifera T. Chen (Family: Fabaceae) is a valuable tree indigenous to Hainan Province and has been gradually cultivated in South China (Sun et al. 2015; Zhao et al. 2020a). In view of its distinctive color, aromatic trunk, and high density, this valuable rosewood tree is particularly popular for manufacturing luxurious furniture, artifacts, and musical instruments (Tao and Wang 2010; Wariss et al. 2018). The heartwood of D. odorifera as an important traditional Chinese medicine in China has been used to treat cardio- and cerebrovascular diseases for a long time (Li et al. 2019; Zhao et al. 2020b). Due to its precious heartwood and medicinal and economic value, D. odorifera has been listed on the IUCN red list since 1998 because of the long-term overexploitation (Wariss et al. 2018; Liu et al. 2019).

As an increasingly popular traditional Chinese medicine, D. odorifera species have attracted much attention from phytochemists. Substantial research has been conducted on this species in recent years, and those studies mainly concerned with its chemical constituents and pharmacological activities of the heartwood (The SN 2017). Some previous studies have focused on the biological activities of the heartwood, such as its antimicrobial (Zhao et al. 2011; Wang et al. 2014), anti-inflammatory (Lee et al. 2014; Choi et al. 2017; Kim et al. 2018), antioxidant (Ma et al. 2013; Sun et al. 2015), anti-platelet aggregation (Tao and Wang 2010), angiogenic (Fan et al. 2017), antitumor (Park et al. 2016; Bastola et al. 2017; Meng et al. 2019) and vasodilatory (Sugiyama et al. 2002; Yang et al. 2013) effects. To date, 175 chemical constituents have been isolated from D. odorifera (Meng et al. 2019; Lu et al. 2019). Among them, flavonoids and volatile oils are the major secondary metabolites of D. odorifera (Zhao et al. 2000). Most studies on the chemical composition of this species have analyzed the heartwood and leaves and reported the isolation of flavonoids, neoflavonoids, isoflavonoids and other flavonoid derivatives (Barnes 2004; Coon et al. 2007). However, no reports on the secondary metabolites from the flowers have been published.

In this study, we extracted the essential oils of D. odorifera flowers, and analyzed using GC-MS. Furthermore, tectorigenin, the main constituent, was isolated first time from the flowers of D. odorifera in this study. The antioxidant activities of the crude extracts as well as tectorigenin were also determined. As reported in previous studies, tectorigenin was also isolated from the heartwood and leaves, and the differences in the tectorigenin content among different organs were determined by using HPLC.

Materials and Methods

General experimental procedures

NMR spectra were recorded on a Bruker Avance-600 NMR spectrometer (Bruker, USA). HR-ESI-MS was carried out on Bruker maXis with an ESI interface and a Q-TOF mass spectrometer (Bruker, USA). The tectorigenin content was analyzed on a Prominence LC-16 HPLC system (Shimadzu, Japan). HPLC analysis of tectorigenin was performed at 40°C using a reversed-phase C₁₈ column (Phenomenex, USA). Acetonitrile and water (30:70, v/v) with 0.01% TFA were used as the mobile phase and pumped at a flow rate of 1.0 mL/min for isocratic elution, with UV detection at 265 nm. Water was purified by an ultrapure water machine (Exceed-Cb-10, Aike, China). Acetonitrile was of HPLC grade.

Plant material and determination of the water content

Leaves, fresh flowers and current-growth branches of D. odorifera were manually collected in the campus of South China Agricultural University (SCAU) from 16-year-old artificially cultivated D. odorifera in May 2018, in Guangzhou, China. The taxonomical identification of the plant materials was performed by Dr. Mingxuan Zheng of College of Forestry and Landscape Architecture (SCAU, Guangzhou, China), where the voucher specimen (SCAULPMH-180518) of the plant was deposited.

The collected plant materials were first cleaned, and the dirt was removed. Then, the xylem and phloem of the current-growth branches were separated. The leaves, xylem and phloem were cut into small pieces and dried in an oven at 50°C. This process was repeated three times for each sample. The material was weighed before (wet weight) and after (dry weight) drying in the oven for several hours. The materials were dried until a constant weight was reached. The water content was calculated as:

[(Mass _{wet weight}−Mass _{dry weight})/Mass _{wet weight}] × 100%

Preparation, GC and GC-MS analysis of the flower essential oil

The volatile oil was extracted from the flowers (0.52 kg) of D. odorifera by hydro-distillation as our previous reported (Feng et al. 2017). GC and GC-MS were carried out by the same column and analysis conditions. The oven temperature was programmed as our previous reported (Feng et al. 2017).

Extraction of secondary metabolites from the flowers

Three different methods were used to extract secondary metabolites from the D. odorifera flowers. Fresh flowers from D. odorifera were extracted three times with ethyl acetate (EtOAc) at room temperature. The EtOAc extracts were dried under vacuum to obtain the crude extracts (sample 1). After extraction of the essential oils, the residue and water were separated by decreasing the temperature to room temperature. The flower residue was also extracted three times with EtOAc at room temperature. The EtOAc extracts were dried under vacuum to obtain sample 2. The water was first concentrated under vacuum and then extracted three times with EtOAc, and the EtOAc extracts were dried under vacuum to obtain sample 3.

Antioxidant activity analysis

Samples 1–3 were further evaluated the antioxidant activities. The radical-scavenging activity was determined with a spectrophotometric microplate method based on the reduction of a methanolic solution of DPPH according to our previous report (Shan et al. 2020).

Extraction and fractionation of tectorigenin

Sample 3 was a yellow solid and mainly contained one compound as identified on the basis of the HPLC analysis. Therefore, sample 3 was purified by recrystallization and repeated until the compound was purified. Finally, the recrystallized compound was chromatographed on a Sephadex LH-20 column with MeOH-CHCl₃ (1:1, V/V) to afford tectorigenin (14 mg).

Preparation of tectorigenin standard solutions

Tectorigenin (1 mg) was accurately weighed and placed in a 2 mL centrifuge tube, where it was dissolved in 1 mL of methanol and the mother solution was 1000 μg/mL. The mother solution was then diluted to 250 μg/mL～0.9765625 μg/mL with methanol using double and half dilution method, and stored at 4°C before use.

Preparation of sample solutions

D. odorifera xylem, phloem and leaves were extracted three times with EtOAc at room temperature to obtain crude extracts. Each sample solution was filtered (pore size, 0.22 μm) and then dissolved in methanol to attain a concentration of 25 mg/mL.

Method validation

The HPLC-UV method was validated according to the procedures described in ICH guidelines Q2 (R1) (ICH Harmonised Tripartite Guideline 2005) and our previous report (Shan et al. 2012) to validate the analytical methods.

Quantitative determination of tectorigenin in leaves and branches

The contents of tectorigenin in the samples, xylem, phloem and leaves of D. odorifera were determined by HPLC-UV. The crude extracts for the HPLC analysis were prepared as described above. The obtained peak area of tectorigenin was substituted into the regression equation to obtain the concentration and the RSD was calculated.

Statistical analysis

All experiments were carried out in triplicate and the data obtained were compared among treatments using one-way analysis of variance (ANOVA SAS 9.4, SAS Institute, Cary, NC). Tukey’s Honest Significant Differences (HSD) tests were conducted after each ANOVA for multiple comparisons. The significance level was determined at p =0.05 for all tests.

Results

Essential oil analysis

TIC 30min

Fig. 1: Total ion chromatogram (TIC) of essential oil from the flowers of D. odorifera

Fig. 2: The chemical structure of tectorigenin

Fig. 1 shows the total ion chromatogram of the essential oil from the flowers of D. odorifera. The yield of volatile oil from the hydro-distillation of D. odorifera flowers was 0.082% (w/w, fresh weight). The GC-MS results are presented in Table 1. Sixteen components were identified in the essential oil from D. odorifera flowers and made up 96.00% of the volatile oil. Of them, the main components were 4-hydroxy-4-methyl-2-pentanone (28.35%), phenethyl alcohol (12.17%), cis-5-ethenyltetrahydro-α, α-5-trimethyl-2-furanmethanol (8.71%), toluene (7.64%), p-xylene (5.93%), benzyl alcohol (5.72%) and ethylbenzene (5.35%), and the relative contents of these compounds accounted for 73.87% of the total oil content. The relative percentages of the remaining components were below 5%. The essential oils from D. odorifera flowers were mainly consisted of alcohols, hydrocarbons, ketones and nitrogenous compounds on the basis of these data shown in Table 1.

Antioxidant activity

Samples 1–3 were submitted a DPPH free radical scavenging assay (Table 2). Sample 3 displayed the highest antioxidant activity with an IC₅₀ of 0.35 µg/mL, which is higher than the IC₅₀ of BHT, 7.14 µg/mL. Interestingly, both samples 1 and 2 displayed weak antioxidant activity, with IC₅₀ values greater than 200 µg/mL. Sample 3 was derived from the water remaining after extraction of the essential oil and contained compounds with high antioxidant activity, prompting further study.

Elucidation of the purified tectorigenin

As described above, sample 3 displayed the highest antioxidant activity and was therefore further purified by recrystallization and a Sephadex LH-20 column. The molecular formula of tectorigenin, C₁₆H₁₂O₆, was assigned by HR-ESI-MS (Fig. S1): m/z 301.0706 [M+H]⁺ (calcd. 301.0707) and m/z 323.0526 [M+Na]⁺ (calcd. 323.0526). After comparing the obtained ¹H and ¹³C NMR (shown in Table 3) and HR-ESI-MS data with those reported in the literature (Lee et al. 2004), this compound was identified as tectorigenin, and its structure is shown in Fig. 2. Tectorigenin was obtained as a yellow powder (MeOH). However, tectorigenin did not show antioxidant activity at 200 μg/mL (results not shown).

Identification of tectorigenin by HPLC-UV

Table 1: Chemical composition of the essential oil from the flowers of D. odorifera

No.

Retention time

Compound

Molecular formula

Molecular weight

RA (%)^a

3.389

Toluene

C₇H₈

7.64

4.470

Acetic acid, butyl ester

C₆H₁₂O₂

116

3.04

4.731

1-Cyclopropyl-1-methyl-ethylamine

C₆H₁₃N

1.58

5.426

4-Hydroxy-4-methyl-2-pentanone

C₆H₁₂O₂

116

28.35

5.879

Ethylbenzene

C₈H₁₀

106

5.35

Table 2: Antioxidant activities

Samples	IC₅₀ (µg/mL)
Sample 1	246.78 ± 0.99b
Sample 2	316.98 ± 2.52a
Sample 3	0.35 ± 0.01d
BHT	7.14 ± 0.08c

Mean ± standard deviation. Values with same letter differ non-significantly (P>0.05)

Table 3: ¹³C-NMR and ¹H-NMR data for tectorigenin (in DMSO-d₆)

Position	1(δ in ppm, J in Hz)
Position	δ_C	δ_H
2	154.0	8.33 (1H, s)
3	121.8	-
4	180.5	-
5	152.7	-
5-OH	-	13.05 (1H, s)
6	131.4	-
7	153.2	-
7-OH	-	9.59 (1H, s)
8	93.8	6.50 (1H, s)
9	157.5	-
10	104.8	-
11	59.9	3.75 (3H, s)
1'	121.2	-
2', 6'	130.1	7.37 (2H, d, J = 8.5 Hz)
3', 5'	115.0	6.82 (2H, d, J = 8.5 Hz)
4'	157.4	-

6.148

p-Xylene

C₈H₁₀

106

5.93

6.965

o-Xylene

C₈H₁₀

106

1.95

12.108

Benzyl alcohol

C₇H₈O

108

5.72

12.380

Benzeneacetaldehyde

C₈H₈O

120

4.01

13.887

cis-5-Ethenyltetrahydro-α,α-5-trimethyl-2-furanmethanol

C₁₀H₁₈O₂

170

8.71

14.291

Linalool

C₁₀H₁₈O

154

1.58

14.696

Phenethyl alcohol

C₈H₁₀O

122

12.17

14.899

Isophorone

C₉H₁₄O

138

2.31

15.520

Benzyl nitrile

C₈H₇N

117

2.19

15.645

2,6,6-Trimethyl-2-cyclohexene-1,4-dione

C₉H₁₂O₂

152

4.01

25.753

2,4-Di-tert-butylphenol

C₁₄H₂₂O

206

1.46

a: RA indicates the relative amount (individual peak area relative to the total peak area)

The chromatograms of the tectorigenin standard, the real sample solution and the mixed standard solution are shown in Fig. 3. Tectorigenin could be distinguished based on its retention times. Fig. 3 (A) shows the HPLC-UV profile of the tectorigenin standard. The retention time of tectorigenin was 18.50 min when isocratically eluted with MeCN-H₂O (30:70) containing 0.01% TFA, with detection at 265 nm. Fig. 3 (B)-(D) shows the HPLC-UV profiles of the EtOAc extracts of the xylem, phloem and leaves, respectively. The tectorigenin peak eluted at 18.50 min for all three samples. A tectorigenin standard was added to crude extracts and analyzed to confirm the peak at 18.50 min. Fig. 3 (E)-(G) shows the HPLC profiles of the crude xylem, phloem and leaf extracts containing the tectorigenin standard. The peak at 18.50 min in Fig. 3 (E)-(G) was significantly higher than that in Fig. 3 (B)-(D). Therefore, the peak at 18.50 min in Fig. 3 (B)-(D) corresponded to tectorigenin.

Calibration curves

Based on the above results, tectorigenin was further selected for quantitative analysis by HPLC-UV. The linear equation was Y = 3398998.33X - 2834.37 (R = 0.9994), where X represent the quantity (μg) of the sample injected for one time, Y represent the peak area, and R is the correlation coefficient. The R value showed a good linearity over a range of 0.009765625 ~ 2.5 μg of injected sample.

Precision

The mean RSDs in the tectorigenin content for intra- and interday detection at three different levels (Table 4) were less than 1.09% and 1.43%, respectively. Because these values are below 2.0%, the method has good reproducibility.

Accuracy

The accuracy was calculated as the means of a standard addition experiment and the result was shown in Table 5. The mean recovery (n = 9) of tectorigenin in xylem, phloem and leaves were 95.99%, 96.87% and 100.50%, respectively, and the mean RSDs were 0.79%, 0.72% and 0.80%, respectively. These satisfactory recoveries and low RSDs confirmed the suitability of this method for the analysis of tectorigenin.

LOD and LOQ

Under the validated HPLC conditions, the LOD and LOQ were determined at an S/N values of approximately 3 and 10, respectively, from injections of 0.9342 and 1.3023 ng, respectively.

Quantitative determination of tectorigenin in leaves and branches

The water contents are shown in Table 6. The water content in leaves was the highest, followed by that in phloem and then that in xylem, with values of 81.05, 49.52 and 34.63%, respectively. The content of tectorigenin was calculated in leaves and branches on the basis of the water content, as shown in Table 7. The content of tectorigenin in the xylem, phloem and leaves was 75.44, 104.26 and 393.11 µg/g, respectively, on a dry weight basis and 49.32, 51.98 and 74.45 µg/g, respectively, on a fresh weight basis.

Fig. 3: HPLC-UV analysis of tectorigenin in xylem, phloem and leaves of D. odorifera

(A) HPLC profile of the tectorigenin standard; (B)-(D) HPLC profiles of the crude xylem, phloem and leaf extracts, respectively (E)-(G) HPLC profiles of the crude xylem, phloem and leaf extracts containing the tectorigenin standard, respectively

Table 4: Precision and RSDs of the determination of tectorigenin by HPLC

Compound	Intraday (n = 5)		Interday (n = 9)
Compound	Concentration (µg/mL)	RSD (%)	Concentration (µg/mL)	RSD (%)
Tectorigenin	3.91	1.09	3.91	1.17
	31.25	0.50	31.25	1.43
	250.00	0.38	250.00	1.12

Table 5: Recoveries and RSDs of tectorigenin in leaves and branches

Sample	Added concentration (μg/mL)	Recovery (%)	RSD (%)
Xylem	0.5 X	95.21	0.44
	1.0 X	98.42	1.45
	2.0 X	94.33	0.49
Phloem	0.5 X	97.41	1.02
	1.0 X	96.38	0.70
	2.0 X	96.83	0.45
Leaf	0.5 X	101.10	1.09
	1.0 X	100.34	0.81
	2.0 X	100.05	0.49

Table 6: Water content of xylem, phloem and leaves

Samples	Water content (%)
Xylem	34.63 ± 0.55c
Phloem	49.52 ± 3.02b
Leaf	81.05 ± 0.78a

Mean ± standard deviation. Values with same letter differ non-significantly (P>0.05)

Table 7: Contents and RSDs of tectorigenin in leaves and branches

Samples	Average (µg/g dry weight)	Average (µg/g fresh weight)	RSD (%)
Xylem	75.44 ± 0.63c	49.32 ± 0.41c	0.83
Phloem	104.26 ± 1.38b	51.98 ± 0.69b	1.32
Leaf	393.11 ± 2.57a	74.45 ± 0.49a	0.65

Mean ± standard deviation. Values with same letter differ non-significantly (P>0.05)

Discussion

Volatile oil is an important component of D. odorifera. To the best of our knowledge, the heartwoods are a valuable source of essential oils that can be used as a precious perfume fixative. In previous studies, the volatile oils obtained from D. odorifera leaves, heartwood and seeds were analyzed and reported. 2-methoxy-4-vinylphenol and n-hexadecanoic acid with the relative percentages 21.73 and 13.97%, respectively, were the major compounds in volatile oil from leaves (Bi et al. 2004). Liu (2009) reported the major components of the volatile oil obtained from heartwood via steam distillation were nerolidol and caryophyllene oxide, and the relative percentages were 57.36 and 22.22%, respectively. Guo et al. (2011) analyzed the chemical composition of the seeds essential oil and the main components were p, p, p-triphenyl phosphine imide and bis(1-methylethyl) peroxide with the relative percentages 35.3 and 16.4%, respectively. As can be seen from the report above, the major constituents in D. odorifera leaves differ from those in the heartwood and seed oil. However, the chemical composition of volatile oil from D. odorifera flowers has not been reported. In this study, the volatile oil of D. odorifera flowers, extracted by steam distillation and analyzed by GC-MS, revealed the following main components: 4-hydroxy-4-methyl-2-pentanone, phenethyl alcohol, cis-5-ethenyltetrahydro-α, α-5-trimethyl-2-furanmethanol, toluene, p-xylene, benzyl alcohol and ethylbenzene. The present analysis also showed that the types and relative contents of the volatile oil components varied greatly in different parts of plant tissues. In addition, the chemical composition also varies according to the extraction method.

Flavonoids are considered the important active principle components in many herbs because of the extensive pharmacological activities (Zhao et al. 2020a). As previously reported, flavonoids are the main secondary metabolites and about 99 flavonoids have been identified from D. odorifera (Zhao et al. 2020b). In previous reports, pharmacological studies revealed flavonoids to be antioxidant components and parts of flavonoids displayed stronger antioxidant activity than those of BHT (Wang et al. 2000; Hou et al. 2011). In this study, the extract of D. odorifera flowers was found to contain tectorigenin, an isoflavone that did not show any antioxidant activity in the DPPH assay. At present, no reports have described the antioxidant activity of tectorigenin. The above results from other studies are consistent with our results in this study. Sample 3 from the flowers of D. odorifera in this study displayed significant in vitro antioxidant activity, but the active components remain unknown. Therefore, the active antioxidant constituents of D. odorifera flowers should be elucidated in the future.

This investigation has shed light on utilization of the flowers from D. odorifera, a Chinese medicinal plant of greater economic and ecological importance, for the study of essential oils and secondary metabolites. This study is the first of its kind in this plant species which involves the use of flowers for biochemical analysis, unlike previous studies which were primarily focused on heartwood. The evidence also suggests that tectorigenin as the major plant metabolite in flower tissues which needs further functional studies and can prove to be very useful.

Conclusion

The is the first study to clarify chemical composition of volatile oil from flowers of D. odorifera and to identify tectorigenin as the key constituent in the flowers secondary metabolites. The main components of the volatile oil from D. odorifera flowers were 4-hydroxy-4-methyl-2-pentanone, phenethyl alcohol, cis-5-ethenyltetrahydro-α, α-5-trimethyl-2-furanmethanol, toluene, p-xylene, benzyl alcohol and ethylbenzene. The distribution of tectorigenin in branches of D. odorifera was also analyzed. The tectorigenin contents in the xylem, phloem and leaves were determined by HPLC to be 75.44 µg/g, 104.26 µg/g and 393.11 µg/g, respectively, on a dry weight basis and 49.32 µg/g, 51.98 µg/g and 74.45 µg/g, respectively, on a fresh weight basis. This information will pave the way for further functional analysis, experimentation, development and application of D. odorifera flowers with keen focus on tectorigenin as an important antioxidant agent.

Acknowledgements

We thank Hao Zhai and Fan Zhang for the suggestions that greatly improved the manuscript. This research was co-financed by the Forestry Science and Technology Innovation Project of Guangdong Province (2020KJCX004) and the Province Natural Science Foundation of Guangdong (2019A1515011554).

Author Contributions

ZM, YW and TS performed the research and recorded the spectra. ML, TC, JL, HH, TS and DW prepared the flower essential oils and performed the GC-MS analysis. ZM, YW and TS isolated and structurally characterized tectorigenin. HS and JW performed the antioxidant assay. TS, TC, JL and HS performed the HPLC analysis of tectorigenin. TS, YW and CT contributed to the data collection process. TS and DW contributed by preparing figures. TS, DW and ZM designed the research. TS, YW, DW and ZM completed the draft of the manuscript. All the authors contributed to the writing, editing and revising of the manuscript.

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Abstract

Introduction

Materials and Methods

General experimental procedures

Plant material and determination of the water content

Preparation, GC and GC-MS analysis of the flower essential oil

Extraction of secondary metabolites from the flowers

Antioxidant activity analysis

Extraction and fractionation of tectorigenin

Preparation of tectorigenin standard solutions

Preparation of sample solutions

Method validation

Quantitative determination of tectorigenin in leaves and branches

Statistical analysis

Results

Essential oil analysis

Antioxidant activity

Elucidation of the purified tectorigenin

Identification of tectorigenin by HPLC-UV

Calibration curves

Precision

Accuracy

LOD and LOQ

Quantitative determination of tectorigenin in leaves and branches

Discussion

Conclusion

Acknowledgements

Author Contributions

References