Antioxidant and cytotoxic properties of protocorm-derived phenol-rich fractions of Dendrobium amoenum

Abstract

Background

Dendrobium amoenum is known for its aesthetic and medicinal values but it is threatened due to loss of wild resources. Plant tissue culture promotes wild resource protection and paves the way for secondary metabolite production. In this study, protocorms developed via in-vitro seed cultivation were used for bioactive secondary metabolite production. The objectives of this study were to evaluate total phenolic and flavonoid contents, to identify the bioactive secondary metabolites, to explore the antioxidants and cytotoxic properties of in-vitro-derived protocorms extracts of D. amoenum.

Methods

Seeds of D. amoenum were cultivated on 10% coconut water, 0.25 and 0.5 mg/L BAP supplemented full-strength and half-strength MS medium to produce protocorms for the isolation of bioactive components. A distinct yellow fraction (DAYF), light-green fraction (DALGF), green fraction (DAGF), and dark-green fraction (DADGF) were obtained from methanol extract on a methanol-based Sephadex LH-20 column. The total phenol and flavonoid contents along with the antioxidant and cytotoxic properties of the fractions were evaluated. The compounds in active DAYF were identified using a GC-MS.

Results

On a full-strength solid MS medium supplemented with 10% coconut water, approximately 95% of the seeds grew into protocorms, while 88.33% did so on a full-strength liquid MS medium. The DAYF had a total phenol content of 206.38 μg of GAE and a total flavonoid content of 101.88 μg of QE. Owing to these high contents, the DAYF inhibited 50% of the DPPH free radicals at a concentration of 63.73 μg/ml. Similarly, it also reduced the growth of HeLa cells by 50% at 67.03 μg/ml and U2OS cells by 50% at 207.40 μg/ml, while it was nontoxic to normal human epithelium cells. Bioactive phenolic compounds 2-methoxy-4-vinylphenol (1), 3,4-dimethoxy-phenol (2), 2-methoxy-4-(1-propenyl)-phenol (3), 2,6-dimethoxy-4-(2-propenyl)-phenol (4), 3-methoxy-1,2-benzenediol (5) were identified in the DAYF.

Conclusion

Protocorms of D. amoenum could serve as sources of bioactive secondary metabolites highlighting their potential in alternative medicine.

Background

Dendrobium is a highly significant orchid genus known for its aesthetic and medicinal value. The genus has a wide distribution ranging from tropical to temperate regions in Asia to Australia and the Pacific, showing impressive species diversity with approximately 1,800 different species exhibiting variations in size, shape, structure, colour and fragrance [1]. In Nepal, there are 30 different Dendrobium species, including D. amoenum Wall. ex Lindl. stand out as a prominent epiphytic orchid found in the Central and Eastern regions at altitudes ranging from 1100 to 2900 m above sea level [2]. This species is highly stared for its beautiful blossoms and medicinal properties, as its stems are used as a tonic for remedies for stomachache, antipyretic, heart diseases, etc. in traditional Chinese medicine [3]. Several compounds including amoenylin, isoamoenylin, moscatilin, bibenzyl derivatives, phenols, phenanthrenes, and sesquiterpenoids have been isolated from D. amoenum and exhibit antioxidant properties [4–6]. The phenolic and flavonoid content-rich extracts of its stems have antioxidant activity [7]. Studies have revealed that the crude extract of D. amoenum contains antioxidants that have cytotoxic effects on human cervical cancer and glioblastoma cells [8].

Plant tissue culture has attracted much attention for plant regeneration and conservation as well as plant-specific bioactive compound production [9]. This technology promotes wild resource protection, promotes the industrialization of plant propagation in horticulture, and provides raw materials applicable to the pharmaceutical industry [10]. Some studies have even emphasized the use of in-vitro tissue culture to produce secondary metabolites [9, 11–13]. Protocorms are highly accumulated tissue developed via in-vitro seed cultivation and are a source of bioactive secondary metabolites [12, 14, 15]. This study used the tissue culture method for the accumulation of bioactive compounds in protocorms via the cultivation of D. amoenum seeds, which could be used as a valuable approach for pharmaceutical industrial production without damaging its wild resources. The objectives of this study were to evaluate the total phenol and flavonoid contents in the protocorms, to identify compounds in the protocorms, and to evaluate the antioxidant and cytotoxic properties of protocorms. In addition, the ability of the medium to respond quickly to seed germination and the development of protocorms was studied, which not only provides a new idea for the accumulation of potentially bioactive compounds in protocorms and seedlings but also lays a foundation for plant regeneration for the conservation of wild resources.

Materials and methods

Plant materials collection, sterilization and seed culture

The mature capsule of Dendrobium amoenum was collected from Nagarkot in the Bhaktapur district of Nepal (27.7236° N, 85.5247° E). Plant was identified by two authors Prof. Dr. Bijaya Pant and Dr. Mukti Ram Paudel. The voucher specimen of the plant’s herbarium was deposited in the Tribhuvan University Central Herbarium (TUCH). The voucher number of this herbarium is DA01. The permission for the plant collection was obtained from the Department of Forest and Soil Conservation of the Ministry of Forest and Environment, Government of Nepal. The capsule was thoroughly washed under running water with 4–5 drops of Tween-20 for 45 min to remove surface dirt. The capsules were then submerged in a 1% sodium hypochlorite solution for 15 min, followed by rinsing with sterile water. The capsule was then immersed in 70% ethanol for 3–5 min and rinsed three more times with sterile water to ensure complete surface sterilization. The powdery seeds extracted from the sterilized capsule were thinly sown on full- and half-strength of Murashige and Skoog (MS) medium each supplemented with 0.25 and 0.5 mg/L of BAP and 10% coconut water, and in full-strength MS liquid medium. Agar-agar (8 g/L) is used to solidify the MS medium. To maintain sterility and prevent bacterial or fungal contamination, all procedures were performed in a laminar airflow cabinet with a flame. The cultured jars containing the seeds were transferred to an incubation room with controlled conditions of 25 ± 2ºC, 78 ± 5% humidity and a photoperiod of 12–16 h. The growth and development of protocorms from seeds were carefully monitored and recorded, and subcultured regularly for protocorms production throughout the incubation period (12 months long).

Preparation of extract and fractions from protocorms

In-vitro cultivated 12-month-old protocorms (approximately 200 g collected from all treatments) were properly shade-dried at room temperature. The dried protocorms were ground into a fine powder and subjected to 72 h of maceration in 90% methanol, effectively extracting the active phytochemicals. Following maceration, the mixture was filtered to collect the methanol filtrate. The crude methanol extract was then concentrated via a rotary evaporator (Eyela, Japan).

The crude methanol extract was subsequently fractionated via a methanol-based Sephadex LH-20 column. Distinct colour bands were collected separately: the yellow fraction (DAYF), light-green fraction (DALGF), green fraction (DAGF), and dark-green fraction (DADGF). Each fraction was further concentrated via a rotary evaporator under reduced pressure at temperatures below 40 °C to prevent the degradation of active compounds and to remove any remaining solvent, resulting in a dry form. The dried fractions were then accurately weighed and stored at 4 °C.

Determination of total phenolic content

The total phenolic content in the methanol fractions was measured using the Folin-Ciocalteau (FC) colourimetric method [16]. Either the fraction sample (1 mg/ml) or gallic acid (0–100 μg/ml) in 75 μl of distilled water was placed in a 96-well plate, followed by the addition of 25 μl of the FC reagent (diluted 1:1 with distilled water). After a 6-minute incubation, 100 μl of 1 M aqueous Na₂CO₃ was added. Absolute methanol was used as the blank. Following a 90-minute incubation at room temperature, the absorbance at 765 nm was measured via a microplate reader (Azure Biosystems). The total phenolic content was calculated via a linear equation observed based on the absorbance of gallic acid at different concentrations (0–100 μg/ml) and expressed as micrograms of gallic acid equivalent per gram of fraction (μg GAE/gm).

Determination of total flavonoid content

The total flavonoid content was determined using the aluminium chloride (AlCl₃) colourimetric technique [17]. First, 250 μl of the methanol fraction (1 mg/ml) was mixed with 750 μl of 10% AlCl₃ and 50 μl of 1 M potassium acetate. The mixture was then diluted with 1.4 ml of distilled water and allowed to stand at room temperature for 30 min. Absolute methanol was used as the blank. The reaction mixture (200 μl) was transferred in triplicate to a 96-well plate, and the absorbance was measured at 415 nm via a microplate reader (Azure Biosystems). The total flavonoid content was calculated via a linear equation derived from the absorbance of quercetin at different concentrations (0–100 μg/ml in methanol) and expressed as micrograms of quercetin equivalent (μg QE/gm) per gram of fraction.

Evaluation of antioxidant activity

The antioxidant activity was assessed via a 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay with slight modification [18]. A 0.2 mM DPPH solution (7.88 mg of DPPH in 100 ml of methanol) was prepared and stored away from direct sunlight. In a 96-well plate, 50 μl of each fraction (at concentrations ranging from 25 to 200 μg/ml in methanol) was mixed with 150 μl of the DPPH solution. Absolute methanol served as the control. After 30 min of incubation at room temperature in the dark, the absorbance of the reaction mixture was measured at 517 nm via a microplate reader (Azure Biosystems). The fraction with the lowest absorbance presented the highest antioxidant activity. The free radical scavenging activity of the samples was calculated via the following equation:

The percentage of radical scavenging activity was plotted on the Y-axis against the concentration on the X-axis. The IC₅₀ value (in μg/ml), which represents the maximal inhibitory concentration required to inhibit 50% of the DPPH radicals, was determined via the linear equation derived from the plot, which was used to determine the antioxidant capacity of the fraction.

Evaluation of the cytotoxic activity

A modified MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) colourimetric was used to evaluate the cytotoxic effects of methanol fractions on three cell lines: a normal epithelial human cell line and two cancer cell lines (HeLa and U2OS). The cell lines were cultured in Eagle minimum essential medium (EMEM) supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin and 1% L-glutamine, and incubated at 37 °C in 5% CO₂. Once 80% confluency was reached, approximately 10,000 cells in the culture medium were transferred to a 96-well plate and incubated for 24 h. The cells were then treated with different concentrations (25, 50, 100 and 200 μg/ml) of methanol fractions and incubated for 48 h. About 10 μl of 5 mg/ml MTT reagent was added to treated cells, and incubated for 4 h. After a 4-hour incubation with the MTT reagent, purple formazan crystals formed. The formazan crystals were dissolved in dimethyl sulfoxide (DMSO), and the absorbance was measured at 595 nm via a microplate reader (Azure Biosystems). The cytotoxic effect of the fractions was assessed by calculating the percentage of cell death compared with that of the control. The IC₅₀ value, which is the maximal inhibitory concentration of the fraction that reduces cell growth by 50%, was determined from the dose-response curve of the percentage of cytotoxic activity. The percentage of cytotoxic activity was calculated via the following formula:

Identification of compounds in the yellow fraction

The compounds in the yellow fraction (DAYF) were identified via a GC-MS-QP2010 Ultra (Shimadzu Europa GmbH, Germany). Owing to their moderate or negligible cytotoxic activity, other fractions were not subjected to GC-MS. The GC-MS employed an electron ionization system with high-energy electrons (70 eV) for spectroscopic detection. The carrier gas was 99.99% pure helium, which flowed at a rate of 0.95 millilitres per minute. The initial temperature was set at 100 °C, held for 10 min, and then increased at a rate of 3 °C per minute. A splitless injection of one microliter of the 1% fraction diluted in methanol was performed. The relative quantity of the compounds was expressed as a percentage of the peak area generated in the chromatogram. The compounds were identified by comparing their spectra with those of the established standards library and using the GC retention times.

Statistical analysis

The time required for protocorm formation (in weeks) and the percentage of seed germination in the culture medium are expressed as the mean ± standard error. The Duncan test (p ≤ 0.05) was used to determine significant differences in seed germination and protocorm development across different media. This test was also applied to evaluate significant differences in total phenol and flavonoid contents, IC₅₀ values of antioxidant activity, and the growth of HeLa, U2OS, and normal cells across different fractions. All the statistical analyses were performed via IBM SPSS v.20.

Results

In-vitro seed germination and protocorm formation

The germination of seeds and their development into protocorms significantly differed across various strengths of MS solid and liquid medium supplemented with BAP (0.25 and 0.5 mg/L) and 10% coconut water (Table 1). Among the tested media, 88.33% of the seeds germinated in MS liquid medium (LMS), with protocorms developing in an average of 5.67 weeks (approx. 5 weeks and 4 days). Additionally, protocorms developed within 6 weeks on a full-strength MS medium (FMS) supplemented with 10% coconut water (CW), where 95% of the seeds germinated (Fig. 1). The lowest seed germination rate (20%) was observed on a half-strength MS medium (HMS) supplemented with 0.25 mg/L BAP. Protocorms on FMS and HMS supplemented with 0.25 mg/L BAP developed after 10 weeks of seed culture. The results indicate that FMS supplemented with 10% CW and LMS are most suitable for developing protocorms from seeds.

Table 1.

In vitro seed germination percentage and protocorm formation

Medium	Protocorm formation (weeks) (Mean ± SE)	Germination percentage (Mean ± SE)
FMS	7.00 ± 0.00b	83.33 ± 0.16b
HMS	7.83 ± 0.16b	66.67 ± 0.33c
LMS	5.67 ± 0.33a	88.33 ± 1.67a
FMS + 10%CW	6.00 ± 0.00a	95.00 ± 0.00a
HMS + 10%CW	7.33 ± 0.33b	78.33 ± 1.67b
FMS + 0.25BAP	10.33 ± 0.33d	23.33 ± 1.67f
FMS + 0.5BAP	9.33 ± 0.33c	41.67 ± 1.67d
HMS + 0.25BAP	10.67 ± 0.33d	20.00 ± 0.00f
HMS + 0.5BAP	7.33 ± 0.33b	31.67 ± 1.67e

The values in a column with different letters are significantly different at p ≤ 0.05 (Duncan test, n = 6)

Fig. 1.

Protocorms developed on full-strength MS media supplemented with 10% coconut water

Total flavonoid and phenolic contents

The methanol fractions presented significant differences in total flavonoid and phenolic contents. The yellow fraction (DAYF) had the highest total flavonoid content (101.88 μg of QE/mg extract), whereas the dark-green fraction (DADGF) had the lowest total flavonoid content (35.21 μg of QE/mg extract). Similarly, the highest total phenolic content was found in the DAYF (206.38 μg of GAE/mg extract), whereas the DADGF had the lowest total phenolic content (74.80 μg of GAE/mg extract) (Table 2). This study revealed that the DAYF had significantly the highest total flavonoid and phenolic contents than other fractions.

Table 2.

Total flavonoid and total phenolic contents in the methanol extract fractions

Fraction of methanol extract	Total flavonoid content (μg QE/mg extract) (Mean ± SE)	Total phenolic content (μg GAE/mg extract) (Mean ± SE)
Yellow fraction (DAYF)	101.88 ± 0.21a	206.38 ± 0.96a
Green fraction (DAGF)	49.30 ± 0.39c	98.62 ± 0.16c
Light-green fraction (DALGF)	79.73 ± 0.57b	177.58 ± 0.14b
Dark-green fraction (DADGF)	35.21 ± 0.57d	74.80 ± 0.28d

The values in a column with different letters are significantly different at p ≤ 0.05 (Duncan test, n = 3)

Antioxidant activity

The antioxidant activities of the four fractions were evaluated via the DPPH free-radical scavenging assay. The antioxidant activity of the fractions was varied, and the mean percentage of DPPH free-radical scavenging activity was calculated for concentrations ranging from 25 to 200 μg/ml. At a concentration of 200 μg/ml, the yellow fraction (DAYF) showed the highest percentage of DPPH free-radical scavenging activity by 83.24%, followed by the light-green fraction (DALGF), the green fraction (DAGF), and the dark-green fraction (DADGF) (Table 3). The results also revealed that the mean percentage of radical scavenging activity was increased with an increase in the concentration of the fractions.

Table 3.

Antioxidant activity of the methanol extract fractions determined via DPPH radical scavenging

Fraction of methanol extract	Percentage of DPPH radical scavenging activity at different concentrations of fraction (μg/ml) (Mean ± SE)				IC50 (μg/ml) (Mean ± SE)
	25	50	100	200
Yellow fraction (DAYF)	22.58 ± 0.39	53.49 ± 0.56	76.98 ± 0.30	83.24 ± 0.44	63.73 ± 0.27a
Green fraction (DAGF)	10.01 ± 0.39	16.93 ± 0.44	38.97 ± 0.25	51.66 ± 0.45	179.77 ± 1.05c
Light-green fraction (DALGF)	21.55 ± 0.47	38.97 ± 0.44	57.19 ± 0.44	69.94 ± 0.53	105.79 ± 0.41b
Dark-green fraction (DADGF)	10.74 ± 0.53	19.85 ± 0.39	35.82 ± 0.61	46.38 ± 0.52	204.28 ± 1.95d

The values in a column of IC₅₀ values with different letters are significantly different at p ≤ 0.05 (Duncan test, n = 3)

The IC₅₀ values, which represent the concentration needed to scavenge 50% of the DPPH free radicals, varied significantly among the fractions. The DAYF had the lowest IC₅₀ value at 63.73 μg/ml, indicating its high antioxidant activity. Conversely, DADGF had the highest IC₅₀ value at 204.28 μg/ml, indicating lower antioxidant activity (Table 3).

Cytotoxicity activity

The cytotoxic effect of fractions on the HeLa, U2OS and normal cell lines varied across different concentrations (25–200 μg/ml). As the concentration of the fractions increased, a greater proportion of cell growth suppression was observed. For example, at a concentration of 200 μg/ml, the yellow fraction (DAYF) inhibited approximately 82.36% of the HeLa cells, followed by the light-green fraction (DALGF) inhibited 81.84% and the green fraction (DAGF) inhibited 79.07% (Table 4). Similarly, at 200 μg/ml, the dark-green fraction (DADGF) inhibited the growth of U2OS cells by 64.76%, whereas DALGF inhibited it by 60.59%.

Table 4.

Cytotoxic effects of the methanol extract fractions on different cell lines

Fraction of methanol extract	Cell line	Percentage of cell growth inhibition at different concentrations of extract (μg/ml) (Mean ± SE)				IC50 (μg/ml) (Mean ± SE)
		25	50	100	200
Yellow fraction (DAYF)	HeLa	7.76 ± 5.27	25.32 ± 7.08	77.82 ± 0.37	79.07 ± 0.70	67.03 ± 2.39a
	U2OS	15.86 ± 6.31	23.22 ± 1.97	38.76 ± 1.53	46.20 ± 0.31	207.40 ± 6.44cc
	Normal	-7.33 ± 10.35	1.07 ± 6.28	5.27 ± 2.62	9.69 ± 2.67	1338.00 ± 38.16bbb*
Green fraction (DAGF)	HeLa	17.36 ± 9.59	23.02 ± 10.03	47.82 ± 8.29	82.36 ± 2.10	110.93 ± 14.20b
	U2OS	7.69 ± 2.41	22.16 ± 1.92	43.50 ± 2.16	58.38 ± 2.20	155.92 ± 4.37bb
	Normal	-16.56 ± 1.78	-4.73 ± 3.57	6.26 ± 4.03	9.92 ± 15.38	997.78 ± 29.83aaa*
Light-green fraction (DALGF)	HeLa	10.13 ± 5.32	20.78 ± 4.54	67.50 ± 5.01	81.84 ± 2.27	78.00 ± 5.32a
	U2OS	13.00 ± 6.03	30.17 ± 2.58	47.83 ± 1.42	60.59 ± 5.34	142.93 ± 10.26bb
	Normal	2.52 ± 4.96	7.25 ± 6.03	9.39 ± 1.77	13.51 ± 4.43	1686.11 ± 22.12ccc*
Dark-green fraction (DADGF)	HeLa	13.42 ± 14.62	16.77 ± 16.47	47.36 ± 9.94	78.15 ± 1.37	114.87 ± 19.05b
	U2OS	18.23 ± 5.15	32.05 ± 10.07	58.63 ± 1.53	64.76 ± 3.41	119.55 ± 4.61aa
	Normal	-12.67 ± 3.43	0.08 ± 6.29	4.27 ± 3.90	7.86 ± 8.98	1280.69 ± 29.24bbb*

The values in a column of IC₅₀ values with different letters are significantly different at p ≤ 0.05 (Duncan test, n = 3), the values with a single letter indicate HeLa cells, the values with two letters indicate U2OS cells, the values with three letters indicate normal epithelial cell lines, and the values with an asterisk (*) in the IC₅₀ of the normal cell line are significantly different from those of the other two cell lines at p ≤ 0.05 (Dunnett test, n = 3)

In contrast, treatment with methanol fractions had a minimal effect on the growth of normal cells, ranging from − 16.56 to 13.51%, indicating negligible cytotoxicity to normal human cells.

In terms of the IC₅₀ values, DAYF and DALGF showed lower values of 67.03 μg/ml and 78.00 μg/ml, respectively, indicating significantly greater cytotoxicity against HeLa cells than DAGF and DADGF. For U2OS cells, DADGF had the lowest IC₅₀ value of 119.55 μg/ml among the fractions, indicating its greater cytotoxicity. The IC₅₀ values for normal cells were equal to or greater than 1000 μg/ml highlighting the significant non-toxicity of fractions to normal cells (Table 4).

Correlations between total phenolic/flavonoid contents and antioxidant/cell growth inhibition

The total phenolic and flavonoid contents of the methanol fractions were significantly correlated with the IC₅₀ value of the antioxidant activity. The Pearson correlation coefficients of -0.987 and − 0.998 indicate a very strong negative relationship between the IC₅₀ of antioxidant activity and the total phenolic/flavonoid contents (Table 5). This suggests that as the total phenolic and flavonoid contents of the fractions increase, the IC₅₀ value of antioxidant activity decreases, indicating greater efficacy in scavenging DPPH radicals.

Table 5.

Correlations between independent variables (total phenolic and flavonoid contents) and dependent variables (antioxidant and cell growth inhibition capacity)

Dependent variables		Independent variables
		Total flavonoid content	Total phenolic content
IC50 of Antioxidant activity	Pearson correlation (r)	-0.998**	-0.987**
	Sig. (2-tailed)	< 0.001	< 0.001
	N	12	12
IC50 of HeLa cells growth inhibition	Pearson correlation (r)	-0.747**	-0.746**
	Sig. (2-tailed)	0.005	0.005
	N	12	12
IC50 of U2OS cells growth inhibition	Pearson correlation (r)	0.800**	0.701*
	Sig. (2-tailed)	0.002	0.011
	N	12	12
IC50 of Normal cells growth inhibition	Pearson correlation (r)	0.476	0.556
	Sig. (2-tailed)	0.117	0.061
	N	12	12

* significant at p < 0.05; ** significant at p < 0.01

Furthermore, the total phenolic and flavonoid contents were strongly negatively correlated (r = -0.746/-0.747) with the IC₅₀ values for the growth inhibition of HeLa cells highlighting their potent ability to inhibit HeLa cell growth. Conversely, these contents exhibited significantly strong positive correlations (r = 0.701/0.800) with the IC₅₀ values for growth inhibition of U2OS cells. These findings indicate that higher phenolic and flavonoid contents increased the IC₅₀ values, suggesting a moderate cytotoxic effect on U2OS cells. However, the phenolic and flavonoid contents showed a weakly positive (not significant) connection with the IC₅₀ values for inhibiting the proliferation of normal cells, indicating that the phenolic and flavonoid contents had no significant impact on the growth of typical normal cell lines (Table 5).

Identified compounds in the yellow fraction

Altogether 27 compounds were identified in the yellow fraction (DAYF) (Table 6) and the chromatogram of the GC-MS is shown in Fig. 2. The major bioactive phenol derivatives (Fig. 3) detected in the DAYF were 2-methoxy-4-vinylphenol (1), 3,4-dimethoxy-phenol (2), 2-methoxy-4-(1-propenyl)-phenol (3), 2,6-dimethoxy-4-(2-propenyl)-phenol (4), 3-methoxy-1,2-benzenediol (5).

Table 6.

Identified compounds in the yellow fraction (DAYF) via GC-MS

S.N.	Compound Name	RT (min)	Content %	Base m/z
1	5-(Hydroxymethyl)-2-furancarboxaldehyde	4.692	25.38	97.10
2	5-[3-(4-Methoxyphenyl)oxaziridin-2-yl]pentan-1-ol	4.867	4.39	135.15
3	3-Methoxy-1,2-benzenediol	4.957	0.98	78.05
4	2-Methoxy-4-vinylphenol	5.176	1.42	150.20
5	3,4-Dimethoxy-phenol	5.386	2.22	154.20
6	2-Methoxy-4-(1-propenyl)-phenol	6.097	0.47	164.20
7	3’,5’-Dimethoxyacetophenone	6.934	3.36	180.20
8	Diethyl Phthalate	7.090	0.20	149.15
9	2,6-Dimethoxy-4-(2-propenyl)-phenol	7.949	0.87	194.15
10	bis(2-Methylpropyl)-1,2-benzenedicarboxylic acid	9.033	2.43	149.15
11	n-Propargyloxycarbonyl-1-methionine-undecyl ester	11.343	0.24	61.00
12	Dansyl hydroxyl-1-proline	11.387	0.39	44.95
13	Octachloro-dibenzofuran	11.620	0.48	48.00
14	n-Tetracosanol-1	11.771	0.86	57.10
15	Heptadecyl-oxirane	12.603	0.24	43.05
16	3-[(Trimethylsilyl)oxy] propyl hexadecanoic acid	12.887	0.22	43.05
17	Tetratetracontane	12.985	1.42	57.10
18	Mono(2-ethylhexyl) 1,2-benzenedicarboxylic acid	13.439	0.42	149.15
19	Octacosyl trifluoroacetate	13.639	0.71	57.10
20	Heneicosane	14.410	1.10	57.10
21	n-Nonadecanol-1	14.451	0.98	69.10
22	1-Octacosanol	15.102	0.66	57.10
23	dl-.alpha.-Tocopherol succinate	15.820	1.24	430.55
24	1-Heptacosanol	15.892	6.92	57.10
25	Octadecamethyl-cyclononasiloxane	16.000	1.11	430.55
26	Methyl (25rs)-3.beta.-acetoxy-5-cholesten-26-oate	17.710	0.39	105.15
27	Stigmast-5-en-3-ol, oleate	18.066	1.69	147.20

Fig. 2.

Chromatogram of the yellow fraction (DAYF)

Fig. 3.

Molecular structure of phenol derivatives identified in the yellow fraction (DAYF)

2-methoxy-4-vinylphenol (1), 3,4-dimethoxy-phenol (2), 2-methoxy-4-(1-propenyl)-phenol (3), 2,6-dimethoxy-4-(2-propenyl)-phenol (4), 3-methoxy-1,2-benzenediol (5)

Discussion

The structure that bridges between an embryo and a seedling during seed germination in a nutrient medium is termed a protocorm. Orchid seeds, lacking endosperm and cotyledons, develop into protocorms before maturing into seedlings [19, 20]. Protocorms typically exhibit a globular or spherical shape and are often green or cream-yellow sometimes featuring foliaceous structures [21]. In the orchid life cycle, protocorms play a crucial role in serving as hosts for symbionts and eventually developing into shoot apical meristems [22, 23].

This study explored the influence of growth regulators such as BAP and coconut water on the multiplication efficiency of Dendrobium amoenum protocorms. BAP and coconut water significantly affect protocorm production because coconut water is rich in cytokinin which plays a significant role in cell division stages during the development of tissues in the protocorms [10–12, 15, 21, 24]. Thus, BAP and coconut water were added to the nutrient medium for crucial seed germination and subsequent development of protocorms [19, 21, 22].

The primary objective of the current study was to produce bioactive secondary metabolites in in-vitro-grown protocorms. Previous research has demonstrated the widespread production of bioactive secondary metabolites in the cells and tissues of in vitro-grown protocorms and plantlets [9, 12, 14, 15]. The synthesis of flavonoid and phenolic compounds in protocorms highlights the ability of plant genes to produce such molecules. The cytokinin in the nutrient medium affects the development of highly organised tissues and may favour the plant genes to produce secondary metabolites during multiplication and differentiation [25]. During the development of cells and tissues, phenolic compounds are synthesized via gene action [26, 27]. Previous studies evaluated the high concentrations of total phenol and flavonoid contents in Dendrobium stems [7, 28]. The methanol extract of D. amoenum, which is rich in bioactive flavonoid and phenol compounds, has demonstrated potent antioxidant and cytotoxic properties in previous studies [7, 8]. The ability of phenolic and flavonoid compounds to be synthesized in the plant tissue culture is supported by previous research on Dendrobium, making plant tissue culture an effective method for producing these compounds without relying on wild plants [9–14].

The yellow fraction of D. amoenum (DAYF) contains significant amounts of biologically active chemicals (Table 6) that potentially contribute to its antioxidant and anticancer properties [29–32]. Antioxidant-rich compounds, including flavonoid and phenol derivatives, play crucial roles in cancer chemoprevention [33]. Phenolic and flavonoid compounds from plants may inhibit cancer cell formation by altering the metabolic activation of carcinogens through xenobiotic-metabolizing enzymes, and certain flavonoids can influence hormone synthesis to suppress malignant cell growth [34, 35].

Conclusions

The full-strength solid MS medium supplemented with coconut water as well as liquid MS medium are effective for protocorm formation from seeds. The yellow fraction has the highest amount of total phenolic and flavonoid contents and has bioactive phenol derivatives. It effectively scavenges more than 50% of the DPPH radicals and significantly inhibits HeLa cell proliferation at low concentration. Importantly, it does not harm normal epithelial cells. Protocorms could serve as a source of bioactive secondary metabolites to pave out their potential in antioxidants and anticancers.

Abbreviations

BAP

6-Benzylaminopurine

CW

Coconut water

DADGF

Dark-green fraction

DAGF

Green fraction

DALGF

Light-green fraction

DAYF

Yellow fraction

DPPH

2,2-Diphenyl-1-picrylhydrazyl

FMS

Full-strength Murashige and Skoog medium

GAE

Gallic acid equivalent

GC-MS

Gas chromatography and mass spectrometry

HeLa

Human cervical cancer cell line

HMS

Half-strength Murashige and Skoog medium

IC₅₀

Half-maximal inhibitory concentration

LMS

Liquid Murashige and Skoog medium

MS

Murashige and Skoog medium

QE

Quercetin equivalent

U2OS

Human osteosarcoma cell line

Author contributions

M.R.P. did conceptualization of the research and methodology, analyzed and interpreted data, and wrote manuscript draft; S.S. did laboratory investigation and wrote manuscript draft; P.R.J. did laboratory investigation; B.P. managed laboratory resources; S.H.W. managed resources and contributed in editing manuscript; P.G. managed laboratory resources; K.P. managed laboratory resources; B.P. did project administration and funding acquisition. All authors read and approved the final manuscript.

Funding

This work was supported by the University Grants Commission, Nepal (Grant No. CRG/077–078/S&T-4). The funder has no role in the conceptualization, design, data collection, analysis, or decision to prepare and publish the manuscript.

Data availability

All data generated or analysed during this study are included in this published article.

Declarations

Ethics approval and consent to participate

The permission for the plant collection was obtained from the Department of Forest and Soil Conservation of the Ministry of Forest and Environment, Government of Nepal (Reference No.: 334 − 079/80).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Clinical trial number

Not applicable.